HK1218428B - Recombinant rsv with silent mutations, vaccines, and methods related thereto - Google Patents

Description

Recombinant RSV with silent mutations, related vaccines and methods

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 61/781,228, filed March 14, 2013, and U.S. Provisional Application No. 61/890,500, filed October 14, 2013, both of which are incorporated by reference in their entirety.

Technical Field

The present application relates to, but is not limited to, recombinant RSV with silent mutations, vaccines related thereto, and methods.

background

Respiratory syncytial virus (RSV) causes lower respiratory tract infections. Immunocompromised patients, premature infants, and children are particularly at risk for severe disease. RSV is the leading cause of viral death in infants. RSV treatment focuses on preventing infection and improving breathing. Palivizumab is a humanized monoclonal antibody that can be administered prophylactically. Palivizumab is ineffective after RSV infection, and protection ends shortly after treatment is stopped. Vaccines are currently unavailable for RSV. Candidate attenuated RSV vaccines have failed due to suboptimal immunogenicity in infants and suboptimal stability that leads to genetic reversion to undesirable wild-type sequences. See Teng, Infectious Disorders–Drug Targets, 2012, 12(2):129-3. Therefore, there is a need to find an attenuated RSV vaccine that is appropriately immunogenic, sufficiently stable, and safe for use in infants.

Due to the redundancy of the genetic code, a single amino acid is encoded by multiple sequences of codons, which are sometimes referred to as synonymous codons. In different species, synonymous codons are used more or less frequently, sometimes referred to as codon bias. Genetically engineering synonymous codons that occur too infrequently into the coding sequence of a gene has been shown to result in a reduced protein translation rate without changes in the amino acid sequence of the protein. Mueller et al. reported on virus attenuation by changes in codon bias. See Science, 2008, 320: 1784. See also WO/2008121992, WO/2006042156, Burns et al., J Virology, 2006, 80(7): 3259 and Mueller et al., J Virology, 2006, 80(19): 9687.

Luongo et al. reported improving the genetic and phenotypic stability of a candidate live attenuated respiratory syncytial virus vaccine by reverse genetics. See Journal of Virology, 2012, 86(19):10792.

Dochow et al. Report independent domains in the paramyxovirus polymerase protein. J Biol Chem, 2012, 287:6878–91.

US Patent No. 8,580,270 reports the RSV F polypeptide sequence. US Patent No. 7,951,384 reports that it covers a VLPRSV vaccine.

The citation of references herein is not an admission that prior art is intended.

Overview

In certain embodiments, present disclosure relates to the polynucleotide sequence of respiratory syncytial virus (RSV).In certain embodiments, present disclosure relates to the nucleic acid and polypeptide comprising the separation or reorganization of the desired nucleotide sequence disclosed herein and mutant.In certain embodiments, there is also provided the RSV (for example, attenuated recombinant RSV) comprising the separation or reorganization of the nucleic acid and polypeptide disclosed herein, and there is also provided the immunogenic composition comprising this type of nucleic acid, polypeptide and RSV genome suitable for use as a vaccine.Also contemplated are the attenuation or inactivation RSV containing these nucleic acids and mutants in the form of nucleic acid (for example, cDNA) copies.

In certain embodiments, present disclosure relates to isolated nucleic acid, recombinant respiratory syncytial virus (RSV) with codon deoptimization, the vaccine produced therefrom and the vaccination method associated therewith.In certain embodiments, recombinant RSV virus comprises gene NS1, NS2, N, P, M, SH, G, F, M2 and L of A2 strain, strain 19, or its Long strain or variant.In certain embodiments, codon deoptimization is in nonstructural genes NS1 and NS2 and optionally in a kind of gene G and optionally in a kind of gene L.In other embodiments, gene SH is deleted.In other embodiments, gene F is mutated, for example, corresponding to a kind of I to V mutation of residue 557 of RSV strain 19F protein.

In certain embodiments, the disclosure relates to isolated nucleic acids encoding reverse optimized genes NS1 and/or NS2 and optionally gene G and optionally gene L of a wild-type human RSV or variant, wherein the nucleotides are substituted such that the codon for Gly is GGT, the codon for Asp is GAT, the codon for Glu is GAA, the codon for His is CAT, the codon for Ile is ATA, the codon for Lys is AAA, the codon for Leu is CTA, the codon for Asn is AAT, the codon for Gln is CAA, the codon for Val is GTA, the codon for Tyr is TAT, or a combination thereof. In certain embodiments, a gene in the isolated nucleic acid further comprises a combination of at least two, three, four, five, six, seven, eight, nine, ten, or all of the individual codons. In certain embodiments, a gene in the isolated nucleic acid comprises at least 20, 30, 40, or 50 or more of these codons.

In certain embodiments, the disclosure relates to isolated nucleic acids as disclosed herein, wherein nucleotides are substituted such that the codon for producing Ala is GCG, the codon for producing Cys is TGT, the codon for producing Phe is TTT, the codon for producing Pro is CCG, the codon for producing Arg is CGT, the codon for producing Ser is TCG, or the codon for producing Thr is ACG or a combination thereof. In certain embodiments, a gene containing the nucleic acid comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, or a combination thereof of these individual codons. In certain embodiments, a gene in the isolated nucleic acid further comprises at least 20, 30, 40, or 50 or more of these codons.

^{27 MTX 28} YX ^{29 X 30} QX ^{31 SX 32 LLGX 33 DLX 34} X ^{35 , wherein X 1 -X 35 are any amino acids or X 1 -X 35 are nucleotide sequences of the present invention . wherein X1} is S or C; ^X2 is S or T; ^X3 is M or V; ^X4 is V or I; ^X5 is F or L; ^X6 is D or N; ^X7 is T or A; ^X8 is H, L or Q; ^X9 is V or T; ^X10 is V or I; ^X11 is D or E; ^X12 is I, A or V; ^X13 is N or D; ^X14 is N or S; ^X15 is V, I or A; ^X16 is Q or R; ^X17 is W or any amino acid; ^X18 is M or L; ^X19 is M or I; ^X20 is P or L; ^X21 is L or V; ^X22 is L, M or I; ^X23 is D or V; ^X24 is K or R; ^X25 is K or R; ^X26 is S or any amino acid; ^X27 is T or V; X ^X28 is N or D; ^X29 is M or I; ^X30 is N or S; ^X31 is L or I; ^X32 is E or D; ^X33 is F or L; ^X34 is N or H; and ^X35 is P or S or is absent.

In certain embodiments, the disclosure relates to nucleic acids disclosed herein that encode NS1 of RSV, such as those identified by NCBI Accession Nos. NP_044589.1, NP_056856.1, P04544.1, AEQ63513.1, AFM55237.1, AFV32554.1, Q86306.1, AFV32528.1, AFM55248.1, AFM95358.1, AFV32568.1, ACY68428.1, CBW45413.1, ACO83290.1, AFM55347.1, CBW45433.1, AEQ634 or variants comprising one, two or three amino acid insertions, deletions, substitutions or conservative substitutions.

In certain embodiments, the disclosure relates to an isolated nucleic acid comprising SEQ ID NO: 6 or SEQ ID NO: 7, or a sequence having 60%, 70%, 80%, 90%, 95% or greater sequence identity thereto.

^{8 X 27 YKKX 28 TEYNTKYGTFPMPIFIX 29 HX 30 GFX 31 ECIGX 32 KPTKHTPIIX 33 KYDLNP , ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​}

wherein X ¹ -X ³³ are any amino acids or

^X1 is D or S; ^X2 is T, A or K; ^X3 is H, S or N; ^X4 is N or P; ^X5 is D, G or E; ^X6 is T or N; ^X7 is P, M, Q, S or A; ^X8 is R or G; ^X9 is M or I; ^X10 is L or M; ^X11 is L, M or I; ^X12 is I, D or E; ^X13 is T or S; ^X14 is I or V; ^X15 is I or T; ^X16 is R or K; ^R17 is D or E; ^R18 is R or K; ^R19 is H or N; ^X20 is R or K; ^X21 is R or K; ^X22 is F or L; ^X23 is T or A; ^X24 is K or N; ^X25 is K or R; ^X26 is T or A; X ^X27 is K or I; ^X28 is T or S; ^X29 is N or any amino acid; ^X30 is D or G; ^X31 is L or I; ^X32 is I or V; and ^X33 is Y or H.

In certain embodiments, the disclosure relates to nucleic acids disclosed herein that encode nucleic acids having, for example, NCBI accession numbers

NP_044590.1, NP_056857.1, CBW45420.1, AFM95337.1, CBW45416.1, CBW4543 0.1, AFV32529.1, Q86305.1, AEQ63383.1, CBW45424.1, AFM55546.1, CBW4544 4.1, P04543.2, AFM55326.1, AFM55425.1, AFM55381.1, AFM55458.1, AFM5521 6.1, AAB59851.1, AEQ63372.1, AFM55337.1, CBW45426.1, AFV32515.1, AFV325 19.1, AAR14260.1, CBW47562.1, AFV32643.1, P24569.1, AFV32657.1, AFI25256.1, CBW45480.1, AFV32605.1, AEQ63580.1, AFV32627.1, AFV32665.1, CBW45482.1, CBW45478.1, CBW45462.1, AEQ63635.1, CBW45448.1, CBW45464.1, CBW45484.1, or a variant comprising one, two, or three amino acid insertions, deletions, substitutions, or conservative substitutions.

In certain embodiments, the disclosure relates to an isolated nucleic acid comprising SEQ ID NO: 9 or SEQ ID NO: 10, or a sequence having 60%, 70%, 80%, 90%, 95% or greater sequence identity thereto.

In certain embodiments, the present disclosure relates to recombinant vectors comprising a nucleic acid disclosed herein.

In certain embodiments, the disclosure relates to an attenuated recombinant RSV comprising a nucleic acid disclosed herein.

In certain embodiments, the disclosure relates to an expression system comprising a vector disclosed herein or an attenuated recombinant RSV disclosed herein.

In certain embodiments, the disclosure relates to vaccines comprising the attenuated recombinant RSV disclosed herein.

In certain embodiments, the present disclosure relates to vaccination methods comprising administering to a subject at risk for RSV infection an effective amount of a vaccine disclosed herein.

In certain embodiments, the subject is less than 2 or 6 months of age, under 1 year of age, was born prematurely, has congenital heart disease or lung disease, is undergoing chemotherapy or a transplant, or has been diagnosed with asthma, congestive heart failure or chronic obstructive pulmonary disease, leukemia, or HIV/AIDS.

In certain embodiments, the vaccine is administered in combination with motavizumab, palivizumab, or another humanized monoclonal antibody directed against an epitope in antigenic site II of the F protein of RSV.

In certain embodiments, the present disclosure relates to vectors disclosed herein, which vectors comprise a bacterial artificial chromosome (BAC) and a nucleic acid sequence comprising respiratory syncytial virus (RSV), and the BAC contains all genes necessary for producing an infectious viral particle in a host cell. The nucleic acid sequence can be a viral genome or antigenome in an operable combination with a regulatory element. Typically, the bacterial artificial chromosome comprises one or more genes in an operable combination with a selective marker (e.g., a gene providing resistance to an antibiotic), the one or more genes being selected from the group consisting of oriS, repE, parA, and parB genes of factor F.

The nucleotide sequence can be the genome or antigenome sequence of a virus (e.g., an RSV strain that is optionally mutated) as provided herein. In certain embodiments, the expression vector is a plasmid comprising an MluI, ClaI, BstB1, SacI restriction endonuclease cleavage site and an AvrII restriction endonuclease cleavage site that is optionally located outside the region of the wild-type viral sequence or outside the sequence encoding the viral gene or outside the viral genome or antigenome. In certain embodiments, the nucleotide sequence further comprises a selective marker or reporter gene that is operably combined therewith, for example, a gene encoding a fluorescent protein.

In certain embodiments, present disclosure relates to the bacterium of the separation comprising one or more of the carriers disclosed herein, and in other embodiments, present disclosure relates to a cell comprising the separation of one or more of the carriers disclosed herein. In certain embodiments, this carrier comprises an RSV antigenome and one or more of the carriers selected from the group consisting of: a carrier of the N protein of encoding RSV, a carrier of the P protein of encoding RSV, a carrier of the L protein of encoding RSV, and a carrier of the M2-1 protein of encoding RSV. Typically, this carrier comprises a regulatory element (for example, a promoter), and the eukaryotic expression of separation activates a nucleic acid or polypeptide of this regulatory element, for example, a polypeptide encoding the transcription of this promoter downstream. In certain embodiments, this promoter is T7, and the polypeptide activating the transcription of this promoter downstream is T7RNA polymerase.

In certain embodiments, the disclosure relates to methods for producing respiratory syncytial virus (RSV) particles, comprising inserting a vector with a BAC gene and an RSV antigenome into an isolated eukaryotic cell, and inserting one or more vectors selected from the group consisting of a vector encoding the N protein of RSV, a vector encoding the P protein of RSV, a vector encoding the L protein of RSV, and a vector encoding the M2-1 protein of RSV into the cell under conditions such that RSV virions are formed. Inserting a vector into a cell can occur by physical injection, electroporation, or mixing the cell with the vector under conditions such that the vector enters the cell.

In certain embodiments, the disclosure relates to the stability of the strain 19F557 mutant virus compared to other strains, and the val at 557 that makes RSV expressing strain 19F even more thermostable. Val at position 557 in other strains may also be stable; therefore, position 557 is important for thermostability. In certain embodiments, the disclosure encompasses other mutations at position 557 (any amino acid, e.g., alanine, valine, isoleucine, leucine) in strain 19F or other RSV strains that improve the thermostability of RSV viruses in any F strain background.

In certain embodiments, the disclosure encompasses RSV F polypeptides comprising an alanine, a valine, a leucine at position 557 (eg, an alanine or a leucine at position 557 of SEQ ID NO: 17).

In certain embodiments, the disclosure relates to a desired sequence of RSV F polypeptides, such as the Strain 19 sequence comprising a valine at position 557, such as SEQ ID NO: 17, and recombinant nucleic acids encoding these polypeptides. In certain embodiments, the disclosure encompasses recombinant vectors comprising nucleic acids encoding these polypeptides and cells comprising the vectors.

In certain embodiments, present disclosure relates to immunogenic compositions, and these compositions comprise recombinant respiratory syncytial virus (RSV), RSV polypeptide, RSV particle, RSV virus-like particle and/or nucleic acid disclosed herein of immune effective dose.In certain embodiments, present disclosure relates to the method for stimulating the immune system of individuality to produce the protective immune response for RSV.In certain embodiments, in a kind of physiologically acceptable carrier, give a kind of RSV, polypeptide and/or nucleic acid disclosed herein of immune effective dose to this individuality.

In certain embodiments, the disclosure relates to pharmaceutical and vaccine products comprising the nucleic acids disclosed herein for the uses disclosed herein.

In certain embodiments, the disclosure relates to the use of a nucleic acid or vector disclosed herein for the manufacture of a medicament for the uses disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows a table with the least used codons in human genes and in specific RSV strains.

Figure 2 shows growth data for kRSV-dNS1h in BEAS-2B (top) and Vero cell lines (bottom). Growth curves for kRSV-A2 (open circles) and kRSV-dNSh (filled circles) at 37°C in HEp-2 (A), Vero cells (B), and BEAS-2B cells (C) infected at an MOI of 0.01, and in differentiated NHBE/ALI cells infected at an MOI of 0.2 (D) or 2.0 (E).

Figure 3 shows data from viral load experiments using certain embodiments disclosed herein. Time course images of NHBE cells infected at an MOI of 0.2, showing mKate2 fluorescence produced by recombinant viruses. *P<0.05

Figure 4 shows a gel after the galK operon was inserted into BAC-RSV by recombineering. MluI digestion. Lane 1, gradient marker. Microprep BAC DNA (lanes 2 to 7). Lane 8, parent BAC-RSV "C2" clone. Lane 9, galK-containing plasmid. The galK operon has an MluI restriction site that serves as a marker for the introduction of galK by homologous recombination.

Figure 5 shows a gel after deletion of the galK operon from BAC-RSV by recombineering. MluI digest of galK-containing plasmid (lane 2), BAC miniprep DNA (lanes 3-7), and parental BAC-RSV clone C2 (lane 8).

6A-6E schematically illustrate the steps for producing BAC-RSV. Three plasmids with RSV segments were generated (see experiment); A) pKBS3 was cut at the BstB1 and Mlul sites for linearization and connected to an oligonucleotide adapter to provide pKBS5; B) pSynRSV#2 with Sac1 and Clal was cut and connected to pKBS5 to provide pKBS5-2; C) pSynRSV#3 with Avr11 and Mlul was cut and connected to pKBS5-2 to provide pKBS5-2-3; D) pSynRSV#1 with BstB1 and Sac1 was cut and connected to pKBS5-2-3 to provide pKBS5-1-2-3; E). Recombineering was used to delete the nucleotides between the two Clal sites to produce pSynRSV-Strain 19F.

Figure 7 shows data indicating the immunogenicity of a RSV strain with an F gene I557 to V mutation. Mice were infected with the indicated doses of A2-K- strain 19F, A2- strain 19F-I557V or A2-K-A2GF and challenged with RSV strain 12-35 after 29 days. Lung viral load was measured on the 4th day after challenge. The dotted line indicates the detection limit.

Fig. 8 shows the data indicating the excellent thermal stability of RSV strains with A2-strain 19F gene I557 to V mutation (SEQ ID NO: 17). Viruses were incubated at the indicated temperature and viral titers were measured every day for 6 days. The results at 4 ° C were statistically significant (P < 0.01) between viruses. The results at 37 ° C showed the same phenotype.

Figure 9 shows a RSV sequence comparison of strain 19, I557V mutation (SEQ ID NO: 17) (query sequence) and a canonical RSV strain 19 sequence (target sequence).

Figure 10 shows a comparison of the RSV sequence of strain 19, I557V mutation (SEQ ID NO: 17) (query sequence) and sequence 61 from US Patent No. 7,951,384 (target sequence).

Figure 11 shows a comparison of the RSV sequence of strain 19, I557V mutation (SEQ ID NO: 17) (query sequence) and sequence 12 from US Patent No. 8,580,270 (target sequence).

Figure 12 shows data on the attenuation, efficacy, and immunogenicity of the embodiments disclosed herein. (A) 6-8 week old BALB/c mice (n=5/group) were intranasally infected with 1.6×105FFU of kRSV-A2 (open circles) or kRSV-dNSh (filled circles) and lung viral titers were measured on day 1, day 2, day 4, day 6, and day 8 after infection. Data represent one of two replicate experiments with similar results. *P<0.05. (B) BALB/c mice were intranasally vaccinated with different doses (105FFU, 104FFU, and 103FFU) of kRSV-A2 (open circles) or kRSV-dNSh (filled circles), or mock infected, and 100 days after vaccination, the mice were challenged with 1.6×106PFU RSV 12-35 strain. Peak lung viral load was measured on day 4 after challenge. Each symbol represents one mouse. Dashed lines (A and B) represent the detection limit for plaque assays. Titers below the detection limit were assigned half the value of the detection limit. (C) BALB/c mice (n = 5/group) were mock infected or infected with 10^FFU of either kRSV-A2 or kRSV-dNSh, and serum nAb titers were measured on the indicated days post-infection. *P < 0.05.

Figure 13 shows data regarding the efficacy of vaccines used in certain embodiments disclosed herein. 6-8 week old BALB/c mice (n=5/group) were mock infected or vaccinated with different indicated doses of kRSV-A2 (open circles) or kRSV-dNSh (filled circles). 28 days later, mice were challenged with (A) 2 x 10⁶ PFU of RSV A2-strain 19 or (B) 5 x 10⁵ PFU of RSV 12-35. Lung viral loads were measured on day 4 post-challenge. Each symbol represents a single mouse. The dotted line represents the detection limit for the plaque assay. Titers below the detection limit were assigned half the value of the detection limit.

Figure 14 shows data on the expression of NS1 and NS2 proteins during RSV infection in cell lines. HEp-2 (A), BEAS-2B (B) and Vero (C) cells were simulated infected or infected with kRSV-A2, kRSV-dNSh or kRSV-dNSv at MOI 5. Twenty hours after infection, NS1 and NS2 protein levels were analyzed by Western blotting and densitometry. Representative blots are shown on the left. Densitometry from 2-3 independent experiments is shown on the right. After being standardized to RSV N protein levels, NS1 and NS2 protein levels expressed by each virus were standardized to protein levels during kRSV-A2 infection and expressed as percentage ± SEM. Unfilled bars represent kRSV-A2, gray bars represent kRSV-dNSv, and black bars represent kRSV-dNSh.

Detailed description

Before describing the present disclosure in more detail, it should be understood that this disclosure is not limited to the particular embodiments described, as such may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and incorporated herein by reference to disclose and describe the methods and/or materials in conjunction with the cited publications. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to an earlier filing date than such publication by reason of prior disclosure. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

As will be clear to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any other embodiments without departing from the scope or spirit of the disclosure. Any recited method may be carried out in the order of events recited or in any other order that is logically possible.

Unless otherwise indicated, the embodiments of the present disclosure will employ techniques of immunology, medicine, organic chemistry, biochemistry, molecular biology, pharmacology, physiology, etc., which are within the skill of the art and are fully explained in the literature.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this specification and the following claims, reference will be made to a number of terms that shall be defined as having the following meanings, unless a contrary intention is evident.

Before describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated.

The terms "protein" and "polypeptide" refer to compounds comprising amino acids linked by peptide bonds and are used interchangeably.

When used in reference to a protein, the term "portion" (as in "a portion of a given protein") refers to fragments of that protein. Fragments can range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.

When used with respect to polypeptides, the term "chimera" refers to the expression product of two or more coding sequences obtained from different genes that have been cloned together and function as a single polypeptide sequence after translation. Chimeric polypeptides are also referred to as "hybrid" polypeptides. These coding sequences include those obtained from organisms of the same or different species.

When used in relation to polypeptides, the term "homolog" or "homologous" refers to a high degree of sequence identity between two polypeptides, or to a high degree of similarity between the three-dimensional structures, or to a high degree of similarity between the active sites and mechanisms of action. In a preferred embodiment, a homolog has greater than 60% sequence identity, and more preferably greater than 75% sequence identity, and still more preferably greater than 90% sequence identity to a reference sequence.

When applied to polypeptides, the term "substantial identity" means that two peptide sequences share at least 80% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity or greater (e.g., 99% sequence identity) when optimally aligned using default gap weights, such as by the programs GAP or BESTFIT. Preferably, residue positions that are not identical differ by conservative amino acid substitutions.

When used in relation to polypeptides, the terms "variant" and "mutant" refer to amino acid sequences that differ from one another in one or more amino acids, typically related polypeptides. The variant may have "conservative" changes, in which the substituted amino acids have similar structural or chemical properties. One type of conservative amino acid substitution refers to the interchangeability of residues with similar side chains. For example, a group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic-hydroxy side chains is serine and threonine; a group of amino acids with amide-containing side chains is asparagine and glutamic acid; a group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids with basic side chains is lysine, arginine, and histidine; and a group of amino acids with sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamic acid. More rarely, variants may have "non-conservative" changes (e.g., replacing glycine with tryptophan). Similar minor variations may also include amino acid deletions or insertions (in other words, additions), or both. Guidance for determining which or how many amino acid residues can be substituted, inserted, or deleted without eliminating biological activity can be found using computer programs well known in the art (e.g., DNAStar software). Variants can be tested in functional assays. Preferred variants have less than 10%, and preferably less than 5%, and still more preferably less than 2% variation (substitutions, deletions, etc.).

The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence comprising the coding sequence necessary to produce RNA, or a polypeptide or its precursor (e.g., proinsulin). A functional polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence, as long as the desired activity or functional properties of the polypeptide (e.g., enzymatic activity, ligand binding, signal transduction, etc.) are retained. When used with respect to a gene, the term "portion" refers to a fragment of the gene. The size of the fragment can range from a few nucleotides to the entire gene sequence minus one nucleotide. Therefore, "nucleotides comprising at least a portion of a gene" can comprise a fragment of the gene or the entire gene.

The term "gene" also encompasses the coding region of a structural gene and includes sequences located adjacent to the coding region at a distance of approximately 1 kb on either end at both the 5' and 3' ends such that the gene corresponds to the length of the full-length mRNA. Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains a coding region interspersed with non-coding sequences called "introns," "intervening regions," or "insertion sequences." Introns are segments of a gene that are transcribed into nuclear RNA (mRNA); introns may contain regulatory elements, such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; therefore, they are not present in messenger RNA (mRNA) transcripts. During translation, mRNA serves to specify the sequence or order of amino acids in a nascent polypeptide.

In addition to containing introns, the genomic form of a gene can also include sequences located at the 5' and 3' ends of these sequences present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript). The 5' flanking region can contain regulatory sequences, such as promoters and enhancers that control or influence the transcription of the gene. The 3' flanking region can contain sequences that direct the termination of transcription, post-transcriptional cleavage, and polyadenylation.

The term "heterologous gene" refers to a gene encoding a factor that is not in its natural environment (i.e., altered by the hand of man). For example, a heterologous gene includes a gene introduced from one species into another species. A heterologous gene also includes a gene that is naturally present in an organism that has been altered in some way (e.g., mutated, added with multiple copies, connected to a non-native promoter or enhancer sequence, etc.). The difference between a heterologous gene and an endogenous plant gene is that the heterologous gene sequence is typically connected to a nucleotide sequence comprising a regulatory element such as a promoter, which is not found to be naturally associated with the gene for the protein encoded by the heterologous gene or with a plant gene sequence in a chromosome, or is associated with a part of a chromosome not found in nature (e.g., a gene expressed in a locus that does not normally express a gene).

The term "polypeptide" refers to a molecule comprising two or more deoxyribonucleotides or ribonucleotides (preferably more than three and typically more than ten). The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. Polynucleotides can be produced in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. The term "oligonucleotide" generally refers to a single-stranded polynucleotide chain of a shorter length, typically less than 30 nucleotides long, but it can also be used interchangeably with the term "polynucleotide."

The term "nucleic acid" refers to a polymer of nucleotides or a polynucleotide as described above. The term is used to refer to a single molecule or a collection of molecules. Nucleic acids can be single-stranded or double-stranded and can include coding regions and various control elements, as described below.

The term "polynucleotide having a nucleotide sequence encoding a gene" or "nucleic acid sequence encoding a specified polypeptide" refers to a nucleic acid sequence comprising the coding region of a gene or, in other words, a nucleic acid sequence encoding a gene product. The coding region may be present in the form of cDNA, genomic DNA or RNA. When present in the form of DNA, the oligonucleotide, polynucleotide or nucleic acid may be single-stranded (i.e., the positive strand) or double-stranded. Suitable control elements, such as enhancers/promoters, splice junctions, polyadenylation signals, etc., may be placed adjacent to the coding region of the gene if necessary to allow proper initiation of transcription and/or correct processing of the original RNA transcript. Alternatively, the coding region used in the expression vector of the present disclosure may contain endogenous enhancers/promoters, splice junctions, intervening sequences, polyadenylation signals, etc., or a combination of both endogenous and exogenous control elements.

When referring to nucleic acid molecules, the term "recombinant" refers to nucleic acid molecules comprising nucleic acid segments joined together by molecular biology techniques. When referring to proteins or polypeptides, the term "recombinant" refers to protein molecules expressed using recombinant nucleic acid molecules.

The terms "complementary" and "complementarity" refer to polynucleotides (i.e., a sequence of nucleotides) that are related by the base pairing rules. For example, the sequence "A-G-T" is complementary to the sequence "T-C-A". Complementarity can be "partial", in which only the bases of some nucleic acids are matched according to the base pairing rules. Alternatively, there can be "complete" or "total" complementarity between multiple nucleic acids. The degree of complementarity between nucleic acid chains has a significant impact on the efficiency and strength of hybridization between nucleic acid chains. This is particularly important in amplification reactions and detection methods that rely on binding between nucleic acids.

When used with respect to nucleic acid, the term "homology" refers to a certain degree of complementarity. There may be partial homology or complete homology (i.e., consistency). "Sequence identity" refers to a measure of dependency between two or more nucleic acids or proteins, and is given as a percentage relative to a total comparison length. Consistency calculations consider those nucleotides or amino acid residues that are consistent or in the same relative position in their respective larger sequences. The calculation of consistency can be performed by the algorithm included in a computer program, such as "GAP" (Genetics Computer Group, Madison, Wisconsin) and "ALIGN" (DNAStar, Madison, Wisconsin). A partially complementary sequence is a sequence that at least partially suppresses (or competes with) a fully complementary sequence from hybridizing with a target nucleic acid - functional terms are referred to as "substantially homologous." The suppression of fully complementary sequence hybridization with a target sequence can be tested using hybridization assays (Southern or Northern blotting, solution hybridization, etc.) under conditions of low stringency. Substantially homologous sequences or probes will compete with and inhibit the binding (i.e., hybridization) of sequences that are completely homologous to the target under conditions of low stringency. This is not to say that conditions of low stringency are such that nonspecific binding is permitted; low stringency conditions require that the binding of two sequences to each other is a specific (i.e., selective) interaction. The lack of nonspecific binding can be tested by using a second target that lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of nonspecific binding, the probe will not hybridize to the second, non-complementary target.

The following terms are used to describe the sequence relationships between two or more polynucleotides: "reference sequence," "sequence identity," "percentage of sequence identity," and "substantial identity." A "reference sequence" is a defined sequence used as a basis for sequence comparison; a reference sequence can be a subset of a larger sequence, for example, a segment of a full-length cDNA sequence given in a sequence listing or can comprise the complete gene sequence. Typically, a reference sequence is at least 20 nucleotides in length, often at least 25 nucleotides in length, and usually at least 50 nucleotides in length. Because two polynucleotides can each (1) contain a sequence that is similar between the two polynucleotides (i.e., a portion of the complete polynucleotide sequence), and (2) can further contain a sequence that diverges significantly from the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing the sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a conceptual segment of at least 20 contiguous nucleotide positions in which a polynucleotide sequence can be compared to a reference sequence of at least 20 contiguous nucleotides, and wherein the portion of the polynucleotide sequence in the comparison window can contain 20% or fewer additions or deletions (i.e., gaps) as compared to the reference sequence (which does not contain additions or deletions), thereby optimally aligning the two sequences. Optimal alignment of sequences for alignment over a comparison window can be performed by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math. 2:482 (1981)), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol. 48:443 (1970)), by the similarity study method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. (U.S.) 85:2444 (1988)), by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA, Wisconsin Genetics Software Package Release 7.0), and by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA, Wisconsin Genetics Software Package Release 7.0). 7.0), Genetics Computer Group, 575 Science Dr., Madison, Wisconsin), or by inspection, and the best alignment produced by each method is selected (i.e., producing the highest homology percentage over the comparison window). The term "sequence identity" means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the comparison window.

In certain embodiments, the term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the comparison window, determining the number of positions at which identical nucleic acid bases (e.g., A, T, C, G, U, or I) appear in the two sequences, thereby obtaining the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

In certain embodiments, sequence "identity" refers to the number of exactly matched amino acids between the two sequences aligned in a sequence alignment (expressed as a percentage), which is calculated using the number of identical positions divided by the number of equivalent positions in the larger sequence or excluding overhangs in the shortest sequence, with internal gaps counted as equivalent positions. For example, the polypeptides GGGGGG and GGGGT have four-fifths or 80% sequence identity. For example, the polypeptides GGGPPP and GGGAPPP have six-sevenths or 85% sequence identity. In certain embodiments, any reference to sequence identity expressed herein can be replaced by sequence similarity. The "similarity" percentage is used to quantify the similarity between the two aligned sequences. This method is the same as determining identity, except that certain amino acids do not have to be identical to have a match. Amino acids can be classified as matches if they are in a group with similar properties according to the following amino acid groups: aromatic - F Y W; hydrophobic - A V I L; positively charged: R K H; negatively charged - D E; Polar - S T N Q.

As used herein, the term "substantial identity" indicates a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence having at least 85% sequence identity, preferably at least 90% to 95% sequence identity, over a comparison window of at least 20 nucleotide positions, often over a comparison window of at least 25-50 nucleotides, compared to a reference sequence, wherein the percent sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence, which polynucleotide sequence may contain deletions or additions totaling 20% or less of the reference sequence over the comparison window. The reference sequence may be a subset of a larger sequence, for example, as a segment of the full-length sequence of a composition claimed in the present disclosure.

When used in reference to a double-stranded nucleic acid sequence such as a cDNA or a genomic clone, the term "substantially homologous" refers to any probe that will hybridize to one or both strands of the double-stranded nucleic acid sequence under conditions of low to high stringency as described above.

When used in reference to a single-stranded nucleic acid sequence, the term "substantially homologous" refers to any probe that hybridizes to (i.e., is the complement of) the single-stranded nucleic acid sequence under conditions of low to high stringency as described above.

The terms "operably combined," "in an operable order," and "operably linked" refer to the linkage of nucleic acid sequences in such a manner as to produce a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule. The terms also refer to the linkage of amino acid sequences in such a manner as to produce a functional protein.

The term "regulatory element" refers to a genetic element that controls some aspect of the expression of a nucleic acid sequence. For example, a promoter is a regulatory element that facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc.

Transcriptional control signals in eukaryotes include "promoter" and "enhancer" elements. Promoters and enhancers consist of short arrays of DNA sequences that specifically interact with cellular proteins involved in transcription (Maniatis et al., Science 236:1237, 1987). Promoter and enhancer elements have been isolated from genes in a variety of eukaryotic sources, including yeast, insects, mammals, or plant cells. Promoter and enhancer elements have also been isolated from viruses and are present in prokaryotes. The selection of specific promoters and enhancers depends on the cell type used to express the target protein. Some eukaryotic promoters and enhancers have a wide host range, while other eukaryotic promoters and enhancers are functional in a limited subset of cell types (for review see Voss et al., Trends Biochem. Sci., 11:287, 1986; and Maniatis et al., ibid., 1987).

As used herein, the term "promoter element," "promoter," or "promoter sequence" refers to a DNA sequence located at the 5' end (before) of the protein coding region of a DNA polymer. Most promoters known in nature are located before the transcription region. The promoter acts as a switch to activate the expression of a gene. If a gene is activated, it is said to be transcribed or participates in transcription. Transcription involves the synthesis of mRNA from the gene. Therefore, the promoter acts as a transcriptional regulatory element and also provides a site for initiating transcription of a gene into mRNA.

Promotor can be tissue-specific or cell-specific.When being used for promotor, term " tissue-specific " refers to under the situation of the expression that does not have identical target nucleotide sequence relatively in different tissue types (for example, leaf), can instruct the promoter of target nucleotide sequence for the selective expression of specific tissue type (for example, seed).The tissue-specificity of promotor can be assessed by for example the following manner: a kind of reporter gene is operably connected to this promoter sequence to produce a kind of report construct, this reporter construct is introduced in the genome of a kind of plant, so that this reporter construct is integrated in each tissue of gained transgenic plant, and detects the expression of this reporter gene (for example, detecting mRNA, albumen or the activity of the protein encoded by this reporter gene) in the different tissues of this transgenic plant.Detecting the expression level of this reporter gene in one or more tissues is higher than the expression level of this reporter gene in other tissues and shows that this promoter has specificity to the tissue that wherein detects larger expression level.As being used for promotor, term " cell-type specific " refers to under the situation of the expression that does not have identical target nucleotide sequence relatively in the different cell types in the same tissue, can instruct the promoter of the selective expression of target nucleotide sequence in specific cell type. "Cell type specificity" when used for a promoter also means a promoter that can promote the selective expression of a target nucleotide sequence in a region within a single tissue. The cell type specificity of a promoter can be assessed using methods well known in the art, such as immunohistochemical staining. In brief, tissue sections are embedded in paraffin and the paraffin sections are reacted with a first antibody specific for a polypeptide product encoded by a target nucleotide sequence whose expression is controlled by the promoter. A labeled (e.g., peroxidase-coupled) second antibody specific for the first antibody is bound to the dissected tissue and specific binding detection is performed by microscopy (e.g., using avidin/biotin).

Promoters can be constitutive or regulatable. When referring to a promoter, the term "constitutive" means that the promoter is capable of directing transcription of an operably linked nucleic acid sequence in the absence of a stimulus (e.g., heat shock, chemicals, light, etc.). Typically, a constitutive promoter is capable of directing expression of a transgene in essentially any cell and any tissue.

In contrast, a "regulatable" or "inducible" promoter is a promoter that is capable of directing a level of transcription of an operably linked nucleic acid sequence in the presence of a stimulus (e.g., heat shock, chemicals, light, etc.) that is different from the level of transcription of the operably linked nucleic acid sequence in the absence of the stimulus.

Enhancers and/or promoters can be "endogenous" or "exogenous" or "heterologous". An "endogenous" enhancer or promoter is one that is naturally associated with a given gene in the genome. An "exogenous" or "heterologous" enhancer or promoter is one that is placed adjacent to a gene by methods of genetic manipulation (i.e., molecular biology techniques) such that transcription of the gene is directed by the associated enhancer or promoter. For example, an endogenous promoter that is operably combined with a first gene can be isolated, removed, and placed in operably combined with a second gene, thereby preparing it as a "heterologous promoter" that is operably combined with the second gene. A variety of such combinations are contemplated (e.g., the first and second genes can be from the same species, or from different species).

Efficient expression of recombinant DNA sequences in eukaryotic cells generally requires the expression of signals that direct the efficient termination and polyadenylation of the resulting transcript. Transcription termination signals are typically present downstream of the polyadenylation signal and are several hundred nucleotides in length. As used herein, the terms "poly(A) site" or "poly(A) sequence" refer to DNA sequences that direct the termination and polyadenylation of nascent RNA transcripts. Efficient polyadenylation of recombinant transcripts is desirable because transcripts lacking a poly(A) tail are unstable and rapidly degraded. The poly(A) signal utilized in an expression vector can be either "heterologous" or "endogenous." An endogenous poly(A) signal naturally occurs in the genome at the 3' end of the coding region of a given gene. A heterologous poly(A) signal is a poly(A) signal isolated from one gene and placed 3' to another gene. One commonly used heterologous poly(A) signal is the SV40 poly(A) signal. This SV40 poly(A) signal is contained on a 237 bp BamHI/BclI restriction fragment and directs termination and polyadenylation.

The term "vector" refers to a nucleic acid molecule that transfers one or more DNA segments from one cell to another. The term "vehicle" is sometimes used interchangeably with "vector."

The term "expression vector" or "expression cassette" refers to a recombinant nucleic acid containing a desired coding sequence or appropriate nucleic acid sequences for expression of an operably linked coding sequence in a particular host organism. Nucleic acid sequences for expression in prokaryotes typically include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

The term "host cell" refers to any cell capable of replicating and/or transcribing and/or translating a heterologous gene. Thus, a "host cell" refers to any eukaryotic or prokaryotic cell (e.g., a bacterial cell such as an E. coli cell, a yeast cell, a mammalian cell, an avian cell, an amphibian cell, a plant cell, a fish cell, and an insect cell), whether located in vitro or in vivo. For example, a host cell can be located in a transgenic animal.

The term "selective marker" refers to a gene encoding an enzyme with activity that confers antibiotic or drug resistance to the cells in which the selective marker is expressed, or that confers expression of a detectable trait (e.g., luminescence or fluorescence). Selective markers can be "positive" or "negative." Examples of positive selective markers include the neomycin phosphotransferase (NPTII) gene that confers resistance to G418 and kanamycin, and the bacterial hygromycin phosphotransferase gene (hyg) that confers resistance to the antibiotic hygromycin. Negative selective markers encode an enzyme activity whose expression is cytotoxic to the cells when grown in an appropriate selective medium. For example, the HSV-tk gene is commonly used as a negative selective marker. The expression of the HSV-tk gene in cells grown in the presence of ganciclovir or acyclovir is cytotoxic; therefore, the growth of cells in a selective medium containing ganciclovir or acyclovir selects cells that are able to express a functional HSV TK enzyme.

The term "reporter gene" refers to a gene that encodes a protein that can be measured. Examples of reporter genes include, but are not limited to, modified katushka, mkate, and mkate2 (see, e.g., Merzlyak et al., Nat. Methods, 2007, 4, 555-557 and Shcherbo et al., Biochem. J., 2008, 418, 567-574), luciferase (see, e.g., de Wet et al., Mol. Cell. Biol. 7:725 (1987) and U.S. Pat. Nos. 6,074,859; 5,976,796; 5,674,713 and 5,618,682; all of which are incorporated herein by reference), green fluorescent protein (e.g., Genbank Accession No. U43284; various GFP variants are commercially available from ClonTech Laboratories, Palo Alto, CA). Laboratories, Palo Alto, Calif.), chloramphenicol acetyltransferase, β-galactosidase, alkaline phosphatase, and horseradish peroxidase.

When referring to a gene, the term "wild-type" refers to a gene that has the characteristics of a gene isolated from a naturally occurring source. When referring to a gene product, the term "wild-type" refers to a gene product that has the characteristics of a gene product isolated from a naturally occurring source. As used herein, the term "naturally occurring" for an object refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified by humans in the laboratory is naturally occurring. A wild-type gene is the gene most frequently observed in a population and is therefore arbitrarily referred to as the "normal" or "wild-type" form of a gene. In contrast, when referring to a gene or gene product, the terms "modified" or "mutated" refer to a gene or gene product that exhibits modifications (i.e., altered characteristics) in sequence and/or functional properties when compared to the wild-type gene or gene product, respectively. It should be noted that naturally occurring mutants can be isolated; these mutants are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.

The term "antisense" or "antigenome" refers to a nucleotide sequence whose nucleotide residue sequence is in the opposite 5' to 3' orientation relative to the nucleotide residue sequence of the sense strand. The "sense strand" of a DNA duplex refers to the strand of the DNA duplex that is transcribed into "sense mRNA" by the cell in its native state. Therefore, an "antisense" sequence is a sequence that has the same sequence as the non-coding strand of a DNA duplex.

The term "isolated" refers to a biological substance such as a virus, nucleic acid or protein, which does not have a component that is usually accompanied or interacts with it in the environment of its natural generation. The isolated material optionally comprises the material not found in its natural environment such as a cell. For example, if the material is in its natural environment such as a cell, the material has been placed in the cell so that it is not natural position (for example, genome or genetic factor) for the material found in the environment. For example, the nucleic acid of natural generation (for example, coding sequence, promoter, enhancer etc.) becomes isolated, if it is introduced into the locus of the genome (for example, vector, such as plasmid or viral vector, or amplicon) for the nucleic acid by the means of non-natural generation. This type of nucleic acid is also referred to as "heterologous" nucleic acid. The isolated virus is in the environment (for example, cell culture system, or purification from cell culture) outside the natural environment (for example, infecting individual nasopharynx) of the wild-type virus.

An "immunologically effective amount" of RSV is an amount sufficient to enhance an individual's (e.g., human) autoimmune response to subsequent exposure to RSV. The level of induced immunity can be monitored, for example, by measuring the amount of neutralizing secretory and/or serum antibodies (e.g., by plaque neutralization, complement fixation, enzyme-linked immunosorbent assay, or microneutralization assay).

A "protective immune response" against RSV refers to a protective immune response exhibited by an individual (eg, a human) against severe lower respiratory tract disease (eg, pneumonia and/or bronchiolitis) when the individual (eg, a human) is subsequently exposed to and/or infected with wild-type RSV.

Recombinant respiratory syncytial virus (RSV) with silent codon usage mutations in nonstructural genes

Candidate live attenuated RSV vaccine has two major obstacles, that is, suboptimal immunogenicity in infants and suboptimal stability causing genetic reversion for wild type and revertant shedding of vaccinee. Virus nonstructural (NS) protein NS1 and NS2 are unique and suppress type I interferon and T cell response. Make NS1/NS2 mutation enhance immunogenicity for vaccine. However, NS1 and NS1/NS2 deletion/null mutant recombinant RSV strains previously developed are overly attenuated, and NS2 null mutant is insufficiently attenuated in vivo.

The mutants disclosed herein overcome the limitations of excessive attenuation and instability. Mutants with partial NS1 and NS2 functions are generated to bridge the attenuation immunogenicity gap for pediatric vaccines. NS1/NS2 mutants are generated by changing the codon usage on the NS1 and NS2 genes using gene synthesis and the RSV BAC rescue system. Codon deoptimization reduces translation efficiency through various mechanisms (e.g., tRNA concentration and mRNA structure). The 84/420nt mutation of NS1 and the 82/375nt mutation of NS2 in a mutant disclosed herein ("dNSh") reduce human codon preference and do not change the amino acid sequence. This virus produces approximately 25% of the wt NS1 level, 25% of the wt NS2 level, and 100% of the wt nucleoprotein level, and replicates similar wt viruses in Vero cells, which are cell lines commonly used to produce attenuated live RSV under GMP conditions (Fig. 2). In addition to reducing NS expression, this method also solves the problem of genetic stability because there are many mutations for reversion.

In certain embodiments, the disclosure relates to vaccines, recombinant RSV genomes, or isolated recombinant nucleic acids encoding RSV NS1, NS2, N, P, M, G, F, M2-1, M2-2, and a codon-deoptimized L gene comprising the NS1 and NS2 genes, wherein the codon deoptimization is configured such that at least one codon for Gly is GGT, the codon for Asp is GAT, at least one codon for Glu is GAA, at least one codon for His is CAT, at least one codon for Ile is ATA, at least one codon for Lys is AAA, at least one codon for Leu is CTA, at least one codon for Asn is AAT, at least one codon for Gln is CAA, at least one codon for Val is GTA, or at least one codon for Tyr is TAT, wherein greater than 25% of the Asp, Glu, His, Ile, Lys, Leu, Asn, Gln, Val, and Tyr amino acids are codon-deoptimized. In certain embodiments, greater than 75% of the amino acids are codon-deoptimized compared to the wild-type sequence, eg, RSV A2 strain 19.

In certain embodiments, the NS1 gene comprises (SEQ ID NO: 6) or a variant thereof having greater than 70%, 80%, 90%, 95%, 97%, 98%, or 99% or greater sequence identity thereto.

In certain embodiments, the NS2 gene comprises (SEQ ID NO: 9) or a variant thereof having greater than 70%, 80%, 90%, 95%, 97%, 98%, or 99% or greater sequence identity thereto.

In certain embodiments, the RSV small hydrophobic (SH) glycoprotein gene is deleted.

In certain embodiments, the nucleic acid has further codon deoptimization of the G gene, wherein the codon deoptimization is configured such that at least one codon for Gly is GGT, the codon for Asp is GAT, at least one codon for Glu is GAA, at least one codon for His is CAT, at least one codon for Ile is ATA, at least one codon for Lys is AAA, at least one codon for Leu is CTA, at least one codon for Asn is AAT, at least one codon for Gln is CAA, at least one codon for Val is GTA, or at least one codon for Tyr is TAT, wherein greater than 25% of the Asp, Glu, His, Ile, Lys, Leu, Asn, Gln, Val, and Tyr amino acids are codon deoptimized.

In certain embodiments, the G gene comprises SEQ ID NO:18ATGTCGAAAAAAAGACCAACGTAACCGCGAAGACGTTAGAACGTACCTGGGATACTCTAAATCATTTACTATTCATATCGTCGTGCCTATATAAGCTAAATCTTAAATCGGTAGCACAAATAACACTATCCATACTGGCGATAATAATCTCGACTTCGCTTATAATAGCAGCGATCATATTTATAGCCTCGGCGAACCATAAAGTCACGCCAACGACTGCGATCATACAAG ATGCGACATCGCAGATAAAGAATACAACGCCAACGTACCTAACCCAAAATCCTCAACTTGGTATCTCGCCCTCGAATCCGTCTGAAATAACATCGCAAATCACGACCATACTAGCGTCCAACGACACCGGGAGTAAAGTCGACCCTACAATCCACGACAGTAAAGACGAAAAACACGACAACGACTCAAACGCAACCCTCGAAGCCGACCACGAAACAACGCCAAAATAAACCACCGA GCAAACCGAATAATGATTTTCACTTTGAAGTATTCAATTTTGTACCCTGTAGCATATGTAGCAATAATCCAACGTGCTGGGCGATCTGTAAAAGAATACCGAACAAAAAACCGGGAAAAAAAACCACGACCAAACCCACGAAAAAACCAACGCTCAAAACAACGAAAAAAGATCCCAAACCGCAAACCACGAAATCAAAAGAAGTACCCACGACCAAACCCACGGAAGAGCCGACCATAAACACGACCAAAACGAACATAATAACTACGCTACTCACGTCCAATACCACGGGAAATCCGGAACTCACGAGTCAAATGGAAACGTTTCACTCGACTTCGTCCGAAGGTAATCCATCGCCTTCGCAAGTCTCGACAACGTCCGAATACCCGTCACAACCGTCATCGCCACCGAACACGCCACGTCAGTAG, or a variant thereof having greater than 70%, 80%, 90%, 95%, 97%, 98% or 99% or greater sequence identity thereto.

In certain embodiments, the G gene comprises SEQ ID NO:19ATGTCGAAAAATAAAGACCAACGTACGGCGAAGACGCTAGAACGTACCTGGGATACGCTAAATCATTTACTATTTATATCGTCGTGCCTATATAAACTAAATCTTAAATCGGTAGCGCAAATAACACTATCGATACTGGCGATAATAATATCGACTTCGCTAATAATAGCAGCGATAATATTTATAGCCTCGGCGAATCATAAAGTCACGCCGACGACTGCGATAATACAAG ATGCGACATCGCAAATAAAGAATACGACGCCAACGTATCTAACCCAAAATCCGCAACTTGGTATATCGCCCTCGAATCCGTCGGAAATAACATCGCAAATAACGACCATACTAGCGTCGACGACACCGGGTGTAAAGTCGACGCTACAATCCACGACGGTAAAGACGAAAAATACGACAACGACGCAAACGCAACCGTCGAAACCGACCACGAAACAACGTCAAAATAAACCACCGT or a variant thereof having greater than 70%, 80%, 90%, 95%, 97%, 98% or 99% or greater sequence identity thereto. CGAAACCGAATAATGATTTTCACTTTGAAGTATTTAATTTTGTACCCTGTTCGATATGTAGCAATAATCCGACGTGCTGGGCGATATGTAAAAGAATACCGAATAAAAAACCGGGAAAAAAAACGACGACCAAACCGACGAAAAAACCAACGCTAAAAACAACGAAAAAAGATCCGAAACCGCAAACCACGAAATCGAAAGAAGTACCCACGACGAAACCCACGGAAGAACCGACCATAAATACGACCAAAACGAATATAATAACTACGCTACTAACGTCCAATACGACGGGAAATCCGGAACTAACGAGTCAAATGGAAACGTTTCATTCGACTTCGTCGGAAGGTAATCCATCGCCGTCGCAAGTCTCGACGACTTCCGAATATCCGTCACAACCGTCGTCGCCACCGAATACGCCACGTCAATAG, or a variant thereof having greater than 70%, 80%, 90%, 95%, 97%, 98% or 99% or greater sequence identity thereto.

In certain embodiments, the G gene comprises SEQ ID NO:20ATGTCGAAAAATAAAGATCAACGTACGGCGAAAACGCTAGAACGTACGTGGGATACGCTAAATCATCTACTATTTATATCGTCGTGTCTATAAACTAAATCTAAAATCGGTAGCGCAAATAACGCTATCGATACTAGCGATAATAATATCGACTTCGCTAATAATAGCGGCGATAATATTTATAGCGTCGGCGAATCATAAAGTAACGCCGACGACGGCGATAATACAAG ATGCGACTTCGCAAATAAAAAATACGACGCCGACGTATCTAACGCAAAATCCGCAACTAGGTATATCGCCGTCGAATCCGTCGGAAATAACGTCGCAAATAACGACGATACTAGCGTCGACGACGCCGGGTGTAAAATCGACGCTACAATCGACGACGGTAAAAACGAAAAATACGACGACGACGCAAACGCAACCGTCGAAACCGACGACGAAACAACGTCAAAATAAACCGCCGT CGAAACCGAATAATGATTTTCATTTTGAAGTATTTAATTTTGTACCGTGTTCGATATGTTCGAATAATCCGACGTGTTGGGCGATATGTAAACGTATACCGAATAAAAAACCGGGTAAAAAAACGACGACGAAACCGACGAAAAAACCGACGCTAAAAACGACGAAAAAAGATCCGAAACCGAAACGACGAAATCGAAAGAAGTACCGACGACGAAACCGACGGAAGAACCGACGA TAAATACGACGAAAACGAATATAATAACGACGCTACTAACGTCGAATACGACGGGTAATCCGGAACTAACGTCGCAAATGGAAACGTTTCATTCGACtTCGTCGGAAGGTAATCCGTCGCCGTCGCAAGTATCGACGACtTCGGAATATCCGTCGCAACCGTCGTCGCCGCCGAATACGCCGCGTCAATAG, or a variant thereof having greater than 70%, 80%, 90%, 95%, 97%, 98% or 99% or greater sequence identity thereto.

In certain embodiments, the F gene encodes valine at position 557 and lysine at position 66. In certain embodiments, the F gene encodes valine at position 557 and the F gene comprises a sequence encoding one or more of the following amino acid sequences. The F gene comprises two, three, four, five, or all of the following amino acid sequences: TTNIMITTIIIVIIVILLSLIAVGLLLYCK (SEQ ID NO: 11),

ARSTPVPILKANAITTILAAVTFCFA(SEQ ID NO:12),AVTFCFASSQNITEEFYQST(SEQ ID NO:13),QSTCSAVSKGYLSALRTGWYTSVITIELSNIKK(SEQ ID NO:14),IKKNKCNGTDAKVKLMKQELDKYKNAV(SEQ ID NO:15), and FPQAEKCKVQSNRVFCDTMYSLTLPSEVNLCNV (SEQ ID NO:16).

In certain embodiments, the F gene comprises two, three, four, five, or all of the following amino acid sequences: (SEQ ID NO: 11), (SEQ ID NO: 12), (SEQ ID NO: 13), (SEQ ID NO: 14), (SEQ ID NO: 15), and (SEQ ID NO: 16).

In certain embodiments, the F gene encodes a valine at position 557 and the F gene encodes one or more of the following amino acids: asparagine at position 8, phenylalanine at position 20, serine at position 35, lysine at position 66, methionine at position 79, lysine at position 124, arginine at position 191, arginine at position 213, glutamate at position 354, lysine at position 357, tyrosine at position 371, valine at position 384, asparagine at position 115, and threonine at position 523.

In certain embodiments, the F gene encodes valine at position 557, lysine at position 66, and methionine at position 79.

In certain embodiments, the F gene encodes valine at position 557, lysine at position 66, and arginine at position 191.

In certain embodiments, the F gene encodes a valine at position 557, a lysine at position 66, an arginine at position 191, and a lysine at position 357.

In certain embodiments, the F gene encodes a valine at position 557, a lysine at position 66, a methionine at position 79, and an asparagine at position 115.

In certain embodiments, the F gene encodes SEQ ID NO:17MELPILKANAITTILAAVTFCFASSQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLMKQELDKYKNAVTELQLLMQSTPAANNRARRELPRFMNYTLNNTKKTNVTLSKKRKRRFLGFLLGVGSAIASGIAVS KVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSRVLDLKNYIDKQLLPIVNKQSCRISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWK LHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAEKCKVQSNRVFCDTMYSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN, or a variant containing one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions, with the proviso that the amino acid at F gene encoding position 557 is valine. In certain embodiments, the amino acid substitutions are conservative substitutions.

In certain embodiments, the disclosure relates to an isolated recombinant nucleic acid comprising an F gene encoding (SEQ ID NO: 17) or a variant containing one or two amino acid substitutions, with the proviso that the F gene encodes valine at position 557 and lysine at position 66.

In certain embodiments, the F gene encodes a valine at position 557 and the F gene encodes one or more of the following amino acids: asparagine at position 8, phenylalanine at position 20, serine at position 35, lysine at position 66, methionine at position 79, lysine at position 124, arginine at position 191, arginine at position 213, glutamate at position 354, lysine at position 357, tyrosine at position 371, valine at position 384, asparagine at position 115, and threonine at position 523.

In certain embodiments, the F gene encodes valine at position 557 and lysine at position 66.

In certain embodiments, the F gene encodes valine at position 557, lysine at position 66, and methionine at position 79.

In certain embodiments, the F gene encodes a valine at position 557, a lysine at position 66, an arginine at position 191, and a lysine at position 357.

In certain embodiments, the F gene encodes a valine at position 557, a lysine at position 66, a methionine at position 79, and an asparagine at position 115.

In certain embodiments, the disclosure relates to a recombinant vector comprising a nucleic acid disclosed herein. In certain embodiments, the disclosure relates to a cell comprising a recombinant vector, recombinant RSV, or attenuated recombinant RSV disclosed herein.

In certain embodiments, the disclosure relates to an F gene encoding (SEQ ID NO: 17) or a variant containing one amino acid substitution, with the proviso that the F gene encodes valine at position 557.

In certain embodiments, the present disclosure relates to a method encoding MELPILKANAITTILAAVTFCFASSQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLMKQELDKYKNAVTELQLLMQSTPAANNRARRELPRFMNYTLNNTKKTNVTLSKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSRVLDLKNYIDKQLLPIVNKQSCRISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQI In certain embodiments, the F gene encodes a valine at position 557, a glutamic acid at position 66, and an arginine at position 191.

In certain embodiments, the disclosure relates to recombinant polypeptides comprising a RSV F protein sequence disclosed herein. In certain embodiments, the disclosure relates to viral particles or virus-like particles produced by recombinant methods comprising a RSV F protein sequence disclosed herein.

In certain embodiments, the disclosure relates to an isolated recombinant nucleic acid comprising an RSV genome OE1 of SEQ ID NO: 1 or a variant thereof having greater than 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater sequence identity.

In certain embodiments, the disclosure relates to an isolated recombinant nucleic acid comprising an RSV genome OE2 of SEQ ID NO:2 or a variant thereof having greater than 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater sequence identity.

In certain embodiments, the disclosure relates to an isolated recombinant nucleic acid comprising an RSV genome OE3 of SEQ ID NO:3 or a variant thereof having greater than 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater sequence identity.

In certain embodiments, the disclosure relates to an isolated recombinant nucleic acid comprising an RSV genome OE4 of SEQ ID NO:4 or a variant thereof having greater than 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater sequence identity.

In certain embodiments, the present disclosure encompasses isolated recombinant nucleic acids comprising the RSV genomes OE1, OE2, OE3, and OE4, wherein one or both of the NS1 and NS2 genes are deleted.

Cultivation of RSV in bacterial artificial chromosomes

Cultivation of RSV in E. coli can be accomplished by utilizing bacterial artificial chromosomes (BACs). A BAC containing the complete antigenome sequence of respiratory syncytial virus (RSV) strain A2, except for the F gene, is disclosed. The F gene is the antigenome sequence of RSV strain 19. Together with a helper plasmid, it can be used in a reverse genetics system to recover infectious viruses. The antigenome sequence on the plasmid can be mutated prior to virus recovery to produce a virus with the desired mutation.

For several reasons, plasmid is an improvement to current RSV antigenome plasmid. Each RSV side joint has a restriction endonuclease cleavage site to allow easy manipulation of any gene. As the basis for viral mutagenesis, this plasmid can be used to design attenuated viruses so that use in vaccines. Before the first RSV gene, an additional gene encoding monomer katushka 2, mKate2 protein has been included in the antigenome. The mKate2 protein is a far-red fluorescent protein that will be expressed together with other RSV genes and will serve as the visual evidence of viral replication. The ribonuclease sequence of the side joint RSV antigenome is also changed and worked in infectious virus production by reverse genetics.

The disclosed carrier allows effective mutagenesis by recombineering.This mutagenesis method needs seldom or even does not need to connect clone, but depends on the recombination mechanism present in the bacterium that carries some gene from bacteriophage.Because RSVcDNA is usually unstable in the cloning vector of high copy number in the bacterium (such as in Escherichia coli (E.coli) that is mainly used for cloning, the single digit copy property of bacterial artificial chromosome reduces this instability, and the instability of this reduction is considered to occur because this single copy property limits the hidden promoter (cryptic promoters) in Escherichia coli identification RSV cDNA and produces the ability of toxic protein.

Respiratory syncytial virus (RSV)

Typically, RSV particles contain a viral genome within a spiral nucleocapsid that is surrounded by matrix proteins and envelope-containing viral glycoproteins. The genome encoding proteins NS1, NS2, N, P, M, SH, G, F, M2-1, M2-2, and L of wild-type RSV. G, F, and SH are glycoproteins. The F gene has been incorporated into various viral vaccines. RSV polymerase activity is composed of a large protein (L) and a phosphoprotein (P). The viral M2-1 protein is used in the transcriptional process and may be a component of the transcriptase complex. The viral N protein is used to encapsidate nascent RNA.

The genome is transcribed and replicated in the host cell's cytoplasm. Host cell transcription typically results in the synthesis of methylated and polyadenylated mRNA. The antigenome is the positive-sense RNA complement of the genome produced during replication, which in turn serves as a template for genome synthesis. Viral genes are flanked by conserved gene start (GS) and gene end (GE) sequences. The 3' and 5' ends of the genome are the leader and trailer nucleotides. The wild-type leader sequence contains a promoter at the 3' end. When the viral polymerase reaches the GE signal, the polymerase polyadenylates the mRNA and releases it, and RNA synthesis is restarted at the next GS signal. It is believed that the L-P complex is responsible for promoter recognition, RNA synthesis, capping and methylation of the 5' end of the mRNA, and polyadenylation of its 3' end. It is believed that the polymerase sometimes dissociates from the gene at the junction. Because the polymerase initiates transcription at the 3' end of the genome, this creates an expression gradient, in which genes at the 3' end of the genome are transcribed more frequently than genes at the 5' end.

In order to replicate the genome, the polymerase does not respond to the cis-acting GE and GS signals and produces the positive RNA complement of the genome, i.e., the antisense genome. At the 3' end of the antisense genome is the complement of the trailing sequence containing the promoter. The polymerase uses this promoter to produce genomic positive RNA. Unlike the mRNA released as naked RNA, the antigenome and genomic RNA are encapsidated by the viral nucleoprotein (N) during synthesis.

In certain embodiments, the present disclosure relates to vectors and nucleic acids containing one or more RSV genes such as wild-type genomes or antigenomes. An example of an RSV antigenome is provided in U.S. Patent No. 6,790,449, which is hereby incorporated by reference. Mention of RSV genes and genomes is expected to include certain mutations, deletions or variant combinations, such as cold passage (cp) and temperature-sensitive (ts) derivatives cpRSV of RSV, such as rA2cp248/404/1030ΔSH. rA2cp248/404ΔSH contains 4 independent attenuated genetic factors: cp, which is based on 5 missense mutations in the N and L proteins and the F glycoprotein, which together confer a non-ts attenuated phenotype to cpRSV; ts248, a missense mutation in the L protein; ts404, a nucleotide substitution in the gene start transcription signal of the M2 gene; and ΔSH, a complete deletion of the SH gene. rA2cp248/404/1030ΔSH contains five independent attenuating genetic elements: those present in rA2cp248/404ΔSH and ts1030, another missense mutation in the L protein. See Karron et al., J Infect Dis., 2005, 191(7):1093-1104, which is hereby incorporated by reference. In certain embodiments, it is contemplated that the RSN antigenome may contain deletions or mutations, or combinations thereof, of nonessential genes (e.g., SH, NS1, NS2, and M2-2 genes).

Bacterial artificial chromosome (BAC)

In certain embodiments, the present disclosure relates to vectors and nucleic acids containing bacterial artificial chromosomes. Bacterial cloning systems for gene mapping and analysis of complex genomes have been disclosed in Shizuya et al., Proceedings of the National Academy of Sciences of the United States of America, 1992, 89: 8794-8797. The BAC system (for bacterial artificial chromosomes) is based on Escherichia coli and its single-copy plasmid F factor, which is described as being useful for cloning large fragments of human DNA. The F factor encodes genes that regulate its own replication, including oriS, repE, parA, and parB. The oriS and repE genes mediate unidirectional replication of the F factor, while parA and parB typically maintain a copy number at one or two levels per E. coli genome. It is expected that genes and chromosomes may contain mutations, deletions, or variants with desired functional properties. BAC vectors (pBAC) typically contain these genes as well as resistance markers and a cloning segment containing a promoter for incorporating the target nucleic acid segment by ligating to a restriction endonuclease site. Exemplary BAC systems include those described in Shizuya and Kouros-Hehr, Keio J Med, 2001, 50(1):26-30, which is hereby incorporated by reference.

People can reconstitute infectious RSV virus from the RSV BAC plasmid disclosed herein. BAC vectors can be transfected to bacteria such as Escherichia coli by electroporation. RSV-BAC disclosed herein can be stably maintained in bacteria, re-isolated from bacteria, and inserted into eukaryotic cells together with one or more vectors expressing N, P, L and M2-1 proteins. These cells produce infectious RSV particles. The generation of infectious RSV is caused by co-transfection of plasmids encoding N, P, L and M2-1 proteins and antigenomes into BHK-21 cells (BSR cells) expressing T7 RNA polymerase under the control of T7 promoter. See Buchholz et al., J Virol., 2000, 74 (3): 1187-1199, which are hereby incorporated by reference.

vaccine

The multiple attenuated RSV strains of the candidate vaccine for intranasal administration have been developed using the chemical mutagenesis of multiple rounds so that multiple mutations are introduced into the virus. Some of the assessment instructions in rodents, chimpanzees, adults and babies are immunogenic and can be attenuated. The increase attenuation of some nucleotide sequence analysis instructions each level of these attenuated viruses is typically associated with two or more new Nucleotide and amino acid replacements.

The present disclosure provides the ability to distinguish accidental silent mutations from those causing phenotypic differences by introducing mutations individually and in various combinations into the genome and antigenome of RSV that infects.This process identification causes mutations such as attenuation, temperature sensitivity, cold acclimatization, small plaque size, host range restriction, etc.Then various combinations can be introduced into the mutation from this menu so that vaccine virus is adjusted to suitable attenuation level as desired.In addition, the present disclosure provides the ability to combine mutations from different virus strains into a strain.

The present disclosure also provides the method for attenuation.For example, the single internal gene of RSV can be replaced by their ox, mouse or other RSV counterparts.This can comprise one or more part or all of NS1, NS2, N, P, M, SH, M2-1, M2-2 and L gene, or the part of G and F gene.Reciprocally, provide by the means that people's attenuated gene is inserted in the cattle RSV genome or antigenome background and produce attenuated live cattle RSV.The people RSV that carries the cattle RSV glycoprotein provides the host range restriction favourable for human vaccine preparation. Bovine RSV sequences useful in the present disclosure are described, for example, in Pastey et al., J. Gen. Viol. 76: 193-197 (1993); Pastey et al., Virus Res. 29: 195-202 (1993); Zamora et al., J. Gen. Viol. 73: 737-741 (1992); Mallipeddi et al., J. Gen. Viol. 74: 2001-2004 (1993); Mallipeddi et al., J. Gen. Viol. 73: 2441-2444 (1992); and Zamora et al., Virus Res. 24: 115-121 (1992), each of which is incorporated herein by reference.

The present disclosure also provides the ability of analyzing other types of attenuated mutations and incorporating them into infection RSV for vaccines or other purposes.For example, the non-pathogenic strain of the adaptation tissue culture of mouse pneumonia virus (the mouse counterpart of RSV) lacks the cytoplasm tail region of G protein (Randhawa et al., Virology (Virology) 207:240-245 (1995)).Equally, RSV glycoprotein F, G and SH respective cytoplasm and transmembrane domain can lack or modify to realize attenuation.

Other sudden changes that are used for infecting RSV of the present invention are included in the sudden change in the cis-acting signal identified in the mutation analysis process of RSV minigenome.For example, the insertion and deletion analysis of leader sequence and trailing sequence and flanking sequence has identified viral promoter and transcription signal, and provides a series of sudden changes that are associated with RNA replication or transcription reduction to varying degrees.The saturation mutagenesis (whereby each position is modified into each nucleotide substitute successively) of these cis-acting signals has also identified many sudden changes that reduce (or increase in a case) RNA replication or transcription.These any sudden changes can be inserted in complete antigenome or genome as described herein.Other sudden changes relate to genomic 3 ' end with its counterpart replacement from antigenome, and this counterpart is associated with the variation in RNA replication and transcription. In addition, intergenic intervals (Collins et al., Proc. Natl. Acad. Sci. USA 83:4594-4598 (1986), hereby incorporated by reference) can be shortened or lengthened or have their sequence content altered, and naturally occurring intergenic overlaps (Collins et al., Proc. Natl. Acad. Sci. USA 84:5134-5138 (1987), hereby incorporated by reference) can be removed or altered into different intergenic intervals by the methods described herein.

In another embodiment, the RSV that can be used for vaccine preparation can be modified easily to adapt to the antigenic variation in circulating virus (comprising antigenic subgroup A and B) and the variation in these subgroups.Typically, modification will be in G and/or F albumen.By the section of whole G or F gene or its coding specific immunogenicity district by replacing the corresponding region in infectious clone or by adding one or more gene copies and be incorporated in RSV genome or antigenome cDNA and make to show some antigenic forms.The progeny virus produced by the RSV cDNA of modification can then be used for the vaccination scheme for novel strain.In addition, the G albumen that comprises RSV subgroup B will widen the response of the wider spectrum to the relative diversity subgroup A and B that covers infected population.

The infectious RSV clone of the present disclosure can also be engineered to enhance its immunogenicity and induce the protection of the larger level than that provided by natural infection, or vice versa, to identify and eliminate the epi-position associated with undesirable immunopathological reaction.The immunogenicity of the enhanced vaccine produced by the present disclosure has solved one in the greatest obstacle of controlling RSV, i.e., the incomplete nature of the immunity induced by natural infection.Other gene can be inserted in the RSV genome or antigenome that is controlled by one group of independent transcriptional signal.Target gene includes those of the protein of coding cytokine (for example, L-2 to IL-15, especially IL-3, IL-6 and IL-7 etc.), gamma-interferon and enrichment T helper cell epitope.Other protein can be expressed as independent protein or as the chimera obtained by copying the second through engineering approaches from one of RSV protein such as SH.This provides the ability to modify and improve the immunne response to RSV quantitatively and qualitatively.

For vaccine use, viruses produced according to the present disclosure can be used directly in vaccine formulations, or lyophilized as desired using lyophilization protocols well known to those skilled in the art. Lyophilized viruses will typically be maintained at approximately 4 degrees Celsius. When ready for use, the lyophilized virus is reconstituted in a stabilizing solution (e.g., saline or containing SPG, Mg, and HEPES) with or without an adjuvant, as further described below.

Therefore, the RSV vaccine of the present disclosure contains an immunogenetically effective amount of RSV prepared as described herein as an active ingredient. The modified virus can be introduced into the host using a physiologically acceptable carrier and/or adjuvant. Useful carriers are well known in the art and include, for example, water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid, etc. The resulting aqueous solution can be packaged for use as is, or lyophilized, and the lyophilized preparation is combined with a sterile solution as mentioned above before administration. The composition can contain pharmaceutically acceptable auxiliary substances as needed for approaching physiological conditions, such as pH regulators and buffers, tension regulators, wetting agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. Acceptable adjuvants include incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide or alum, which are materials well known in the art.

When immunization is carried out with RSV compositions described herein by aerosol, drops, oral, topical or other approaches, the host's immune system responds to the vaccine by producing antibodies specific to RSV viral proteins, such as F and G glycoproteins. Due to the vaccination, the host becomes at least partially or completely immune to RSV infection, or is resistant to developing moderate or severe RSV infection, particularly lower respiratory tract RSV infection.

The host to which the vaccine is administered can be any mammal susceptible to RSV infection or closely related viral infection, and the host is capable of producing a protective immune response to the antigens of the inoculated strain. Thus, suitable hosts include humans, non-human primates, cattle, horses, pigs, sheep, goats, rabbits, rodents, etc. Therefore, the present disclosure provides a method for producing vaccines for various human and veterinary uses.

The vaccine composition containing the RSV of the present disclosure is given to a host susceptible to RSV infection or otherwise having RSV infection risk to enhance the host's autoimmune response ability. Such amount is defined as "immunogenic effective dose". In this use, accurate amount depends equally on the host's health and body weight, mode of administration, formulation properties. Vaccine formulations should provide the RSV of the present disclosure modified enough to effectively protect the host patient from the amount of serious or life-threatening RSV infection.

The RSV produced according to this disclosure can be combined with the virus of other subgroups or strains to realize the protection for multiple RSV subgroups or strains, or the protective epitope of these strains can be engineered into a kind of virus as described herein.Typically, different viruses will be mixtures and give simultaneously, but can also give separately.For example, because the difference of the F glycoprotein of these two RSV subgroups in amino acid sequence is only about 11%, this similarity is the basis of the cross-protective immune response observed in the animal that stimulates with RSV or F antigen immunity and with heterologous strain.Therefore, the different strains of identical or different subgroups can be defended with a kind of strain immunity.

In some cases, it may be desirable to combine the RSV vaccine of the present disclosure with the vaccine for inducing the protective response of other antigens, particularly other children's viruses.For example, the RSV vaccine of the present disclosure can be such as described in the et al., such as Clements, Clinical Microbiology Journal (J.Clin.Microbiol.) 29:1175-1182 (1991) incorporated herein by reference in the parainfluenza virus vaccine and give simultaneously.On the other hand of the present disclosure, RSV can be used as a carrier for the protective antigen of other respiratory pathogens such as parainfluenza virus, in the form of being incorporated into the sequence encoding these protective antigens for producing the RSV genome or antigenome for infectious RSV as described herein.

The single or multiple administration of the vaccine composition of the present disclosure can be performed. In neonates and infants, multiple sequential administrations may be required to cause enough immunity levels. Administration can be started in the first month of life, or before about two months old, typically no later than six months old, and at intervals throughout childhood, such as at intervals of two months, six months, one year or two years, as necessary for maintaining enough natural (wild type) RSV infection defense levels. Equally, adults who are particularly susceptible to repeated or serious RSV infections, such as health care workers, daily care workers, family members of young children, the elderly (more than 55, 60 or 65 years old), individuality with impaired cardiopulmonary function, may need multiple immunizations to establish and/or maintain protective immune responses. The level of induced immunity can be monitored by measuring the amount of neutralization secretion and serum antibodies, and dosage can be adjusted or repeated vaccinations can be used to maintain desired protection levels if necessary. In addition, different vaccine viruses may be beneficial to different recipient groups. For example, the engineered RSV strains expressing the additional proteins of enriched T cell epitopes may be particularly beneficial to adults compared to infants.

In another aspect of the present disclosure, RSV is used as a carrier for the transient gene therapy of respiratory tract. According to the present embodiment, recombinant RSV genome or antigenome is incorporated into the sequence that can encode target gene product.Target gene product is subject to the control of identical or different promoters, and this promoter control RSV is expressed.To be given the patient by the infectious RSV that produces of co-expression recombinant RSV genome or antigenome and N, P, L and M2-1 albumen and the sequence that comprises encoding target gene product.Administration typically is given the respiratory tract of the patient being treated by aerosol, nebulizer or other local application.Give recombinant RSV with the amount that is enough to cause the expression of the treatment or prevention level of desired gene product. Examples of representative gene products administered in this manner include those encoding, for example, interleukin-2, interleukin-4, gamma-interferon, GM-CSF, G-CSF, erythropoietin, and other cytokines, glucocerebrosidase, phenylalanine hydroxylase, cystic fibrosis transmembrane conductance regulator (CFTR), hypoxanthine-guanine phosphoribosyltransferase, cytotoxins, tumor suppressor genes, antisense RNA, and vaccine antigens, for example.

In certain embodiments, the present disclosure relates to immunogenic compositions (e.g., vaccines) comprising an immunogenic amount of a recombinant RSV of the invention (e.g., a live attenuated recombinant RSV or an inactivated non-replicating RSV), an immunogenic amount of a polypeptide disclosed herein, and/or an immunogenic amount of a nucleic acid disclosed herein.

In certain embodiments, present disclosure relates to the method for stimulating the immune system of individuality to produce the protective immune response for RSV.In these methods, the nucleic acid disclosed here of the recombinant RSV disclosed here of immune effective amount, the polypeptide disclosed here of immune effective amount and/or immune effective amount is given to individuality with physiologically acceptable carrier.

Typically, carrier or excipient is a pharmaceutically acceptable carrier or excipient, such as sterile water, saline solution, buffered saline solution, glucose aqueous solution, glycerol aqueous solution, ethanol or a combination thereof. The preparation of such solutions ensuring sterility, pH, isotonicity and stability is achieved according to the scheme established in this area. Generally, carrier or excipient are selected so as to minimize allergy and other adverse effects, and so as to be suitable for specific route of administration, such as subcutaneous, intramuscular, intranasal, oral, topical etc. The aqueous solution gained can, for example, be packaged for use as is, or lyophilized, and before administration, the lyophilized preparation is combined with a sterile solution.

In certain embodiments, RSV (or RSV components) is administered in an amount sufficient to stimulate an immune response specific to one or more strains of RSV (e.g., an immunologically effective amount of RSV or RSV components is administered). Preferably, the administration of RSV elicits a protective immune response. The dosage and method for eliciting a protective antiviral immune response can be adapted to produce a protective immune response against RSV, and these dosages and methods are known to those skilled in the art. See, for example, U.S. Patent No. 5,922,326; Wright et al. (1982) Infect. Immun. 37: 397-400; Kim et al. (1973) Pediatrics 52: 56-63; and Wright et al. (1976) J. Pediatr. 88: 931-936. In some embodiments, the present invention provides the vaccine of the present invention.For example, virus can provide (for example, often give dosage 103-106 pfu (plaque forming unit) in the scope of often giving dosage 104-105 pfu).Typically, dosage will be adjusted according to for example age, physical condition, body weight, sex, diet, mode of administration and time and other clinical factors.Prophylactic vaccine preparation can for example be given whole body by subcutaneous or intramuscular injection using needle and syringe or needleless injection device.Preferably, vaccine preparation is for example by drops, aerosol (for example, large particle aerosol (greater than about 10 microns)), or is sprayed into upper respiratory tract and intranasal administration.Although any above-mentioned delivery route all causes protective systemic immune response, intranasal administration gives the additional benefit of mucosal immunity that causes at virus entry site.For intranasal administration, attenuated live virus vaccine is normally preferred, for example attenuated, cold acclimatization and/or temperature-sensitive recombinant RSV, for example chimeric recombinant RSV. As an alternative or supplement to live attenuated virus vaccines, for example, killed virus vaccines, nucleic acid vaccines, and/or polypeptide subunit vaccines can be used, as suggested by Walsh et al. (1987) J. Infect. Dis. 155: 1198-1204 and Murphy et al. (1990) Vaccine 8: 497-502.

In certain embodiments, attenuated recombinant RSV is used as in a vaccine and is sufficiently attenuated so that symptoms of infection, or at least symptoms of severe infection, do not occur in most individuals immunized (or otherwise infected) with the attenuated RSV - in embodiments where viral components (e.g., nucleic acids or polypeptides herein) are used as vaccines or immunogenic components. However, virulence is typically sufficiently abolished so that mild or severe lower respiratory tract infections generally do not occur in vaccinated hosts or occasional hosts.

In some embodiments, the present invention provides the present invention and/or the protective immune response of the present invention.Though it is preferred to stimulate protective immune response with a single dose, additional dosage can be given by identical or different approach, to reach desired preventive effect.In neonates and infants, for example, multiple administrations may be needed to cause enough immunity levels.Can be administered at intervals throughout the childhood, as necessary for maintaining enough wild-type RSV infection defense levels.Equally, the adult who is particularly susceptible to repeated or serious RSV infection, for example, as the individuality of family members, the elderly and cardiopulmonary impairment of health care workers, daily care workers, young children, may need multiple immunity to set up and/or maintain protective immune response.The level of induced immunity can be monitored by measuring the amount of secretion and serum antibody in the virus, and adjusts dosage or repeated vaccination to maintain desired protection level if necessary.

Alternatively, the immune response can be stimulated by targeting dendritic cells with the virus in vitro or in vivo. For example, the proliferating dendritic cells are exposed to the virus in sufficient amounts and for a sufficient period of time to allow the dendritic cells to capture RSV antigens. The cells are then transferred to the subject to be vaccinated by standard intravenous transplantation methods.

Optionally, the formulation for the prophylactic administration of RSV also contains one or more adjuvants for enhancing the immune response to RSV antigens. Suitable adjuvants include, for example, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oils or hydrocarbon emulsions, BCG (BCG), Corynebacterium brevis, and the synthetic adjuvant QS-21.

If necessary, RSV preventive vaccine administration can be combined with the administration of one or more immunostimulatory molecules. Immunostimulatory molecules include various cytokines, lymphokines and chemokines with immunostimulation, immunoenhancement and proinflammatory 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 factors, Flt3 ligand, B7.1; B7.2 and the like. Immunostimulatory molecules can be given in the same preparation as RSV, or can be given alone. Protein or an expression vector encoding the protein can be given to produce an immunostimulatory effect.

Although vaccination of an individual with an attenuated RSV of a particular strain of a particular subgroup can induce cross-protection against RSV of different strains and/or subgroups, cross-protection can be enhanced, if desired, by vaccinating the individual with attenuated RSV from at least two strains (e.g., each representing a different subgroup). Similarly, attenuated RSV vaccines can optionally be combined with vaccines that induce protective immune responses against other infectious agents.

experiment

A2-strain 19F-I557V virus is immunogenic in BALB/c mice

This is shown in Figure 7, and it illustrates that this virus induces higher levels of RSV neutralizing serum antibodies than RSV A2 and RSV A2-strain 19F.Fig. 7 B shows, even if it is low input dose, when exciting 29 days after primary infection, this virus also provides the full protection exciting for RSV heterologous strain.For other two strains of RSV, A2-K-strain 19F and A2-K-A2GF, do not see the full protection under this low dose immunity, and this allows breakthrough to infect again.These two viruses are similar to A2-strain 19F-I557V, except F protein, show that the I557V F protein by this virus encoding is important for this phenotype.

In addition to being immunogenic (Figure 7A), the A2-strain 19F-I557V virus is thermostable. Viral thermostability is measured as the ability of the virus to maintain titer over multiple days when incubated at 4°C or 37°C. The results showed that the virus was more thermostable than the A2-K-A2GF virus at both tested temperatures and more stable than the A2-strain 19F at 4°C. As mentioned above, the F gene is the only difference between the two viruses, indicating that this unique F protein is responsible for this phenotype.

The A2-strain 19F RSV strain is more stable than the A2 strain, and Val at position 557 in the context of the 19F protein makes the virus even more stable. Val at position 557 in other strains may also stabilize position 557 and stability. In certain embodiments, the present disclosure encompasses other mutations at position 557 (any amino acid, e.g., alanine, valine, isoleucine, leucine) that affect the thermal stability of the virus in any F strain background.

Production and growth attenuation of recombinant RSV harboring NS1 and NS2 codon-silent mutations

Codons that are rare in humans were used to prepare recombinant RSVs having NS1 and NS2 genes, hereinafter referred to as dNS1h and dNS2h. Codons that are rare in RSV were used to prepare recombinant RSVs having NS1 and NS2 genes, hereinafter referred to as dNS1v and dNS2v. Figure 1 provides a table for determining the optimal sequence. Recombinant RSVs were prepared with the following nucleotide sequences for the NS1 and NS2 genes. It is important to note that before testing the codons, it was unpredictable if rare human codons or rare codons would produce the desired candidate RSV vaccine. Experiments using codons that are rare in RSV sequences had the unintended and adverse effect of increased expression. Using codons that are rare in human sequences had the expected effect of increased expression. Experiments comparing NS codons that are rare in human sequences with NS codons that are rare in RSV sequences showed that codons that are rare in human sequences are preferred for vaccine development.

The dNS1h nucleotide sequence (SEQ ID NO: 6) differs from NS1 in wild-type A2 in 84 of 420 nucleotides (20%) and 68 of 140 codons (48%).

SEQ ID NO:6

ATGGGTTCGAATTCGCTATCGATGATAAAAGTACGTCTACAAAATCTATTTGATAATGATGAAGTAGCGCTACTAAAAATAACGTGTTATACGGATAAACTAATACATCTAACGAATGCGCTAGCGAAAGCGGTAATACATACGATAAAACTAAATGGTATAGTATTTGTACATGTAATAACGTCGTCGGATATATGTCCGAATAATAAT ATAGTAGTAAAATCGAATTTTACGACGATGCCGGTACTACAAAATGGTGGTTATATATGGGAAATGATGGAACTAACGCATTGTTCGCAACCGAATGGTCTACTAGATGATAATTGTGAAATAAAATTTTCGAAAAAAACTATCGGATTCGACGATGACGAATTATATGAATCAACTATCGGAACTACTAGGTTTTGATCTAAATCCGTAA

The dNS1v nucleotide sequence (SEQ ID NO: 7) differs from NS1 in wild-type A2 by 145 of 420 nucleotides (34%) and 122 of 140 codons (87%).

SEQ ID NO:7

ATGGGGTCGAACTCGCTCTCGATGATCAAGGTCCGCCTCCAGAATCTCTTCGACAACGACGAGGTCGCGCTCCTCAAGATCACGTGTTACACGGACAAGCTCATCCACCTCACGAACGCGCTCGCGAAGGCGGTCATCCACACGATCAAGCTCAACGGGATCGTCTTCGTCCACGTCATCACGTCGTCGGACATCTGTCCGAACAACAAC ATCGTCGTCAAGTCGAACTTCACGACGATGCCGGTCCTCCAGAACGGGGGGTACATCTGGGAGATGATGGAGCTCACGCACTGTTCGCAGCCGAACGGGTCCCTCGACGACAACTGTGAGATCAAGTTCTCGAAGAAGCTCTCGGACTCGACGATGACGAACTACATGAACCAGCTCTCGGAGCTCCTCGGGTTCGACCTCAACCCGTAA

The dNS2h nucleotide sequence (SEQ ID NO: 9) differs from NS1 in wild-type A2 by 82 of 420 nucleotides (21%) and 73 of 140 codons (58%).

SEQ ID NO:9

ATGGATACGACGCATAATGATAATACGCCGCAACGTCTAATGATAACGGATATGCGTCCGCTATCGCTAGAAACGATAATAACGTCGCTAACGCGTGATATAATAACGCATAAATTTATATATCTAATAAATCATGAATGTATAGTACGTAAACTAGATGAACGTCAAGCGACGTTTACGTTTCTAG TAAATTATGAAATGAAACTACTACATAAAGTAGGTTCGACGAAATATAAAAAATATACGGAATATAATACGAAATATGGTACGTTTCCGATGCCGATATTTATAAATCATGATGGTTTTCTAGAATGTATAGGTATAAAACCGACGAAACATACGCCGATAATATATAAATATGATCTAAATCCGTAA

The dNS2v nucleotide sequence (SEQ ID NO: 10) differs from NS1 in wild-type A2 by 103 of 420 nucleotides (27%) and 92 of 140 codons (73%).

SEQ ID NO: 10

ATGGACACGACGCACAACGACAACACGCCGCAGCGCCTCATGATCACGGACATGCGCCCGCTCTCGCTCGAGACGATCATCACGTCGCTCACGCGCGACATCATCACGCACAAGTTCATCTACCTCATCAACCACGAGTGTATCGTCCGCAAGCTCGACGAGCGCCAGGCGACGTTCACGTTCCTCG TCAACTACGAGATGAAGCTCCTCCACAAGGTCGGGTCGACGAAGTACAAGAAGTACACGGAGTACAACACGAAGTACGGGACGTTCCCGATGCCGATCTTCATCAACCACGACGGGTTCCTCGAGTGTATCGGGATCAAGCCGACGAAGCACACGCCGATCATCTACAAGTACGACCTCAACCCGTAA

The 60-70% confluent BEAS-2B cell line was infected with the recombinant virus as described above at an MOI (multiplicity of infection) of 0.01 (i.e., one infectious virus particle was present for every 100 cells). This was done by first counting the cells before infection, calculating the total number of cells in each well, and then calculating the infectious dose of each virus. The infection was carried out at room temperature for 1 hour and then washed away. The infected cells were left in a 37°C incubator under 5% CO2 for up to 96 hours. After incubation and freezing for 12, 24, 48, 72, and 96 hours, samples were taken. After collecting samples at all time points, the amount of virus in each sample was determined by titrating the Vero cell line according to a standard protocol, and the low degree (FFU/mL, which means fluorescent focus forming units per milliliter) was calculated for each sample. Because the virus used has a red fluorescent gene in the genome, the infected cells were counted under a fluorescence microscope to provide fluorescent focus forming units. Each data point represents duplicate samples from two independent experiments.

As shown in Figure 2, the growth of kRSV-dNS1h (human deoptimized NS1+NS2 virus) was attenuated in the BEAS-2B cell line at 72 and 96 hours post-infection. This is believed to be due to reduced NS1 and NS2 proteins compared to wild-type virus.

Expression of RSV from plasmids designed for low copy number

Infectious recombinant RSV (rRSV) can be recovered from the plasmid of transfection. The co-expression of RSV N, P, L and M2-1 protein and full-length antigenomic RNA is sufficient for RSV replication. Infectious RSV can be produced by co-transfection of a plasmid encoding N, P, L and M2-1 protein and antigenomic cDNA into BHK-21 cells (BSR cells) expressing T7 RNA polymerase under the control of a T7 promoter. Current research laboratories typically use RSV antigenomic cDNA (medium copy number, 15-20 copies per Escherichia coli) cloned in plasmid pBR322. In order to maintain the antigenomic cDNA in this plasmid, bacteria are grown at 30°C and under low ventilation. However, plasmid rearrangement and cloning loss are often experienced.

It was found that the fraction of RSV cDNA containing the viral attachment glycoprotein (G) and fusion (F) genes could not be cloned in a pUC-based plasmid (500-700 plasmid copies in E. coli). This fragment was cloned in a low copy number (approximately 5 copies per E. coli) plasmid called pLG338-30.5. Plasmid pLG338-30 was developed to increase the stability of cloned lentiviral glycoproteins. Cunningham et al., Gene, 1993, 124, 93-98. It is hypothesized that cDNA instability in E. coli stems from the presence of a hidden E. coli transcript promoter in the viral glycoprotein sequence. Therefore, cDNA instability in "promoterless" plasmids in bacteria may occur because abnormal proteins are expressed from hidden promoters, resulting in toxicity that is exacerbated by plasmid copy number.

An antigenomic plasmid containing the RSV strain A2 genome in which the A2F gene was replaced by the strain 19F gene was generated. This was derived from an antigenomic plasmid first described in Collins et al., Proc. Natl. Acad. Sci. USA, 1995, 92(25):11563–11567 and U.S. Pat. No. 6,790,449 (hereby incorporated by reference). The antigenomic plasmid was digested from the plasmid vector and ligated into pKBS3BAC.

GalK recombineering reagent is available from NCI and successfully established BAC-RSV reverse genetics scheme (Fig. 4 and 5).Referring to http://web.ncifcrf.gov/research/brb/recombineeringInformation.aspx, it is hereby combined by reference.The ability of RSV for producing mutants is enhanced by the sudden change of RSV cDNA of BAC recombineering.Another benefit of this system is to enhance the stability of full-length antigenome cDNA in BAC vector.

The BAC-based RSV antigenome vectors were propagated in E. coli at 32°C and 250 RPM without any observed vector rearrangement or clonal loss. Therefore, BAC-RSV not only enables manipulation by recombineering but also facilitates RSV reverse genetics overall due to the elimination of genetic instability.

RSV antigenome in BAC vector (pSynkRSV_strain 19F construction)

RSV-BAC pSynkRSV_ strain 19F contains the katushka gene (mKate2, fluorescent protein) of modification and the restriction site for standard cloning method conveniently.In order to set up pSynkRSV, three nucleic acid fragments were synthesized by Gene Art, a company that synthesizes DNA. Subsequently these three fragments must be placed together in bacterial artificial chromosome (BAC). These three fragments are called pSynkRSV-BstBI_SacI (#1), pSynkRSV-SacI_ClaI (#2) and pSynkRSV-ClaI_MluI (#3). Use plasmid pKBS3 as the skeleton for building pSynkRSV. Referring to Fig. 6 A-E. pSynkRSV contains the copy number regulating in the bacterium and the bacterial artificial chromosome sequence required for subregion.

To insert the three synthetic segments, oligonucleotide adaptors were placed between two existing restriction endonuclease cleavage sites in pKBS3, BstBI and MluI.

The overhangs were designed so that the adapter would be connected to pKBS3 at the BstBI and MluI sites. The underlined sequences indicate restriction sites: from right to left, SacI, ClaI, and AvrII. This produced a multiple cloning site containing the restriction sites BstBI, SacI, ClaI, AvrII, and MluI (in that order), and a plasmid called pKBS5. See Figure 6A. One person cut and connected the SacI_ClaI segment (#2) from Genentech to pKBS5. See Figure 6B. The next person used the enzymes AvrII and MluI (due to the inactivated ClaI restriction site in pSynkRSV-ClaI_MluI, ClaI could not be used again) to cut and connect segment #3. See Figure 6C. In this regard, plasmid pKBS5 contains the Genentech sequence from SacI to ClaI, some intervening nucleotides (less than 10), and the Genentech sequence from AvrII to MluI. One person used BstBI and SacI to cut and connect segment #1. Referring to Figure 6 D. This RSV BAC contains approximately 10 undesirable nucleotides (from segments #2 and #3) between the two ClaI sites. Recombineering is used to delete those nucleotides, thereby producing pSynkRSV_Strain 19F. Referring to Figure 6 E. These three segments should be connected in this order to avoid potential interference from multiple restriction sites.

Recombinant respiratory syncytial virus (RSV) as a live attenuated vaccine (LAV)

Four expression plasmids are generated, one expressing RSV nucleoprotein (N), one expressing RSV phosphoprotein (P), one expressing RSV matrix 2 ORF1 protein (M2-1), and one expressing RSV large polymerase (L) - pA2-Nopt, pA2-Popt, pA2-M2-lopt, and pA2-Lopt. The fact reflected by this term is that these genes are the A2 strain of RSV, and these cDNAs are optimized for human codon bias to increase expression levels in mammalian cells. Recovering recombinant RSV from cDNA includes five components: full-length RNA (for example, provided by pSynk-RSV119F) and RSV N, P, M2-1, and L proteins. Four auxiliary plasmids, pA2-Nopt, pA2-Popt, pA2-M2-lopt, and pA2-Lopt, can be used to drive RSV rescue.

A recombinant respiratory syncytial virus strain, A2-strain 19F, was generated with a point mutation at residue F557, changing isoleucine to valine (virus name: A2-strain 19F-I557V). A protein expression plasmid encoding the strain 19F protein with the same leucine to valine mutation at position 557 was also generated (protein name – strain 19F-I557V). A2-strain 19F-I557V exhibited increased thermal stability at both 4°C and 37°C compared to the A2-strain 19F parental virus. This increased stability may contribute to the enhanced neutralizing antibody induction and protection elicited by A2-strain 19F-I557V relative to A2-strain 19F.

The development of live attenuated RSV vaccines has been hampered by low RSV immunogenicity and limited genomic stability in infants and young children (who constitute the target population).An ideal vaccine would be immunogenic and genetically and thermally stable and safe for vaccination of infants and young children.

RSV nonstructural (NS) protein 1 and 2 (NS1 and NS2) are associated with the inhibition of host cell interferon pathway, and therefore potentially limit the immunogenicity of the virus. Small hydrophobic (SH) glycoprotein forms cationic pores in the membrane, adjusts the host apoptosis pathway and inhibits tumor necrosis factor-alpha (TNF-alpha) signal transduction. SH, NS1 and NS2 are dispensable for viral replication. However, NS1 and NS2 deletions together cause excessive attenuation. The deletion of SH protein has no obvious effect on the decay of the experimental candidate vaccine currently being evaluated. However, SH deletion enhances RSV replication in vitro and may enhance downstream genes, such as the expression of antigenic G and F genes.

The candidate RSV vaccine disclosed herein combines multiple technologies to overcome the challenges of poor immunogenicity and limited genetic and thermal stability in safe viral vaccine candidates. RSV LAV 0E1 combines limited expression of the immunosuppressive proteins NS1 and NS2 (by codon deoptimization) and the SH protein (by deletion) without the possibility of rapid reversal in a stable and immunogenic viral background.

The vaccine candidate was generated using BAC-based RSV reverse genetics codon inversion of the nonstructural (NS) genes NS1 and NS2 and the A2-strain 19F gene containing a mutation at residue 557, as well as deletion of the RSV small hydrophobic (SH) glycoprotein.

OE1 viral genome (SEQ ID NO: 1)

Candidate RSV vaccine genotypes:

A2-mKate2-dNSh-ΔSH-A2G-Strain 19F-I557V (tagged)

and A2-dNSh-ΔSH-A2G-line 19F-I557V (untagged)

The RSV attachment glycoprotein (G) is a heavily glycosylated protein that exists in two variant forms: membrane-bound and secreted.

Studies have shown that it plays a role in inhibiting toll-like receptor activation and its

The secreted form may serve as an immune antigen bait. In addition to RSV F, G protein

is also immunogenic, however, due in part to its extensive glycosylation, it is

A poor antigen for the production of neutralizing antibodies. RSV G is essential for viral replication, but

The deletion resulted in excessive attenuation. Therefore, G can be considered a non-essential virulence gene.

The RSV A2G protein sequence was replaced with a gene containing the M48I mutation and 50% codon deoptimization [dGm(50%)] into the context of the RSV LAV OE1 viral genome. The OE2 viral context includes codon deoptimization of the nonstructural (NS) genes NS1 and NS2 and the A2-strain 19F gene containing a mutation at amino acid residue 557, as well as a deletion of the RSV small hydrophobic (SH) glycoprotein.

OE2 viral genome (SEQ ID NO: 2)

Candidate RSV vaccine genotypes:

A2-mKate2-dNSh-ΔSH-dGm (50%)-line 19F-I557V (tagged)

and A2-dNSh-ΔSH-dGm (50%)-line 19F-I557V (untagged)

RSV LAV OE2 combines reduced expression of the immunosuppressive glycoprotein G by 50% codon deoptimization of the codon set, 100% codon deoptimization of the immunomodulatory proteins NS1 and NS2, and deletion of the SH protein without the possibility of rapid reversal in a stable and immunogenic viral background.

In the third vaccine candidate, the RSV A2G protein sequence was replaced with one containing the M48I mutation and 75% codon deoptimization [dGm(75%)] into the context of the RSV LAV OE1 viral genome. The OE3 viral context includes codon deoptimization of the nonstructural (NS) genes NS1 and NS2 and the A2-strain 19F gene containing a mutation at residue 557, as well as a deletion of the RSV small hydrophobic (SH) glycoprotein.

OE3 viral genome (SEQ ID NO: 3)

Candidate RSV vaccine genotypes:

A2-mKate2-dNSh-ΔSH-dGm (75%)-line 19F-I557V (tagged)

and A2-dNSh-ΔSH-dGm (75%)-line 19F-I557V (untagged)

RSV LAV OE3 combines reduced expression of the immunosuppressive glycoprotein G by 75% codon deoptimization, 100% codon deoptimization of the immunomodulatory proteins NS1 and NS2, and deletion of the SH protein without the possibility of rapid reversion in a stable and immunogenic viral background.

The RSV A2G protein sequence was generated containing the M48I mutation and 100% codon inverse optimization [dGm (100%)] into the context of the RSV LAV OE1 viral genome. The OE4 viral context includes codon inverse optimization of the nonstructural (NS) genes NS1 and NS2 and the A2-strain 19F gene containing a mutation at residue 557, as well as a deletion of the RSV small hydrophobic (SH) glycoprotein.

OE4 viral genome (SEQ ID NO: 4)

Candidate RSV vaccine genotypes:

A2-mKate2-dNSh-ΔSH-dGm (100%)-line 19F-I557V (tagged)

and A2-dNSh-ΔSH-dGm (100%)-line 19F-I557V (untagged).

Claims (10)

1. An isolated recombinant nucleic acid comprising a nucleotide sequence encoding a nonstructural protein 1 (NS1) of wild-type human respiratory syncytial virus, wherein the nucleotide sequence encoding NS1 is composed of SEQ ID NO:6. 2. A recombinant vector comprising the isolated recombinant nucleic acid as described in claim 1. 3. An attenuated recombinant respiratory syncytial virus comprising the recombinant nucleic acid isolated as described in claim 1. 4. An expression system comprising the recombinant vector as described in claim 2 or the attenuated recombinant respiratory syncytial virus as described in claim 3. 5. A vaccine comprising the attenuated recombinant respiratory syncytial virus as described in claim 3. 6. The vaccine of claim 5, wherein the vaccine is intended for use in subjects who are less than 6 months old, under 1 year old, premature, have congenital heart disease or lung disease, have undergone chemotherapy or transplantation, or have been diagnosed with asthma, congestive heart failure or chronic obstructive pulmonary disease, leukemia, are elderly, or have HIV/AIDS. 7. A medicament comprising the vaccine of claim 5 in combination with another humanized monoclonal antibody against an epitope at the A antigen site of the F protein of respiratory syncytial virus, namely, movizumab, palilizumab, or another humanized monoclonal antibody. 8. Use of the vaccine of claim 5 or claim 6 for the preparation of a medicament for treating respiratory syncytial virus infection, wherein the medicament is used to treat subjects who are less than 6 months old, under 1 year old, premature, have congenital heart disease or lung disease, have undergone chemotherapy or transplantation, or have been diagnosed with asthma, congestive heart failure or chronic obstructive pulmonary disease, leukemia, are elderly, or are HIV/AIDS. 9. The use of claim 8, wherein the drug is administered in combination with another humanized monoclonal antibody against an epitope at the A antigen site of the F protein of respiratory syncytial virus. 10. The attenuated recombinant respiratory syncytial virus of claim 3, further comprising SEQ ID NO: 19.