Vaccine candidates for human respiratory syncytial virus (RSV) having attenuated phenotypes

Abstract

To provide live attenuated human respiratory syncytial virus (RSV) strains that replicate efficiently in vitro, and are maximally immunogenic, attenuated, and refractory to de-attenuation in vivo.SOLUTION: Provided herein are presumptively de-attenuating mutations that are useful, either individually or in combinations that may include other known mutations, in producing recombinant strains of human respiratory syncytial virus (RSV) exhibiting attenuation phenotypes. Also provided herein is a novel RSV construct, Min_L-NPM2-1(N88K)L, which exhibits an attenuated phenotype and is stable and as immunogenic as wild type RSV. The recombinant RSV strains described herein are suitable for use as live-attenuated RSV vaccines. Exemplary vaccine candidates are described herein. Also provided are polynucleotide sequences capable of encoding the described viruses, and methods for producing and using the viruses.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 399,133, filed September 23, 2016, and U.S. Provisional Application No. 62 / 400,476, filed September 27, 2016, both of which are incorporated by reference in their entireties for all purposes.

[0002] Reference to sequence listing This application contains a sequence listing that has been submitted as an electronic text file entitled "Sequence_Listing_6137NIAID-65-PCT_ST25.txt", having a size of 98 kb and created on September 20, 2017. The information contained in this electronic file is incorporated herein in its entirety pursuant to 37 CFR § 1.52(e)(5).

[0003] Government Power The United States Government has certain rights in this invention.

[0004] FIELD OF THEINVENTION The presently disclosed subject matter is respiratory syncytial virus (RSV) and its attenuated, mutant strains suitable for use as vaccines. [Background technology]

[0005] 2. Background of the Invention Human respiratory syncytial virus (RSV) infects nearly everyone worldwide during infancy and is responsible for substantial mortality and morbidity (for general reviews, see Collins and Graham, 2008, J Virol. 82:2040-2055; Collins and Melero, 2011, Virus Res 162: 80-99; Collins and Karron, 2013, Fields Virology 6th Edition, pp 1086-1123; Collins, et al., 2013, Curr Top Microbiol Immunol 372:3-38). In the United States alone, RSV is responsible for 75,000-125,000 hospitalizations annually, and conservative estimates indicate that RSV causes 64 million childhood infections and over 160,000 childhood deaths worldwide each year. Another hallmark of RSV is that severe infection during infancy is followed in some individuals by prolonged airway dysfunction, including a predisposition to airway reactivity, that often persists for years and may extend into adolescence and beyond. RSV infection may exacerbate asthma and may play a role in asthma induction.

[0006] RSV is a negative-stranded RNA virus of the pneumoviridae family. The genome of RSV is a single, negative-sense strand of 15.2 kilobase RNA, which is transcribed by the viral polymerase into 10 mRNAs by a sequential start-stop mechanism that initiates a single viral promoter at the 3' end of the genome. Each mRNA encodes one major protein, except for the M2 mRNA, which has two overlapping open reading frames (ORFs) that encode two separate proteins, M2-1 and M2-2. The 11 RSV proteins are the RNA-binding nucleoprotein (N), phosphoprotein (P), large polymerase protein (L), attachment glycoprotein (G), fusion protein (F), small hydrophobic (SH) surface glycoprotein, internal matrix protein (M), two nonstructural proteins NS1 and NS2, as well as the M2-1 and M2-2 proteins. The RSV gene order is 3'-NS1-NS2-NPM-SH-GF-M2-L. Each gene is flanked by short conserved transcriptional signals, termed gene start (GS) signals, present at the upstream end of each gene and involved in transcription of the respective gene, and gene end (GE) signals, present at the downstream end of each gene and involved in synthesis of a polyA tail and subsequent mRNA release.

[0007] The RSV F and G proteins are the only RSV proteins known to induce RSV neutralizing antibodies and are the major protective antigens. The F protein is generally considered to be a more effective neutralizing and protective antigen than the G protein. F is also relatively well conserved among RSV strains, while the G protein can differ substantially. Differences in G are the major factor separating RSV strains into two antigenic subgroups, A and B (about 53% and about 90% amino acid sequence identity for G and F in the two subgroups, respectively). The tools and methods of the present invention focus on RSV strain A2 of subgroup A, but can be easily applied to other strains of either subgroup.

[0008] Vaccines and antivirals against RSV are in preclinical and clinical development by numerous investigators; however, no vaccines or antivirals for routine use against RSV are commercially available.

[0009] Development of an RSV vaccine has been ongoing since the 1960s but has been complicated by a number of factors. For example, immunization of RSV-naive infants with inactivated RSV primes disease exacerbation following subsequent natural RSV infection, and studies in laboratory animals indicate that disease exacerbation is also associated with purified RSV subunit vaccines. However, RSV disease exacerbation has not been observed in association with live or live-vectored RSV vaccines, an important observation that has been confirmed in multiple clinical trials (Wright, et al., 2007, Vaccine 25:7372-7378). Therefore, inactivated and subunit vaccines are contraindicated in infants and young children, whereas appropriately attenuated live and live-vectored vaccines are acceptable for use in this population, which is the primary vaccine target population.

[0010] Another obstacle to immune protection is that RSV replicates and causes disease in superficial cells of the respiratory airway lumen, where the effectiveness of immune protection is reduced. Therefore, immune control of RSV infection is inefficient and often incomplete, making it important that RSV vaccines are as immunogenic as possible. Another obstacle to RSV vaccines is that the strength of the protective immune response is roughly proportional to the degree of viral replication (and antigen production). Therefore, the attenuation of RSV required to create a live vaccine generally involves reduced replication and antigen synthesis, and a concomitant reduction in immunogenicity, and therefore it is essential to identify a replication level that is well tolerated while still being sufficiently immunogenic.

[0011] Another aspect of RSV vaccine development is that the virus does not replicate efficiently in most experimental animals, such as rodents and monkeys. Chimpanzees, although more tolerated, are no longer available for RSV research. Therefore, RSV vaccine development is highly dependent on clinical trials from the early stages of development. In addition, RSV grows only to moderate titers in cell culture and often exists in long fibers that are difficult to purify. In addition, RSV can easily lose infectivity during handling.

[0012] Another obstacle is the difficulty of identifying and developing attenuating mutations. The appropriate mutation must be attenuating in vivo, but in vitro replication should be minimally limiting, as this is essential for efficient vaccine production. Yet another obstacle is the genetic instability that is characteristic of RNA viruses, whereby attenuating mutations can revert to a wild-type (wt) assignment or another assignment that confers a non-attenuated phenotype.

[0013] The combined approach of sequence design and synthetic biology allows the production of DNA molecules with a wide range of targeted modifications. Synonymous genome recoding, in which one or more ORFs of a pathogenic microorganism are modified at the nucleotide level without affecting the amino acid coding, is now being extensively evaluated, especially for RNA viruses, for pathogen fitness reduction and the creation of potential live-attenuated vaccines. The main strategies for attenuation by synonymous genome recoding are codon deoptimization (CD), codon pair deoptimization (CPD) and increased dinucleotide CpG and UpA content, which is usually the result of CD and CPD.

[0014] A deoptimized viral genome contains tens to thousands of silent nucleotide mutations in one or more ORFs. Presumably, attenuation is the sum of many individual mutations. This mutational diversity is predicted to confer stability against substantial deattenuation, since a large number of mutations would present a substantial barrier to reversion to virulence. In principle, any single-site reversion would yield only a very small selective advantage against the backdrop of thousands of attenuating mutations. The most likely route to reversion that can be imagined under this model is the gradual accumulation of many individual mutations, providing a slow progression to deattenuation.

[0015] To date, genetic stability studies of large-scale deoptimized viruses have shown that deattenuation appears to be low indeed, suggesting that these viruses are genetically stable. However, an important limitation of these studies is that the deoptimized viruses have not generally been subjected to the strong selective pressures that favor the growth of viruses carrying deattenuating mutations. Summary of the Invention [Problem to be solved by the invention]

[0016] Therefore, there continues to be a need for live attenuated RSV strains that replicate efficiently in vitro, are maximally immunogenic, are attenuated, and are resistant to in vivo deattenuation. [Means for solving the problem]

[0017] Summary of the Invention Disclosed herein are presumatively de-attenuating mutations in vitro that are useful alone or in combination with other known mutations to generate recombinant strains of human respiratory syncytial virus (RSV) that exhibit an attenuated phenotype in vivo. Also disclosed herein are novel live attenuated RSV strains suitable for use as RSV vaccines. Also provided herein are methods and compositions related to the expression of the disclosed viruses. For example, disclosed are isolated polynucleotide molecules that include nucleic acid sequences encoding the genome or antigenome of the described viruses.

[0018] In one embodiment, the invention includes an isolated polynucleotide molecule encoding a recombinant respiratory syncytial virus (RSV) variant having an attenuated phenotype comprising a RSV genome or antigenome sequence, wherein the RSV genome or antigenome is modified by a mutation at a position corresponding to T1166 of SEQ ID NO:11 in the L ORF of the L protein.

[0019] In some embodiments, the RSV genome or antigenome is further modified by a mutation selected from the group consisting of: (i) a mutation in the M2-1 ORF of the M2-1 protein at a position corresponding to N88 or A73 of SEQ ID NO: 9; (ii) a mutation in the N ORF of the N protein at a position corresponding to K136 of SEQ ID NO: 3; (iii) a mutation in the P ORF of the P protein at a position corresponding to E114 of SEQ ID NO: 4; and (iv) a combination thereof. In some embodiments, the RSV genome or antigenome is further modified by a mutation selected from the group consisting of: (i) a mutation in the M2-1 ORF of the M2-1 protein at a position corresponding to N88 of SEQ ID NO: 9; (ii) a mutation in the N ORF of the N protein at a position corresponding to K136 of SEQ ID NO: 3; (iii) a mutation in the P ORF of the P protein at a position corresponding to E114 of SEQ ID NO: 4; and (iv) a combination thereof. In some embodiments, the RSV genome or antigenome is further modified by a mutation selected from the group consisting of: (i) a mutation in the M2-1 ORF of the M2-1 protein at a position corresponding to A73 of SEQ ID NO:9; (ii) a mutation in the N ORF of the N protein at a position corresponding to K136 of SEQ ID NO:3; (iii) a mutation in the P ORF of the P protein at a position corresponding to E114 of SEQ ID NO:4; and (iv) a combination thereof. In some embodiments, the RSV genome or antigenome is modified by at least two of the mutations (i)-(iii). In some embodiments, the RSV genome or antigenome is modified by all of the mutations (i)-(iii).

[0020] In some embodiments, the mutation at the position corresponding to T1166 of SEQ ID NO: 11 in the L ORF of the L protein is T1166I. In some embodiments, (a) the mutation at the position corresponding to N88 of SEQ ID NO: 9 in the M2-1 ORF of the M2-1 protein is N88K, and the mutation at the position corresponding to A73 of SEQ ID NO: 9 in the M2-1 ORF of the M2-1 protein is A73S; (b) the mutation at the position corresponding to K136 of SEQ ID NO: 3 in the N ORF of the N protein is K136R; and (c) the mutation at the position corresponding to E114 of SEQ ID NO: 4 in the P ORF of the P protein is E114V. In some embodiments, the RSV genome or antigenome is modified by at least two of mutations a-c. In some embodiments, the RSV genome or antigenome is modified by all of mutations a-c. In some embodiments, the mutation at the position corresponding to T1166 of SEQ ID NO: 11 in the L ORF of the L protein is T1166I.

[0021] In some embodiments, the RSV genome or antigenome is modified by mutations corresponding to T1166I of SEQ ID NO: 11 in the L protein, N88K of SEQ ID NO: 9 in the M2-1 protein, K136R of SEQ ID NO: 3 in the N protein, and E114V of SEQ ID NO: 4 in the P protein. In some embodiments, the RSV genome or antigenome is modified by mutations corresponding to T1166I of SEQ ID NO: 11 in the L protein, A73S of the M2-1 protein of SEQ ID NO: 9, K136R of SEQ ID NO: 3 in the N protein, and E114V of SEQ ID NO: 4 in the P protein.

[0022] In another embodiment, the invention includes an isolated polynucleotide molecule encoding a recombinant respiratory syncytial virus (RSV) variant having an attenuated phenotype comprising a RSV genome or antigenome sequence, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions set forth in Table S1. In one embodiment, the RSV genome or antigenome is modified by one or more mutations selected from the positions set forth in Table S1-A. In one embodiment, the RSV genome or antigenome is modified by one or more mutations selected from the positions set forth in Table S1-B.

[0023] In another embodiment, the invention includes an isolated polynucleotide molecule encoding a recombinant respiratory syncytial virus (RSV) variant having an attenuated phenotype comprising a RSV genome or antigenome sequence, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions set forth in Table S2. In one embodiment, the RSV genome or antigenome is modified by one or more mutations selected from the positions set forth in Table S2-A. In one embodiment, the RSV genome or antigenome is modified by one or more mutations selected from the positions set forth in Table S2-B.

[0024] In another embodiment, the invention includes an isolated polynucleotide molecule encoding a recombinant RSV variant having an attenuated phenotype comprising a respiratory syncytial virus (RSV) genome or antigenome sequence, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions set forth in Table S3. In one embodiment, the RSV genome or antigenome is modified by one or more mutations selected from the positions set forth in Table S3-A. In one embodiment, the RSV genome or antigenome is modified by one or more mutations selected from the positions set forth in Table S3-B.

[0025] In some embodiments, the RSV genome or antigenome comprises a deletion in at least one protein selected from M2-2, NS1, and NS2. In some embodiments, the RSV genome or antigenome is codon-pair deoptimized. In some embodiments, the L-ORF of the RSV genome or antigenome is codon-pair deoptimized.

[0026] In certain embodiments, the invention comprises a polynucleotide molecule comprising a nucleotide sequence that is at least about 80% identical to the nucleotide sequence of SEQ ID NO: 14. In certain embodiments, the invention comprises a polynucleotide molecule comprising a nucleotide sequence that is at least about 90% identical to the nucleotide sequence of SEQ ID NO: 14. In certain embodiments, the invention comprises a polynucleotide molecule comprising a nucleotide sequence that is at least about 95% identical to the nucleotide sequence of SEQ ID NO: 14. In certain embodiments, the invention comprises a polynucleotide molecule comprising the nucleotide sequence of SEQ ID NO: 14.

[0027] In some embodiments, the invention includes a vector comprising the isolated polynucleotide molecule described above. In some embodiments, the invention includes a cell comprising the isolated polynucleotide molecule described above.

[0028] In some embodiments, the present invention comprises a pharmaceutical composition comprising an immunologically effective amount of a recombinant RSV variant encoded by the above-mentioned isolated polynucleotide molecule.In some embodiments, the present invention comprises a method for vaccinating a subject against RSV, comprising administering the pharmaceutical composition.In some embodiments, the present invention comprises a method for inducing an immune response, comprising administering the pharmaceutical composition.In some embodiments, the pharmaceutical composition is administered intranasally.In some embodiments, the pharmaceutical composition is administered by injection, aerosol delivery, nasal spray or nasal drops.

[0029] In some embodiments, the present invention includes a live attenuated RSV vaccine comprising a recombinant RSV variant encoded by the isolated polynucleotide. In some embodiments, the present invention includes a pharmaceutical composition comprising a RSV vaccine. In some embodiments, the present invention includes a method of producing a vaccine comprising expressing the isolated polynucleotide molecule. [Brief description of the drawings]

[0030] [Figure 1-1] Figure 1A-G shows that Min_FLC was phenotypically stable during the temperature stress test, whereas Min_L was not. (A) Genetic maps of Min_L and Min_FLC showing the ORFs that were wt (gray) or CPD (black). The number of mutations introduced into each virus in Vero cells and the shutoff temperature (TSH) are shown. (B-E) Incubation temperature and virus yield at each passage level during serial passaging in the temperature stress test are shown. Replicate cultures of Vero cells in T25 flasks were infected with the indicated viruses at an MOI of 0.1, and when the viral cytopathic effect was extensive or cells began to detach (for passaging of Min_FLC at 37°C and beyond, C), flasks were harvested and clarified cultures were passaged 1:5 in new flasks. Each starting replicate flask initiated an individual series of passages (lineage). Aliquots of clarified cultures were frozen for titering and sequence analysis. (B, C) Temperature stress test of Min_FLC. Two control flasks inoculated with Min_FLC (B) were passaged 18 times at the permissive temperature of 32 °C. Ten further replicates (C) were passaged at 32-40 °C for two passages at each temperature. (D, E) Temperature stress test of Min_L. Two control flasks inoculated with Min_L (D) were passaged 8 times at the permissive temperature of 32 °C. Ten further replicates (E) were passaged at 37-40 °C for two passages at each temperature. Lineages #3 and 8 are shown. (F, G) Accumulation of the most abundant mutations (>30% of reads at at least one passage) in lineages #3 (F) and #8 (G) during the passage series as determined by deep sequencing (for detailed data, see Tables S2 and S3). [Figure 1-2] Same as above.

[0031] [Figure 2-1] Figures 2A-C show the M2-1 mutations [A73S] and [N88K] segregating into different virus subpopulations. (A) The percentage of deep sequencing reads containing the M2-1 mutations [A73S] or [N88K] at P6 (second passage at 39°C) for each of the 10 lineages from the experiment in Figure 1E. (B) Loss of association of the M2-1 mutations [A73S] and [N88K] as illustrated by the percentage of deep sequencing reads containing the combined intended assignments at codons 73 (wt vs. [A73S]) and 88 (wt vs. [N88K]) in the same read; based on the reads from the experiment in Figure 1F spanning both codons. (C) The degree of linkage of the M2-1 mutations [A73S] and [N88K] and other mutations during the first four passages of lineage #3 as determined by PacBio sequencing of contiguous reads corresponding to an 8.2-kb region of the RSV genome from the 3' end to the middle of the M2-2 ORF (Fig. 1E and F). Four major virus subpopulations were identified, showing associated mutations on the same genome. [Figure 2-2] Same as above. [Figure 2-3] Same as above.

[0032] [Figure 3-1]Figure 3A-D shows the effect of specific mutations on temperature sensitivity and in vitro replication of Min_L derivatives. Five major mutations identified in lineage #3 (N[K136R], P[E114V], L[T1166I], M2-1[N88K] and M2-1[A73S], Figure 1F) were individually and in combination inserted by site-directed mutagenesis and reverse genetics into Min_L for phenotypic analysis. Min_L-derived viruses were named based on the gene name carrying the introduced mutation, with the M2-1 mutation identified in brackets. (A) Mutations are shown on the viral genome map. (B) TSH determined by efficiency of plaque formation at 32°C, 35°C, 36°C, 37°C, 38°C, 39°C and 40°C using published methods. Experiments were performed four times with viruses #1, 5, 12 and 14, three times with viruses #2, 3, 7, 8 and 9, two times with viruses #4 and 11, and one time with viruses #6, 10 and 13 (bar graphs: center and range). (C, D) Min_L-derived mutants replication in vitro. Vero cells were infected at 32°C and 37°C (MOI of 0.01). Titers correspond to the mean of two replicate titrations for each time point. Standard deviations are shown. Due to the large number of viruses, the analysis was split into experiments #1 (C) and #2 (D). [Figure 3-2] Same as above.

[0033] [Figure 4-1]Figure 4A-G show the effect of specific mutations on RNA synthesis and plaque size of Min_L derivatives. (A-E) Replicate cultures of Vero cells were infected with the indicated viruses (MOI of 3). Cultures were harvested every 4 h from 4 to 24 h postinfection for cell-associated RNA, protein and virus. (A) Positive-sense viral RNA (i.e., mRNA + antigenome) was quantified in triplicate by strand-specific RT-qPCR. Data are shown for P. qPCR results were analyzed using the comparative threshold cycle (ΔCt) method, normalized to 18S rRNA and expressed as log2 fold increase over Min_L at the 4 h time point. (B) Quantification of P protein expression by Western blotting. (C) Quantification of L mRNA + antigenome by strand-specific RT-qPCR (fold increase relative to the 4 h time point postinfection, calculated separately for each virus because different primer-probe sets were required for the wt L gene in wt rRSV and the CPD L gene present in Min_L and its derivatives). For Wt L and CPD L, data are derived from three and four different primer-probe sets, respectively, designed along the L ORF, and median and range are shown. (D) Quantification of cell-associated genomic RNA by strand-specific RT-qPCR, expressed as fold increase over the 4-h time point post-infection with Min_L. (E) Viral titers from cultures incubated at 32°C and 37°C and assayed at 32°C are shown. (F-G) Viral plaque sizes. Vero cells were infected with 30 pfu / 2 cm2 well of wt rRSV, Min_L and Min_L-derived mutants and incubated for 12 days at 32°C under methylcellulose. Plaques were visualized by immunostaining and quantified by IR imaging (Licor) using Image J. F) Representative diagram of viral plaque sizes. G) Plaque size distribution for the indicated viruses. A minimum of 1000 plaques / virus was measured (*=p≦0.05). [Figure 4-2] Same as above.

[0034] [Figure 5-1]Figure 5A-D shows the analysis of Min_L derivatives in rodents, which shows the differential effect of M2-1 mutations A73S and N88K and identifies an improved vaccine candidate NPM2-1[N88K]L. Replication of Min_L, Min_L mutants and wt rRSV at days 4 (A) and 5 (B) post-infection in mice or day 3 (C) post-infection in hamsters is shown. Groups of 20 mice (AB) or 18 hamsters (C) were infected intranasally with 106 pfu of the indicated virus / animal. At days 4 (A), 5 (B) and 10 (data not shown) post-infection in the mouse study or day 3 (C) in the hamster study, RSV titers in the nasal turbinates (NT) and lungs were determined as described in the Experimental Methods section. The limit of detection is indicated by a dotted line. D) RSV neutralizing antibodies at day 26 in hamsters from 9 hamsters / group. Plaque reduction neutralizing antibody titers of 60% (PRNT60) were determined as previously described. Statistical differences compared to wt rRSV are indicated above each graph; statistical differences between Min_L and Min_L-derived mutants are indicated in brackets (*p≦0.05, **p≦0.01, ***p≦0.001 and ****p≦0.0001). [Figure 5-2] Same as above.

[0035] [Figure 6-1] 6A-D show molecular modeling of the effect of deattenuating mutations on the M2-1 tetramer. (A) Plan view of the wt M2-1 tetramer. (B) Zoom-in of a region of the wt tetramer that includes amino acids A73 and N88. (C) Molecular dynamics snapshot of the S73 mutation proximal region. The [A73S] mutation is indicated and the arrow indicates the predicted novel hydrogen bond. (D) Molecular dynamics snapshot of the K88 mutant region. The [N88K] mutation is indicated and the arrow indicates the predicted novel salt bridge. [Figure 6-2] Same as above.

[0036] [Figure 7-1]Figure 7A-B shows the minimal accumulation of adventitious mutations in Min_FLC during 18 passages at 32 °C. Min_FLC was subjected to 18 passages at 32 °C. At the end of the 18th passage, viral RNA was extracted from lineages #1 (A) and #2 (B) and complete genomes were amplified by overlapping RT-PCR and analyzed by deep sequencing (Ion Torrent). Adventitious mutations (which are not specifically shown) are indicated by bars showing their genomic location and relative abundance. WT genes are colored in grey and CPD genes in black. [Figure 7-2] Same as above.

[0037] [Figure 8-1] Figure 8A-B shows the minimal accumulation of adventitious mutations in Min_L during six passages at 32 °C. Min_L was serially passaged six times in Vero cells at 32 °C. At the end of the sixth passage, viral RNA was extracted from lineages #1 (A) and #2 (B) and complete genomes were amplified by overlapping RT-PCR and analyzed by deep sequencing (Ion Torrent). Adventitious mutations are indicated by bars showing their genomic location and relative abundance; specific nucleotide changes are not shown. WT genes are colored in grey and CPD genes in black. [Figure 8-2] Same as above.

[0038] [Figure 9-1] Figure 9 shows the contribution of specific mutations to the phenotype of Min_L derivatives: RT-qPCR of cell-associated positive-sense RNA (mRNA + antigenome). Shown here are RT-qPCR data during infection of Vero cells with wt rRSV, Min_L and Min_L-derivatives for NS1, NS2, N, P, M, SH, G, F and M2 mRNA. [Figure 9-2] Same as above.

[0039] [Figure 10-1]Figure 10 shows the contribution of specific mutations to the phenotype of Min_L derivatives: protein expression of Min_L and Min_L-derived mutants. Vero cells were infected with Min_L, M2-1[A73S], M2-1[N88K], PM2-1[N88K], NPM2-1[N88K]L or wt rRSV at an MOI of 3 pfu / cell at 32°C or 37°C. Total cell lysates were harvested from one well of a 6-well plate into NuPage LDS sample buffer (Life Technologies) every 4 hours from 4 to 24 hours postinfection. Western blot analysis of NS1, NS2, N, P, G, F and M2-1 was performed as described in the Materials and Methods section. GAPDH protein was used as a loading control. Membranes were scanned with an Odyssey® Infrared Imaging System. Incidence data were analyzed using Odyssey software, version 3.0. For quantification of the identified RSV proteins of interest, background fluorescence was corrected and values ​​shown represent the median fluorescence intensity per protein band. [Figure 10-2] Same as above.

[0040] [Figure 11]Figure 11A-B shows the TSH of Min_FLC derived mutants. The effect of the mutations responsible for the loss of temperature sensitivity of Min_L on Min_FLC temperature sensitivity was tested. A) To do so, the mutations identified in Min_L lineages #3 (in N, P, M2-1 [N88K] and L genes) or #8 (M2-1 mutation [A73S]) were reintroduced into the Min_FLC backbone alone or in the combinations indicated, and the derived cDNA was fully sequenced by Sanger sequencing. Viruses were rescued by reverse genetics, passaged once, and viral stocks of P2 were titered. Due to the low viral titers of most viral stocks, only the mutant virus Min_FLC_M2-1 [A73S] was fully sequenced by Sanger sequencing. B) The ts phenotype of some of these Min_FLC derived mutants was assessed by the plaque formation efficiency at 32°C, 35°C, 36°C, 37°C, 38°C, 39°C and 40°C. Plaque assays were performed in duplicate on Vero cells and incubated in sealed tubs in a temperature-controlled water bath at various temperatures as described above. Experiments were performed in duplicate. Median and standard deviation are shown.

[0041] [Figure 12-1] Figures 12A-C show that NPM2-1[N88K]L is phenotypically stable under temperature stress testing. A) Schematic of RSV genome organization. Abbreviated gene names are shown. Genes with wt or CPD ORFs are shown in grey and black, respectively. Mutations in N, P, M2-1 and L identified in lineage #3 and introduced into the Min_L backbone to generate NPM2-1[N88K]L virus are shown by bars in the viral genome. B) Final virus titers from 32°C and C) temperature stress passages (increasing temperatures are shown below the x-axis). Each symbol represents one copy. [Figure 12-2] Same as above.

[0042] [Figure 13-1] 13 shows the amino acid sequences of RSV proteins NS1, NS2, N, P, M, SH, G, F, M2-1, M2-2 and L, which are represented by SEQ ID NOs: 1-11, respectively. [Figure 13-2] Same as above.

[0043] [Figure 14-1] 14 shows the nucleotide sequence of recombinant RSV Min_L-NPM2-1[N88K]L, which is represented by SEQ ID NO:14. [Figure 14-2] Same as above. [Figure 14-3] Same as above. [Figure 14-4] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] Detailed Description Provided herein are recombinant RSV strains suitable for use as live, attenuated vaccines in humans. The RSV strains can be generated by the introduction of one or more mutations in the RSV genome or antigenome sequence selected from the positions listed below and in Tables S1, S2, and S3. These mutations were identified by evaluation of phenotypic reversion of deoptimized human respiratory syncytial virus (RSV) vaccine candidates in the context of strong selective pressure.

[0045] A codon pair deoptimized (CPD) version of RSV was attenuated and temperature sensitive. During serial passages at gradually increasing temperatures, the CPD RSV containing 2,692 synonymous mutations in 9 of 11 ORFs, designated Min_FLC, did not lose temperature sensitivity and was genetically and phenotypically stable for 7 months of passage in vitro at the permissive temperature of 32°C, as well as at increasing temperatures during passaging. This is strong evidence of the stability of Min_FLC and confirms the safety of multigene CPDs for live attenuated vaccine development of RSV and related viruses, provided that extensive CPDs are used.

[0046] However, a CPD RSV deoptimized only for the polymerase L ORF, designated Min_L, was highly stable at 32°C, but surprisingly, despite the numerous changes involved in its CPD, it rapidly lost substantial attenuation and evolved to circumvent the temperature-sensitive restriction. Comprehensive sequence analysis of the virus population identified a large number of different potentially deattenuating mutations in the L ORF, many of which surprisingly appeared in other ORFs not attached to the CPD. Notably, deep sequencing of the Min_L lineage identified mutations not specifically in the CPD L ORF, as expected, but rather in all but the NS2 ORF. Surprisingly, many of the mutations in L occurred at nucleotides and codons that were not involved in the CPD. These are shown in Tables S1, S2 and S3.

[0047] Some of these putative deattenuating mutations were found to have the surprising effect that, although they were deattenuating in vitro, when introduced into Min_L together with other putative deattenuating mutations, they were further attenuated in vivo than Min_L.

[0048] In one exemplary embodiment, detailed below (the nucleotide sequence is shown in FIG. 14), Min_L-NPM2-1[N88K]L (also referred to herein as NPM2-1[N88K]L) was more attenuated than Min_L in vivo, rather than deattenuated. Moreover, the NPM2-1[N88K]L virus was highly attenuated in vivo, yet surprisingly as immunogenic as wild-type RSV. Moreover, it did not acquire any significant mutations during further stress testing (see FIG. 12), and therefore was more genetically stable than Min_L. Therefore, Min_L-NPM2-1[N88K]L represents a substantial improvement over Min_L as a vaccine candidate for the following reasons: it was significantly more attenuated in vivo than Min_L, while being as immunogenic as wt RSV. It did not accumulate additional mutations when passaged in stress tests at 39-40°C. It showed increased replication compared to Min_L in Vero cells, which is important for vaccine production. Furthermore, as detailed below, the virus is highly resistant to acquisition of the deattenuated M2-1[A73S] mutation in a hamster model because the M2-1[N88K] and [A73S] mutations are incompatible.

[0049] Thus, provided herein are recombinant RSV strains having an attenuated phenotype comprising an RSV genome or antigenome sequence, wherein the RSV genome or antigenome is modified by one or more mutations selected from Tables S1, S2, or S3. The mutations listed in Tables S1, S2, or S3 are putative deattenuating mutations, but surprisingly can confer an attenuated phenotype in vivo. The mutations listed in Tables S1, S2, and S3 that are present in ≧25% read data are shown in Tables S1-A, S2-A, and S3-A, respectively, and the most abundant mutations present in ≧50% read data are shown in Tables S1-B, S2-B, and S3-B, respectively.

[0050] In some embodiments, the present invention includes an isolated polynucleotide molecule encoding a recombinant respiratory syncytial virus (RSV) variant having an attenuated phenotype comprising a RSV genome or antigenome sequence, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions set forth in Table S1. In some embodiments, the RSV genome or antigenome is modified by one or more mutations selected from the positions set forth in Table S1-A. In some embodiments, the RSV genome or antigenome is modified by one or more mutations selected from the positions set forth in Table S1-B.

[0051] In some embodiments, the invention includes an isolated polynucleotide molecule encoding a recombinant respiratory syncytial virus (RSV) variant having an attenuated phenotype comprising a RSV genome or antigenome sequence, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions set forth in Table S2. In some embodiments, the RSV genome or antigenome is modified by one or more mutations selected from the positions set forth in Table S2-A. In some embodiments, the RSV genome or antigenome is modified by one or more mutations selected from the positions set forth in Table S2-B.

[0052] In some embodiments, the invention includes an isolated polynucleotide molecule encoding a recombinant respiratory syncytial virus (RSV) variant having an attenuated phenotype comprising a RSV genome or antigenome sequence, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions set forth in Table S3. In some embodiments, the RSV genome or antigenome is modified by one or more mutations selected from the positions set forth in Table S3-A. In some embodiments, the RSV genome or antigenome is modified by one or more mutations selected from the positions set forth in Table S3-B.

[0053] In some embodiments, the RSV genome or antigenome can be modified by a mutation at a position corresponding to or at the codon encoding amino acid residue 1166 in the L ORF of the L protein. In some embodiments, the mutation in the L ORF can be at a position corresponding to T1166 of the L protein shown in the sequence of Figure 13 (SEQ ID NO: 11). In some embodiments, the mutation can encode an amino acid other than threonine at that position. In some embodiments, the mutation can encode isoleucine at that position. This mutation, T1166I, is listed in Tables S1, S1-A and S1-B.

[0054] In some embodiments, the RSV genome or antigenome can be further modified by one or more additional mutations. The additional mutations can be made in either the L ORF or other ORFs. For example, in some embodiments, one or more additional mutations can be made in the M2-1 ORF, the N ORF, or the P ORF.

[0055] In some embodiments, the additional mutation can be made at a position corresponding to or at the codon encoding amino acid residue 88 or 73 in the M2-1 ORF of the M2-1 protein. In some embodiments, the mutation in the M2-1 ORF can be made at a position corresponding to N88 or A73 of the M2-1 protein shown in the sequence of FIG. 13 (SEQ ID NO: 9). In some embodiments, the additional mutation in the M2-1 ORF can be at a position corresponding to position N88 of the M2-1 protein, which can encode an amino acid other than asparagine at that position. In some embodiments, it can encode lysine at that position (N88K). In some embodiments, the additional mutation in the M2-1 ORF can be at a position corresponding to position A73 of the M2-1 protein, which can encode an amino acid other than alanine at that position. In some embodiments, the mutation in the codon encoding amino acid residue 73 of the M2-1 protein can encode serine at that position (A73S).

[0056] In some embodiments, the additional mutation can be made at a position corresponding to or at the codon encoding amino acid residue 136 in the N ORF of the N protein. In some embodiments, the mutation in the N ORF can be at a position corresponding to K136 of the N protein shown in the sequence of Figure 13 (SEQ ID NO:3) and can encode an amino acid other than lysine at that position. In some embodiments, the mutation in the codon encoding amino acid residue 136 of the N protein can encode arginine at that position (K136R).

[0057] In certain embodiments, an additional mutation can be made in the codon encoding amino acid residue 114 in the P ORF of the P protein. In certain embodiments, the mutation in the P ORF can be at a position corresponding to E114 of the P protein shown in the sequence of Figure 13 (SEQ ID NO:4) and can encode an amino acid other than glutamic acid at that position. In certain embodiments, a mutation in the codon encoding amino acid residue 136 of the P protein can encode valine at that position (E114V).

[0058] In some embodiments, the RSV genome or antigenome can be modified to include at least two of the above mutations. For example, it can include at least two mutations at positions corresponding to N88 or A73 in the M2-1 protein, K136 in the N protein, E114 in the P protein, and T1166 in the L protein. In some embodiments, the RSV genome or antigenome can be modified to include all four of the above mutations. Thus, for example, in some embodiments, it can include mutations at positions corresponding to N88 in the M2-1 protein, K136 in the N protein, E114 in the P protein, and T1166 in the L protein. In some embodiments, it can include mutations at positions corresponding to A73 in the M2-1 protein, K136 in the N protein, E114 in the P protein, and T1166 in the L protein.

[0059] In some embodiments, the isolated polynucleotide molecule can include a RSV genome or antigenome modified by mutations corresponding to or encoding N88K in the M2-1 protein, K136R in the N protein, E114V in the P protein, and T1166I in the L protein. In some embodiments, the isolated polynucleotide molecule can include a RSV genome or antigenome modified by mutations corresponding to or encoding A73S in the M2-1 protein, K136R in the N protein, E114V in the P protein, and T1166I in the L protein.

[0060] In some embodiments, the RSV genome or antigenome can be deoptimized. Thus, in some embodiments, the attenuated RSV described herein is produced by introducing codon changes in the viral genome that are not optimally processed by host cells. Most of these mutations do not change the amino acids of the resulting proteins encoded by the viral genome, thus allowing the production of viruses with the same antigenic properties as wild-type viruses. However, it should be understood that extensive non-coding changes to the codons of the viral genome can result in selective pressure to generate one or more amino acid mutations in the viruses described herein.

[0061] This replacement of synonymous codons alters various parameters in the genome, including codon bias, codon pair bias, density of deoptimized codons and deoptimized codon pairs, RNA secondary structure, CpG dinucleotide content, C+G content, translation frameshift sites, translation pause sites, presence or absence of tissue-specific microRNA recognition sequences, or any combination thereof. The main strategies for attenuation by synonymous genome recoding are codon deoptimization (CD), codon pair deoptimization (CPD), and increased dinucleotide CpG and UpA content, which is usually the result of CD and CPD.

[0062] In some embodiments, any one of the ORFs of RSV, including NS1, NS2, N, P, M, SH, G, F, M2-1, M2-2, and L, can be codon-pair deoptimized. In some embodiments, any two or more of the ORFs of RSV can be codon-pair deoptimized. In some embodiments, any three or more of the ORFs of RSV can be codon-pair deoptimized. In some embodiments, any four or more of the ORFs of RSV can be codon-pair deoptimized. In some embodiments, any five or more of the ORFs of RSV can be codon-pair deoptimized. In some embodiments, any six or more of the ORFs of RSV can be codon-pair deoptimized. In some embodiments, any seven or more of the ORFs of RSV can be codon-pair deoptimized. In some embodiments, any eight or more of the ORFs of RSV can be codon-pair deoptimized. In some embodiments, any nine or more of the ORFs of RSV can be codon-pair deoptimized. In some embodiments, any ten or more of the ORFs of RSV can be codon-pair deoptimized. In some embodiments, any of the ORFs of RSV can be codon-pair deoptimized. In some embodiments, the L ORF of RSV can be codon-pair deoptimized. In some embodiments, the NS1, NS2, N, P, M and SH ORFs of RSV can be codon-pair deoptimized. In some embodiments, the G and F ORFs of RSV can be codon-pair deoptimized. In some embodiments, the NS1, NS2, N, P, M, SH, G, F and L ORFs of RSV can be codon-pair deoptimized.

[0063] In some embodiments, the isolated polynucleotide molecule may comprise a nucleotide sequence that is at least about 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%, 99% or 100% identical (or any percent identity therebetween) to the nucleotide sequence of SEQ ID NO: 14. In some embodiments, the isolated polynucleotide molecule may comprise a nucleotide sequence that is at least about 80% identical to the nucleotide sequence of SEQ ID NO: 14. In some embodiments, the isolated polynucleotide molecule may comprise a nucleotide sequence that is at least about 90% identical to the nucleotide sequence of SEQ ID NO: 14. In some embodiments, the isolated polynucleotide molecule may comprise a nucleotide sequence that is at least about 95% identical to the nucleotide sequence of SEQ ID NO: 14. In certain embodiments, the isolated polynucleotide molecule can comprise an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO:14.

[0064] In some embodiments, the described viruses can be combined with known attenuating mutations of RSV and related viruses to produce a stepwise attenuated phenotype.Many such mutations are known in the art and are encompassed by the present invention.For example, in some embodiments, the RSV genome or antigenome can be modified by deletions in the M2-2 ORF, NS1 ORF, or NS2 ORF.

[0065] Given that various strains of RSV exist (e.g., RSV A2, RSV B1, RSV Long), one skilled in the art will recognize that a strain of RSV may have a nucleotide or amino acid insertion or deletion that changes the position of a residue. For example, if another RSV strain's protein has two additional amino acids at the upstream end of the protein compared to strain A2, this will increase the amino acid numbering of the downstream residue by two compared to strain A2. However, due to the high degree of sequence identity of these strains, one skilled in the art can determine the position of the corresponding sequence by simply aligning the nucleotide or amino acid sequence of the A2 control strain with the sequence of the strain in question. Thus, although the amino acids and nucleotide positions described herein are specifically numbered in this disclosure, it should be understood that they may correspond to other positions when sequence shifts occur or due to sequence diversity between viral strains. In comparing proteins or protein segments or genes or genomes or genome segments of two or more related viruses, "corresponding" amino acids or nucleotide residues are those that are considered to be exactly or approximately equivalent in function in different species.

[0066] The numbering used in this disclosure corresponds to the amino acid sequence of the wild-type RSV A2 strain (GenBank accession number M74568, expressly incorporated herein), and all nucleotide sequences depicted are in the positive sense. The amino acid sequences of the eleven RSV proteins NS1, NS2, N, P, M, SH, G, F, M2-1, M2-2, and L are shown in Figure 13 and set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11, respectively.

[0067] In one embodiment of the invention, the recombinant RSV strain can be derived from a recombinant of strain A2 designated D46. The complete sequence of D46 is set forth in U.S. Patent 6,790,449 (GenBank accession number KT992094, expressly incorporated herein). (In some circumstances and publications, the parent virus and sequence are designated D53 rather than D46, but this is a book-keeping difference referring to the strain of bacteria used to propagate the antigenomic cDNA and has no other known significance or effect. For purposes of the present invention, D46 and D53 are interchangeable.) The nucleotide sequence of D46 differs from the sequence of RSV A2 strain M74568 by 25 nucleotide positions, including a 1 nt insertion at position 1099.

[0068] Additional mutations may be further introduced in combination with the above-defined mutations to construct additional virus strains with desired characteristics. For example, the added mutations may specify different strengths of attenuation and thus result in incremental attenuation. Thus, a candidate vaccine strain may be further attenuated by incorporating at least one, preferably two or more, different attenuating mutations, such as those identified from a panel of known, biologically derived mutant RSV strains. A number of such mutations are described herein by way of example. From this exemplary panel, a large "book" of attenuating mutations may be made, where each mutation may be combined with any other mutation in the panel to calibrate the level of attenuation and other desired phenotypes. Additional attenuating mutations may be identified in non-RSV negative stranded RNA viruses and incorporated into the RSV mutants of the invention by mapping the mutations to the corresponding, homologous sites in the recipient RSV genome or antigenome and mutating (identical or conservative) existing sequences in the recipient to the mutant genotype. Additional useful mutations can be determined empirically by mutational analysis using recombinant minigenome systems and infectious viruses as described in the references incorporated herein.

[0069] The recombinant RSV vaccine strain of the present invention was produced using a recombinant DNA-based technique called reverse genetics (Collins, et al. 1995. Proc Natl Acad Sci USA 92:11563-11567). This system allows for the de novo acquisition of complete infectious virus from cDNA in the appropriate cell substrate under defined conditions. Reverse genetics provides a means for the introduction of planned mutations into the RSV genome via a cDNA intermediate. Specific attenuating mutations were characterized in preclinical trials and combined to achieve the desired level of attenuation. The derivation of vaccine virus from cDNA minimizes the risk of contamination with adventitious contaminants and aids in concise and well-documented passage history. Once acquired, the engineered virus strain is propagated in a manner similar to biologically derived viruses. As a result of the passage and amplification, the vaccine virus is free of recombinant DNA from the initial acquisition.

[0070] Recombinant virus strains containing various combinations of mutations described herein are for illustrative purposes only and are not intended to limit the scope of the invention. For example, in some embodiments, the recombinant RSV strains of the invention further contain deletions of non-translated sequences. In some embodiments, such deletions occur at the downstream end of the SH gene, resulting in a mutation referred to herein as the "6120 mutation." It includes the deletion of 112 nucleotides in the downstream non-translated region of the SH gene and the introduction of five translationally silent point mutations in the last three codons and the stop codon of the SH gene (Bukreyev, et al. 2001. J Virol 75:12128-12140). The presence of the term "LID" or "6120" in the name of the recombinant virus indicates that the recombinant virus contains the 6120 mutation.

[0071] The 6120 mutation stabilizes the antigenomic cDNA in bacteria and is therefore easier to manipulate and prepare. In wt RSV, this mutation was previously found to contribute to a 5-fold increase in replication efficiency in vitro (Bukreyev, et al. 2001. J Virol 75:12128-12140), but was not thought to increase replication efficiency in vivo.

[0072] The 6120 mutation was associated with increased replication in seronegative infants and children. Therefore, the 6120 mutation provided another means for shifting the level of attenuation. Sequence deletions can also include any equivalent genomic sequence that does not contain a critical cis-acting signal, although the 6120 mutation in the downstream untranslated region of the SH gene is an example (Collins and Karron. 2013. Fields Virology 6th Edition, pp 1086-1123). Genomic regions that are candidates for deletion include, but are not limited to, untranslated regions in other genes, intergenic regions, and trailer regions.

[0073] In some embodiments, the recombinant RSV strain can contain the "cp" mutation, which refers to a series of five amino acid substitutions in three proteins (N (V267I), F (E218A and T523I), and L (C319Y and H1690Y)) that together (by themselves) contribute to an approximately 10-fold reduction in replication and reduced disease in seronegative chimpanzees (Whitehead, et al. 1998. J Virol 72:4467-4471). We have previously shown that the cp mutation is associated with a moderately attenuated phenotype (Whitehead, et al. 1999. J Virol 72:4467-4471).

[0074] Furthermore, previous analysis of six biological viruses induced by chemical mutagenesis of cpRSV and selected for a temperature-sensitive (ts) phenotype yielded a total of six independent mutations that each conferred a ts attenuation phenotype and could be used in various combinations. Five of these resulted in amino acid substitutions in the L protein and were named by virus number rather than sequence position: "955" (N43I), "530" (F521L), "248" (Q831L), "1009" (M1169V) and "1030" (Y1321N) (Juhasz, et al. 1999. Vaccine 17:1416-1424; Collins, et al. 1999. Adv Virus Res 54:423-451; Firestone, et al. 1996. Virology 225:419-422; Whitehead, et al. 1999. J Virol 73:871-877). The sixth mutation (designated "404") was a single nucleotide change in the gene start transcription signal of the M2 gene (GGGGCAAATA to GGGGCAAACA, mRNA sense) (Whitehead, et al. 1998. Virology 247:232-239). We have recently used reverse genetics to increase the genetic stability of the 248 and 1030 mutations (Luongo, et al. 2009. Vaccine 27:5667-5676; Luongo, et al. 2012. J Virol 86:10792-10804). In addition, we created a novel attenuating mutation by deleting codon 1313 in the L protein and combining it with an I1314L substitution to increase genetic stability (Luongo, et al. 2013. J Virol 87:1985-1996).

[0075] In certain embodiments, the recombinant strains can contain one or more changes in the F protein, such as the "HEK" mutation, which includes two amino acid substitutions in the F protein, namely K66E and Q101P (described in Connors, et al. 1995. Virology 208:478-484; Whitehead, et al. 1998. J Virol 72:4467-4471). Introduction of the HEK amino acid assignments into the strain A2 F sequence of the present invention results in an F protein amino acid sequence identical to an early passage (human embryonic kidney cell passage 7, HEK-7) of the original clinical isolate of strain A2 (Connors, et al. 1995. Virology 208:478-484; Whitehead, et al. 1998. J Virol 72:4467-4471). It results in a much less fusogenic F protein, which is believed to represent the phenotype of the original A2 strain clinical isolate (Liang et al. J Virol 2015 89:9499-9510). The HEK F protein also forms a more stable trimer (Liang et al. J Virol 2015 89:9499-9510). This may provide a more reliable and immunogenic form of the RSV F protein, possibly enriched for the highly immunogenic pre-fusion conformation (McLellan et al. Science 2013 340(6136):1113-7; Science 2013 342(6158):592-8). Therefore, mutations may be introduced that have effects in addition to the effect on the strength of viral replication.

[0076] In certain embodiments, the recombinant strain can contain one or more changes in the L protein, such as the stabilized 1030 or "1030s" mutations, including 1321K(AAA) / S1313(TCA) (Luongo, et al. 2012. J Virol 86:10792-10804).

[0077] In certain embodiments, the recombinant strain may contain one or more changes in the N protein, such as amino substitutions like T24A. Deletions of the SH, NS1, and NS2 genes, individually and in combination, retain the ability to replicate in cell culture but progressively generate a virus that is attenuated in vivo in the following order: SH < NS2 < NS1 (Bukreyev, et al. 1997. J Virol 71:8973-8982; Whitehead, et al. 1999. J Virol 73:3438-3442; Teng, et al. 2000. J Virol 74:9317-9321). Thus, deletions or other mutations in the SH, NS2, or NS1 genes or their ORF portions can be combined with the mutations described herein. For example, in certain embodiments, the recombinant strain may contain one or more changes in the SH protein, including the disappearance or removal of the SH protein. In certain embodiments, the viral strain may contain a deletion in the SH gene. For example, in certain embodiments, the viral strain contains a 419 nucleotide deletion at positions 4197-4615 (4198-4616), herein referred to as the “ΔSH” mutation. This deletion results in a deletion at the end of the M gene, a deletion in the region between the M / SH genes, and a deletion of the SH ORF, as shown in FIG. 6. In certain embodiments, the recombinant strain may contain one or more changes in the NS1 or NS2 protein, including the disappearance or removal of these proteins. In certain embodiments, the mutation may be an amino substitution such as K51R in the NS2 protein.

[0078] Various properties may be introduced into the RSV strain of the present invention that change the characteristics of the virus in ways other than attenuation. For example, codon optimization of the ORFs encoding proteins may be performed. The major protective antigens F and G may result in increased antigen synthesis. The F and / or G protein genes may be shifted upstream (closer to the promoter) to increase expression. The F and / or G protein amino acid sequences may be modified to represent currently circulating strains or early passage clinical isolates, which may be particularly important in the case of diverse G proteins. Deletions or substitutions may be introduced into the protein to improve immunogenicity or other desired properties. For example, the CX3C fractalkine motif in the G protein should be removed to improve immunogenicity (Chirkova et al. J Virol 2013 87:13466-13479).

[0079] For example, in one embodiment, the nucleotide sequence encoding the G protein of RSV can be replaced with the nucleotide sequence of clinical isolate A / Maryland / 001 / 11. In one embodiment, the nucleotide sequence encoding the F protein of RSV can be replaced with the nucleotide sequence of clinical isolate A / Maryland / 001 / 11, e.g., F001.

[0080] In some embodiments, the native or naturally occurring nucleotide sequence encoding a protein of RSV can be replaced with a codon-optimized sequence designed to increase expression in a selected host, particularly humans. For example, in some embodiments, the nucleotide sequence encoding the F protein of RSV can be replaced with a codon-optimized sequence. In some embodiments, the nucleotide sequence encoding the F protein of RSV can be replaced with a codon-optimized sequence from clinical isolate A / Maryland / 001 / 11. In some embodiments, the nucleotide sequence encoding the G protein of RSV can be replaced with a codon-optimized nucleotide sequence from clinical isolate A / Maryland / 001 / 11.

[0081] Yet another embodiment of the present invention includes changing gene position or gene order. For example, NS1, NS2, SH and G genes can be deleted individually or NS1 and NS2 genes can be deleted together, thereby shifting the position of each downstream gene relative to the viral promoter. For example, when NS1 and NS2 are deleted together, N moves from gene 3 to gene 1, P moves from gene 4 to gene 2, etc. Alternatively, the deletion of any other gene in the gene order only affects the position (relative to the promoter) of the gene located further downstream. For example, SH occupies position 6 in the wild-type virus, and its deletion does not affect M (or any other upstream gene) at position 5, but moves G from position 7 to position 6 relative to the promoter. It should also be noted that gene deletions can also occur (rarely) in biologically derived mutant viruses. For example, subgroup B RSV, which has been extensively passaged in cell culture, naturally lacks the SH and G genes (Karron et al. Proc. Natl. Acad. Sci. USA 94:13961 13966, 1997; incorporated herein by reference).

[0082] Gene order shift modifications (i.e., positional modifications that move one or more genes in a recombinant viral genome to a more promoter-proximal or promoter-distal position) result in viruses with altered biological properties. For example, RSV lacking NS1, NS2, SH, G, NS1 and NS2 together, or SH and G together, has been shown to be attenuated in vitro, in vivo, or both. In particular, the G and F genes, both alone and in tandem, can be shifted to a more promoter-proximal position relative to their wild-type gene order. These two proteins normally occupy positions 7 (G) and 8 (F) in the RSV gene order (NS1-NS2-NPM-SH-G-FM2-L). In some embodiments, the order of the nucleotide sequences encoding the G and F proteins can be reversed relative to the naturally occurring order.

[0083] In addition to the above mutations, the attenuated viruses of the invention can incorporate heterologous coding or non-coding nucleotide sequences from any RSV or RSV-like virus, such as human, bovine, ovine, murine (pneumonia virus of mice) or avian (turkey rhinotracheitis virus) pneumovirus or other enveloped viruses, such as parainfluenza virus (PIV). An example of a heterologous sequence is a RSV sequence from one human RSV strain combined with a sequence from a different human RSV strain. Alternatively, the RSV may incorporate sequences from two or more wild-type or mutant human RSV subgroups, such as a combination of human RSV subgroup A and subgroup B sequences. In yet other embodiments, one or more human RSV coding or non-coding polynucleotides are replaced with a counterpart sequence from a heterologous RSV or non-RSV virus to generate a novel attenuated vaccine strain.

[0084] In addition to the recombinant RSV with the specific mutations and combinations of these mutations described herein, the disclosed viruses can be further modified as recognized by those skilled in the art.For example, the recombinant RSV can have one or more of its proteins deleted or mutated otherwise, or a heterologous gene from a different organism can be added to its genome or antigenome, so that the recombinant RSV expresses or incorporates that protein when it infects and replicates in a cell.Furthermore, those skilled in the art will recognize that other previously reported mutations known to have an effect on RSV can be combined with any one or more of the mutations described herein to produce a recombinant RSV with desired attenuation or stability characteristics.

[0085] In certain embodiments, the mutations described herein, when used alone or in combination with other mutations, can provide different levels of viral attenuation, providing the ability to tailor the balance between attenuation and immunogenicity, and providing a more stable genotype than the parent virus.

[0086] Further representative viruses from those described herein can be evaluated in cell culture for infectivity, replication kinetics, yield, protein expression efficiency and genetic stability using the method described and described herein in the examples using exemplary recombinant strains. Further representative strains can be evaluated in rodents and non-human primates for infectivity, replication kinetics, yield, immunogenicity and genetic stability. These incomplete test systems may not reliably detect all differences in replication, but some substantial differences can be detected. Recombinant strains can also be evaluated directly in seronegative pups without the prior step of evaluation in adults and seropositive pups. This can be done, for example, in groups of 10 vaccine recipients and 5 placebo recipients, which is a small number that allows for simultaneous evaluation of multiple candidates. Candidates can be evaluated for vaccine virus infectivity, replication kinetics, shedding, tolerability, immunogenicity and genetic stability immediately after immunization, and vaccines can be investigated for safety, RSV disease and changes in RSV-specific serum antibodies during the next RSV season, as described in Karron, et al. 2015, Science Transl Med 2015 7(312):312ral75, the entirety of which is incorporated herein by reference. Thus, analysis of selected representative viruses can provide a relatively fast screen for triaging candidates to identify the most suitable ones.

[0087] Reference to a protein or peptide includes its naturally occurring form and any fragment, domain, or homolog of such a protein. As used herein, the term "homolog" is used to refer to a protein or peptide that differs from a naturally occurring protein or peptide (i.e., a "prototype" or "wild-type" protein) by minor modifications of the naturally occurring protein or peptide, but retains the basic protein and side chain structure of the naturally occurring form. Such changes include, but are not limited to, changes in one or a few amino acid side chains; changes in one or a few amino acids, including deletions (e.g., truncated forms of the protein or peptide), insertions, and / or substitutions; changes in the stereochemistry of one or a few atoms; and / or minor derivatizations, including, but not limited to, methylation, glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation. Homologs have enhanced, decreased, or substantially similar properties compared to the naturally occurring protein or peptide. A homologue of a protein can comprise, consist essentially of, or consist of an amino acid sequence that is at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical (or any % identity in integer increments between 45% and 99%) to the amino acid sequence of the reference protein.

[0088] In some embodiments of the invention, a selected gene segment, such as one encoding a selected protein or protein region from RSV (e.g., a cytoplasmic tail, a transmembrane or ectodomain, an epitope site or region, a binding site or region, an active site or active site-containing region, etc.), can be replaced with a counterpart gene segment from the same or a different RSV or other source to generate a novel recombinant with a desired phenotypic change compared to a wild-type or parental RSV strain. For example, this type of recombinant can express a chimeric protein having a cytoplasmic tail and / or a transmembrane domain of one RSV fused to an ectodomain of another RSV. Another example of this type of recombinant expresses a duplicate protein region, such as a duplicate immunogenic region. As used herein, a "counterpart" gene, gene segment, protein or protein region is generally from a heterologous source (e.g., representing the same (i.e., homologous or allelic) gene or gene segment from a different RSV gene or in a different RSV strain). Exemplary counterparts selected in the present invention share overall structural characteristics, e.g., each counterpart may encode equivalent structural "domains," such as a cytoplasmic domain, a transmembrane domain, an ectodomain, a binding site or region, an epitope site or region, and the like. Counterpart domains and their encoding gene segments encompass a collection of species with a wide range of size and amino acid (or nucleotide) sequence diversity, the range being defined by the common biological activity within the domain or gene segment variants. For example, two selected protein domains encoded by counterpart gene segments in the present invention may share substantially the same qualitative activity, such as transmembrane function, specific binding activity, providing an immunological recognition site, and the like. More generally, the specific biological activity shared between counterparts, e.g., between selected protein segments or proteins, is substantially similar in quantitative terms, i.e., does not vary by more than 30%, preferably more than 20%, and more preferably more than 5-10% in the quantitative activity profile.

[0089] In another embodiment of the invention, the infectious RSV produced from the cDNA expression genome or antigenome can be any RSV or RSV-like strain, such as human, bovine, murine, etc., or any pneumovirus or metapneumovirus, such as murine pneumonia virus or avian metapneumovirus. To generate a protective immune response, the RSV strain can be endogenous to the subject to be immunized, for example, human RSV is used to immunize humans. The endogenous RSV genome or antigenome can, in any event, be modified to express RSV genes or gene segments from a combination of various sources, e.g., combinations of genes or gene segments from different RSV species, subgroups or strains, or from RSV and other respiratory pathogens, such as human parainfluenza viruses (PIVs) (see, e.g., Hoffman et al. J. Virol. 71:4272-4277 (1997); Durbin et al. Virology 235(2):323-32 (1997); Murphy et al. U.S. Patent Application 60 / 047,575, filed May 23, 1997, and the following plasmids for generating infectious PIV clones: p3 / 7(131) (ATCC 97990); p3 / 7(131)2G (ATCC 97889); and p218(131) (ATCC 97991); each of which is incorporated herein by reference in its entirety by reference in its entirety. The strain was deposited under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC), 1-1000-1 San Jose Blvd., Manassas, Va. 20110-2209, USA, on April 18, 1997, and assigned the above-mentioned accession number.

[0090] In one embodiment of the present invention, recombinant RSV is provided in which individual internal genes of human RSV are replaced with counterparts or foreign genes from, for example, bovine or other RSV counterparts or other respiratory pathogens such as PIV. The replacement, deletion, etc. of RSV genes or gene segments in the present invention can include part or all of one or more of the NS1, NS2, N, P, M, SH and L genes or the non-immunogenic portions of the M2-1 open reading frame or the G and F genes. Human RSV cis-acting sequences, such as promoters or transcription signals, can also be replaced with, for example, their bovine RSV counterparts. Reciprocally, the insertion of human attenuating genes or cis-acting sequences into the bovine RSV genome or antigenome background can provide a means to produce live attenuated bovine RSV.

[0091] Therefore, an infectious recombinant RSV intended for administration to humans is a human RSV that has been modified to contain genes, such as from bovine RSV or PIV, for purposes of attenuation. For example, the insertion of a gene or gene segment from PIV provides a bivalent vaccine against both PIV and RSV. Alternatively, a heterologous RSV species, subgroup or strain or another respiratory pathogen, such as PIV, can be modified to contain genes that, for example, encode epitopes or proteins that induce protection against human RSV infection. For example, a human RSV glycoprotein gene is substituted for a bovine glycoprotein gene, and the resulting bovine RSV, carrying a human RSV surface glycoprotein and with limited replicative capacity in a human host due to the residual bovine genetic background, can induce a protective immune response in humans against human RSV strains.

[0092] The ability to analyze and incorporate other types of attenuating mutations into infectious RSV for vaccine development has expanded to a wide collection of targeted changes in RSV clones.For example, any RSV gene that is not essentially for growth can be deleted or otherwise modified to obtain the desired effect on virulence, pathogenesis, immunogenicity and other phenotypic characteristics.In addition, a variety of other genetic modifications can be made to recombinant RSV genomes or antigenomes to incorporate infectious recombinant RSV alone or together with one or more attenuating point mutations adopted from biologically derived mutant RSV.

[0093] As used herein, "heterologous genes" refers to genes taken from different RSV strains or types or from non-RSV sources. These heterologous genes can be, in whole or in part, altered gene order, removed gene duplications, RSV genomic promoters replaced with their antigenomic counterparts, portions of genes removed or replaced, and entire genes deleted. Different or additional modifications in the sequence can be made to facilitate manipulation, such as the insertion of unique restriction sites in various intergenic regions (e.g., the unique Stul site between the G and F genes) or elsewhere. Non-translated gene sequences can be removed to increase the capacity for inserting foreign sequences.

[0094] The deletion, insertion, substitution and other mutations involved in the change of the entire viral gene or gene segment in the recombinant RSV of the present invention produce highly stable vaccine candidates, which is particularly important in the case of immunosuppressed individuals. Many of these mutations result in the attenuation of the resulting vaccine strain, while others specify different types of desired phenotypic changes. For example, certain viral genes are known that code for proteins that specifically interfere with host immunity (see, for example, Kato et al., EMBO. J. 16:578-87 (1997)). It is expected that the removal of such genes in vaccine viruses will enhance virulence and pathogenesis and / or improve immunogenicity.

[0095] Other mutations in the RSV of the present invention are the replacement of the 3' end of the genome with its counterpart from the antigenome, which is associated with changes in RNA replication and transcription. Additionally, the methods described herein may shorten or extend or alter the sequence content of intergenic regions (Collins et al., Proc. Natl. Acad. Sci. USA 83:4594-4598 (1986)), and may remove or alter naturally occurring gene duplications (Collins et al., Proc. Natl. Acad. Sci. USA 84:5134-5138 (1987)) into different intergenic regions.

[0096] In another embodiment, the sequence surrounding the translation start site (preferably including nucleotide position -3) of a selected RSV gene is modified, either alone or in combination with the introduction of an upstream start codon, to regulate RSV gene expression by determining up- or down-regulation of translation.

[0097] Alternatively, or in combination with other RSV modifications disclosed herein, RSV gene expression can be regulated by altering the transcriptional GS signal of a selected gene of the virus. In one exemplary embodiment, the GS signal of NS2 is modified to include certain mutations in order to superimpose ts restriction on viral replication.

[0098] Yet another RSV clone in the present invention incorporates modification to transcription GE signal.For example, the RSV clone is provided that replaces or mutates the GE signal of NS1 and NS2 genes with respect to its N gene, resulting in reduced read-through mRNA levels and increased protein expression from downstream genes.The resulting recombinant virus shows increased growth kinetics and plaque size, and provides the modification of RSV growth properties by, for example, modifying cis-acting regulatory elements in the RSV genome.

[0099] In other embodiments, the expression of G protein can be increased by modifying G mRNA. G protein is expressed as both a membrane-bound form and a secreted form, the latter form being expressed by translation initiation at an initiation site within the G gene translation open reading frame. The secreted form can account for as much as half of the expressed G protein. Deletion of the internal initiation site (e.g., by sequence modification, deletion, etc.) alone or in conjunction with the sequence organization of the upstream initiation site results in the desired change in G protein expression. Deletion of the secreted form of G protein also improves the quality of the host immune response to the exemplary recombinant RSV, since the soluble form of G protein is believed to act as a "decoy" for neutralizing antibody capture. Soluble G protein has also been associated with enhanced immunopathology due to preferential stimulation of Th2-biased responses.

[0100] In a related embodiment, the expression level of a RSV gene can be modified at the transcriptional level. In some embodiments, the position of a selected gene in the RSV genetic map can be changed to a more promoter-proximal or promoter-distal position, thereby allowing the gene to be expressed less or more efficiently, respectively. This embodiment allows for the regulation of expression of a specific gene to be achieved, resulting in a 2-fold, more commonly 4-fold, and up to 10-fold or greater decrease or increase in gene expression compared to wild-type levels. In one example, the NS2 gene (second in the order of the RSV genetic map) is replaced with the SH gene (sixth in the order), resulting in the expected decrease in NS2 expression. Increases in expression of a selected RSV gene by positional changes can be achieved up to 10-fold, 30-fold, 50-fold, 100-fold, or greater, often with a counterbalanced decrease in the expression level of the replaced gene.

[0101] In some exemplary embodiments, the F and G genes can be translocated, either alone or together, to more promoter-proximal or promoter-distal sites in the (recombinant) RSV genetic map to achieve higher or lower levels of gene expression, respectively. These and other translocation changes result in new RSV clones with attenuated phenotypes, for example, due to reduced expression of selected viral proteins involved in RNA replication. In yet other embodiments, RSV useful in vaccine formulations can be conveniently modified by allowing circulating viruses to accommodate antigenic drift. Generally, the modification is to the G and / or F proteins. The entire G or F gene or a segment encoding a particular immunogenic region thereof is incorporated into the RSV genome or antigenome cDNA by replacing the corresponding region in the infectious clone or adding more than one copy of the gene, resulting in several antigenic forms.

[0102] Progeny virus produced from the modified RSV cDNA is then used in vaccination protocols against emerging strains. Furthermore, the inclusion of the G protein gene of RSV subgroup B as a gene addition broadens the response to cover a broader spectrum of the relatively diverse subgroup A and B strains present in the human population.

[0103] Infectious RSV clones of the invention can be engineered by the methods and compositions disclosed herein to increase their immunogenicity and induce greater levels of protection than are provided by infection with wild-type RSV or incompletely attenuated parental viruses or clones. For example, immunogenic epitopes from heterologous RSV strains or types or non-RSV sources, such as PIV, can be added with appropriate nucleotide changes in the polynucleotide sequence encoding the RSV genome or antigenome. Recombinant RSV can also be engineered to identify and remove epitopes (e.g., by amino acid insertion, substitution, or deletion) associated with undesirable immunopathological reactions. In other embodiments, additional genes can be inserted into or adjacent to the RSV genome or antigenome under the control of an independent set of transcriptional signals. Genes of interest can include, but are not limited to, those encoding cytokines (e.g., IL-2 to IL-15, particularly IL-2, IL-6, and IL-12), gamma-interferon, and proteins rich in T-helper cell epitopes. Additional proteins can be expressed as separate proteins or as chimeras engineered from a second copy of one of the RSV proteins, such as SH. This provides the ability to modify and improve the immune response to RSV, both quantitatively and qualitatively.

[0104] In addition to the above modifications to recombinant RSV, different or additional modifications in the RSV clone can be made to facilitate manipulation, such as the insertion of unique restriction sites into various intergenic regions (e.g., the unique Stul site between the G and F genes) or elsewhere. Non-translated gene sequences can be removed to increase the capacity for the insertion of foreign sequences.

[0105] The introduction of such mutations into an infectious RSV clone can be accomplished by a variety of well-known methods. An "infectious clone" refers to a cDNA or product thereof, synthetic or otherwise, that can be transcribed into genomic or antigenomic RNA capable of producing infectious virus. The term "infectious" refers to a virus or viral structure that can replicate in cultured cells or animal or human hosts to produce progeny viruses or viral structures that may be of the same activity. Thus, mutations can be introduced into a cDNA copy of the genome or antigenome by conventional techniques (e.g., site-directed mutagenesis). The use of antigenome or genome cDNA subfragments to construct a complete antigenome or genome cDNA is well known to those of skill in the art and has the advantage that each region can be manipulated separately (small cDNAs are easier to manipulate than larger ones) and then easily assembled into a complete cDNA. Thus, a complete antigenome or genome cDNA, or any subfragments thereof, can be used as a template for oligonucleotide-directed mutagenesis. The mutated subfragments can then be assembled into a complete antigenome or genome cDNA. Mutations can vary from single nucleotide changes to large pieces of cDNA that encompass one or more genes or genomic regions.

[0106] Recombinant RSV can be produced by intracellular co-expression of cDNA encoding the RSV genomic RNA and viral proteins required for transcription, replication, and nucleocapsid production. Plasmids encoding other RSV proteins can also be included among these essential proteins. Alternatively, RNA can be synthesized in an in vitro transcription reaction and transfected into cultured cells.

[0107] Thus, also described herein are isolated polynucleotides that encode mutant viruses, generate genomes or antigenomes, express genomes or antigenomes, or encode various proteins useful in in vitro recombinant RSV production. Polynucleotides comprising any of the sequences of the SEQ ID NOs described herein are encompassed by the invention. Further included are polynucleotides having a sequence consisting of or essentially comprising any of the above sequences, polynucleotides having a sequence which is at least about 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%, 99% or 100% identical (or any percent identity in between) to any of the above SEQ ID NOs, as well as polynucleotides which hybridize to or are the complement of the above molecules.

[0108] These polynucleotides can be included in or expressed by a vector to produce recombinant RSV. Thus, cells transfected with isolated polynucleotides or vectors are also within the scope of the present invention and are exemplified herein. Thus, in some embodiments, the present invention includes vectors that contain the isolated polynucleotide molecules. In some embodiments, the present invention includes cells that contain the isolated polynucleotide molecules.

[0109] In related aspects of the invention, compositions (e.g., isolated polynucleotides and vectors incorporating RSV-encoding cDNA) and methods are provided for producing isolated, infectious, recombinant RSV carrying attenuating mutations. Included in these aspects of the invention are novel, isolated polynucleotide molecules, including RSV genomes or antigenomes modified as described herein, and vectors incorporating such molecules. Also provided are the same or different expression vectors that include isolated polynucleotide molecules encoding one or more RSV proteins. These proteins are also expressed directly from genomic or antigenomic cDNA. The vectors are preferably expressed or co-expressed in cells or cell-free lysates, thereby producing mutant RSV particles or component virus particles.

[0110] In one embodiment, the invention includes a method for producing one or more purified RSV proteins, comprising infecting a host cell permissive for RSV infection with a recombinant RSV strain under conditions that allow RSV reproduction in the infected cell. After a period of replication in culture, the cells are lysed and recombinant RSV is isolated therefrom. One or more desired RSV proteins are purified after virus isolation to obtain one or more RSV proteins for vaccine, diagnostic and other uses.

[0111] The above methods and compositions for producing attenuated recombinant RSV mutants yield infectious virus or component virus particles or derivatives thereof. Infectious virus is comparable to authentic RSV virus particles and is similarly infectious. It can directly infect fresh cells. Infectious component virus particles are generally small components of virus particles that can initiate infection under appropriate conditions. For example, nucleocapsids containing genomic or antigenomic RNA and N, P, L and M2-1 proteins are examples of component virus particles that can initiate infection if introduced into the cytoplasm of a cell. The component virus particles provided within the present invention include virus particles that lack one or more proteins, protein segments or other viral components that are not essential for infectivity.

[0112] In another embodiment, the present invention provides a cell or cell-free lysate comprising an expression vector comprising an isolated polynucleotide molecule encoding the attenuated recombinant RSV genome or antigenome described above and an expression vector (the same or different vectors) comprising one or more isolated polynucleotide molecules encoding the N, P, L and RNA polymerase elongation factor proteins of RSV. One or more of these proteins may also be expressed from the genome or antigenome cDNA. Upon expression, the genome or antigenome and the N, P, L and RNA polymerase elongation factor proteins combine to produce infectious RSV virus or component virus particles.

[0113] The recombinant RSV of the present invention is useful in various compositions to generate a desired immune response against RSV in a host susceptible to RSV infection. The attenuated rRSV strains of the present invention are capable of eliciting a protective immune response in an infected human host with sufficient attenuation so as not to cause unacceptable symptoms of severe respiratory disease in immunized hosts. The attenuated virus or virus component particles may be present in cell culture supernatants, isolated from the culture, or partially or completely purified. The virus may also be lyophilized and, if desired, combined with a variety of other components for storage or delivery to the host.

[0114] In another embodiment of the invention, recombinant RSV strains can be used as "vectors" for protective antigens from other pathogens, particularly respiratory tract pathogens such as parainfluenza virus (PIV). For example, a recombinant RSV carrying the T1166I mutation can be engineered to incorporate sequences encoding protective antigens from PIV to produce an infectious, attenuated vaccine virus.

[0115] In some embodiments, the present invention includes a pharmaceutical composition comprising an immunologically effective amount of a recombinant RSV variant encoded by the isolated polynucleotide molecule described above. In some embodiments, the present invention includes a method of vaccinating a subject or inducing an immune response, comprising administering the pharmaceutical composition. The composition may be administered in any suitable manner, including, but not limited to, injection, aerosol delivery, nasal spray, nasal drops, oral inoculation, or topical application. In some embodiments, the composition may be administered by injection, aerosol delivery, nasal spray, nasal drops. The composition may be administered intranasally or subcutaneously or intramuscularly. In some embodiments, the composition may be administered intranasally. The administration methods and routes are described in more detail below.

[0116] In a related embodiment, the invention provides a method of stimulating the immune system of an individual to induce an immune response to RSV in a mammalian subject, the method comprising administering an immunologically sufficient or effective amount of an immunogenic formulation of attenuated RSV in a physiologically acceptable carrier and / or adjuvant.

[0117] In some embodiments, the invention includes a live attenuated RSV vaccine comprising a recombinant RSV variant encoded by the isolated polynucleotide molecule described above. In some embodiments, the invention includes a pharmaceutical composition comprising a RSV vaccine. In a related embodiment, the invention includes a method of producing a vaccine comprising expressing the isolated polynucleotide molecule described above.

[0118] The vaccine may comprise a physiologically acceptable carrier and / or adjuvant and isolated attenuated recombinant RSV particles or component virus particles. In one embodiment, the vaccine consists of an attenuated recombinant RSV having at least one, and preferably two or more, of the mutations or other nucleotide modifications described herein to achieve an appropriate balance of attenuation and immunogenicity.

[0119] To select candidate vaccine viruses from the host of recombinant RSV strains provided herein, the criteria of viability, efficient in vitro replication, in vivo attenuation, immunogenicity and phenotypic stability are determined by well-known methods. The most desirable viruses for the vaccines of the present invention must maintain viability, replicate well under acceptable conditions in vitro to allow vaccine production, have a stable attenuated phenotype, be well tolerated, be able to replicate in immunized hosts (even at low levels), and efficiently induce the production of sufficient immune responses in the vaccine to confer protection against severe disease caused by subsequent infection with wild-type virus. Apparently, the RSV mutants known and reported thus far do not meet all of these criteria. Indeed, contrary to predictions from results reported with known attenuated RSV, the viruses of the present invention are not only more viable and attenuated than previous mutants, but also more genetically stable in vivo than previously tested mutants.

[0120] A number of cell lines that allow RSV growth may be used to propagate RSV virus for vaccine use and other purposes. RSV grows in a variety of human and animal cells. Preferred cell lines for propagating attenuated RS virus for vaccine use include DBSFRhL-2, MRC-5 and Vero cells. Highest virus yields are usually achieved in epithelial cell lines such as Vero cells. Cells are inoculated with virus at an infection multiplicity generally ranging from about 0.001 to 1.0 or higher and cultured under conditions permissive for virus replication, e.g., at about 30-37°C, for about 3-10 days or the time required for the virus to achieve a suitable titer. Temperature-sensitive viruses are often grown using 32°C as the "permissive temperature." The virus is removed from the cell culture and cellular components are generally separated by well-known clarification methods, e.g., centrifugation, and may be further purified as desired by methods well known to those of skill in the art.

[0121] RSV that has been attenuated as described herein can be tested in a variety of well-known and commonly accepted in vitro and in vivo models to confirm appropriate attenuation, resistance to phenotypic reversion, and immunogenicity for vaccine use. In in vitro assays, modified viruses capable of propagating attenuated, biologically derived, or recombinant RSV are tested for temperature sensitivity or "ts phenotype" of viral replication and for small plaque phenotype. The modified viruses are further tested in animal models of RSV infection. Various animal models (e.g., mice, cotton rats, and primates) have been described and are known to those skilled in the art.

[0122] According to the above description and the following examples, the present invention also provides an isolated, infectious RSV composition for use in a vaccine. The attenuated virus that is a component of the vaccine is in an isolated and generally purified form. By isolated, we mean RSV that is outside the natural environment of the wild type virus, such as the nasopharynx of an infected individual. More generally, isolated is intended to include the attenuated virus as a component of a cell culture or other artificial medium. For example, the attenuated RSV of the present invention can be produced by an infected cell culture that is isolated from the cell culture and added to a stabilizer.

[0123] The RSV vaccine of the present invention comprises as an active ingredient an immunogenically effective amount of RSV produced as described herein. Biologically derived or recombinant RSV may be used directly in the vaccine formulation. Biologically derived or recombinant modified viruses may be introduced into the host together with 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, and the like. The resulting aqueous solution is packaged for use as is or in frozen form or lyophilized to be thawed prior to use, as described above, and the lyophilized preparation is combined with a sterile solution prior to administration. The compositions may contain pharma- ceutically acceptable auxiliary substances as necessary to approximate physiological conditions, including, but not limited to, pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and the like, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sucrose, magnesium sulfate, phosphate buffer, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, sorbitan monolaurate, and triethanolamine oleate. Acceptable adjuvants include incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum, which are materials well known to those skilled in the art. A preferred adjuvant is also Stimulon adjuvant. TM QS-21 (Aquila Biopharmaceuticals, Inc., Worchester, Mass.), MPL TM (3-0-deacylated monophosphoryl lipid A; RIBI ImmunoChem Research, Inc., Hamilton, Mont.) and interleukin-12 (Genetics Institute, Cambridge, Mass.).

[0124] Upon immunization with the RSV vaccine composition, the host responds to the vaccine by producing antibodies specific to RSV viral proteins, such as the F and G glycoproteins. In addition, innate and cell-mediated immune responses are elicited, which can provide antiviral effectors and modulate the immune response. As a result of vaccination, the host is at least partially or completely immune to RSV infection or is resistant to developing moderate or severe RSV disease, particularly in the lower respiratory tract.

[0125] The host to which the vaccine is administered can be any mammal that is susceptible to infection with RSV or closely related viruses and can produce a protective immune response against the antigen of the vaccinating strain.Therefore, suitable hosts include humans, non-human primates, cows, horses, pigs, sheep, goats, lagomorphs, rodents, such as mice or cotton rats.Thus, the present invention provides a method for making vaccines for a variety of human and animal uses.

[0126] The vaccine composition comprising the attenuated RSV of the present invention is administered to a subject susceptible or otherwise at risk for RSV infection in an "immunogenically effective dose" that is sufficient to induce or enhance the individual's immune response capability against RSV. The RSV vaccine composition may be administered by any suitable method, including, but not limited to, injection, aerosol delivery, nasal spray, nasal drops, oral inoculation, or topical application. For human subjects, the attenuated virus of the present invention is administered by a well-established human RSV vaccine protocol (Karron et al. JID 191:1093-104, 2005). That is, an adult or child is inoculated intranasally with an immunogenically effective dose of droplets of the RSV vaccine, typically in a volume of 0.5 ml of a physiologically acceptable diluent or carrier. This has the advantages of simplicity and safety compared to parenteral immunization with a non-replicating vaccine. It also provides local respiratory tract immune direct stimulation, which plays a major role in resistance to RSV. Furthermore, this vaccination regimen effectively circumvents the immunosuppressive effects of RSV-specific maternally derived serum antibodies, which are typically seen at a very early age, and parenteral administration of RSV antigens can be associated with immunopathological complications that have not been observed with live virus.

[0127] In some embodiments, the vaccine can be administered intranasally or subcutaneously or intramuscularly.In some embodiments, the vaccine can be administered to the upper respiratory tract.This can be carried out by any suitable method, including but not limited to spray, droplet or aerosol delivery.Often, the composition is administered to individuals who are seronegative for antibodies against RSV or have transplacental acquired maternal antibodies against RSV.

[0128] For any given subject, the exact amount of RSV vaccine administered and the timing and repetition of administration will be determined by a variety of factors, including the patient's health and weight, the mode of administration, the nature of the formulation, etc. Dosages are generally approximately 3.0 log of virus per patient. 10 ~About 6.0 logs 10 In the range of plaque forming units ("PFU") or higher, more commonly about 4.0 log per patient10 ~5.0log 10 In one embodiment, the virus is about 5.0 log per patient. 10 ~6.0log 10 PFUs may be administered during infancy, such as 1-6 months, with one or more additional booster doses administered 2-6 months or more later. In other embodiments, young infants receive about 5.0 log per patient. 10 ~6.0log 10 Doses of PFU are given at approximately 2 months, 4 months, and 6 months, which are the recommended administration times for many other pediatric vaccines. In yet other embodiments, an additional booster dose is administered at approximately 10-15 months. In any event, the vaccine formulation should provide a sufficient amount of the attenuated RSV of the invention to efficiently stimulate or elicit an anti-RSV immune response (an "effective amount").

[0129] In some embodiments, the vaccine comprises an attenuated recombinant RSV virus that induces an immune response against a single RSV strain or antigenic subgroup, such as A or B, or against multiple RSV strains or subgroups. In this regard, rRSV can be combined in a vaccine formulation with other RSV vaccine strains or subgroups that have different immunogenic characteristics for more effective protection against single or multiple RSV strains or subgroups. They can be administered in a vaccine mixture or separately in a coordinated treatment protocol to induce more effective protection against single or multiple RSV strains or subgroups.

[0130] The resulting immune response may be characterized in a variety of ways. These include taking samples of nasal washes or serum for analysis of RSV-specific antibodies, which may be detected by complement fixation tests, plaque neutralization, enzyme-linked immunosorbent assays, luciferase immunoprecipitation assays, and flow cytometry. Additionally, immune responses may be detected by cytokine assays in nasal washes or serum, ELISPOT of immune cells from either source, quantitative RT-PCR or microarray analysis of nasal wash or serum samples, and restimulating immune cells from nasal washes or serum by re-exposure to viral antigens in vitro and analyzing for production or presentation of cytokines, surface markers or other immune correlates, or for cytotoxic activity against indicator target cells presenting RSV antigens by flow cytometry. In this regard, individuals are also monitored for signs and symptoms of upper respiratory disease.

[0131] In some embodiments, newborns and infants are given multiple doses of the RSV vaccine to induce a sufficient level of immunity. Administration is given within the first month of life and at regular intervals throughout childhood, such as 2 months, 4 months, 6 months, 1 year, and 2 years, as long as necessary to maintain a sufficient level of protection against natural RSV infection. In other embodiments, adults who are particularly susceptible to frequent or severe RSV infections, such as health care workers, childcare workers, young children, the elderly, and family members of individuals with cardiopulmonary insufficiency, are given multiple doses of the RSV vaccine to establish and / or maintain a protective immune response. The level of induced immunity can be monitored by measuring the amount of neutralizing secretory and serum antibodies, and dosages are adjusted or vaccinations are repeated as necessary to maintain the desired level of protection. Additionally, different vaccine viruses are indicated for administration to different recipient groups. For example, engineered RSV strains expressing additional proteins rich in cytokines or T cell epitopes may be particularly advantageous for adults, but not for infants. The vaccines produced according to the present invention can be combined with viruses of other subgroups or strains of RSV to provide protection against multiple RSV subgroups or strains, or selected gene segments of these strains, for example, encoding protective epitopes, can be engineered into an RSV clone as described herein. In such an embodiment, different viruses can be mixed and administered simultaneously or provided in separate preparations and administered separately. For example, the F glycoproteins of two RSV subgroups differ in amino acid sequence by only about 11%, and this similarity is the basis for the cross-protective immune response seen in animals immunized with RSV or F antigen and challenged with a heterologous strain. Thus, immunization with a single strain can also provide protection against different strains of the same or different subgroups.

[0132] The level of attenuation of the vaccine virus can be determined, for example, by quantifying the amount of virus present in the respiratory tract of an immunized host and comparing it to the amount of wild-type RSV or other attenuated RS viruses being evaluated as candidate vaccine strains. For example, the attenuated viruses of the present invention have a greater degree of restriction of replication in the upper respiratory tract of a highly susceptible host, such as a chimpanzee, compared to the replication level of the wild-type virus, e.g., 10-1000 times less. To further reduce the rhinorrhea associated with viral replication in the upper respiratory tract, an ideal vaccine candidate virus should exhibit restricted levels of replication in both the upper and lower respiratory tracts. However, the attenuated viruses of the present invention must be sufficiently infectious and immunogenic in humans to provide protection in vaccinated individuals. Methods for determining RSV levels in the nasopharynx of an infected host are well known in the literature. Samples are obtained by aspiration or washing of nasopharyngeal secretions, and the virus is quantified by tissue culture or other laboratory methods. See, e.g., Belshe et al., J. Med. Virology 1:157-162 (1977), Friedewald et al., J. Amer. Med. Assoc. 204:690-694 (1968); Gharpure et al., J. Virol. 3:414-421 (1969); and Wright et al., Arch. Ges. Virusforsch. 41:238-247 (1973). The virus can be conveniently located in the nasopharynx of a host animal, such as a chimpanzee.

[0133] The present invention also provides a method for producing infectious RSV from one or more isolated polynucleotides, e.g., one or more cDNAs. According to the present invention, a cDNA encoding a RSV genome or antigenome is constructed by intracellular or in vitro co-expression with viral proteins necessary for infectious RSV formation. "RSV antigenome" refers to an isolated positive-sense polynucleotide molecule that serves as a template for the synthesis of progeny RSV genomes. Preferably, a cDNA is constructed that is a positive-sense version of the RSV genome, corresponding to a replicative intermediate RNA or antigenome, to minimize the possibility of hybridizing with a positive-sense transcript of complementary sequences encoding proteins necessary to produce a transcription, replicative nucleocapsid-encoding sequence, i.e., the N, P, L and M2-1 proteins.

[0134] The native RSV genome generally comprises a negative-sense polynucleotide molecule, which encodes eleven viral proteins, namely nonstructural proteins NS1 and NS2, N, P, matrix (M), small hydrophobic (SH), glycoprotein (G), fusion (F), M2-1, M2-2 and L, via complementary viral mRNA, substantially as described in Mink et al., Virology 185: 615-624 (1991), Stec et al., Virology 183: 273-287 (1991) and Connors et al., Virol. 208:478-484 (1995). For purposes of the present invention, the genome or antigenome of the recombinant RSV of the present invention need only contain the genes or portions thereof necessary to render the virus or component virus particles encoded thereby infectious. Furthermore, the genes or portions thereof may be provided by more than one polynucleotide molecule, i.e., the genes may be provided by complementation, etc., from another nucleotide molecule.

[0135] Recombinant RSV refers to an RSV or RSV-like virus or component virus particle derived or propagated directly or indirectly from a recombinant expression system or a virus or component virus particle produced therefrom. Recombinant expression systems use recombinant expression vectors that contain an operably linked transcription unit that includes at least one genetic element or elements that have a regulatory role in RSV gene expression, such as a promoter, a structural or coding sequence that is transcribed into RSV RNA, and a collection of appropriate transcription start and stop sequences.

[0136] To produce infectious RSV from a cDNA-expressed genome or antigenome, the genome or antigenome is co-expressed with RSV proteins necessary to (i) produce nucleocapsids capable of RNA replication and (ii) render the progeny nucleocapsids competent for both RNA replication and transcription. Transcription by the genome nucleocapsid provides other RSV proteins to initiate productive infection. Additional RSV proteins required for productive infection can also be provided by co-expression.

[0137] Another means of constructing a cDNA encoding a portion thereof is reverse transcription PCR, using improved PCR conditions that reduce the number of subunit cDNA components to one or two pieces (e.g., as described in Cheng et al., Proc. Natl. Acad. Sci. USA 91:5695-5699 (1994); Samal et al., J. Virol 70:5075-5082 (1996)). In other embodiments, different promoters (e.g., T3, SP6) or different ribozymes (e.g., those of hepatitis delta virus) can be used. Different DNA vectors (e.g., cosmids) can be used for propagation to better accommodate larger sized genomes or antigenomes.

[0138] The N, P, L and M2-1 proteins may be encoded by one or more expression vectors, which may be the same or separate from those encoding the genome or antigenome, and various combinations thereof. Additional proteins may be optionally included, encoded by their own vectors or vectors encoding the N, P, L or M2-1 proteins or complete genomes or antigenomes. Expression of the genome or antigenome and proteins from a transfecting plasmid may be achieved, for example, by infection transfection or transduction with an expression system for T7 RNA polymerase, for example, by placing each cDNA under the control of the promoter of T7 RNA polymerase, provided by a vaccinia virus MVA strain recombinant expressing T7 RNA polymerase (Wyatt et al., Virology, 210:202-205 (1995)). Viral proteins and / or T7 RNA polymerase may also be provided by transfection of transformed mammalian cells or by transfection of preformed mRNA or proteins.

[0139] In summary, the materials, information and methods described herein provide a series of attenuated strains with progressively attenuated phenotypes and provide guidance for selecting suitable vaccine candidate strains based on clinical benchmarks.

[0140] Although various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of these embodiments may be made by those skilled in the art. However, it should be clearly understood that such modifications and adaptations are within the scope of the present invention, as set forth in the appended claims. The following examples are provided for illustrative purposes and are not intended to limit the scope of the present invention.

[0141] Each publication, sequence or other reference disclosed below and elsewhere herein is hereby incorporated by reference in its entirety to the extent not inconsistent with this disclosure. U.S. Provisional Application No. 62 / 399,133, filed Sep. 23, 2016, entitled "Improved codon-pair-deoptimized vaccine candidates for human respiratory syncytial virus," U.S. Provisional Application No. 62 / 400,476, filed Sep. 27, 2016, entitled "Vaccine candidates for respiratory syncytial virus (RSV) having attenuated phenotypes," and U.S. Publication No. 2015-036862, entitled "Attenuation of human respiratory syncytial virus by genome scale codon-pair deoptimization," are hereby incorporated by reference in their entireties. EXAMPLES

[0142] material and method The following materials and methods were used in the examples below.

[0143] Virus harvest and titration Vero cells were scraped into tissue culture medium, vortexed for 30 seconds, clarified by low-speed centrifugation, and flash frozen. Viral titers of the clarified fluids were determined by immune plaque assay on Vero cells at 32°C.

[0144] Viral stocks were made by scraping infected cells into the medium, followed by vortexing for 30 s and clarifying the supernatant by centrifugation. Viral aliquots were flash frozen and stored at -80°C. Viral titers were determined by plaque assay on Vero cells with a 0.8% methylcellulose overlay. After 10–12 days of incubation at 32°C, plates were fixed with 80% cold methanol and plaques were visualized by immunostaining with a cocktail of three RSV-specific monoclonal antibodies. Titers were expressed as pfu / ml. Viral RNA was isolated from all viral stocks and sequence analysis of the viral genome was performed from overlapping RT-PCR fragments by Sanger sequencing to confirm that the genomic sequences of the recombinant viruses were correct and free of adventitious mutations. The only sequences that were not directly confirmed for each genome were the outermost primer positions, i.e., nucleotides 1–23 and 15,174–15,222.

[0145] Ion Torrent Deep Sequencing Purified viral RNA from clarified cultures was copied into eight overlapping fragments spanning the viral genome. Libraries were prepared according to Ion Torrent protocols, loaded onto semiconductor sequencing chips, and sequenced with a Personal Genome Machine (Ion Torrent). Nucleotide variants were called if they occurred >50-fold with a mean read depth of 1000× and a P-value <10-7 (quality score >70).

[0146] Viral RNA was extracted using a Qiagen viral RNA extraction kit from the indicated aliquots of viruses that were passaged during the Min_L or Min_FLC temperature stress test. Viral RNA was reverse transcribed using superscript II RT (Life Technologies) according to the manufacturer's recommendations. cDNA was then amplified by PCR using RSV-specific primers and pfx DNA polymerase enzyme (Life Technologies) of eight overlapping fragments covering the entire viral genome. Each PCR product was purified using a QIAquick PCR purification kit (Qiagen).

[0147] Equal amounts of DNA from each of the 8 PCR reactions were pooled into a 1.5 ml LoBind tube (Eppendorf). The DNA was subjected to enzymatic shearing using ShearEnzyme (Ion Torrent) at 37 °C for 30 min in a heat block. The sheared DNA was then purified using 1.8 volumes of Agencourt magnetic beads (Beckman). The Agencourt beads were washed twice with 0.2 ml of 70% ethanol, air dried for 5 min, and resuspended in 20-30 μl of 10 mM Tris-HCl pH 7.5 buffer, followed by incubation at room temperature for 5 min. The DNA was recovered from the supernatant by placing the 1.5 ml LoBind tube containing the Agencourt beads on a magnetic rack for 2 min. The DNA was treated with end-repair enzymes (Ion Torrent) according to the manufacturer's instructions. The end-repaired DNA was purified with 1.8 volumes of Agencourt beads as above and recovered on a magnetic rack.

[0148] Approximately 100 ng of repaired DNA from each sample was used to ligate with specific barcode and sequencing adapters in a 20 μl reaction volume with ligase and buffer (Ion Torrent) according to the manufacturer's instructions. The ligation reaction was carried out at room temperature for 30 min and stopped by adding 4 μl of 0.5 M EDTA pH 8.0. Equal volumes of the different ligated DNA libraries were then combined in a 1.5 ml LoBind tube and purified with 1.8 volumes of Agencount as described above to obtain DNA libraries. The DNA was further subjected to nick translation using Bst 2.0 DNA polymerase and buffer (NEB). The digested DNA was purified using a spin column MinElute kit (Qiagen).

[0149] Approximately 100 ng of the DNA library was then added to a PCR mix using Platinum High Fidelity DNA Polymerase Master Mix (Life Technologies), followed by two cycles of 10 min at 95°C, followed by two cycles of 30 s at 95°C, 30 s at 58°C, and 30 s at 72°C. The PCR products were further purified with 1.8 volumes of Agencourt as above, and DNA was obtained using a magnetic rack. DNA was quantified using the Qubit system (Invitrogen).

[0150] Approximately 70 million DNA molecules in 1 ml of PCR solution were mixed with a fixed ratio (0.5-1.0) of Ion sphere particles (ISP) (Ion Torrent) in the presence of PCR reaction mix and oil (Ion Torrent) to form droplets of tens of millions of emulsion particles. These droplets were passed through a OneTouch (Ion Torrent) closed capillary PCR plate, which allows liquid and particles to pass continuously through the plate to perform emulsion PCR amplification. The ISPs were obtained by OneTouch centrifugation into a pair of collection tubes. At the end of the OneTouch emulsion PCR, the collection tubes were centrifuged at 15,000g for 3 min to remove most of the supernatant. The ISPs were washed once with 1 ml wash buffer (Ion Torrent) and centrifuged at 15,500g for 3 min to remove most of the supernatant. The ISPs containing amplified DNA were separated from the ISPs without DNA by centrifugation with Dynabeads® MyOne TM Further enrichment was performed by incubating with streptavidin C1 magnetic beads for 10 min at room temperature on a rotating rack. Enriched IPS were obtained by placing the tubes on a magnetic rack for 2 min, washing twice with 0.2 ml of wash buffer by pipetting, placing on a magnetic rack for 2 min, and discarding the supernatant. ISP was purified by Dynabeads® MyOne TMStreptavidin C1 magnetic beads were eluted by incubation with 0.4 ml 0.125N NaOH and 0.1% Tween 20 for 7 min at room temperature in a rotating rack. The eluted ISP was washed twice with wash buffer and centrifuged at 15,500 g for 4 min to remove most of the supernatant. The ISP was resuspended by pipetting and transferred to Dynabeads® MyOne TM Place on magnetic rack for 2 minutes to remove last traces of beads.

[0151] 100 μl of the solution was transferred to a new tube when the final library of ISPs was ready for QC testing and sequencing. For sequencing, the ISPs were centrifuged at 15,500 g for 3 min and most of the supernatant was removed. The ISPs were resuspended by pipetting and transferred to a 0.2 ml PCR tube containing 150 μl Annealing Buffer. 5 μl Control Ion Spheres TM (Ion Torrent) was added to the ISP mix and centrifuged for 3 min at 15,500 g to remove most of the supernatant from the top, leaving 15 μl at the bottom, followed by the addition of 12 μl sequencing primer, denaturing and annealing for 2 min at 95° C. and 2 min at 37° C. 3 μl DNA polymerase (Ion Torrent) was added and the samples were loaded onto semiconductor sequencing chips 316 or 318 (Ion Torrent) and DNA sequencing was performed on a Personal Genome Machine (PGM) (Ion Torrent).

[0152] DNA sequences were analyzed against Min_FLC or Min_L control sequences using VariantCaller 3.2 software from Ion Torrent on the Ion Torrent Server. The analysis pipeline was set to default somatic variant placement. Nucleotide variants were analyzed as previously described if the variants had an average read depth of 1000× and a P value <10 -7A call was made if it occurred >50x (quality score >70). Raw read data were also manually verified using the genome browser IVG (The Broad Institute).

[0153] Deep sequencing of long PCR fragments Purified viral RNA from cultures was reverse transcribed using the Maxima H minus first strand cDNA synthesis kit (Thermo Scientific). cDNA was generated using RSV-specific primers and the SequalPrep long PCR kit (Life Technologies) to generate a PCR product of 8.2 kb of the genome extending from the 3' end to the middle of the M2-2 ORF. DNA template libraries were prepared, sequenced, and analyzed using PacBio kits and instrumentation and CluCon software (https: / / github.com / mpsbpbi / clusteringConsensus).

[0154] Mutations that were not coexistent or occurred in the M2-1 and P genes during the first four passages of Min_L lineage #3 at 38°C and 39°C were examined by deep sequencing. To do so, viral RNA from aliquots of virus from these passages was extracted using the Qiagen virus extraction kit as described above. The viral RNA was then reverse transcribed using the Maxima H minus first strand cDNA synthesis kit (Thermo Scientific) as recommended by the manufacturer. An 8.2 kb PCR product was generated, covering the region from the 3' end of the genome to the M2-2 gene, using RSV-specific primers and the SequalPrep long PCR kit (Life Technologies). Created .

[0155] To prepare the PacBio SMRTbell DNA template library, the PCR products were purified as described above and then concentrated using 0.45 volumes of AMPure PB magnetic beads. To bind the DNA to the beads, the mixture was mixed at 2000 rpm for 10 minutes at room temperature on a VWR vortex mixer. After a brief spin to pellet the beads, each tube was placed in a magnetic bead rack and the supernatant was carefully discarded. The beads were then washed twice with 1.5 ml of freshly prepared 70% ethanol. After removing the ethanol, the bead pellet was allowed to dry for about 1 minute. The tubes were then removed from the magnetic bead rack and centrifuged to pellet the beads. The DNA was then eluted using Pacific Biosciences Elution buffer. To repair any DNA damage, the concentrated DNA was incubated in DNA damage repair buffer, NAD+, ATP high, dNTPs and DNA damage repair enzyme mix in LoBind tubes for 20 minutes at 37°C. The DNA was then incubated with DNA end repair mix for 5 minutes at 25°C. After the reaction, the DNA was purified using AMPure PB beads as described above, and the beads were eluted in 30 μl of elution buffer.

[0156] The end-repaired DNA was then annealed to the blunt-end adapters in a reaction containing the adapters, buffer, ATP and ligase for 15 min at 25° C. The ligase was inactivated at 65° C. for 10 min. A final 1-h, 37° C. exonuclease step was included to remove any failed ligation products. The SMRTbell DNA template library was then purified 3-fold using AMPure PB beads as described above.

[0157] After purification, SMRTbell library templates were sequenced on SMRT cells using the PacBio DNA Polymerase Binding Kit P6 on a PacBio RSII instrument. Each sample library was sequenced on 2 SMRT cells using MagBead loading with a video collection time of 240 min.

[0158] Data was analyzed using CluCon software (https: / / github.com / mpsbpbi / clusteringConsensus). All reads were aligned with the control Min_L sequence. Only reads spanning 99% (>8161 bases) of the complete target were analyzed; yielding an average of 32,738 near-full-length reads per read time point (24,131 minimum reads, 41,118 maximum reads). The algorithm identifies variant positions by examining the alignments and finding positions with low observed frequency that cannot be statistically explained by random noise. The complete topological haplotype is then estimated by matching what is sequenced in each of the near-full-length reads at the variant position. Statistical tests are used to discard "noisy" haplotypes or those that are simply explained by other truly observed haplotypes that are corrupted by sequencing noise.

[0159] Determination of the temperature shutoff of CPD rRSV The ts phenotype of each of the rRSV viruses was assessed by plaque formation efficiency at 32°C, 35°C, 36°C, 37°C, 38°C, 39°C and 40°C. Plaque assays were performed in duplicate on Vero cells and incubated in sealed caskets in temperature-controlled water baths at various temperatures as described previously. The shutoff temperature (T SH ) is defined as the lowest restrictive temperature at which there is a reduction in plaque number compared to 32°C that is more than 100-fold greater than that observed with wt RSV at these two temperatures.

[0160] In vitro virus replication kinetics. Multicycle and single cycle growth kinetics were performed on confluent monolayers of Vero cells in 6-well plates at both 32°C and 37°C.

[0161] In multicycle growth kinetic experiments, Vero cells were infected in duplicate with the indicated viruses at an MOI of 0.01 pfu / cell. On days 0-14, virus was harvested by scraping infected cells into the medium, followed by vortexing for 30 seconds and clarification of the supernatant by centrifugation. Virus inocula and daily aliquots were snap frozen and stored at -80°C. Virus titers were determined by plaque assay as described above.

[0162] In single cycle growth kinetic experiments, three wells of Vero cells in a 6-well plate were infected with the indicated viruses at an MOI of 3 pfu / well at 32°C or 37°C. Every 4 h from 4 to 24 h postinfection, cell-associated RNA was obtained from one well using the RNeasy mini kit (Qiagen) according to the manufacturer's instructions. For Western blot analysis, total cell lysates were collected in NuPage LDS sample buffer (Life Technologies) and then homogenized using QIAshredder spin columns (Qiagen). Finally, the last well was used for virus capture and virus titer determination as described above.

[0163] Strand-specific rRSV RNA quantification Cell-associated RNA from single-cycle replication experiments was used to specifically quantitate viral negative-sense (genomic) and positive-sense (mRNA and antigenomic) RNA as previously described. qPCR results were analyzed using the comparative threshold cycle (ΔCt) method, normalized to 18S rRNA, and then expressed as log2 fold increase over the indicated control samples.

[0164] Cell-associated RNA from single-cycle replication experiments was used to specifically quantify viral negative-sense (genomic) and positive-sense (mRNA and antigenomic) RNA as previously described. Taqman assays for antigenomic / mRNA specific for each of the 11 wt RSV genes were designed using Primer Express 3.0 software (Life Technologies). In particular, for the L gene, three different Taqman assays were designed for the wt sequence and four for the CPD sequence.

[0165] One microgram of DNA digested RNA was reverse transcribed using Superscript III (Life Technologies) in a 20 μl reaction using tagged first strand primers specific for the genome or antigenome / mRNA. After 5-fold dilution, each of the cDNAs was amplified in triplicate with a tag-specific primer, a second gene-specific primer and a probe. Therefore, only cDNAs containing the tagged RT primer sequence were amplified. The probe sequence was RSV gene specific. To normalize the results, 18S rRNA was quantified in parallel using first strand cDNA produced with random primers and a standard 18S rRNA Taqman assay (Applied Biosystems). qPCR results were analyzed using the comparative threshold cycle (ΔCt) method, normalized to 18S rRNA, and then expressed as log2 fold increase over Min_L at 4 hours, except for wt L quantification, where data was expressed as fold increase over wt at 4 hours. A negative control without first strand primer was included in each strand-specific qPCR to demonstrate the absence of non-specific priming during first strand cDNA synthesis.

[0166] Western blot analysis.Cell lysates prepared from the single-cycle infection experiments described above were resolved on NuPAGE 4-12% Bis-Tris SDS-PAGE gels in MES electrophoresis buffer (Life Technologies) alongside Odyssey Two-Color Protein Molecular Weight Marker (Li-Cor). Thirty micrograms of protein were transferred to PVDF-F membranes (Millipore) in 1x NuPAGE buffer. Membranes were blocked with Odyssey blocking buffer (LI-COR) and incubated with primary antibodies in the presence of 0.1% Tween-20. The primary antibodies and dilutions used were as follows: mouse anti-RSVN, P, G, F and M2-1 monoclonal antibodies (1:1,000) were purchased from Abcam; rabbit polyclonal antiserum recognizing both NS1 and NS2 was raised by peptide immunization of rabbits (Abgent) with a synthetic peptide representing the C-terminal 14 amino acids of NS2 (the C-termini of NS2 and NS1 are identical in the last 4 amino acids, which likely explains the cross-reactivity); rabbit anti-GAPDH polyclonal antibody (1:200) was used as a loading control (Santa-Cruz Biotechnologies, Inc.). Secondary antibodies used at a dilution of 1:15,000 were goat anti-rabbit IgG IRDye 680 (Li-Cor) and goat anti-mouse IgG IRDye 800 (Li-Cor). Membranes were scanned with an Odyssey® Infrared Imaging System. Data was analyzed using Odyssey software, version 3.0 (Li-Cor). For quantification of the identified RSV proteins of interest, the fluorescent signals were background corrected. Numbers indicate the median fluorescence intensity of each protein band.

[0167] Plaque size determination. Viral plaque size was determined by plaque assay in Vero cells using 24-well plates. Vero cell monolayers were inoculated with 30 pfu / well of pre-titered and sequenced virus stocks. After 2 h of adsorption, a 0.8% methylcellulose overlay was added to each well. After 12 days of incubation at 32°C, plates were fixed with 80% cold methanol. Wells were then incubated with a cocktail of three RSV-specific monoclonal antibodies (Bukreyev et al. 2001) in blocking buffer (Odyssey buffer, Licor) for 1 h. After washing with blocking buffer, plaques were stained with goat anti-mouse IRdye 680LT (Licor) secondary antibody and plaques were visualized using the Odyssey® Infrared Imaging System. Images were analyzed using Image J and the area of ​​more than 1000 plaques per virus was measured and expressed in pixels2. Viral plaque size distributions were compared for statistical significance using the Kolmogorov-Smirnov test followed by Bonferroni correction (Prism 6.0, GraphPad). Data sets were considered statistically significant only at p ≤ 0.05.

[0168] CPD rRSV replication assessment in mice and hamsters Animal studies were approved by the NIAID Animal Care and Use Committee and were performed using methods previously described.

[0169] All animal studies were approved by the National Institutes of Health (NIH) Institutional Animal Care and Use Committee (ACUC). CPD virus replication was assessed in the upper and lower respiratory tract of 6-week-old BALB / c mice as previously described. Groups of 20 mice were injected with 10 6Mice were inoculated intranasally with pfu of wt rRSV, Min_L, M2-1[A73S], M2-1[N88K] or NPM2-1[N88K]L. On days 4 and 5, eight mice from each group were sacrificed by carbon dioxide inhalation. The remaining four mice from each group were sacrificed on day 10. Nasal turbinate (NT) and lung tissues were harvested and homogenized separately in Leibovitz (L)-15 medium containing 1× SPG, 2% L-glutamine, 0.06 mg / mL ciprofloxacin, 0.06 mg / mL clindamycin phosphate, 0.05 mg / mL gentamicin, and 0.0025 mg / mL amphotericin B. Viral titers were determined in duplicate by incubation at 32°C in Vero cells as described above. Viral detection limits were 100 and 50 pfu / g for NT and lung specimens, respectively.

[0170] CPD virus replication was evaluated in the upper and lower respiratory tract of 6-week-old Golden Syrian hamsters, and immunogenicity was also tested. On day 0, groups of 18 hamsters were inoculated with 10 6 Mice were inoculated intranasally with pfu of wt rRSV, Min_L, M2-1[A73S], M2-1[N88K], or NPM2-1[N88K]L.

[0171] On day 3, which corresponds to the peak of wt rRSV replication in hamsters, 9 hamsters from each group were sacrificed by carbon dioxide inhalation. NT and lung tissues were harvested and homogenized as described above. Viral titers were determined in duplicate by incubation at 32°C in Vero cells as described above. The limit of virus detection was 50 pfu / g in NT and lung.

[0172] Two days before immunization and on day 26 after immunization, blood from 9 hamsters / group was collected for serum collection and RSV antibody titer determination. 6Hamsters were challenged intranasally with pfu of wt rRSV. Three days after challenge, hamsters were sacrificed by carbon dioxide inhalation. NT and lung tissues were harvested and wt rRSV titers were determined in duplicate by incubation at 32° C. in Vero cells as described above.

[0173] Molecular dynamics analysis of M2-1 tetramer Mutations were made to the crystal structure of the transcription terminator M2-1 protein of human RSV (PDB ID 4C3D) using the SYBYL program (Certara). Molecular dynamics simulations were performed using the NAMD program (v.2.9).

[0174] Mutations were made to the crystal structure of the transcription termination conformation M2-1 protein of human RSV (PDB ID 4C3D) using the SYBYL program (Certara, St. Louis, MO). Mutants or wt RSVM2-1 were analyzed using the VMD program to determine the TIP3P water molecules and Na + and Cl - Explicitly solvated with counterions. All-atom, isobaric-isothermal (1 atm, 310 K) molecular dynamics simulations were performed with periodic boundary conditions using the NAMD program (v.2.9) on a Biowulf Linux cluster at the National Institutes of Health, Bethesda, MD (http: / / hpc.nih.gov) after explicit solvation and energy minimization, followed by warming in 10 K increments up to 310 K. Electrostatic interactions were calculated using Particle-Mesh Ewald summation. The CHARMM27 force field was used with CHARMM atom types and charges. For all simulations, a 2 femtosecond built-in time step was used with a 12 Å cutoff. The temperature was kept at 310 K using Langevin dynamics, and the pressure was controlled using a modified Nose-Huber-Langevin. Simulations were run for 100 ns.

[0175] statistical analysisPlaque size distribution was analyzed by the Kolmogorov-Smirnov test followed by Bonferroni correction. Viral replication and antibody responses in animal experiments were analyzed using the nonparametric Kruskal-Wallis test followed by Dunn post-hoc test. 10 Transformations were used on data sets when necessary to obtain equal standard deviations between groups. Statistics were performed using Prism 6 (GraphPad Software). Data were considered significant only when p<0.05.

[0176] Example 1: Production of Min_L and Min_FLC RSV constructs. The design of the CPD RSV genes and the construction and rescue of Min_L and Min_FLC have been previously described in US Publication US2015-0368622 and Le Nouen et al. (2014). The CPD ORF was designed based on RSV strain A2 using a previously described computational algorithm (Coleman et al. 2008 and Mueller et al. 2010). Min_L contains the CPD L ORF, which shows 1,378 silent mutations compared to the wild-type (wt) L ORF. Min_FLC (for full-length clones) contains all CPD ORFs except M2-1 and M2-2, which were left unmodified because these overlapping ORFs participate in combined stop-start translation that is dependent on sequences (and possibly secondary structures) that are currently incompletely defined. Min_FLC contains 2,692 silent mutations compared to wt RSV (Figure 1A). The amino acid sequences of Min_L and Min_FLC are identical to those of wt RSV. The viruses were constructed using the RSV6120 backbone with a 112-nt deletion in the downstream NTR of the H gene and 5 silent nucleotide point mutations involving the last 3 codons and the stop codon of the SH ORF. These changes in the SH gene stabilize RSV cDNA during propagation in E. coli (Bukreyev et al. 2004). The wt RSV in this study was the 6120 virus. Min_L and Min_FLC virus stocks were fully sequenced by Sanger and Ion Torrent deep sequencing and found to be free of adventitious mutations. The nucleotide sequence of Min_FLC is shown in SEQ ID NO:12, and the nucleotide sequence of Min_L is shown in SEQ ID NO:13.

[0177] Example 2: Codon pair deoptimization (CPD) of multiple RSV genes resulting in a highly stable temperature sensitive (Ts) phenotype restricted to replication at 32-34°C. As described above, Min_FLC (for full-length clones) is a mutant in which 9 of 11 RSVORFs (except M2-1 and M2-2) are CPDs, resulting in a total of 2,692 silent mutations (Figure 1A). Min_FLC is highly temperature sensitive and can be expressed by a temperature-blocking factor (T SH ) plaques at 35°C, whereas wild-type (wt) rRSV readily forms plaques at 40°C. SH is defined as the least restrictive temperature at which the difference in titer compared to 32°C is ≧100-fold reduction compared to the difference in titer observed with wt RSV at these two temperatures.

[0178] To test Min_FLC stability, a temperature stress test was used, which represents a surrogate model of genetic stability during viral replication and spreads from the cool upper respiratory tract to the warm lower respiratory tract. Ten independent 25 cm 2 Duplicate flasks were infected with Min_FLC at an initial MOI of 0.1 plaque-forming units (pfu) / cell and serially passaged at increasing temperatures for a total of 18 passages representing 7 months of continuous culture (flasks were incubated at the starting temperature described until extensive cytopathology was observed. Virus was collected and serially passaged at increasing restrictive temperatures (1°C temperature increase every other passage). Two further replicate flasks were infected as controls and passaged in parallel at the permissive temperature of 32°C (Figure 1B-C). One ml (out of a total of 5 ml) of supernatant was used to inoculate the next passage. After each passage, aliquots were frozen for titering and sequence analysis by Sanger sequencing and / or deep sequencing as described. Viral titers were determined by plaque assay at the permissive temperature (32°C).

[0179] At 32°C, Min_FLC is 10 6 ~10 7pfu / ml (Fig. 1B). Deep sequencing of the complete genome of two control lineages after 18 passages showed only low levels of sporadic mutations (Fig. 7), indicating that Min_FLC is genetically stable under permissive conditions. In flasks incubated at increasing temperatures, Min_FLC replicated efficiently at 32°C and 33°C (10 6 ~10 7 pfu / ml, Fig. 1C). However, after the first passage at 34°C (P5), viral replication was reduced 200-fold in all 10 lineages, and by the end of the second passage at 35°C (P8), virus was undetectable in 9 lineages. In the 10th lineage, virus was undetectable by the end of the first passage at 37°C (P11). In contrast, as noted, wt rRSV showed no growth restriction at temperatures up to at least 40°C.

[0180] Therefore, Min_FLC was highly restricted, if not inactive, at temperatures above 34–35°C (the latter being its T SH As a result, Min_FLC cannot escape its Ts phenotype under stress conditions and is phenotypically stable. Sequencing was not performed on Min_FLC samples passaged under increasing restrictive temperatures due to the rapid decrease in titer. These results meet the expectations of phenotypic stability for CPD viruses.

[0181] Example 3: Temperature stress on Min_L virus promoted the emergence of multiple mutations in multiple genes. The Min_L virus, in which the L ORF alone (representing 48% of the assembled RSVORFs) was a CPD, gave rise to 1,378 silent mutations (51% more changes than in Min_FLC). SH Ten replicate flasks were infected with Min_L and serially passaged at gradually increasing temperatures for a total of eight passages, corresponding to two months of continuous culture, and two further replicate flasks were infected as controls and passaged in parallel at 32 °C (Figure 1D-E).

[0182] As expected, Min_L efficiently grew at each passage (10 7 pfu / ml) (Fig. 1D). Sequence analysis of RNA from control lineages by deep sequencing at P6 (Fig. 8) and by Sanger sequencing at P8 (data not shown) showed only sporadic, low-level mutations. In 10 lineages passaged at increasing temperatures, the titer of Min_L in 9 flasks decreased by about 20-fold at the end of P1 (37°C) (Fig. 1E). However, during the second passage at 37°C, the titer of the same 9 lineages increased by about 200-fold, indicating that selection and growth of temperature-adapted mutants had already occurred. After P3 (38°C), the virus titer of all 10 lineages decreased constantly: at P8 (second passage at 40°C), the virus was undetectable in 7 lineages, and in the other 2 lineages, the titer was extremely low (20 pfu / ml each). The remaining lineage (#3, colored green) had a titer of 500 pfu / ml. Therefore, various Min_L lineages appear to undergo some loss of temperature-sensitive phenotype but are ultimately highly restricted at 40°C.

[0183] Whole genome deep sequencing was performed on each of the 10 differentiation lineages passaged through increasing temperatures at P6 (second passage at 39°C), when viral replication was still detectable in each lineage. Mutations present in ≥45% of the sequencing reads are shown in Table 1. Remarkably, many of these striking mutations were in genes that were not subjected to CPD. Notably, of these 23 striking mutations, 21 were distributed in 6 ORFs (P, M, SH, G, M2-1, and L) and 2 in extragenic regions. Of the 23 mutations, 11 (48%) and 5 (22%) occurred in the M2-1 and L ORFs, respectively. Of the 21 mutations present in the ORFs, all but one were missense mutations, suggesting a bias for amino acid changes. This positive selection of amino acid changes suggests that at least part of the adaptation of Min_L to selective stress involves structure / function changes in various viral proteins. Some mutations were common across differentiation lineages. In particular, a mutation in the antitermination transcription factor M2-1 [A73S] was prominent in 8 of 10 lineages. Other M2-1 mutations (N88K) and L (A1479T) were prominent in 2 lineages. M2-1 was the only gene with at least one prominent mutation in all lineages.

[0184] Table S1 shows mutations that were present in ≥5% of reads for P6 samples from the same experiment. At this lower cutoff, more mutations were prominent in all genes except NS2. Similar to the prominent mutations shown in Table 1, these less prominent mutations were mostly missense mutations. In the CPD L ORF, only 17 of the total 31 mutations (55%) involved nts or codons modified in the CPD (Table S1).

[0185] Whole genome deep sequencing analysis was performed to evaluate the transient occurrence of mutations in the complete passage series of lineages #3 and #8, which are the most interesting because they maintained the highest titers during stress testing (Figure 1E) and therefore have the greatest deattenuation. The occurrence and frequency of the more abundant mutations are shown graphically in Figure 1F (lineage #3) and G (lineage #8). A more detailed list of mutations is shown in Tables S2 and S3.

[0186] In both lineages, a single mutation ([A73S] in M2-1) appeared at P1 (13% of each lineage) and increased at P2 (37% and 51% in lineages #3 and 8, respectively). From P2, the two lineages followed different evolutionary trajectories. In lineage #3, between P2 and P3, the frequency of the M2-1 mutation [A73S] began to decrease (30%), whereas 10 other mutations in M2-1 appeared, constituting roughly 15–30% of the population (Table S2). One of these M2-1 mutations, namely [N88K], became abundant at P4 (71%) and closely co-occurred with the equally abundant (66%) mutation [E114V] in P (Figure 1F). The other M2-1 mutations decreased and became undetectable from P5 onwards, suggesting a selective sweep. Two additional prominent mutations were acquired at P6 (N[K136R]) and P7 (L[T1166I]). In lineage #8, the mutation in M2-1 [A73S] was fixed at P4 (88%). At P2, two additional mutations (5' trailer region and L) were acquired and became prominent and fixed by the end of P4. After the first passage at 40°C (P7), some additional mutations were acquired, three of which became prominent by the end of P8; one silent in L, one silent in N, and one in P [E113G].

[0187] Example 4: Two mutations, N88K and A73S, in the antitermination transcription factor M2-1 are significant but incompatible. All 10 lineages at P6 had the M2-1 mutation [A73S] or [N88K] (Table 1, Figure 2A). Therefore, these two M2-1 mutations were considered segregated. Furthermore, the disappearance of the [A73S] mutation during the passage series of lineage #3 was accompanied by the appearance and increase of [N88K] until the latter was present in the complete population (Figure 1F). We reevaluated the deep sequencing results of lineage #3 and scored only the reads that spanned both positions 73 and 88 in M2-1, thus providing a combined analysis. At P3 and P4, only 1% of the reads had both mutations (Figure 2B), suggesting that these two mutations in M2-1 are incompatible in the same genome and therefore constitute two separate virus populations.

[0188] To further characterize the dynamics of the main virus population in lineage #3, the association of the major mutations that emerged during the first four passages was investigated using PacBio long reads, a single molecule sequencing that provides complete read data for the entire 8.2 kb region from the 3' genomic end to the middle of the M2-2 ORF. This showed that the first four passages contained four major virus subpopulations (Figure 2C). One was the original Min_L virus, which gradually decreased with passage. The other subpopulation, which had only the M2-1 mutation [A73S], peaked at P2 and nearly disappeared at P4. The other had 7 mutations (3 synonymous, 4 nonsynonymous) in M2-1, which appeared together at P2, reached a maximum at P3 (about 20%), and then disappeared. Finally, the fourth subpopulation had the P[E114V] and M2-1[N88K] mutations, which appeared together at P3 and became prominent at P4.

[0189] Example 5: Introduction of mutations N[K136R], P[E114V], M2-1[N88K], M2-1[A73S] and L[T1166I] into Min_L. Direct identification of the mutations responsible for the temperature sensitivity of Min_L was sought by introducing into Min_L, individually and in combination, the major mutations identified in lineage #3, namely N[K136R], P[E114V], M2-1[N88K] and L[T1166I] (Fig. 1F), as well as one of the prominent mutations in replication #8, the M2-1 mutation [A73S] (Fig. 1G). The resulting 12 viruses (Fig. 3A) were recovered and fully sequenced to confirm the correct sequence and the absence of additional mutations.

[0190] This was performed using the Quickchange Lightning Site-directed Mutagenesis kit (Agilent) according to the manufacturer's recommendations. The cDNA was fully sequenced by Sanger sequencing using a set of specific primers. CPD viruses carrying the targeted mutations were then rescued from the cDNA as previously described. Briefly, BSR T7 / 5 cells were transfected using Lipofectamine 2000 (Life Technologies) and a plasmid mixture containing 5 μg of full-length cDNA, 2 μg each of pTM1-N and pTM1-P, and 1 μg each of pTM1-M2-1 and pTM1-L. After overnight incubation at 37°C, the transfected cells were harvested by scraping into the medium and added to a subconfluent monolayer of Vero cells and incubated at 32°C. Rescued viruses were harvested 11–14 days after transfection.

[0191] Introduction of the N[K136R] or P[E114V] mutation alone significantly reduced T SH Interestingly, introduction of M2-1[A73S] or [N88K] alone resulted in an approximately 1°C increase in T SH induced a 2°C increase in T, suggesting that either of these two M2-1 mutations alone has the greatest role in deattenuating Min_L. The combination of N or P mutation with M2-1[N88K] induced a 2°C increase in T SH The combination of N, P and M2-1[N88K] mutations gave an additional, small increase in T compared to Min_L (average of 2.5°C for three independent experiments). SH (40°C) and the addition of the L mutation did not result in a further increase. SH We described the additive role of N, P, and M2-1[N88K] mutations in increasing T of Min_L. SH did not confer any increase to , suggesting their incompatibility, as predicted based on the deep sequencing results.

[0192] The effect of these mutations on the kinetics and efficiency of Min_L replication in Vero cells was examined (Fig. 3C-D). The effect of the mutations was more pronounced at 37°C (Fig. 3C,D, right panels) than at 32°C (left panels), as expected from temperature-sensitive mutations. The N and P mutations alone and in combination had only a minor effect on virus replication compared to Min_L. In contrast, introduction of either the M2-1[N88K] or [A73S] mutations alone resulted in a substantial increase in replication, which was largely unaffected by further addition of the N, P, and L mutations. Furthermore, viruses carrying both the incompatible M2-1[A73S] and [N88K] mutations replicated equally or less efficiently than Min_L at 37°C and 32°C, respectively (Fig. 3D, left and right panels). Therefore, the M2-1[N88K] or [A73S] mutations have a major role in restoring the ability of Min_L to replicate in Vero cells, although they were incompatible.

[0193] Furthermore, we tested the effect of the introduced mutations on the kinetics of viral gene transcription, viral genomic RNA synthesis, protein expression and viral particle production in a single infection cycle (Figure 4A-E). Vero cells were infected with the indicated viruses at an MOI of 3 pfu / cell and samples were taken every 4 h for analysis up to 24 h.

[0194] Analysis of accumulation of the nine small RSV mRNAs (i.e., all except L) was performed with positive-sense specific RT-qPCR assays specific for each mRNA. Data for a generally representative P mRNA are shown in FIG. 4A, and the complete data set for these nine mRNAs is shown in FIG. 9. In general, transcription was greatly reduced at 37° C. with Min_L compared to wt rRSV. Introduction of any of the M2-1 mutations in Min_L resulted in a substantial restoration of transcription. Further addition of the N, P, and L mutations to M2-1[N88K] provided further modest, but largely consistent, increases. Western blot analysis showed that, as expected, viral protein accumulation occurred later than mRNA, but otherwise the pattern was similar to mRNA accumulation (FIGS. 4B and 10).

[0195] Accumulation of RSV L mRNA (Fig. 4C) was examined by positive-sense-specific, L-specific RT-qPCR. At 32°C, basal levels of L mRNA were observed in Min_L-infected cells, but did not increase substantially with time, in contrast to the progressive increase with time observed for wt L mRNA. Extensive sequence differences in the wt and CPD L genes necessitated the use of different primer pairs for wt rRSV and Min_L derivatives, preventing direct comparison of relative abundance at various time points. At 37°C, CPD L mRNA was undetectable, indicating a strong restriction at this temperature. Addition of the M2-1 [N88K] or [A73S] mutations to Min_L partially restored CPD L gene transcription at both 32°C and 37°C. Further inclusion of N, P, and L mutations further increased L gene expression.

[0196] Cell-associated genomic RNA production by Min_L (Fig. 4D) was nearly undetectable at 32° C. or 37° C., but was detected by M2-1[A73S] and M2-1[N88K] at 24 h postinfection, and increased amounts were detected in NPM2-1[N88K]L-infected cells at 32° C. and 37° C. Genomic RNA production by wt rRSV was detectable from 12 h postinfection at both 32° C. and 37° C. and was higher than that of NPM2-1[N88K]L.

[0197] The production of infectious virus particles was paralleled by the accumulation of genomic RNA (Fig. 4E). At 32°C, Min_L virus titers began to increase only 24 h after infection, and no increase was observed at 37°C. M2-1[A73S] and M2-1[N88K] virus particles began to accumulate earlier (20 h after infection at both temperatures) and at higher levels (6- and 110-fold higher at 32°C and 37°C, respectively) than Min_L particles. NPM2-1[N88K]L virus production was first detected 16 h after infection at both temperatures and was more abundant than Min_L virus production (9- and 300-fold higher at 32°C and 37°C, respectively). Infectious wt rRSV was first observed 12 h after infection at both temperatures (Fig. 4E) and at higher levels than NPM2-1[N88K]L (10-fold higher at both 32°C and 37°C).

[0198] The size of plaques produced in Vero cells was measured as an additional parameter of viral fitness (Fig. 4F-G). wt rRSV produced plaques of significantly larger size than Min_L (p<0.05). Addition of the M2-1[A73S] or [N88K] mutations to Min_L increased viral fitness and resulted in plaque sizes that were not significantly different from wt rRSV (p>0.05 vs. wt rRSV). Plaques induced by M2-1[A73S][N88K] were smaller than Min_L plaques, further confirming that these two M2-1 mutations are incompatible.

[0199] Therefore, the two most prominent mutations acquired under stress were two missense mutations ([A73S] and [N88K]) in the M2-1 ORF encoding the RSV transcriptional antiterminator. Reintroduction of either of these mutations by reverse genetics rescued a substantial portion of the replicative fitness of Min_L at 37°C and increased viral gene transcription, protein expression, particle production and plaque size. These two M2-1 mutations partially restored transcription of the CPD L gene at 37°C, otherwise below detection levels at this temperature. Partial restoration of L gene expression was predicted to increase polymerase production, but was not monitored directly here due to its low amount and lack of available antibodies. We hypothesized that increased L protein production would subsequently increase transcription of all RSV genes, indirectly increasing viral protein synthesis, increasing RNA replication and ultimately indirectly increasing progeny virus production. These effects on the accumulation of viral mRNA, protein, genomic RNA and progeny virions were indeed observed. Therefore, acquisition of either of the two mutations in M2-1 renders Min_L adaptive at 37°C by increasing CPD L gene transcription.

[0200] The mechanism behind rescued CPD L gene expression by two M2-1 mutations is unknown. The RSVM2-1 protein is required for efficient synthesis of full-length mRNA, which is prematurely terminated in its absence. The M2-1 protein also increases the synthesis of polycistronic read-through mRNAs, which bind nascent mRNAs by cotranscription and prevent their termination by the viral polymerase. In addition, the M2-1 protein directly binds P. Binding of P and RNA to M2-1 was found to be mutually exclusive due to partially overlapping interaction surfaces. A73 and N88 are distant from the RNA / P binding interface but could possibly be passageways for pre-existing nascent RNA molecules. A simple model is a 1,378-nt change introduced in the CPD affecting the L gene template such that the efficiency of transcriptional elongation of nascent L mRNA is reduced. L transcription partially rescued the M2-1 mutation via some effect on the polymerase complex. The most prominent mutations acquired under stress were most frequent in the M2-1 ORF, but were also found in the P, N, and L ORFs, all of which encode viral proteins involved in RNA synthesis. These additional N, P, and L mutations further increased the efficiency of CPD L gene transcription, presumably by also increasing the efficiency of transcriptional elongation of the CPD L gene.

[0201] Example 6: Computer-based molecular dynamics simulations (MDS). Computer-based molecular dynamics simulations (MDS) were used to test the possible effect of the M2-1 [A73S] and [N88K] mutations on the M2-1 structure (Figure 6). The M2-1 tetramer is shown in Figure 6A, with specific illustrations in panels B, C, and D. In the wt M2-1 tetramer, a salt bridge is predicted to exist between K19 of one monomer and D116 of an adjacent monomer. These amino acids are shown as red and cyan monomers (Figure 6B). MDS suggests that the salt bridge helps stabilize the interaction between adjacent monomers. The A73 residue of the third monomer is in close proximity but is predicted not to be involved in the interaction. When A73 is changed to serine ([A73S], Figure 6C), the salt bridge between K19 and D116 is predicted to be maintained. Moreover, unlike alanine, the serine at codon 73 forms a hydrogen bond with K19 and is predicted to form a hydrogen bond with D116 in a certain MDS time frame (not shown). Therefore, S73 provides a novel stabilizing bond between each adjacent monomer. The predicted effect of the N88K mutation is increased stability within a monomer rather than between monomers. Notably, in the wt M2-1 tetramer structure, N88 is predicted to form a hydrogen bond with S82 (Figure 6B). In contrast, the lysine residue at codon 88 is predicted to form an intramonomer salt bridge with E70 (Figure 6D). K88 no longer interacts with S82. Furthermore, the hydrophobic carbon chain of K88 is predicted to form numerous intramonomer van der Waals interactions with L74. Therefore, the prominent M2-1 mutations acquired during the stress test are predicted to form novel interactions within (A73S) and (N88K) and (N88K) of the M2-1 monomer. This increased stability likely contributes to the rescue transcription of the CPD L gene. Interestingly, this increased stability is predicted not to be maintained when both mutations are present together. In fact, these two mutations likely form an H-bond pair between the side chains of S73 and K88, which leads to a decrease in the mobility of the loop in which the K88 residue resides. This decreased mobility explains the incompatibility of these two mutations.

[0202] Interestingly, the mutations found in P ([E113G] and [E114V]) were found to localize to the M2-1 interaction domain with P. Mutations at these two positions were shown to increase the affinity of P for M2-1. This study further supports the theory that compensatory mutations act by increasing the stability of the ribonucleoprotein complex, which we hypothesize may promote transcription of the CPD L gene.

[0203] As described, a single mutation (A73S) in the M2-1 gene, which appeared in the first passage of Min_L at 37°C and was seen in 8 of 10 cultures, was sufficient to rescue Min_L replication at that temperature. Moreover, this single mutation contributed to the increased replication of Min_L in hamsters. We expected that deattenuation of the CPD ORF would involve multiple changes in the CPD sequence, resulting in incremental deattenuation. However, this study showed that single mutations in different genes were sufficient to produce substantial deattenuation. Thus, deoptimization involving multiple nt changes did not necessarily provide a stable attenuated phenotype.

[0204] Example 7: Introduction of deattenuating mutations from Min_L to Min_FLC. The major mutations introduced into Min_L, i.e., N[K136R], P[E114V], M2-1[N88K], M2-1[A73S] and L[T1166I], were introduced into Min_FLC in various combinations, and the viral titers after recovery (Figure 11A) and T SH (Figure 11B). The M2-1[N88K] and [A73S] mutations were assessed for their effect on viral titers or T SH The combination of N, P and M2-1[N88K] mutations did not individually increase the fitness of Min_FLC as measured by T SH Although this resulted in a 2°C increase in temperature, the virus only grew to low titers.

[0205] Surprisingly, introduction of the L[T1166I] mutation into Min_FLC, either alone or in combination with one or more other mutations, prevented the recovery. Hence, none of these mutations improved the overall fitness of Min_FLC, despite it carrying the same CPD L gene as Min_L. This result suggests that multiple CPD ORFs enhance phenotypic stability under selective pressure.

[0206] Example 8: Evaluation of Min_L derivatives in mice and hamsters. Replication of Min_L derivatives was evaluated in vivo (Figure 5). BALB / c mice were infected with 10 6 pfu. Nasal turbinates (NT) and lungs were harvested on days 4 (n=8 / virus), 5 (n=8), and 10 (n=4) postinfection (pi). At the peak of viral replication (day 5 pi; Fig. 5B), virus was only observed in the NT of 2 mice infected with Min_L and 3 mice infected with M2-1[N88K]. M2-1[A73S] replication was observed in 4 of 8 mice and was comparable to wt rRSV. NPM2-1[N88K]L replication was not observed in the NT of any mice. Replication of M2-1[N88K] and M2-1[A73S] was slightly reduced compared to Min_L in the lungs on day 5, but not statistically significant, and NPM2-1[N88K]L replication was strongly suppressed in the lungs compared to Min_L. Day 10 titers are not shown because virus was recovered from only two animals and at trace levels in the M2-1[A73S] group.

[0207] The same set of viruses was compared in hamsters (Fig. 5C). On day 3, NT and lungs were collected from 9 hamsters per virus. In NT, Min_L replication was approximately 100-fold reduced compared to wt rRSV (p ≤ 0.01). Replication of M2-1[N88K] was mildly increased compared to Min_L, but maintained significant attenuation compared to wt rRSV. In contrast, titers of M2-1[A73S] were further increased compared to Min_L and were not statistically different from wt rRSV. Interestingly, replication of NPM2-1[N88K]L in NT was reduced compared to Min_L. In lung, Min_L and M2-1[N88K] were detected in only 1 of 9 hamsters with each virus, and replication of NPM2-1[N88K]L was undetectable. In contrast, M2-1[A73S] was increased compared to Min_L, with 5 of 9 hamsters showing approximately 10 2 Thus, in hamsters, the mutation M2-1[A73S] increased the replication of the deattenuated marker Min_L, the M2-1[N88K] mutation did not affect the replication of Min_L, and the combination of the N, P, L, and M2-1[N88K] mutations reduced replication.

[0208] Despite the marked restriction of replication, Min_L and Min_L-derived viruses induced antibody titers that were not statistically different from those induced by wt rRSV (Fig. 5D). M2-1[A73S] virus induced significantly higher levels of RSV-neutralizing serum antibodies than Min_L and M21-1[N88K]. Interestingly, NPM2-1[N88K]L virus, despite its highly restricted replication, also induced RSV-neutralizing antibodies comparable to wt rRSV. On day 31, hamsters were challenged IN with wt rRSV, and NT and lungs were collected 3 days postchallenge. No detectable challenge virus replication was detected except for trace amounts of virus in one animal in the Min_L group (not shown).

[0209] Example 9: Genetic stability of Min_L-NPM2-1[N88K]L virus. The observation that the NPM2-1[N88K]L virus was more highly attenuated than Min_L, yet as immunogenic as wt rRSV, identified this virus as a promising vaccine candidate. Its stability was therefore assessed in a temperature stress study involving four passages at 39°C and four passages at 40°C, corresponding to two months of serial passages (Figure 12). Sanger sequencing of the complete genome of the final passage of 10 different stress differentiation lines and two control flasks did not detect any massive mutations (not shown). This indicates that the introduction of the N, P, M2-1[N88K] and L mutations into Min_L to create the promising NPM2-1[N88K]L virus conferred genetic stability. The nucleotide sequence of Min_L-NPM2-1[N88K]L is shown in Figure 14 and represented by SEQ ID NO:14.

[0210] [Table 1] a Percentage of reads with the indicated mutation; only mutations present in ≥45% of reads are shown. Nucleotide numbering is based on RSV sequence M74568 (biological wt RSV strain A2). Mutations present in ≥5% of reads from the same experiment are shown in Table S1. b A mutation involving a converting codon as part of the CPD of the L ORF. c Mutations involving the nucleotide position that is changed as part of the CPD of the L ORF.

[0211] [Table 2] [Table 3] [Table 4] [Table 5] a Percentage of reads with mutations shown; only mutations present in ≥5% of reads are shown. Mutations detected in ≥50% of reads are highlighted in yellow, and mutations detected in 25-49% of reads are highlighted in green. Nucleotide numbering is based on RSV sequence M74568. b The mutations involving the altered codon as part of the CPD of L . c A mutation involving an altered nucleotide as part of the CPD of L. d The mutation contains a nucleotide that is altered as part of the CPD of L and is restored in the wt sequence.

[0212] [Table 6] a Percentage of reads with the indicated mutation; only mutations present in 1% of reads are shown. Nucleotide numbering is based on RSV sequence M74568. b The mutations involving the altered codon as part of the CPD of L . c A mutation involving an altered nucleotide as part of the CPD of L.

[0213] [Table 7] aPercentage of reads with the indicated mutation; only mutations present in 1% of reads are shown. Nucleotide numbering is based on RSV sequence M74568. b The mutations involving the altered codon as part of the CPD of L . c A mutation involving an altered nucleotide as part of the CPD of L.

[0214] [Table 8] [Table 9] a Percentage of reads with mutations indicated; only mutations detected in ≥5% of reads at one passage for at least two consecutive passages are shown. Temperatures of specific passages are indicated in brackets. Mutations detected in ≥50% of reads at a passage are highlighted in yellow, and mutations detected in 25-49% of reads are highlighted in green. Nucleotide numbering is based on the RSV sequence M74568. b The mutations involving the altered codon as part of the CPD of L . c A mutation involving an altered nucleotide as part of the CPD of L. d The mutation contains a nucleotide that is altered as part of the CPD of L and is restored in the wt sequence.

[0215] [Table 10] a Percentage of reads with the indicated mutation; only mutations detected in ≧25% of reads in passage 1 for at least 2 consecutive passages are shown. Temperatures of specific passages are indicated in brackets. Nucleotide numbering is based on the RSV sequence M74568. b The mutations involving the altered codon as part of the CPD of L . c A mutation involving an altered nucleotide as part of the CPD of L. d The mutation contains a nucleotide that is altered as part of the CPD of L and is restored in the wt sequence.

[0216] [Table 11] a Percentage of reads with the indicated mutation; only mutations detected in >50% of reads in passage 1 for at least 2 consecutive passages are shown. Temperatures of specific passages are indicated in brackets. Nucleotide numbering is based on the RSV sequence M74568. b The mutations involving the altered codon as part of the CPD of L .

[0217] [Table 12] [Table 13] a Percentage of reads with mutations indicated; only mutations detected in ≥5% of reads at one passage for at least two consecutive passages are shown. Temperatures of specific passages are indicated in brackets. Mutations detected in ≥50% of reads at a passage are highlighted in yellow, and mutations detected in 25-49% of reads are highlighted in green. Nucleotide numbering is based on the RSV sequence M74568. bThe mutations involving the altered codon as part of the CPD of L . c A mutation involving an altered nucleotide as part of the CPD of L. d The mutation contains a nucleotide that is altered as part of the CPD of L and is restored in the wt sequence.

[0218] [Table 14] a Percentage of reads with the indicated mutation; only mutations detected in ≧25% of reads in passage 1 for at least 2 consecutive passages are shown. Temperatures of specific passages are indicated in brackets. Nucleotide numbering is based on the RSV sequence M74568.

[0219] [Table 15] a Percentage of reads with the indicated mutation; only mutations detected in >50% of reads in passage 1 for at least 2 consecutive passages are shown. Temperatures of specific passages are indicated in brackets. Nucleotide numbering is based on the RSV sequence M74568.

[0220] References 1. Abil Z, Xiong X, & Zhao H (2015) Synthetic biology for therapeutic applications. Mol Pharm 12(2):322-331. 2. Martinez MA, Jordan-Paiz A, Franco S, & Nevot M (2015) Synonymous Virus Genome Recoding as a Tool to Impact Viral Fitness. Trends Microbiol. 3. Gaunt E, et al. (2016) Elevation of CpG frequencies in influenza A genome attenuates pathogenicity but enhances host response to infection. Elife 5. 4. Nogales A, et al. (2014) Influenza A virus attenuation by codon deoptimization of the NS gene for vaccine development. J Virol 88(18):10525-10540. 5. Broadbent AJ, et al. (2015) Evaluation of the attenuation, immunogenicity, and efficacy of a live virus vaccine generated by codon-pair bias de-optimization of the 2009 pandemic H1N1 influenza virus, in ferrets. Vaccine. 6. Cheng BY, Ortiz-Riano E, Nogales A, de la Torre JC, & Martinez-Sobrido L (2015) Development of live-attenuated arenavirus vaccines based on codon deoptimization. J Virol 89(7):3523-3533. 7. Diaz-San Segundo F, et al. (2015) Synonymous deoptimization of the foot-and-mouth disease virus causes attenuation in vivo while inducing a strong neutralizing antibody response. J Virol. 8. Coleman JR, et al. (2008) Virus attenuation by genome-scale changes in codon pair bias. Science 320(5884):1784-1787. 9. Yang C, Skiena S, Futcher B, Mueller S, & Wimmer E (2013) Deliberate reduction of hemagglutinin and neuraminidase expression of influenza virus leads to an ultraprotective live vaccine in mice. Proc Natl Acad Sci U S A 110(23):9481-9486. 10. Kunec D & Osterrieder N (2015) Codon Pair Bias Is a Direct Consequence of Dinucleotide Bias. Cell reports. 11. Tulloch F, Atkinson NJ, Evans DJ, Ryan MD, & Simmonds P (2014) RNA virus attenuation by codon pair deoptimisation is an artefact of increases in CpG / UpA dinucleotide frequencies. Elife 3:e04531. 12. Shen SH, et al. (2015) Large-scale recoding of an arbovirus genome to rebalance its insect versus mammalian preference. Proc Natl Acad Sci U S A 112(15):4749-4754. 13. Lauring AS, Jones JO, & Andino R (2010) Rationalizing the development of live attenuated virus vaccines. Nat Biotechnol 28(6):573-579. 14. Hanley KA (2011) The double-edged sword: How evolution can make or break a live-attenuated virus vaccine. Evolution (N Y) 4(4):635-643. 15. Bull JJ (2015) Evolutionary reversion of live viral vaccines: Can genetic engineering subdue it? Virus Evolution 1(1):vev005. 16. Burns CC, et al. (2006) Modulation of poliovirus replicative fitness in HeLa cells by deoptimization of synonymous codon usage in the capsid region. J Virol 80(7):3259-3272. 17. Mueller S, Papamichail D, Coleman JR, Skiena S, & Wimmer E (2006) Reduction of the rate of poliovirus protein synthesis through large-scale codon deoptimization causes attenuation of viral virulence by lowering specific infectivity. J Virol 80(19):9687-9696. 18. Bull JJ, Molineux IJ, & Wilke CO (2012) Slow fitness recovery in a codon-modified viral genome. Mol Biol Evol 29(10):2997-3004. 19. Nougairede A, et al. (2013) Random codon re-encoding induces stable reduction of replicative fitness of Chikungunya virus in primate and mosquito cells. PLoS Pathog 9(2):e1003172. 20. Vabret N, et al. (2014) Large-scale nucleotide optimization of simian immunodeficiency virus reduces its capacity to stimulate type I interferon in vitro. J Virol 88(8):4161-4172. 21. Meng J, Lee S, Hotard AL, & Moore ML (2014) Refining the balance of attenuation and immunogenicity of respiratory syncytial virus by targeted codon deoptimization of virulence genes. MBio 5(5):e01704-01714. 22. Ni YY, et al. (2014) Computer-aided codon-pairs deoptimization of the major envelope GP5 gene attenuates porcine reproductive and respiratory syndrome virus. Virology 450-451:132-139. 23. Le Nouen C, et al. (2014) Attenuation of human respiratory syncytial virus by genome-scale codon-pair deoptimization. Proc Natl Acad Sci U S A 111(36):13169-13174. 24. White MD, Bosio CM, Duplantis BN, & Nano FE (2011) Human body temperature and new approaches to constructing temperature-sensitive bacterial vaccines. Cellular and molecular life sciences : CMLS 68(18):3019-3031. 25. Nielsen R (2005) Molecular signatures of natural selection. Annu Rev Genet 39:197-218. 26. Fearns R & Collins PL (1999) Role of the M2-1 transcription antitermination protein of respiratory syncytial virus in sequential transcription. J Virol 73(7):5852-5864. 27. Tanner SJ, et al. (2014) Crystal structure of the essential transcription antiterminator M2-1 protein of human respiratory syncytial virus and implications of its phosphorylation. Proc Natl Acad Sci U S A 111(4):1580-1585. 28. Tran TL, et al. (2009) The respiratory syncytial virus M2-1 protein forms tetramers and interacts with RNA and P in a competitive manner. J Virol 83(13):6363-6374. 29. Blondot ML, et al. (2012) Structure and functional analysis of the RNA- and viral phosphoprotein-binding domain of respiratory syncytial virus M2-1 protein. PLoS Pathog 8(5):e1002734. 30. Mason SW, et al. (2003) Interaction between human respiratory syncytial virus (RSV) M2-1 and P proteins is required for reconstitution of M2-1-dependent RSV minigenome activity. J Virol 77(19):10670-10676. 31. Chapman MA, et al. (2011) Initial genome sequencing and analysis of multiple myeloma. Nature 471(7339):467-472. 32. Mueller S, et al. (2010) Live attenuated influenza virus vaccines by computer-aided rational design. Nat Biotechnol 28(7):723-726. 33. Bukreyev A, Belyakov IM, Berzofsky JA, Murphy BR, & Collins PL (2001) Granulocyte-macrophage colony-stimulating factor expressed by recombinant respiratory syncytial virus attenuates viral replication and increases the level of pulmonary antigen-presenting cells. J Virol 75(24):12128-12140. 34. Rothberg JM, et al. (2011) An integrated semiconductor device enabling non-optical genome sequencing. Nature 475(7356):348-352. 35. Buchholz UJ, Finke S, & Conzelmann KK (1999) Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter. J Virol 73(1):251-259. 35. Collins PL, et al. (1995) Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development. Proc Natl Acad Sci U S A 92(25):11563-11567. 37. Crowe JE, Jr., Collins PL, London WT, Chanock RM, & Murphy BR (1993) A comparison in chimpanzees of the immunogenicity and efficacy of live attenuated respiratory syncytial virus (RSV) temperature-sensitive mutant vaccines and vaccinia virus recombinants that express the surface glycoproteins of RSV. Vaccine 11(14):1395-1404. 38. Liang B, et al. (2015) Enhanced Neutralizing Antibody Response Induced by Respiratory Syncytial Virus Pre-fusion F Protein Expressed by a Vaccine Candidate. J Virol. 39. Humphrey W, Dalke A, & Schulten K (1996) VMD: visual molecular dynamics. J Mol Graph 14(1):33-38, 27-38. 40. Phillips JC, et al. (2005) Scalable molecular dynamics with NAMD. J Comput Chem 26(16):1781-1802. 41. Brooks BR, et al. (2009) CHARMM: the biomolecular simulation program. J Comput Chem 30(10):1545-1614.

Claims

1. An isolated polynucleotide molecule encoding a recombinant respiratory syncytial virus (RSV) variant having an attenuated phenotype comprising a RSV genome or antigenome sequence, wherein the RSV genome or antigenome is modified by a mutation at a position corresponding to T1166 of SEQ ID NO:11 in the L ORF of the L protein.

2. RSV genome or antigenome i. A mutation in the M2-1 ORF of the M2-1 protein at a position corresponding to N88 or A73 of SEQ ID NO:9 ii. a mutation in the N ORF of the N protein at a position corresponding to K136 of SEQ ID NO:3; iii. a mutation in the P ORF of the P protein at a position corresponding to E114 of SEQ ID NO:4; and iv. Combinations of these 2. The isolated polynucleotide molecule of claim 1, further modified with a mutation selected from the group consisting of:

3. RSV genome or antigenome i. a mutation in the M2-1 ORF of the M2-1 protein at a position corresponding to N88 of SEQ ID NO:9; ii. a mutation in the N ORF of the N protein at a position corresponding to K136 of SEQ ID NO:3; iii. a mutation in the P ORF of the P protein at a position corresponding to E114 of SEQ ID NO:4; and iv. Combinations of these 3. The isolated polynucleotide molecule of claim 2, further modified with a mutation selected from the group consisting of:

4. RSV genome or antigenome i. a mutation in the M2-1 ORF of the M2-1 protein at a position corresponding to A73 of SEQ ID NO:9; ii. a mutation in the N ORF of the N protein at a position corresponding to K136 of SEQ ID NO:3; iii. a mutation in the P ORF of the P protein at a position corresponding to E114 of SEQ ID NO:4; and iv. Combinations of these 3. The isolated polynucleotide molecule of claim 2, further modified with a mutation selected from the group consisting of:

5. The isolated polynucleotide molecule of any one of claims 2 to 4, wherein the RSV genome or antigenome is modified by at least two of the mutations (i) to (iii).

6. The isolated polynucleotide molecule of any one of claims 2 to 5, wherein the RSV genome or antigenome is modified by all of the mutations (i) to (iii).

7. The isolated polynucleotide molecule of any one of claims 1 to 6, wherein the mutation at the position corresponding to T1166 of SEQ ID NO: 11 in the L ORF of the L protein is T1166I.

8. a. the mutation at the position corresponding to N88 in SEQ ID NO:9 in the M2-1 ORF of the M2-1 protein is N88K, and the mutation at the position corresponding to A73 in SEQ ID NO:9 in the M2-1 ORF of the M2-1 protein is A73S; b. the mutation at the position corresponding to K136 of SEQ ID NO:3 in the N ORF of the N protein is K136R; and c. the mutation at the position corresponding to E114 of SEQ ID NO:4 in the P ORF of the P protein is E114V; An isolated polynucleotide molecule according to any one of claims 1 to 7.

9. The isolated polynucleotide molecule of claim 8, wherein the RSV genome or antigenome is modified by at least two of mutations a to c.

10. The isolated polynucleotide molecule of claim 8, wherein the RSV genome or antigenome is modified with all of mutations a to c.

11. The isolated polynucleotide molecule of any of claims 1 to 10, wherein the RSV genome or antigenome is modified by corresponding mutations selected from the group consisting of: (1) T1166I in the L protein, N88K in the M2-1 protein, K136R in the N protein, and E114V in the P protein; and (2) T1166I in the L protein, A73S in the M2-1 protein, K136R in the N protein, and E114V in the P protein.

12. The isolated polynucleotide molecule of any of claims 1 to 11, wherein the RSV genome or antigenome comprises a deletion in at least one of the proteins selected from M2-2, NS1 and NS2.

13. The isolated polynucleotide molecule of any one of claims 1 to 12, wherein the RSV genome or antigenome is codon pair deoptimized.

14. The isolated polynucleotide molecule of any one of claims 1 to 13, wherein the L ORF genome or antigenome of RSV is codon pair deoptimized.

15. A vector comprising the isolated polynucleotide molecule of any one of claims 1 to 14.

16. A cell comprising the isolated polynucleotide of any one of claims 1 to 15.

17. A pharmaceutical composition comprising an immunologically effective amount of a recombinant RSV variant encoded by the isolated polynucleotide molecule of any one of claims 1-14.

18. 20. A method of vaccinating a subject against RSV comprising administering the pharmaceutical composition of claim 17.

19. 20. A method of inducing an immune response comprising administering the pharmaceutical composition of claim 17.

20. 20. The method of claim 18 or 19, wherein the pharmaceutical composition is administered intranasally.

21. 21. The method of claim 20, wherein the pharmaceutical composition is administered by injection, aerosol delivery, nasal spray, or nasal drops.

22. 15. A live attenuated RSV vaccine comprising a recombinant RSV variant encoded by the isolated polynucleotide of any of claims 1-14.

23. 23. A pharmaceutical composition comprising the RSV vaccine of claim 22.

24. A method for producing a vaccine according to claim 22, comprising expressing an isolated polynucleotide molecule according to any one of claims 1 to 14.

25. An isolated polynucleotide molecule encoding a recombinant respiratory syncytial virus (RSV) variant having an attenuated phenotype comprising a RSV genome or antigenome sequence, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S1.

26. 26. The isolated polynucleotide molecule of claim 25, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S1-A.

27. The isolated polynucleotide molecule of claim 25 or 26, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S1-B.

28. An isolated polynucleotide molecule encoding a recombinant respiratory syncytial virus (RSV) variant having an attenuated phenotype comprising a RSV genome or antigenome sequence, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S2.

29. 29. The isolated polynucleotide molecule of claim 28, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S2-A.

30. 30. The isolated polynucleotide molecule of claim 28 or 29, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S2-B.

31. An isolated polynucleotide molecule encoding a recombinant respiratory syncytial virus (RSV) variant having an attenuated phenotype comprising a RSV genome or antigenome sequence, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S3.

32. The isolated polynucleotide molecule of claim 31, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S3-A.

33. The isolated polynucleotide molecule of claim 31 or 32, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S3-B.

34. 34. The isolated polynucleotide molecule of any of claims 25 to 33, wherein the RSV genome or antigenome comprises a deletion in at least one of the proteins selected from M2-2, NS1 and NS2.

35. The isolated polynucleotide molecule of any of claims 25 to 34, wherein the RSV genome or antigenome is codon pair deoptimized.

36. The isolated polynucleotide molecule of any of claims 25 to 35, wherein the L ORF genome or antigenome of RSV is codon pair deoptimized.

37. A vector comprising the isolated polynucleotide molecule of any one of claims 25 to 36.

38. A cell comprising the isolated polynucleotide of any one of claims 25 to 36.

39. A pharmaceutical composition comprising an immunologically effective amount of a recombinant RSV variant encoded by the isolated polynucleotide molecule of any one of claims 25-36.

40. 40. A method of vaccinating a subject against RSV comprising administering the pharmaceutical composition of claim 39.

41. 40. A method of eliciting an immune response comprising administering the pharmaceutical composition of claim 39.

42. 42. The method of claim 40 or 41, wherein the pharmaceutical composition is administered intranasally.

43. 43. The method of claim 42, wherein the pharmaceutical composition is administered by injection, aerosol delivery, nasal spray, or nasal drops.

44. A live attenuated RSV vaccine comprising a recombinant RSV variant encoded by the isolated polynucleotide of any of claims 25-36.

45. 45. A pharmaceutical composition comprising the RSV vaccine of claim 44.

46. A method of producing a vaccine according to claim 44, comprising expressing an isolated polynucleotide molecule according to any one of claims 25 to 36.

47. 37. The isolated polynucleotide molecule of any of claims 1-3, 5-14 and 25-36, comprising a nucleotide sequence which is at least about 80% identical to the nucleotide sequence of SEQ ID NO:

14.

48. 37. The isolated polynucleotide molecule of any of claims 1-3, 5-14 and 25-36, comprising a nucleotide sequence which is at least about 90% identical to the nucleotide sequence of SEQ ID NO:

14.

49. An isolated polynucleotide molecule which is at least about 80% identical to the nucleotide sequence of SEQ ID NO:

14.

50. 50. The isolated polynucleotide molecule of claim 49, which is at least about 90% identical to the nucleotide sequence of SEQ ID NO:

14.

51. 50. The isolated polynucleotide molecule of claim 49, which is at least about 95% identical to the nucleotide sequence of SEQ ID NO:

14.

52. 50. The isolated polynucleotide molecule of claim 49, comprising the nucleotide sequence of SEQ ID NO:

14.

53. A vector comprising the isolated polynucleotide molecule of any one of claims 47 to 52.

54. A cell comprising the isolated polynucleotide of any one of claims 47 to 52.

55. A pharmaceutical composition comprising an immunologically effective amount of a recombinant RSV variant encoded by the isolated polynucleotide molecule of any of claims 47-52.

56. 56. A method of vaccinating a subject against RSV comprising administering the pharmaceutical composition of claim 55.

57. 56. A method of eliciting an immune response comprising administering the pharmaceutical composition of claim 55.

58. 58. The method of claim 56 or 57, wherein the pharmaceutical composition is administered intranasally.

59. 59. The method of claim 58, wherein the pharmaceutical composition is administered by injection, aerosol delivery, nasal spray, or nasal drops.

60. 53. A live attenuated RSV vaccine comprising a recombinant RSV variant encoded by the isolated polynucleotide of any of claims 47-52.

61. 61. A pharmaceutical composition comprising the RSV vaccine of claim 60.

62. A method of producing a vaccine according to claim 60, comprising expressing an isolated polynucleotide molecule according to any one of claims 47 to 52.

Images