RSV vaccine bearing a polynucleotide which is de-optimised and de-attenuated

Abstract

Provided is a polynucleotide encoding a respiratory syncytial virus (RSV) variant having an attenuated phenotype comprising a modified RSV genome or antigenome that encodes a mutant RSV NS1, P, or M2-1 protein that differ from their parental counterpart at one or more amino acid residues. The invention also relates to methods of vaccinating an animal with the RSV variant or a pharmaceutical composition containing the RSV variant or inducing an immune response by administering the RSV variant to an animal, and further relates to methods of producing an RSV variant vaccine.

Description

RSV VACCINE BEARING A POLYNUCLEOTIDE WHICH IS DE-OPTIMISED AND DE-ATTENUATEDCROSS-REFERENCE TO RELATED APPLICATION

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 610,282, filed December 14, 2023. which is incorporated by reference in its entirety herein.STATEMENT REGARDINGFEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under project number 1ZIAAI 000372-40 by the National Institutes of Health, National Institute Allergy and Infectious Diseases. The Government has certain rights in the invention.INCORPORATION -BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0003] Incorporated by reference in its entirety herein is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: One 166,947 Byte Extensible Markup Language (XML) file named “772294.xml,” created on December 10, 2024.BACKGROUND OF THE INVENTION

[0004] Respiratory syncytial virus (RSV, also known as orthopneumovirus) belongs to the Pneumoviridae family of RNA viruses, and formerly belonged to the Paramyxoviridae family. RSV is an enveloped virus with a linear negative-sense RNA genome. Accordingly, the RNA genome is first transcribed before it is translated. The genome contains 10 genes in the order 3’-NSl-NS2-N-P-M-SH-G-F-M2-L-5’ encoding 11 proteins, namely two non- structural proteins (NS1 and NS2), the RNA-binding nucleocapsid protein (N). the phosphoprotein (P), the internal matrix protein (M), the small hydrophobic surface glycoprotein (SH), the attachment glycoprotein (G), the fusion protein (F), two proteins encoded from overlapping open reading frames of the M2 gene (M2-1 and M2-2), and the large polymerase protein (L).

[0005] RSV is a widespread pathogen, known to cause respiratory tract infections which can lead to serious illness and even death, particularly in young children, older adults, and immunosuppressed individuals. RSV is estimated to have caused worldwide more than 33 million lower respiratory tract illnesses in children under 5 years of age, three million hospitalizations, and nearly 200.000 childhood deaths annually, with many deaths occurring in developing countries. However, despite RSV’s prevalence and the dangers associated with pediatric RSV infections in infants and young children, no RSV vaccine with the ability to induce active immunity' in this age group has been successfully developed and licensed to date. Accordingly, there is a need for RSV vaccines, such as those based on the disclosures herein.BRIEF SUMMARY OF THE INVENTION

[0006] An aspect of the invention provides a polynucleotide encoding a recombinant RSV variant having an attenuated phenotype comprising a modified RSV genome or antigenome, wherein NS1, NS2, N, P, M, SH, and L ORFs of the modified RSV genome or antigenome are codon-pair deoptimized.

[0007] An aspect of the invention provides a multivalent RSV vaccine composition comprising a recombinant RSV variant of an aspect of the invention, a second recombinant RSV variant of an aspect of the invention, and, optionally, one or more additional recombinant RSV variants of an aspect of the invention, wherein the first, second, and optional additional recombinant RSV variants have different nucleotide sequences.

[0008] An aspect of the invention provides a recombinant RSV variant comprising a polynucleotide of an aspect of the invention.

[0009] An aspect of the invention provides a pharmaceutical composition comprising the recombinant RSV variant of an aspect of the invention and at least one excipient.

[0010] An aspect of the invention provides a multivalent vaccine composition comprising at least one recombinant RSV variant of an aspect of the invention and at least one antigen from a non-RSV virus.

[0011] An aspect of the invention provides a method of vaccinating an animal, comprising administering a pharmaceutical composition of an aspect of the invention or the multivalent vaccine composition of an aspect of the invention to an animal.

[0012] An aspect of the invention provides a method of inducing an immune response in an animal, comprising administering a recombinant RSV variant of an aspect of theinvention, a pharmaceutical composition of an aspect of the invention, or a multivalent RSV vaccine composition of an aspect of the invention to an animal.

[0013] An aspect of the invention provides a method of producing a recombinant RSV variant vaccine, comprising expressing the polynucleotide of an aspect of the invention in a cell.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1A are schematics showing aspects of the invention. The genome map of Min AL virus compared to wild type (wt) RSV and Min A, Min L and Min FLC viruses. The sections of the virus with codon pair deoptimization (CPD) are shown in black and the wt open reading frames (ORFs) are shown in grey. The number of silent nucleotide substitutions introduced by CPD in each construct is indicated to the right.

[0015] Figure IB shows a graph showing the total number of CpG (left) and UpA (right) dinucleotides in the ORFs of the indicated viruses.

[0016] Figure 1C is a set of graphs show ing the results of multi-cycle replication kinetics in Vero cells. Cell monolayers in six-well plates were infected using an MOI of 0.01 pfu / cell with the indicated viruses and incubated at 32 °C (left panel) or 37 °C (right panel). At 24 h intervals, cells from duplicate wells for each vims were scraped into the medium, vortexed to release cell-associated virus, the suspensions w ere clarified by low-speed centrifugation, and the media supernatants w ere harvested, aliquoted, and snap-frozen. Virus titers were determined later by immunoplaque assay at 32 °C. Titers correspond to the mean of two replicate titrations of virus from each of two replicate wells at each timepoint. Day 0 titers correspond to the back-titration of the inocula.

[0017] Figure ID is a graph showing plaque sizes. Vero cells in six-well plates were infected with 250 pfu / well of the indicated virus and incubated under methylcellulose at 32 °C. At day seven post infection (pi), plates were fixed and stained with a mixture of three anti -RSV F MAbs and a PE-labeled secondary antibody. The plaque area (in pm2) was evaluated on an average of 3,451 (+1,200) plaques per vims (**= p<0.01, ****= p<0.0001, Wilcoxon rank test with continuity’ correction post hoc test).

[0018] Figure 2A is a graph showing the results of an in-vitro stress test of Min AL, an aspect of the invention. Twelve replicate monolayer cultures of Vero cells in 25 cm2flasks were inoculated at a multiplicity' of infection (MOI) of 0. 1 pfu / cell with Min AL and serially passaged in parallel. Each flask represented a separate lineage. Two lineages of the tenlineages were passaged 18 times at the permissive temperature of 32 °C as controls. When extensive syncytia were observed or when cells started to detach (typically between six and 11 days pi), cells were scraped into the medium and the supernatants were harvested and clarified by low-speed centrifugation. One ml of the total of five clarified ml was used to inoculate the following passage. The remaining supernatants were aliquoted and snap frozen in dry ice for subsequent titration at 32 °C and sequence analysis. The passage number and corresponding temperatures of incubation are indicated, as well as the virus titer of the initial inoculum and passage harvests. Each lineage is represented separately by a different symbol. The arrow indicates the passage number used for viral RNA extraction and sequence analysis.

[0019] Figure 2B is a graph showing the results of the in-vitro stress test of Min AL. an aspect of the invention. For these 10 lineages (of the twelve described above in the figure description for Figure 2A), the temperature of incubation was increased by one °C after every other passage from 32 °C to 40 °C for a total of 18 passages. The passages incubated at the 36 °C shut-off temperature of Min AL (TSH. P9 and 10) are indicated with an arrow. The arrow also identifies the passage number used for viral RNA extraction and sequence analysis.

[0020] Figure 3A shows a set of schematics showing prominent mutations identified from the in-vitro stress test incrementally reduce the temperature sensitivity of Min A. Genome maps of Min AL and derivatives containing the reintroduction of one or more prominent (>50% abundance) mutations that were identified by whole-genome deep sequencing of passage 14 (P14) of lineages #1, #3 and #7 (Table 3). Reintroduced mutations are identified underneath the genome of each Min AL derivative. The two viruses at the top contain the indicated mutations from lineage #1; the next three viruses contain the indicated mutations from lineage #3, and the bottom two viruses contain the indicated mutations from lineage #7.

[0021] Figure 3B shows a set of graphs showing an example of multi-cycle replication kinetics of Min AL and derivatives. Replicate Vero monolayers in six-well plates were infected using an of MOI of 0.01 pfu / cell with wt RSV, Min AL, P14 supernatant from lineages #1, 3 or 7 of the in-vitro stress test, or Min AL derivatives containing the indicated reintroduced mutations, and incubated at 32 °C. Titers for Min AL, its derivatives, and wt RSV are the means with standard deviation of two replicate titrations each of two replicate wells, harvested at each timepoint as described in the legend of Figure 1C. Due to limited sample availability, titers of the P14 lineages #1 and #3 are the means of two replicate titrations of one well at every other time point. Day 0 titers are the back titration of theinocula. Min AL derivatives containing mutations from lineages #1 and #7 (left and right panels) were evaluated in a first experiment, and Min AL derivatives containing mutations from lineage #3 (middle panel) were evaluated in a second independent experiment. Wt RSV and Min AL were included in both experiments.

[0022] Figure 3C shows a set of graphs showing an example of multi-cycle replication kinetics of Min AL and derivatives. Replicate Vero monolayers in six-well plates were infected using an of MOI of 0.01 pfu / cell with wt RSV, Min AL, P14 supernatant from lineages #1, 3 or 7 of the in-vitro stress test, or Min AL derivatives containing the indicated reintroduced mutations, and incubated at 37 °C. Titers for Min AL. its derivatives, and wt RSV are the means with standard deviation of two replicate titrations each of two replicate wells, harvested at each timepoint as described in the legend of Figure 1C. Due to limited sample availability, titers of the P14 lineages #1 and #3 are the means of two replicate titrations of one well at every' other time point. Day 0 titers are the back titration of the inocula. Min AL derivatives containing mutations from lineages #1 and #7 (left and right panels) were evaluated in a first experiment, and Min AL derivatives containing mutations from lineage #3 (middle panel) were evaluated in a second independent experiment. Wt RSV and Min AL were included in both experiments.

[0023] Figure 4 shows a set of graphs showing prominent mutations identified from the in-vitro stress test incrementally rescued RNA synthesis by Min AL. Vero cell monolayers in six-well plates were inoculated with an MOI of 3 pfu / cell with the indicated viruses at 37 °C and the total cell-associated RNA was harvested at 24 and 48 hpi. Positive-sense RSV RNAs (primarily mRNA, with a small content of antigenome) were quantified in triplicate by strand-specific RT-qPCR using tagged primers. Data for the wt and CPD ORFs are shown with solid and hatched bars, respectively. Note that the sequences of the CPD ORFs (N, P, and L) differed from the wt ORFs and therefore necessitated the use of separate primers and probes. Thus, taqman results involving the CPD N, P and L ORFs of the Min AL-derived viruses could not be directly compared to those of wt RSV. The accumulation of negativesense genomic RNA also was evaluated using a strand-specific RT-qPCR assay with tagged primers and a probe specific to the M2-1 ORF, which was the unchanged wt sequence in all viruses and permitted direct comparisons. Data were normalized to 18S ribosomal (r)RNA and expressed as logio fold increase over Min AL at the 24 hour pi time point.

[0024] Figure 5A shows a graph showing that prominent mutations identified from the in- vitro stress test incrementally and partially rescued protein expression by Min AL. Thisfigure shows data from the experiment in Figure 4 combined with two additional independent repeat experiments. Additional replicate Vero cell monolayers from the single-cycle infection experiment described in Figure 4 (MOI of 3 pfu / cell, 37 °C) and from the two independent repeat experiments were harvested at 48 hpi (one well per virus per time point) for analysis of viral protein expression by flow cytometry. Cells were permeabilized, immunostained for RSV N, P, G, and F protein expression, and evaluated by flow cytometry to determine the percentage of cells expressing the N, P, G, and F proteins. Each experiment is represented by a symbol, and means and standard deviations are shown.

[0025] Figure 5B is a set of graphs showing the level of expression of N, P, G, and F proteins (expressed as median fluorescence intensity; MFI) in the N+P+G+F+cells as described above in regards to Figure 5A. Each experiment is represented by a symbol, and means and standard deviations are shown.

[0026] Figure 5C is a set of graphs showing images of gels from Western Blots. Cell lysates were prepared and analyzed by Western blotting to evaluate the expression of P and F with tubulin used as a loading control. Ladder is a molecular w eight marker.

[0027] Figure 5D is a set of graphs showing additional replicate Vero cell monolayers from the experiment described in Figure 4 w ere harvested by scraping at 24 and 48 hpi (one well per vims per time point), vortexed, clarified, and titered in duplicate by immunoplaque assay to determine virus titer.

[0028] Figure 6A is a timeline of a hamster experiment using an aspect of the invention. Groups of 16 five- to six -w eek old golden Syrian hamsters were inoculated intranasally (IN) with 6 logio pfu per hamster of the indicated virus. An additional group of eight animals were kept uninfected as controls. At 3 days post-infection (dpi), eight hamsters per group were euthanized and nasal turbinates (NT) and lungs were harvested and homogenized, and viral titers were determined by immunoplaque assay. Serum was collected at day -2 and 28 dpi for evaluation of the anti-RSV antibody response. At 32 dpi, all remaining hamsters including the group of eight non-immunized control hamsters were challenged with wt RSV. At 3 days post challenge (dpc), all hamsters were euthanized, bronchoalveolar lavages (BAL) were collected for evaluation of the mucosal antibody response in the lower airways and NT and lungs w ere harvested for evaluation of the replication of the challenge wt RSV virus.

[0029] Figure 6B is a set of graphs showing replication of wt RSV, Min AL and derivatives in NT and lungs at 3 dpi from eight hamsters per group. Harvested tissues were homogenized, clarified, aliquoted, snap frozen in dry ice and stored at -80 °C. Titers weredetermined by immunoplaque assay and expressed as pfu / g of tissue. The limit of detection is 50 pfu / g of tissue (dotted line). Viruses were grouped by lineage (Li), i.e., the number of the in-vitro stress test lineage in which the specific mutations were identified. The median, min, and max values, 25thand 75thquartile, and individual values are shown. Statistical differences in comparison to wt RSV are indicated at the top of each graph, while differences between Min AL and its derivatives are indicated in brackets (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, Kruskall Wallis test with Dunns post hoc test).

[0030] Figure 7A is a set of graphs showing Min AL and derivatives induced robust serum antibody responses to the prefusion form of the RSV fusion protein F (preF) in hamsters. Sera from eight remaining hamsters per group including the group of nonimmunized hamsters were collected at 28 dpi to evaluate the anti-RSV antibody response. Serum anti-RSV preF IgG (left panel) and IgA (right panel) titers were determined by ELISA (IgG) or dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) ELISA (IgA). The limit of detection is two logio.

[0031] Figure 7B is a set of graphs showing Min AL and derivatives induced robust serum antibody responses to the RSV attachment glycoprotein G in hamsters. Sera from eight remaining hamsters per group including the group of non-immunized hamsters were collected at 28 dpi to evaluate the anti-RSV antibody response. Serum anti-RSV G IgG (left panel) and IgA (right panel) titers were determined by ELISA (IgG) or dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) ELISA (IgA). The limit of detection is two logio.

[0032] Figure 7C shows that Min AL and derivatives induced robust serum RSV neutralizing antibody responses in hamsters. 60% plaque-reduction neutralizing antibodytiters (PRNTeo) were determined on Vero cells by RSV plaque-reduction neutralization assay, performed in the presence of complement. Viruses are grouped by lineage (Li), i.e., the number of the in-vitro stress test lineage in which the specific mutations were identified. The median, min. and max values, 25thand 75thquartile, and individual values are shown. Statistical differences to wt RSV are indicated at the top of each graph, while differences between Min AL and its derivatives are indicated in brackets (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, One-way ANOVA with Tukey post test).

[0033] Figure 8A is a set of graphs showing that Min AL and derivatives induced robust mucosal antibody responses in the airways of hamsters. At 32 dpi, eight hamsters per group including the group of eight non-immunized control hamsters were challenged IN with 6logio pfu of wt RSV. At day 3 dpc, animals were euthanized and bronchoalveolar lavage (BAL), NT and lung tissues were collected from each animal. Anti-RSV pre F TgG (left panel) and IgA (right panel) titers in BAL were determined by ELISA (IgG) or DELFIA ELISA (IgA). The limit of detection is one logio. The median, min, and max values, 25thand 75thquartile, and individual values are shown. Viruses are grouped by lineage (Li), i.e., the number of the in-vitro test lineage in which the specific mutations were identified. Statistical differences to wt RSV are indicated at the top of the graph, while differences between Min AL and derivatives are indicated in brackets (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Kruskall Wallis test with Dunns post hoc test or One-way ANOVA with Tukey post test).

[0034] Figure 8B is a set of graphs showing Min AL and derivatives induced robust mucosal antibody responses in the airways of hamsters. At 32 dpi, eight hamsters per group including the group of eight non-immunized control hamsters were challenged IN with 6 logio pfu of wt RSV. At day 3 dpc, animals were euthanized and bronchoalveolar lavage (BAL), NT and lung tissues were collected from each animal. Anti-RSV G IgG (left panel) and IgA (right panel) titers in BAL were determined by ELISA (IgG) or DELFIA ELISA (IgA). The limit of detection is one logio. The median, min, and max values, 25thand 75thquartile, and individual values are shown. Viruses are grouped by lineage (Li), i.e., the number of the in-vitro test lineage in which the specific mutations were identified. Statistical differences to wt RSV are indicated at the top of the graph, while differences between Min AL and derivatives are indicated in brackets (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Kruskall Wallis test with Dunns post hoc test or One-way ANOVA with Tukey post test).

[0035] Figure 8C is a set of graphs showing replication of wt RSV challenge virus that was evaluated by plaque assay from NT or lung tissues harvested at day 3 pc. The median, min, and max values, 25thand 75thquartile, and individual values are shown. Viruses are grouped by lineage (Li), i.e.. the number of the in-vitro test lineage in which the specific mutations were identified. Statistical differences to wt RSV are indicated at the top of the graph, while differences between Min AL and derivatives are indicated in brackets (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, Kruskall Wallis test with Dunns post hoc test or One-way ANOVA with Tukey post test).

[0036] Figure 9 is a set of graphs showing Min AL derivatives that were genetically stable during an in-vitro stress test. The stability of four Min AL-derived viruses (Min ALM2-I[I87K], Min AL M2-l [I87K]+2. Min AL P[G26D] and Min AL P[G26D]+3) was evaluated in an in vitro stress test. Five replicate cultures of Vero cells in 25 cm2flasks were inoculated using an initial MOI of 0.1 pfu / cell. Two replicate cultures (dashed lines) were passaged eight times at the permissive temperature of 32 °C. Three replicate cultures (solid lines) were incubated for four passages at one degree below the indicated virus TSH, followed by four additional passages at the virus TSH, representing two months of culture. Each lineage was represented by a different symbol. Flasks were harvested when extensive syncytia were observed or when the cells started to detach. One of five ml of clarified fluids from the previous passage was used to infect the following passage of fresh cells. Furthermore, aliquots of clarified virus from each lineage were snap frozen for virus titration by plaque assay at 32 °C at the end of the experiment and sequencing. Whole genome Sanger sequencing was performed at the end of P8 for the lineages that were passaged at 32 °C. Whole genome Sanger sequencing of two of three Min AL M2-1[I87K] lineages (upper left panel, gray square and circle) was done at the end of P8 while sequencing of the third lineage (gray triangle) was done at P7 instead of P8 due to low virus titer at P8. Due to low replication, whole genome sequencing of the other three stressed Min AL derived viruses could not be done, but the sharp drop in replication was indicative of stability.

[0037] Figures 10A-10B are a set of graphs showing expression of inflammatory cytokines in the lungs of hamsters at day 3 following IN inoculation. From the hamster experiment, in which eight animals per group were sacrificed on day 3 post-inoculation and lung homogenates prepared, seven aliquots of clarified lung supernatants were chosen at random from the Min AL and wt RSV groups, as well as two RSV groups from uninoculated hamsters derived from a previous study (Liu, et al., PLoS Pathog., 19(6): el 011057 (2023)) and processed to purify total RNA. The RNAs were reverse-transcribed using random primers, and expression of 13 inflammation-related-genes was evaluated by hamster-specific Taqman assays. The QPCR data were analyzed by the comparative threshold cycle (A CT) method, normalized to beta-actin and expressed as fold-increase over the mean expression of each evaluated gene determined from the two uninoculated control hamsters (dashed line). In each graph, the median, min, and max values, 25thand 75thquartile, and individual values are shown (*=p<0.05; **=p<0.01, Mann- Whitney test).

[0038] Figures 11 A-l IB are a set of graphs showing expression of inflammatory cytokines in the lungs of hamsters at day 3 post-challenge with wt RSV. At 32 dpi, eight hamsters per group including the group of eight non-immunized control hamsters werechallenged IN with 6 logio pfu of wt RSV. At day 3 pc. animals were euthanized and bronchoalveolar lavage (BAL), NT and lung tissues were collected from each animal. After challenge w ith wt RSV, seven of eight non-immunized control hamsters that exhibited high levels of wt RSV challenge virus replication were selected (Figure 8C, right panel), and seven Min AL-immunized animals were selected randomly. Lung homogenates from these animals w ere processed to purify total RNA, together with lung homogenates from two control hamsters of the same source and age range from a previous study. RNAs were reverse transcribed using random primers, and expression of 13 inflammation-related-genes w as evaluated by hamsterspecific Taqman assays. The qPCR data were analyzed by the comparative threshold cycle ( ACT) method, normalized to beta-actin and expressed as fold-increase over the mean expression of each evaluated gene determined from the two uninoculated control hamsters (dashed line). In each graph, the median, min, and max values, 25thand 75thquartile, and individual values are shown (**=p<0.01; ***=p<0.001, Mann-Whitney test).DETAILED DESCRIPTION OF THE INVENTION

[0039] An aspect of the invention provides a polynucleotide encoding a recombinant RSV variant having an attenuated phenotype comprising a modified RSV genome or antigenome, wherein NS1, NS2, N, P, M, SH, and L ORFs of the modified RSV genome or antigenome are codon-pair deoptimized.Codon Pair Deoptimization

[0040] In some aspects, the genome or antigenome of the attenuated RSV variant is codon-pair deoptimized (CPD). CPD, along with codon deoptimization (CD) and increasing the dinucleotide CpG and UpA content, are techniques for modifying the nucleotide sequence of a virus that can lead to attenuation of the virus. In CD, the nucleotide sequence encoding a virus is modified to change one or more codons within an open reading frame (ORF) of a gene in a way that the amino acid encoded by the new codon is still the same as the amino acid encoded by the original codon, i.e., CD involves the insertion of synonymous mutations into ORFs. This process can affect certain characteristics of the nucleotide sequence, including codon bias, codon pair bias, CpG dinucleotide content, C+G content, density of deoptimized codons and deoptimized codon pairs. RNA secondary structure, translation frame sites, translation pause sites, the presence or absence of tissue specific microRNA recognition sequences, or any combination thereof.

[0041] CPD is based on the observation that certain codon pairs appear more or less frequently than expected. For example, the codon pair alanine-glutamate is encoded by the nucleotide bases GCC GAA and GCA GAG. If these codon pairs appeared randomly, then one would expect to see GCC GAA half of the time and GCA GAG half of the time. However, GCC GAA is strongly unrepresented, appearing only 1 / 7* as often as GCA GAG. Without wishing to be bound to any particular theory, the existence of this codon pair bias is thought to stem from the effect certain codon pairs have on mRNA stability or synthesis, translation efficiency (some tRNA pairs interact less efficiently on the ribosome) and / or innate immunity (potentially a consequence of dinucleotide bias, insofar as the immune system seeks to suppress TLR ligands CpG and UpA).

[0042] Codon pair bias has been exploited to prepare weakened, i.e., attenuated, virus strains via CPD. See, e.g., U.S. Patent No. 9,957,486, incorporated by reference in its entirety herein. With the advent of synthetic biology, including the increased availability and affordability of large-scale custom DNA synthesis, synonymous mutations to the nucleotide sequence of a virus’s ORFs can be made in large numbers to take advantage of codon pair bias to attenuate the strain, by, e.g., reduce the replicative fitness of the resulting virus. In other words, CPD can now be applied on a genomic level. An advantage of using CPD as a technique for generating an attenuated RSV strain is that the probability of reversion to virulence is presumably extremely low when a large number of mutations are made in the strain. Following CPD, the nucleotide sequence containing the genome or antigenome of a CPD RSV variant encodes the same amino acid sequence as the genome or antigenome of a parental and / or wild-type RSV strain. However, other mutations can be introduced into the genome or antigenome of the CPD RSV variant, such that the genome or antigenome of the CPD RSV variant no longer encodes the same amino acid sequence as the genome or antigenome of the parental and / or wild-type RSV strain. Similarity' on the amino acid level between a RSV variant and a parental and / or wild-ty pe RSV strain is desirable because increased similarity between the sequences results in an increased likelihood that the CPD and parental and / or wild-type RSV strains will exhibit many or even all of the same epitopes. Inasmuch as cellular and humoral immunity are induced by such epitopes, CPD RSV variants desirably resemble parental and / or wild-type RSV strains on the amino acid level, at least in part.

[0043] Accordingly, in some aspects, the inventive polynucleotide comprises a modified RSV genome or antigenome that is codon-pair deoptimized. In certain aspects, the CPD RSVvariant strain and the corresponding parental and / or wild-type strain encode the same amino acid sequence. However, identity at the amino acid level is not required. Thus, in other aspects, the amino acid sequence encoded by the polynucleotide encoding the genome or antigenome of the CPD RSV variant is, or is at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the ammo acid sequence encoded by the polynucleotide encoding the genome or antigenome of a wildtype RSV strain.

[0044] Nucleotide or amino acid sequence ‘"identity,’' as referenced herein, can be determined by comparing a nucleotide or ammo acid sequence of interest to a reference nucleotide or amino acid sequence. The percent identity is the number of nucleotides or amino acid residues that are the same (i.e., that are identical) as between the optimally aligned sequence of interest and the reference sequence divided by the length of the longest sequence (i.e., the length of either the sequence of interest or the reference sequence, whichever is longer). Alignment of sequences and calculation of percent identity' can be performed using available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, BLASTp, BLASTn, and the like) and FASTA programs (e.g.. FASTA3x, FASTM, and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms also are disclosed in, for example, Altschul et al., J. Mol. Biol., 215(3): 403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10') 3770-3775 (2009), Durbin et al., eds.. Biological Sequence Analysis: Probalistic Models of Proteins and Nucleic Acids , Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics, 21 (7): 951 -960 (2005), Altschul et al.. Nucleic Acids Res., 25(17): 3389-3402 (1997), and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)). Percent (%) identity of sequences can be also calculated, for example, as 100 x [(identical positions) / min(TGA, TGB)], where TGA and TGB are the sum of the number of residues and internal gap positions in peptide sequences A and B in the alignment that minimizes TGA and TGB. See, e.g., Russell et al., J. Mol. Biol., 244: 332-350 (1994).

[0045] In some aspects, a computer program calculates the location and number of mutations within one more ORFs. i.e.. nucleotide sequence, of an RSV genome or antigenome to generate a desired RSV CPD genome or antigenome nucleotide sequence.See, for example, Coleman et al., Science. 320(5884): 1784-1787 (2008). Such programs can generate under-represented codon pairs (i.e., deoptimize codon pairs) while leaving codon usage and nucleotide frequency unchanged.

[0046] Accordingly, in some aspects, the codon usage and / or nucleotide frequency in one or more ORFs, i.e., nucleotide sequences, in the genome or antigenome of a RSV variant is the same as the codon usage and / or nucleotide frequency in the corresponding one or more ORFs in the genome or antigenome of a parental and / or wild-tj pe RSV strain. In other aspects, the codon usage and / or nucleotide frequency in one or more ORFs in the genome of a RSV variant is different than the codon usage and / or nucleotide frequency in the corresponding one or more ORFs in the genome or antigenome of a parental and / or wild-type RSV strain. In some aspects, the codon usage and / or nucleotide frequency in all ORFs in the genome or antigenome of a RSV variant is about the same as in all ORFs in the genome or antigenome of a parental and / or wild-type RSV strain. In a preferred aspect, the codon usage and / or nucleotide frequency of the ORFs in the genome of a RSV variant coding for RSV proteins NS1, NS2, N, P, M. and SH is about the same as in the corresponding ORFs in the genome or antigenome of a parental and / or wild-type RSV strain.

[0047] Moreover, using CPD, the level of attenuation of the virus can be modulated to a desirable level by adjusting the number of mutations introduced into the nucleotide sequence encoding one or more ORFs of the viral proteins. Accordingly, in some aspects, the polynucleotide comprising the genome or antigenome of the CPD RSV variant contains 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200. 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650. 1700, 1750, 1800, 1850, 1900. 1950. 2000, 2050, 2100, 2150. 2200, 2250, 2300, 2350. 2400. 2450, 2500, 2550, 2600, 2650, or 2700 synonymous mutations, or synonymous mutations in a range bounded by any two of the foregoing values, in comparison to the genomic or antigenomic sequence of a parental and / or wild-type RSV strain. In certain aspects, the polynucleotide comprising the genome or antigenome of the CPD RSV variant is recombinant, isolated, and / or not naturally occurring, i.e., not found in nature.

[0048] In some aspects, the mutations described herein, when used either alone or in combination with another mutation, may provide for different levels of virus attenuation, providing the ability to adjust the balance between attenuation and immunogenicity, and provide a more stable genotype than that of the parental virus.

[0049] The level of atenuation of vaccine virus may be determined by. for example, quantifying the amount of virus present in the respiratory tract of an immunized host and comparing the amount to that produced by parental and / or wild-type RSV or other atenuated RSV viruses which have been evaluated as candidate vaccine strains. For example, the atenuated virus of the invention will 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 levels of replication of parental and / or wild-type virus, e.g., 10- to 1000-fold less. In order to further reduce the development of rhinorrhea, which is associated with the replication of virus in the upper respiratory’ tract, an ideal vaccine candidate virus should exhibit a restricted level of replication in both the upper and lower respiratory tract. The RSV variant disclosed herein, to be effective, should be sufficiently infectious and immunogenic in humans to confer protection in vaccinated individuals. The vims can conveniently be measured in the nasopharynx of host animals, such as chimpanzees. Methods for determining levels of RSV in the nasopharynx of an infected host are well known in the literature. Specimens are obtained by aspiration or washing out of nasopharyngeal secretions and virus quantified in tissue culture or other by laboratory procedure. See, for example, 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 : 248-247 (1973).

[0050] In some aspects, the RSV variant may comprise other known atenuating mutations of RSV and / or related viruses to yield other atenuation phenotypes. A number of such mutations are known in the art. For instance, in some aspects, the M2 -2 ORF, the NS1 ORF or the NS2 ORF may be partially or completely deleted from the CPD RSV genome or antigenome.

[0051] In some aspects, the inventive polynucleotide which encodes a recombinant respiratory syncytial virus (RSV) variant having an atenuated phenotype comprises a modified RSV genome or antigenome that encodes mutant RSV NS1, NS2, N, P, M. SH, and / or L proteins that differ from parental RSV NS1, NS2, N, P, M, M2-1, M2-2, SH, and / or L proteins at one or more amino acid residues, wherein the nucleotide sequence of the modified RSV genome or antigenome encoding one or more of RSV NS1, NS2, N, P, M, SH, and L proteins have about 70% to about 95% identity with the nucleotide sequence of a parental and / or wild-type RSV genome or antigenome encoding the same one or more of RSV NS1, NS2, N, P, M, SH, and L proteins.

[0052] In some aspects, the polynucleotide comprising the nucleotide sequence of the CPD RSV genome or antigenome encoding one or more of RSV proteins NS 1, NS2, N, P, M, SH, G, F, M2-1, M2-2, and L has at least 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 percent identity with a nucleotide sequence of a parental and / or wild-type RSV genome encoding the same one or more of RSV proteins NS1, NS2, N, P, M, SH, G, F, M2-1, M2 -2, and L. In some aspects, the polynucleotide comprising the nucleotide sequence of the CPD RSV genome or antigenome encoding one or more of RSV proteins NS1, NS2, N, P, M, and SH has at least 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 percent identity with a nucleotide sequence of a parental and / or wild-type RSV genome encoding the same one or more of RSV proteins NS1, NS2, N, P, M, and SH.

[0053] In an aspect of the invention, NS1, NS2, N, P, M, SH, and L ORFs of the modified RSV genome or antigenome are CPD. In an aspect of the invention, the remaining ORFs that are not specifically indicated as codon pair deoptimized are not codon pair deoptimized. In an aspect of the invention, G, F, M2-1 or M2-2 ORFs, and any combination thereof, are not CPD.

[0054] In an aspect of the invention, the sequence of the polynucleotide encoding a recombinant RSV variant is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to SEQ ID NO: 12 (Min AL).

[0055] In certain aspects, the NS1, NS2, N, P. M. SH, and L ORFs of the modified RSV genome or antigenome that have been codon pair deoptimized each independently have a codon-pair-bias value of less than about 0.0. In another aspect, the NS1, NS2, N, P, M, SH, and L ORFs that have been codon pair deoptimized each independently have a codon-pair- bias value of less than about -0.05. In certain aspects, the NS1, NS2, N, P, M, SH, and L ORFs that have been codon pair deoptimized each independently have a codon-pair-bias value of less than about -0. 10. In certain aspects, the NS1, NS2, N, P, M, SH, and L ORFs that have been codon pair deoptimized each independently have a codon-pair-bias value of between about -0.10 to -0.45.

[0056] In an aspect of the invention, the NS 1 ORF has been codon pair deoptimized and has a codon-pair-bias value of between about -0.10 to about -0.45. In an aspect of theinvention, the NS1 ORF has been codon pair deoptimized and has a codon-pair-bias value of between about -0.10 to about -0.20. In an aspect of the invention, the NS1 ORF has been codon pair deoptimized and has a codon-pair-bias value of between about -0.12 to about - 0. 16. In an aspect of the invention, the NS1 ORF has been codon pair deoptimized and has a codon-pair-bias value of about -0. 14.

[0057] In an aspect of the invention, the NS2 ORF has been codon pair deoptimized and has a codon-pair-bias value of between about -0.10 to about -0.45. In an aspect of the invention, the NS2 ORF has been codon pair deoptimized and has a codon-pair-bias value of between about -0.10 to about -0.20. In an aspect of the invention, the NS2 ORF has been codon pair deoptimized and has a codon-pair-bias value of between about -0.20 to about - 0.24. In an aspect of the invention, the NS2 ORF has been codon pair deoptimized and has a codon-pair-bias value of about -0.22.

[0058] In an aspect of the invention, the N ORF has been codon pair deoptimized and has a codon-pair- bias value of between about -0. 10 to about -0.45. In an aspect of the invention, the N ORF has been codon pair deoptimized and has a codon-pair-bias value of between about -0.25 to about -0.35. In an aspect of the invention, the N ORF has been codon pair deoptimized and has a codon-pair-bias value of between about -0.29 to about -0.33. In an aspect of the invention, the N ORF has been codon pair deoptimized and has a codon-pair- bias value of about -0.31.

[0059] In an aspect of the invention, the P ORF has been codon pair deoptimized and has a codon-pair-bias value of between about -0. 10 to about -0.45. In an aspect of the invention, the P ORF has been codon pair deoptimized and has a codon-pair-bias value of between about -0.20 to about -0.30. In an aspect of the invention, the P ORF has been codon pair deoptimized and has a codon-pair-bias value of between about -0.22 to about -0.26. In an aspect of the invention, the P ORF has been codon pair deoptimized and has a codon-pair- bias value of about -0.24.

[0060] In an aspect of the invention, the M ORF has been codon pair deoptimized and has a codon-pair-bias value of between about -0. 10 to about -0.45. In an aspect of the invention, the M ORF has been codon pair deoptimized and has a codon-pair-bias value of between about -0.25 to about -0.35. In an aspect of the invention, the M ORF has been codon pair deoptimized and has a codon-pair-bias value of between about -0.29 to about -0.33. In an aspect of the invention, the M ORF has been codon pair deoptimized and has a codon-pair- bias value of about -0.31.

[0061] In an aspect of the invention, the SH ORF has been codon pair deoptimized and has a codon-pair-bias value of between about -0. 10 to about -0.45. In an aspect of the invention, the SH ORF has been codon pair deoptimized and has a codon-pair-bias value of between about -0.15 to about -0.25. In an aspect of the invention, the SH ORF has been codon pair deoptimized and has a codon-pair-bias value of between about -0.16 to about - 0.20. In an aspect of the invention, the SH ORF has been codon pair deoptimized and has a codon-pair-bias value of about -0.18.

[0062] In an aspect of the invention, the L ORF has been codon pair deoptimized and has a codon-pair- bias value of between about -0. 10 to about -0.45. In an aspect of the invention, the L ORF has been codon pair deoptimized and has a codon-pair-bias value of between about -0.25 to about -0.35. In an aspect of the invention, the L ORF has been codon pair deoptimized and has a codon-pair-bias value of between about -0.27 to about -0.31. In an aspect of the invention, the L ORF has been codon pair deoptimized and has a codon-pair- bias value of about -0.29.

[0063] Codon pair-bias values discussed herein were calculated according to the algorithms set forth in Coleman et al., Science, 320(5884): 1784-1787 (2008).

[0064] In certain aspects, the NS 1, NS2, N, P, M, SH, and L ORFs of the modified RSV genome or antigenome each independently have a codon pair bias (CPB) score reduction of at least about 0.01. In certain aspects, the NS 1, NS2, N. P, M. SH, and L ORFs of the modified RSV genome or antigenome each independently have a codon pair bias (CPB) score reduction of about 0.1. In certain aspects, the NS1, NS2, N, P, M, SH, and L ORFs of the modified RSV genome or antigenome each independently have a codon pair bias (CPB) score reduction of about 0.2. In certain aspects, the NS1, NS2, N, P, M, SH, and L ORFs of the modified RSV genome or antigenome each independently have a codon pair bias (CPB) score reduction of about 0.3. In certain aspects, the NS 1, NS2, N, P, M, SH, and L ORFs of the modified RSV genome or antigenome each independently have a codon pair bias (CPB) score reduction of about 0.4.

[0065] In certain aspects, the ORF, i.e., nucleotide sequence, encoding the NS 1 protein in the genome or antigenome of the RSV variant is codon pair deoptimized. In certain aspects, the nucleotide sequence of the modified RSV genome encoding RSV protein NS 1 has about 75% to about 95% identity with the ORF, i.e., nucleotide sequence, of the parental and / or wild-type RSV genome encoding RSV protein NS I. In further aspects, the nucleotide sequence of the modified RSV genome encoding RSV NS1 protein has about 87% identitywi th the nucleotide sequence of the parental and / or wild-type RSV genome encoding RSV protein NS 1.

[0066] In certain aspects, the ORF, i.e., nucleotide sequence, encoding the NS2 protein in the genome or antigenome of the RSV variant is codon pair deoptimized. In certain aspects, the nucleotide sequence of the modified RSV genome encoding RSV protein NS2 has about 75% to about 95% identity with the ORF, i.e., nucleotide sequence, of the parental and / or wild- t pe RSV genome encoding RSV protein NS2. In further aspects, the nucleotide sequence of the modified RSV genome encoding RSV NS2 protein has about 88% identity with the nucleotide sequence of the parental and / or wild-type RSV genome encoding RSV protein NS2.

[0067] In certain aspects, the ORF, i.e., nucleotide sequence, encoding the N protein in the genome or antigenome of the RSV variant is CPD. In certain aspects, the nucleotide sequence of the modified RSV genome encoding RSV protein N has about 70% to about 90% identity with the ORF, i.e.. nucleotide sequence, of the parental and / or wild-type RSV genome encoding RSV protein N. In certain aspects, the nucleotide sequence of the modified RSV genome encoding RSV N protein has about 80% identity with the nucleotide sequence of the parental and / or wild-type RSV genome encoding RSV protein N.

[0068] In certain aspects, the ORF, i.e., nucleotide sequence, encoding the P protein in the genome or antigenome of the RSV variant is codon pair deoptimized. In certain aspects, the nucleotide sequence of the modified RSV genome encoding RSV protein P has about 75% to about 95% identity w ith the ORF, i.e., nucleotide sequence, of the parental and / or wild-type RSV genome encoding RSV protein P. In certain aspects, the nucleotide sequence of the modified RSV genome encoding RSV NS1 protein has about 84% identity with the nucleotide sequence of the parental and / or wild-type RSV genome encoding RSV protein P.

[0069] In certain aspects, the ORF, i.e., nucleotide sequence, encoding the M protein in the genome or antigenome of the RSV variant is codon pair deoptimized. In certain aspects, the nucleotide sequence of the modified RSV genome encoding RSV protein M has about 75% to about 95% identity with the ORF, i.e., nucleotide sequence, of the parental and / or wild-type RSV genome encoding RSV protein M. In certain aspects, the nucleotide sequence of the modified RSV genome encoding RSV M protein has about 83% identity with the nucleotide sequence of the parental and / or wild-type RSV genome encoding RSV protein M.

[0070] In certain aspects, the ORF, i.e., nucleotide sequence, encoding the SH protein in the genome or antigenome of the RSV variant is codon pair deoptimized. In certain aspects,the nucleotide sequence of the modified RSV genome encoding RSV protein SH has about 85% to about 95% identity with the ORF, i.e., nucleotide sequence, of the parental and / or wild- type RSV genome encoding RSV protein SH. In certain aspects, the nucleotide sequence of the modified RSV genome encoding RSV SH protein has about 92% identity with the nucleotide sequence of the parental and / or wild-type RSV genome encoding RSV protein SH.

[0071] In certain aspects, an amino acid sequence of the one or more of RSV proteins NS1, NS2, N, P, M, and SH encoded by the nucleotide sequence of the modified RSV genome or antigenome is identical to an amino acid sequence of the same one or more of RSV proteins NS 1, NS2. N, P. M. and SH encoded by the nucleotide sequence of the parental and / or wild-type RSV genome or antigenome, except at the one or more amino acid residues where the mutant RSV protein P differs from the parental and / or wild-type RSV protein P. In certain aspects, the amino acid sequence of the one or more of RSV proteins NS1, NS2, N. P, M, and SH encoded by the nucleotide sequence of the modified RSV genome or antigenome is, or is at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to an amino acid sequence of the same one or more of RSV proteins NS1, NS2, N. P, M and SH encoded by the nucleotide sequence of the parental and / or wild-type RSV genome or antigenome. In some aspects, when the amino acid sequence encoded by the nucleotide sequence of the modified RSV genome or antigenome encodes two or more RSV proteins, percent identity is calculated using the combined amino acid sequence of the tw o or more RSV proteins. In other aspects, percent identity is calculated for each of the two or more RSV proteins individually, and the percent identity for each protein must be at least the recited percent identity.

[0072] As stated previously, the probability of reversion to virulence (i.e., de-attenuation) is presumed to be low when a large number of mutations is made in a RSV variant, such as a CPD RSV variant. However, in order to study de-attenuation, it is advantageous to subject the virus of interest to strong selective pressure. A limitation of previous de-attenuation studies of other CPD viruses was a lack of such strong selective pressure. See, e.g., Bull, et al., Mol. Biol, and Evol., 29(10): 2997-3004 (2012); Bums, et al., J. Virol., 80(19): 9687- 9696 (2006); Cheng, et al.. Virology, 501 : 35-46 (2015); Coleman, et al., Science, 320(5884): 1784-7 (2008); Meng, et al.. MBio, 5.5: 301704-14 (2014); Mueller, et al., J. Virol., 80(19): 9687-96 (2006), Ni, et al., Virology, 450: 132-139 (2014); Nougairede, et al., PLoSPathogens 9.2 (2013). Given that at least some CPD RSV variants are temperature sensitive, such RSV variants provide an excellent subject for studying de-attenuation of CPD viruses. See, e.g., U.S. Patent Application Publication 2019 / 0233476 Al, incorporated by reference in its entirety herein. Temperature sensitive viruses have a shut-off temperature, at which they fail to continue replicating.

[0073] By serially culturing such CPD RSV viruses in vitro while exposing them to step- wise increases in temperature from a permissive temperature, i.e., a temperature at which the virus can continue to replicate, to temperatures approaching and reaching the viruses’ shutoff temperatures, mutations can be identified that rescue replication in those CPD RSV viruses near or at their previous shut-off temperatures.

[0074] Using this technique, the inventors identified mutations in RSV proteins NS1, N, P, G, F, M2-1, M2 -2, and L that rescued replication in certain CPD RSV strains. When these de-attenuating mutations were introduced back into the original CPD RSV strains, it was surprisingly found that the resulting RSV variants exhibited increased attenuation, increased genetic stability, and / or increased immunogenicity in comparison to the original CPD RSV strains that did not contain any of the presumably de-attenuating mutations.Polynucleotide of RSV Variant Having An Attenuated PhenotypeNS1 Mutations

[0075] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV NS1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 22 at at least position 5. In certain aspects, the residue at position 5 of the amino acid sequence is threonine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV NS1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 22 only at position 5, wherein the residue at position 5 of the amino acid sequence is threonine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV NS1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 22 only at position 5, wherein the residue at position 5 of the amino acid sequence is threonine, and wherein the ORFs encoded by the NS 1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in theaforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD.

[0076] In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G, F, and M2 genes are not CPD. In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV NS1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 22 at at least position 46. In certain aspects, the residue at position 46 of the amino acid sequence is methionine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV NS1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 22 only at position 46, wherein the residue at position 46 of the amino acid sequence is methionine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV NS1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 22 only at position 46, wherein the residue at position 46 of the amino acid sequence is methionine, and wherein the NS1, NS2, N. P. M. SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G, F, and M2 genes are not CPD.

[0077] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV NS1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 22 at at least position 97. In certain aspects, the residue at position 97 of the amino acid sequence is glutamine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV N S 1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 22 only at position 97, wherein the residue at position 97 of the amino acid sequence is glutamine, and preferably wherein the modified RSV genomeor antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV NS1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 22 only at position 97. wherein the residue at position 97 of the amino acid sequence is glutamine, and wherein the NS1, NS2, N. P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G, F, and M2 genes are not CPD.

[0078] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV NS1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 22 at at least position 110. In certain aspects, the residue at position 110 of the amino acid sequence is glycine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV NS1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 22 only at position 110, wherein the residue at position 110 of the amino acid sequence is glycine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV NS1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 22 only at position 110, wherein the residue at position 110 of the amino acid sequence is glycine, and wherein the NS 1 , NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G, F, and M2 genes are not CPD.N mutations

[0079] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV N protein with an amino acid sequence that differs fromthe amino acid sequence set forth in SEQ ID NO: 23 at at least position 50. In certain aspects, the residue at position 50 of the amino acid sequence is leucine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV N protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 23 only at position 50. wherein the residue at position 50 of the amino acid sequence is leucine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV N protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 23 only at position 50, wherein the residue at position 50 of the amino acid sequence is leucine, and wherein the NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G, F, and M2 genes are not CPD.

[0080] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV N protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 23 at at least position 53. In certain aspects, the residue at position 53 of the amino acid sequence is valine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV N protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 23 only at position 53, wherein the residue at position 53 of the amino acid sequence is valine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV N protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 23 only at position 53, wherein the residue at position 53 of the amino acid sequence is valine, and wherein the NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspectof the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G, F, and M2 genes are not CPD.

[0081] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV N protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 23 at at least position 136. In certain aspects, the residue at position 136 of the amino acid sequence is arginine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV N protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 23 only at position 136, wherein the residue at position 136 of the amino acid sequence is arginine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV N protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 23 only at position 136, wherein the residue at position 136 of the amino acid sequence is arginine, and wherein the ORFs encoded by the NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G. F, and M2 genes are not CPD.

[0082] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV N protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 23 at at least position 280. In certain aspects, the residue at position 280 of the amino acid sequence is glutamic acid. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV N protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 23 only at position 280, wherein the residue at position 280 of the amino acid sequence is glutamic acid, and preferably wherein the modified RSV genome or antigenome comprises at least one ORF that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV Nprotein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 23 only at position 280, wherein the residue at position 280 of the amino acid sequence is glutamic acid, and wherein the NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G. F, and M2 genes are not CPD.P Mutations

[0083] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV P protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 24 at at least position 19. In certain aspects, the residue at position 19 of the amino acid sequence is isoleucine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV P protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 24 only at position 19, wherein the residue at position 19 of the amino acid sequence is isoleucine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV P protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 24 only at position 19, wherein the residue at position 19 of the amino acid sequence is isoleucine, and wherein the ORFs encoded by NS 1 , NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned poly nucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned poly nucleotide, ORFs encoded by G, F, and M2 genes are not CPD.

[0084] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV P protein with an amino acid sequence that differs fromthe amino acid sequence set forth in SEQ ID NO: 24 at at least position 26. In certain aspects, the residue at position 26 of the amino acid sequence is aspartic acid. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV P protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 24 only at position 26, wherein the residue at position 26 of the amino acid sequence is aspartic acid, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV P protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 24 only at position 26, wherein the residue at position 26 of the amino acid sequence is aspartic acid, and wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G, F, and M2 genes are not CPD.

[0085] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV P protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 24 at at least position 27. In certain aspects, the residue at position 27 of the amino acid sequence is glutamine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV P protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 24 only at position 27, wherein the residue at position 27 of the amino acid sequence is glutamine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV P protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 24 only at position 27, wherein the residue at position 27 of the amino acid sequence is glutamine, and wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the Fgene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G, F, and M2 genes are not CPD.

[0086] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV P protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 24 at at least position 32. In certain aspects, the residue at position 32 of the amino acid sequence is glutamine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV P protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 24 only at position 32, wherein the residue at position 32 of the amino acid sequence is glutamine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV P protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 24 only at position 32, wherein the residue at position 32 of the amino acid sequence is glutamine, and wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide. ORFs encoded by G. F, and M2 genes are not CPD.

[0087] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV P protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 24 at at least position 237. In certain aspects, the residue at position 237 of the amino acid sequence is serine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV P protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 24 only at position 237, wherein the residue at position 237 of the amino acid sequence is serine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotidecomprising a modified RSV genome or antigenome encodes a mutant RSV P protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 24 only at position 237, wherein the residue at position 237 of the amino acid sequence is serine, and wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G. F, and M2 genes are not CPD.

[0088] In an aspect of the invention, the modified RSV genome or antigenome comprises SEQ ID NO: 30. In an aspect of the invention, the modified RSV genome or antigenome comprises SEQ ID NO: 30, except that the modified RSV genome or antigenome comprises mutation c2334a (thethnucleotide of the start signal is adenine, GGGGCAAAT, SEQ ID NO: 30). SEQ ID NO: 30, or mutated SEQ ID NO: 30 with the 5thnucleotide of the start signal being adenine, may be in the P gene start signal.

[0089] In an aspect of the invention, the modified RSV genome or antigenome comprises SEQ ID NO: 20. In an aspect of the invention, the modified RSV genome or antigenome comprises SEQ ID NO: 20, except that the modified RSV genome or antigenome comprises mutation a2337t (the 8thnucleotide of the signal is thymine, GGGGCAAAT, SEQ ID NO: 20). SEQ ID NO: 20, or mutated SEQ ID NO: 20 with the 5thnucleotide of the start signal being thymine, may be in the P gene start signal.

[0090] In an aspect of the invention, the modified RSV genome or antigenome comprises mutation a2344g in the P gene. In an aspect of the invention, the modified RSV genome or antigenome comprises mutation a2344g in the 5’ UTR region of the P gene.G Mutations

[0091] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV G protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 27 at at least position 198. In certain aspects, the residue at position 198 of the amino acid sequence is threonine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV G protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 27 only at position 198, wherein the residue at position198 of the amino acid sequence is threonine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV G protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 27 only at position 198, wherein the residue at position 198 of the amino acid sequence is threonine, and wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G, F, and M2 genes are not CPD.F Mutations

[0092] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV F protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 28 at at least position 425. In certain aspects, the residue at position 425 of the amino acid sequence is threonine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV F protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 28 only at position 425, wherein the residue at position 425 of the amino acid sequence is threonine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV F protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 28 only at position 425, wherein the residue at position 425 of the amino acid sequence is threonine, and wherein the ORFs encoded by NS1, NS2, N. P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of theinventions, in the aforementioned polynucleotide. ORFs encoded by G. F, and M2 genes are not CPD.M2-1 Mutations

[0093] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 at at least position 14. In certain aspects, the residue at position 14 of the amino acid sequence is arginine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 14. wherein the residue at position 14 of the amino acid sequence is arginine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 14, wherein the residue at position 14 of the amino acid sequence is arginine, and wherein the ORFs encoded by NS 1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G, F, and M2 genes are not CPD.

[0094] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 at at least position 50. In certain aspects, the residue at position 50 of the amino acid sequence is isoleucine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 50, wherein the residue at position 50 of the amino acid sequence is isoleucine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forthin SEQ ID NO: 25 only at position 50, wherein the residue at position 50 of the amino acid sequence is isoleucine, and wherein the ORFs encoded by NS1 , NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G, F, and M2 genes are not CPD.

[0095] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 at at least position 69. In certain aspects, the residue at position 69 of the amino acid sequence is alanine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 69, wherein the residue at position 69 of the amino acid sequence is alanine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 69, wherein the residue at position 69 of the amino acid sequence is alanine, and wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G, F, and M2 genes are not CPD.

[0096] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 at at least position 70. In certain aspects, the residue at position 70 of the amino acid sequence is aspartic acid. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes amutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 70, wherein the residue at position 70 of the amino acid sequence is aspartic acid, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 70, wherein the residue at position 70 of the amino acid sequence is aspartic acid, and wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide. ORFs encoded by G. F, and M2 genes are not CPD.

[0097] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 at at least position 73. In certain aspects, the residue at position 73 of the amino acid sequence is serine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 73, wherein the residue at position 73 of the amino acid sequence is serine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 73, wherein the residue at position 73 of the amino acid sequence is serine, and wherein the ORFs encoded by NS 1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned poly nucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / orM2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G, F, and M2 genes are not CPD.

[0098] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 at at least position 73. In certain aspects, the residue at position 73 of the amino acid sequence is alanine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 73, wherein the residue at position 73 of the amino acid sequence is alanine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 73, wherein the residue at position 73 of the amino acid sequence is alanine, and wherein the ORFs encoded by NS 1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned poly nucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G, F, and M2 genes are not CPD.

[0099] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 at at least position 76. In certain aspects, the residue at position 76 of the amino acid sequence is alanine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 76, wherein the residue at position 76 of the amino acid sequence is alanine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 76, wherein the residue at position 76 of the amino acid sequence is alanine, andwherein the ORFs encoded by NS 1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G, F, and M2 genes are not CPD.

[0100] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 at at least position 87. In certain aspects, the residue at position 87 of the amino acid sequence is lysine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 87. wherein the residue at position 87 of the amino acid sequence is lysine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 87, wherein the residue at position 87 of the ammo acid sequence is lysine, and wherein the ORFs encoded by NS 1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G, F, and M2 genes are not CPD.

[0101] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 at at least position 88. In certain aspects, the residue at position 88 of the amino acid sequence is threonine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 88, wherein the residue at position 88of the amino acid sequence is threonine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. Tn a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 88, wherein the residue at position 88 of the amino acid sequence is threonine, and wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G, F, and M2 genes are not CPD.

[0102] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 at at least position 88. In certain aspects, the residue at position 88 of the amino acid sequence is serine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 88, wherein the residue at position 88 of the amino acid sequence is serine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 88, wherein the residue at position 88 of the amino acid sequence is serine, and wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G. F, and M2 genes are not CPD.

[0103] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 at at least position 90. In certain aspects, the residue at position 90 of the amino acid sequence is threonine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 90, wherein the residue at position 90 of the amino acid sequence is threonine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 90, wherein the residue at position 90 of the amino acid sequence is threonine, and wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide. ORFs encoded by G. F, and M2 genes are not CPD.

[0104] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 at at least position 90. In certain aspects, the residue at position 90 of the amino acid sequence is isoleucine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 90, wherein the residue at position 90 of the amino acid sequence is isoleucine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 90. wherein the residue at position 90 of the amino acid sequence is isoleucine, and wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and Lgenes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G, F, and M2 genes are not CPD.

[0105] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 at at least position 91. In certain aspects, the residue at position 91 of the amino acid sequence is threonine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 91, wherein the residue at position 91 of the amino acid sequence is threonine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 91. wherein the residue at position 91 of the amino acid sequence is threonine, and wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G, F, and M2 genes are not CPD.

[0106] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 at at least position 98. In certain aspects, the residue at position 98 of the amino acid sequence is serine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence setforth in SEQ ID NO: 25 only at position 98. wherein the residue at position 98 of the amino acid sequence is serine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 98, wherein the residue at position 98 of the amino acid sequence is serine, and wherein the ORFs encoded by NS 1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G, F, and M2 genes are not CPD.

[0107] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 at at least position 188. In certain aspects, the residue at position 188 of the amino acid sequence is alanine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 188, wherein the residue at position 188 of the amino acid sequence is alanine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 188, wherein the residue at position 188 of the amino acid sequence is alanine, and wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide. ORFs encoded by G. F, and M2 genes are not CPD.

[0108] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-2 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 10 at at least position 4. In certain aspects, the residue at position 4 of the amino acid sequence is leucine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-2 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 10 only at position 4, wherein the residue at position 4 of the amino acid sequence is leucine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV M2-2 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 10 only at position 4, wherein the residue at position 4 of the amino acid sequence is leucine, and wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G. F, and M2 genes are not CPD.L Mutations

[0109] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV L protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 29 at at least position 171. In certain aspects, the residue at position 171 of the amino acid sequence is leucine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV L protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 29 only at position 171, wherein the residue at position 171 of the amino acid sequence is leucine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV L protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 29 only at position 171. wherein the residue at position 171 of the amino acid sequence is leucine, and wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and L genes of themodified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G, F, and M2 genes are not CPD.

[0110] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV L protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 29 at at least position 761. In certain aspects, the residue at position 761 of the amino acid sequence is asparagine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV L protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 29 only at position 761, wherein the residue at position 761 of the amino acid sequence is asparagine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV L protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 29 only at position 761. wherein the residue at position 761 of the amino acid sequence is asparagine, and wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned poly nucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned poly nucleotide, ORFs encoded by G, F, and M2 genes are not CPD.

[0111] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV L protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 29 at at least position 1,249. In certain aspects, the residue at position 1,249 of the amino acid sequence is lysine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV L protein with an amino acid sequence that differs from the amino acid sequenceset forth in SEQ ID NO: 29 only at position 1.249, wherein the residue at position 1,249 of the amino acid sequence is lysine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV L protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 29 only at position 1,249, wherein the residue at position 1,249 of the amino acid sequence is lysine, and wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G. F, and M2 genes are not CPD.

[0112] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV L protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 29 at at least position 1,510. In certain aspects, the residue at position 1,510 of the amino acid sequence is serine. In a further aspect, the polynucleotide compnsing a modified RSV genome or antigenome encodes a mutant RSV L protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 29 only at position 1,510, wherein the residue at position 1,510 of the amino acid sequence is serine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV L protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 29 only at position 1,510, wherein the residue at position 1,510 of the amino acid sequence is serine, and wherein the ORFs encoded by NS 1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of theinventions, in the aforementioned polynucleotide. ORFs encoded by G. F, and M2 genes are not CPD.

[0113] In certain aspects, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV L protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 29 at at least position 2,076. In certain aspects, the residue at position 2,076 of the amino acid sequence is serine. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV L protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 29 only at position 2.076, wherein the residue at position 2,076 of the amino acid sequence is serine, and preferably wherein the modified RSV genome or antigenome comprises at least one gene that is CPD. In a further aspect, the polynucleotide comprising a modified RSV genome or antigenome encodes a mutant RSV L protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 29 only at position 2,076, wherein the residue at position 2,076 of the amino acid sequence is serine, and wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and / or M2-2 ORFs encoded by the M2 gene are not CPD. In an aspect of the inventions, in the aforementioned polynucleotide, ORFs encoded by G, F, and M2 genes are not CPD.

[0114] In an aspect of the invention, the modified RSV genome or antigenome comprisesSEQ ID NO: 21 . In an aspect of the invention, the modified RSV genome or antigenome comprises SEQ ID NO: 21, except that the modified RSV genome or antigenome comprises mutation a8494g (the 4thnucleotide of the signal is guanine, GGGACAAAAT, SEQ ID NO: 21). SEQ ID NO: 21, or mutated SEQ ID NO: 21 with the 4thnucleotide of the start signal being guanine, may be in the L gene start signal.

[0115] In an aspect of the invention, the polynucleotide comprising a modified RSV genome or antigenome encodes:(a) a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 at position 87, optionally wherein the residue at position 87 of the amino acid sequence is lysine;(b) a mutant RSV NS 1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 22 at position 46, optionally wherein the residue at position 46 of the amino acid sequence is methionine;(c) a mutant RSV P gene start signal with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 19 at position 70, wherein the residue at position 19 of the amino acid sequence is adenosine; or(d) any combination of (a)-(c). and optionally wherein the ORFs encoded by NS1, NS2, N. P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and M2-2 ORFs as encoded by the M2 gene are not CPD.

[0116] In an aspect of the invention, the polynucleotide comprising a modified RSV genome or antigenome encodes:(a) a mutant RSV P protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 22 at position 26, optionally wherein the residue at position 26 of the amino acid sequence is aspartic acid;(b) a mutant RSV M2- 1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 at position 88, optionally wherein the residue at position 88 of the amino acid sequence is threonine;(c) a mutant RSV L gene start signal with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 21 at position 4. optionally wherein the residue at position 4 of the amino acid sequence is guanosine;(d) a mutant RSV M2-2 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 26 at position 4, optionally wherein the residue at position 4 of the amino acid sequence is leucine; or(e) any combination of (a)-(d), and optionally wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and M2-2 ORFs as encoded by the M2 gene are not CPD.

[0117] In an aspect of the invention, the polynucleotide comprising a modified RSV genome or antigenome encodes:(a) a mutant RSV NS 1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 22 at position 5, optionally wherein the residue at position 5 of the amino acid sequence is threonine;(b) a mutant RSV N protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 23 at position 50, optionally wherein the residue at position 50 of the amino acid sequence is leucine;(c) a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 at position 70, optionally wherein the residue at position 70 of the amino acid sequence is aspartic acid;(d) a mutant RSV L protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 29 at position 1,249, optionally wherein the residue at position 1.249 of the amino acid sequence is lysine; or(e) any combination of (a)-(d). and optionally wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and M2-2 ORFs as encoded by the M2 gene are not CPD.

[0118] In an aspect of the invention, the polynucleotide comprising a modified RSV genome or antigenome encodes:(e) a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 87, optionally wherein the residue at position 87 of the amino acid sequence is lysine;(!) a mutant RSV NS 1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 22 only at position 46, optionally wherein the residue at position 46 of the amino acid sequence is methionine;(g) a mutant RSV P gene start signal with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 19 only at position 70, wherein the residue at position 19 of the amino acid sequence is adenosine; or(h) any combination of (a)-(c), and optionally wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and M2 -2 ORFs as encoded by the M2 gene are not CPD.

[0119] In an aspect of the invention, the polynucleotide comprising a modified RSV genome or antigenome encodes:(f) a mutant RSV P protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 22 only at position 26, optionally wherein the residue at position 26 of the amino acid sequence is aspartic acid;(g) a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 88, optionally wherein the residue at position 88 of the amino acid sequence is threonine;(h) a mutant RSV L gene start signal with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 21 only at position 4, optionally wherein the residue at position 4 of the amino acid sequence is guanosine;(i) a mutant RSV M2-2 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 26 only at position 4, optionally wherein the residue at position 4 of the amino acid sequence is leucine; or(j) any combination of (a)-(d). and optionally wherein the ORFs encoded by NS1, NS2, N. P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and M2-2 ORFs as encoded by the M2 gene are not CPD.

[0120] In an aspect of the invention, the polynucleotide comprising a modified RSV genome or antigenome encodes:(f) a mutant RSV NS 1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 22 only at position 5, optionally wherein the residue at position 5 of the amino acid sequence is threonine;(g) a mutant RSV N protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 23 only at position 50, optionally w herein the residue at position 50 of the amino acid sequence is leucine;(h) a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 only at position 70, optionally wherein the residue at position 70 of the amino acid sequence is aspartic acid;(i) a mutant RSV L protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 29 only at position 1,249, optionally wherein the residue at position 1,249 of the amino acid sequence is lysine; or(j) any combination of (a)-(d). and optionally wherein the ORFs encoded by NS1 , NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned poly nucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and M2-2 ORFs as encoded by the M2 gene are not CPD.

[0121] In an aspect of the invention, the polynucleotide comprising a modified RSV genome or antigenome encodes:(i) a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 at at least position 87, optionally wherein the residue at position 87 of the amino acid sequence is lysine;(j) a mutant RSV NS 1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 22 at at least position 46, optionally wherein the residue at position 46 of the amino acid sequence is methionine;(k) a mutant RSV P gene start signal with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 19 at at least position 70, wherein the residue at position 19 of the amino acid sequence is adenosine; or(l) any combination of (a)-(c), and optionally wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and M2. -2 ORFs as encoded by the M2 gene are not CPD.

[0122] In an aspect of the invention, the polynucleotide comprising a modified RSV genome or antigenome encodes:(k) a mutant RSV P protein with an ammo acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 22 at at least position 26, optionally wherein the residue at position 26 of the amino acid sequence is aspartic acid;(l) a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 at at least position 88, optionally wherein the residue at position 88 of the amino acid sequence is threonine;(m) a mutant RSV L gene start signal with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 21 at at least position 4, optionally wherein the residue at position 4 of the amino acid sequence is guanosine;(n) a mutant RSV M2-2 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 26 at at least position 4, optionally wherein the residue at position 4 of the amino acid sequence is leucine; or(o) any combination of (a)-(d), and optionally wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and M2-2 ORFs as encoded by the M2 gene are not CPD

[0123] In an aspect of the invention, the polynucleotide comprising a modified RSV genome or antigenome encodes:(k) a mutant RSV NS 1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 22 at at least position 5, optionally wherein the residue at position 5 of the amino acid sequence is threonine;(l) a mutant RSV N protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 23 at at least position 50, optionally wherein the residue at position 50 of the amino acid sequence is leucine;(m) a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 at at least position 70, optionally wherein the residue at position 70 of the amino acid sequence is aspartic acid;(n) a mutant RSV L protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 29 at at least position 1,249, optionally wherein the residue at position 1,249 of the amino acid sequence is lysine; or(o) any combination of (a)-(d). and optionally wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD. Further, in an aspect of the invention, in the aforementioned poly nucleotide, the ORF encoded by the G gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the ORF encoded by the F gene is not CPD. In an aspect of the invention, in the aforementioned polynucleotide, the M2-1 and M2 -2 ORFs as encoded by the M2 gene are not CPD.

[0124] In an aspect of the invention, the sequence of the polynucleotide encoding a recombinant RSV variant is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%. 98.2%. 98.3%. 98.4%. 98.5%. 98.6%. 98.7%. 98.8%. 98.9%. 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to SEQ ID NO: 13 (Min AL-M2-1[I87K]).

[0125] In an aspect of the invention, the sequence of the polynucleotide encoding a recombinant RSV variant is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to SEQ ID NO: 14 (Min AL-M2-l[I87K]+2).

[0126] In an aspect of the invention, the sequence of the polynucleotide encoding a recombinant RSV variant is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%,99.3%. 99.4%. 99.5%. 99.6%. 99.7%, 99.8%, or 99.9% identical to SEQ ID NO: 15 (Min AL-P[G26D] SEQ ID NO: 15).

[0127] In an aspect of the invention, the sequence of the polynucleotide encoding a recombinant RSV variant is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to SEQ ID NO: 16 (Min AL-P[G26D]+2).

[0128] In an aspect of the invention, the sequence of the polynucleotide encoding a recombinant RSV variant is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%. 98.2%. 98.3%. 98.4%. 98.5%. 98.6%. 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to SEQ ID NO: 17 (Min AL-P[G26D]+3).

[0129] In an aspect of the invention, the sequence of the polynucleotide encoding a recombinant RSV variant is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to SEQ ID NO: 18 (Min AL-M2-l[E70D]).

[0130] In an aspect of the invention, the sequence of the polynucleotide encoding a recombinant RSV variant is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to SEQ ID NO: 19 (Min AL-M2-l[E70D]+3).

[0131] As used herein, the term "wild-type” refers to any naturally occurring RSV strain, including those isolated from a natural source, such as a mammalian subject. Exemplary wild- t pe RSV strain subgroups include, but are not limited to, human RSV subgroups A and B, which can be further classified into genotypes, such as Al. A2, A3, A4, A5, A6, A6, and other designations such as GAI-7. SAA1, NA1-4. and ON1-2, as well as Bl. B2, B3, B4, and other designations such as GB1-4, SABI-4, URU1-2, and BAI-10. Exemplary specific strains include RSV A2, RSV Long, RSV 8-60 and RSV 18537. The amino acid position numbering used herein is based on the amino acid sequence of the wild-tj pe RSV A2 strain (GenBank accession number M74568. which is incorporated by reference herein) and all nucleotide sequences described are in positive-sense. The amino acid sequences of RSVproteins NS 1, N. P, M2-1, M2-2, G. F, L, and NS2 are represented by SEQ ID NOs: 22-29 and 32, respectively.

[0132] The term “wild-type” is further intended to encompass the recombinant version of RSV strain A2 that is called D46. The complete sequence of D46 is shown in U.S. Patent No. 6,790,449 (GenBank accession number KT992094. which is incorporated by reference herein). In some instances and publications, the parent virus and sequence is called D53 rather than D46, a book-keeping difference that refers to the strain of bacteria used to propagate the antigenomic cDNA and has no other known significance or effect. For the purposes of this disclosure, D46 and D53 are interchangeable. The nucleotide sequence of D46 differs from the sequence of RSV A2 strain M74568 in 25 nucleotide positions, which includes a 1-nt insert at position 1099.

[0133] The terms “parent” and “parental” used in the context of a virus, protein, or polynucleotide denotes the virus, protein, or polynucleotide from which another virus is derived. In some aspects, the derived virus is made by recombinant means, or by culturing the parent virus under conditions that give rise to a mutation, and thus a different virus. In some aspects, the terms refer to viral genomes and protein encoding sequences from which new' sequences, which may be more or less attenuated, are derived. In some aspects, the parent (or parental) viruses and sequences are wild type or naturally occurring prototypes or isolates of variants for which it is desired to obtain a more highly attenuated virus. In certain other aspects, the parent (or parental) viruses are mutants specifically created or selected in the laboratory' on the basis of real or perceived desirable properties. Accordingly, in certain aspects, the parent (or parental) viruses that are candidates for attenuation are mutants of w ild type. In other aspects, the parent (or parental) viruses are naturally occurring viruses that have deletions, insertions, amino acid substitutions and the like. In further aspects, the parent (or parental) viruses are mutants which have codon substitutions.

[0134] Those skilled in the art will recognize that the polynucleotide comprising the genome or antigenome of certain RSV variants may have nucleotide insertions or deletions that alter the encoded amino acid sequence, which in some cases can alter the position of one or more amino acid residues. For example, if a protein of another RSV strain had, in comparison w ith strain A2, two additional amino acids in the upstream end of the protein, this would cause the amino acid numbering of downstream residues relative to strain A2 to increase by an increment of two. How ever, because these strains share a large degree of sequence identity', those skilled in the art would be able to determine the location ofcorresponding sequences by simply aligning the nucleotide or amino acid sequence of the A2 reference strain with that of the strain in question. Therefore, it should be understood that the amino acid and nucleotide positions described herein, though specifically enumerated in the context of this disclosure, can correspond to other positions when a sequence shift has occurred or due to sequence variation between virus strains. In the comparison of a protein, or protein segment, or ORF, or gene, or genome, or genome segment between two or more related viruses, a “corresponding” amino acid or nucleotide residue is one that is exactly or approximately equivalent in function in the different RSV species.

[0135] In some aspects, the amino acid sequence encoded by the genome or antigenome of the RSV variant may contain additional differences from the amino acid sequence encoded by the genome or antigenome of a parental and / or wild-type RSV strain. For instance, in some aspects, the amino acid sequence encoded by the genome or antigenome of the RSV variant may comprise one or more changes in the F protein, e g., the “HEK” mutation, which comprises two amino acid substitutions in the F protein, namely K66E and Q101P (described in Connors, et al.. Virology’, 208: 478-484 (1995); Whitehead, et al., J. Virol., 72: 4467-4471 (1998)). The introduction of the HEK amino acid assignments into the strain A2 F sequence of this disclosure results in an F protein amino acid sequence that is identical to that of an early-passage (human embryonic kidney cell passage 7. HEK-7) of the original clinical isolate of strain A2 (Connors, et al.. Virology. 208: 478-484 (1995); Whitehead, et al., J. Virol., 72: 4467-4471 (1998)). It results in an F protein that is much less fusogenic and is believed to represent the phenotype of the original A2 strain clinical isolate (Liang, et al., J. Virol., 89: 9499-9510 (2015)). The HEK F protein also forms a more stable trimer (Liang, et al., J. Virol., 89: 9499-9510 (2015)). This may provide a more authentic and immunogenic form of the RSV F protein, possibly enriched for the highly immunogenic pre-fusion conformation (McLellan, et al., Science, 340(6136): 1113-1117 (2013); McLellan, et al., Science, 342(6158): 592-598 (2013)). Thus, mutations can be introduced with effects additional to effects on the magnitude of virus replication.

[0136] In some aspects the amino acid sequence encoded by the genome or antigenome of the RSV variant may comprise one or more changes in the L protein, e.g., the stabilized 1030 or the “1030s” mutation which comprises 1321K(AAA) / 51313(TCA) (Luongo, et al., J. Virol., 86: 10792-10804 (2012)).

[0137] In some aspects the amino acid sequence encoded by the genome or antigenome of the RSV variant may comprise one or more changes in the N protein, for example, an amino substitution such as T24A.

[0138] Deletion of the SH, NS1, and NS2 genes individually and in combination has been shown to yield viruses that retain their ability to replicate in cell culture but are attenuated in vivo in the following order of increasing magnitude: SH<NS2<NS1 (Bukreyev, et al., J.Virol., 71 : 8973-8982 (1997); Whitehead et al., J. Virol.. 73: 3438-3442 (1999); Teng et al., J. Virol., 74: 9317-9321 (2000)). Therefore, in some aspects, the genome or antigenome of the RSV variant comprises deletion or other mutations of the SH, NS2, or NS1 genes, or parts of their ORFs, is combined with one or more mutations described herein. For example, in some aspects, the amino acid sequence encoded by the genome or antigenome of the RSV variant may comprise one or more changes in the SH protein, including an ablation or elimination of the SH protein. In some aspects, the genome or antigenome of the RSV variant comprises a deletion in the SH gene. In some aspects, the genome or antigenome of the RSV variant comprises a 419 nucleotide deletion at position 4197-4615 (4198-4616 of), denoted herein as the “ASH” mutation. This deletion results in the deletion of M gene-end, M / SH intergenic region, and deletion of the SH ORF. In some aspects, the genome or antigenome of the RSV variant comprises one or more changes in the NS1 or the NS2 protein, which may result in an ablation or elimination of the protein. In some aspects, the mutation encodes an amino substitution such as K51R in the NS2 protein.

[0139] In some aspects, the genome or antigenome of the RSV variant encodes the “cp” mutation. This mutation refers to a set of five amino acid substitutions in three proteins (N (V2671). F (E218A and T5231). and L (C319Y and H1690Y)) which confer an approximate 10-fold reduction in replication in seronegative chimpanzees and a reduction in illness (Whitehead, et al., J. Virol., 72: 4467-4471 (1998)). The cp mutation has been associated with a moderate attenuation phenotype (Whitehead, et al.. J. Virol., 72: 4467-4471 (1999)).

[0140] In some aspects, the genome or antigenome of the RSV variant encodes one or more amino acid substitutions in the L protein, including N43I, F521L, Q831L, Ml 169V, and / or Y1321N. Each substitution independently confers atemperature sensitive phenotype (i.e., an attenuated phenotype) and can optionally be combined with other modifications to the nucleotide sequence of the RSV variant, such as a single nucleotide change in the genestart transcription signal of the M2 gene (GGGGCAAATA [SEQ ID NO: 30] toGGGGCAAACA [SEQ ID NO: 31], mRNA-sense), or the deletion of codon 1313 and amino acid substitution I1314L within the L protein.

[0141] Shifts in gene order (i.e., positional modifications moving one or more genes to a more promoter-proximal or promoter-distal location in the recombinant viral genome) in the genome or antigenome of the RSV variant can result in RSV viruses with altered biological properties. For example, RSV strains lacking NS1, NS2, SH, or G individually, or NS1 and NS2 together, or SH and G together have been shown to be attenuated in vitro, in vivo, or both. In particular, the G and F genes may be shifted, singly and in tandem, to a more promoter-proximal position relative to their parental and / or wild-type gene order. These two proteins normally occupy positions 7 (G) and 8 (F) in the RSV gene order (NS1-NS2-N-P-M- SH-G-F-M2-1-M2-2-L). In some aspects, the order of the nucleotide sequences encoding the G and the F proteins may be reversed relative to the naturally occurring order.

[0142] In addition to the herein described mutations, in some aspects, the polynucleotides of the invention can incorporate heterologous, coding or non-coding nucleotide sequences from any RSV or RSV-like virus, e.g., human, bovine, ovine, murine (pneumonia virus of mice), or avian (turkey rhinotracheitis virus) pneumovirus, or from another enveloped virus, e. g., parainfluenza virus (PIV). Exemplary heterologous sequences include RSV nucleotide sequences from one human RSV strain combined with nucleotide sequences from a different human RSV strain. Alternatively, the RSV may incorporate nucleotide sequences from two or more, parental, wild-type, and / or mutant human RSV subgroups, for example, a combination of human RSV subgroup A and subgroup B sequences. In still further aspects, one or more human RSV coding or non-coding polynucleotides are substituted with a counterpart sequence from a heterologous RSV or non-RSV virus.

[0143] In addition to the polynucleotides and resulting RSV variants described herein, the disclosed viruses may be modified further as would be appreciated by those skilled in the art. For instance, the genome or antigenome of the RSV variant may have the ORF for one or more proteins removed or otherwise mutated or a heterologous gene from a different organism may be added thereto so that the genome or antigenome of the CPD RSV expresses or incorporates that protein upon infecting a cell and replicating. Furthermore, those skilled in the art will recognize that other previously defined mutations known to have an effect on RSV may be combined with one or more of any of the mutations described herein to produce a CPD RSV with desirable attenuation or stability characteristics.

[0144] In other aspects, yet further modifications can be incorporated into the genome or antigenome of the RSV variants that affect the strains’ characteristics in ways other than attenuation. For instance, the one or more ORFs encoding RSV proteins may be codon- optimized within the context of the requirements for the RSV variants as described herein. Major protective antigens F and G can result in increased antigen synthesis. The F and / or G protein gene may be shifted upstream (i.e., closer to the promoter) to increase expression. The amino acid sequences encoding F and / or G protein can be modified to represent currently -circulating strains, which can be particularly important in the case of the divergent G protein, or to represent early-passage clinical isolates. Deletions or substitutions may be introduced into the nucleotide sequence encoding the G protein to obtain improved immunogenicity or other desired properties. For example, the CX3C fractalkine motif in the G protein might be ablated to improve immunogenicity (Chirkova, et al., J. Virol., 87: 13466- 13479 (2013)).

[0145] In some aspects, the genome or antigenome of the CPD RSV comprises the nucleotide sequence of an ORF which has not been codon pair deoptimized and which has been replaced with a nucleotide sequence from a clinical isolate. For instance, the nucleotide sequence of an ORF encoding the RSV G protein may be replaced with a nucleotide sequence from a clinical isolate, such as A / Maryland / 001 / 11. In some aspects, the nucleotide sequence encoding the RSV F protein may be replaced with a nucleotide sequence from a clinical isolate, such as A / Maryland / 001 / 11.

[0146] In some aspects, a native or naturally occurring nucleotide sequence encoding one or more proteins of the RSV variant is replaced with a codon optimized sequence designed for increased expression in a selected host, for instance in humans. In some aspects, the nucleotide sequence encoding the RSV F protein is replaced with a codon optimized sequence. In some aspects, the nucleotide sequence of the ORF encoding the RSV F protein is replaced with the codon optimized sequence from a clinical isolate such as A / Maryland / 001 / 11. In some aspects, the nucleotide sequence encoding the RSV G protein is replaced with the codon optimized nucleotide sequence from a clinical isolate, such as A / Maryland / 001 / 11.

[0147] In some aspects, the genome or antigenome of the RSV variants further comprise a deletion of one or more non-translated sequences. In an aspect, a portion of the downstream end of the SH gene is deleted, resulting in a mutation referred to as the “6120 Mutation” herein. The 6120 Mutation includes deletion of 112 nucleotides of thedownstream non-translated region of the SH gene and the introduction of five translationally- silent point mutations in the last three codons and the termination codon of the SH gene (Bukreyev, et al., J. Virol., 15'. 12128-12140 (2001)). Presence of the term “LID” or “6120” in a recombinant virus name indicates that the recombinant virus contains the 6120 mutation.

[0148] The 6120 Mutation stabilizes the antigenomic cDNA in bacteria so that it can be more easily manipulated and prepared. In wild-type RSV strains, this mutation has been found to confer a 5-fold increase in replication efficiency in vitro (Bukreyev, et al., J. Virol., 75: 12128-12140 (2001)), whereas it is not believed to increase replication efficiency in vivo.

[0149] Moreover, the deletion of sequence exemplified by the 6120 Mutation in the downstream non-translated region of the SH gene, may involve any comparable genome sequence that does not contain a critical cis-acting signal (Collins and Karron, Fields Virology, 6th Edition (2013), pages 1086-1123). Genome regions that are candidates for deletion include, but are not limited to, non-translated regions in other genes, in the intergenic regions, and in the trailer region.

[0150] In certain aspects, one or more genes of the genome or antigenome of the CPD RSV are replaced with, e.g., a bovine or other RSV counterpart, or with a counterpart or foreign gene from another respiratory pathogen such as PIV. Substitutions, deletions, and other modifications of RSV genes or gene segments in this context can include part or all of one or more of the NS1. NS2, N, P, M, SH, and L genes, or the M2-1 ORFs, or non- immunogenic parts of the G and F genes. Also, human RSV cis-acting sequences, such as promoter or transcription signals, can be replaced with, for example, their bovine RSV counterpart. In other aspects, RSV variants comprise human attenuating genes or cis-acting sequences inserted into a bovine RSV genome or antigenome background.

[0151] Accordingly, RSV variants encoded by the polypeptide of the invention which is intended for administration to humans can be a human RSV that has been modified to contain genes from, for example, a bovine RSV or a PIV, such as for the purpose of attenuation. For example, by inserting a gene or gene segment from PIV, a bivalent vaccine to both PIV and RSV is provided. Alternatively, a heterologous RSV species, subgroup, or strain, or a distinct respiratory pathogen such as PIV, may be modified, e.g., to contain genes that encode epitopes or proteins which elicit protection against human RSV infection. For example, the human RSV glycoprotein genes can be substituted for the bovine glycoprotein genes such that the resulting bovine RSV. which now bears the human RSV surface glycoproteins and would retain a restricted ability to replicate in a human host due to the remaining bovinegenetic background, elicits a protective immune response in humans against human RSV strains.

[0152] In certain aspects, a selected gene segment, such as one encoding a selected protein or protein region (for instance, a cytoplasmic tail, transmembrane domain or ectodomain, an epitope, a binding site or region, or an active site or region containing an active site) from one RSV strain, can be substituted for a counterpart gene segment from the same or different RSV strain or other source, to yield novel recombinants having desired phenoty pic changes compared to parental and / or wild-ty pe RSV strains. Such resulting strains may, for example, express a chimeric protein having a cytoplasmic tail and / or transmembrane domain of one RSV fused to an ectodomain of another RSV. Other exemplary aspects of this type express duplicate protein regions, such as duplicate immunogenic regions.

[0153] As used herein, “counterpart'’ genes, gene segments, proteins, or protein regions, are typically from heterologous sources (for example, from different RSV genes, or representing the same (i.e., homologous or allelic) gene or gene segment in different RSV strains). Generally, counterparts selected in this context share gross structural features, for example each counterpart may encode a comparable structural “domain,” such as a cytoplasmic domain, transmembrane domain, ectodomain, binding site or region, or epitope. Counterpart domains and their encoding gene segments embrace an assemblage of species having a range of size and amino acid (or nucleotide) sequence variations, which range is defined by a common biological activity7among the domain or gene segment variants.

[0154] For example, in an aspect, two selected protein domains encoded by counterpart gene segments may share substantially the same qualitative activity, such as providing a membrane spanning function, a specific binding activity', or an immunological recognition site. More ty pically, a specific biological activity shared between counterparts, for example, between selected protein segments or proteins, will be substantially similar in quantitative terms, i.e., they will not vary in respective quantitative activity profiles by more than 30%. preferably by no more than 20%, more preferably by no more than 5-10%.

[0155] In some aspects, the RSV variant produced from a cDNA-expressed genome or antigenome can be any of the RSV or RSV-like strains, such as, human, bovine, or murine, or of any pneumovirus or metapneumovirus, such as pneumonia virus of mice or avian metapneumovirus. To elicit a protective immune response, the RSV variant may be one which is endogenous to the subject being immunized, such as human RSV being used toimmunize humans. The genome or antigenome of endogenous RSV can be modified, however, to express RSV genes or gene segments from a combination of different sources, such as a combination of genes or gene segments from different RSV species, subgroups, or strains, or from an RSV and another respiratory pathogen such as human parainfluenza virus (PIV) (see, for example, Hoffman, et al., J. Virol.. 71 : 4272-4277 (1997); Durbin, et al., Virology. 235(2): 323-32 (1997); U.S. Patent 7,208,161; WO 1998 / 053078; and the following plasmids for producing infectious PIV clones: p3 / 7(l 31 ) (ATCC 97990); p3 / 7(131)2G(ATCC 97889); and p218(131) (ATCC 97991); each deposited Apr. 18, 1997 under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC) of 10801 University Blvd.. Manassas. Va. 20110-2209. U.S.A., and accorded the aforementioned accession numbers).RSV Variant Having An Attenuated Phenotype

[0156] The invention provides an RSV variant having an attenuated phenotype that is encoded by the inventive polynucleotide as described herein.

[0157] The inventive RSV variant may be virus particle or a subviral particle. It may be present in a cell culture supernatant, isolated from the culture, or partially or completely purified. The RSV variant may also be ly ophilized, and can be combined with a variety of other components for storage or delivery to a host, as desired.

[0158] The RSV variant of the invention is useful in various compositions to generate a desired immune response against RSV in a host susceptible to RSV infection. RSV variants of the invention are capable of eliciting a protective immune response in an infected human host, yet are sufficiently attenuated so as to not cause unacceptable symptoms of severe respiratory disease in the immunized host. In some aspects, a live attenuated RSV vaccine comprises the RSV variant of the invention.

[0159] To select candidate vaccine viruses from the host of RSV variants provided herein, the criteria of viability, efficient replication in vitro, attenuation in vivo, immunogenicity, and phenotypic stability are determined according to well-known methods. The most desirable RSV viruses, in regards to generation of RSV vaccines and pharmaceutical compositions, should maintain viability, replicate sufficiently well in vitro under permissive conditions to make vaccine manufacture possible, have a stable attenuation phenotype, be well-tol erated, exhibit replication in an immunized host (albeit at lower levels), and effectively elicit production of an immune response in a vaccine sufficient to confer protection against serious disease caused by subsequent infection from wild-type virus. TheRSV variants of the invention meet these criteria by exhibiting strong immunogenicity in vivo, at or near levels elicited by wild-type RSV, while still exhibiting stable attenuated replication.

[0160] RSV variants as described herein can be tested in various well known and generally accepted in vitro and in vivo models to confirm adequate attenuation, resistance to phenotypic reversion, and immunogenicity for vaccine use. The RSV variants, which can be a multiply attenuated, biologically derived or recombinant RSV, can be tested in in vitro assays for temperature sensitivity of virus replication or “ts phenotype’' and for the small plaque phenotype. The RSV variants may be further tested in animal models of RSV infection. A variety of animal models (e.g.. murine, hamster, cotton rat, and primate) have been described and are known to those skilled in the art.

[0161] In an aspect of the invention, an RSV variant may be employed as a “vector” for protective antigens of other pathogens, particularly respiratory' tract pathogens such as parainfluenza virus (NV). For example, a recombinant RSV having a T11661 mutation may be prepared which incorporates sequences that encode protective antigens from PIV to produce infectious, attenuated vaccine virus.Production of RSV Variants Having Attenuated Phenotype

[0162] The invention provides a method for producing the inventive polynucleotide or inventive RSV variant as described herein.

[0163] The inventive polynucleotide or inventive RSV variant can be prepared by any suitable production technique, many of which are known in the art. For example, the inventive polynucleotide can be inserted into a suitable vector, which is used to transform a suitable host cell, e.g., a host cell permissive of RSV infection, which is replicated in a suitable culture, and then expressed to produce the inventive RSV variant. As such, the invention includes a vector comprising the inventive polynucleotide or inventive RSV variant, as well as a host cell transfected or transformed w ith the inventive polynucleotide or inventive RSV variant, e.g., by use of the inventive vector.

[0164] The inventive RSV variant can be produced from one or more isolated polynucleotides, for instance, one or more cDNAs. In an aspect, cDNA encoding a RSV variant genome or antigenome is constructed for intracellular expression. In another aspect, cDNA encoding all or part of a RSV variant genome or antigenome is coexpressed in vitro with the necessary viral proteins to form RSV variant. “RSV antigenome” refers to an isolated positive-sense polynucleotide molecule which serves as the template for thesynthesis of progeny RSV genome. A cDNA is preferably constructed which is a positivesense version of the RSV genome, corresponding to the replicative intermediate RNA, or antigenome, so as to minimize the possibility of hybridizing with positive-sense transcripts of the complementing sequences that encode proteins necessary to generate a transcribing, replicating nucleocapsid, i.e., sequences that encode N, P, L, and M2-1 proteins.

[0165] In certain aspects, the invention provides a method for producing one or more purified RSV protein(s) which involves infecting a host cell permissive of RSV infection with a RSV variant under conditions that allow for RSV propagation in the infected cell. After a period of replication in culture, the cells are lysed, and the RSV is isolated therefrom. In other aspects, one or more desired RSV proteins are purified after isolation of the virus, yielding one or more RSV proteins for vaccine, diagnostic, and other uses.

[0166] To propagate an RSV variant virus for vaccine use and other purposes, a number of different cell lines which allow for RSV growth may be used. 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 with epithelial cell lines such as Vero cells. Cells are typically inoculated with virus at a multiplicity of infection ranging from about 0.001 to 1.0, or more and are cultivated under conditions permissive for replication of the virus, e.g., at about 30-37° C and for about 3-10 days, or as long as necessary for the virus to reach an adequate titer. Temperature-sensitive viruses often are grown using 32° C. as the ‘'permissive temperature.” Virus is removed from cell culture and separated from cellular components, typically by well-known clarification procedures, such as centrifugation, and may be further purified as desired using procedures well known to those skilled in the art.

[0167] The herein described method for producing attenuated recombinant RSV mutants can be used to yield infectious viral or subviral particles, or derivatives thereof. An infectious virus is comparable to the wild-type RSV virus particle and is infectious “as is.” An infectious virus can directly infect fresh cells. An infectious subviral particle typically is a subcomponent of the virus particle which can initiate an infection under appropriate conditions. For example, a nucleocapsid containing the genomic or antigenomic RNA and the N, P, L, and M2-1 proteins is an example of a subviral particle which can initiate an infection if introduced into the cytoplasm of cells. Subviral particles provided by an aspect of the invention include viral particles which lack one or more protein(s), protein segment(s). or other viral component(s) not essential for infectivity.

[0168] Other aspects provide a cell or a cell-free lysate containing an expression vector which comprises the inventive polynucleotide, and an expression vector (the same or different vector) comprising one or more isolated polynucleotide molecules encoding the N, P, L, and M2-2 proteins of RSV. In further aspects, one or more of these proteins is expressed from genome or antigenome cDNA. Upon expression, the genome or antigenome and N, P, L, and M2-2 proteins combine to produce an infectious RSV viral or sub-viral particle.Pharmaceutical Composition

[0169] An aspect of the invention provides a pharmaceutical composition comprising the inventive RSV variant and at least one excipient. The inventive pharmaceutical composition desirably comprises an immunologically effective amount of the inventive RSV variant. In some aspects, a live attenuated RSV vaccine comprises the inventive pharmaceutical composition.

[0170] The inventive pharmaceutical composition can be prepared in any suitable manner, many of which are known in the art. The excipient can be any suitable excipient, such as a carrier. Suitable carriers include, for example, buffers, stabilizers, diluents, preservatives, and / or solubilizers, and can also be formulated to facilitate sustained release. Diluents include water, saline, dextrose, ethanol, glycerol, and the like. Additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin, among others. Other suitable vaccine vehicles and additives, including those that are particularly useful in formulating modified live vaccines, are known or will be apparent to those skilled in the art. See, e.g., Remington's Pharmaceutical Science, 18th ed., Mack Publishing (1990). which is incorporated herein by reference.

[0171] The inventive pharmaceutical composition may comprise one or more additional immunomodulatory components such as, for instance, an adjuvant or cytokine, among others. Adjuvants that can be used in the compositions include, but are not limited to, the RIBI adjuvant system (Ribi Inc., Hamilton, Mont ), alum, mineral gels such as aluminum hydroxide gel, oil-in-water emulsions, water-in-oil emulsions such as, for example, Freund's complete and incomplete adjuvants, Block copolymer (CytRx, Atlanta Ga.), QS-21 (Cambridge Biotech Inc., Cambridge Mass.), SAF-M (Chiron, Emeryville Calif), AMPHIGEN™ adjuvant, saponin. Quil A or other saponin fraction, monophosphoryl lipid A, ionic polysaccharides, and Avridine lipid-amine adjuvant. Non-limiting examples of oil-in- water emulsions useful in the vaccine of the invention include modified SEAM62 and SEAM1 / 2 formulations. Modified SEAM62 is an oil-in-water emulsion containing 5% (v / v) squalene (Sigma), 1% (v / v) SPAN™ 85 detergent (ICI Surfactants), 0.7% (v / v) TWEEN™ 80 detergent (ICI Surfactants), 2.5% (v / v) ethanol, 200 iig / ml Quil A, 100 pg / ml cholesterol, and 0.5% (v / v) lecithin. Modified SEAM 1 / 2 is an oil-in-water emulsion comprising 5% (v / v) squalene, 1% (v / v) SPAN™ 85 detergent, 0.7% (v / v) Tween 80 detergent, 2.5% (v / v) ethanol, 100 pg / ml Quil A, and 50 pg / ml cholesterol. Other immunomodulatory agents that can be included in the composition include, for example, one or more interleukins, interferons, or other known cytokines. Additional adjuvant systems permit for the combination of both T-helper and B-cell epitopes, resulting in one or more types of covalent T-B epitope linked structures, which may be additionally hpidated. such as those described in WO 2006 / 084319, WO 2004 / 014957, and WO 2004 / 014956.

[0172] The inventive pharmaceutical composition contains as an active ingredient an immunogenically effective amount of a RSV variant as described herein. Biologically derived or recombinant RSV strains can be administered directly to a host as a vaccine used directly in a vaccine formulation or composition. The biologically derived or recombinantly modified virus may be introduced into a host with a physiologically acceptable carrier and / or adjuvant. Useful carriers are well known in the art and include, for example, w ater, buffered water, saline, e.g., 0.4% saline, glycine, 0.3% glycine, hyaluronic acid, and the like. The resulting aqueous solutions can be packaged for use “as is” provided in frozen form that is thawed prior to use, or lyophilized, with the lyophilized preparation being combined with a sterile solution prior to administration. The pharmaceutical compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, which include, but are not limited to, pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sucrose, magnesium sulfate, phosphate buffers, HEPES (4-(2-hydroxyethyl)-l -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 in the art. Preferred adjuvants also include STIMULON™ QS-21 immunostimulatory saponin (Aquila Biopharmaceuticals, Inc., Worchester, Mass.), MPL™ (3-O-deacylated monophosphoryl lipid A; RIM ImmunoChem Research, Inc., Hamilton.Mont), and interleukin- 12 (Genetics Institute. Cambridge, Mass.).Multivalent Vaccine Compositions

[0173] An aspect of the invention provides a multivalent RSV vaccine composition comprising a first RSV variant of the invention, a second RSV variant of the invention, and, optionally, one or more additional RSV variants of the invention, wherein the first, second, and optional additional RSV variants have different nucleotide sequences.

[0174] An aspect of the invention provides multivalent vaccine compositions comprising at least one recombinant RSV variant of an aspect of the invention, and at least one antigen from a non-RSV virus. The non-RSV virus can be any suitable virus. In an aspect of the invention, the non-RSV virus is parainfluenza viruses (PIV). In another aspect of the invention, the non-RSV virus is human metapneumovirus (HMPV).

[0175] In an aspect of the invention, multivalent vaccine compositions comprise at least one recombinant RSV variant of an aspect of the invention, and at least one antigen from a PIV. In another aspect of the invention, multivalent vaccine compositions comprise at least one recombinant RSV variant of an aspect of the invention, and at least one antigen from a HMPV. In an aspect of the invention, multivalent vaccine compositions comprise at least one recombinant RSV variant of an aspect of the invention, at least one antigen from a PIV, and at least one antigen from a HMPV. In an aspect of the invention, the PIV can be HPIV. In a further aspect of the invention, the HPIV can be HPIV type 3 (HPIV3).

[0176] The inventive multivalent vaccine compositions can comprise one or more excipients or other components as described herein for the inventive pharmaceutical composition.

[0177] In some aspects, the vaccine or pharmaceutical composition comprises an RSV variant that elicits an immune response against a single RSV strain or antigenic subgroup, e.g., A or B, or against multiple RSV strains or subgroups. In this regard, an RSV variant can be combined in vaccine formulations with other RSV vaccine strains or subgroups having different immunogenic characteristics for more effective protection against one or multiple RSV strains or subgroups. The vaccine or pharmaceutical composition may be administered in a vaccine mixture, or administered separately in a coordinated treatment protocol to elicit more effective protection against one RSV strain, or against multiple RSV strains or subgroups.Vaccination Method

[0178] The invention provides a method of vaccinating an animal. The vaccination method comprises administering the inventive RSV variant, preferably in the form of theinventive pharmaceutical composition, to an animal. Further provided is a method of inducing an immune response comprising administering the vaccine or pharmaceutical composition.

[0179] In certain aspects, a live attenuated RSV variant vaccine (or RSV variant pharmaceutical composition) is administered, wherein the vaccine or pharmaceutical composition comprises the RSV variant encoded by a polynucleotide of the invention as described herein.

[0180] The vaccine and pharmaceutical compositions may be administered by any suitable method, including but not limited to, via injection, nasal spray, nasal droplets, topical application, aerosol delivery, or oral inoculation. In some aspects, the compositions may be administered intranasally or subcutaneously or intramuscularly. In some aspects, the compositions may be administered to the upper respiratory tract. The compositions can be administered to an individual seronegative for antibodies to RSV or possessing transplacentally acquired maternal antibodies to RSV.

[0181] The animal to which the vaccine or pharmaceutical composition is administered can be any mammal susceptible to infection by RSV or a closely related virus and capable of generating a protective immune response to antigens of the vaccine strain. Thus, suitable animals include humans, non-human primates, bovine, equine, swine, ovine, caprine, lagamorph. rodents, such as mice or cotton rats. etc. Accordingly, the invention provides methods for creating vaccines for a variety of human and veterinary uses.

[0182] In the case of humans, the RSV variant can be administered according to well established human vaccine protocols (Karron et al., JID, 191 : 1093-104 (2005)). Briefly, adults or children are inoculated intranasally via droplet with an immunogenically effective dose of RSV vaccine, typically in a volume of 0.5 ml of a physiologically acceptable diluent or carrier. This has the advantage of simplicity and safety’ compared to parenteral immunization with a non-replicating vaccine. It also provides direct stimulation of local respiratory tract immunity, which plays a major role in resistance to RSV. Further, this mode of vaccination effectively bypasses the immunosuppressive effects of RSV specific maternally -derived serum antibodies, which typically are found in the very young. Also, while the parenteral administration of RSV antigens can sometimes be associated with immunopathologic complications, this has never been observed with a live virus.

[0183] For humans, the precise amount of RSV variant vaccine administered and the timing and repetition of administration will be determined by various factors, including thepatient's state of health and weight, the mode of administration, and the nature of the formulation. Dosages will generally range from about 3.0 logl 0 to about 6.0 logl 0 plaque forming units (“PFU”) or more of virus per patient, more commonly from about 4.0 log 10 to 5.0 loglO PFU virus per patient. In one aspect, about 5.0 loglO to 6.0 loglO PFU per patient may be administered during infancy, such as between 1 and 6 months of age. and one or more additional booster doses could be given 2-6 months or more later. In another aspect, young infants could be given a dose of about 5.0 loglO to 6.0 loglO PFU per patient at approximately 2, 4, and 6 months of age, which is the recommended time of administration of a number of other childhood vaccines. In still another aspect, an additional booster dose could be administered at approximately 10-15 months of age. The vaccine formulations and pharmaceutical compositions should provide a quantity of RS V variant of the invention sufficient to effectively stimulate or induce an anti-RSV immune response (an “immunogenically effective amount”).

[0184] In some aspects, neonates and infants are given multiple doses of RSV vaccine to elicit sufficient levels of immunity. Administration may begin within the first month of life, and at intervals throughout childhood, such as at two months, four months, six months, one year and two years, as necessary to maintain sufficient levels of protection against natural RSV infection. In other aspects, adults who are particularly susceptible to repeated or serious RSV infection, such as, for example, health care workers, day care workers, family members of young children, the elderly, individuals with compromised cardiopulmonary function, are given multiple doses of RSV vaccine to establish and / or maintain protective immune responses. Levels of induced immunity can be monitored by measuring amounts of neutralizing secretory and serum antibodies, and dosages adjusted or vaccinations repeated as necessary to maintain desired levels of protection. Further, different vaccine viruses may be indicated for administration to different recipient groups. For example, an engineered RSV strain expressing a cytokine or an additional protein rich in T cell epitopes may be particularly advantageous for adults rather than for infants. Vaccines produced in accordance with the present invention can be combined with viruses of the other subgroup or strains of RSV to achieve protection against multiple RSV subgroups or strains, or selected gene segments encoding, for example, protective epitopes of these strains can be engineered into one RSV variant clone as described herein. In such aspects, the different viruses can be in admixture and administered simultaneously or present in separate preparations and administered separately. For example, as the F glycoproteins of the two RSV subgroupsdiffer by only about 11% in amino acid sequence, this similarity is the basis for a cross- protective immune response as observed in animals immunized with RSV or F antigen and challenged with a heterologous strain. Thus, immunization with one strain may protect against different strains of the same or different subgroup.

[0185] Upon immunization with a RSV vaccine composition, the host responds to the vaccine by producing antibodies specific for RSV virus proteins, for example, F and G glycoproteins. In addition, innate and cell-mediated immune responses are induced, which can provide antiviral effectors as well as regulating the immune response. As a result of the vaccination the host becomes at least partially or completely immune to RSV infection, or resistant to developing moderate or severe RSV disease, particularly of the lower respiratory tract.

[0186] The resulting immune response can be characterized by a variety of methods. These include taking samples of nasal washes or sera for analysis of RSV-specific antibodies, which can be detected by tests including, but not limited to. complement fixation, plaque neutralization, enzyme-linked immunosorbent assay, luciferase-immunoprecipitation assay, and flow cytometry. In addition, immune responses can be detected by assay of cytokines in nasal washes or sera, ELISPOT of immune cells from either source, quantitative RT-PCR or microarray analysis of nasal wash or serum samples, and restimulation of immune cells from nasal washes or serum by re-exposure to viral antigen in vitro and analysis for the production or display of cytokines, surface markers, or other immune correlates measured by flow cytometry7or for cytotoxic activity against indicator target cells displaying RSV antigens. In this regard, individuals are also monitored for signs and symptoms of upper respiratory7illness.Method of Inducing an Immune Response

[0187] An aspect of the invention provides a method of inducing an immune response in an animal. Another aspect of the invention provides a method of inducing an immune response to RSV in an animal. The method comprises administering the inventive RSV variant to an animal. The inventive RSV variant can be administered in the same forms and / or same ways as described herein for the inventive vaccination method.

[0188] Provided herein is a method for stimulating the immune system of an individual to elicit an immune response against RSV in a mammalian subj ect. The method comprises administering an immunogenic formulation of an immunologically sufficient or effective amount of a RSV variant in a physiologically acceptable carrier and / or adjuvant.

[0189] The RSV variant of the invention is useful in various compositions to generate a desired immune response against RSV in a host susceptible to RSV infection. Attenuated variant RSV strains of the invention are capable of eliciting a protective immune response in an infected human host, yet are sufficiently attenuated so as to not cause unacceptable symptoms of severe respiratory disease in the immunized host. The attenuated virus or subviral particle may be present in a cell culture supernatant, isolated from the culture, or partially or completely purified. The virus may also be lyophilized, and can be combined with a variety' of other components for storage or delivery to a host, as desired.

[0190] In an aspect of the invention, the RSV variant has a shutoff temperature for plaque formation of about 37 °C or above (i.e., about 37 °C or above, about 38 °C or above, about 39 °C or above, about 40 °C or above, about 41 °C or above, or about 42 °C or above). The “shutoff temperature,” as used herein, is defined as the lowest temperature at which there is a reduction in plaque number compared to 32 °C that is 100-fold or greater than that observed for wt RSV between the two temperatures.Method of Producing RSV Vaccine

[0191] The invention provides a method of producing an RSV vaccine. The method comprises expressing the polynucleotide of the invention as described herein in a cell. The aspects of the method, e.g., the nature of the cell, are the same as described herein for the production of the inventive RSV variant.

[0192] As used herein, the terms “recipient,” “individual,” “subject,” “host,” and “patient” are used interchangeably and refer to any mammalian subject for whom vaccination is desired (e.g.. humans). “Mammal” and “animal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, camels, etc. In certain aspects, the mammal is human.Examples of Non-Limiting Aspects of the Disclosure

[0193] Aspects, including embodiments, of the invention described herein may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered (1 )-(34) are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to providesupport for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:

[0194] (1) A polynucleotide encoding a recombinant respiratory syncytial virus (RSV) variant having an attenuated phenotype comprising a modified RSV genome or antigenome, wherein NS1, NS2, N, P, M. SH, and L ORFs of the modified RSV genome or antigenome are codon-pair deoptimized.

[0195] (2) The polynucleotide of aspect 1, wherein each of the NS1, NS2, N, P, M, SH, and L ORFs of the modified RSV genome or antigenome polynucleotide independently comprise a codon pair bias (CPB) score reduction of at least 0.01.

[0196] (3) The polynucleotide of aspect 1 or 2, wherein each of the NS 1. NS2. N, P. M,SH, and L ORFs of the modified RSV genome or antigenome polynucleotide independently comprise a CPB score of at least -0.01.

[0197] (4) The polynucleotide of any one of aspects 1-3, wherein the nucleotide sequence of the modified RSV genome or antigenome encoding one or more of RSV proteins NS1, NS2, N, P, M, SH, and L has about 70% to about 95% identity with the nucleotide sequence of the parental RSV genome or antigenome encoding the same one or more of RSV proteins NS1, NS2, N, P, M, SH, and L.

[0198] (5) The polynucleotide of any one of aspects 1-4, wherein the nucleotide sequence of the modified RSV genome or antigenome encoding RSV NS1 protein has at least about 95% identity to SEQ ID NO: 1.

[0199] (6) The polynucleotide of any one of aspects 1-5, wherein the nucleotide sequence of the modified RSV genome or antigenome encoding RSV NS2 protein has at least about 95% identity to SEQ ID NO: 2.

[0200] (7) The polynucleotide of any one of aspects 1-6, wherein the nucleotide sequence of the modified RSV genome or antigenome encoding RSV N protein has at least about 95% identity to SEQ ID NO: 3.

[0201] (8) The polynucleotide of any one of aspects 1-7, wherein the nucleotide sequence of the modified RSV genome or antigenome encoding RSV P protein has at least about 95% identity to SEQ ID NO: 4.

[0202] (9) The polynucleotide of any one of aspects 1-8, wherein the nucleotide sequence of the modified RSV genome or antigenome encoding RSV M protein has at least about 95% identity to SEQ ID NO: 5.

[0203] (10) The polynucleotide of any one of aspects 1-9, wherein the nucleotide sequence of the modified RSV genome or antigenome encoding RSV SH protein has at least about 95% identity to SEQ ID NO: 6.

[0204] (11) The polynucleotide of any one of aspects 1-10, wherein the nucleotide sequence of the modified RSV genome or antigenome encoding RSV L protein has at least about 95% identity to SEQ ID NO: 11.

[0205] (12) The polynucleotide of any one of aspects 1-11, wherein the amino acid sequence of the one or more of RSV proteins NS1, NS2, N, P, M, M2-1, M2-2, SH, and L encoded by the nucleotide sequence of the modified RSV genome or antigenome is at least 99% identical to the amino acid sequence of the same one or more of RSV proteins NS1. NS2, N, P, M, M2-1, M2-2, SH, and L encoded by the nucleotide sequence of the parental RSV genome or antigenome.

[0206] (13) The polynucleotide of aspect 12, wherein the RSV M2-1 protein has an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 at at least position 87.

[0207] (14) The polynucleotide of aspect 13, wherein the residue at position 87 ofSEQ ID NO: 25 is lysine.

[0208] (15) The polynucleotide of any one of aspects 12-14, wherein a residue at position 46 of RSV NS 1 protein SEQ ID NO: 22 is methionine, and the 5thnucleotide in the RSV P gene start signal SEQ ID NO: 19 is changed to an adenosine residue.

[0209] (16) The polynucleotide of any one of aspects 13-15, wherein the RSV P protein has an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 24 at at least position 26.

[0210] (17) The polynucleotide of aspect 16, wherein the residue at position 26 ofSEQ ID NO: 24 is aspartic acid.

[0211] (18) The polynucleotide of any one of aspects 13-16, wherein the residue at position 4 of the RSV M2 -2 protein SEQ ID NO: 26 is a leucine, and the 4thnucleotide in SEQ ID NO: 21 is changed to a guanosine residue.

[0212] (19) The polynucleotide of any one of aspects 13-18, wherein the residue at position 4 of the RSV M2 -2 protein SEQ ID NO: 26 is a leucine, and the residue at position 88 of the M2-1 SEQ ID NO: 25 protein is a threonine residue, and the 4thnucleotide in SEQ ID NO: 21 is changed to a guanosine residue.

[0213] (20) The polynucleotide of any one of aspects 1-18. wherein the polynucleotide comprising a modified RSV genome or anti genome encodes:(a) a mutant RSV NS 1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 22 at position 5, optionally wherein the residue at position 5 of the amino acid sequence is threonine;(b) a mutant RSV N protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 23 at position 50, optionally wherein the residue at position 50 of the amino acid sequence is leucine;(c) a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 at position 70. optionally wherein the residue at position 70 of the amino acid sequence is aspartic acid;(d) a mutant RSV L protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 29 at position 1,249, optionally wherein the residue at position 1.249 of the amino acid sequence is lysine; or(e) any combination of (a)-(d), and optionally wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD.

[0214] (21) A recombinant RSV variant comprising the isolated polynucleotide of any one of aspects 1-20.

[0215] (22) A pharmaceutical composition comprising the recombinant RSV variant of aspect 21 and at least one excipient.

[0216] (23) A multivalent RSV vaccine composition comprising a recombinantRSV variant of aspect 21, a second recombinant RSV variant of aspect 21, and, optionally, one or more additional recombinant RSV variants of aspect 21, wherein the first, second, and optional additional recombinant RSV variants have different nucleotide sequences.

[0217] (24) A multivalent vaccine composition comprising at least one recombinant RSV variant of aspect 21 and at least one antigen from a non-RSV virus.

[0218] (25) The multivalent vaccine composition of aspect 24, wherein the at least one antigen from a non-RSV virus is a parainfluenza viruses (PIV).

[0219] (26) The multivalent vaccine composition of aspect 24 or 25, further comprising an antigen from human metapneumovirus (HMPV).

[0220] (27) A method of vaccinating an animal, comprising administering the pharmaceutical composition of aspect 22 or the multivalent vaccine composition of aspects 23-26 to an animal.

[0221] (28) A method of inducing an immune response in an animal, comprising administering the recombinant RSV variant of aspect 21, the pharmaceutical composition of aspect 22, or the multivalent RSV vaccine composition of aspects 23-26 to an animal.

[0222] (29) The method of aspect 27 or 28, wherein the recombinant RSV variant does not increase the level of cytokine protein expression in the animal.

[0223] (30) The method of any one of aspects 27-29, wherein the recombinantRSV variant is administered via injection, nasal spray, nasal droplets, topical application, aerosol delivery, or oral inoculation.

[0224] (31) The method of any one of aspects 27-29, wherein the recombinantRSV variant has a shutoff temperature for plaque formation of 37 °C or above.

[0225] (32) The method of any one of aspects 27-31. wherein the animal is a mammal.

[0226] (33) The method of any one of aspects 27-31, wherein the animal is a human.

[0227] (34) A method of producing a recombinant RSV variant vaccine, comprising expressing the polynucleotide of any of aspects 1-20 in a cell.

[0228] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.EXAMPLES

[0229] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

[0230] Cell lines. African green monkey kidney (Vero) cells were grown in OptiMEM (Gibco-Life Technologies) with 5% fetal bovine serum (FBS, Hyclone) and 1% L-glutamine (Gibco-Life Technologies). Vero cells were maintained in 2% FBS during experiments. Baby hamster kidney cells that constitutively express the T7 polymerase (BSR T7 / 5) (Buchholz, et al.. J. Virol., 73(1): 261-9 (1999)) were grown in GMEM media (Gibco-Life Technologies) with 10% FBS and 2% non-essential amino acids (Gibco-Life Technologies). Every other cell passage, 2% of gentamicin (Quality Biological) was added to the media to maintain selection for the T7-polymerase-expressing cells. Both cell lines were maintained at 37 °C with 5% CO2.

[0231] Viruses. Min A, Min L, and Min FLC viruses were described previously (Le Nouen, et al., PNAS USA, 111(36): 13169-13174 (2014)). The parent virus of the CPD RS Vs is the recombinant virus RSV D46 / 6120. RSV D46 / 6120 was derived from the original recombinant version of wt RSV called D46, which has a 15,223-nucleotide genome (Genbank accession number KT992094). RSV D46 / 6120 has an identical sequence except that it contains a 112-nucleotide deletion in the 3’ non-translated region of the SH gene and five silent nucleotide point mutations in the last three codons and termination codon of the SH ORF. These mutations were previously shown to stabilize the RSV cDNA during propagation in E. coll without any effects on the replication in vitro and in mice (Bukreyev, et al.. J. Virol., 75(24): 12128-40 (2001)). The sequence numbering of RS V used herein is based on KT992094.

[0232] Construction and rescue of Min AL and derivatives. The Min AL antigenomic cDNA was generated by replacing the wt L ORF in Min A by the CPD L ORF from Min L using BamHI and KasI restriction sites. Mutations of interest identified during serial passage of Min AL were introduced into the Min AL antigenomic cDNA using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies) following the manufacturer’s recommendations. Min AL and derivatives were recovered by reverse genetics as previously described (Le Nouen. et al., PNAS USA, 111(36): 13169-13174 (2014)). A second passage of the rescued viruses was performed on Vero cells to generate working P2 vims stocks. The consensus sequence of these vims stocks was confirmed by Sanger or Illumina deep sequencing using overlapping reverse transcribed PCR amplicons generated using previously described primers (Le Nouen, et al.. PNAS USA, 111(36): 13169- 13174 (2014)).

[0233] Virus titration by immunoplaque assay. As previously described (Le Nouen, et al., PNAS USA, 11 1(36): 13169-13174 (2014)), duplicate wells of Vero cells in 24-well plates were infected with 10-fold serial dilutions of virus for two h at 32 °C. After seven to 10 days incubation at 32 °C, cells were fixed with 80% cold methanol and immunostained using a mixture of three RSV F-specific monoclonal antibodies (MAbs) (Murphy, et al., Vaccine, 8(5): 497-502 (1990)), and further incubated with a polyclonal anti-mouse-IgG antibody conjugated to horseradish peroxidase to visualize the plaques. Virus titers are expressed in pfu / ml.

[0234] Multi-cycle growth kinetics. Two sets of subconfluent wells of Vero cells in six- well plates were inoculated each in duplicate with the indicated viruses using an MOI of 0.01 pfu / ml. One set of plates was incubated at 32 °C for 12 days while the other set was incubated at 37 °C for 10 days. Virus was harvested on the indicated days pi by scraping and harvesting the cells with the media. The cell and media suspensions were vortexed three times for 10 sec each and the supernatant was clarified by centrifugation at 1,200 rpm for 5 min at 4 °C. Aliquots of viruses w ere snap frozen in dry ice and stored at -80 °C before titration by plaque assay.

[0235] Characterization of plaque size. As previously described (Chen, et al., PLoSPathog., 17(12): el010191 (2021)), Vero cell monolayers in six-well plates were inoculated with 250 pfu per well of virus and incubated at 32 °C for seven days with an overlay containing 0.8% methylcellulose. On day 7, plates were fixed with cold 80% methanol and incubated with a cocktail of three anti-RSV F MAbs (Murphy, et al., Vaccine, 8(5): 497-502 (1990)) in Odyssey Blocking Buffer in PBS (Li-Cor), washed, and incubated with an R- phycoerythrin goat anti-mouse IgG(H+L) secondary antibody (Thermofisher). Plaques were visualized using the Celigo imager (Nexcelcom Bioscience) and the plaque size (pm2) w as analyzed using Celigo software. An average of 3,451 (+1,200) individual plaques were analyzed per virus.

[0236] Illumina whole-genome deep sequencing. Viral RNA was isolated from clarified supernatants collected at the end of passage 14 (stressed lineages) or 18 (control lineages) of the Min AL in-vitro stress test using the QIAamp Viral RNA extraction kit (Qiagen). Then, the viral RNA was reverse transcribed using Superscript II Reverse Transcriptase (RT, ThermoFisher) and the cDNA was amplified by PCR using overlapping RSV-specific primers and a high-fidelity DNA polymerase (pfx DNA polymerase, Thermofisher) as described previously (Le Nouen, et al., PNAS USA, 114(3): E386-E95 (2017)). OverlappingPCR amplicons were purified using the QIAquick PCR Purification kit (Qiagen) and sequenced at the NIH Intramural Sequencing Center (NISC) using Illumina MiSeq. Only the sequences corresponding to the outer-most primers (nucleotides 1-23 and 15, 174-15, 223) were not directly determined.

[0237] Then, a reference sequence of the parental Min AL genome was prepared using the Bowtie 2 software (Langmead, et al., Nat. Methods, 9(4): 357-9 (2012)). Sequencing reads of Min AL and Min AL lineages were received as fastq files from NISC and were aligned to the Min AL reference sequence using Bow tie 2 with the very sensitive local parameter. Aligned reads were next converted from SAM to BAM files and then sorted based on genome position using SAMtools (Li, et al.. Bioinformatics. 25(16):2078-9 (2009)).

[0238] Sequencing reads were then sorted by their position on the flow cell and PCR duplicates w ere marked and removed using Picard Tools with a maximum optical duplicate pixel distance of 2.500. After sorting reads and removing duplicates, the depth of coverage was measured with SAMtools (average coverage per lineage was 7,530 +775 reads / nt) and single nucleotide variants were detected using LoFreq (Wilm, et al.. Nucleic Acids Res., 40(22): 11189-11201 (2012)) with the default parameters. Analysis focused on prominent mutations, defined as detected in >30% of the sequencing reads.

[0239] Single-cycle infections. As previously described (Chen, et al.. PLoS Pathog., 17(12): el010191 (2021); Le Nouen, et al., PNAS USA. 114(3): E386-E95 (2017)). replicate monolayers of Vero cells in six-well plates were infected using an MOI of 3 pfu / cell at 32 °C or 37 °C with the indicated viruses. Two hours after inoculation, the monolayers were washed twice with PBS and replenished with fresh media. At 24 and 48 hpi, four wells per virus were harvested: (i) one well was processed for analysis of cell-associated viral RNA by strand-specific RT-qPCR, (ii) one well was harvested for analysis of viral protein expression by flow' cytometiy. (iii) one well was harvested for Western blot analysis, and (iv) the last well was harvested to quantify virus titer by plaque assay as described above for the multicycle replication experiments.

[0240] Strand-specific RT-qPCR. The cell-associated RNA from infected Vero cells from single-cycle infections was extracted using the RNeasy mini kit (Qiagen) following the manufacturer’s recommendations. Then, RNA was subjected to strand-specific RT-qPCR to quantify viral positive-sense (mRNA and antigenome) RNA and negative-sense genomic RNA, as described previously (Le Nouen. et al.. PNAS USA, 111(36): 13169-13174 (2014); Chen, et al., PLoS Pathog., 17(12): el 010191 (2021)) with minor modifications. Briefly, fivehundred ng of DNAse-treated RNA were reverse transcribed using SuperScript III First- Strand Synthesis System (Thermofisher) and a primer specific either to antigenomic / mRNA or genomic RNA. Each RT primer was linked to an unrelated oligonucleotide tag (Le Nouen, et al., PNAS USA, 114(3): E386-E95 (2017)) to provide for specific amplification of the RT product at the following PCR step. Then, the cDNA was amplified by QPCR in triplicate with a primer corresponding to the oligonucleotide tag, a gene-specific reverse primer, and a probe. QPCR data were analyzed using the comparative threshold cycle (ACt) method, normalized to 18S rRNA internal control that had also been subjected to RT-qPCR in parallel using random first-strand primers and a 18S rRNA Taqman assay (Thermofisher). Data was analyzed using the relative quantification software on the Thermofisher Connect platform and expressed as logio fold increase over the Min AL 24-h time point.

[0241] Flow cytometry. NQ cells from single-cycle infections (MOI of 3 pfu / cell, 32 °C or 37 °C) were harvested at 48 hpi, stained using the Live / Dead Fixable Near-IR or Blue Dead Cell dye (Thermofisher), washed with PBS. and fixed and permeabilized using BD Cytofix / Cy toperm (BD Biosciences). Then, cells were stained for 20 min at RT in Perm / Wash buffer (BD Biosciences) containing a cocktail of pre-titrated anti-RSV antibodies; namely a fluorescein isothiocy anate (FITC)-labeled anti-RSV P monoclonal antibody (Abeam), an allophycocyanin (APC)-labeled anti-RSV N monoclonal antibody (Imgenex). a brilliant violet 421 (BV421)-labeled anti-RSV G antibody (Abeam), and a Biotin-labeled anti-RSV F monoclonal antibody (Millipore). After incubation, cells w ere washed three times with Perm / Wash Buffer and incubated with a streptavidin-PE-labeled secondary' antibody (Thermofisher) for 20 min at room temperature. The cells were washed three times with Perm / Wash Buffer and resuspended in PBS. Data were acquired using a BD flow cytometer Symphony (BD Biosciences) and analyzed using FlowJo 10.8. The expression of the virus proteins N, P, G, and F was analyzed on single live cells.

[0242] Western blot analysis. Infected Vero cells from single-cycle infections were harvested in IX NuPage LDS sample buffer (Thermofisher) diluted in PBS containing protease inhibitor (Roche). Cell lysates were homogenized using a QIAshredder spin column (Qiagen) and protein concentrations were determined by BCA assay (Pierce BCA Protein Assay kit).

[0243] Thirty’ pg of proteins were denatured in a final composition of IX NuPAGE LDS Sample Buffer (ThermoFisher) and IX NuPAGE Sample Reducing Agent (Invitrogen) by heating at 90 °C for 10 min before being subjected to electrophoresis on NuPAGE 4-15%Mini-PROTEAN TGX Gels (Bio-Rad) with IX TGS Running Buffer (Bio-Rad). Odyssey Protein Molecular Weight Marker (Li-Cor) was run in parallel. Proteins were transferred to PVDF membranes using the iBlot 2 Gel Transfer Device (ThermoFisher) and stained with a primary mouse anti-RSV F (abeam, ab43812), or a mouse anti-RSV P (abeam, ab94965) antibody. The rabbit anti-Tubulin (abeam. ab52866n) antibody was used as a loading control. The secondary antibodies used were goat anti-rabbit IgG IRDye 680RD (at 1: 15,000), and goat anti-mouse IgG IRDye 800CW (1: 10,000, Li-Cor). Membranes were scanned using Odyssey software, version 3.0 (Li-Cor).

[0244] Hamster experiment. The hamster experiment was approved by the NIH Institutional Animal Care and Use Committee (IACUC). At day -2, serum was collected from 104 five-to-six-week old male golden Syrian hamsters (Mesocricetus auratus') (Envigo). On day 0, six groups of 16 hamsters were inoculated IN under isoflurane anesthesia with 6 logio pfu of wt RSV, Min AL, or the indicated Min AL-derivatives. A last group of eight hamsters were left uninfected as control.

[0245] At 3 days post-infection (pi), which corresponds to the peak of replication of wt RSV in hamsters, eight hamsters from each inoculated group were euthanized by carbon dioxide inhalation. Nasal turbinates (NT) and lungs were harvested and homogenized separately in Leibovitz (L-15) medium, and the suspensions were clarified by low-speed centrifugation and aliquots were flash-frozen and stored at -80 °C. Virus titers were determined later in duplicate by immunoplaque assay on Vero cells at 32 °C using a methylcellulose layer containing 1% Amphotericin B, 0.1% Gentamicin, and 0.06 mg / mL clindamycin phosphate. The limit of virus detection was 50 pfu / g in both the NT and lungs.

[0246] At 28 dpi, sera was isolated from the blood of all remaining hamsters to evaluate the RSV-specific antibody response. All sera were heated at 56 °C for 30 min to inactivate complement prior to serology assays. The 60% plaque reduction neutralizing antibody titer (PRNTeo) was evaluated using a complement-enhanced immunoplaque assay as described previously (Le Nouen, et al.. PNAS USA, 111(36): 13169-13174 (2014)).

[0247] At 32 dpi, the remaining hamsters were challenged IN with 6 logio pfu of wt RSV. Three days after challenge, hamsters were euthanized by carbon dioxide inhalation and NT, bronchoalveolar lavages (BAL) and lung tissues were harvested. Wt RSV virus titers in NT and lung tissues were determined in duplicate by plaque assay on Vero cells incubated at 32 °C as described above. BAL samples were heated at 56 °C for 30 min to inactivate complement prior to serology assays.

[0248] Expression of inflammatory and antiviral genes in lungs of hamsters. One hundred j l of lung homogenate was mixed with 300 j l of TRIzol LS (Thermo Fisher) and total RNA was extracted using Phasemaker Tubes Complete System (Thermo Fisher) and PureLink RNA Mini Kit (Thermo Fisher). RNA from lung homogenates of two control hamsters (non-immunized and non-challenged) was derived from a previous study (McLellan, et al.. Science, 342(6158): 592-598 (2013)). Then, 7 pl of the extracted RNA was reverse transcribed into cDNA using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher). TaqMan assays (Thermo Fisher) specific for hamster's (Mesocricetus auratus) inflammatory and antiviral genes (Liu, et al.. PLoS Pathog., 19(6): el 011057 (2023)) were performed using the TaqMan Fast Advanced Master Mix (Thermo Fisher) as previously described (McLellan, et al., Science, 342(6158): 592-598 (2013)). Hamster p-actin was used as a housekeeping gene. qPCR results were analyzed using the comparative threshold cycle (AACT) method, normalized to p-actin, and expressed as fold changes over the average expression of the two uninfected, unchallenged hamsters.

[0249] Expt ■ession and purification of the prefusion form of RSV F and ectodomain of G.The DS-CAV1 prefusion-stabilized form of the RSV F protein (pre-F, A2 strain) was expressed from a previously described cDNA (McLellan, et al., Science, 342(6158):592-598 (2013)) that was the kind gift of Drs. Peter Kwong and Barney Graham (VRC, NIAID, NIH). This molecule contained aa 1-513 inclusive of the F protein linked at the C-terminal end to the T4-phage fibritin trimerization domain (foldon) sequence for trimerization of F and a 6X His tag for purification. This plasmid was transfected into Expi293 cells using expifectamine. On day 6 post-transfection, the supernatant containing the F protein was harvested and clarified by centrifugation followed by filtration through a 0.2 pm PES membrane. Then, the supernatant was mixed with Ni-NTA Agarose resin (Thermofisher) for two to three hours at 4 °C with slow rotation. The mixture of cell supematant / resin was poured onto a gravity -flow column (BioRad, cat# 7372512) and washed three times with binding buffer (50 mM Tris-Hcl, 400 mM NaCl). The pre-F protein was eluted using the binding buffer containing 300 mM imidazole (Sigma-Aldrich), concentrated using an Ami con filter, and purified by size exclusion chromatography in PBS using a Superdex 200 Increase 10 / 300 GL column (Cytiva). The purified pre-F protein was concentrated by centrifugation using an Amicon filter and aliquots were snap-frozen in liquid nitrogen and stored at -80 °C until use.

[0250] The sequence encoding the ectodomain of RSV G (A2 strain) was codon- optimized for expression in human cells (Genscript) and cloned under the control of the CMV promoter in the pcDNA3. l(-) vector between the Xbal and Kpnl sites. The construct encodes from the 5'-end to the 3’-end (i) the prolactin signal sequence, (ii) aa 65-298 (234 aa) of RSV G A2, (hi) the Avi tag sequence, (iv) the human rhinovirus 3C protease cleavage site, (v) an 8X His tag and (vi) the Strep-tag II. The resulting pcDNA3. 1-RSV G A2 vector was transfected into Expi293 cells using expifectamine. On day 6 post-transfection, the media supernatant that contained the G protein was clarified by centrifugation further clarified through a 0.2 pm PES vacuum filter. Then, the clarified supernatant was supplemented with 20 mM final Tris-HCl pH 8 and 300 mM final NaCl and mixed with Ni-NTA Agarose resin (Thermofisher) for two to three hours at 4 °C with slow rotation. The mixture was poured onto a gravity' flow column (BioRad), washed three times with binding buffer (20 mM Tris- Hcl, 300 mM NaCl), and the G protein was eluted using binding buffer containing 75 mM imidazole. The eluted G protein was concentrated using an Amicon filter and purified by size exclusion chromatography in PBS using a Superdex 200 Increase 10 / 300 GL column (Cytiva). Aliquots of the purified G protein were snap-frozen in liquid nitrogen and stored at -80 °C until use.

[0251] Dual IgG- and IgA-specific DELFIA ELISA. The levels of binding antibodies against the RSV pre F or G protein in the serum and BAL of hamsters were determined by a dual IgG- and IgA-specific dissociation-enhanced lanthanide fluorescent (DELFIA) time resolved fluorescence (TRF) immunoassay. Black 96-well assay plates (MaxiSorp, ThermoFisher, cat# 437111) were coated with 100 pl / well of pre F or G protein (0.5 pg / ml for serum samples, 1 pg / ml for BAL samples) in carbonate coating buffer [50 mM; 3.7 g sodium bicarbonate and 0.64 g sodium carbonate (Sigma) in 1 L water] overnight at 4 °C. Then, plates were washed three times in washing buffer [IxDPBS (Gibco) containing 0. 1% IGEPAL CA-630, Millipore Sigma] using a BioTek 405 micro-plate washer and blocked with 250 pl of blocking buffer [IxDPBS containing 5% dry milk (W / V) (Carnation nonfat dry milk, Nestle)] overnight at 4 °C and washed once.

[0252] Sera and BAL samples were diluted 1: 100 and 1 : 10. respectively, followed by 10 three-fold serial dilutions in a dilution plate (Costar, cat# 3799) using sample dilution buffer (lxDPBS+5% dry milk+0.2% IGEPAL). Then, 100 pl of diluted samples were transferred from the dilution plate to the coated black plates in duplicate. After 1 h incubation at roomtemperature on a rotating shaker, plates were washed three times with 250 pl washing buffer, replenished with 250 pl washing buffer, and placed on a rotating shaker for another 20 min. After washing, 100 pl per well of a secondary antibody mixture [(goat anti -hamster IgG(H+L)-HRP, 1 : 10,000 dilution, Invitrogen, cat# PA 1-29426, and rabbit anti -hamster IgA- biotin 1: 1,000 dilution (1 :500 for BAL samples), Brookwood biomedical, cat# Sab3002a)] diluted in dilution buffer was added per well. Then, plates were incubated for 1 h at room temperature on a rotating shaker, washed three times as described above, and 100 pl per well of streptavidin-europium (PerkinElmer: 1244-360) diluted 1 :2000 in PBS+0.2% IGEPAL was added. After 1 hour incubation at room temperature, plates were washed three time as above and 50 pl Pierce ECL (ThermoFisher, cat# 32106) per well was added. After 10 min incubation, plates were read on a Synergy neo (BioTek) plate reader for luminescence to detect IgG. After reading, plates were washed again as described above and 100 ml per well of pre-warmed enhancement solution (PerkinElmer, cat# 4001-0010) was added and plates were further incubated for 20 min on a rocking shaker. Finally, plates were read with the Synergy neo plate reader using a program for time-resolved fluorescence with excitation wavelengths of 360 / 40 nm and emission filters of 620 / 40 nm to detect IgA.

[0253] The data were processed as follows: (i) the mean reading for each sample from duplicate wells was calculated, (ii), the mean reading from the blank samples was subtracted from the mean reading for each sample, (iv) the cut-off value was set to the blank mean plus three standard deviations of blank readings. Then, the IgG and IgA titers of each sample were determined by interpolating the sigmoid standard curve generated on Prism 9.0.

[0254] Statistical analysis. Distributions of the virus plaque area in Figure ID were compared for statistical significance using the Wilcoxon rank test with continuity correction post hoc test. In Figures 6B. 7A-7C, and 8A-8C, significant differences among data sets were evaluated using parametric one-way ANOVA with the Tukey post hoc tests for normally distributed data sets or the non-parametric Kruskall Wallis test with Dunns post hoc test for non-normal data sets. A logio transformation was applied to data sets when necessary7to obtain equal standard deviations among groups, a necessary requirement of both tests. In Figures 10A-10B and 11A-1 IB, significant differences among data sets were evaluated using the non-parametric Mann- Whitney test. Statistics were performed on the Prism 8 version GraphPad Software). Data were only considered significant at p<0.05.

[0255] Construction and rescue of Min AL. The new CPD virus Min AL was created by combining the CPD ORFs of Min A (CPD NS 1, NS2. N, P. M, SH) and Min L (CPD L)(Figure 1 A). Thus, Min AL contains CPD versions of seven of the 11 RSV ORFs. with a total of 2,073 silent mutations within ORFs. This represents a 3- and 1 5-fold increase over Min A (695 silent mutations) and Min L (1,378 silent mutations; Figure 1A), respectively.

[0256] Analysis of the CpG and UpA content revealed that wt RSV full-length genome contained 87 CpGs and 1,462 UpAs while Min AL full-length genome contained 426 CpGs and 1,770 UpAs. When the analysis was focused on the combined ORFs, wt RSV contains 79 CpGs and 1,312 UpAs, Min A contains 205 CpGs and 1,418 UpAs, Min L contains 292 CpGs and 1,514 UpAs, and Min AL contains 418 CpGs and 1,620 UpAs (Figure IB). The codon pair scores for the wt ORFs and the CPD ORFs are shown below in Table 1. Min AL was readily rescued by reverse genetics as previously described (Le Nouen, et al., PNAS USA, 1 11(36): 13169-13174 (2014)) and passaged once to prepare working stocks, and Illumina deep-sequencing of these stocks confirmed that the recovered genomes were free of any adventitious mutations with an abundance of >1% of reads.Table 1

[0257] Min AL combines temperature sensitivity! from Min A and Min L. The replication of Min AL in comparison to the previous CPD viruses was evaluated in multi-cycle in-vitro growth kinetics experiments (Figure 1C). Vero cells were infected with Min A, Min L, Min AL, Min FLC or wt RSV using an multiplicity of infection (MOI) of 0.01 plaque forming units (pfu) / cell and incubated at 32 °C, a relatively permissive temperature for temperaturesensitive mutants that also simulates the temperature of the upper respiratory7tract, or the more-restrictive temperature of 37 °C that simulates the temperature of the lower respiratory tract and core body. Virus replication was evaluated for 12 and 10 days, respectively.

[0258] At 32 °C (Figure 1C, left panel), wt RSV replicated efficiently, reaching 7.4 logic pfu / ml by day 6 post-infection (pi), as is typically observed. Replication of all CPD RSVs was reduced and delayed, with replication of Min FLC being the most affected, reaching a maximum peak titer of 6.0 logic pfu / ml by day 12 pi. Replication of the new Min AL virus was comparable to Min L, and slightly more efficient than Min A, reaching a peak titer of 6.7 logic pfu / ml on day 12 pi. At 37 °C (Figure 1C, right panel), wt RSV replicated efficiently and reached a peak titer on day 3 pi (7.2 logic pfu / ml). As previously described (Le Nouen, et al., PATS' USA, 111(36): 13169-13174 (2014)), replication of Min A and Min L was strongly reduced (by 48-fold and 1000-fold, respectively) and only a baseline replication of Min FLC was detected. The replication of Min AL was reduced compared to Min L by about 60-fold, indicating that the new virus was more temperature sensitive than Min A and Min L.

[0259] The plaque sizes of wt and CPD RSVs at 32 °C on Vero cells were evaluated following incubation for seven days under under a methylcellulose overlay (Figure ID). Compared to wt RSV, the plaque sizes of all CPD RSVs were significantly reduced by 3- to 5-fold (p<0.0001). The plaques formed by Min AL appeared to be slightly smaller than Min L, slightly larger than Min A, and even larger than Min FLC, but the differences were not significant.

[0260] The shut off temperature (TSH) for plaque formation by Min AL w as then determined, defined as the lowest temperature at which there is a reduction in plaque number compared to 32 °C that is 100-fold or greater than that observed for wt RSV between the two temperatures (Table 4 [Temperature sensitivity of wt RSV and the indicated CPD RSVs on Vero cells]). Wt RSV efficiently formed plaques at 40 °C as typically observed, while the TSH of Min A, Min L and Min FLC were 40 °C. 38 °C and 36 °C, respectively, as previously described (Le Nouen, et al., PNAS USA, 1 1 1 (36): 13169-13174 (2014)). The TSH of Mm AL was found to be 36 °C, suggesting that its temperature sensitivity phenotype had contributions from both Min A and Min L.Table 2aThe ts phenotype for each virus was evaluated by assessing virus growth on Vero at the indicated temperatures utilizing temperature controlled water-baths. For viruses with a ts phenoty pe, the shut-off temperatures (TSH) are underlined and listed in boldface at the right. TSH is defined as the lowest restrictive temperature at which there is a reduction in plaque number compared to 32 °C that is 100-fold or greater than that observed for wt RSV at the two temperatures. The ts phenotype is defined as having a TSH of 40 °C or less.

[0261] Genetic and phenotypic stability of Min AL when serially passaged at increasing temperature (in-vitro stress test). Since Min AL exhibited a strong temperature sensitivity, the genetic stability and the stability of the temperature-sensitivity phenotype of Min AL in an in-vitro stress test (Figure 2) was evaluated. Twelve 25 cm2flasks of Vero cells were inoculated with Min AL using an initial MOI of 0. 1 pfu / cell. Each flask represented an independent lineage. Ten lineages were passaged in parallel starting at the permissive temperature of 32 °C, with an increase in temperature of 1 °C following every second passage up to 40 °C, for a total of 18 passages representing approximately 4-5 months of continuous culture. In parallel, two additional lineages were passaged 18 times at 32 °C as controls. Cell monolayers were harvested when the cytopathic effect was extensive, and one- fifth of the clarified culture medium was passaged. The remainder of the clarified medium was snap frozen in aliquots on dry ice for subsequent titration and whole-genome Illumina deep sequencing.

[0262] For the two control lineages of Min AL passaged at 32 °C, the virus replicated efficiently for all 18 passages, reaching titers that varied between 5.5 and 7.0 logio pfu / ml (Figure 2A). Virus titers of each of the ten Min AL lineages passaged at increasing temperatures increased by 3-fold during the first two passages at 32 °C, reaching on average 6.9 logio pfu / ml, similar to the control lineages (Figure 2B). From P3 to P8 (33 °C to 35 °C), titers in each lineage decreased steadily by about 30-fold on average to 5.5 logio pfu / ml. Then, from P9 to P14 (first passage at 36 °C to second passage at 38 °C), replication was variable among the lineages. Titers in six of the 10 lineages continued to decrease between6- and 1,800-fold between P9 to P14; the titers in one lineage remained unchanged from P9 to P14; and titers in two lineages increased by 2- to 4-fold from P9 to P14. From P15 to P16 (the two passages at 39 °C), titers sharply decreased in all lineages, ranging from 4. 1 logio pfu / ml to below the limit of detection. Finally, at P17 and P18 (the two passages at 40 °C), replication of all Min AL lineages was below the limit of detection. The observation that most of the lineages replicated efficiently beyond the Tsn for Min AL of 36 °C, but that none of them replicated at 40 °C, suggested that they had a partial loss of the ts phenotype.

[0263] Min AL lineages acquired multiple, different point mutations in both CPD and wt ORFs during the in-vitro stress test. To identify mutations that might be responsible for the partial loss of the ts phenotype during the in-vitro stress test, viral RNA was extracted from clarified cell culture supernatants harvested at the end of P14 (second passage at 38 °C), corresponding to the last passage at which replication was still detected in most of the lineages (Figure 2B, red arrow). Viral RNA also was extracted from supernatants from the two control lineages at Pl 8. The viral RNAs were amplified by overlapping RT-PCRs using RSV-specific primers. PCR amplicons covering the whole genome, with the exception of the outer-most primers at the 3’- and 5’-ends (nucleotides 1-23 and 15, 174-15, 223), were sequenced by Illumina deep sequencing. PCR amplicons were not obtained for P14 of lineages #2 and #4, presumably due to low viral titers, and thus these two lineages were not evaluated further.

[0264] Sequence analysis of these eight '‘stressed” lineages (#1, 3, 5, 6, 7, 8, 9 and 10) identified a total of 38 different prominent (>30% of the sequencing reads) point mutations (Table 3). Of these 38 different mutations, three were present in two lineages each and one in three lineages; the remaining 34 mutations were present in only one lineage each. Each lineage had 3-8 mutations with >30% abundance.

[0265] Of the 38 prominent mutations from the stressed lineages, only three were in nonprotein-coding regions: namely, in the P gene-start signal (c2334a, the 5thnucleotide of the signal GGGGCAAAT, SEQ ID NO: 30, found in two lineages), the 5 untranslated region (UTR) of the P gene (a2344g), and the L gene-start signal (a8494g, the 4thnucleotide of the signal GGGACAAAAT, SEQ ID NO: 21). The remaining 35 mutations (92%) were in ORFs; of these, 25 (71%) were missense mutations, suggesting a selective pressure for aa change. Of these 25 missense mutations, four (16%) were in NS1, three (12%) in N. four (16%) in P, one (4%) in F. 11 (44%) in M2-1. one (4%) in M2-2 and one (4%) in L. Thus. Min AL lineages passaged under increasing temperature acquired missense mutations in theCPD NS1, N, P and L ORFs but also in the F. M2-1 and M2-2 ORFs which had not been subjected to CPD, with about half of the missense mutations found in M2-1 .After 18 passages of the two control lineages at the permissive temperature of 32 °C, only a single point mutation of >30% abundance was identified, occurring in one of the two lineages (Table 3, Ctl). This mutation was located in the P gene-start signal (a2337t at the 8hnucleotide of the signal GGGGCAAAT, SEQ ID NO: 20). Thus, Min AL was genetically stable at the permissive temperature.

[0266] Reintroduction of mutations identified at P14 of the in-vitro stress test (second passage at 38 °C into the Min AL backbone. The mutations responsible for the partial loss of the phenotype of Min AL during the in-vitro stress test (Table 3 [Mutations detected at a frequency of >30% in eight of the ten Min AL lineages at the end of passage 14 (2ndpassage at 38°C) as well as in each of the two controls at passage 18 (32 °C)]) were then identified. To do so. four lineages (#1, 3, 6 and 7, Table 3) were considered that replicated the most efficiently at P14 (titers >5.0 logio pfu / ml) and thus might be expected to contain mutations with the strongest compensatory effects on the ts phenotype and virus replication. Of these four lineages, lineage #6 contained six missense mutations, with none present at >45% of the reads, suggesting that this lineage contained mixed virus populations that might be difficult to study. Thus, this lineage was not evaluated further. Lineage #1 contained three prominent mutations that each was >50% of the reads, present in the NS1 ORF (I46M), the P gene-start signal (c2334a), and the M2-1 ORF (I87K). Lineage #3 contained eight prominent mutations, three of which were of >50% abundance: namely, one each in the P ORF (G26D), the M2-2 ORF (P4L), and the L gene-start signal (a8494g). Lineage #7 contained five prominent mutations, all of >90% abundance. Four of these were missense mutations in the NS 1 ORF (S5T), the N ORF (M50L), the M2-1 ORF (E70D), and the L ORF (N1249K), and the other was a silent mutation in the L ORF.Table 3‘ indicates that the amino acid mutation is not applicable for this particular mutation as the given mutation is localized in a non-translated region.*Percentage of reads with the indicated mutation; only mutations present in >30% of the reads are shown.Missense mutations and mutations in non-translated regions present in >50% of the reads are bolded.Nucleotide numbering is based on RSV sequence KT992094.^Mutations involving a codon that had been changed as part of CPD of NS1, NS2, N, P, M, SH, or L^Mutations involving a nucleotide that had been changed as part of CPD ofNS l, NS2, N, P, M, SH or L.§Mutation involving a nucleotide that had been changed as part of CPD ofNSl, NS2, N, P, M, SH or L and that restored wt sequence.

[0267] Based on these observations, two derivatives of Min AL were made using mutations from lineage #1 (Figure 3 A, top): one derivative contained the mutation M2- 1 [I87K] alone (virus Min AL-M2-1[I87K]) and a second derivative contained this mutation plus two additional mutations, namely NS1 [I46M] and the P gene-start signal mutation (c2334a) (virus Min AL-M2[I87K]+2).

[0268] Three additional derivatives of Min AL were made using mutations from lineage #3 (Figure 3A, middle): one derivative contained the mutation P[G26D] alone (virus Min AL-P[G26D]); a second derivative contained this mutation plus two additional mutations, namely M2-2[P4L] and the L gene-start signal mutation (a8494g) (virus Min AL- P[G26D]+2); and a third derivative contained these three mutations combined with the M2- 1 [N88T] mutation (Mm AL-P[G26D]+3).

[0269] Two additional derivatives of Min AL were made using mutations from lineage #7 (Figure 3 A, botom): one derivative contained the mutation M2-1 [E70D] alone (virus Min AL-M2-l[E70D]), and a second derivative contained this mutation combined with three additional mutations, namely NS1[S5T], N[M50L] and L[N1249K] (Min AL-M2- l[E70D]+3).

[0270] All seven Min AL-derived viruses were readily rescued by reverse genetics and Sanger sequencing confirmed that the working virus stocks were free of any adventitious mutations.

[0271] Mutations identified in P14 of the in-vitro stress test increased multi-cycle Min AL replication in vitro. The effects of the introduced P14 mutations on the multicycle replication of Min AL at 32 °C and 37 °C were evaluated. Vero cells were infected with an MOI of 0.01 pfu / cell and virus replication was monitored for 10 to 12 days (Figures 3B-3C). Due to the large number of viruses to evaluate, two separate multi-cycle replication experiments were done. Min AL-derived viruses containing mutations from lineage #1 or #7 as well as the corresponding P14 of lineage #1 and #7 were evaluated in the same experiment (Figures 3B- C, left and right panels), while Min AL-derived viruses containing mutations from lineage #3 as well as P14 of lineage #3 were evaluated in a separate experiment (Figures 3B-C, center panels). Wt RSV and Min AL were included in both experiments. Cell monolayers were harvested by scraping, vortexing to release cell-associated virus, and clarification of medium supernatants by centrifugation for analysis.

[0272] At 32 °C (Figure 3B), wt RSV reached peak titers of 6.9-7. 1 logio pfu / ml on days 11-12 pi. Replication of Min AL was delayed and w as maximal on day 12 pi (6.2-6.4 logic pfu / ml). Min AL-M2-1 [I87K] from lineage #1 and all three Min AL-derived viruses from lineage #3 replicated to higher titers than Min AL, achieving peak titers similar to or surpassing those of wt RSV (6.9 to 7.5 logic pfu / ml). Min AL-M2-l[I87K]+2 and viruses from lineage #7 replicated to similar titers as Min AL (6.2 logic pfu / ml).

[0273] At 37 °C (Figure 3C), differences were much more pronounced. Wt RSV reached peak titers of 6.4 to 7.2 logic pfu / ml. In contrast, Min AL replicated poorly, reaching titers below 2.5 logic pfu / ml. The incremental reintroduction of the selected P14 mutations increased replication of Min AL incrementally in the case of lineages #3 and #7. The individual mutations M2-1[I87K], P[G26D], and M2-l [E70D] from lineage #1, #3 and #7. respectively, increased Min AL titers by 128-. 9-, and 54-fold, respectively. Reintroduction of the additional set of mutations from each lineage recovered Min AL replicationincrementally to P14 levels in the case of lineages #3 and #7. This showed that these mutations indeed were responsible for the partial loss of the ts phenotype of these Min AL lineages.

[0274] To determine whether the introduced P14 mutations reduced the temperature sensitivity of Min AL, their effect on the Tsn of Min AL was evaluated in a plaque assay on Vero cells at temperatures ranging from 32 °C to 40 °C (Table 4 [Temperature sensitivity of Min AL and derivatives on Vero cells]). This showed that, indeed, the incremental reintroduction of the P14 mutations from lineages #1 and #7 increased the TSH of Min AL from 36 °C to 39 °C, and the reintroduction of the P14 lineage #3 mutations increased the TSH to 40 °C, confirming the compensatory effect of the P 14 mutations on the ts phenotype of Min AL.Table 4aFor viruses with a ts phenotype, the shut-off temperatures (TSH) are underlined and listed in boldface at the right. See Table 3 for definition of TSH. The ts phenotype is defined as having a TSII of 40°C or less.

[0275] Single-cycle virus replication experiments to analyze the effects ofP14 mutations on viral RNA, protein synthesis, and viral yield by Min AL. The effect of the P 14 mutations on the expression of cell-associated viral RNA and proteins was evaluated, as well as singlecycle viral yield, by Min AL derivatives during single-cycle replication experiments at 37 °C (Figures 4 and 5), the temperature that had been shown in Figures 1C and 3C to have substantial differential effects on the replication of Min AL and its derivatives. To do so, quadruplicate wells of Vero cells per time point were infected with an MOI of 3 pfu / cell with wt RSV, Min AL, or the Min AL-derived viruses and incubated at 37 °C. Due to the low titer of Min AL M2-l[E70D]+3 stock (representing lineage #7), this virus was not included in these evaluations. At 24 and 48 hpi. one well was harvested for total cell-associated RNA in order to evaluate the accumulation of positive- and negative-sense viral RNA (Figure 4); at 48 hpi, one well was used to harvest cells and evaluate viral protein expression by flow cytometry (Figures 5A-B); at 48 hpi, one well was used to collect cell lysates and evaluate protein expression by Western blot analysis (Figure 5C); and at 24 and 48 hpi, one well was used to harvest cell suspensions that were vortexed. clarified, and evaluated by plaque assay to measure single-cycle viral yield (Figure 5D). The results are described in the next two sections.

[0276] Single-cycle virus replication: P14 mutations increased the accumulation of positive-sense viral RNAs by Min AL to wt RSV levels. From the single-cycle replication experiment described above (using Vero cells infected with an MOI of 3 pfu / cell at 37 °C) viral RNA was extracted from cell lysates at 24 and 48 hpi, and gene-specific RT-qPCR taqman assays were used to quantify the accumulation of positive-sense N, P, G, F, M2 and L RNA as well as negative-sense genomic RNA (Figure 4). Note that intracellular positivesense RSV RNA at these time points generally contains approximately 95% mRNA and 5% antigenomic RNA, and thus this assay was primarily for viral transcription. Because the nt sequences of the CPD and wt ORFs of the N, P, and L gene differed due to the CPD, different taqman assays had to be used to quantify’ CPD RNAs (Figure 4, hatched bars) and wt RNAs (solid bars), thus preventing direct comparison between Min AL and its derivatives with wt RSV based on these three genes. However, direct comparison between all viruses waspossible for the G, F, and M2 genes because they were unchanged in all viruses, as well as for the genomic RNA, which was quantified based on the M2 sequence that also was unchanged in all viruses.

[0277] Cells infected with Min AL at 37 °C had low levels of accumulation of the various positive-sense viral RNAs at 24 hpi (not shown), indicating that the Min AL polymerase complex was inefficient at this temperature. For the other viruses at 24 and 48 hpi, the level of accumulation of each RNA was normalized relative to the Min AL value at 24 hpi taken as 1.0 (Figure 4). This analysis showed that the incremental addition of P14 mutations from lineages #1, 3, or 7 to Min AL increased the expression of all of the evaluated CPD and wt RNAs. and the complete sets of major mutations from lineages #1 (Min AL-M2-l[I87K]+2) or #3 (Min AL-M2-l[G26D]+3) had the strongest effects. Indeed, at 48 hpi, the levels of positive sense G, F, and M2 RNA in cells infected with Min AL-M2-l[I87K]+2 (lineage #1) or Min AL-M2-l[G26D]+3 (lineage #3) were between 24- and 800-fold increase compared to Min AL at 24 hpi, and were comparable to wt RSV. This indicated that the sets of major P14 mutations increased the accumulation of positive-sense mRNAs by Min AL derivatives to levels that approached or equaled those of wt RSV at 24 and, especially, at 48 hpi. In case of lineage #7, only the effect of the M2-l[E70D] mutation to wt RSV was compared because of the low titer of the other Min AL derivative bearing lineage #7 mutations. Its addition to Min AL moderately increased the synthesis of the positive-sense RNAs. similarly to the increases observed after addition of M2-1[I87K], an M2-1 mutation identified in lineage #1.

[0278] The accumulation of cell-associated negative-sense genomic RNA, representative of RNA replication, was also evaluated. Accumulation of genomic RNA was undetectable between 24 and 48 hpi in cells infected with Min AL. Similarly to the positive-sense RNA, the incremental addition of the selected Pl 4 mutations from lineages #1 , 3, and 7 into Min AL increased the accumulation of negative-sense genomic RNA, and the complete sets of major mutations from lineage #1 or #3 had the strongest effect (30-fold increase compared to Min AL). However, the levels of genomic RNA in cells infected with Min AL-derived viruses remained 8- to 10-fold lower than in wt RSV-infected cells.

[0279] Single-cycle virus replication: P14 mutations increased Min A protein expression and viral yield. From the single-cycle virus replication experiments described above (using Vero cells infected with an MOI of 3 pfu / cell at 37 °C), the effects on Min AL of the P 14 mutations were also evaluated on cell-associated viral protein expression and single-cycle viral yield. Viral protein expression was evaluated at 48 hpi by flow cytometry (Figures 5A-B). Data in Figures 5A and B are derived from additional wells in the experiment described in Figure 4 as well as from two additional independent experiments (n=3 total).

[0280] For flow cytometric analysis, the infected Vero cells were harvested at 48 hpi, then fixed and stained under permeabilizing condition with a cocktail of fluochrome-labeled monoclonal antibodies to evaluate the expression of N, P. G, and F proteins. Gating and analysis was done on live single N+P+F+G+cells.

[0281] At 48 hpi, only 3% on average of the cells infected with Min AL were positive for all four proteins by flow cytometry (Figure 5A). Similarly to the viral gene expression, the incremental addition of P14 mutations from each lineage into Min AL increased the frequency of cells expressing all four viral proteins. The addition of the full set of selected P14 mutations from lineage #1 or #3 had the strongest effect, resulting in 51% to 63% of the cells expressing all four proteins. This remained lower than wt RSV, for which 97% of cells expressed all four viral proteins.

[0282] The median level of expression of N, P, G and F protein in N+P+G+F+cells at this 48-hpi time point was also determined (expressed as median fluorescence intensity; MFI) (Figure 5B). Since the levels of viral mRNAs in cells infected with Min AL-derived viruses bearing the complete set of lineage #1 or #3 mutations had been found to approach or equal the levels for wt RSV at 48 hpi (Figure 4). it was of particular interest to determine whether the levels of expression of the viral proteins similarly approached or equaled those of wt RSV. In general, at 48 hpi, the MFIs of all four viral proteins remained substantially lower in cells infected with Min AL or derivatives than in cells infected with wt RSV (note that in Figure 5B, the level of expression of Min AL is set at 1.0 and values for the Min AL derivatives and wt RSV are expressed as fold-increases). For example, the MFI of the RSV N protein was about 5-fold lower in Min AL-infected cells than in wt RSV-infected cells, and the addition of lineage #1, 3, or 7 P14 mutations to Min AL did not substantially increase the N MFI closer to wt RSV levels. The level of expression of the P protein was about two-fold increase in wt RSV-infected cells compared to cells infected with Min AL or derivatives. Interestingly, in cells infected with the Min AL M2-l [I87K]+2 virus, which included the c2334a mutation in the P gene start signal, P protein expression was further reduced to less than half of that of Min AL (Figure 5B). In the case of the G and F proteins, which w ere encoded by ORFs not modified by CPD, the incremental addition of mutations from each lineage into Min AL increased the protein expression in infected cells by about 2- to 2.5-fold for the viruses that contained all the mutations from lineage #1 or #3. However, G and Fexpression still remained lower compared to wt RSV infected cells that showed 5- to 7-fold higher G and F expression than Min AL-infected cells.

[0283] From the single-cycle replication experiment described above, additional wells were harvested at 48 hpi for Western blot analysis using monoclonal antibodies against the RSV F and P proteins (Figure 5C) and G (G data not shown,). G was only detected in Min AL P[G26D]+3 and wt RSV-infected cells and was predominantly detected as a diffuse band between 100 and 150 kDa, corresponding to the full-length, fully -glycosylated form. A few less-abundant bands (30-70 kDa) were also detected in wt RSV infected cells. These bands might represent intermediates with incomplete O-glycosylation or truncated versions of the G protein that are mostly present on virions released from infected Vero cells (Kwilas, et al.. J Virol., 83(20): 10710-8 (2009)). The analysis used secondary antibodies tagged with infrared fluorochromes, and blots are shown in Figure 5C. The blots were quantified by scanning, and the observed levels of P, F, and G proteins (G data not shown) were consistent with the flow cytometry results of Figures 5 A and B.

[0284] The evaluation of the expression level of RSV G (referenced in the previous paragraph) was completed as a follow^ up assay to the RSV F and P protein assay. Specifically, additional replicate Vero cell monolayers from the single-cycle infection experiment described in Figures 4A-4C (MOI of 3 pfu / cell, 37°C) were harvested at 48 hpi (one well per virus per time point) for analysis of viral protein expression by Western blot. Cell lysates were prepared and analyzed by Western blotting using an anti-RSV G monoclonal antibody (RSV 133) that detects the full-length as w ell as the truncated form of G (Haid, et al., J. Virol. 2015; 90(6): 3065-73 (2015)). The staining of tubulin was not included to avoid interference with the detection of possible sizes of RSV G. however the same amount of protein as in Figure 5C was used.

[0285] Single-cycle viral yields of Min AL and derivatives w ere evaluated by harvesting monolayers at 24 and 48 hpi by scraping, vortexing to release cell-associated virus, clarification of the supernatants by centrifugation, and analysis of the supernatants by immunoplaque assay. The results showed that the incremental addition of mutations from each lineage to Min AL increased virus replication, following a trend comparable to that of the accumulation of viral genomic RNA and protein expression. How ever, the viral titers of the Min AL derivatives were at least 10-fold less at both time points compared to wt RSV.

[0286] Min AL and derivatives are highly attenuated in hamsters. The replication, immunogenicity, and protective efficacy of Min AL and derivatives in hamsters wasevaluated next (see Figure 6A for the timeline of the experiment). Due to low titers of virus stocks, three Min AL derivatives were not included for analysis: Min AL P[G26D]+2 (lineage #3), Min AL M2-l[E70D] (lineage #7), and Min AL M2-l [E70D]+3 (lineage #7). Groups of 16 golden Syrian hamsters were inoculated IN with 6.0 logic pfu of the indicated virus. Virus replication in nasal turbinates (NT) and lungs was evaluated from eight hamsters per group on day 3 pi.

[0287] In the NT, wt RSV replicated efficiently to a geometric mean titer (GMT) of 4.7 logic pfu / g. Replication of Min AL was reduced by about 10-fold compared to wt RSV. Interestingly, the restriction of replication of Min AL increased with the incremental addition of the lineage #1 mutations. Specifically, introduction of mutation M2-1[I87K] alone (virus Min AL-M2[I87K]) reduced Min AL titers by about 3-fold and the further addition of the NS1[I46M] and P gene start (c2334a) mutations (virus Min AL-M2[I87K]+2) reduced Min AL replication by about 70-fold (p<0.01 compared to Min AL). The introduction of the lineage #3 mutations (viruses Min AL-P[G26D] and Min AL-P[G26D]+3) did not affect replication of Min AL in NT.

[0288] In the lungs, wt RSV replicated to a GMT of 3.9 logic pfu / g, as typically observed. No or baseline replication of Min AL, Min AL M2-1 [I87K] (lineage #1) and Min AL P[G26D] (lineage #3) was detected, showing that Min AL and derivatives were highly attenuated in the lung (p<0.0001 compared to wt RSV). However, the inclusion of further mutations from lineage #1 (Min AL M2-l[I87K]+2) and #3 (Min AL P[G26D]+3) partly rescued Min AL replication, with virus replication detectable in the lungs of 3 of 8 hamsters and 5 of 8 hamsters, respectively.

[0289] The expression of 13 inflammation-related genes by RT-qPCR at day 3 pi in the lungs of seven of the eight hamsters that were inoculated with Min AL or wt RSV was also evaluated (Figures 10A-10B). Data are expressed as fold-increase over the expression determined from two unimmunized hamsters. This showed that, although wt RSV replicated to approximately 3.9 logic pfu / g in the lungs on day 3 (Figure 5B), it induced only a moderate inflammatory response, as only the interferon-induced gene MX-2 and the pro-inflammatory cytokine IL-lp w ere increased in lung tissues (10- and 5-fold increase compared to unimmunized animals, respectively). Min AL did not replicate to detectable levels in the lungs, as shown in Figure 5B, and the level of expression of these two cytokines was lower in Min AL-inoculated animals (p<0.05 compared to wt RSV for the expression of MX-2;Figures 10A-10B).

[0290] Min AL and derivatives induce robust serum anti-RSV antibodies. The immunogenicity of Min AL and derivatives was evaluated (Figures 7 and 8). In the hamster experiment described above, sera were collected at day 27 pi from the eight remaining hamsters per group and the levels of IgG and IgA antibodies binding to the prefusion form of RSV F (pre F) or RSV G were determined by ELISA using recombinantly-expressed purified pre F and G proteins (Figure 7).

[0291] Surprisingly, despite the strong restriction in replication in hamsters, Min AL induced high titers of serum anti-pre F IgG and IgA that were comparable to those induced by wt RSV (Figure 7A). With the exception of the most attenuated Min AL-derived virus (Min AL M2-1 [I87K]+2). which induced the lowest titers of anti-pre F IgG and IgA antibodies, the other Min AL derivatives induced high titers of serum anti-pre F IgG and IgA that also were comparable to those induced by wt RSV. Min AL and derivatives also induced robust levels of serum anti-RSV G IgG and IgA antibodies, however the titers induced by Min AL were significantly lower than those induced by wt RSV (p<0.01 and p<0.001 for IgG and IgA, respectively, Figure 7B). Among the Min AL derivatives, Min AL M2-1 [I87K] and Min AL P[G26D]+3 induced higher titers of serum anti-RSV G IgG and IgA than Min AL, whereas the titers induced by Min AL P[G26D]+3 were not significantly different than those induced by wt RSV.

[0292] The serum 60%-plaque-reduction RS V-neutralizing antibody titers (PRNTeo) induced by the viruses was also determined (Figure 7C). Min AL and derivatives induced robust mean PRNTeo ranging from 7.2 to 9.3 log2 (for Min AL M2-1 [I87K]+2 and Min AL P[G26D]+3, respectively) albeit these titers were significantly lower than those induced by wt RSV (10.8 log2: p<0.05 to p<0.0001).

[0293] Min AL and derivatives induced robust mucosal anti-RSV antibodies and were fully protective against wt RSV challenge. In the hamster experiment described above, the remaining hamsters were challenged on day 32 with 6.0 logio pfu of wt RSV (Figure 6A) and, at day 3 post challenge (pc), all animals were necropsied. Bronchoalveolar lavages (BAL) were collected to evaluate the levels of anti-RSV pre F and G IgG and IgA binding antibodies in the lower airways (Figures 8A and 8B). In addition, NT and lung tissues were collected, homogenized, and subjected to immunoplaque assay to determine the level of replication of challenge wt RSV (Figure 8C).

[0294] Remarkably, although the replication of Min AL and derivatives had been low or undetectable in the lungs of immunized hamsters, as described above, these viruses (with theexception of the most attenuated Min AL M2-1 [I87K]+2 virus) induced robust titers of anti- RSV pre-F IgG and IgA in the lungs that were not significantly different than to those induced by wt RSV (Figure 8A). Similarly to the serum, Min AL and derivatives induced detectable but lower levels of anti-G IgG and IgA than wt RSV (Figure 8B). As expected, no anti-RSV pre-F and G IgG and IgA antibodies were detected in the lungs of control mock- immunized hamsters that were challenged with wt RSV (Figures 8A and 8B).

[0295] The protective efficacy of Min AL and derivatives against wt RSV challenge at day 3 pc was evaluated next (Figure 8C). Wt RSV replicated efficiently in the NT and lung tissues of hamsters from the mock-immunized group. However, with the exception of Min AL M2-l[I87K]+2, the most-attenuated virus. Min AL and the other derivatives were fully protective against detectable wt RSV challenge replication in NT and lung tissues.

[0296] To confirm that Min AL-immunized hamsters were protected against inflammatory responses after challenge, the expression of 13 inflammation-related genes by RT-qPCR on day 3 pc were also evaluated. Lungs from 7 mock-immunized animals with robust RSV challenge virus replication and 7 Min AL-immunized animals (no detectable challenge virus replication in this group) were selected. Data were expressed as fold-increase over the expression determined from two unimmunized unchallenged hamsters (Liu, et al., PLoS Pathog., 19(6): el011057 (2023)). Similarly to the results from Figures 10A-10B. only the interferon-inducible gene Mx-2 and the pro-inflammatory cytokine gene IL-1 [3 were increased in mock-immunized animals on day 3 after RSV challenge (9- and 3-fold increase compared to unimmunized unchallenged animals, respectively; p<0.001 and p<0.01 for Mx-2 and IL-1(3 (Figures 11A-B), indicative of only a moderate inflammatory response in mock- immunized animals after RSV challenge. However, in Min AL-immunized animals, the level of expression of the 13 inflammation related genes after wt RSV challenge was comparable to unimmunized unchallenged animals, indicating that Min AL was protective against RSV challenge, with no sign of inflammation after challenge.

[0297] Min AL-derived viruses are genetically and phenotypically stable in vitro.Finally, genetic stability was evaluated, and the stability of the temperature-sensitivity phenotype, of the four Min AL-derivatives that had been evaluated in the hamster experiment described above by subjecting them to an in vitro stress test (Figure 9). Five 25-cm2flasks of Vero cells per virus were inoculated using an initial MOI of 0.1 pfu / cell. Three lineages per virus (in blue) were passaged four times in parallel at a temperature that was one degree below the virus TSH (indicated in Figure 9). The incubation temperature was increased byone °C (to the virus TSH) and four additional passages were made, for a total of eight passages, corresponding to approximately two months of continuous culture. The other two control lineages (in black) were passaged 8 times in parallel at the permissive temperature of 32 °C.

[0298] All four Min AL-derived viruses replicated efficiently at the 32 °C control temperature, reaching final titers at the end of P8 of between 4.7 and 7.4 logio pfu / ml (Figure 9). Whole-genome Sanger sequencing using viral RNA isolated from supernatants harvested at P8 from all lineages at 32 °C showed that none of the Min AL derivatives accumulated prominent mutations.

[0299] For the lineages passaged at increasing temperature (Figure 9), the Min AL M2- 1 [I87K] lineages replicated efficiently at one degree below its TSH (37 °C), reaching 7.0-7.3 logio pfu / ml at P4 (upper left panel). The titers subsequently decreased during the four passages at the virus TSH. Whole-genome Sanger sequencing of viral RNA isolated from supernatants harvested at P7 for one lineage and at P8 for the two other lineages did not reveal any prominent mutations. The three other Min AL-derivatives, (Min AL-M2- l[I87K]+2, Figure 9, upper right panel; MinAL P[G26D], lower left panel; and Min AL P[G26D]+3, lower right panel), exhibited strongly reduced replication when passaged at one degree below their respective TSH and were completely restricted at their respective TSH, indicating that they did not acquire mutations that rescued replication. Overall this experiment indicated that Min AL derivatives were genetically and phenotypically stable when serially passaged at the permissive temperature of 32 °C and at their respective TSH.

[0300] CPD RSV vaccine candidate, Min AL. w as created which contained the CPD NS 1. NS2, N, P, M, SH, and L ORFs of Min A combined w ith the CPD L ORF of Min L, while the G, F and M2 ORFs were kept unchanged. Min AL replicated efficiently at the permissive temperature of 32 °C in Vero cells and w as genetically stable when serially passaged at this temperature, which is an important consideration for vaccine manufacture. Similar to previous RSV derivatives containing CPD ORFs (Le Nouen. et al., PNAS USA, 111(36): 13169-13174 (2014)), Min AL w as temperature sensitive: the TSH of Min AL w as 36 °C, which is lower than that of Min A (40° C) or Min L (37°C), suggesting that Min AL had contributions to temperature-sensitivity from both parents. When serially passaged at increasing temperatures in an in-vitro stress test, Min AL retained greater temperaturesensitivity compared to Min A and Min L: none of the Min AL lineages had any detectable replication at 40 °C, a temperature at which the replication of stressed Min A lineages wasbarely affected and the replication of most of the Min L stressed lineages was reduced but still substantial for at least one passage (Chen, et al., PI. S Pathog., 17(12): el 010191 (2021 ); Le Nouen, et al., PNAS USA, 114(3): E386-E95 (2017)). Whole-genome deep sequencing of lineages of Min AL from P14 of the in-vitro stress test revealed that, under temperature stress, Min AL acquired a diverse array of 38 different prominent point mutations (>30% abundance. Table 3) occurring in four CPD genes (NS1, N, P, L) and four wt genes (G, F, M2-1, and M2-2). The mutations were predominantly missense mutations (27 of 38). Only a few mutations were found in more than one lineage, and thus the set of P14 mutations for each lineage were mostly unique. In previous studies, sequence analysis of passage levels of stress tests of Min L and Min A similarly identified many different mutations in both CPD and wt ORFs (Chen, et al., PLoS Pathog., 17(12): el010191 (2021); Le Nouen, et al., PNAS USA, 114(3): E386-E95 (2017)). Despite the apparent diversity, the P14 mutations of the present study had a common feature: they mostly involved genes involved in viral RNA synthesis. Most of the missense mutations (25 of 27) were present in the genes encoding proteins of the polymerase / nucleocapsid complex. Four other missense mutations were in NS 1, which previously was suggested by minigenome experiments to strongly affect RNA synthesis (Atreya et al., J. Virol., 1998;72(2): 1452-1461 (1998)). Thus, only one of the 27 missense mutation was in a protein without a proposed role in RNA synthesis, namely a single mutation in F (S425T).

[0301] The greatest numbers of missense mutations were in P and M2-1, with four and 11 mutations, respectively. Five of the eight stressed Min AL lineages sequenced at P14 (lineages #3, 5, 6, 8, and 10, Table 3) contained one or more of the four P missense mutations. All four of these P mutations were present in an N-terminal segment comprising aa 19-31 inclusive. Interestingly, missense mutations in this region of P were previously identified in stressed lineages of Min A and w ere found to rescue its replication at 40 °C (Chen, et al., PLoS Pathog., 17(12): el010191 (2021)). Each of the eight stressed Min AL lineages sequenced at P14 in the present study also contained one or more of the 11 M2-1 mutations. All 11 of these M2-1 mutations were present in the N-terminal half of the protein, between aa 14 and 98, inclusive. These included mutations that were identical (A73S) or very similar (N88T, N88S) to mutations in M2-1 ([A73S] and [N88K]) that were identified previously in all 10 lineages in the stress test of Min L and were shown to rescue Min L replication at temperatures up to 40 °C (Le Nouen, et al., PNAS USA. 1 14(3): E386-E95 (2017)).

[0302] In previous molecular-dynamic simulations, mutations in the N-terminal domain of P were predicted to increase its flexibility, which might facilitate the interactions of P with N, resulting in increased efficiency of the RSV polymerase complex (Chen, et al., PLoS Pathog., 17(12): el010191 (2021)). Also, previous molecular dynamic simulations of the [A73S] and [N88K] mutations identified in the M2-1 protein of stressed Min L lineages suggested that they increased the stability within or between M2-1 monomers, which also might increase the efficiency of the RSV polymerase complex (Le Nouen, et al., PNAS USA, 114(3): E386-E95 (2017)). It was hypothesized that P14 mutations in polymerase components increase the activity of the polymerase on a per-molecule basis, and were selected during the in-vitro stress test because they partly compensate for reduced viral gene expression due to CPD.

[0303] Three P14 mutations from the eight stressed lineages were found outside of ORFs. Of these, two were found in transcription gene-start signals. One mutation was in the P genestart signal (c2334a at the 5thnucleotide of the signal GGGGCAAAT, lineages #1 and 9); in a previous study, saturation mutagenesis of a gene-start signal in a minigenome showed that this mutation reduced transcription to 57% (Kuo, et al., J. Virol., 71(7): 4944-4953 (1997)). The second mutation was in the L gene-start signal (a8494g at the 4thnucleotide of the signal GGGACAAAAT, lineage #3): the previous minigenome experiment indicated that A or G should be essentially equivalent at this position (Kuo et al.. J. Virol., 71(7): 4944-4953 (1997)). The sole mutation identified in one of the control lineages also involved a mutation in the P gene-start signal (a2337t at the 8thnucleotide of the signal GGGGCAAAT); in the previous minigenome experiment this mutation reduced mRNA synthesis by about 90% (Kuo, et al., J.4944-4953 (1997)). Thus, for four of the five lineages with a point mutation in non-ORF sequence, the mutation was in a transcription gene-start signal, and was in the P or L gene that encodes polymerase components. Since these mutations occur in transcription signals, and indeed in signals controlling transcription of two key components of the polymerase / nucleocapsid complex, they might serve to alter expression of these components in order to compensate for CPD. The reduced activity predicted for the two mutations in the P gene-start signal would be contrary to the idea that they cause increased expression of polymerase components. However, since the polymerase components in the Min AL derivatives also acquired missense mutations, the effects of the non-ORF mutations in the gene-start signals also may be different than the wt situation.

[0304] A functional analysis of some the prominent missense mutations was performed, as well as two gene-start mutations, from P14 of lineages #1, 3, and 7, by introducing them in various combinations into the Min AL backbone and evaluating effects on temperaturesensitivity, viral transcription, viral protein expression, and virus replication. Three Min AL derivatives were made by the reintroduction of a single M2-1 or P mutation into Min AL. Further derivatives were made by the further addition of two or three additional mutations from the respective lineages. The reintroduction of single M2-1 or P mutations into Min AL resulted in a partial rescue of replication at 37 °C. When additional mutations from the respective lineages were added, in the case of lineages #3 and #7. virus replication at 37 °C was increased to the same level as the respective P14 pools from the in-vitro stress test. This showed that these sets of mutations indeed were responsible for rescuing Min AL replication at increased temperatures, and that the largest sets of reintroduced mutations from lineage #3 and #7 gave full rescue to P14 levels.

[0305] The reintroduction of single M2-1 or P mutations into Min AL resulted in a partial rescue of expression of positive-sense RNA at 37 °C. The addition of further P14 mutations increased the accumulation of positive-sense RNAs to levels approaching or equaling that of wt RSV at 24 hpi and, in particular, 48 hpi. This supports the idea that these mutations increased the activity of the viral polymerase for the expression of positive-sense RNA, which mainly is mRNA. Since the M2-1 protein is generally known to function in transcription and not RNA replication, mutations in M2-1 in particular seem likely candidates to be involved in increasing the expression of positive-sense RNA.

[0306] In response to the incremental addition of P14 mutations to Min AL, the accumulation of cell-associated viral proteins at 48 hpi was increased but remained substantially below that of wt RSV. Inefficient translation of CPD ORFs is one likely factor. Instability of CPD mRNAs is another potential factor, although the stabilities of CPD and non-CPD versions of viral genes could not be directly compared because the sequence differences necessitated different primers and probes. In addition, Min AL M2-1 [I87K]+2 (lineage #1) had a substantial reduction in accumulation of the P protein. This virus contained the c2334a mutation in the P gene-start signal, which reduced mRNA synthesis by 50% in a minigenome study (Kuo, et al., J. Virol., 71(7): 4944-4953 (1997)) and thus might be responsible for the observed reduction in P protein expression. Although a corresponding reduction in positive-sense P RNA was not observed by RT-QPCR, this might reflect inefficient detection of P mRNA against the background of readthrough mRNAs andantigenome, or might indicate that the mutation acted at the level of translation. Another factor in the lesser accumulation of viral proteins is that the increase in viral mRNAs to levels comparable to wt RSV was more evident at 48 hpi than 24 hpi, and increases in the accumulation of viral proteins would be expected to lag temporally behind that of mRNAs and thus could be proportionately lower at 48 hpi. It also is possible that, due to their increased contents of CpG and UpA, Min AL and its derivatives induced increased innate immune reactions in vitro that caused a global reduction in the synthesis of viral proteins. While increased innate responses were not observed for Min AL in vivo in the hamster experiment, the situation could be different for the Min AL derivatives in vitro.

[0307] The accumulation of negative-sense genomic RNA was increased by addition of the P14 mutations, with the full sets of mutations being more effective than the single M2-1 and P mutations. However, the level of accumulation of genomic RNA remained lower than that of wt RSV. This is not surprising, since RNA replication by non-segmented negativestrand RNA viruses depends on concurrent viral protein synthesis, which was reduced as discussed above. The production of infectious virus during single-cycle replication similarly was increased by the single multiple P14 mutations, and even more by multiple P14 mutations, but remained below the levels of wt RSV. This likely reflects the reduced accumulation of viral proteins and genomic RNA by the Min AL derivatives compared to wt RSV.

[0308] When inoculated IN into hamsters, Min AL replicated in the NT to levels comparable to those previously observed with Min A and Min L (Chen, et al., PLoS Pathog., 17(12): el010191 (2021); Le Nouen, et al., PNAS USA, 114(3): E386-E95 (2017)).Surprisingly, the addition to Min AL of the P 14 mutations, which had partially rescued replication of Min AL in vitro at 37 °C, did not have any effect on replication in the NT (lineage #3 mutations) or caused a decrease (lineage #1 mutations which included the mutation in the P gene-start signal). In the lungs, Min AL did not replicate to detectable levels, while in previous studies Min A and Min L replicated at low levels (Chen, et al., PLoS Pathog., 17(12): el010191 (2021); Le Nouen, et al., PNAS USA, 114(3): E386-E95 (2017)). The more attenuated phenotype of Min AL compared to Min A and Min L in the lungs likely reflects, at least in part, the increased temperature sensitivity' of Min AL compared to its tw o parents (the body temperature of the Syrian hamster is 37 °C). Increased temperature sensitivity is thought to preferentially restrict viral replication in the lower respiratory tract due to its higher temperature compared to the upper respiratory tract, and thereby is thoughtto provide additional safety in the lungs of infants. The greater attenuation of Min AL due to its increased temperature sensitivity compared to Min A and Min L is also supported by the increase in replication in the lungs by the addition of mutations that increase the Tsu, such as in Min AL M2-l[I87K]+2 and Min AL P[G26D]+3, for which the TSH was increased by 3 °C and 4 °C to 39 °C and 40 °C, respectively, compared to Min AL.

[0309] Despite their high level of attenuation, Min AL and derivatives induced robust systemic and mucosal anti-pre F and anti-G IgG and IgA antibodies in hamsters.Surprisingly, with the exception of Min AL M2-l [I87K]+2 that contained the P gene-start mutation, the levels of anti-pre F antibodies in both the serum and BAL of hamsters infected with Min AL or its derivatives were comparable to those induced by wt RSV. However, the levels of anti-G antibodies in both the serum and BAL were lower than those induced by wt RSV. The reason behind the differences between the levels of anti-pre F and anti-G antibodies is unknown and remains to be determined. The levels of serum anti-pre F and anti-G antibodies correlated well with the robust anti-RSV serum antibody titers induced by Min AL and derivatives. Despite their greatly reduced levels of replication in hamsters, especially in the lungs, the systemic and mucosal antibody titers induced by Min AL derivatives were just slightly low er than those induced by wt RSV, suggesting that in some cases, the immunogenicity of the Min AL variants was stronger than expected based on their level of replication. One could speculate that the increase in CpG and UpA resulting from CPD may be immunostimulatory, compensating for some loss of immunogenicity due to the low' level of replication of Min AL variants. The cytokine response in the lungs of hamsters inoculated with these candidates w as also evaluated, however, cytokine induction in the lungs of the Min AL infected hamsters were not detected, likely due to the complete restriction of Min AL replication in the lower airways. Furthermore, with the exception of Min AL M2- l[I87K]+2 that w as over-attenuated, Min AL and its derivatives w ere fully protective against wt RSV challenge in both the upper and lower airways.

[0310] Amino acids may be referred to herein by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0311] The terms “about” and “around,” as used herein to modify a numerical value, indicate a close range surrounding the numerical value. Thus, if “X” is the value, “about X” or “around X” indicates a value of from 0.9X to 1.1X, e.g., from 0.95X to 1.05X or from0.99X to 1.0 IX. A reference to “about X” or “around X” specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1 .03X, 1 .04X, and 1.05X. Accordingly, “about X” and “around X” are intended to teach and provide written description support for a claim limitation of, e.g., “0.98X.”

[0312] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0313] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly- contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0314] Preferred embodiments or aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments or aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modificationsand equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIMS:

1. A polynucleotide encoding a recombinant respiratory syncy tial virus (RSV) variant having an attenuated phenotype comprising a modified RSV genome or antigenome, wherein NS1, NS2, N, P, M. SH, and L ORFs of the modified RSV genome or antigenome are codon-pair deoptimized.

2. The polynucleotide of claim 1. wherein each of the NS 1, NS2, N. P, M. SH, and L ORFs of the modified RSV genome or antigenome polynucleotide independently comprise a codon pair bias (CPB) score reduction of at least 0.01.

3. The polynucleotide of claim 1 or 2, wherein each of the NS1, NS2, N, P, M, SH, and L ORFs of the modified RSV genome or antigenome polynucleotide independently comprise a CPB score of at least -0.01.

4. The polynucleotide of any one of claims 1-3, wherein the nucleotide sequence of the modified RSV genome or antigenome encoding one or more of RSV proteins NS1, NS2, N, P, M, SH, and L has about 70% to about 95% identity with the nucleotide sequence of the parental RSV genome or antigenome encoding the same one or more of RSV proteins NS 1, NS2, N, P, M, SH, and L.

5. The polynucleotide of any one of claims 1-4, wherein the nucleotide sequence of the modified RSV genome or antigenome encoding RSV NS1 protein has at least about 95% identity to SEQ ID NO: 1.

6. The polynucleotide of any one of claims 1-5, wherein the nucleotide sequence of the modified RSV genome or antigenome encoding RSV NS2 protein has at least about 95% identity to SEQ ID NO: 2.

7. The polynucleotide of any one of claims 1-6, wherein the nucleotide sequence of the modified RSV genome or antigenome encoding RSV N protein has at least about 95% identity to SEQ ID NO: 3.

8. The polynucleotide of any one of claims 1-7, wherein the nucleotide sequence of the modified RSV genome or antigenome encoding RSV P protein has at least about 95% identity to SEQ ID NO: 4.

9. The polynucleotide of any one of claims 1-8, wherein the nucleotide sequence of the modified RSV genome or antigenome encoding RSV M protein has at least about 95% identity to SEQ ID NO: 5.

10. The polynucleotide of any one of claims 1-9, wherein the nucleotide sequence of the modified RSV genome or antigenome encoding RSV SH protein has at least about 95% identity to SEQ ID NO: 6.

11. The polynucleotide of any one of claims 1-10, wherein the nucleotide sequence of the modified RSV genome or antigenome encoding RSV L protein has at least about 95% identity to SEQ ID NO: 11.

12. The polynucleotide of any one of claims 1-11, wherein the amino acid sequence of the one or more of RSV proteins NS1, NS2, N, P, M, M2-1, M2-2, SH, and L encoded by the nucleotide sequence of the modified RSV genome or antigenome is at least 99% identical to the amino acid sequence of the same one or more of RSV proteins NS1. NS2, N, P, M, M2-1. M2-2, SH, and L encoded by the nucleotide sequence of the parental RSV genome or antigenome.

13. The polynucleotide of claim 12, wherein the RSV M2-1 protein has an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 at at least position 87.

14. The polynucleotide of claim 13, wherein the residue at position 87 of SEQ ID NO: 25 is lysine.

15. The polynucleotide of any one of claims 12-14, wherein a residue at position 46 of RSV NS1 protein SEQ ID NO: 22 is methionine, and the 5thnucleotide in the RSV P gene start signal SEQ ID NO: 20 is changed to an adenosine residue.

16. The polynucleotide of any one of claims 13-15, wherein the RSV P protein has an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 24 at at least position 26.

17. The polynucleotide of claim 16, wherein the residue at position 26 of SEQ ID NO: 24 is aspartic acid.

18. The polynucleotide of any one of claims 13-17, wherein the residue at position 4 of the RSV M2-2 protein SEQ ID NO: 26 is a leucine, and the 4thnucleotide in SEQ ID NO: 21 is changed to a guanosine residue.

19. The polynucleotide of any one of claims 13-18, wherein the residue at position 4 of the RSV M2-2 protein SEQ ID NO: 26 is a leucine, residue at position 88 of the M2-1 SEQ ID NO: 25 protein is a threonine residue, and the 4thnucleotide in SEQ ID NO: 21 is changed to a guanosine residue.

20. The polynucleotide of any one of claims 1-19, wherein the polynucleotide comprising a modified RSV genome or antigenome encodes:(a) a mutant RSV NS 1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 22 at position 5, optionally wherein the residue at position 5 of the amino acid sequence is threonine;(b) a mutant RSV N protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 23 at position 50, optionally wherein the residue at position 50 of the amino acid sequence is leucine;(c) a mutant RSV M2-1 protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 25 at position 70, optionally wherein the residue at position 70 of the amino acid sequence is aspartic acid;(d) a mutant RSV L protein with an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 29 at position 1,249, optionally wherein the residue at position 1.249 of the amino acid sequence is lysine; or(e) any combination of (a)-(d). and optionally wherein the ORFs encoded by NS1, NS2, N, P, M, SH, and L genes of the modified RSV genome or antigenome are each CPD.

21. A recombinant RSV variant comprising the isolated polynucleotide of any of claims 1-20.

22. A pharmaceutical composition comprising the recombinant RSV variant of claim 21 and at least one excipient.

23. A multivalent RSV vaccine composition comprising a recombinant RSV variant of claim 21, a second recombinant RSV variant of claim 22, and, optionally, one ormore additional recombinant RSV variants of claim 21. wherein the first, second, and optional additional recombinant RSV variants have different nucleotide sequences.

24. A multivalent vaccine composition comprising at least one recombinant RSV variant of claim 21 and at least one antigen from a non-RSV virus.

25. The multivalent vaccine composition of claim 24, wherein the at least one antigen from a non-RSV virus is a parainfluenza viruses (PIV).

26. The multivalent vaccine composition of claim 24 or 25, further comprising an antigen from human metapneumovirus (HMPV).

27. A method of vaccinating an animal, comprising administering the pharmaceutical composition of claim 22 or the multivalent vaccine composition of claims 23- 26 to an animal.

28. A method of inducing an immune response in an animal, comprising administering the recombinant RSV variant of claim 21, the pharmaceutical composition of claim 22, or the multivalent RSV vaccine composition of claims 23-26 to an animal.

29. The method of claim 27 or 28, wherein the recombinant RSV variant does not increase the level of cytokine protein expression in the animal.

30. The method of any one of claims 27-29, wherein the recombinant RSV variant is administered via injection, nasal spray, nasal droplets, topical application, aerosol delivery, or oral inoculation.

31. The method of any one of claims 27-30, wherein the recombinant RSV variant has a shutoff temperature for plaque formation of 37 °C or above.

32. The method of any one of claims 27-31, wherein the animal is a mammal.

33. The method of any one of claims 27-31, wherein the animal is a human.

34. A method of producing a recombinant RSV variant vaccine, comprising expressing the polynucleotide of any of claims 1-20 in a cell.

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