Vaccine candidate for human respiratory multinuclear virus (RSV) with attenuated phenotype

Abstract

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

Description

Technical Field

[0001] Cross-reference of related applications This application claims priority based on U.S. Provisional Application No. 62 / 399,133 filed on September 23, 2016 and U.S. Provisional Application No. 62 / 400,476 filed on September 27, 2016, both of which are hereby incorporated by reference in their entirety for all purposes.

[0002] Reference to sequence listings This application includes a Sequence Listing filed as an electronic text file named "Sequence_Listing_6137NIAID-65-PCT_ST25.txt" having a size of 98 kb and created on September 20, 2017. The information contained in the electronic file is hereby incorporated by reference in its entirety in accordance with 37 CFR §1.52(e)(5).

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

[0004] Field of Invention The subject matter disclosed herein is a respiratory syncytial virus (RSV) and its attenuated, mutant strains suitable for use as a vaccine.

Background Art

[0005] Background of the Invention Human respiratory multinuclear virus (RSV) infects almost everyone worldwide during infancy and is a major cause of mortality and morbidity (see Collins and Graham, 2008, J Virol. 82:2040-2055; Collins and Melero, 2011, Virus Res 162: 80-99; Collins and Karron, 2013, Fields Virology 6th Edition, pp 1086-1123; Collins, et al., 2013, Curr Top Microbiol Immunol 372:3-38 for a general review). In the United States alone, RSV accounts for 75,000 to 125,000 hospitalizations annually, and even conservative estimates suggest that RSV causes 64 million childhood infections and more than 160,000 childhood deaths worldwide each year. Other notable features of RSV include a severe infection in infancy that can lead to persistent airway dysfunction, including a predisposition to airway reactivity, which in some individuals can often last for several years and even into adolescence. RSV infection can exacerbate asthma and may be involved in asthma triggering.

[0006] RSV is a negative-sense RNA virus belonging to the Pneumovirus family. The RSV genome is a single, negative-sense strand of 15.2 kilobase RNA, which is transcribed into 10 mRNAs by viral polymerase via a sequential start-stop mechanism that initiates a single viral promoter at the 3' end of the genome. Each mRNA encodes one major protein, except for the M2 mRNA, which has two duplicated open reading frames (ORFs) encoding two separate proteins, M2-1 and M2-2. The 11 RSV proteins are RNA-binding nucleoprotein (N), phosphoprotein (P), macropolymerase protein (L), adherent glycoprotein (G), fusion protein (F), small hydrophobic (SH) surface glycoprotein, internal matrix protein (M), two non-structural proteins NS1 and NS2, and M2-1 and M2-2 proteins. The RSV gene order is 3'-NS1-NS2-NPM-SH-GF-M2-L. Each gene has a gene start (GS) signal located at its upstream end, which is involved in the transcription of that gene, and a short-conserved transcription signal called a gene stop (GE) signal located at its downstream end, which is involved in the synthesis of the poly(A) tail and subsequent mRNA release.

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

[0008] Vaccines and antiviral agents against RSV are in preclinical and clinical development by numerous researchers; however, no vaccine or antiviral agent for routine use against RSV is commercially available.

[0009] The development of RSV vaccines has been ongoing since the 1960s, but is complicated by numerous factors. For example, immunization of RSV-unsensitized infants with inactivated RSV triggers subsequent disease exacerbations due to spontaneous RSV infection, and studies in laboratory animals show that disease exacerbations are also associated with purified RSV subunit vaccines. However, RSV disease exacerbations have not been observed in association with live or live-vectored RSV vaccines, a crucial observation confirmed in numerous clinical trials (Wright, et al., 2007, Vaccine 25:7372-7378). Therefore, inactivated and subunit vaccines are contraindicated in infants and young children, while appropriately attenuated live and live-vectored vaccines are acceptable for use in this population, which is the primary vaccine target population.

[0010] Another impairment to immunoprotection is that RSV replicates in superficial cells of the respiratory tract lumen, where immunoprotection is reduced, leading to disease. Therefore, immune control of RSV infection is inefficient and often incomplete, making it crucial that RSV vaccines be as immunogenic as possible. Another impairment to RSV vaccines is that the strength of the protective immune response is roughly proportional to the degree of viral replication (and antigen production). Therefore, attenuation of RSV necessary to produce a viable vaccine generally involves a decrease in replication and antigen synthesis, and a simultaneous decrease in immunogenicity; thus, it is essential to identify a replication level that is sufficiently immunogenic while exhibiting good tolerability.

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

[0012] Another obstacle is the difficulty in identifying and developing attenuating mutations. While the appropriate mutation must be attenuating in vivo, in vitro replication should be minimally restricted, as this is essential for efficient vaccine production. Yet another obstacle is the genetic instability inherent in RNA viruses, which allows attenuating mutations to revert to wild-type (wt) assignments or other assignments conferring a non-attenuating phenotype.

[0013] A combined approach of sequence design and synthetic biology enables the production of DNA molecules with broad targeted modifications. Synonymous genome recoding, in which one or more ORFs of pathogenic microorganisms are modified at the nucleotide level without affecting amino acid encoding, is now being extensively evaluated, particularly for RNA viruses, for reducing pathogen fitness and producing promising viable attenuated vaccines. The main strategies for attenuation by synonymous genome recoding are codon de-optimization (CD), codon-pair de-optimization (CPD), and increased dinucleotide CpG and UpA content (which are usually consequences of CD and CPD).

[0014] Deoptimized viral genomes contain tens to thousands of silent nucleotide mutations in one or more ORFs. Presumably, attenuation is the sum of numerous individual mutations. This mutational diversity is predicted to confer substantial stability toward deattenuation, as the numerous mutations present a significant barrier to reverting to pathogenicity. In principle, against the backdrop of thousands of attenuating mutations, any single-site revert offers only a very small selective advantage. Under this model, the most likely path toward reverting is the gradual accumulation of numerous individual mutations, providing a slow progression toward deattenuation.

[0015] To date, large-scale genetic stability studies of deoptimized viruses have shown that deattenuation appears to be actually low, suggesting that these viruses are genetically stable. However, a significant limitation of these studies is that deoptimized viruses are not subjected to the strong selective pressures that generally favor the proliferation of viruses with deattenuating mutations. [Overview of the project] [Problems that the invention aims to solve]

[0016] Therefore, there is a continuing demand for viable, attenuated RSV strains that replicate efficiently in vitro, are maximally immunogenic, are attenuated, and are resistant to in vivo deattenuation. [Means for solving the problem]

[0017] Summary of the Invention Disclosed herein are in vitro presumatively de-attenuating mutations useful, alone or in combination with other known mutations, for the production of recombinant human respiratory multinuclear virus (RSV) strains exhibiting an attenuated phenotype in vivo. Also disclosed herein are novel viable attenuated RSV strains suitable for use as RSV vaccines. Furthermore, methods and compositions related to the expression of the disclosed virus are provided. For example, isolated polynucleotide molecules containing nucleic acid sequences encoding the genome or antigenome of the described virus are disclosed. The present invention may provide the following embodiments. [Section 1] An isolated polynucleotide molecule encoding a recombinant RSV variant having an attenuated phenotype, comprising a respiratory multinuclear virus (RSV) genome or antigenome sequence, wherein the RSV genome or antigenome is modified by a mutation at the position corresponding to T1166 of SEQ ID NO: 11 in the L ORF of the L protein. [Section 2] RSV genome or antigenome i. Mutations in the M2-1 ORF of the M2-1 protein at positions corresponding to N88 or A73 of SEQ ID NO: 9 ii. Mutation at the position corresponding to K136 of SEQ ID NO: 3 in the N ORF of the N protein; iii. Mutations in the P ORF of the P protein at the position corresponding to E114 of SEQ ID NO: 4; and iv. These combinations An isolated polynucleotide molecule as described in item 1, further modified by mutations selected from the group consisting of the following. [Section 3] RSV genome or antigenome i. Mutation in the M2-1 ORF of the M2-1 protein at the position corresponding to N88 of SEQ ID NO: 9; ii. Mutation at the position corresponding to K136 of SEQ ID NO: 3 in the N ORF of the N protein; iii. Mutations in the P ORF of the P protein at the position corresponding to E114 of SEQ ID NO: 4; and iv. These combinations An isolated polynucleotide molecule as described in item 2, further modified by selected mutations from the above. [Section 4] RSV genome or antigenome i. Mutation in the M2-1 ORF of the M2-1 protein at the position corresponding to A73 of SEQ ID NO: 9; ii. Mutation at the position corresponding to K136 of SEQ ID NO: 3 in the N ORF of the N protein; iii. Mutations in the P ORF of the P protein at the position corresponding to E114 of SEQ ID NO: 4; and iv. These combinations An isolated polynucleotide molecule as described in item 2, further modified by selected mutations from the above. [Section 5] An isolated polynucleotide molecule as described in any of sections 2-4, wherein the RSV genome or antigenome is modified by at least two of the mutations (i)-(iii). [Section 6] An isolated polynucleotide molecule as described in any of sections 2-5, wherein the RSV genome or antigenome is modified by all of mutations (i)-(iii). [Section 7] An isolated polynucleotide molecule as described in any of items 1-6, wherein the mutation in the L ORF of the L protein corresponds to T1166 in SEQ ID NO: 11, and is T1166I. [Section 8] a. A mutation in the M2-1 ORF of the M2-1 protein at the position corresponding to N88 of SEQ ID NO: 9 is N88K, and a mutation in the M2-1 ORF of the M2-1 protein at the position corresponding to A73 of SEQ ID NO: 9 is A73S; b. The mutation at the position corresponding to K136 in Sequence ID No. 3 in the N ORF of the N protein is K136R; and c. The mutation in the P ORF of the P protein at the position corresponding to E114 in SEQ ID NO: 4 is E114V. An isolated polynucleotide molecule as described in any of items 1 to 7. [Section 9] An isolated polynucleotide molecule as described in item 8, wherein the RSV genome or antigenome is modified by at least two of mutations a to c. [Section 10] An isolated polynucleotide molecule as described in item 8, wherein the RSV genome or antigenome is modified by all of mutations a-c. [Section 11] An isolated polynucleotide molecule as described in any of items 1 to 10, wherein the RSV genome or antigenome is modified by corresponding mutations selected from the group consisting of (1) T1166I in the L protein, N88K in the M2-1 protein, K136R in the N protein, and E114V in the P protein; and (2) T1166I in the L protein, A73S in the M2-1 protein, K136R in the N protein, and E114V in the P protein. [Section 12] An isolated polynucleotide molecule as described in any of items 1 to 11, wherein the RSV genome or antigenome contains a deletion in at least one protein selected from M2-2, NS1, and NS2. [Section 13] An isolated polynucleotide molecule as described in any of items 1-12, wherein the RSV genome or antigenome has been codon-pairing optimized. [Section 14] An isolated polynucleotide molecule as described in any of items 1-13, in which the L ORF genome or antigenome of an RSV has been codon-pairing optimized. [Section 15] A vector containing an isolated polynucleotide molecule as described in any of items 1 to 14. [Section 16] A cell containing an isolated polynucleotide as described in any of items 1 to 15. [Section 17] A pharmaceutical composition comprising a recombinant RSV variant encoded by an isolated polynucleotide molecule as described in any of the sections 1 to 14 of the section on immunoeffective amounts. [Section 18] A method of vaccinating a subject against RSV, comprising administering the pharmaceutical composition described in item 17. [Section 19] A method for inducing an immune response, comprising administering the pharmaceutical composition described in item 17. [Section 20] The method according to item 18 or 19, wherein the pharmaceutical composition is administered intranasally. [Section 21] The method according to item 20, wherein the pharmaceutical composition is administered by injection, aerosol delivery, nasal spray, or nasal instillation. [Section 22] A viable attenuated RSV vaccine containing a recombinant RSV variant encoded by any of the isolated polynucleotides listed in items 1-14. [Section 23] A pharmaceutical composition comprising the RSV vaccine described in item 22. [Section 24] A method for producing the vaccine described in item 22, comprising expressing an isolated polynucleotide molecule described in any of items 1 to 14. [Section 25] An isolated polynucleotide molecule encoding a recombinant RSV variant having an attenuated phenotype containing a respiratory multinuclear virus (RSV) genome or antigenome sequence, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S1. [Section 26] An isolated polynucleotide molecule as described in item 25, modified by one or more mutations selected from the positions shown in Table S1-A, which are either an RSV genome or an antigenome. [Section 27] An isolated polynucleotide molecule as described in item 25 or 26, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S1-B. [Section 28] An isolated polynucleotide molecule encoding a recombinant RSV variant having an attenuated phenotype containing a respiratory multinuclear virus (RSV) genome or antigenome sequence, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S2. [Section 29] An isolated polynucleotide molecule as described in item 28, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S2-A. [Section 30] An isolated polynucleotide molecule as described in section 28 or 29, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S2-B. [Section 31] An isolated polynucleotide molecule encoding a recombinant RSV variant having an attenuated phenotype containing a respiratory multinuclear virus (RSV) genome or antigenome sequence, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S3. [Section 32] An isolated polynucleotide molecule as described in item 31, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S3-A. [Section 33] An isolated polynucleotide molecule as described in item 31 or 32, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S3-B. [Section 34] An isolated polynucleotide molecule as described in any of sections 25-33, wherein the RSV genome or antigenome contains a deletion in at least one protein selected from M2-2, NS1, and NS2. [Section 35] An isolated polynucleotide molecule as described in any of sections 25-34, wherein the RSV genome or antigenome has been codon-pairing optimized. [Section 36] An isolated polynucleotide molecule as described in any of sections 25-35, wherein the L ORF genome or antigenome of the RSV is codon-pairing optimized. [Section 37] A vector containing an isolated polynucleotide molecule as described in any of items 25-36. [Section 38] A cell containing an isolated polynucleotide as described in any of items 25-36. [Section 39] A pharmaceutical composition comprising a recombinant RSV variant encoded by an isolated polynucleotide molecule as described in any of sections 25 to 36 of the section on immunoeffective amounts. [Section 40] A method of vaccinating a subject against RSV, comprising administering the pharmaceutical composition described in item 39. [Section 41] A method for inducing an immune response, comprising administering the pharmaceutical composition described in item 39. [Section 42] The method according to item 40 or 41, wherein the pharmaceutical composition is administered intranasally. [Section 43] The method according to item 42, wherein the pharmaceutical composition is administered by injection, aerosol delivery, nasal spray, or nasal instillation. [Section 44] A viable attenuated RSV vaccine containing a recombinant RSV variant encoded by an isolated polynucleotide as described in any of items 25-36. [Section 45] A pharmaceutical composition comprising the RSV vaccine described in item 44. [Section 46] A method for producing the vaccine described in item 44, comprising expressing an isolated polynucleotide molecule described in any of items 25 to 36. [Section 47] An isolated polynucleotide molecule as described in any of sections 1-3, 5-14, and 25-36, comprising a nucleotide sequence that is at least approximately 80% identical to the nucleotide sequence of SEQ ID NO: 14. [Section 48] An isolated polynucleotide molecule as described in any of sections 1-3, 5-14, and 25-36, comprising a nucleotide sequence that is at least approximately 90% identical to the nucleotide sequence of SEQ ID NO: 14. [Section 49] An isolated polynucleotide molecule whose nucleotide sequence is at least approximately 80% identical to that of Sequence ID No. 14. [Section 50] An isolated polynucleotide molecule as described in item 49, which is at least approximately 90% identical to the nucleotide sequence of SEQ ID NO: 14. [Section 51] An isolated polynucleotide molecule as described in item 49, which is at least approximately 95% identical to the nucleotide sequence of SEQ ID NO: 14. [Section 52] An isolated polynucleotide molecule as described in item 49, containing the nucleotide sequence of SEQ ID NO: 14. [Section 53] A vector containing an isolated polynucleotide molecule as described in any of sections 47-52. [Section 54] A cell containing an isolated polynucleotide as described in any of sections 47-52. [Section 55] A pharmaceutical composition comprising a recombinant RSV variant encoded by an isolated polynucleotide molecule as described in any of sections 47 to 52 of the section on immunoeffective amounts. [Section 56] A method of vaccinating a subject against RSV, comprising administering the pharmaceutical composition described in item 55. [Section 57] A method for inducing an immune response, comprising administering the pharmaceutical composition described in item 55. [Section 58] The method according to item 56 or 57, wherein the pharmaceutical composition is administered intranasally. [Section 59] The method according to item 58, wherein the pharmaceutical composition is administered by injection, aerosol delivery, nasal spray, or nasal instillation. [Section 60] A viable attenuated RSV vaccine comprising a recombinant RSV variant encoded by an isolated polynucleotide as described in any of items 47-52. [Section 61] A pharmaceutical composition comprising the RSV vaccine described in item 60. [Section 62] A method for producing the vaccine according to claim 60, comprising expressing an isolated polynucleotide molecule as described in any of claims 47 to 52.

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

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

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

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

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

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

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

[0025] In one embodiment, the RSV genome or antigenome contains a deletion in at least one of the proteins selected from M2-2, NS1, and NS2. In one embodiment, the RSV genome or antigenome is codon-pair-optimized. In one embodiment, the L-ORF of the RSV genome or antigenome is codon-pair-optimized.

[0026] In one embodiment, the present invention includes a polynucleotide molecule containing a nucleotide sequence that is at least about 80% identical to the nucleotide sequence of SEQ ID NO: 14. In another embodiment, the present invention includes a polynucleotide molecule containing a nucleotide sequence that is at least about 90% identical to the nucleotide sequence of SEQ ID NO: 14. In yet another embodiment, the present invention includes a polynucleotide molecule containing a nucleotide sequence that is at least about 95% identical to the nucleotide sequence of SEQ ID NO: 14. In yet another embodiment, the present invention includes a polynucleotide molecule containing the nucleotide sequence of SEQ ID NO: 14.

[0027] In one embodiment, the present invention comprises a vector containing the above-mentioned isolated polynucleotide molecule. In another embodiment, the present invention comprises a cell containing the above-mentioned isolated polynucleotide molecule.

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

[0029] In one embodiment, the present invention includes a viable attenuated RSV vaccine comprising a recombinant RSV variant encoded by the isolated polynucleotide described above. In one embodiment, the present invention includes a pharmaceutical composition comprising the RSV vaccine. In one embodiment, the present invention includes a method for producing a vaccine, comprising expressing the isolated polynucleotide molecule described above. [Brief explanation of the drawing]

[0030] [Figure 1-1] Figures 1A-G show that Min_FLC was phenotypically stable during the temperature stress test, while Min_L was not. (A) Genetic maps of Min_L and Min_FLC showing ORFs that are wt (gray) or CPD (black). Shows the number of mutations introduced into each virus in Vero cells and the cutoff temperature (TSH). (B-E) Shows the incubation temperature and virus yield at each passage level during continuous passage in the temperature stress test. Isotyped Vero cells in T25 flasks were infected with the described virus at an MOI of 0.1. When the viral cytotoxic effect was widespread or when cells began to detach (C for passage of Min_FLC above 37°C), the flasks were collected and the purified culture medium was passaged in a 1:5 ratio in new flasks. Each initial isotyped flask initiated an individual series of passages (lineage). A certain amount of the purified culture medium was frozen for titer analysis and sequence analysis. (B, C) Temperature stress tests of Min_FLC. Two control flasks (B) inoculated with Min_FLC were passaged 18 times at an acceptable temperature of 32°C. Ten further replicas (C) were passaged at 32–40°C, with two passages at each temperature. (D, E) Temperature stress tests of Min_L. Two control flasks (D) inoculated with Min_L were passaged 8 times at an acceptable temperature of 32°C. Ten further replicas (E) were passaged at 37–40°C, with two passages at each temperature. Differentiation series #3 and #8 are shown. (F, G) Accumulation of the most abundant mutations (at least >30% of read data in one passage) in differentiation series #3 (F) and #8 (G) between passage series determined by deep sequencing (see Tables S2 and S3 for detailed data). [Figure 1-2] Same as above.

[0031] [Figure 2-1] Figures 2A–C show the M2-1 mutations [A73S] and [N88K] isolated in different viral subpopulations. (A) Percentage of deep sequencing read data containing the M2-1 mutation [A73S] or [N88K] at P6 (second passage at 39°C) for each of the 10 differentiation series from the experiment in Figure 1E. (B) Loss of association between the M2-1 mutations [A73S] and [N88K], explained by the percentage of deep sequencing read data containing the intended assignment combinations at codons 73 (wt vs. [A73S]) and 88 (wt vs. [N88K]) in the same read data; based on read data from the experiment in Figure 1F spanning both codons. (C) Degree of association between the M2-1 mutations [A73S] and [N88K] and other mutations during the first four passages of lineage #3, determined by PacBio sequencing of sequential read data corresponding to an 8.2kb region of the RSV genome from the 3' end to the center of the M2-2 ORF (Figure 1E and F). Four major viral subpopulations were identified, showing related mutations on the same genome. [Figure 2-2] Same as above. [Figure 2-3] Same as above.

[0032] [Figure 3-1]Figures 3A-D show the effects of specific mutations on temperature sensitivity and in vitro replication of Min_L derivatives. Five major mutations identified in lineage #3 (N[K136R], P[E114V], L[T1166I], M2-1[N88K], and M2-1[A73S], Figure 1F) were individually and combined into Min_L via site-directed mutagenesis and reverse genetics for phenotypic analysis. Min_L-derived viruses were named based on the gene names carrying the transmutations, with M2-1 mutations identified in parentheses. (A) Mutations are shown on a viral genome map. (B) TSH determined by plaque formation efficiency at 32°C, 35°C, 36°C, 37°C, 38°C, 39°C, and 40°C using published methods. The experiment was performed four times with viruses #1, 5, 12, and 14, three times with viruses #2, 3, 7, 8, and 9, twice with viruses #4 and 11, and once with viruses #6, 10, and 13 (bar graph: center and range). (C, D) shows in vitro replication of Min_L-derived mutants. Vero cells were infected at 32°C and 37°C (MOI of 0.01). Titers correspond to the mean of two repeated titer measurements of two replications at each time point. Standard deviation is shown. Due to the large number of viruses, the analysis was divided into experiment #1 (C) and #2 (D). [Figure 3-2] Same as above.

[0033] [Figure 4-1]Figures 4A-G show the effects of specific mutations of Min_L derivatives on RNA synthesis and plaque size. (A-E) Isomorphic cultures of Vero cells were infected with the described viruses (MOI of 3). Cultures were collected every 4 hours from 4 to 24 hours post-infection for cell-related RNA, proteins, and viruses. (A) Positive sense viral RNA (i.e., mRNA + antigenome) was quantified in triplicates by strand-specific RT-qPCR. Data for P are shown. QPCR results were analyzed using the comparative threshold cycle (ΔCt) method, normalized to 18S rRNA, and expressed as a log2 increase factor exceeding Min_L at 4 hours. (B) Quantification of P protein expression by Western blotting. (C) Quantification of L mRNA + antigenome by strand-specific RT-qPCR (increase factor relative to 4 hours post-infection; calculated separately for each virus because different primer-probe sets were required for the wt L gene in wt rRSV and the CPD L gene present in Min_L and its derivatives). For Wt L and CPD L, data are derived from three and four different primer-probe sets, respectively, designed according to the L ORF, and median and range values ​​are shown. (D) Quantification of cell-associated genomic RNA by strand-specific RT-qPCR, expressed as the increase factor beyond 4 hours post-infection for Min_L. (E) Viral titers from cultures incubated at 32°C and 37°C and assayed at 32°C. (FG) Viral plaque size. Vero cells were infected with wt rRSV, Min_L and Min_L-derived mutants in 30 pfu / 2cm2 wells and incubated under methylcellulose at 32°C for 12 days. Plaques were visualized by immunostaining and quantified by IR contrast (Licor) using Image J. F) Representative diagram of viral plaque size. G) Distribution of viral plaque size shown. Minimum 1000 plaques / virus were measured (*=p≦0.05). [Figure 4-2] Same as above.

[0034] [Figure 5-1]Figures 5A-D show the analysis of Min_L derivatives in rodents, which demonstrate the different effects of M2-1 mutants A73S and N88K, identifying the improved vaccine candidate NPM2-1[N88K]L. The figures show replication of Min_L, Min_L mutants, and wt rRSV at post-infection day 4 (A) and day 5 (B) in mice or post-infection day 3 (C) in hamsters. Groups of 20 mice (AB) or 18 hamsters (C) were intranasally infected with 106 pfu of the virus / animal. RSV titers in the nasal turbinates (NT) and lungs were determined at post-infection day 4 (A), day 5 (B), and day 10 (data not shown) in the mouse study, or day 3 (C) in the hamster study, as described in the experimental methods section. Detection limits are indicated by dotted lines. D) RSV neutralizing antibodies at day 26 in hamsters from 9 hamsters / group. The 60% plaque reduction neutralizing antibody titer (PRNT60) was determined as previously described. Statistical differences compared to wt rRSV are shown at the top of each graph; statistical differences between Min_L and Min_L-derived mutants are shown in parentheses (*p≦0.05, **p≦0.01, ***p≦0.001, and ****p≦0.0001). [Figure 5-2] Same as above.

[0035] [Figure 6-1] Figures 6A-D show molecular modeling of the effects of deattenuation mutations on the M2-1 tetramer. (A) Plan view of the wt M2-1 tetramer. (B) Magnified view of one region of the wt tetramer containing amino acids A73 and N88. (C) Molecular dynamics snapshot of the proximal region of the S73 mutation. The [A73S] mutation is shown, and the arrow indicates the predicted novel hydrogen bond. (D) Molecular dynamics snapshot of the K88 mutant region. The [N88K] mutation is shown, and the arrow indicates the predicted novel salt bridge. [Figure 6-2] Same as above.

[0036] [Figure 7-1]Figures 7A-B show the minimum accumulation of non-determinate mutations in Min_FLC over 18 passages at 32°C. Min_FLC was attached to 18 passages at 32°C. At the end of the 18th passage, viral RNA was extracted from lineages #1(A) and #2(B), the complete genome was amplified by duplicate RT-PCR, and analyzed by deep sequencing (Ion Torrent). Non-determinate mutations (which are not specifically shown) are indicated by bars showing their genomic location and relative abundance. WT genes are colored gray, and CPD genes are colored black. [Figure 7-2] Same as above.

[0037] [Figure 8-1] Figures 8A-B show the minimum accumulation of non-determinate mutations in Min_L during 6 passages at 32°C. Min_L was passaged 6 times consecutively in Vero cells at 32°C. At the end of the 6th passage, viral RNA was extracted from lineages #1(A) and #2(B), the complete genome was amplified by duplicate RT-PCR, and analyzed by deep sequencing (Ion Torrent). Non-determinate mutations are shown by bars indicating their genomic location and relative abundance; specific nucleotide changes are not shown. WT genes are colored gray, and CPD genes are colored black. [Figure 8-2] Same as above.

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

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

[0040] [Figure 11]Figures 11A-B show the TSH of Min_FLC-derived mutants. The effect of mutations involved in the loss of temperature sensitivity in Min_L on Min_FLC temperature sensitivity was tested. A) To do so, mutations identified in Min_L lineage #3 (in the N, P, M2-1[N88K] and L genes) or #8 (M2-1 mutation[A73S]) were reintroduced into the Min_FLC backbone, either individually or in the combinations described, and the resulting cDNA was completely sequenced by Sanger sequencing. The viruses were rescued by reverse genetics, passaged once, and the P2 viral stocks were titrated. Due to the low viral titers of most viral stocks, only the mutant virus Min_FLC_M2-1[A73S] was completely sequenced by Sanger sequencing. B) Several ts phenotypes of these Min_FLC-derived mutants were evaluated by the high rate of plaque formation at 32°C, 35°C, 36°C, 37°C, 38°C, 39°C, and 40°C. Plaque assays were performed in duplicate using Vero cells and incubated in sealed containers in temperature-controlled water baths at various temperatures as described above. The experiment was performed twice. Median and standard deviation are shown.

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

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

[0043] [Figure 14-1] Figure 14 shows the nucleotide sequence of recombinant RSV Min_L-NPM2-1[N88K]L, as indicated by sequence number 14. [Figure 14-2] Same as above. [Figure 14-3] Same as above. [Figure 14-4] Same as above. [Modes for carrying out the invention]

[0044] Detailed description Provided herein are recombinant RSV strains suitable for use as attenuated and viable vaccines in humans. RSV strains can be produced by introducing one or more mutations into the RSV genome or antigenome sequence selected from the locations listed below and in Tables S1, S2, and S3. These mutations were identified by evaluating the phenotypic reversion of deoptimized human respiratory multinuclear virus (RSV) vaccine candidates under strong selective pressure.

[0045] The codon-pair deoptimization (CPD) version of RSV was attenuated and temperature-sensitive. During continuous passage with gradually increasing temperature, a CPD RSV named Min_FLC, containing 2,692 synonymous mutations in 9 out of 11 ORFs, retained temperature sensitivity and remained genetically and phenotypically stable for 7 months in vitro under the tolerable temperature of 32°C and under increasing temperature conditions during passage. This provides strong evidence of the stability of Min_FLC and confirms the safety of multi-gene CPD for the development of viable attenuated vaccines for RSV and related viruses, provided that widespread CPD is used.

[0046] However, the CPD RSV, named Min_L, which had only the polymerase L ORF deoptimized, was highly stable at 32°C, but surprisingly, despite numerous changes involved in its CPD, it rapidly lost substantial attenuation and evolved to circumvent temperature sensitivity limitations. Comprehensive sequencing analysis of the viral population identified numerous different potentially deattenuated mutations in the L ORF, many of which, surprisingly, appeared in other ORFs not associated with CPD. In particular, deep sequencing of the Min_L differentiation lineage identified mutations not specifically in the CPD L ORF, but rather in all ORFs except the NS2 ORF, as predicted. Surprisingly, many of the mutations in L occurred in nucleotides and codons not involved in CPD. These are shown in Tables S1, S2, and S3.

[0047] Some of these putative deattenuating mutations were found to be deattenuating in vitro, but when introduced into Min_L along with other putative deattenuating mutations, they were found to have the surprising effect of being even more attenuating than Min_L in vivo.

[0048] In one exemplary embodiment, as detailed below (nucleotide sequence shown in Figure 14), Min_L-NPM2-1[N88K]L (also referred to here as NPM2-1[N88K]L) was attenuated more than Min_L in vivo, rather than being deattenuated. Furthermore, while the NPM2-1[N88K]L virus was highly attenuated in vivo, it surprisingly exhibited immunogenicity comparable to wild-type RSV. Moreover, it acquired no significant mutations during further stress testing (see Figure 12) and was therefore genetically more stable than Min_L. For these reasons, Min_L-NPM2-1[N88K]L shows considerable improvement over Min_L as a vaccine candidate for the following reasons: It was significantly more attenuated in vivo than Min_L while exhibiting immunogenicity comparable to wt RSV. It did not accumulate additional mutations when passaged in stress tests at 39-40°C. It showed increased replication in Vero cells compared to Min_L, which is important for vaccine production. Furthermore, as detailed below, this virus is highly resistant to acquiring the deattenuated M2-1[A73S] mutation in the hamster model, as the M2-1[N88K] and [A73S] mutations are incompatible.

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

[0050] In one embodiment, the present invention comprises an isolated polynucleotide molecule encoding a recombinant RSV variant having an attenuated phenotype, comprising a respiratory multinuclear virus (RSV) genome or antigenome sequence, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S1. In another embodiment, the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S1-A. In yet another embodiment, the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S1-B.

[0051] In one embodiment, the present invention comprises an isolated polynucleotide molecule encoding a recombinant RSV variant having an attenuated phenotype, comprising a respiratory multinuclear virus (RSV) genome or antigenome sequence, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S2. In another embodiment, the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S2-A. In yet another embodiment, the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S2-B.

[0052] In one embodiment, the present invention comprises an isolated polynucleotide molecule encoding a recombinant RSV variant having an attenuated phenotype, comprising a respiratory multinuclear virus (RSV) genome or antigenome sequence, wherein the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S3. In one embodiment, the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S3-A. In another embodiment, the RSV genome or antigenome is modified by one or more mutations selected from the positions shown in Table S3-B.

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

[0054] In one embodiment, the RSV genome or antigenome may be further modified by one or more additional mutations. The additional mutations may occur in any of the L ORFs or other ORFs. For example, in one embodiment, one or more additional mutations may occur in the M2-1 ORF, N ORF, or P ORF.

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

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

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

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

[0059] In one embodiment, the isolated polynucleotide molecule may include an RSV genome or antigenome modified by mutations corresponding to or encoding N88K in the M2-1 protein, K136R in the N protein, E114V in the P protein, and T1166I in the L protein.

[0060] In some embodiments, the RSV genome or antigenome can be deoptimized. Therefore, in some embodiments, the attenuated RSV described herein is produced by introducing codon changes in the viral genome that are not optimally processed by the host cell. Most of these mutations do not alter the amino acids of the resulting protein encoded by the viral genome, thus enabling the production of a virus with the same antigenic properties as the wild-type virus. However, it should be understood that extensive non-coding changes to codons in the viral genome can create a selective pressure that induces one or more amino acid mutations in the virus described herein.

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

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

[0063] In one embodiment, an isolated polynucleotide molecule may contain a nucleotide sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any identity percentage in between). In one embodiment, an isolated polynucleotide molecule may contain a nucleotide sequence that is at least about 80% identical to the nucleotide sequence of SEQ ID NO: 14. In one embodiment, an isolated polynucleotide molecule may contain a nucleotide sequence that is at least about 90% identical to the nucleotide sequence of SEQ ID NO: 14. In one embodiment, an isolated polynucleotide molecule may contain a nucleotide sequence that is at least about 95% identical to the nucleotide sequence of SEQ ID NO: 14. In one embodiment, the isolated polynucleotide molecule may include an isolated polynucleotide containing the nucleotide sequence of SEQ ID NO: 14.

[0064] In one embodiment, the described virus, in combination with known attenuating mutations of RSV and related viruses, can produce a stepwise attenuation phenotype. Numerous such mutations are known in the art and are included in the present invention. For example, in one embodiment, the RSV genome or antigenome may be modified by deletions in M2-2 ORFs, NS1 ORFs, or NS2 ORFs.

[0065] Given the existence of various RSV strains (e.g., RSV A2, RSV B1, RSV Long), those skilled in the art will recognize that some RSV strains may have nucleotide or amino acid insertions or deletions that alter the position of certain residues. For example, if the protein of another RSV strain has two additional amino acids at its upstream end compared to strain A2, this would increase the amino acid numbering of the downstream residues by two compared to strain A2. However, due to the high degree of sequence identity among these strains, those skilled in the art can determine the position of the corresponding sequence simply by aligning the nucleotide or amino acid sequence of the A2 control strain with the sequence of the strain in question. Therefore, while the amino acid and nucleotide positions described herein are specifically numbered in this disclosure, it should be understood that they may correspond to other positions when sequence shifts occur or due to sequence diversity between viral strains. In comparisons of proteins or protein segments or genes or genomes or genomic segments of two or more related viruses, “corresponding” amino acids or nucleotide residues are those that are considered to have exactly or approximately equivalent function in different species.

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

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

[0068] Additional mutations may be introduced in combination with the mutations defined above to construct further viral strains with desired characteristics. For example, the added mutations may identify different attenuation intensities and thus result in a gradual increase in attenuation. Therefore, candidate vaccine strains may be further attenuated by incorporating at least one, preferably two or more, different attenuating mutations, such as those identified from a group of known, biologically induced mutant RSV strains. Numerous such mutations are described here as examples. From this exemplary group, a large “list” of attenuating mutations can be made, where each mutation may be combined with any other mutation within the group to scale the levels of attenuation and other desired phenotypes. Additional attenuating mutations have been identified in non-RSV negative-strand RNA viruses and may be incorporated into the RSV variant of the present invention by mapping the mutation to a corresponding homologous site in the recipient RSV genome or antigenome and mutating the existing sequence in the recipient to the mutant genotype (identical or conserved mutation). Further useful mutations can be empirically determined by mutation analysis using recombinant minigenome systems and infectious viruses, as described in the references incorporated herein.

[0069] The recombinant RSV vaccine strain of the present invention was produced using a recombinant DNA-based technique called reverse genetics (Collins, et al. 1995. Proc Natl Acad Sci USA 92:11563-11567). This system allows for the de novo acquisition of a complete infectious virus from cDNA in a suitable cell substrate under specified conditions. Reverse genetics provides a means for introducing planned mutations into the RSV genome via cDNA intermediates. Specific attenuating mutations were characterized in preclinical studies and combined to achieve the desired level of attenuation. Induction of vaccine virus from cDNA minimizes the risk of contamination by accidental contaminants and helps to concisely and thoroughly document the passage history. Once acquired, the engineered virus strain is propagated in the same manner as biologically induced viruses. As a result of passage and amplification, the vaccine virus does not contain recombinant DNA from the initial acquisition.

[0070] The recombinant virus strains containing various combinations of mutations described herein are for illustrative purposes only and are not intended to limit the scope of the present invention. For example, in one embodiment, the recombinant RSV strain of the present invention further includes a deletion of an untranslated sequence. In one embodiment, such a deletion occurs at the downstream end of the SH gene and results in a mutation referred to hereby as the “6120 mutation.” This involves the deletion of 112 nucleotides in the downstream untranslated region of the SH gene and the introduction of five translationally silent point mutations in the last three codons and the stop codon of the SH gene (Bukreyev, et al. 2001. J Virol 75:12128-12140). The presence of the terms “LID” or “6120” in the recombinant virus name indicates that the recombinant virus contains the 6120 mutation.

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

[0072] The 6120 mutation was associated with increased replication in seronegative infants and children. Therefore, the 6120 mutation provided another means for shifting the attenuation level. Furthermore, sequence deletions, exemplified by the 6120 mutation in the downstream untranslated region of the SH gene, can include any equivalent genomic sequence that does not, in principle, contain important cis-acting signals (Collins and Karron. 2013. Fields Virology 6th Edition, pp 1086-1123). Candidate genomic regions for deletion include, but are not limited to, untranslated regions in other genes, intergeneric regions, and trailer regions.

[0073] In one embodiment, recombinant RSV strains may contain “cp” mutations. These mutations, together (or by themselves), refer to a series of five-amino acid substitutions in three proteins (N(V267I), F(E218A and T523I), and L(C319Y and H1690Y)) that contribute to approximately a tenfold reduction in replication and disease in seronegative chimpanzees (Whitehead, et al. 1998. J Virol 72:4467-4471). We previously showed that cp mutations are associated with a moderately attenuated phenotype (Whitehead, et al. 1999. J Virol 72:4467-4471).

[0074] Furthermore, previous analyses of six biological viruses selected for their temperature-sensitive (ts) phenotype, induced by chemical mutations in cpRSV, resulted in a total of six independent mutations, each conferring a ts attenuated phenotype and usable in various combinations. Five of these resulted in amino acid substitutions in the L protein, and these were named by their viral numbers rather than their sequence positions: “955” (N43I), “530” (F521L), “248” (Q831L), “1009” (M1169V), and “1030” (Y1321N) (Juhasz, et al. 1999. Vaccine 17:1416-1424; Collins, et al. 1999. Adv Virus Res 54:423-451; Firestone, et al. 1996. Virology 225:419-422; Whitehead, et al. 1999. J Virol 73:871-877). The sixth mutation (referred to as "404") was a single nucleotide change in the gene initiation transcription signal of the M2 gene (GGGGCAAATA to GGGGCAAACA, mRNA sense) (Whitehead, et al. 1998. Virology 247:232-239). We recently increased the genetic stability of the 248 and 1030 mutations using reverse genetics (Luongo, et al. 2009. Vaccine 27:5667-5676; Luongo, et al. 2012. J Virol 86:10792-10804). Furthermore, we created novel attenuating mutations by deleting codon 1313 in the L protein and combining this with the I1314L substitution to increase genetic stability (Luongo, et al. 2013. J Virol 87:1985-1996).

[0075] In one embodiment, the recombinant strain may contain one or more changes in the F protein, such as the “HEK” mutation, which includes two amino acid substitutions in the F protein, namely K66E and Q101P (as described in Connors, et al. 1995. Virology 208:478-484; Whitehead, et al. 1998. J Virol 72:4467-4471). The introduction of the HEK amino acid assignment into the F sequence of strain A2 of the present invention results in an F protein amino acid sequence identical to that of the early passage of the original clinical isolate of strain A2 (human embryonic kidney cell passage 7, HEK-7) (Connors, et al. 1995. Virology 208:478-484; Whitehead, et al. 1998. J Virol 72:4467-4471). This results in a much less fusionable F protein, which is thought to represent the phenotype of the original A2 strain clinical isolate (Liang et al. J Virol 2015 89:9499-9510). The HEK F protein also forms a more stable trimer (Liang et al. J Virol 2015 89:9499-9510). This may provide a more reliable and immunogenic form of the RSV F protein, enriched with what is likely a highly immunogenic prefusion structure (McLellan et al. Science 2013 340(6136):1113-7; Science 2013 342(6158):592-8). Therefore, in addition to its effect on the intensity of viral replication, it may introduce effective mutations.

[0076] In one embodiment, the recombinant strain may contain one or more changes in the L protein, such as a stabilizing 1030 or “1030s” mutation including 1321K(AAA) / S1313(TCA) (Luongo, et al. 2012. J Virol 86:10792-10804).

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

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

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

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

[0081] A further aspect of the present invention involves altering the position or order of genes. For example, the NS1, NS2, SH, and G genes may be deleted individually, or the NS1 and NS2 genes may be deleted together, thereby shifting the position of each downstream gene relative to the viral promoter. For example, when both NS1 and NS2 are deleted, N moves from position 3 to position 1, and P moves from position 4 to position 2, and so on. Alternatively, the deletion of any other gene in the gene order may only affect the position of further downstream genes (relative to the promoter). For example, SH occupies position 6 in the wild-type virus, and its deletion does not affect M at position 5 (or any other upstream gene), but moves G from position 7 to position 6 relative to the promoter. It should also be noted that gene deletions may also occur (rarely) in biologically induced mutant viruses. For example, subgroup B RSVs, which are extensively passaged in cell cultures, spontaneously lack the SH and G genes (Karron et al. Proc. Natl. Acad. Sci. USA 94:13961 13966, 1997; incorporated herein by reference).

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

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

[0084] In addition to recombinant RSV having the specific mutations and combinations thereof described herein, the disclosed viruses may be further modified as will be recognized by those skilled in the art. For example, recombinant RSV may have one or more of its proteins deleted or otherwise mutated, or heterologous genes from different organisms added to its genome or antigenome, so that when recombinant RSV infects and replicates in cells, it may express or incorporate its proteins. Furthermore, those skilled in the art will recognize that other previously reported mutations known to be effective against RSV may be combined with any one or more of the mutations described herein to produce recombinant RSV with desired attenuation or stability properties.

[0085] In one embodiment, the mutations described herein, when used alone or in combination with other mutations, can provide varying levels of viral attenuation, offer the ability to adjust the balance between attenuation and immunogenicity, and provide a more stable genotype than the parent virus.

[0086] Further representative viruses from those described herein may be evaluated in cell culture for infectivity, replication dynamics, yield, protein expression efficiency, and genetic stability using exemplary recombinant strains and the methods described herein. Further representative strains may be evaluated in rodents and non-human primates for infectivity, replication dynamics, yield, immunogenicity, and genetic stability. While these imperfect test systems may not reliably detect all differences in replication, specific substantial differences may be detected. Recombinant strains may also be evaluated directly in seronegative offspring without prior evaluation in adults and seronegative offspring. This can be done, for example, with a group of 10 vaccine recipients and 5 placebo recipients, which is a small number that allows for simultaneous evaluation of multiple candidates. Candidates can be evaluated immediately after immunization for vaccine virus infectivity, replication dynamics, shedding, tolerability, immunogenicity, and genetic stability, and the vaccine can be investigated for safety, RSV disease, and changes in RSV-specific serum antibodies during the next RSV epidemic, as described in Karron, et al. 2015, Science Transl Med 2015 7(312):312ra175, which is incorporated herein by reference. Therefore, analysis of selected representative viruses can provide a relatively rapid selection for candidate triage to identify the most optimal one.

[0087] Descriptions of proteins or peptides include their naturally occurring forms and any fragments, domains, or homologs of such proteins. The term “homolog” as used herein is used to refer to a protein or peptide that, through minor modifications to a naturally occurring protein or peptide, differs from the naturally occurring protein or peptide (i.e., the “prototype” or “wild-type” protein) but retains the basic protein and side-chain structure of the naturally occurring form. Such modifications include changes in one or more amino acid side chains; changes in one or more amino acids, including deletions (e.g., cleavage forms of proteins or peptides), insertions, and / or substitutions; stereochemical changes of one or more atoms; and / or minor derivatizations, including but not limited to methylation, glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, and amidation. Homologs may have enhanced, reduced, or substantially similar properties compared to the naturally occurring protein or peptide. A homolog of a protein may contain, essentially consist of, or be composed of, an amino acid sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical (or any percentage identical in integer increments between 45% and 99%) to the amino acid sequence of a control protein.

[0088] In one embodiment of the present invention, a selected gene segment, such as one encoding a selected protein or protein region from an RSV (e.g., a cytoplasmic tail, transmembrane domain or extramembrane domain, epitope site or region, binding site or region, active site or active site-containing region), can be replaced with a counterpart gene segment from the same or a different RSV or other source to create a novel recombination with a desired phenotypic change compared to a wild-type or parental RSV strain. For example, this type of recombination may express a chimeric protein having the cytoplasmic tail and / or transmembrane domain of one RSV fused to the extramembrane domain of another RSV. Another example of this type of recombination is the expression of a duplicate protein region, such as a duplicate immunogenicity region. The “counterpart” gene, gene segment, protein, or protein region used herein is generally from a different source (e.g., from a different RSV gene or representing an identical (i.e., homologous or allelic) gene or gene segment in a different RSV strain). The typical counterparts selected in the present invention share overall structural characteristics, and each counterpart may encode equivalent structural “domains,” such as cytoplasmic domains, transmembrane domains, extracellular domains, binding sites or regions, epitope sites or regions, etc. The counterpart domains and their encoding gene segments encompass a collection of species with a wide range of size and amino acid (or nucleotide) sequence diversity, the range of which is defined by the common biological activity within the domain or gene segment variant. For example, two selected protein domains encoded by counterpart gene segments in the present invention may share substantially the same qualitative activity, such as transmembrane function, specific binding activity, or provision of an immunological recognition site. More generally, the specific biological activity shared between counterparts, for example between selected protein segments or proteins, is substantially similar in terms of quantity, i.e., it does not change by more than 30%, preferably more than 20%, and more preferably more than 5-10% in the quantitative activity profile.

[0089] In another aspect of the present invention, the infectious RSV produced from a cDNA-expressed genome or antigenome may be any RSV or RSV-like strain, e.g., human, bovine, mouse, etc., or any pneumovirus or metapneumovirus, e.g., mouse pneumonia virus or trimeta-pneumovirus. To produce a protective immune response, the RSV strain may be endogenous to the target being immunized, for example, human RSV may be used for human immunization. The genome or antigenome of endogenous RSV can, in any case, be modified to express RSV genes or gene segments from various combinations of origins, e.g., combinations of genes or gene segments from different RSV species, subgroups, or strains, or from other respiratory pathogens such as RSV and human parainfluenza virus (PIV) (see, e.g., Hoffman et al. J. Virol. 71:4272-4277 (1997); Durbin et al. Virology 235(2):323-32 (1997); Murphy et al. U.S. Patent Application 60 / 047,575 filed May 23, 1997 and the following plasmids for infectious PIV cloning: g:p3 / 7(131)(ATCC 97990); p3 / 7(131)2G(ATCC 97889); and p218(131)(ATCC 97991); respectively, 10801 University It was deposited with the American Type Culture Collection (ATCC) at Blvd., Manassas, Va. 20110-2209, USA, on April 18, 1997, under the Budapest Convention, and was assigned the above accession number.

[0090] In one embodiment of the present invention, recombinant RSV is provided in which individual internal genes of human RSV are replaced, for example, with counterparts from bovine or other RSV counterparts or other respiratory pathogens such as PIV, or with exogenous genes. Substitutions, deletions, etc., of RSV genes or gene segments in the present invention may include one or more parts or all of the nonimmunogenic portions of the NS1, NS2, N, P, M, SH, and L genes or the M2-1 open reading frame or the G and F genes. In addition, human RSV cis-acting sequences, such as promoters or transcription signals, may be replaced, for example, with their bovine RSV counterparts. Conversely, means for producing viable attenuated bovine RSV may be provided by inserting human attenuating genes or cis-acting sequences into a bovine RSV genome or antigenomic background.

[0091] Therefore, infectious recombinant RSV intended for administration to humans is human RSV that has been modified to include genes, for example, from bovine RSV or PIV, for the purpose of attenuation. For example, insertion of a gene or gene segment from PIV provides a bivalent vaccine against both PIV and RSV. Alternatively, another respiratory pathogen, such as a heterologous RSV species, subgroup or strain, or PIV, may be modified to include genes encoding epitopes or proteins that induce protection against human RSV infection, for example. For example, bovine RSV obtained by replacing the bovine glycoprotein gene with the human RSV glycoprotein gene, carrying the human RSV surface glycoprotein, and having its replication ability in the human host limited due to the remaining bovine genetic background, may induce a protective immune response in humans against human RSV strains.

[0092] The ability to analyze and incorporate other types of attenuating mutations into infectious RSV for vaccine development extends to a broader range of targeted changes in RSV clones. For example, arbitrary RSV genes that are not inherently for proliferation can be removed or otherwise modified to obtain desired effects on pathogenicity, pathogenesis, immunogenicity, and other phenotypic features. Furthermore, a variety of other genetic modifications can be made to recombinant RSV genomes or antigenomes to incorporate infectious recombinant RSV alone or together with one or more attenuating point mutations adopted from biologically induced mutant RSV.

[0093] The term "heterogene" as used herein refers to genes taken from different RSV strains or types or non-RSV sources. These heterogenes may, in whole or in part, alter gene sequences, removed gene duplications, RSV genome promoters replaced with their antigenomic counterparts, removed or substituted gene portions, or deleted whole genes. Different or additional modifications in the sequences may be made to facilitate manipulation, such as the insertion of unique restriction sites in various intergene regions (e.g., a unique Stul site between the G and F genes) or elsewhere. Uncoding gene sequences may be removed to increase the volume for foreign sequence insertions.

[0094] Deletions, insertions, substitutions, and other mutations involving alterations of the viral gene or entire gene segment in the recombinant RSV of the present invention result in highly stable vaccine candidates, which is particularly important in immunosuppressed individuals. Many of these mutations lead to attenuation of the resulting vaccine strain, while others identify different types of desired phenotypic changes. For example, certain viral genes encoding proteins that specifically interfere with host immunity are known (see, e.g., Kato et al., EMBO. J. 16:578-87 (1997)). Removal of such genes in vaccine viruses is expected to enhance pathogenicity and pathogenesis and / or improve immunogenicity.

[0095] Other mutations within the RSV of the present invention are replacements of the 3' end of the genome, in its counterpart from the antigenome, relating to changes in RNA replication and transcription. Furthermore, intergeneric regions (Collins et al., Proc. Natl. Acad. Sci. USA 83:4594-4598 (1986)) may be shortened or lengthened or their sequence content altered in the manner described herein, and naturally occurring gene duplications (Collins et al., Proc. Natl. Acad. Sci. USA 84:5134-5138 (1987)) may be removed or replaced with different intergeneric regions.

[0096] In another embodiment, the sequence around the translation initiation site (preferably including the nucleotide at position -3) of a selected RSV gene is modified, either alone or in combination with the introduction of an upstream start codon, thereby regulating RSV gene expression by specifying upregulation or downregulation of translation.

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

[0098] Further RSV clones within the present invention incorporate modifications to transcriptional GE signals. For example, RSV clones are provided in which the GE signals of the NS1 and NS2 genes are substituted or mutated with respect to their N genes, resulting in a decrease in readthrough mRNA levels and an increase in protein expression from downstream genes. The resulting recombinant viruses exhibit increased proliferation dynamics and increased plaque size, and, as an example, modification of cis-acting regulatory elements in the RSV genome provides a modification of RSV proliferation properties.

[0099] In another embodiment, G protein expression can be increased by modification of G mRNA. G proteins are expressed in both membrane-bound and secreted forms, the latter of which is expressed by translation initiation at an initiation site within the G gene translation open reading frame. The secreted form can account for about half of the expressed G protein. Deletion of an internal initiation site (e.g., by sequence modification or deletion) alone or in conjunction with sequence modifications of the upstream initiation site results in desired changes in G protein expression. Deletion of the secreted form of a G protein also improves the quality of the host immune response to exemplary recombinant RSV, as the soluble form of the G protein is thought to act as a “decoy” for neutralizing antibody capture. Furthermore, soluble G proteins have been associated with enhancement of immunopathology by preferential stimulation of the Th2 bias response.

[0100] In related embodiments, RSV gene expression levels can be modified at the transcriptional level. In some embodiments, the location of a selected gene on the RSV gene map can be changed to a location more proximal to or distal to the promoter, thereby reducing or increasing the efficiency of gene expression, respectively. This embodiment can achieve regulation of specific gene expression to a 2x, more commonly 4x, up to 10x, or more decrease or increase in gene expression compared to wild-type levels. For example, the NS2 gene (second in order on the RSV gene map) is replaced with the SH gene (sixth in order), resulting in a predicted decrease in NS2 expression. The increase in selected RSV gene expression due to the positional change can be achieved up to 10x, 30x, 50x, 100x, or more, often accompanied by a reciprocal, balanced decrease in the expression level of the substituted gene.

[0101] In one exemplary embodiment, the F and G genes, individually or together, can be transposed to more proximal or distal regions of the promoter within the (recombinant) RSV gene map to achieve higher or lower levels of gene expression, respectively. These and other transposition changes result in novel RSV clones with attenuated phenotypes, for example, by reducing the expression of a select viral protein involved in RNA replication. In yet another embodiment, RSV useful for vaccine formulations can be easily modified by accommodating antigenic drift in the circulating virus. Generally, such modification is to the G and / or F proteins. The entire G or F gene or a segment encoding a specific immunogenic region is incorporated into the RSV genome or antigenomic cDNA by substitution of the corresponding region in the infectious clone or by adding more than one copy of the gene, resulting in several antigenic forms.

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

[0103] The infectious RSV clones of the present invention may be manipulated by the methods and compositions disclosed herein to increase their immunogenicity and induce a greater level of protection provided by infection with wild-type RSV or an incompletely attenuated parental virus or clone. For example, immunogenic epitopes from heterologous RSV strains or types or non-RSV sources such as PIVs may be added to appropriate nucleotide changes in the polynucleotide sequence encoding the RSV genome or antigenome. Recombinant RSVs may also be manipulated for the identification and removal of epitopes associated with undesirable immunopathological reactions (e.g., by amino acid insertions, substitutions, or deletions). In other embodiments, additional genes may be inserted into or placed adjacent to the RSV genome or antigenome under the control of an independent set of transcriptional signals. The target genes may include, but are not limited to, those encoding cytokines (e.g., IL-2 to IL-15, particularly IL-2, IL-6, and IL-12), gamma-interferon, and proteins rich in cytokines (e.g., IL-2 to IL-15, particularly IL-2, IL-6, and IL-12), gamma-interferon, and T helper cell epitopes. Additional proteins may be expressed as separate proteins or as chimeric proteins engineered from a second copy of one RSV protein, such as SH. This provides the ability to modify and improve the immune response to RSV, both quantitatively and qualitatively.

[0104] In addition to the modifications described above to recombinant RSV, different or additional modifications in the RSV clone may be made to facilitate manipulation, such as the insertion of unique restriction sites into various intergenetic regions (e.g., a unique Stul site between the G and F genes) or anywhere else. Uncoding gene sequences may be removed to increase the volume for foreign sequence insertions.

[0105] The introduction of mutations into infectious RSV clones can be achieved by a variety of well-known methods. “Infectious clone” refers to a synthesized or other cDNA or its product that can be transcribed into a genome or antigenomic RNA capable of producing infectious viruses. The term “infectious” refers to a virus or viral structure that can replicate in cultured cells or animals or human hosts to produce progeny viruses or viral structures of the same activity. Therefore, mutations can be introduced into a cDNA copy of a genome or antigenomic by conventional techniques (e.g., site-directed mutagenesis). The use of antigenomic or genomic cDNA subfragments to construct a complete antigenomic or genomic cDNA is well known to those skilled in the art, and has the advantage that each region can be manipulated separately (smaller cDNAs are easier to manipulate than larger ones) and then readily assembled into a complete cDNA. Therefore, a complete antigenomic or genomic cDNA or any of its subfragments can be used as a template for oligonucleotide-directed mutagenesis. Mutant subfragments can then be assembled into a complete antigenomic or genomic cDNA. Mutations can range from single nucleotide changes to large cDNA fragments containing one or more genes or genomic regions.

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

[0107] Accordingly, also described herein are isolated polynucleotides that encode mutant viruses, construct genomes or antigenomes, express genomes or antigenomes, or encode various proteins useful for the in vitro synthesis of recombinant RSV. Polynucleotides containing any sequence of the sequence numbers described herein are included in the present invention. Further encompassing are sequences consisting of or essentially containing any of the sequences, polynucleotides having sequences that are at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any identity %) in between, and polynucleotides that hybridize with or are complements of the molecules.

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

[0109] In related embodiments of the present invention, compositions (e.g., isolated polynucleotides and vectors incorporating RSV-coding cDNA) and methods for producing isolated infectious recombinant RSV carrying attenuating mutations are provided. Included in these embodiments of the present invention are novel, isolated polynucleotide molecules and vectors incorporating such molecules, comprising the RSV genome or antigenome modified as described herein. Also provided are identical or different expression vectors comprising isolated polynucleotide molecules encoding one or more RSV proteins. These proteins are also expressed directly from the genome or antigenomic cDNA. The vectors are preferably expressed or co-expressed in cells or cell-free lysates to produce mutant RSV particles or viral component particles.

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

[0111] The above methods and compositions for producing attenuated recombinant RSV variants yield infectious viruses or viral component particles or derivatives thereof. Infectious viruses are comparable to and similarly infectious to reliable RSV viral particles. They can directly infect fresh cells. Infectious viral component particles are generally smaller components of viral particles that can initiate infection under appropriate conditions. For example, a nucleocapsid containing genome or antigenomic RNA and N, P, L, and M2-1 proteins is an example of a viral component particle that can initiate infection once introduced into the cytoplasm of a cell. Viral component particles provided in this invention include viral particles lacking one or more proteins, protein segments, or other viral components that are not essential for infectivity.

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

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

[0114] In another aspect of the present invention, recombinant RSV strains can be used as “vectors” for protective antigens against other pathogens, particularly respiratory pathogens such as parainfluenza virus (PIV). For example, recombinant RSV having the T1166I mutation can be engineered to incorporate a sequence encoding a protective antigen from PIV in order to produce an infectious, attenuated vaccine virus.

[0115] In one embodiment, the present invention comprises a pharmaceutical composition comprising an immunoassayable amount of a recombinant RSV variant encoded by the above-mentioned isolated polynucleotide molecule. In one embodiment, the present invention comprises a method of vaccinating a subject or inducing an immune response, comprising administering the pharmaceutical composition. The composition may be administered by any suitable method, including but not limited to injection, aerosol delivery, nasal spray, nasal drop, oral administration, or topical application. In one embodiment, it may be administered by injection, aerosol delivery, nasal spray, or nasal drop. The composition may be administered intranasally, subcutaneously, or intramuscularly. In one embodiment, it may be administered intranasally. The methods and routes of administration are described in further detail below.

[0116] In a related embodiment, the present invention provides a method for stimulating the immune system of an individual to induce an immune response to RSV in a mammalian subject. The method comprises administering an immunogenic preparation of attenuated RSV in an immunosufficient or effective amount in a physiologically acceptable carrier and / or adjuvant.

[0117] In one embodiment, the present invention includes a viable attenuated RSV vaccine comprising a recombinant RSV variant encoded by the isolated polynucleotide molecule. In another embodiment, the present invention includes a pharmaceutical composition comprising the RSV vaccine. In a related embodiment, the present invention includes a method for producing a vaccine, comprising expressing the isolated polynucleotide molecule.

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

[0119] To select candidate vaccine viruses from the hosts of the recombinant RSV strains provided herein, criteria for viability, efficient in vitro replication, in vivo attenuation, immunogenicity, and phenotypic stability are determined by well-known methods. The viruses most desirable for the vaccine of the present invention must maintain viability, replicate sufficiently under conditions acceptable in vitro to enable vaccine production, have a stable attenuated phenotype, be well-tolerated, replicate in an immunized host (even at low levels), and efficiently induce in the vaccine the production of a sufficient immune response to confer protection against serious illness caused by subsequent infection with wild-type viruses. Clearly, none of the RSV variants known and reported to date meet all of these criteria. In fact, contrary to predictions from results reported with known attenuated RSVs, the viruses of the present invention are not only more viable and attenuated than the aforementioned variants, but are also genetically more stable in vivo than the previously tested variants.

[0120] Numerous cell lines capable of RSV growth can be used to propagate RSV virus for vaccine use and other purposes. RSV grows in a variety of human and animal cells. Preferred cell lines for propagating attenuated RSV for vaccine use include DBSFRhL-2, MRC-5, and Vero cells. The highest viral yield is usually achieved in epithelial cell lines such as Vero cells. Cells are generally inoculated with the virus with an infection efficiency in the range of about 0.001 to 1.0 or higher and cultured under conditions acceptable for viral replication, e.g., at about 30 to 37°C, for about 3 to 10 days or the time required for the virus to achieve an appropriate titer. Temperature-sensitive viruses are often grown using 32°C as the acceptable temperature. The virus is taken from the cell culture, and the cellular components can be separated by generally known purification methods, e.g., centrifugation, and further purified as desired by methods well known to those skilled in the art.

[0121] As described herein, attenuated RSV 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. In in vitro assays, modified viruses capable of propagating attenuated, biologically induced, or recombinant RSV are tested for temperature sensitivity of viral replication or “ts phenotype” and for small plaque phenotype. Modified viruses are further tested in animal models of RSV infection. Various animal models (e.g., mouse, cotton rat, and primate) have been described and are known to those skilled in the art.

[0122] Based on the above description and the following examples, the present invention also provides isolated, infectious RSV compositions for vaccine use. Attenuated viruses, which are components of vaccines, are isolated and generally purified forms. Isolation means RSV present outside of the natural environment of wild-type viruses, such as the nasopharynx of an infected individual. More generally, isolation is intended to include attenuated viruses as components of cell cultures or other artificial media. For example, the attenuated RSV of the present invention can be produced by an infected cell culture, isolated from the cell culture and added to a stabilizer.

[0123] The RSV vaccine of the present invention contains, as an active ingredient, an effective amount of RSV as an immunogen produced as described herein. Biologically induced or recombinant RSV may be used directly in the vaccine formulation. Biologically induced or recombinantly modified viruses may be introduced into a host together with a physiologically acceptable carrier and / or adjuvant. Useful carriers are well known in the art and include, for example, water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid, etc. The resulting aqueous solution is packaged for use as is, in a frozen form to be thawed before use, or lyophilized, as described above, and the lyophilized preparation is combined with a sterile solution before administration. The composition includes pharmaceutically acceptable adjuvants as needed to approximate physiological conditions, which include, but are not limited to, pH adjusters and buffers, tonicity adjusters, and wetting agents, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sucrose, magnesium sulfate, phosphate buffer, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, sorbitan monolaurate, and triethanolamine oleate. Acceptable adjuvants include incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum, which are substances well known to those skilled in the art. A preferred adjuvant is also Stimulon. TM QS-21 (Aquila Biopharmaceuticals, Inc., Worchester, Mass.), MPL TM This product contains (3-0-deacylated monophosphoryl lipid A; RIBI ImmunoChem Research, Inc., Hamilton, Mont.) and interleukin-12 (Genetics Institute, Cambridge, Mass.).

[0124] Immunization with the RSV vaccine composition causes the host to respond to the vaccine by producing antibodies specific to RSV viral proteins, such as F and G glycoproteins. Furthermore, a spontaneous and cell-mediated immune response is induced, which provides antiviral effectors and can modulate the immune response. As a result of vaccination, the host becomes at least partially or completely immunized to RSV infection or becomes resistant to the development of moderate or severe RSV disease, particularly in the lower respiratory tract.

[0125] The host to which the vaccine is administered can be any mammal that is susceptible to infection with RSV or a closely related virus and capable of producing a protective immune response to the antigen of the vaccinated strain. Therefore, suitable hosts include humans, non-human primates, cattle, horses, pigs, sheep, goats, rabbits, rodents, such as mice or cotton rats. Accordingly, the present invention provides a method for producing vaccines for diverse human and animal use.

[0126] The vaccine composition containing the attenuated RSV of the present invention is administered to subjects susceptible to or otherwise at risk of RSV infection in an “immunogen-effective dose” sufficient to induce or enhance the individual’s ability to respond to RSV. The RSV vaccine composition may be administered by any suitable method, including but not limited to injection, aerosol delivery, nasal spray, nasal drops, oral administration, or topical application. In human subjects, the attenuated virus of the present invention is administered according to a well-established human RSV vaccine protocol (Karron et al. JID 191:1093-104, 2005). That is, an immunogen-effective dose of the RSV vaccine is administered intranasally to adults or children in a volume of generally 0.5 ml of physiologically acceptable diluent or carrier. This has advantages in terms of simplicity and safety compared to non-enteral immunization with non-replicating vaccines. It also provides direct local respiratory tract immune stimulation, which plays a major role in resistance to RSV. Furthermore, this vaccination method efficiently bypasses the immunosuppressive effects of RSV-specific maternal-derived serum antibodies, which are generally observed in very young children. Additionally, non-enteral administration of RSV antigen can be associated with immunopathological complications, which have not been observed with viable viruses.

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

[0128] For all subjects, the precise dose, timing, and frequency of RSV vaccine administration are determined by various factors, including the patient's health status and weight, administration method, and formulation properties. The dose is generally approximately 3.0 log of the virus per patient. 10 ~Approximately 6.0 log 10 Plaque-forming units ("PFUs") or larger, more generally about 4.0 log per patient.10 ~5.0 log 10 It is a PFU virus. In certain embodiments, about 5.0 log 10 ~6.0 log 10 PFU per patient is administered during infancy, such as 1 to 6 months, and one or more additional immunizing doses can be administered 2 to 6 months or more later. In other embodiments, the young infant receives about 5.0 log 10 ~6.0 log 10 PFU doses at approximately 2 months, 4 months, and 6 months, which are the recommended administration seasons for many other pediatric vaccines. In yet other embodiments, additional immunizing doses are administered at approximately 10 - 15 months. In any case, the vaccine formulation should provide a sufficient amount ("effective amount") of the attenuated RSV of the present invention for efficient stimulation or induction of an anti - RSV immune response.

[0129] In certain embodiments, the vaccine comprises an attenuated recombinant RSV virus that elicits an immune response against a single RSV strain or antigenic subgroup, e.g., A or B, or multiple RSV strains or subgroups. In this regard, rRSV can be combined in a vaccine formulation with other RSV vaccine strains or subgroups having different immunogenic characteristics for more effective protection against a single or multiple RSV strains or subgroups. They can be administered in a vaccine mixture or separately in a co - administration protocol for inducing more effective protection against a single RSV strain or multiple RSV strains or subgroups.

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

[0131] In one embodiment, neonates and infants are given multiple doses of RSV vaccine to induce a sufficient level of immunity. Doses are administered within the first month of life and then at regular intervals throughout childhood, such as 2 months, 4 months, 6 months, 1 year, and 2 years, as long as necessary to maintain a sufficient level of protection against natural RSV infection. In another embodiment, adults particularly susceptible to frequent or severe RSV infections, such as healthcare workers, childcare workers, infants, the elderly, and family members of individuals with cardiopulmonary failure, are given multiple doses of RSV vaccine to establish and / or maintain a protective immune response. The level of induced immunity can be monitored by measuring the amount of neutralizing secretion and serum antibodies, and the dose may be adjusted or vaccination repeated as needed to maintain the desired level of protection. Furthermore, different vaccine viruses are indicated for administration to different recipient groups. For example, engineered RSV strains expressing additional proteins rich in cytokines or T-cell epitopes may be particularly advantageous for adults rather than infants. The vaccine produced according to the present invention can be combined with viruses of other subgroups or strains of RSV to provide protection against multiple RSV subgroups or strains, or a selective gene segment encoding a protective epitope, for example, in these strains can be manipulated into a certain RSV clone as described herein. In such embodiments, different viruses can be mixed and administered simultaneously or provided in separate preparations and administered separately. For example, the F glycoproteins of two RSV subgroups differ by only about 11% in amino acid sequence, and this similarity is the basis for the cross-protective immune response observed in animals immunized with RSV or the F antigen and then attacked with a different strain. Therefore, immunization with a single strain may also provide protection against different strains of the same or different subgroups.

[0132] The attenuation level of a vaccine virus can be determined, for example, by quantifying the amount of virus present in the respiratory tract of an immunized host and comparing it to the amount of wild-type RSV or other attenuated RSV strains evaluated as candidate vaccine strains. For example, the attenuated virus of the present invention exhibits a greater degree of limitation of replication in the upper respiratory tract of highly susceptible hosts such as chimpanzees compared to the replication level of the wild-type virus, for example, 10 to 1000 times less. To further reduce nasal discharge associated with viral replication in the upper respiratory tract, an ideal vaccine candidate virus must exhibit limitation of replication levels in both the upper and lower respiratory tracts. However, the attenuated virus of the present invention must be sufficiently infectious and immunogenic in humans to provide protection in vaccinated individuals. Methods for determining RSV levels in the nasopharynx of an infected host are well known in the literature. Samples are obtained by aspiration or nasopharyngeal secretion washing, and the virus is quantified by tissue culture or other laboratory methods. 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. 41:238-247 (1973). The virus can be easily identified in the nasopharynx of host animals such as chimpanzees.

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

[0134] The natural RSV genome generally contains negative-sense polynucleotide molecules, which, via complementary viral mRNA, encode 11 viral proteins, namely the non-structural proteins NSL and NS2, N, P, matrix (M), small hydrophobic (SH), glycoprotein (G), fusion (F), M2-1, M2-2, and L, as described substantially in Mink et al., Virology 185: 615-624 (1991), Stec et al., Virology 183: 273-287 (1991), and Connors et al., Virol. 208: 478-484 (1995). For the purposes of the present invention, the recombinant RSV genome or antigenome of the present invention requires only the genes or parts thereof necessary to make the virus or viral component particles encoded thereby infectious. Furthermore, the genes or parts thereof may be provided by more than one polynucleotide molecule, i.e., the genes may be provided by complementation from another nucleotide molecule, for example.

[0135] Recombinant RSV means RSV or RSV-like viruses or viral component particles that are directly or indirectly derived from or propagated by a recombinant expression system or viruses or viral component particles produced therefrom. The recombinant expression system uses a recombinant expression vector that includes a manipulably bound transcriptional unit comprising at least one or more genetic elements having a regulatory role in RSV gene expression, e.g., a promoter, a structural or coding sequence transcribed into RSVRNA, and appropriate transcription start and stop sequences.

[0136] To produce infectious RSV from a cDNA-expressed genome or antigenome, the genome or antigenome is co-expressed with RSV proteins necessary to (i) produce RNA-replicable nucleocapsids and (ii) make the progeny nucleocapsids eligible for both RNA replication and transcription. Transcription by the genomic nucleocapsids provides other RSV proteins and initiates productive infection. Further RSV proteins necessary for productive infection can also be supplied through co-expression.

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

[0138] The N, P, L, and M2-1 proteins can be encoded by one or more expression vectors identical or separable from those encoding the genome or antigenome, and various combinations thereof. Additional proteins encoded by the vector itself or by vectors encoding the N, P, L, or M2-1 proteins or the complete genome or antigenome may optionally be included. Expression of the genome or antigenome and proteins from a transfect plasmid can be achieved, for example, by infectious transfection or transduction in a T7 RNA polymerase expression system, for example, by placing each cDNA under the control of a T7 RNA polymerase promoter, provided by a recombinant vaccinia virus MVA strain expressing T7 RNA polymerase (Wyatt et al., Virology, 210:202-205 (1995)). Viral proteins and / or T7 RNA polymerase can also be provided by transfection of transformed mammalian cells or pre-formed mRNA or proteins.

[0139] In summary, the materials, information, and methods described herein provide a series of attenuated strains having a stepwise attenuation phenotype and provide guidance for selecting a suitable vaccine candidate strain based on clinical benchmarks.

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

[0141] The publications, sequences, or other citations disclosed below and elsewhere in this Specified Publication are incorporated herein by reference in their entirety to the extent that they do not contradict this Disclosure. U.S. Provisional Application 62 / 399,133, not filed September 23, 2016, entitled “Improved codon-pair-deoptimized vaccine candidates for human respiratory syncytial virus”; U.S. Provisional Application 62 / 400,476, filed September 27, 2016, entitled “Vaccine candidates for respiratory syncytial virus (RSV) having attenuated phenotypes”; and U.S. Publication 2015-036862, entitled “Attenuation of human respiratory syncytial virus by genome scale codon-pair deoptimization,” are incorporated herein by reference in their entirety. [Examples]

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

[0143] Virus collection and titer measurement Vero cells were scraped onto tissue culture medium, vortexed for 30 seconds, purified by low-speed centrifugation, and flash-frozen. The viral titer of the purified fluid was determined by an immunoplaque assay of Vero cells at 32°C.

[0144] Virus stocks were prepared by scraping infected cells onto culture medium, vortexing for 30 seconds, and purifying the supernatant by centrifugation. Virus aliquots were flash-frozen and stored at -80°C. Viral titers were determined by a plaque assay using Vero cells with a 0.8% methylcellulose stratified bed. After incubation at 32°C for 10–12 days, plates were fixed with 80% cold methanol, and plaques were visualized by immunostaining with a cocktail of 3RSV-specific monoclonal antibodies. Titer was expressed as pfu / ml. Viral RNA was isolated from the entire virus stock, and viral genome sequencing was performed from duplicated RT-PCR fragments by Sanger sequencing to confirm that the recombinant virus genome sequence was correct and free of indeterminate mutations. The only sequences not directly confirmed for each genome were at the positions of the outermost primers, i.e., nucleotides 1–23 and 15,174–15,222.

[0145] Ion Torrent Deep Sequencing Purified viral RNA from the purified culture medium was copied into eight duplicate fragments spanning the viral genome. The library was prepared according to the Ion Torrent protocol, loaded onto a semiconductor sequencing chip, and sequenced using a Personal Genome Machine (Ion Torrent). Nucleotide variants were called if they occurred at a mean read depth of 1000 and a p-value < 10⁻⁷ (quality score > 70) and a factor of > 50.

[0146] Viral RNA was extracted using the Qiagen Viral RNA Extraction Kit to obtain aliquots of the virus, which were then passaged through temperature stress tests in Min_L or Min_FLC. The viral RNA was reverse transcribed using Superscript II RT (Life Technologies) as recommended by the manufacturer. The cDNA was then amplified by PCR using RSV-specific primers for eight duplicate fragments covering the entire viral genome and pfx DNA polymerase enzyme (Life Technologies). Each PCR product was purified using the QIAquick PCR Purification Kit (Qiagen).

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

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

[0149] Next, approximately 100 ng of the DNA library was added to the PCR mix using Platinum High Fidelity DNA Polymerase Master Mix (Life Technologies), followed by PCR amplification at 95°C for 10 minutes for 2 cycles, then at 95°C for 30 seconds for 2 cycles, at 58°C for 30 seconds, and at 72°C for 30 seconds. The PCR product was further purified in 1.8 volumes of Agencourt as described above, and the DNA was obtained using a magnetic rack. The DNA was quantified using the Qubit system (Invitrogen).

[0150] Approximately 70 million DNA molecules in 1 ml of PCR solution were mixed with Ion sphere particles (ISPs) (Ion Torrent) at a fixed ratio (0.5-1.0) in the presence of PCR reaction mix and oil (Ion Torrent) to form droplets of tens of millions of emulsion particles. These droplets were passed through a OneTouch (Ion Torrent) sealed capillary PCR plate, allowing for continuous passage of liquid and particles through the plate, thus enabling emulsion PCR amplification. The ISPs were collected in a pair of collection tubes using OneTouch centrifugation. At the end of OneTouch emulsion PCR, the collection tubes were centrifuged at 15,000 g for 3 minutes to remove most of the supernatant. The ISPs were washed once with 1 ml of washing buffer (Ion Torrent) and centrifuged at 15,500 g for 3 minutes to remove most of the supernatant. ISPs containing amplified DNA were separated from ISPs without DNA using Dynabeads® MyOne TM The IPS was further enriched by incubation with streptavidin C1 magnetic beads in a rotating rack at room temperature for 10 minutes. The enriched IPS was obtained by placing the tube in a magnetic rack for 2 minutes, washing it twice by pipetting with 0.2 ml of washing buffer, placing it in the magnetic rack for 2 minutes, and discarding the supernatant. The IPS was obtained from Dynabeads® MyOne. TMElution was performed by incubating Streptavidin C1 magnetic beads with 0.4 ml of 0.125 N NaOH and 0.1% Tween 20 for 7 minutes in a rotating rack at room temperature. The eluted ISP was washed twice with wash buffer and centrifuged at 15,500 g for 4 minutes to remove most of the supernatant. The ISP was resuspended by pipetting and Dynabeads® MyOne TM To remove the last traces of the beads, it was placed on a magnetic rack for 2 minutes.

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

[0152] The DNA sequences were analyzed using VariantCaller 3.2 software from Ion Torrent on the Ion Torrent Server against the Min_FLC or Min_L control sequences. The analysis pipeline was set to the default somatic variant configuration. Nucleotide variants were called as described above with a variant having an average read depth of 1000× and a P-value < 10 -7If a quality score > 70 occurred and the multiplication factor was > 50, it was called. The raw read data was also manually verified using the genome browser IVG (The Broad Institute).

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

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

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

[0156] Next, the end-repair DNA was annealed for 15 minutes at 25°C in a reaction involving a blunt-end adapter, the adapter, buffer, ATP, and ligase. The ligase was inactivated at 65°C for 10 minutes. Finally, an exonuclease step at 37°C for 1 hour was included to remove all failed ligation products. The SMRTbell DNA template library was then purified 3-fold using AMPure PB beads as described above.

[0157] After purification, the SMRTbell library template was sequenced in SMRT cells using the PacBio DNA Polymerase Binding Kit P6 on a PacBio RSII instrument. Each sample library was sequenced in two SMRT cells using MagBead loading with a video acquisition time of 240 minutes.

[0158] The data was analyzed using CluCon software (https: / / github.com / mpsbpbi / clusteringConsensus). All read data were aligned with the control Min_L sequence. Only read data covering 99% of the complete target (8161 bases or more) was analyzed; this resulted in an average of 32,738 near-full-length read data per read point (minimum read data 24,131, maximum read data 41,118). The algorithm identifies variant locations by testing the alignment and finding locations where low frequencies were observed that could not be statistically explained by accidental noise. The complete phase haplotype is then estimated by matching what was sequenced in each near-full-length read data at the variant location. Statistical tests are used to discard “noisy” haplotypes or those that can be simply explained by other truly observed haplotypes altered by sequencing noise.

[0159] Determination of temperature cutoff for CPD rRSV The ts phenotype of each rRSV virus was evaluated by plaque formation efficiency at 32°C, 35°C, 36°C, 37°C, 38°C, 39°C, and 40°C. Plaque assays were performed in duplicate using Vero cells and incubated in sealed casks in temperature-controlled water baths at various temperatures as described above. Cutoff temperature (T SH ) is defined as the minimum limit temperature at which there is a reduction in plaque count compared to 32°C that is more than 100 times greater than what is observed in wt RSV at these two temperatures.

[0160] In vitro virus replication dynamics. Multicycle and monocycle proliferation dynamics were analyzed in 6-well plates at both 32°C and 37°C using a confluent monolayer of Vero cells.

[0161] In multi-cycle proliferation dynamics experiments, Vero cells were infected with the described virus at a duplicate MOI of 0.01 pfu / cell. From day 0 to 14, the virus was obtained by scraping infected cells onto culture medium, followed by vortexing for 30 seconds, and the supernatant was purified by centrifugation. The virus inoculation material and daily aliquots were flash-frozen and stored at -80°C. Viral titers were determined by plaque assay as described above.

[0162] In a single-cycle growth dynamics experiment, Vero cells in 3 wells of a 6-well plate were infected with the described virus at 32°C or 37°C at an MOI of 3 pfu / well. Cell-associated RNA was collected from one well every 4 hours from 4 to 24 hours post-infection using an RNeasy mini-kit (Qiagen) according to the manufacturer's instructions. For Western blot analysis, total cell lysate was collected in NuPage LDS sample buffer (Life Technologies) and then homogenized using a QIAshredder spin column (Qiagen). Finally, the last well was used for virus acquisition and viral titer determination as described above.

[0163] Strand-specific rRSV RNA quantification As previously described, viral negative sense (genomic) and positive sense (mRNA and antigenomic) RNA were specifically quantified using cell-associated RNA derived from single-cycle replication experiments. qPCR results were analyzed using the comparative threshold cycle (ΔCt) method, normalized to 18S rRNA, and then expressed as the log2 increase factor above the control sample.

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

[0165] 1 μg of DNA-digested RNA was reverse transcribed using Superscript III (Life Technologies) in a 20 μl reaction using tagged first-strand primers specific to the genome or antigenome / mRNA. After 5-fold dilution, each cDNA was tripletically amplified with tag-specific primers, second-gene-specific primers, and probes. Therefore, only cDNAs containing tagged RT primer sequences were amplified. The probe sequences were RSV gene-specific. To normalize the results, 18S rRNA was quantified in parallel using the Taqman assay (Applied Biosystems) with first-strand cDNA produced with random primers and the standard 18S rRNA. qPCR results were analyzed using the comparative threshold cycle (ΔCt) method, normalized for 18S rRNA, and then expressed as log2 increase factors exceeding Min_L at 4 hours, except for wt L quantification, where the increase factor exceeding wt was expressed as the increase factor exceeding wt at 4 hours. A negative control without first-strand primers was included in each strand-specific qPCR to demonstrate the absence of nonspecific priming during first-strand cDNA synthesis.

[0166] Western blot analysis.Cell lysates prepared from the above single-cycle infection experiment were separated on a NuPAGE 4-12% Bis-Tris SDS-PAGE gel using MES electrophoresis buffer (Life Technologies) in parallel with Odyssey Two-Color Protein Molecular Weight Marker (Li-Cor). 30 μg of protein was transferred to a PVDF-F membrane (Millipore) in 1× NuPAGE buffer. The membrane was blocked with Odyssey barrier buffer (Li-Cor) and incubated with primary antibody in the presence of 0.1% Tween-20. The primary antibodies and dilutions used were as follows: mouse anti-RSVN, P, G, F, and M2-1 monoclonal antibodies (1:1,000) were purchased from Abcam; rabbit polyclonal antiserum recognizing both NS1 and NS2 was produced by peptide immunization of rabbits (Abgent) with a synthetic peptide representing the C-terminal 14 amino acids of NS2 (the last 4 amino acids of the C-terminuses of NS2 and NS1 are identical, which likely explains cross-reactivity); rabbit anti-GAPDH polyclonal antibody (1:200) was used as a loading control (Santa-Cruz Biotechnologies, Inc.). The secondary antibodies used at a 1:15,000 dilution were goat anti-rabbit IgG IRDye 680 (Li-Cor) and goat anti-mouse IgG IRDye 800 (Li-Cor). Membranes were scanned using the Odyssey® Infrared Imaging System. The data were analyzed using Odyssey software, version 3.0 (Li-Cor). Fluorescence signals were background-corrected for quantification of the target identified RSV protein. The numerical values ​​represent the median fluorescence intensity of each protein band.

[0167] Plaque size determination. Viral plaque size was determined using a plaque assay with Vero cells in a 24-well plate. A Vero cell monolayer was inoculated into a pre-tited and sequenced virus stock at 30 pfu / well. After 2 hours of adsorption, a 0.8% methylcellulose strut was added to each well. After incubation at 32°C for 12 days, the plate was fixed with 80% cold methanol. The wells were then incubated for 1 hour in a cocktail of three RSV-specific monoclonal antibodies (Bukreyev et al. 2001) and a barrier buffer (Odyssey buffer, Licor). After washing with barrier buffer, the plaques were stained with goat anti-mouse IRdye 680LT (Licor) secondary antibody, and the plaques were visualized using the Odyssey® Infrared Imaging System. The images were analyzed using Image J, and the area of ​​more than 1000 plaques per virus was measured and represented in pixels 2. The distribution of viral plaque sizes was compared for statistical significance using the Kolmogorov-Smirnov test, followed by the Bonferroni correction (Prism 6.0, GraphPad). For the dataset, statistical significance was considered only at p ≤ 0.05.

[0168] Evaluation of CPD rRSV replication in mice and hamsters The animal studies were approved by the NIAID Animal Care and Use Committee and conducted using the method described above.

[0169] The entire animal study was approved by the National Institutes of Health (NIH) Institutional Animal Care and Use Committee (ACUC). CPD virus replication was evaluated in the upper and lower respiratory tracts of 6-week-old BALB / c mice as previously described. In groups of 20 mice, under isoflurane anesthesia, 10 6Mice were inoculated intranasally with wt rRSV, Min_L, M2-1[A73S], M2-1[N88K], or NPM2-1[N88K] L of pfu. On days 4 and 5, 8 mice from each group were sacrificed by carbon dioxide inhalation. The remaining 4 mice in each group were sacrificed on day 10. Nasal turbinates (NT) and lung tissues were collected and homogenized separately in Leibovitz (L)-15 medium containing 1×SPG, 2% L-glutamine, 0.06 mg / mL ciprofloxacin, 0.06 mg / mL clindamycin phosphate, 0.05 mg / mL gentamicin, and 0.0025 mg / mL amphotericin B. Virus titers were determined in duplicate by incubating with Vero cells at 32 °C as described above. The virus detection limits were 100 and 50 pfu / g in NT and lung specimens, respectively.

[0170] Replication of the CPD virus was evaluated in the upper and lower respiratory tracts of 6-week-old Golden Syrian hamsters, and immunogenicity was also tested. On day 0, a group of 18 hamsters was inoculated intranasally with 10 6 pfu of wt rRSV, Min_L, M2-1[A73S], M2-1[N88K], or NPM2-1[N88K] L under methoxyflurane anesthesia.

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

[0172] Two days before immunization and on day 26 after immunization, blood from 9 hamsters / group was collected for serum collection and measurement of RSV antibody titers. On day 31, 10 6PFU wt rRSV was administered intranasally. Three days after administration, the hamsters were sacrificed by carbon dioxide inhalation. NT and lung tissue were collected, and wt rRSV titers were determined by duplication by incubation in Vero cells at 32°C, as described above.

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

[0174] The mutations were traced to the M2-1 protein (PDB ID 4C3D), the transcription termination structure of human RSV, using the SYBYL program (Certara, St. Louis, MO). The mutant or wt RSVM2-1 was then traced to the TIP3P water molecule and Na using the VMD program. + and Cl - The counterions were explicitly solvated. All-atom, isobaric-isothermal (1 atm, 310K) molecular dynamics simulations were performed with periodic boundary conditions using the NAMD program (v.2.9) on the Biowulf Linux cluster at the National Institutes of Health, Bethesda, MD (http: / / hpc.nih.gov), followed by heating in 10K increments up to 310K, after explicit solvation and energy minimization. Electrostatic interactions were calculated using Particle-Mesh Ewald summation. CHARMM27 force fields were used with CHARMM atomic types and charges. A 2-femtosecond embedded time step was used for all simulations, with a 12Å cutoff. Langevin dynamics were used to maintain the temperature at 310K, and modified No-Hoover-Langevin dynamics were used to control the pressure. Simulations were run for 100 nanoseconds.

[0175] statistical analysisPlaque size distribution was analyzed using the Kolmogorov-Smirnov test, followed by the Bonferroni correction. Viral replication and antibody response in animal experiments were analyzed using the nonparametric Kruskal-Wallis test and the Dunn post-hoc test. 10 The transformation was applied to the dataset when it was necessary to obtain equal standard deviations between groups. Statistics were performed using Prism 6 (GraphPad software). Data were considered significant only when p < 0.05.

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

[0177] Example 2: Codon-pair de-optimization (CPD) of multiple RSV genes resulting in an extremely stable temperature-sensitive (Ts) phenotype limited to replication at 32-34°C. As described above, Min_FLC (for full-length clones) is a mutant in which 9 out of 11 RSVORFs (excluding M2-1 and M2-2) are CPDs, resulting in a total of 2,692 silent mutations (Figure 1A). Min_FLC is highly temperature sensitive, and the cutoff temperature (T SH Plaque formation occurs at 35°C, while wild-type (wt)rRSV readily forms plaque at 40°C. SH This is defined as the minimum limit temperature at which the difference in titer observed in wt RSV at these two temperatures is greater than or equal to 100 times compared to 32°C.

[0178] To test Min_FLC stability, a temperature stress test was used, representing a surrogate model of genetic stability during viral replication, spreading from the cold upper respiratory tract to the warm lower respiratory tract. Ten independent Vero cells were subjected to 25 cm² of stress. 2 Replication flasks were infected with Min_FLC at an initial MOI of 0.1 plaque-forming units (pfu) / cell and continuously passaged in 18 passages with gradually increasing temperature, representing 7 months of continuous culture (flasks were incubated at the described starting temperature until extensive cytopathology was observed. Viruses were collected and continuously passaged while increasing the limiting temperature (1°C temperature increase every other passage)). Two additional replication flasks were infected as controls and passaged in parallel at an acceptable temperature of 32°C (Figure 1B-C). 1 ml (out of a total of 5 ml) of supernatant was used to inoculate the next passage. After each passage, aliquots were frozen for titer determination and sequence analysis by Sanger sequencing and / or deep sequencing as described. Viral titers were determined by plaque assay at an acceptable temperature (32°C).

[0179] At 32℃, Min_FLC is 10 6 ~10 7The cells continued to replicate up to a titer of pfu / ml (Figure 1B). Deep sequencing of the complete genomes of two control differentiation lines after 18 passages showed only low levels of sporadic mutations (Figure 7), demonstrating that Min_FLC is genetically stable under tolerable conditions. In a flask incubated with increasing temperature, Min_FLC replicated efficiently at 32°C and 33°C (10 6 ~10 7 pfu / ml, Figure 1C). However, after the first passage at 34°C (P5), viral replication decreased 200-fold in all 10 differentiation lines, and at the end of the second passage at 35°C (P8), the virus was undetectable in 9 differentiation lines. In the 10th lineage, the virus was not detected at the end of the first passage at 37°C (P11). In contrast, as described, wt rRSV did not show any limitation of growth at temperatures up to at least 40°C.

[0180] Therefore, Min_FLC is highly limited, even if not inert, at temperatures above 34-35°C (the latter is its T SH (Therefore, Min_FLC is phenotypic stable, as it cannot escape its Ts phenotype under stress conditions.) Sequencing was not performed on Min_FLC samples passed under restricted temperature increases due to the rapid decrease in titer. These results meet expectations regarding phenotypic stability for CPD viruses.

[0181] Example 3: Temperature stress on the Min_L virus promoted the emergence of multiple mutations in multiple genes. Min_L viruses, where L ORF alone (representing 48% of the aggregate RSVORF) was CPD, resulted in 1,378 silent mutations (about 51% more than Min_FLC). Min_L was induced at 37°C T SH The following was done: Ten replication flasks were infected with Min_L and continuously subcultured for a total of eight passages, corresponding to two months of continuous culture, while gradually increasing the temperature. Two additional replication flasks were infected as controls and subcultured in parallel at 32°C (Figure 1D-E).

[0182] As expected, Min_L was efficient in each passage at 32°C (10 7 The virus was replicated (pfu / ml) (Figure 1D). Sequence analysis of RNA from control differentiation lines by deep sequencing at P6 (Figure 8) and Sanger sequencing at P8 (data not shown) showed only sporadic, low-level mutations. In 10 differentiation lines passaged with increasing temperature, the Min_L titer in 9 flasks decreased approximately 20-fold at the end of P1 (37°C) (Figure 1E). However, during the second passage at 37°C, the titer of the same 9 differentiation lines increased approximately 200-fold, indicating that selection and growth of temperature-adapted mutants had already occurred. After P3 (38°C), the viral titer of all 10 differentiation lines decreased consistently: at P8 (second passage at 40°C), the virus was undetectable in 7 differentiation lines, and the titer was extremely low in the other 2 differentiation lines (20 pfu / ml each). The remaining lineage (#3, colored green) had a titer of 500 pfu / ml. Therefore, various Min_L differentiation lines appeared to be subject to partial loss of temperature-sensitive phenotypes, but were ultimately very strongly restricted at 40°C.

[0183] Whole-genome deep sequencing was performed in each of the 10 differentiated lineages, which were passaged at increasing temperatures, at P6 (second passage at 39°C), where viral replication was still detectable in each lineage. Table 1 shows the mutations present in ≥45% of the sequencing read data. Notably, many of these significant mutations occurred in genes that were not assigned to CPD. In particular, of these 23 significant mutations, 21 were distributed in 6ORFs (P, M, SH, G, M2-1, and L) and 2 in extragenetic regions. Of the 23 mutations, 11 (48%) and 5 (22%) occurred in M2-1 and L ORFs, respectively. Of the 21 mutations present in the ORFs, all but one were missense mutations, suggesting a bias in amino acid changes. This positive selection of amino acid changes suggests that at least part of Min_L's adaptation to selective stress is involved in structural / functional changes in various viral proteins. Some mutations were common across the multiple differentiated lineages. In particular, the mutation in the anti-termination transcription factor M2-1 [A73S] was prominent in 8 out of 10 differentiation lineages. Mutations in other M2-1 variants (N88K) and L (A1479T) were prominent in 2 differentiation lineages. M2-1 was the only gene with one or more prominent mutations across all lineages.

[0184] Table S1 shows the mutations present in ≥5% of the P6 sample read data from the same experiment. At this low cutoff, more mutations were prominent in all genes except NS2. Similar to the prominent mutations shown in Table 1, these less prominent mutations were mostly missense mutations. In the CPD L ORF, only 17 out of 31 mutations (55%) were involved in nt or codons modified during CPD (Table S1).

[0185] Whole-genome deep sequencing analysis was performed to evaluate the transient appearance of mutations in the fully passaged series of differentiation lineages #3 and #8, which were of the most interesting nature for maintaining the highest titer during stress testing (Figure 1E) and therefore possessing maximum deattenuation. The appearance and frequency of more abundant mutations are graphically shown in Figures 1F (Lineage #3) and G (Lineage #8). A more detailed list of mutations is shown in Tables S2 and S3.

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

[0187] Example 4: Two mutations in the anti-termination transcription factor M2-1, N88K and A73S, are significant but incompatible. All 10 differentiating lineages in P6 possessed either the M2-1 mutation [A73S] or [N88K] (Table 1, Figure 2A). Therefore, these two M2-1 mutations were considered isolated. Furthermore, the disappearance of the [A73S] mutation in the passage series of lineage #3 coexisted with the appearance and increase of [N88K] until the latter was present in a complete population (Figure 1F). Deep sequencing results for lineage #3 were re-evaluated, and only read data extending to both positions 73 and 88 in M2-1 were scored to provide a combined analysis. In P3 and P4, only 1% of the read data contained both mutations (Figure 2B), suggesting that these two mutations in M2-1 are incompatible within the same genome and therefore constitute two separate viral populations.

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

[0189] Example 5: Mutations N[K136R], P[E114V], M2-1[N88K], M2-1[A73S], and L[T1166I] were introduced into Min_L. The direct identification of the temperature-sensitive mutations in Min_L was explored by introducing the major mutations identified in lineage #3, namely N[K136R], P[E114V], M2-1[N88K], and L[T1166I] (Figure 1F), as well as the M2-1 mutation [A73S] (Figure 1G), one of the prominent mutations in replication #8, into Min_L individually and in combination. The resulting 12 viruses (Figure 3A) were collected, completely sequenced, and their accurate sequences and the absence of further mutations were confirmed.

[0190] This was performed using the Quickchange Lightning Site-directed Mutagenesis Kit (Agilent) as recommended by the manufacturer. The cDNA was fully sequenced by Sanger sequencing using a set of specific primers. The CPD virus with the target mutation was then rescued from the cDNA as previously described. Specifically, BSR T7 / 5 cells were transfected with Lipofectamine 2000 (Life Technologies) and a plasmid mixture containing 5 μg of full-length cDNA, 2 μg each of pTM1-N and pTM1-P, and 1 μg each of pTM1-M2-1 and pTM1-L. After overnight incubation at 37°C, the transfected cells were collected by scraping them onto culture medium and added to a subconfluent monolayer of Vero cells, which was incubated at 32°C. The rescued virus was collected 11–14 days post-transfection.

[0191] Introducing the N[K136R] or P[E114V] mutation alone is more effective than introducing Min_L. SH This resulted in an increase of approximately 1°C (Figure 3B), and L[T1166I] alone had no effect. Interestingly, the introduction of M2-1[A73S] or [N88K] alone was effective. SH This induced a 2°C increase, suggesting that either of these two M2-1 mutations alone plays the greatest role in deattenuating Min_L. The combination of an N or P mutation with M2-1[N88K] suggests that T SH This gave a further, small increase (average 2.5°C across three independent experiments). The combination of N, P, and M2-1[N88K] mutations gave T compared to Min_L. SH This induced a 3°C increase (to 40°C), and the addition of the L mutation did not cause a further increase. This is the T gene from lineage #3. SH The additive roles of N, P, and M2-1[N88K] mutations in the elevation were explained. The combination of M2-1[A73S] and [N88K] is associated with the T of Min_L. SH It does not imply any increase and suggests their incompatibility, as predicted based on the deep sequencing results.

[0192] The effects of these mutations on the dynamics and efficiency of Min_L replication in Vero cells were tested (Figures 3C-D). As predicted from temperature-sensitive mutations, the effects of the mutations were more pronounced at 37°C (Figures 3C, D, right panel) than at 32°C (left panel). N and P mutations alone and in combination had only a slight effect on viral replication compared to Min_L. In contrast, the introduction of either the M2-1[N88K] or [A73S] mutation alone resulted in a substantial increase in replication, which was largely unaffected by the addition of further N, P, and L mutations. Furthermore, viruses carrying both incompatible M2-1[A73S] and [N88K] mutations replicated at or below the efficiency of Min_L at 37°C and 32°C, respectively (Figure 3D, left and right panels). Therefore, while M2-1[N88K] or [A73S] mutations play a major role in restoring the ability of Min_L to replicate in Vero cells, they were incompatible.

[0193] Furthermore, the effects of transmutations on the dynamics of viral gene transcription, viral genomic RNA synthesis, protein expression, and viral particle production in a single infection cycle were examined (Figures 4A-E). Vero cells were infected with the described virus at an MOI of 3 pfu / cell, and samples were collected every 4 hours for analysis up to 24 hours.

[0194] The accumulation of nine small RSV mRNAs (i.e., all except L) was analyzed using positive sense-specific RT-qPCR assays specific to each mRNA. Generally, representative P mRNA data is shown in Figure 4A, and the complete datasets for these nine mRNAs are shown in Figure 9. Generally, transcription was significantly reduced at 37°C in Min_L compared to wt rRSV. Introduction of any of the M2-1 mutations in Min_L resulted in a substantial recovery of transcription. Further addition of N, P, and L mutations to M2-1[N88K] provided a more moderate but largely consistent increase. Western blot analysis showed, as expected, viral protein accumulation occurred later than mRNA, but otherwise, the pattern was similar to mRNA accumulation (Figures 4B and 10).

[0195] The accumulation of RSVL mRNA by positive sense-specific, L-specific RT-qPCR (Figure 4C) was examined. At 32°C, basal levels of L mRNA were observed in Min_L-infected cells, but there was no substantial increase over time, in contrast to the gradual increase observed with wt L mRNA. Extensive sequence differences in the wt and CPD L genes required the use of different primer pairs for wt rRSV and Min_L derivatives, preventing direct comparison of relative abundances at various time points. At 37°C, CPD L mRNA was undetectable, indicating strong limitation at this temperature. Addition of the M2-1[N88K] or [A73S] mutation to Min_L partially restored CPD L gene transcription at both 32°C and 37°C. Further inclusion of N, P, and L mutations further increased L gene expression.

[0196] Cell-associated genomic RNA production by Min_L (Figure 4D) was almost undetectable at 32°C or 37°C, but was detectable by M2-1[A73S] and M2-1[N88K] at 32°C and 37°C 24 hours after infection, and an increased amount was detected in NPM2-1[N88K]L-infected cells. Genomic RNA production by wt rRSV began to be detected from 12 hours after infection at both 32°C and 37°C, and was higher compared to NPM2-1[N88K]L.

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

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

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

[0200] The mechanism behind rescue CPD L gene expression by two M2-1 mutations is unknown. The RSVM2-1 protein is required for efficient synthesis of full-length mRNA; without it, premature termination occurs. The M2-1 protein also increases the synthesis of polycistronic readthrough mRNA, which binds to nascent mRNA via co-transcription and prevents termination by viral polymerase. Furthermore, the M2-1 protein directly binds to P. The binding of P and RNA to M2-1 was found to be mutually exclusive due to partially overlapping interaction surfaces. A73 and N88 are away from the RNA / P binding interface but may be pathways for existing nascent RNA molecules. A simple model is a 1,378nt change introduced during CPD that affects the L gene template so as to reduce the transcriptional elongation efficiency of nascent L mRNA. L transcription partially recovered the M2-1 mutation via some effect on the polymerase complex. Significant mutations acquired under stress are most frequently observed in M2-1 ORFs, but are also found in P, N, and L ORFs, all of which encode viral proteins involved in RNA synthesis. These additional N, P, and L mutations further increased CPD L gene transcription efficiency, likely by also increasing the efficiency of CPD L gene transcription elongation.

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

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

[0203] As described, a single mutation (A73S) in the M2-1 gene, which appeared in the first passage of Min_L at 37°C and was observed in 8 out of 10 cultures, was sufficient to rescue Min_L replication at that temperature. Furthermore, this single mutation contributed to increased Min_L replication in hamsters. We anticipated that deattenuation of CPD ORFs would involve multiple changes in the CPD sequence, leading to gradual deattenuation. However, this study showed that a single mutation in a different gene was sufficient to produce substantial deattenuation. Therefore, deoptimization involving numerous nt changes did not necessarily provide a stable attenuated phenotype.

[0204] Example 7: Introduction of a deattenuating mutation from Min_L to Min_FLC. The major mutations introduced into Min_L, namely N[K136R], P[E114V], M2-1[N88K], M2-1[A73S], and L[T1166I], were introduced into Min_FLC in various combinations, and the viral titers after recovery (Figure 11A) and T SH (Figure 11B) was evaluated. The M2-1[N88K] and [A73S] mutations were evaluated for viral titer or T SH Measurements showed that the fitness of Min_FLC did not increase individually. The combination of N, P, and M2-1[N88K] mutations was T SH Although this resulted in a 2°C increase, the virus only proliferated to a low titer.

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

[0206] Example 8: Evaluation of Min_L derivatives in mice and hamsters. Replication of Min_L derivatives was evaluated in vivo (Figure 5). BALB / c mice were subjected to 10% of each virus. 6 Intranasal infection (IN) was induced with pfu. Nasal turbinates (NT) and lungs were collected on post-infection (PI) days 4 (n=8 / virus), 5 (n=8), and 10 (n=4). At the peak of viral replication (5 days post-infection; Figure 5B), the virus was observed only in the NTs of 2 mice infected with Min_L and 3 mice infected with M2-1[N88K]. M2-1[A73S] replication was observed in 4 out of 8 mice and was comparable to wt rRSV. NPM2-1[N88K]L replication was not observed in the NTs of any of the mice. In the lungs on day 5, replication of M2-1[N88K] and M2-1[A73S] was slightly reduced compared to Min_L, but not statistically significant, and NPM2-1[N88K]L replication was strongly suppressed in the lungs compared to Min_L. The titer on day 10 is not shown because the virus was recovered from only two animals, and in the M2-1[A73S] group, it was at a trace level.

[0207] The same set of viruses was compared in hamsters (Figure 5C). On day 3, NT and lung samples were collected from 9 hamsters per virus. In NT, Min_L replication was approximately 100-fold reduced compared to wt rRSV (p ≤ 0.01). M2-1[N88K] replication was slightly increased compared to Min_L, but maintained significant attenuation compared to wt rRSV. In contrast, the titer of M2-1[A73S] was even more increased compared to Min_L and showed no statistically significant difference from wt rRSV. Interestingly, NPM2-1[N88K]L replication in NT was reduced compared to Min_L. In the lungs, Min_L and M2-1[N88K] were detected in only 1 out of 9 hamsters for each virus, and NPM2-1[N88K]L replication was undetectable. In contrast, the number of M2-1[A73S] doubles increased compared to Min_L, with 5 out of 9 hamsters being approximately 10 2 The virus exhibited pfu / g replication. Therefore, in hamsters, the M2-1[A73S] mutation increased the replication of Min_L, a deattenuation marker, the M2-1[N88K] mutation did not affect Min_L replication, and combinations of N, P, L, and M2-1[N88K] mutations decreased replication.

[0208] Despite significant replication limitations, Min_L and Min_L-derived viruses induced antibody titers that were statistically comparable to those induced by wt rRSV (Figure 5D). M2-1[A73S] virus induced significantly higher levels of RSV neutralizing serum antibodies than Min_L and M21-1[N88K]. Interestingly, NPM2-1[N88K]L virus, despite highly limited replication, also induced RSV neutralizing antibodies comparable to wt rRSV. On day 31, hamsters were exposed to wt rRSV in vitro, and NT and lung tissue were collected 3 days after exposure. No detectable exposure-induced viral replication was detected (not shown) except for trace amounts of virus in one animal in the Min_L group.

[0209] Example 9: Genetic stability of Min_L-NPM2-1[N88K]L virus. The NPM2-1[N88K]L virus was identified as a promising vaccine candidate based on observations that it was significantly attenuated compared to Min_L while still possessing comparable immunogenicity to wt rRSV. Therefore, its stability was evaluated in temperature stress tests, including four passages at 39°C and four passages at 40°C, corresponding to two months of continuous passage (Figure 12). Sanger sequencing of the complete genomes of the final passages from 10 different stress differentiation lines and two control flasks did not detect (or show) any significant mutations. This indicates that the introduction of N, P, M2-1[N88K], and L mutations into Min_L to create the promising NPM2-1[N88K]L virus provided genetic stability. The nucleotide sequence of Min_L-NPM2-1[N88K]L is shown in Figure 14 and is represented by Sequence ID No. 14.

[0210] [Table 1] a The percentage of read data containing the indicated mutation is shown; only mutations present in ≥45% of read data are indicated. Nucleotide numbering is based on RSV sequence M74568 (biological wt RSV strain A2). Table S1 shows mutations present in ≥5% of read data from the same experiment. b A mutation that includes codons that are converted as part of the CPD of L ORF. c A mutation involving altered nucleotide positions as part of the CPD of L ORF.

[0211] [Table 2] [Table 3] [Table 4] [Table 5] a The percentage of read data containing the indicated mutation is shown; only mutations present in ≥5% of the read data are shown. Mutations detected in ≥50% of the read data are highlighted in yellow, and mutations detected in 25-49% of the read data are highlighted in green. Nucleotide numbering is based on the RSV sequence M74568. b A mutation involving a codon that is altered as part of the CPD of L. c A mutation involving nucleotides that are altered as part of L's CPD. d This mutation, which is part of the CPD of L, involves nucleotides that are restored in the wt sequence.

[0212] [Table 6] a Percentage of read data containing the indicated mutation; only mutations present in 1% of read data are shown. Nucleotide numbering is based on the RSV sequence M74568. b A mutation involving a codon that is altered as part of the CPD of L. c A mutation involving nucleotides that are altered as part of L's CPD.

[0213] [Table 7] aPercentage of read data containing the indicated mutation; only mutations present in 1% of read data are shown. Nucleotide numbering is based on the RSV sequence M74568. b A mutation involving a codon that is altered as part of the CPD of L. c A mutation involving nucleotides that are altered as part of L's CPD.

[0214] [Table 8] [Table 9] a The percentage of read data containing the indicated mutation; only mutations detected in ≥5% of read data in at least two consecutive passages are shown. The temperature of the specific passage is shown in parentheses. Mutations detected in ≥50% of read data in a given passage are highlighted in yellow, and mutations detected in 25-49% of read data are highlighted in green. Nucleotide numbering is based on the RSV sequence M74568. b A mutation involving a codon that is altered as part of the CPD of L. c A mutation involving nucleotides that are altered as part of L's CPD. d This mutation, which is part of the CPD of L, involves nucleotides that are restored in the wt sequence.

[0215] [Table 10] a The percentage of read data containing the mutation shown; only mutations detected in ≥25% of read data in at least two consecutive passages are shown. The specific passage temperature is shown in parentheses. Nucleotide numbering is based on the RSV sequence M74568. b A mutation involving a codon that is altered as part of the CPD of L. c A mutation involving nucleotides that are altered as part of L's CPD. d This mutation, which is part of the CPD of L, involves nucleotides that are restored in the wt sequence.

[0216] [Table 11] a The percentage of read data containing the mutation shown; only mutations detected in ≥50% of read data in at least two consecutive passages are shown. The specific passage temperature is shown in parentheses. Nucleotide numbering is based on the RSV sequence M74568. b A mutation involving a codon that is altered as part of the CPD of L.

[0217] [Table 12] [Table 13] a The percentage of read data containing the indicated mutation; only mutations detected in ≥5% of read data in at least two consecutive passages are shown. The temperature of the specific passage is shown in parentheses. Mutations detected in ≥50% of read data in a given passage are highlighted in yellow, and mutations detected in 25-49% of read data are highlighted in green. Nucleotide numbering is based on the RSV sequence M74568. bA mutation involving a codon that is altered as part of the CPD of L. c A mutation involving nucleotides that are altered as part of L's CPD. d This mutation, which is part of the CPD of L, involves nucleotides that are restored in the wt sequence.

[0218] [Table 14] a The percentage of read data containing the mutation shown; only mutations detected in ≥25% of read data in at least two consecutive passages are shown. The specific passage temperature is shown in parentheses. Nucleotide numbering is based on the RSV sequence M74568.

[0219] [Table 15] a The percentage of read data containing the mutation shown; only mutations detected in ≥50% of read data in at least two consecutive passages are shown. The specific passage temperature is shown in parentheses. Nucleotide numbering is based on the RSV sequence M74568.

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Claims

1. An isolated polynucleotide molecule encoding a recombinant RSV variant having an attenuated phenotype, comprising a respiratory multinuclear virus (RSV) genome or antigenome sequence, wherein the RSV genome or antigenome is modified by a missense mutation at a position corresponding to T1166 of SEQ ID NO: 11 in the L ORF of the L protein, the missense mutation being T1166I of the L protein in SEQ ID NO: 11, and the RSV genome or antigenome encoding an L protein comprising an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:

11.

2. The isolated polynucleotide molecule according to claim 1, wherein the missense mutation is the result of a single nucleotide change in the second codon nucleotide for T1166 at position 11995 of the positive strand of the RSV sequence M74568.

3. The isolated polynucleotide molecule according to claim 1, wherein the RSV genome or antigenome is further modified by a missense mutation at the position corresponding to N88 of SEQ ID NO: 9 in the M2-1 ORF of the M2-1 protein, a missense mutation at the position corresponding to A73 of SEQ ID NO: 9 in the M2-1 ORF of the M2-1 protein, a missense mutation at the position corresponding to K136 of SEQ ID NO: 3 in the N ORF of the N protein, a missense mutation at the position corresponding to E114 of SEQ ID NO: 4 in the P ORF of the P protein, or any combination thereof.

4. The isolated polynucleotide molecule according to claim 3, wherein the missense mutation in the M2-1 ORF of the M2-1 protein at the position corresponding to N88 of SEQ ID NO: 9 is N88K, the missense mutation in the M2-1 ORF of the M2-1 protein at the position corresponding to A73 of SEQ ID NO: 9 is A73S, the missense mutation in the N ORF of the N protein at the position corresponding to K136 of SEQ ID NO: 3 is K136R, the missense mutation in the P ORF of the P protein at the position corresponding to E114 of SEQ ID NO: 4 is E114V, or any combination thereof.

5. The isolated polynucleotide molecule according to claim 1, wherein the RSV genome or antigenome is further modified by two or more of the following: a missense mutation at the position corresponding to N88 of SEQ ID NO: 9 in the M2-1 ORF of the M2-1 protein; a missense mutation at the position corresponding to A73 of SEQ ID NO: 9 in the M2-1 ORF of the M2-1 protein; a missense mutation at the position corresponding to K136 of SEQ ID NO: 3 in the N ORF of the N protein; and a missense mutation at the position corresponding to E114 of SEQ ID NO: 4 in the P ORF of the P protein.

6. The isolated polynucleotide molecule according to claim 5, wherein the two or more missense mutations include a first missense mutation in a first RSV protein and a second missense mutation in a second RSV protein.

7. The isolated polynucleotide molecule according to claim 5, wherein the missense mutation in the M2-1 ORF of the M2-1 protein at the position corresponding to N88 of SEQ ID NO: 9 is N88K, the missense mutation in the M2-1 ORF of the M2-1 protein at the position corresponding to A73 of SEQ ID NO: 9 is A73S, the missense mutation in the N ORF of the N protein at the position corresponding to K136 of SEQ ID NO: 3 is K136R, the missense mutation in the P ORF of the P protein at the position corresponding to E114 of SEQ ID NO: 4 is E114V, or any combination thereof.

8. The isolated polynucleotide molecule according to claim 1, wherein the RSV genome or antigenome comprises deletions of one or more ORF codons in at least one of M2-2 ORF, NS1 ORF, and NS2 ORF.

9. The isolated polynucleotide molecule according to claim 1, wherein the RSV genome or antigenome is codon-pairing optimized.

10. The isolated polynucleotide molecule according to claim 1, wherein the L ORF of the RSV genome or antigenome is codon-pairing optimized.

11. A vector comprising the isolated polynucleotide molecule described in claim 1.

12. A cell comprising the isolated polynucleotide described in claim 1.

13. A pharmaceutical composition comprising an immunoassayable amount of a recombinant RSV variant encoded by the isolated polynucleotide molecule described in claim 1.

14. The pharmaceutical composition according to claim 13 for vaccinating a target against RSV.

15. A pharmaceutical composition according to claim 13 for inducing an immune response.

16. The pharmaceutical composition according to claim 14 or 15, wherein the pharmaceutical composition is administered intranasally.

17. The pharmaceutical composition according to claim 14 or 15, wherein the pharmaceutical composition is administered by injection, aerosol delivery, nasal spray, or nasal instillation.

18. A viable attenuated RSV vaccine comprising a recombinant RSV variant encoded by any isolated polynucleotide of claim 1.

19. A pharmaceutical composition comprising the RSV vaccine described in claim 18.

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