Recombinant human parainfluenza virus 1 (HPIV1) expressing a chimeric RSV / HPIV1 F protein and uses thereof

Abstract

To provide recombinant paramyxoviruses including a viral genome encoding a heterologous gene.SOLUTION: In several embodiments, the recombinant paramyxovirus is a recombinant parainfluenza virus, such as a recombinant PIV3 including a viral genome encoding a heterologous respiratory syncytial virus F ectodomain linked to the transmembrane domain and the cytoplasmic tail of the F protein from the PIV3. Nucleic acid molecules including the genome of recombinant paramyxoviruses are also provided. The recombinant viruses may advantageously be used in vaccine formulations, e.g., for vaccines against a parainfluenza virus and a respiratory syncytial virus.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 105,667, filed January 20, 2015, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to a recombinant paramyxovirus comprising a viral genome comprising a heterologous gene encoding an antigen of a heterologous virus. For example, the recombinant paramyxovirus can be a recombinant parainfluenza virus (PIV) comprising a genome comprising a heterologous gene encoding a respiratory syncytial virus (RSV) fusion (F) protein. [Background technology]

[0003] Paramyxoviruses are a family of negative-strand, single-stranded RNA viruses that are the primary cause of numerous animal and human deaths worldwide each year. Paramyxoviruses include the subfamily Paramyxovirinae and the subfamily Pneumovirinae. Respiratory syncytial virus (RSV) is an enveloped, non-segmented, negative-strand RNA virus in the genus Paramyxoviridae, subfamily Pneumovirinae. It is the most common cause of bronchiolitis and pneumonia in children in their first year of life. RSV also causes recurrent infections, including severe lower respiratory tract disease, at any age, particularly in the elderly or those with compromised heart, lung, or immune systems. Passive immunization is currently used to prevent severe disease caused by RSV infection, particularly in premature infants and infants with bronchopulmonary dysplasia or congenital heart disease. Despite the burden of RSV infection in certain populations, the development of an effective RSV vaccine remains elusive.

[0004] Parainfluenza viruses (PIVs), like RSV, are other enveloped, non-segmented, negative-strand RNA viruses in the Paramyxoviridae family. However, PIVs are in the Paramyxovirinae subfamily. PIVs include members of the Respirovirus genus (including PIV1, PIV3, and Sendaivirus) and the Rubulavirus genus (including PIV2, PIV4, and PIV5). Furthermore, members of the Avulavirus genus (including Newcastle disease virus, NDV) have historically been referred to as PIVs and may be operationally considered identical. Human parainfluenza viruses (HPIVs, serotypes 1, 2, and 3) are second only to RSV in causing severe respiratory infections in infants and children worldwide, with HPIV3 being the most significant of the HPIVs in terms of disease impact. The HPIV genome is approximately 15.5 kb, containing the gene sequence 3'-NPMF-HN-L. Each gene encodes a separate mRNA encoding the major proteins: N, nucleoprotein; P, phosphoprotein; M, matrix protein; F, fusion glycoprotein; HN, hemagglutinin-neuraminidase glycoprotein; and L, large polymerase protein. The P gene contains one or more additional open reading frames (ORFs) encoding accessory proteins. As with RSV, the development of an effective HPIV vaccine remains elusive. Summary of the Invention [Problem to be solved by the invention]

[0005] Recombinant paramyxoviruses comprising a viral genome encoding a heterologous gene are provided. In some embodiments, the recombinant paramyxovirus comprises a viral genome comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain linked to the cytoplasmic tail (CT) or transmembrane domain (TM) of the paramyxovirus F protein and a recombinant RSV F ectodomain linked to the CT. The paramyxovirus may be, for example, a recombinant human / bovine parainfluenza virus 3 (B / HPIV3), a recombinant human parainfluenza virus 1 (HPIV1), a recombinant human parainfluenza virus 2 (HPIV2), a recombinant human parainfluenza virus 3 (HPIV3), or a recombinant bovine parainfluenza virus 3 (BPIV3). [Means for solving the problem]

[0006] Surprisingly, replacing the TM and CT of the heterologous RSV F protein with the corresponding TM and CT of the paramyxovirus F protein resulted in a greater diversity of RSV F ectodomain integration into the recombinant paramyxovirus envelope, significantly increasing the induction of immune responses against the ectodomain when the recombinant paramyxovirus was administered to a subject. Furthermore, the induction of virus-neutralizing serum antibodies was significantly increased in both quantity and quality. Thus, in some embodiments, the disclosed recombinant paramyxovirus can be included in an immunogenic composition to induce a bivalent immune response against the paramyxovirus and the heterologous RSV F protein.

[0007] The RSV F ectodomain encoded by the heterologous gene can be derived from the human RSV F protein. In some embodiments, the RSV F ectodomain can contain one or more amino acid substitutions (such as "DS-Cav1" substitutions, S155C, S290C, S190F, and V207L) to stabilize the ectodomain in the RSV F pre-fusion conformation. In further embodiments, the RSV F ectodomain can contain another amino acid substitution to increase ectodomain expression or integration in the viral envelope (such as "HEK" substitutions, K66E and Q101P).

[0008] In one non-limiting embodiment, the recombinant paramyxovirus may be a recombinant B / HPIV3, wherein the RSV F ectodomain is linked to the TM and CT from the BPIV3 F protein. In some such embodiments, the RSV F ectodomain linked to the TM and CT from the BPIV3 F protein comprises the amino acid sequence set forth in SEQ ID NO: 21, or an amino acid sequence at least 90% identical to SEQ ID NO: 21.

[0009] In some embodiments, the recombinant paramyxovirus is a recombinant PIV that comprises, from upstream to downstream, a PIV genomic promoter followed by a viral genome comprising the N, P, M, F, HN, and L genes. In some such embodiments, the heterologous gene contained in the viral genome can be located between the genomic promoter and the gene encoding the N protein or between the gene encoding the N protein and the gene encoding the P protein.

[0010] In further embodiments, the heterologous gene contained in the viral genome of the recombinant paramyxovirus can be codon-optimized for expression in human cells. In further embodiments, the recombinant paramyxovirus can be an attenuated virus. In other embodiments, the added gene and its encoded protein can provide the attenuation required for the vaccine candidate.

[0011] Immunogenic compositions comprising the recombinant paramyxovirus are also provided. The compositions may further comprise an adjuvant. Also disclosed are methods for generating an immune response in a subject by administering an effective amount of the disclosed recombinant paramyxovirus to the subject. Also provided are isolated nucleic acid molecules comprising the viral genome of any of the recombinant paramyxoviruses disclosed herein.

[0012] The foregoing and other features and advantages of the present disclosure will become more apparent from the following detailed description of several embodiments that proceeds with reference to the accompanying drawings. [Brief explanation of the drawings]

[0013] [Figure 1]

[0033] Figure 1 shows the construction of rB / HPIV3 vectors expressing versions of the RSV F protein containing non-HEK or HEK amino acid assignments. The F ORF was codon-optimized for human expression using the GeneArt (GA) algorithm. The constructs were designated non-HEK / GA-opt and HEK / GA-opt. HEK (66E, 101P) and non-HEK (66K, 101Q) amino acid assignments are indicated with asterisks. Other annotations: S, signal sequence; p27, 27kJ protein fragment released by cleavage activation; FP, fusion peptide; TM, transmembrane; CT, cytoplasmic tail. The RSV F ORF was inserted into the second genomic location between the N and P genes of the B / HPIV3 vector under the control of the BPIV3 gene start and gene end transcription signals. The rB / HPIV3 vector contains the N, P, M, and L genes (derived from BPIV3) and the F and NH genes (derived from HPIV3). The same vector genome location and vector transcription signal was used for all of the other rB / HPIV3 vectors expressing the RSV F protein described in Figures 1-35. [Figure 2] Figures 2A and 2B show that the presence of the HEK assignment in the RSV F protein resulted in increased protein expression and decreased protein trimer mobility in polyacrylamide gel electrophoresis compared to that of non-HEK F proteins. Vero cells were infected with vectors expressing HEK or non-HEK RSV F (derived from the GA-optimized ORF, as shown in Figure 1) at an MOI of 10 TCID50 at 32 °C. Cell lysates were prepared 48 hours post-infection. Equal amounts of cell lysates were analyzed by electrophoresis after boiling and reduction (A) or without boiling and reduction (B). Denatured and reduced RSV F monomers were detected with a commercially available RSV F-specific mouse monoclonal antibody (A). Native RSV F trimers were detected with a polyclonal antibody raised in rabbits by repeated immunization with sucrose-purified RSV particles (B). [Figure 3] Figures 3A and 3B are photographs of syncytia formation in Vero cell monolayers infected with rB / HPIV3 vectors expressing non-HEK or HEK RSV F proteins. Cells were infected at an MOI of 10 TCID50 at 32°C with (A) non-HEK or (B) rB / HPIV3 expressing GA-codon-optimized RSV F (see Figure 1) in HEK configuration. Images of infected cells were taken 48 hours post-infection. Representative syncytia are marked with dashed outlines. [Figure 4]

[0023] Figure 1 shows the construction of rB / HPIV3 vectors expressing RSV F ORFs containing HEK assignments, codon-optimized for human expression using different algorithms. ORFs encoding the RSV F protein with HEK assignments were optimized for human codon usage using the GA algorithm (HEK / GA-opt, shown in Figure 1), the DNA2.0 algorithm (HEK / D2-opt), or the GenScript (GS) algorithm (HEK / GS-opt). These codon-optimized ORFs were compared with non-optimized versions of the RSV F ORF, non-HEK (non-HEK / non-opt). These RSV F ORFs were inserted into the rB / HPIV3 vector at the exact same location and with the same vector signals as in Figure 1. [Figure 5]Figures 5A and 5B show the increased in vitro expression of the RSV F protein from the rB / HPIV3 vector due to HEK assignment and codon optimization. RSV F expression in (A) Vero and (B) LLC-MK2 cells was assessed by Western blot analysis. Cells were infected with the indicated rB / HPIV3 vectors at an MOI of 10 TCID50 at 32°C, and cell lysates were collected 48 hours post-infection. Lysates were subjected to gel electrophoresis under reducing and denaturing conditions and analyzed by Western blotting. Proteins were visualized by reaction with fluorescent antibodies and detected by infrared imaging. A total of three wells per virus were performed. A monoclonal antibody specific for RSV F was used to detect the uncleaved F0 precursor and the cleaved F1 subunit. RSV F1 band density was quantified and normalized to the band density of the non-HEK / non-opt sample, indicated as "1." HPIV3 HN protein expression was also determined as an internal control for vector protein expression to ensure equivalent MOI and replication; β-actin was used as a loading control. [Figure 6] Photographs show the effect of codon optimization of the HEK and F ORF on syncytia formation in vector-infected Vero cell monolayers. Cells were mock infected (mock) or infected with an empty rB / HPIV3 vector (empty B / H3) or an rB / HPIV3 vector expressing the RSV F ORF, which was non-HEK and non-optimized (non-HEK / non-opt), HEK and GA-optimized (HEK / GA-opt), HEK and DNA2.0-optimized (HEK / D2-opt), or HEK and GS-optimized (HEK / GS-opt). Infections were performed at an MOI of 10 TCID50 at 32°C, and images were acquired 48 hours postinfection. Representative syncytia are indicated by dashed outlines in some panels. [Figure 7]Figures 7A and 7B are graphs showing the multicycle in vitro replication of rB / HPIV3 vectors expressing HEK or non-HEK RSV F proteins derived from non-optimized or codon-optimized ORFs. (A) LLC-MK2 and (B) Vero cells were infected in triplicate at 32°C with an empty rB / HPIV3 vector (empty B / H3) or a vector expressing the RSV F ORF, at an MOI of 0.01 TCID50. The vectors were non-HEK-containing and non-optimized (non-HEK / non-opt), non-HEK-containing and GA-optimized (non-HEK / GA-opt), HEK-containing and GA-optimized (HEK / GA-opt), or HEK-containing and GS-optimized (HEK / GS-opt). Aliquots of culture supernatant were collected at 24-hour intervals for 6 days, and virus titers were determined by limiting dilution assay on LLC-MK2 cells at 32°C and reported as TCID50 / ml. The mean titers ± SEM from three independent experiments are shown. [Figure 8]Figures 8A and 8B are graphs showing replication in hamsters of rB / HPIV3 vectors expressing HEK or non-HEK RSV F proteins from non-optimized or codon-optimized ORFs. Golden Syrian hamsters were infected intranasally (IN) with a 0.1 ml inoculum containing 10 TCID of the indicated rB / HPIV3 vector or 10 PFU of wt RSV (strain A2). Hamsters were sacrificed on days 3 and 5 post-infection (n=6 per virus per day), and (A) nasal turbinates and (B) lungs were removed and homogenized. Virus titers were determined by limiting dilution on LLC-MK2 (rB / HPIV3 vector) or Vero (RSV) cells at 32°C; open and closed circles indicate titers from animals sacrificed on days 3 and 5, respectively. Each symbol corresponds to an individual animal, and the mean titer for each group is shown by the dashed and solid horizontal lines for days 3 and 5, respectively. The limit of detection (LOD) was 1.5 log TCID per gram of tissue, indicated by the dashed line. rB / HPIV3 vectors were titrated by limiting dilution assay in LLC-MK2 cells and reported as TCID / g; RSV was titrated by plaque assay in Vero cells and reported as PFU / g. [Figure 9]Figure 1 shows serum RSV-neutralizing antibody titers from hamsters infected with rB / HPIV3 vectors expressing HEK or non-HEK RSV F proteins derived from non-optimized or codon-optimized ORFs. Hamsters (n = 6 animals per virus) were inoculated IN with a 0.1 ml inoculum containing 10 TCID of the indicated rB / HPIV3 vector or 10 PFU of wt RSV. Serum samples were collected 28 days post-immunization, and RSV-neutralizing antibody titers were determined using a 60% plaque reduction neutralization test (PRNT60) performed on Vero cells at 32°C in the presence of guinea pig complement. Each symbol represents an individual animal. The height of each bar corresponds to the mean titer for each group. Mean titer values ​​are shown above the bars. The standard error of the mean is indicated by a horizontal line. The limit of detection for the neutralization assay was the reciprocal log2 PRNT60 of 5.3, indicated by the dashed line. [Figure 10] Figures 10A and 10B are graphs showing the protection of immunized hamsters against RSV challenge. Hamsters (n = 6 animals per virus) immunized with the indicated rB / HPIV3 vectors or with wt RSV as shown in Figure 9 were challenged IN with a 0.1 ml inoculum containing 10 PFU of wt RSV 31 days after immunization. Three days after challenge, hamsters were euthanized, and (A) nasal turbinates and (B) lungs were collected. RSV titers in tissue homogenates were determined by plaque assay in Vero cells. Each symbol corresponds to an individual animal, and the mean virus titer for the group is shown as a horizontal line. The detection limit of the assay was log10 2.7 PFU per gram of tissue, as indicated by the dashed line. [Figure 11]Schematic diagram of the construction of rB / HPIV3 vectors expressing secreted (Ecto), post-fusion, and stabilized pre-fusion RSV F proteins. Each of these modified proteins contains HEK assignments and is expressed from a GA-optimized (for human expression) ORF. Annotations: S, signal sequence; p27, 27kJ protein fragment released by cleavage activation; FP, fusion peptide; TM, transmembrane; CT, cytoplasmic tail. The HEK / GA-opt construct expresses full-length RSV F. The ectodomain, or "ecto" form, consists of amino acids 1-513 of the RSV F protein; it lacks the CT and TM anchors and will be available for secretion. The "post-fusion" form was derived from the ectodomain (1-513 aa) by further deleting the first 10 aa (FP; 137-146 aa) from the N-terminal domain of the fusion peptide (McLellan et al., 2011, J Virol 85:7788-96). "DS" and "DS-Cav1" are two versions of the full-length RSV F protein stabilized in pre-fusion form by the S155C / S290C mutation (DS) or the DS and S190F / V207L ​​(Cav1) mutations (McLellan et al., 2013, Science 342: 931). ORFs encoding these various forms of RSV F were inserted into the rB / HPIV3 vector at the same positions and with the same vector signals as shown in Figures 1 and 4. [Figure 12]Figures 12A and 12B are graphs showing the multi-cycle in vitro replication of rB / HPIV3 vectors expressing secreted post-fusion and stabilized pre-fusion RSV F proteins. (A) LLC-MK2 and (B) Vero cells were infected with an empty rB / HPIV3 vector (empty B / H3) at an MOI of 0.01 TCID50 or with the indicated constructs: HEK / GA-opt; Ecto; post-fusion; and DS (see Figure 11 for an explanation). Viral replication over a 6-day period at 32 ° C was determined by collecting culture supernatant samples at 24-hour intervals and performing virus titration by limiting dilution in LLC-MK2 cells. For a diagram of mutant proteins, see Figure 11. An asterisk * indicates that all these RSV F constructs were HEK and GA optimized. [Figure 13] Figures 13A and 13B show the in vitro expression of secreted (Ecto), post-fusion, and stabilized pre-fusion RSV F proteins from rB / HPIV3 vectors. Vero cells were infected with the indicated rB / HPIV3 vectors at an MOI of 10 TCID50 or with wt RSV at an MOI of 10 PFU. Infected cells were incubated at (A) 32°C or (B) 37°C for 48 hours. (A) Culture supernatants and lysates from cells infected with rB / HPIV3 vectors expressing Ecto or HEK / GA-opt, or with wt RSV after fusion, and (B) lysates from cells infected with rB / HPIV3 vectors carrying non-HEK / non-opt, HEK / GA-opt, DS, or DS-Cav1 RSV F were collected and analyzed for RSV F expression by Western blot. Constructs marked with an asterisk * contained HEK assignment and were GA-optimized. [Figure 14]14A and 14B are graphs showing the replication in hamsters of rB / HPIV3 vectors expressing secreted (Ecto), post-fusion, and stabilized pre-fusion RSV F proteins. Hamsters were infected IN with 10 TCID of the indicated rB / HPIV3 vector or with a 0.1 ml inoculum containing 10 PFU of wt RSV. Hamsters were euthanized on days 3 and 5 post-infection (n=6 per virus per day), and (A) nasal turbinates and (B) lungs were removed and homogenized. Virus titers were determined by limiting dilution at 32°C in LLC-MK2 cells (rB / HPIV3 vector) or Vero (RSV) cells; open and closed circles indicate titers for animals sacrificed on days 3 and 5, respectively. Each symbol represents an individual animal, and the mean titer for each group is indicated by a dashed or solid horizontal line for days 3 and 5, respectively. The mean titer on day 5 is shown at the top. rB / HPIV3 vectors were titrated by limiting dilution assay in LLC-MK2 cells and reported as TCID50 / g; RSV was titrated by plaque assay in Vero cells and reported as PFU / g. The limit of detection (LOD) is 1.5 log10 TCID50 per g of tissue, indicated by the dotted line. Statistical significance of differences between peak titers was determined by Tukey-Kramer test and is indicated by an asterisk; *, P ≤ 0.05; **, P ≤ 0.01; or ***, P ≤ 0.001. Constructs indicated with an asterisk * contained HEK assignment and were GA-optimized for human expression. [Figure 15]Figures 15A and 15B are graphs showing serum RSV-neutralizing antibody titers from hamsters infected with rB / HPIV3 vectors expressing secreted (Ecto), post-fusion, and stabilized pre-fusion RSV F proteins. Hamsters (n = 6 animals per virus) were inoculated IN with 10 TCID of the indicated rB / HPIV3 vector or with a 0.1 ml inoculum containing 10 PFU of wt RSV. Serum samples were collected 28 days post-immunization, and RSV-neutralizing antibody titers were determined in a 60% plaque reduction neutralization test (PRNT60) performed in Vero cells at 32°C with (A) and without (B) the addition of guinea pig complement. The height of each bar represents the mean titer. The mean titer value is shown above the bar. The standard error of the mean is indicated by a horizontal line. The detection limit of the neutralization assay is indicated by a dashed line. The mean neutralization titer is below the limit of detection. Statistical significance of differences between groups was determined by Tukey-Kramer test and is indicated by an asterisk; *, P ≤ 0.05; **, P ≤ 0.01; or ***, P ≤ 0.001; or ns, P > 0.05. [Figure 16] Figures 16A and 16B are graphs showing the protection of immunized hamsters against RSV challenge. Hamsters immunized as shown in Figure 15 (n = 6 animals per virus) were challenged IN 31 days after immunization with a 0.1 ml inoculum containing 10 PFU of wt RSV. Three days after challenge, the hamsters were euthanized, and (A) nasal turbinates and (B) lungs were collected. RSV titers in tissue homogenates were determined by plaque assay on Vero cells at 32°C. Each symbol corresponds to an individual animal, and the mean virus titer for that group is shown as a horizontal line. The detection limit of the assay was log 10 2.7 PFU per gram of tissue, as indicated by the dotted line. [Figure 17A]Figure 1 shows the construction of rB / HPIV3 vectors expressing engineered versions of the RSV F protein to enhance integration into vector particles. (A) Structure of the F protein. (B) The cytoplasmic tail (CT), transmembrane (TM) domain, and adjacent regions of the RSV F protein (amino acid assignments in black) and the BPIV3 F protein (bold) with the indicated amino acid sequence positions. Each of these modified proteins contains an HEK assignment and was expressed from a GA-optimized ORF. The HEK / GA-opt construct expressed the full-length RSV F protein. "B3CT" has the CT (amino acid sequence positions 551-574) of the RSV F protein replaced with the CT (positions 515-540, bold) of the BPIV3 F protein. "B3TMCT" has both the TM and CT (positions 530-574) of the RSV F protein replaced with the TM and CT (positions 494-540, bold) of the BPIV3 F protein. "DS / B3CT", "DS / B3TMCT", "DS-Cav1 / B3CT", and "DS-Cav1 / B3TMCT" are versions of B3CT and B3TMCT containing DS or DS-Cav1 mutations designed to stabilize the pre-fusion conformation. ORFs encoding these various forms of the RSV F protein were inserted into the rB / HPIV3 vector at the same positions and with the same vector signals as described in Figures 1, 4, and 11. [Figure 17B]Figure 1 shows the construction of rB / HPIV3 vectors expressing engineered versions of the RSV F protein to enhance integration into vector particles. (A) Structure of the F protein. (B) The cytoplasmic tail (CT), transmembrane (TM) domain, and adjacent regions of the RSV F protein (amino acid assignments in black) and the BPIV3 F protein (bold) with the indicated amino acid sequence positions. Each of these modified proteins contains an HEK assignment and was expressed from a GA-optimized ORF. The HEK / GA-opt construct expressed the full-length RSV F protein. "B3CT" has the CT (amino acid sequence positions 551-574) of the RSV F protein replaced with the CT (positions 515-540, bold) of the BPIV3 F protein. "B3TMCT" has both the TM and CT (positions 530-574) of the RSV F protein replaced with the TM and CT (positions 494-540, bold) of the BPIV3 F protein. "DS / B3CT", "DS / B3TMCT", "DS-Cav1 / B3CT", and "DS-Cav1 / B3TMCT" are versions of B3CT and B3TMCT containing DS or DS-Cav1 mutations designed to stabilize the pre-fusion conformation. ORFs encoding these various forms of the RSV F protein were inserted into the rB / HPIV3 vector at the same positions and with the same vector signals as described in Figures 1, 4, and 11. [Figure 18]Figures 18A and 18B show the incorporation of the B3CT and B3TMCT versions of the RSV F protein into rB / HPIV3 vector particles. LLC-MK2 cells were infected with the indicated rB / HPIV3 vectors at an MOI of 0.01 TCID50 at 32°C. Culture supernatants were collected 6-7 days postinfection, clarified by low-speed centrifugation, and subjected to centrifugation on a 10%-30% sucrose gradient to obtain partially purified vector particles. Additional Vero cells were infected with wt RSV at an MOI of 0.01 PFU and treated in the same manner. The protein concentration of the sucrose-purified preparation was determined using a standard commercially available kit. (A) Western blot assessment of the packaging efficiency of RSV F protein into rB / HPIV3 particles. To compare the relative amounts of RSV F in the particles, 0.5 μg of sucrose-purified particles were lysed, denatured, reduced, and subjected to Western blot analysis. The HPIV3 HN and BPIV3 N proteins of the vector particles were quantified for comparison. (B) The packaging efficiency of each form of RSV F into its respective vector particles was calculated by normalizing its band density to that of the BPIV3 N protein. The lane order is the same as in Part A. The packaging efficiency of various forms of RSV F is shown relative to the native F protein, which is set to "1." The packaging efficiency of the B3CT and B3TMCT forms of RSV F into vector particles was determined to be similar to that of RSV F into RSV particles, because the amount of modified RSV F protein per 0.5 μg of vector particles (lanes 3, 4, 6, and 7) was similar to the amount of native RSV F protein per 0.5 μg of RSV particles (lane 5). The construct indicated by an asterisk * contains HEK assignments and is GA-codon-optimized for human expression. [Figure 19-1]Figures 19A-19F visualize the incorporation of the B3CT and B3TMCT versions of the RSV F protein into rB / HPIV3 particles by transmission electron microscopy (TEM). Sucrose-purified virus was labeled with an RSV F-specific mouse monoclonal antibody and a mouse IgG-specific secondary antibody (labeled with 6 nm gold particles). Virions and gold particles were visualized by TEM. Representative images of (A) RSV, (B) empty rB / HPIV3 vector (empty B / H3), (C) HEK / GA-opt-expressing vector, (D) B3CT-expressing vector, (E) B3TMCT-expressing vector, and (F) DS / B3TMCT-expressing vector are shown. Arrows point to scattered gold particles in HEK / GA-opt virions (C). Substantially higher amounts of gold particles associated with the vector particles are evident in D, E, and F. [Figure 19-2] Continuation of Figure 19-1. [Figure 20] Figures 20A and 20B are graphs showing the multicycle in vitro replication of rB / HPIV3 vectors expressing the B3CT and B3TMCT versions of the RSV F protein. (A) LLC-MK2 and (B) Vero cells were infected at 32°C with an empty rB / HPIV3 vector (empty B / H3) or vectors expressing HEK / GA-opt, B3CT (upper panel), B3TMCT (upper panel), DS / B3CT (lower panel), or DS / B3TMCT (lower panel) at an MOI of 0.01 TCID50. Aliquots of culture supernatant were collected at 24-hour intervals for 6 days, and viral titers were determined by limiting dilution assay in LLC-MK2 cells at 32°C and reported as TCID50 / ml. Constructs indicated with an asterisk * contain HEK assignments and are GA-codon-optimized for human expression. The multiplicity of infection in the assay was 0.01. [Figure 21]Figures 21A and 21B show the in vitro expression of the B3CT and B3TMCT versions of the RSV F protein with or without DS or DS-Cav1 mutations that stabilize the pre-fusion form of the RSV F protein. (A) Expression of B3CT and B3TMCT; and (B) Expression of DS and DS-Cav1 (combined with B3CT and B3TMCT). Vero cells were infected with the indicated rB / HPIV3 vectors at an MOI of 10 TCID50 or RSV at an MOI of 10 PFU. Infected cells were incubated at (A) 32 ° C or (B) 37 ° C for 48 hours. Cell lysates were analyzed for RSV F expression by Western blot. The HPIV3 HN protein was used as a control for equivalent vector replication; GAPDH was used as a loading control. Constructs indicated by an asterisk * contain HEK assignments and are GA-codon-optimized for human expression. [Figure 22] Figure 1 shows syncytia formation in Vero cell monolayers infected with rB / HPIV3 vectors expressing the B3CT or B3TMCT versions of the RSV F protein with or without DS mutations that stabilize the prefusion form of the RSV F protein. Vero cells were infected with rB / HPIV3 vectors expressing the indicated versions of the RSV F protein at an MOI of 10 TCID50 and incubated at 32°C. Images were acquired 48 hours post-infection. Constructs indicated by an asterisk * contain HEK assignments and are GA-codon-optimized for human expression. [Figure 23]Figures 23A and 23B show the replication in hamsters of rB / HPIV3 vectors expressing the B3CT or B3TMCT versions of the RSV F protein, with or without DS mutations that stabilize the prefusion form of the RSV F protein. Hamsters were infected IN with 0.1 ml of inoculum containing 10 TCID of the rB / HPIV3 vector or 10 PFU of wt RSV. Hamsters were euthanized on days 3 and 5 post-infection (6 per virus per day), and (A) nasal turbinates and (B) lungs were removed and homogenized, and virus titers were determined by limiting dilution in LLC-MK2 (rB / HPIV3 vector) or Vero (RSV) cells at 32 ° C. The open and closed circles indicate the titers of animals sacrificed on days 3 and 5, respectively. Each symbol corresponds to an individual animal, and the mean titer for each group is indicated by a dashed or solid horizontal line for days 3 and 5, respectively. The mean titer on day 5 is indicated at the top. rB / HPIV3 vectors were titrated by limiting dilution assay in LLC-MK2 cells and reported as TCID50 / g; RSV was titrated by plaque assay in Vero cells and reported as PFU / g. The limit of detection (LOD) is indicated by the dotted line and is 1.5 log10 TCID50 per g of tissue. Statistical significance of differences between peak titers was determined by Tukey-Kramer test and indicated by asterisks (*, P ≤ 0.05; **, P ≤ 0.01; or ***, P ≤ 0.001). Constructs indicated by an asterisk * along the x-axis contain HEK assignments and are GA-codon-optimized for human expression. Constructs containing the DS-Cav1 modification were not tested as they were not available at the time of this experiment. [Figure 24]Figures 24A and 24B are graphs showing serum RSV-neutralizing antibody titers from hamsters infected with rB / HPIV3 vectors expressing the B3CT or B3TMCT versions of the RSV F protein, with or without DS mutations that stabilize the prefusion form of the RSV F protein. Hamsters (n = 6 animals per virus) were inoculated IN with a 0.1 ml inoculum containing 10 TCID of the indicated rB / HPIV3 vector or 10 PFU of wt RSV. Serum samples were collected 28 days post-immunization, and antibody titers were determined by a 60% plaque reduction neutralization test (PRNT60) with (A) or without (B) the addition of guinea pig complement. The height of each bar corresponds to the mean titer, shown along with the SEM. The mean titer value is shown above the bar. The detection limit of the neutralization assay is indicated by a dotted line. Statistical significance of differences in mean titers was determined by Tukey-Kramer test and is indicated by asterisks (*, P ≤ 0.05; **, P ≤ 0.01; ns, P ≥ 0.05). ND, neutralization titer was below the limit of detection. Constructs indicated by an asterisk * along the x-axis contain HEK assignments and are GA-codon-optimized for human expression. [Figure 25] Figures 25A and 25B are graphs showing the protection of immunized hamsters against RSV challenge. Immunized hamsters (n=6 animals per virus) as shown in Figure 24 were challenged IN 31 days post-immunization with a 0.1 ml inoculum containing 10 PFU of wt RSV. Three days after challenge, the hamsters were euthanized, and (A) nasal turbinates and (B) lungs were collected. RSV titers in tissue homogenates were determined by plaque assay in Vero cells at 32°C. Each symbol corresponds to an individual animal, and the mean virus titer for the group is shown as the horizontal line. The detection limit of the assay was log102.7 PFU per gram of tissue, indicated by the dotted line. [Figure 26]This table shows the stability of RSV F expression by rB / HPIV3 vectors during replication in hamsters. The percentage of recovered vectors expressing RSV F in the nasal turbinates and lungs on days 3 and 5 post-immunization was determined by double-stain plaque assay of vectors recovered directly from tissue homogenates. Results are presented for individual animals. The percentage of rB / HPIV3 expressing RSV F protein among the tested specimens is shown. Specimens expressing 100% RSV F protein are colored yellow; specimens expressing 90-99% RSV F are colored green. Specimens expressing 80-89% RSV F are colored orange; specimens expressing less than 79% RSV F are colored red. Specimens that did not produce plaques due to low titers are marked "NA." If the total number of plaques produced in a sample was less than 10, the number of plaques was recorded in parentheses as "p=X" (X equals the number of plaques). [Figure 27] 1 is a table showing the temperature-sensitive phenotype of B / HPIV3 vectors. The indicated vectors were evaluated for their ability to form plaques in LLC-MK2 cells at the temperatures indicated. A ≥ 100-fold reduction in plaque formation is indicative of temperature sensitivity. The lowest such restrictive temperature for each virus is shown in bold and underlined and is referred to as the shut-off temperature. [Figure 28] Figure 1 shows rB / HPIV3 constructs evaluated for attenuation and immunogenicity in non-human primates (rhesus macaques). Rhesus macaques were infected by combined IN and intratracheal routes in groups of 5, 5, and 4 animals, respectively, with 10 TCID per site of the following constructs: non-HEK / non-opt; HEK / GA-opt / DS; and HEK / GA-opt / DS / B3TMCT. [Figure 29]Figures 29A and 29B are graphs showing rB / HPIV3 vector replication in rhesus macaques. Rhesus macaques were infected with the indicated rB / HPIV3 vectors as described in Figure 28. Vector replication in the respiratory tract was assessed by collecting (A) nasopharyngeal swabs and (B) tracheal washes on the indicated days and determining virus titers by limiting dilution assay. The limit of detection is 1.2 log10 TCID50 / mL, shown as the dotted line. [Figure 30] Graph showing serum HPIV3-neutralizing antibody titers induced by rB / HPIV3 vector. Monkey sera were collected 0, 14, 21, 28, 35, and 56 days after immunization, and HPIV3-neutralizing antibody titers were determined in a 60% plaque reduction neutralization test (PRNT60) in the presence of added guinea pig complement. The detection limit of the neutralization assay is indicated by the dotted line. The day of RSV challenge is indicated. [Figure 31] Graphs showing serum RSV-neutralizing antibody titers induced by rB / HPIV3 vectors. Monkey sera were collected on days 0, 14, 21, 28, 35, and 56 post-immunization (Figure 31). RSV neutralizing antibody titers were determined at all time points by a 60% plaque reduction neutralization test (PRNT60) in the presence of added guinea pig complement (Figure 32). RSV neutralizing antibody titers were determined on day 28 post-immunization by a 60% plaque reduction neutralization test (PRNT60) in the absence of added complement (Figure 33). The detection limit of the neutralization assay is indicated by a dotted line. Statistically significant differences in mean titers were determined by Tukey-Kramer test and are indicated by asterisks (**, P ≤ 0.01; ***, P ≤ 0.001). The day of RSV challenge is indicated. [Figure 32]Graphs showing serum RSV-neutralizing antibody titers induced by rB / HPIV3 vectors. Monkey sera were collected on days 0, 14, 21, 28, 35, and 56 post-immunization (Figure 31). RSV neutralizing antibody titers were determined at all time points by a 60% plaque reduction neutralization test (PRNT60) in the presence of added guinea pig complement (Figure 32). RSV neutralizing antibody titers were determined on day 28 post-immunization by a 60% plaque reduction neutralization test (PRNT60) in the absence of added complement (Figure 33). The detection limit of the neutralization assay is indicated by a dotted line. Statistically significant differences in mean titers were determined by Tukey-Kramer test and are indicated by asterisks (**, P ≤ 0.01; ***, P ≤ 0.001). The day of RSV challenge is indicated. [Figure 33] 1 shows the stability of RSV F expression by rB / HPIV3 vectors during replication in rhesus macaques. The percentage of recovered vectors expressing RSV F in nasopharyngeal swabs from days 4, 5, and 6 post-immunization was determined by dual-stain plaque assay. The percentage of rB / HPIV3 expressing RSV F among the tested specimens is shown. Specimens with 100% RSV F-expressing virus are colored yellow; specimens with 99-90% RSV F-expressing virus are colored green; specimens that did not produce plaques due to low titers are marked "NA." [Figure 34]

[0033] Figure 1 shows the construction of an rB / HPIV3 vector expressing a secreted version of the HEK / GS-opt / DS-Cav1 RSV F protein containing a C-terminal "fold-on" sequence. The RSV F protein, containing HEK assignments and expressed from a GS-codon-optimized (for human expression) ORF (with the DS-Cav1 mutation), was engineered to contain the N-terminal 513 amino acids of the F protein (i.e., lacking the TM and CT domains) fused to the indicated 4-amino acid linker and 27-amino acid fold-on sequence from T4 phage (SEQ ID NO: 132, Efimov et al. 1994, J Mol Biol 242:470-486; Miroshnikov et al. 1998, Protein Eng. 11:329-332). The ORF was inserted into the rB / HPIV3 vector at the same position and with the same vector signals as those described in Figures 1, 4, 11, and 17. [Figure 35] 1 is a table showing a summary of exemplary rB / HPIV3 vectors expressing RSV F, annotated to indicate the constructs evaluated in two different studies in hamsters and two different studies in rhesus monkeys in Example 1. [Figure 36]Schematic diagram of the construction of antigenomic cDNAs of HPIV1 CD170 and LY942A mutations containing RSV F gene inserts at the first (F1), second (F2), or third (F3) genomic positions. The rHPIV1 backbone used for RSV F expression contained two attenuating mutations: a CD170 mutation in the P / C gene (indicated by *) or an LY942A mutation in the L gene (indicated by ·). For the HPIV1-F1 construct, the RSV F gene was inserted into the first genomic position in front of the HPIV1 N gene at an MluI site located in the untranslated region upstream of the N gene. For HPIV1-F2, the RSV F gene was inserted between the HPIV1 N and P genes at an AscI site located in the untranslated region upstream of the P gene. For HPIV1-F3, the RSV F gene was cloned between the HPIV1 P and M genes at a NotI site located in the untranslated region downstream of the P gene. For all constructs, the RSV F ORF was codon-optimized for human expression and contained HEK amino acid assignments. A copy of the N gene end (GE), intergenic (IG) CTT triplet, and P gene start (GS) sequence was added after (F1, F2) or before (F3) the RSV F insert so that it was under the control of the HPIV1 transcription signal. The sequences of SEQ ID NOs: 138-140 are shown adjacent to the RSV F insert under HPIV1-F1; the sequences of SEQ ID NOs: 141-143 are shown adjacent to the RSV F insert under HPIV1-F2; and the sequences of SEQ ID NOs: 144-145 are shown adjacent to the RSV F insert under HPIV1-F3. [Figure 37-1]Figures 37A-37D are graphs showing the multistage replication of HPIV1 / RSV-F virus in Vero (37A and 37C) and LLC-MK2 (37B and 37D) cells. Triplicate wells of cell monolayers in 6-well plates were infected with HPIV1 CΔ170 (A and B) or LY942A (C and D) viruses expressing RSV F (F1, F2, or F3) in parallel with wt HPIV1, HPIV1 LY942A, and HPIV1 CΔ170 at an MOI of 0.01 TCID. Cultures were incubated at 32°C. Aliquots of cell culture medium were collected at 24-hour intervals, and virus titers (log10 TCID / ml) were determined by serial dilution and hemadsorption assay on LLC-MK2 cells at 32°C. The mean titers and standard error of the mean (SEM) are shown. Statistically significant differences between titers of each virus relative to wt HPIV1 at 2 days postinfection were determined using one-way ANOVA with Tukey's multiple comparison test and are indicated with asterisks as follows: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p < 0.0001. [Figure 37-2] Continuation of Figure 37-1. [Figure 38-1]Figures 38A-38C show Western blot analysis of RSV F and HPIV1 vector protein expression. Vero cells were infected with the indicated viruses at an MOI of 5. Forty-eight hours postinfection, cells were lysed with SDS sample buffer. All samples were denatured, reduced, and subjected to SDS-PAGE and Western blot analysis. Proteins were transferred to PVDF membranes and probed with either an RSV F-specific mouse monoclonal antibody or HPIV1 N-, P-, HN-, or F-specific polyclonal antibodies (raised by immunizing rabbits separately with synthetic peptides corresponding to each protein). Bound antibodies were visualized using corresponding anti-mouse (IRDye 680LT) and anti-rabbit (IRDye 800CW) antibodies conjugated to infrared dyes. Images were obtained by scanning the blots using an Odyssey infrared imaging system. Images shown are from a single experiment, representative of three independent experiments. (B and C) The intensities of protein bands for the rHPIV1 CΔ170 (B) and rHPIV1 LY942A (C) constructs were quantified for three independent experiments, and expression is shown relative to the F3 virus, which was set at 1.0. Plots show data as mean ± SEM from three independent experiments analyzed by one-way ANOVA with Dunnett's multiple comparison test using 95% confidence intervals. HPIV1 protein expression by F1, F2, and F3 viruses was statistically compared to that of their corresponding empty vector backbones. *, p<0.05; **, p<0.01; ***, p<0.001. [Figure 38-2] Continuation of Figure 38-1. [Figure 39]Figures 39A-39I are photographs showing cytopathic effects and syncytia formation in LLC-MK2 cell monolayers infected with rHPIV1 vectors expressing RSV F. MK2 cells were infected at an MOI of 0.01 TCID50 and incubated for 5 days. Images were acquired at 40x magnification using phase contrast light microscopy. Micrographs of (A) rHPIV1CΔ170-F1; (B) rHPIV1CΔ170-F2; (C) rHPIV1CΔ170-F3; (D) rHPIV1CΔ170; (E) rHPIV1LY942A-F1; (F) rHPIV1LY942A-F2; (G) rHPIV1LY942A-F3; (H) rHPIV1LY942A; and (I) wt HPIV1 are shown. [Figure 40]Figures 40A and 40B are graphs showing the replication of HPIV1 vectors expressing RSV F in the nasal turbinates (40A) and lungs (40B) of hamsters. Hamsters were inoculated intranasally with 10 TCID of wt HPIV1, rHPIV1 CD170, or rHPIV1 LY942A empty vector, rHPIV1 CD170, or rHPIV1 LY942A expressing RSV F from three genomic locations (F1, F2, or F3), rHPIV1-CR84GCD170HN553ALY942A (a previously described HPIV1 vaccine candidate (Bartlett et al. 2007 Virol J 4:6)), or rB / HPIV3-F2, a chimeric bovine / human PIV3 expressing RSV F from a second location (also known as HEK / GA-opt, see Figure 1). Viral titers were determined by hemadsorption assay in LLC-MK2 cells and reported as Log10 TCID50 / g of tissue. Titers for individual animals (six per group) are shown for days 3 (Δ) and 5 (·), with each symbol representing an individual animal. Mean values ​​for each group are shown in bold for day 3 and in italics for day 5. The limit of detection (LOD) was 1.5 log10 TCID50 / ml and is indicated by the dotted line across the bottom of each graph. Statistical significance of differences between each virus versus wt HPIV1 (red asterisk) or rB / HPIV3-F2 (top bar) was confirmed by one-way ANOVA at 95% confidence intervals using Tukey's multiple comparison test for days 3 and 5 postinfection (pi). *, p ≤ 0.05; ***, p ≤ 0.001; ****, p ≤ 0.0001; or ns, no significant difference. [Figure 41]Figures 41A and 41B are graphs showing protection against wt RSV challenge virus replication in the nasal turbinates (41A) and lungs (41B) of immunized hamsters. Hamsters (n=6) in each group were challenged intranasally with 10 PFU of wt RSV A2 30 days post-immunization. Nasal turbinates and lungs were collected from euthanized animals 3 days post-challenge, and virus titers were determined for each sample by RSV-specific plaque assay on Vero cells and reported as Log10 PFU per gram of tissue. The mean values ​​for each group are indicated by bold numbers and horizontal bars. Statistical significance of differences between viruses was determined by one-way ANOVA with 95% confidence intervals using Tukey's multiple comparison test and is indicated by *, p<0.05; **, p<0.01; ****p<0.0001; or ns, not significant. [Figure 42] 1 is a table illustrating attenuating mutations induced in the HPIV1 backbone in the P / C or L ORF. Nucleotide changes (deletions or substitutions) in the wt sequence are underlined. [Figure 43] 1 is a table illustrating the temperature sensitivity of recombinant viruses to LLC-MK2 cell monolayers. For temperature sensitivity, bold underlined values ​​indicate the virus shutoff temperature, indicating a temperature-sensitive phenotype defined as the lowest restrictive temperature, where the mean log reduction in virus titer at a given temperature versus 32°C was 2.0 log or greater than that of wt rHPIV1 at the same two temperatures. For monolayers, serial dilutions of each indicated virus on LLC-MK2 cells were incubated at various temperatures for 7 days. Viral titers were determined by hemadsorption using guinea pig red blood cells and reported as Log TCID / ml with a detection limit of 1.2. [Figure 44]1 shows the percentage of virus populations expressing RSV F after in vivo replication. The percentage of virus populations expressing RSV F after in vivo replication (stability) was determined by immunofluorescence double-staining plaque assay. Vero cells were infected with serially diluted tissue homogenates (144 samples total) from nasal turbinates or lungs of infected hamsters (n = 6 per virus) collected on days 3 and 5 post-infection (pi) and incubated for 6 days under a methylcellulose overlay. Viral plaques were stained using mouse monoclonal anti-RSV F and goat polyclonal anti-HPIV1 specific antibodies, followed by detection with the corresponding infrared dye-conjugated secondary antibodies. The percentage of plaques expressing both RSV F and HPIV1 antigens is shown. The stability of HPIV1 CD170-F1, -F2, and F3 in lung samples, and the stability of HPIV1 LY942A-F1, -F2, and F3 in URT and lungs could not be tested due to their lack of replication in these tissues. The number in parentheses indicates the RSV F expression status for a total of 6 hamsters per virus. ND, no plaques detected. [Figure 45]This table lists results showing that immunization of hamsters with rHPIV1 expressing RSV F induces serum neutralizing antibodies to RSV. Groups of 6-week-old hamsters (n=6) were immunized intranasally with 10 TCID of each indicated virus contained in a 0.1 ml inoculum. Serum samples were collected before immunization and 28 days after immunization. Antibody titers to RSV and HPIV1 were determined using a 60% plaque reduction neutralization test (PRNT60) using green fluorescent protein (GFP)- or enhanced GFP (eGFP)-expressing viruses (rRSV-eGFPM or HPIV1-GFP), and neutralizing antibody titers were expressed as the mean reciprocal log2 ± SE. Based on the initial serum dilution used in the assay, the PRNT60 assay has a titer detection limit of 3.3 and 1.0 reciprocal log2 PRNT60 for RSV and HPIV1, respectively. Statistical significance of differences between groups for RSV antibody titers was determined by one-way ANOVA using Tukey's multiple comparison test (p<0.05), and significance of differences for HPIV1 antibody titers was determined by unpaired t-test. Mean neutralizing antibody titers were categorized into groups (shown in parentheses as A, B, C, and D). Mean antibody titers for treatment groups with different letters are statistically different from each other; titers with two letters are not statistically different from the titers with either letter. [Figure 46]Graphs showing multi-cycle in vitro replication of rB / HPIV3 vectors expressing GA-optimized (GA-opt) prefusion forms of RSV F with DS-Cav1 mutations. (Figure 46) Vero and (Figure 47) LLC-MK2 cells were infected in triplicate at 32°C at an MOI of 0.01 TCID50 with an empty rB / HPIV3 vector (empty B / H3) or a vector expressing the RSV F ORF, where the RSV F ORF was either HEK-containing, GA-opt, and contained the DS-Cav1 prefusion stabilizing mutation (HEK / GA-opt / DS-Cav1), or HEK-containing, GA-opt, and contained the DS-Cav1 mutation and the BPIV3-specific TM and CT domains as a potential packaging signal (HEK / GA-opt / DS-Cav1 / B3TMCT). Aliquots of culture supernatant were collected at 24-hour intervals for 6 days, and virus titers were determined by limiting dilution assay in LLC-MK2 cells at 32°C and reported as TCID50 / ml. Mean titers ± SEM from three independent experiments are shown. [Figure 47] Graphs showing multi-cycle in vitro replication of rB / HPIV3 vectors expressing GA-optimized (GA-opt) prefusion forms of RSV F with DS-Cav1 mutations. (Figure 46) Vero and (Figure 47) LLC-MK2 cells were infected in triplicate at 32°C at an MOI of 0.01 TCID50 with an empty rB / HPIV3 vector (empty B / H3) or a vector expressing the RSV F ORF, where the RSV F ORF was either HEK-containing, GA-opt, and contained the DS-Cav1 prefusion stabilizing mutation (HEK / GA-opt / DS-Cav1), or HEK-containing, GA-opt, and contained the DS-Cav1 mutation and the BPIV3-specific TM and CT domains as a potential packaging signal (HEK / GA-opt / DS-Cav1 / B3TMCT). Aliquots of culture supernatant were collected at 24-hour intervals for 6 days, and virus titers were determined by limiting dilution assay in LLC-MK2 cells at 32°C and reported as TCID50 / ml. Mean titers ± SEM from three independent experiments are shown. [Figure 48]Figures 48A and 48B are graphs showing multi-cycle in vitro replication of rB / HPIV3 vectors expressing GS-optimized (GS-opt) RSV F with different modifications. (A) Vero and (B) LLC-MK2 cells were infected in triplicate at 32°C at an MOI of 0.01 TCID50 with an empty rB / HPIV3 vector (empty B / H3) or a vector expressing the RSV F ORF, which was either HEK-containing and GS-opt RSV F (HEK / GS-opt), or HEK-containing, GS-opt and carrying a DS-Cav1 prefusion stabilizing mutation (HEK / GS-opt / DS-Cav1), or HEK-containing, GS-opt and carrying a DS-Cav1 mutation and BPIV3-specific TM and CT domains (HEK / GS-opt / DS-Cav1 / B3TMCT), or a truncated RSV F of 1 to 513 amino acids fused to a 4-amino acid linker and a 27-amino acid oligomerization sequence derived from T4 phage. F, which is HEK-containing, GS-opt, and carries the DS-Cav1 mutation (HEK / GS-opt / DS-Cav1 / (1-513)Foldon). Aliquots of culture supernatant were collected at 24-hour intervals for 6 days, and virus titers were determined by limiting dilution assay in LLC-MK2 cells at 32°C and reported as TCID50 / ml. Mean titers ± SEM from three independent experiments are shown. [Figure 49]Figures 49A-49D are graphs showing a comparison of multi-cycle in vitro replication of rB / HPIV3 vectors expressing GS-opt and GA-opt RSV F. Figures 49A and 49B: (A) Vero and (B) LLC-MK2 cells were infected in triplicate at 32°C at an MOI of 0.01 TCID50 with an empty rB / HPIV3 vector (empty B / H3) or a vector expressing the RSV F ORF, where the RSV F ORF was HEK-containing, GS-opt, and carrying the DS-Cav1 mutation (HEK / GS-opt / DS-Cav1), or HEK-containing, GA-opt, and carrying the DS-Cav1 mutation (HEK / GA-opt / DS-Cav1). Figures 49C and 49D: (C) Vero and (D) LLC-MK2 cells were infected at an MOI of 0.01 TCID50 at 32 ° C. with an empty rB / HPIV3 vector (empty B / H3) or a vector expressing the RSV F ORF, where the RSV F ORF was HEK-containing, GS-opt, and carried the DS-Cav1 and B3TMCT modifications (HEK / GS-opt / DS-Cav1 / B3TMCT), or HEK-containing, GA-opt, and contained the DS-Cav1 and B3TMCT modifications (HEK / GA-opt / DS-Cav1 / B3TMCT). Aliquots of culture supernatant were collected at 24-hour intervals for 6 days, and virus titers were determined by limiting dilution assay in LLC-MK2 cells at 32 ° C. and reported as TCID50 / ml. The mean titer ± SEM from three independent experiments is shown. [Figure 50]Figures 50A-50C show the expression of various modified forms of RSV F by rB / HPIV3 vectors in cell culture. (A) Vero and (B, C) LLC-MK2 cells were infected with an empty rB / HPIV3 vector (lane 1), or an rB / HPIV3 vector expressing the indicated modified forms of RSV F (lanes 2-5 and 8), or wild-type RSV (wt RSV, lane 6) at an MOI of 3 PFU / cell, or were left uninfected (mock, lane 7). Infected Vero (A) and LLC-MK2 (B) cells were incubated at 32°C, and LLC-MK2 (C) cells were incubated at 37°C. Vero cell lysates and culture supernatants were collected at 48 hpi and subjected to Western blot analysis for RSV F expression. RSV F was detected as cleaved F1 and / or uncleaved F0 forms. BPIV3 N was used as an internal control for vector protein expression; GAPDH was used as a loading control. [Figure 51]Figures 51A and 51B show replication of rB / HPIV3 vectors in the upper and lower respiratory tracts of hamsters. Hamsters were infected IN with a 0.1 ml inoculum containing 10 TCID of the indicated rB / HPIV3 vector or 10 PFU of wt RSV. Hamsters were euthanized on days 4 and 5 postinfection (n=6 per virus per day), (A) nasal turbinates and (B) lungs were removed and homogenized, and virus titers were determined by limiting dilution in LLC-MK2 cells at 32°C and reported as TCID / g (rB / HPIV3 vector) or by plaque assay in Vero cells at 32°C and reported as PFU / g (wt RSV). The limit of detection (LOD) is 1.5 log TCID per g of tissue, indicated by the dotted line. Open and closed circles indicate titers from individual animals sacrificed on days 4 and 5, respectively. The mean titers for each group are indicated by dashed and solid horizontal lines for days 4 and 5, respectively. The mean titer values ​​for days 4 and 5 are shown at the top. The mean virus titers on day 5 were assigned to different groups using the Tukey-Kramer test: mean titers with different letters are statistically different (p<0.05), while titers with two letters are not significantly different from the titer indicated by either letter. [Figure 52]52A-52C are graphs showing serum RSV-neutralizing antibody titers from hamsters infected with the indicated rB / HPIV3 vectors expressing GA-opt or GS-opt RSV F proteins, with or without the DS, DS-Cav1, or B3TMCT modification. Hamsters (n=6 animals per virus) were inoculated IN with a 0.1 ml inoculum containing 10 TCID of the indicated rB / HPIV3 vector or 10 PFU of wt RSV. Serum samples were collected 28 days post-immunization, and antibody titers were determined by 60% plaque reduction neutralization test (PRNT60) with (FIG. 52) and without (FIG. 53) the addition of guinea pig complement. The height of each bar corresponds to the mean titer, along with the SEM. The mean titer value is indicated above the bar. Pairwise Student's t-tests were used to assess statistical significance between values: values ​​indicated by vertical bars at each of three horizontal lines above the mean titer were compared pairwise to each other and recorded as significantly different (*, p<0.05) or not significantly different (ns). The detection limit of the neutralization assay is indicated by a dotted line. ND, neutralization titer was below the detection limit. [Figure 53]52A-52C are graphs showing serum RSV-neutralizing antibody titers from hamsters infected with the indicated rB / HPIV3 vectors expressing GA-opt or GS-opt RSV F proteins, with or without the DS, DS-Cav1, or B3TMCT modification. Hamsters (n=6 animals per virus) were inoculated IN with a 0.1 ml inoculum containing 10 TCID of the indicated rB / HPIV3 vector or 10 PFU of wt RSV. Serum samples were collected 28 days post-immunization, and antibody titers were determined by 60% plaque reduction neutralization test (PRNT60) with (FIG. 52) and without (FIG. 53) the addition of guinea pig complement. The height of each bar corresponds to the mean titer, along with the SEM. The mean titer value is indicated above the bar. Pairwise Student's t-tests were used to assess statistical significance between values: values ​​indicated by vertical bars at each of three horizontal lines above the mean titer were compared pairwise to each other and recorded as significantly different (*, p<0.05) or not significantly different (ns). The detection limit of the neutralization assay is indicated by a dotted line. ND, neutralization titer was below the detection limit. [Figure 54]Figures 54A and 54B are graphs showing protection against RSV challenge in hamsters immunized with the indicated rB / HPIV3 vectors. Hamsters immunized as shown in Figure 53 (n=6 animals per immunization group) were challenged IN 30 days post-immunization with a 0.1 ml inoculum containing 10 PFU of wt RSV. Three days after challenge, the hamsters were euthanized and (A) nasal turbinates and (B) lungs were collected. RSV titers in tissue homogenates were determined by plaque assay in Vero cells at 37°C. Each symbol corresponds to an individual animal, and the mean virus titer for the group is shown above the symbol and is represented as a short horizontal line. Pairwise Student's t-tests were used to assess statistical significance between values: for each horizontal line above the mean titer, values ​​indicated by vertical bars were compared pairwise to each other and recorded as significant (*, p<0.05) or not significantly different (ns). The detection limit of the assay was log101.7 PFU / g of tissue, shown as the dotted line. [Figure 55] Figure 1 shows rB / HPIV3 constructs evaluated for attenuation and immunogenicity in non-human primates (rhesus macaques). Rhesus macaques were infected by combined IN and intratracheal routes in groups of 4, 6, and 6 animals, respectively, using 10 TCID per site of the following constructs: HEK / GA-opt / DS / B3TMCT; HEK / GA-opt / DS-Cav1 / B3TMCT; and HEK / GS-opt / DS-Cav1 / B3TMCT. [Figure 56] Figures 56A and 56B are graphs showing rB / HPIV3 vector replication in rhesus macaques. Rhesus macaques were infected with the rB / HPIV3 vectors shown in Figure 55. Vector replication in the respiratory tract was assessed by collecting (A) nasopharyngeal swabs and (B) tracheal washes on the indicated days and determining virus titers by limiting dilution assay. The limit of detection is 1.2 log10 TCID50 / mL, shown as the dotted line. [Figure 57]Figures 57A and 57B are graphs showing serum RSV-neutralizing antibody titers induced by rB / HPIV3 vectors. Sera were collected 0, 14, 21, and 28 days post-immunization from the experiments shown in Figures 55 and 56. Figure 57A: RSV neutralizing antibody titers at the indicated time points were determined by a 60% plaque reduction neutralization test (PRNT60) in the presence of added guinea pig complement. Statistical significance of differences between mean titers at each time point was determined by pairwise Student's t-test (ns, P>0.05). Figure 57A: RSV neutralizing antibody titers 28 days after immunization were determined by PRNT60 in the absence of added complement. The detection limit for the neutralization assay is indicated by the dotted line. Statistical significance of differences between mean titers at each time point was determined by pairwise Student's t-test (ns, P>0.05). [Figure 58] Figures 58A and 58B show the construction of an rB / HPIV3 vector expressing HEK / GS-opt / DS-Cav1 / B3TMCT from the pre-N locus, as well as modifications to the amino acid sequence of the HPIV3 HN protein to achieve increased phenotypic stability of the vector. Figure 58A: Insertion of the HEK / GS-opt / DS-Cav1 / B3TMCT insert into the first gene locus of rB / HPIV3. Figure 58A: Modification of the HPIV3 HN gene to confer increased phenotypic stability. The HN gene in the original recombinant HPIV3, generated by reverse genetics (Durbin et al., Virology 235:323-332, 1997), had two engineered nucleotide substitutions in the HN gene at antigenomic positions 7913 and 7915 (resulting in the amino acid substitution P370T) and a spontaneous mutation at antigenomic position 7593 (resulting in the amino acid substitution T263I). Here, these mutations were reverted to the "wild-type" assignment, ie, to that found in the biologically derived HPIV3 strain JS (Genbank Z11575.1; Stokes et al. Virus Res 25:91-103. 1992). [Figure 59]Figures 59A and 59B show intracellular expression of RSV F and vector proteins by vectors expressing various versions of the RSV F protein at the first gene position (pre-N) or the second gene position (NP). Analysis of rB / HPIV3-wt HN-HEK / GS-opt / DS-Cav1 / B3TMCT / pre-N, the construct depicted in Figure 58A. Vero (Figure 59A) and LLC-MK2 (Figure 59B) cells were infected with the empty rB / HPIV3 vector (empty B / H3, lane 1), or the wtHN / HEK / GS-opt / DS-Cav1 / B3TMCT / pre-N construct (pool CL20a, CL24a, lanes 3 and 4), or a vector with the same version of RSV F inserted into the second (NP) position (HEK / GS-opt / DS-Cav1 / B3TMCT / NP, lane 5), or a vector with a non-HEK / non-opt version of RSV F inserted into the pre-N position (lane 6), or RSV (lane 2), or mock-infected (lane 7). The vectors were infected at an MOI of 10 TCID50 / cell, and wt RSV was infected at an MOI of 3 PFU / cell. Infected monolayers were incubated at 32°C. Cell lysates were collected at 48 hpi and subjected to Western blot analysis. RSV F was detected in the form of cleaved F1 and / or uncleaved F0. BPIV3 N and P proteins were used to assess the effect on vector protein expression. GAPDH was used as a loading control. [Figure 60] HPIV1 vector: The amino acid sequence positions of the cytoplasmic tail (CT), transmembrane (TM) domain, and adjacent regions of the RSV F protein ectodomain (strain A2, amino acid assignments) and the HPIV1 F protein (bold). RSV-F-TMCT is a chimeric protein consisting of the RSV F protein ectodomain linked to the TM and CT domains of the HPIV1 F protein. [Figure 61]61A and 61B show the construction of the HPIV1-CΔ170 vector expressing a version of the RSV F protein designed to be stabilized in the pre-fusion conformation (DS-Cav1) and to have increased integration into HPIV1 vector particles. Each of these modified RSV F inserts contains HEK assignments (HEK) and has been codon-optimized by GS for human expression (GS-opt). The RSV F insert was engineered to be stabilized in the pre-fusion conformation with DS and Cav1 mutations (DS-Cav1) alone (upper constructs in Figures 61 and 62), or with further modification by replacing the TMCT domain with a domain from HPIV1 F (TMCT, lower constructs in Figures 61 and 62). The resulting HEK / GS-opt / DS-Cav1 and HEK / GS-opt / DS-Cav1 / TMCT versions of RSV F were modified with flanking sequences and inserted into the HPIV1-CΔ170 vector (see Example 2 for a description of the HPIV1 vector and CΔ170 mutations) at either the first gene position (MluI site) (Figure 61) or the second gene position (AscI site) (Figure 62). In each case, RSV F was under the control of the HPIV1 transcription signal for expression as a separate mRNA. Nucleotide numbering refers to the complete antigenome RNA sequence of the final construct. The sequences of SEQ ID NOs: 146 and 147 are shown adjacent to the RSV F insert below the diagram for F1 / HEK / GS-opt / DS-Cav1, F1 / HEK / GS-opt / DS-Cav1 / TMCT, F2 / HEK / GS-opt / DS-Cav1, and F2 / HEK / GS-opt / DS-Cav1 / TMCT. [Figure 62]61A and 61B show the construction of the HPIV1-CΔ170 vector expressing a version of the RSV F protein designed to be stabilized in the pre-fusion conformation (DS-Cav1) and to have increased integration into HPIV1 vector particles. Each of these modified RSV F inserts contains HEK assignments (HEK) and has been codon-optimized by GS for human expression (GS-opt). The RSV F insert was engineered to be stabilized in the pre-fusion conformation with DS and Cav1 mutations (DS-Cav1) alone (upper constructs in Figures 61 and 62), or with further modification by replacing the TMCT domain with a domain from HPIV1 F (TMCT, lower constructs in Figures 61 and 62). The resulting HEK / GS-opt / DS-Cav1 and HEK / GS-opt / DS-Cav1 / TMCT versions of RSV F were modified with flanking sequences and inserted into the HPIV1-CΔ170 vector (see Example 2 for a description of the HPIV1 vector and CΔ170 mutations) at either the first gene position (MluI site) (Figure 61) or the second gene position (AscI site) (Figure 62). In each case, RSV F was under the control of the HPIV1 transcription signal for expression as a separate mRNA. Nucleotide numbering refers to the complete antigenome RNA sequence of the final construct. The sequences of SEQ ID NOs: 146 and 147 are shown adjacent to the RSV F insert below the diagram for F1 / HEK / GS-opt / DS-Cav1, F1 / HEK / GS-opt / DS-Cav1 / TMCT, F2 / HEK / GS-opt / DS-Cav1, and F2 / HEK / GS-opt / DS-Cav1 / TMCT. [Figure 63]Figure 1 shows the kinetics of multicycle propagation in Vero cells of the rHPIV1-CΔ170 vector expressing RSV F stabilized in a prefusion conformation (DS-Cav1) without or with TMCT derived from the HPIV1 F protein. Vero cells were infected in triplicate with the constructs at an MOI of 0.01 and incubated at 32°C for 7 days. At 24-hour intervals, 0.5 mL of culture supernatant was collected for a total of 3 mL over 7 days. After sample collection, 0.5 mL of fresh medium was added to each culture to restore the original volume. Virus titration of the collected samples was performed by hemadsorption assay on LLC-MK2 cells, and values ​​are plotted as the mean ± SEM. [Figure 64] This figure shows the incorporation of RSV F protein stabilized in the prefusion conformation (DS-Cav1) into HPIV1-CΔ170 virion particles, without or with TMCT derived from HPIV1 F protein. The indicated viral constructs (HEK / GS-opt designation omitted for brevity) were propagated in LLC-MK2 cells, and virions were purified by sucrose gradient centrifugation. The protein concentration of purified virus was determined by BCA assay. A total of 1 μg of protein from each purified virus was dissolved in RIPA lysis buffer, reduced, denatured, and subjected to SDS-PAGE and Western blot analysis. RSV F (top panel) and HPIV1 proteins (second, third, and fourth panels) were detected with mouse monoclonal and rabbit polyclonal HPIV1-peptide-specific (N, F, and HN) antibodies, respectively. Bound primary antibody was detected using an infrared-labeled secondary antibody. The chimeric RSV-F-DS-Cav1 / TMCT protein is visible in lanes 2 and 4 (fourth panel) because anti-peptide serum specific for the HPIV1 F protein was raised using a synthetic peptide containing the C-terminal 18 amino acids of the CT domain and therefore reacts with the RSV F protein containing the HPIV1 F protein TMCT domain. [Figure 65]This figure shows the expression of RSV F protein stabilized in the prefusion conformation (DS-Cav1) in infected Vero cells without and with TMCT derived from HPIV1 F protein. Vero cell monolayers in 6-well plates were inoculated with the indicated viruses, including wt HPIV1 and the rHPIV1-CΔ170 empty vector control (the designation HEK / GS-opt was omitted for brevity), at an MOI of 5 and incubated at 32°C for 48 hours. Cell lysates were prepared by dissolving the monolayers in 200 μL of LDS sample buffer. Protein samples were reduced and denatured, and 45 μL of each sample was electrophoresed, followed by protein transfer to a PVDF membrane. RSV F and HPIV1 proteins were detected using the same primary and secondary antibodies described in Figure 64. [Figure 66] 1 is a sequence diagram of the cytoplasmic tail (CT), transmembrane (TM) domain, and adjacent regions of the RSV F protein (amino acid assignment) and the ectodomain of the HPIV3 F protein (bold), with the amino acid sequence positions indicated. RSV-F-H3TMCT is a chimeric protein consisting of the ectodomain of the RSV F protein linked to the TM and CT domains of the HPIV3 F protein. [Figure 67A]

[0039] Figure 58B shows the construction of an rHPIV3 vector expressing a version of the RSV F protein designed to be stabilized in the prefusion conformation (DS-Cav1) and to have increased integration into rHPIV3 vector particles. This vector was the wild-type rHPIV3 strain JS, which was modified to contain the amino acid assignments 263T and 370P in the HN protein, which were found to confer phenotypic stability to the vector (see Figure 58B). Additionally, the rHPIV3 vector was modified by creating a BlpI site at positions 103-119 for insertion of RSV F (or potentially any other insert) at gene position 1 (A, top construct) or an AscI site at positions 1675-1682 for insertion of RSV F at gene position 2 (B, top construct). Each modified RSV F insert contained HEK assignments (HEK) and was codon-optimized by GS for human expression (GS-opt). The RSV F insert was further engineered to be stabilized in the pre-fusion conformation by the DS and Cav1 mutations (DS Cav1) alone (A and B, second construct) or by further modification by replacing its TMCT domain with that from rHPIV3 F (H3TMCT, A and B, third construct). The resulting HEK / GS-opt / DS-Cav1 and HEK / GS-opt / DS-Cav1 / H3TMCT versions of RSV F were modified with flanking sequences and inserted into (A) the first gene position (BlpI site) or (B) the second gene position (AscI site) of wt rHPIV3 JS. In each case, RSV F was under the control of the HPIV3 transcription signal for expression as a separate mRNA. Nucleotide numbering refers to the complete antigenome RNA sequence of the final construct. The sequence of SEQ ID NO: 148 is shown below the schematic of rHPIV3 wt-JS.The sequences of SEQ ID NOs: 149 and 150 are shown adjacent to the RSV F insert below the schematic diagrams of F1 / HEK / GS-opt / DS-Cav1, F1 / HEK / GS-opt / DS-Cav1 / H3TMCT, F2 / HEK / GS-opt / DS-Cav1, and F2 / HEK / GS-opt / DS-Cav1 / H3TMCT. [Figure 67B]

[0039] Figure 58B shows the construction of an rHPIV3 vector expressing a version of the RSV F protein designed to be stabilized in the prefusion conformation (DS-Cav1) and to have increased integration into rHPIV3 vector particles. This vector was the wild-type rHPIV3 strain JS, which was modified to contain the amino acid assignments 263T and 370P in the HN protein, which were found to confer phenotypic stability to the vector (see Figure 58B). Additionally, the rHPIV3 vector was modified by creating a BlpI site at positions 103-119 for insertion of RSV F (or potentially any other insert) at gene position 1 (A, top construct) or an AscI site at positions 1675-1682 for insertion of RSV F at gene position 2 (B, top construct). Each modified RSV F insert contained HEK assignments (HEK) and was codon-optimized by GS for human expression (GS-opt). The RSV F insert was further engineered to be stabilized in the pre-fusion conformation by the DS and Cav1 mutations (DS Cav1) alone (A and B, second construct) or by further modification by replacing its TMCT domain with that from rHPIV3 F (H3TMCT, A and B, third construct). The resulting HEK / GS-opt / DS-Cav1 and HEK / GS-opt / DS-Cav1 / H3TMCT versions of RSV F were modified with flanking sequences and inserted into (A) the first gene position (BlpI site) or (B) the second gene position (AscI site) of wt rHPIV3 JS. In each case, RSV F was under the control of the HPIV3 transcription signal for expression as a separate mRNA. Nucleotide numbering refers to the complete antigenome RNA sequence of the final construct. The sequence of SEQ ID NO: 148 is shown below the schematic of rHPIV3 wt-JS.The sequences of SEQ ID NOs: 149 and 150 are shown adjacent to the RSV F insert below the schematic diagrams of F1 / HEK / GS-opt / DS-Cav1, F1 / HEK / GS-opt / DS-Cav1 / H3TMCT, F2 / HEK / GS-opt / DS-Cav1, and F2 / HEK / GS-opt / DS-Cav1 / H3TMCT. DETAILED DESCRIPTION OF THE INVENTION

[0014] Sequence Listing The nucleic acid and amino acid sequences listed in the accompanying Sequence Listing are shown using standard letter abbreviations for nucleotide bases and three-letter codes for amino acids as defined in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. The Sequence Listing is incorporated herein by reference. The data was submitted as an ASCII text file in the form of a file named "Sequence.txt" (approximately 344kb) created on January 19, 2016.

[0015] Previous studies (Zimmer et al. J Virol 2005 79:10467-77) evaluated the expression of the RSV F protein from a heterologous gene in Sendai virus, the murine counterpart of HPIV1 and closely related to HPIV3. The studies showed that very little RSV F protein is incorporated into Sendai virus vector particles. The researchers compared the CT or CT and TM of the RSV F protein with the corresponding sequences from the Sendai F protein, which were then expressed as foreign RSV F proteins. The substitutions were made with the assumption that they would improve the efficiency of the F protein's interaction with vector particles. These modifications actually increased the incorporation of the engineered RSV F into Sendai particles, but only when the Sendai F protein gene was also deleted. The need to delete the vector F protein is not compatible with generating infectious attenuated viruses for vaccination and also removes one of the vector protective antigens that may be required to generate a bivalent vaccine.

[0016] As disclosed herein, the RSV F protein, including the RSV F TM and CT, is incorporated into vector particles only in trace amounts when expressed by rB / HPIV3, HPIV3, or HPIV1. However, replacing the TM and CT of the heterologous RSV F protein with the corresponding TM and CT of the paramyxovirus F protein provides a diverse increase in RSV F ectodomain incorporation into the recombinant paramyxovirus envelope, resulting in packaging of RSV F into vectors as efficient (e.g., B / HPIV3) or more efficient (e.g., HPIV1) than RSV itself per μg of purified virion. This was true when the TM and CT were replaced together or when the CT was replaced alone. However, the unexpected effect of increasing the fusogenicity of chimeric RSV F specific to CT alone provides guidance that TMCT is preferred.

[0017] Efficient packaging of RSV F into vector particles significantly enhanced the induction of immune responses against the ectodomain (containing all of the neutralizing epitopes) when the recombinant paramyxovirus was administered to subjects. Unexpectedly, virus-neutralizing serum antibody responses were qualitatively significantly enhanced, as assessed by comparing RSV neutralizing activity in vitro in the absence (measuring strongly neutralizing antibodies) or presence (enhancing neutralization by weakly or non-neutralizing antibodies) of complement. This unexpected qualitative enhancement of antibodies is particularly important for RSV, which is notorious for inducing incomplete immune protection. Expression and efficient packaging of a foreign glycoprotein containing the TMCT domain of the vector glycoprotein clearly had the potential to disrupt vector replication and morphogenesis; however, constructs in which this effect was minimal were provided.

[0018] To further enhance immunogenicity, the stabilization of the RSV F protein in the pre-fusion conformation was evaluated. Pre-fusion stabilization also naturally resulted in an increase in strong neutralizing antibodies, suggesting the stabilization of neutralizing epitopes. In hamster models, the effect of pre-fusion stabilization on increasing immunogenicity and protection appeared to be additive to the effective packaging conferred by TMCT. However, when evaluated in non-human primates, the effect of packaging appeared to be greater than that of pre-fusion stabilization.

[0019] Given the challenge of achieving protection against RSV, maximal immunogenicity is desirable. Extensive experimentation has explored other aspects of vector and insert constructs (e.g., the use of various insertion sites, the use of codon optimization, and the use of early passage RSV F protein sequences). was found to result in increased expression of RSV F and reduced cytopathic effects of syncytia formation mediated by the highly fusogenic RSV F protein.

[0020] It is noteworthy that a prototype vaccine virus based on rB / HPIV3 expressing an unmodified RSV F protein, which had disappointing RSV immunogenicity in clinical trials (Bernstein et al. 2012. Pediatric Infectious Disease Journal 31:109-114), was confirmed by the methods of the present disclosure to induce RSV-neutralizing serum antibodies with low-quality neutralizing activity in vitro only in the presence of added complement. In contrast, the disclosed construct induced high titers of serum antibodies capable of effectively neutralizing RSV in African green monkeys in vitro in the absence of complement.

[0021] I. Terminology Summary Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology are provided by Benjamin Lewin, Genes X, Jones & Bartlett Publishers, 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, Wiley-VCH Publishing, Vol. 16, 2008; and other similar references.

[0022] As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural unless the context clearly indicates otherwise. For example, the term "an antigen" includes singular or multiple antigens and can be considered equivalent to the phrase "at least one antigen." As used herein, the term "comprises" means "includes." It should be further understood that any and all base sizes or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and provided for convenience, unless otherwise specified. Although many methods and materials similar or equivalent to those described herein can be used, specific suitable methods and materials are described herein. In case of conflict, the present specification, including the explanations of terms, will control. Furthermore, the materials, methods, and examples are merely illustrative and are not intended to be limiting. In order to facilitate review of various embodiments, the following explanations of terms are provided:

[0023] Adjuvant: A medium used to enhance antigenicity. Adjuvants include suspensions of minerals (alum, aluminum hydroxide, or aluminum phosphate) to which antigens are adsorbed; or water-in-oil emulsions, e.g., antigen solutions emulsified in mineral oil (Freund's incomplete adjuvant), sometimes containing killed mycobacteria to further enhance antigenicity (to inhibit antigen degradation and / or induce macrophage influx) (Freund's complete adjuvant). Immunostimulatory oligonucleotides (e.g., those containing CpG motifs) can also be used as adjuvants. Adjuvants include biological molecules ("biological adjuvants"), such as costimulatory molecules. Exemplary adjuvants include IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL, immune stimulating complex (ISCOM) matrix and toll-like receptor (TLR) agonists such as TLR-9 agonists, poly I:C or poly ICLC. Those skilled in the art are familiar with adjuvants (see, for example, Singh (ed.) Vaccine Adjuvants and Delivery Systems. Wiley-Interscience, 2007). Adjuvants can be used in combination with the disclosed recombinants.

[0024] Administration: The introduction of a composition into a subject by a selected route. Administration can be local or systemic. For example, if the selected route is intranasal, the composition (such as a composition comprising a disclosed recombinant paramyxovirus) is administered by introducing the composition into the nasal passage of the subject. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (e.g., topical), intranasal, vaginal, and inhalation routes.

[0025] Amino acid substitution: The replacement (i.e., deletion) of an amino acid in a polypeptide with a different amino acid or with no amino acid. In some instances, an amino acid in a polypeptide is replaced with an amino acid from a homologous polypeptide. For example, an amino acid in a recombinant group A RSV F polypeptide is replaced with the corresponding amino acid from a group B RSV F polypeptide. Reference to the "66E" amino acid in the RSV F protein refers to a RSV F protein containing a glutamic acid residue at position 66. The amino acid may be present due to a substitution from the reference sequence. Reference to a "K66E" substitution in the RSV F protein refers to a RSV F protein containing a glutamic acid residue at position 66 substituted for a lysine residue in the reference (e.g., native) sequence.

[0026] Attenuated: A paramyxovirus having an "attenuated" or "attenuated phenotype" refers to a paramyxovirus that has reduced virulence compared to a reference wild-type paramyxovirus under similar infection conditions. Attenuation is usually associated with reduced viral replication compared to the replication of a reference wild-type paramyxovirus under similar infection conditions; therefore, "attenuation" and "restricted replication" are often used synonymously. In some hosts (usually non-native hosts, including laboratory animals), disease is not evident during infection with the reference paramyxovirus in question, and restricted viral replication can be used as a surrogate marker of attenuation. In some embodiments, an attenuated recombinant paramyxovirus (e.g., RSV, PIV3) exhibits at least about a 10-fold or greater reduction in viral titer in the upper or lower respiratory tract of a mammal, such as at least about a 100-fold or greater reduction, compared to the unattenuated wild-type viral titer in the upper or lower respiratory tract, respectively, of a mammal of the same species under the same infection conditions. Examples of mammals include, but are not limited to, humans, mice, rabbits, rats, hamsters such as golden hamsters (Mesocricetus auratus), and non-human primates such as plains monkeys (Ceroptihecus aethiops). Attenuated paramyxoviruses can exhibit distinct phenotypes, including, but not limited to, altered growth, temperature-sensitive growth, host range-restricted growth, or altered plaque size.

[0027] Cytoplasmic tail (CT): The continuous region of a transmembrane protein that includes the terminus (either N- or C-terminus) of the protein and extends from the cytoplasmic surface of the cell membrane or the viral envelope into the cytoplasm of the cell or enveloped virus. In the case of type I transmembrane proteins, the CT includes the C-terminus of the protein. In the case of type II transmembrane proteins, the CT includes the N-terminus of the protein.

[0028] Degenerate variant: In the context of this disclosure, a "degenerate variant" refers to a polynucleotide that encodes a polypeptide that comprises a sequence that is degenerate as a result of the genetic code. There are 20 naturally occurring amino acids, most of which are specified by more than one codon. Thus, all degenerate nucleotide sequences that encode a peptide are included, as long as the amino acid sequence of the peptide encoded by the nucleotide sequence is unchanged.

[0029] Gene: A nucleic acid sequence, usually a DNA sequence that includes regulatory and coding sequences required for transcription of RNA, whether mRNA or not. For example, a gene may contain a promoter, one or more enhancers or silencers, a sequence encoding an RNA and / or a polypeptide. The nucleic acid sequence may include a nucleic acid sequence that regulates the expression of mRNA, downstream regulatory sequences and, optionally, other nucleic acid sequences involved in regulating the expression of mRNA.

[0030] Heterologous: originates from a different gene source. A heterologous gene contained in a recombinant viral genome is a gene that is not derived from the viral genome. In one specific, non-limiting example, a heterologous gene encoding the ectodomain of the RSV F protein is contained in the genome of a recombinant PIV vector. Methods for introducing heterologous genes into viral vectors are well known in the art and are described herein.

[0031] Host cell: A cell in which a vector can be propagated and its nucleic acid expressed. The cell can be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parent cell since mutations may occur during replication. However, such progeny are included when the term "host cell" is used.

[0032] Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response"). In one embodiment, the immune response is a T cell response, such as a CD4+ or CD8+ response. In another embodiment, the response is a B cell response, resulting in the production of specific antibodies.

[0033] Immunogen: A compound, composition, or substance capable of stimulating antibody production or a T-cell response in an animal, including compositions injected or absorbed into an animal. An immunogen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous antigens such as the disclosed recombinant paramyxoviruses. Administration of an immunogen to a subject can lead to protective immunity against the pathogen of interest.

[0034] Immunogenic composition: A composition comprising an immunogen that induces a measurable T cell response to an antigen or induces a measurable B cell response (such as antibody production) to an antigen, the immunogen being contained on or encoded by a nucleic acid molecule contained in the immunogen. In one example, the immunogenic composition is a composition comprising a disclosed recombinant paramyxovirus that, when administered to a subject, induces a measurable CTL response to RSV and / or PIV or induces a measurable B cell response (such as antibody production) to RSV and / or PIV. The immunogenic composition may comprise an isolated recombinant paramyxovirus as disclosed herein. For in vivo use, the immunogenic composition typically comprises a recombinant paramyxovirus in a pharmaceutically acceptable carrier and may also include other agents, such as an adjuvant.

[0035] Isolated: An "isolated" biological component is one that has been substantially separated or purified from other biological components, such as other chromosomal and extrachromosomal DNA, RNA, and other biological components in which the component naturally occurs, such as proteins. "Isolated" proteins, peptides, nucleic acids, and viruses include those purified by standard purification methods. Isolation does not require absolute purity and can include proteins, peptides, nucleic acids, or viral molecules that are at least 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% isolated.

[0036] Linked: The terms "linked," "linked," and "linked" refer to the joining of two molecules into one continuous molecule, e.g., the joining of two polypeptides into one continuous polypeptide by recombinant means. Reference to a gene encoding a type I membrane protein comprising an RSV F ectodomain "linked" with the TM and CT of a heterologous F protein refers to the expression of the gene from N to C terminally linked to the RSV F ectodomain, TM, and This refers to the genetic linkage between a nucleic acid sequence encoding the RSV F ectodomain and a nucleic acid sequence encoding the TM and CT of a heterologous F protein in a gene by recombinant means, leading to the production of a protein containing CT. In some embodiments, the C-terminal residue of the RSV F ectodomain can be directly linked to the N-terminal residue of the TM (via a peptide bond). In some embodiments, the C-terminal residue of the RSV F ectodomain can be indirectly linked to the N-terminal residue of the TM via a peptide linker (such as a glycine-serine linker).

[0037] Linker: A bifunctional molecule that can be used to link two molecules into one contiguous molecule. A non-limiting example of a peptide linker is a glycine-serine linker.

[0038] Native protein, sequence, or disulfide bond: A polypeptide, sequence, or disulfide bond that has not been modified, for example, by selective mutation. For example, selective mutation to focus the antigenicity of an antigen against a target epitope or to introduce a disulfide bond into a protein that does not exist in the native protein. A native protein or native sequence is also called a wild-type protein or wild-type sequence. A non-native disulfide bond is a disulfide bond that does not exist in the native protein, for example, a disulfide bond that forms in a protein due to the introduction of one or more cysteine ​​residues into the protein by genetic engineering.

[0039] Nucleic acid molecule: A polymeric form of nucleotides that can include both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. Nucleotide refers to ribonucleotides, deoxynucleotides, or modified forms of any type of nucleotide. The terms "nucleic acid molecule" are synonymous with "nucleic acid" and "polynucleotide" herein. Nucleic acid molecules are typically at least 10 bases in length, unless otherwise specified. This term includes single- and double-stranded forms of DNA. Polynucleotides can contain either or both naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide linkages.

[0040] Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked nucleic acid sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

[0041] Paramyxovirus: A family of enveloped, non-segmented, negative-strand, single-stranded RNA viruses. Examples of paramyxoviruses include, but are not limited to, human parainfluenza viruses (HPIVs), including types 1, 2, 3, 4A, and 4B (HPIV1, HPIV2, HPIV3, HPIV4A, and HPIV4B, respectively), murine parainfluenza type 1 (Sendai virus, MPIV1), bovine parainfluenza virus type 3 (BPIV3), parainfluenza virus 5 (PIV5, formerly known as simian virus 5, SV5), simian virus 41 (SV41), and mumps virus. HPIV1, HPIV3, MPIV1, and BPIV3 are classified in the genus Respirovirus. HPIV2, HPIV4, SV5, SV41, and mumps virus are classified in the genus Rubulavirus. MPIV1, PIV5, and BPIV3 are animal relatives of HPIV1, HPIV2, and HPIV3, respectively (Chancock et al., Parainfluenza Viruses, Knipe et al. (eds.), pp. 1341-1379, Lippincott et al., 2001). (Incott Williams & Wilkins, Philadelphia, 2001). HPIV1, HPIV2, and HPIV3 represent distinct serotypes and do not induce significant cross-immunity. HPIVs are causative agents of respiratory infections such as croup, pneumonia, and bronchitis.

[0042] Parainfluenza virus (PIV): Several enveloped, non-segmented, negative-strand, single-stranded RNA viruses from the Paramyxoviridae family are descriptively grouped together. This includes all members of the Respirovirus genus (e.g., HPIV1, HPIV3) and some members of the Rubulavirus genus (e.g., HPIV2, HPIV4, PIV5). Members of the Avulavirus genus (e.g., NDV) have historically been called PIVs and are considered part of this group. HPIV serotypes 1, 2, and 3 are second only to RSV in causing severe respiratory infections in infants and children worldwide, with HPIV3 being the most significant HPIV in terms of disease impact. PIVs are composed of two structural modules: (1) an internal ribonucleoprotein core or nucleocapsid containing the viral genome and (2) an outer, roughly spherical lipoprotein envelope. The PIV viral genome is approximately 15,000 nucleotides long and encodes at least eight polypeptides. These proteins include the nucleocapsid structural protein (NP, NC, or N, depending on the genus), phosphoprotein (P), matrix protein (M), fusion glycoprotein (F), hemagglutinin-neuraminidase glycoprotein (HN), large polymerase protein (L), and C and D proteins. The P gene contains one or more additional open reading frames (ORFs) that encode accessory proteins. The gene order is 3'-NPMF-HN-L-5', with each gene encoding a distinct protein that encodes an mRNA. Exemplary PIV strain sequences, such as those of HPIV1, HPIV2, HPIV3, and BPIV3 viruses, are known to those of skill in the art.

[0043] Pharmaceutically acceptable carriers: Pharmaceutically acceptable carriers that are useful are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed immunogens.

[0044] Generally, the nature of the carrier will vary depending on the particular mode of administration being used. For example, parenteral formulations usually contain injectable fluids containing pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solution, aqueous dextrose, glycerol, or the like as a vehicle. Solid compositions (e.g., in powder, pill, tablet, or capsule form) can include conventional non-toxic solid carriers, such as pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate. In certain embodiments suitable for administration to a subject, the carrier is sterile and / or suspended or otherwise contained in a unit dosage form containing one or more measured doses of a composition suitable for inducing the desired immune response. This is also achieved by applying the drug for its therapeutic use. The unit dosage form can be, for example, in a sealed vial containing sterile contents or a syringe for injection into a subject, or lyophilized or in a solid or sustained release dosage form for subsequent solubilization and administration.

[0045] Polypeptide: Any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). "Polypeptide" applies to amino acid polymers, including naturally occurring and non-naturally occurring amino acid polymers, and , in which one or more amino acid residues are unnatural amino acids, e.g., artificial chemical mimetics of the corresponding naturally occurring amino acids. "Residue" refers to an amino acid or amino acid mimetic incorporated into a polypeptide by an amide bond or amide bond mimetic. Polypeptides have an amino-terminal (N-terminal) end and a carboxy-terminal (C-terminal) end. "Polypeptide" is used interchangeably with peptide or protein and is used herein to refer to a polymer of amino acid residues.

[0046] Prime-boost vaccination: immunotherapy involves administering a first immunogenic composition (primer vaccine) to a subject, followed by a second immunogenic composition (booster vaccine) to induce an immune response. A booster vaccine is administered to a subject after the primer vaccine; those skilled in the art will understand the appropriate time interval between the administration of the primer vaccine and the booster vaccine, and examples of such time frames are disclosed herein. The prime-boost protocol can also include further administration, for example, a second boost.

[0047] Recombinant: A recombinant nucleic acid molecule is one that has a sequence that is not found in nature, e.g., contains one or more nucleic acid substitutions, deletions, or insertions, and / or has a sequence that is made by the artificial combination of two otherwise separate segments of sequence, which can be accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acid, e.g., by genetic engineering techniques.

[0048] A recombinant virus is one that contains a genome that includes a recombinant nucleic acid molecule.

[0049] A recombinant protein is one that has a sequence that does not occur in nature or that is created by the artificial combination of two otherwise separate segments of sequence. In some embodiments, a recombinant protein is encoded by a heterologous (e.g., recombinant) nucleic acid that has been introduced into a host cell, such as a bacterial or eukaryotic cell, or into the genome of a recombinant virus.

[0050] Respiratory syncytial virus (RSV): An enveloped, non-segmented, negative-sense, single-stranded RNA virus of the Paramyxoviridae family. The RSV genome is approximately 15,000 nucleotides long and contains 10 genes encoding 11 proteins, including glycoproteins SH, G, and F. The F protein mediates fusion, allowing viral entry into the cytoplasm and also promotes syncytial formation. Two antigenic subgroups of human RSV strains, A and B subgroups, have been described, based primarily on antigenic differences in the G glycoprotein. RSV strains of other species, including bovine RSV, are also known. Exemplary RSV strain sequences are known to those of skill in the art. Additionally, several models of human RSV infection are available, including model organisms infected with species-specific RSV, such as the use of model organisms infected with hRSV as well as bRSV infection in cattle (see, e.g., Bern et al., Am J Physiol Lung Cell Mol Physiol, 301:L148~L156, 2011; and Nam and Kun (eds.) Respiratory Syncytial Virus: Prevention, Diagnosis and Treatment Nova Biomedical Nova Science Publisher, 2011; and Cane (ed.) Respiratory Syncytial Virus Elsevier Science, 2007).

[0051] RSV fusion (F) protein: An RSV envelope glycoprotein that promotes fusion of viral and cellular membranes. In reality, the RSV F protein is initially synthesized as a single polypeptide precursor, designated F0, approximately 574 amino acids in length. F0 contains an N-terminal signal peptide that directs localization to the endoplasmic reticulum, where the signal peptide (approximately F The first 22 residues of F0 are proteolytically cleaved. The remaining F0 residues oligomerize to form a trimer, which also contains two conserved furin consensus cleavage sequences (approximately F0 positions 109 / 110 and 136 / 137; e.g., RARR 109(SEQ ID NO: 1, residues 106-109) and RKRR 136 (SEQ ID NO: 1, residues 133-136)) is proteolytically processed by cellular proteases to excise the pep27 polypeptide and generate two disulfide-linked fragments, F1 and F2. The smaller of these fragments, F2, originates from the N-terminal portion of the F precursor and contains approximately residues 26-109 of F. The larger of these fragments, F1, contains the C-terminal portion of the F precursor (approximately residues 137-574), including the extracellular / luminal region (approximately residues 137-529), TM (approximately residues 530-550), and CT (approximately residues 551-574) at the C-terminus.

[0052] The three F2-F1 promoters oligomerize in the mature F protein, which adopts a metastable "pre-fusion" conformation that is triggered to undergo a conformational change (to a "post-fusion" conformation) upon contact with the target cell membrane. This conformational change exposes a hydrophobic sequence, known as the fusion peptide, located at the N-terminus of the F1 polypeptide, which binds to the host cell membrane and promotes fusion of the viral or infected cell membrane with the target cell membrane.

[0053] The extracellular portion of the RSV F protein is the RSV F ectodomain, which contains the F2 protein and the F1 ectodomain. The RSV F ectodomain trimer is composed of three RSV Contains the F ectodomain protein complex.

[0054] The RSV F protein adopts a "pre-fusion" conformation prior to the induction of membrane fusion events that lead to the transition of RSV F to a post-fusion conformation and subsequent processing into the mature RSV F protein in the secretory system. The three-dimensional structure of an exemplary RSV F protein in a pre-fusion conformation is known and is disclosed, for example, in WO2014160463, which is incorporated herein by reference. In the pre-fusion state, the RSV F protein contains RSV F residues 62-69 and 196-209, and also contains the epitopes of the D25 and AM22 monoclonal antibodies, known as "antigenic sites." Φ The recombinant RSV contains an antigenic site at its membrane-distal apical end, called the "prefusion conformation." Thus, the recombinant RSV is stabilized in the prefusion conformation. The F protein binds to the pre-fusion conformation of the RSV F protein but not to the post-fusion conformation, e.g., an antigenic site. Φ It is specifically bound by antibodies that specifically bind to epitopes within the F-line fusion domain, such as the D25 or AM22 antibodies. Additional RSV F-line fusion-specific antibodies include the 5C4 and MPE8 antibodies.

[0055] Sequence identity: The similarity between amino acid sequences is alternatively expressed in terms of the similarity between the sequences, called sequence identity. Sequence identity is often measured in terms of percent identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Polypeptide homologs, orthologs, or variants have a relatively high degree of sequence identity when aligned using standard methods.

[0056] Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151~3, 1989;Corpet et al., Nuc. Acids Res.16:10881~90, 1988;Huang et al.Comp and Pearson et al., Meth. Mol. Biol. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, present a detailed discussion of sequence alignment methods and homology calculations.

[0057] Once aligned, the number of matches is determined by counting the number of positions where the same nucleotide or amino acid residue exists in both sequences. The percent sequence identity is determined by dividing the number of matches by either the length of the sequence shown in the identified sequence or by the specified length (such as 100 consecutive nucleotides or amino acid residues from the sequence shown in the identified sequence), and then multiplying the resulting value by 100. For example, a peptide sequence that has 1166 matches when aligned with a test sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1166÷1554*100=75.0). The percent sequence identity value is rounded to one decimal place. For example, 75.11, 75.12, 75.13 and 75.14 will round down to 75.1, while 75.15, 75.16, 75.17, 75.18 and 75.19 will round up to 75.2. Length values ​​are always integers.

[0058] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD), and on the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. A description of how to use this program to determine sequence identity is available on the Internet at the NCBI website.

[0059] Homologs and variants of polypeptides (such as the RSV F ectodomain) are typically characterized by having at least about 75% sequence identity, e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity across the entire length of the amino acid sequence of interest. Proteins with even greater similarity to the reference sequence will exhibit increased percentages of identity, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, when assessed by this method. When less than the entire sequence is compared for sequence identity, homologs and variants typically have at least 80% sequence identity over a short window of 10 to 20 amino acids, and may have at least 85%, or at least 90%, or 95% sequence identity, depending on the similarity to the reference sequence. Methods for determining sequence identity over such short windows are available on the internet at the NCBI website. Those skilled in the art will understand that these sequence identity ranges are provided merely for guidance; it is entirely possible that potentially significant homologs can be obtained that lie outside the ranges provided.

[0060] For the sequence comparison of nucleic acid sequences, a sequence usually serves as a reference sequence to which test sequences are compared.Using sequence comparison algorithm, test and reference sequences are input into a computer, and subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Default program parameters are used.Methods for aligning sequences for comparison are well known in the art.Optimal alignment of sequences for comparison can be found, for example, in Smith & Waterman, Adv.Appl.Mat. h.2:482, 1981 by the local homology algorithm of Needleman & By the homology alignment algorithm of Wunsch, J.Mol.Biol.48:443,1970, by the similarity search method of Pearson & Lipman, Proc.Nat'l.Acad.Sci.USA 85:2444,1988, by computer implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (for example, see Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, detailed appendix 104, 2013)). One example of a useful algorithm is PILEUP. PILEUP uses the progressive alignment simplification method of Feng & Doolittle, J. Mol. Evol. 35:351-360, 1987. The method used is similar to that described by Higgins & Sharp, CABIOS 5:151-153, 1989. Using PILEUP, a reference sequence is compared to other test sequences to determine percent sequence identity using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained, for example, from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al., Nuc. Acids Res. 12:387-395, 1984).

[0061] Another example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., J. Mol. Biol. 215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402, 1977. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, alignment (B) of 50, prediction (E) of 10, M=5, N=-4, and a comparison of both strands. The BLASTP program (for amino acid sequences) uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989). Oligonucleotides are linear polynucleotide sequences up to about 100 nucleotide bases in length.

[0062] As used herein, references to "at least 90% identity" refer to "at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% identity" to the specified reference sequence.

[0063] Subject: A category that includes living multicellular vertebrates, humans and non-human mammals. In one example, the subject is a human. In a specific example, the subject is a newborn. In another example, the subject that needs to inhibit RSV infection is selected. For example, the subject is either not infected and at risk of RSV infection, or infected and in need of treatment.

[0064] transmembrane domain (TM) e.g., the lipid bilayer of a cell or virus or virus-like particle The amino acid sequence spanning the lipid bilayer of the transmembrane domain can be used to anchor the antigen to the membrane. In some instances, the transmembrane domain is the RSV F transmembrane domain.

[0065] Vaccine: A preparation of immunogenic material capable of stimulating an immune response administered to prevent, ameliorate, or treat infection or other types of disease. Immunogenic material may include attenuated or killed microorganisms (such as bacteria or viruses) or antigenic proteins, peptides, or DNA derived therefrom. Attenuated vaccines are pathogenic organisms that have been modified to produce less pathogenic forms but still retain the ability to elicit antibodies and cellular immunity against the pathogenic forms. Inactivated (killed) vaccines are formerly pathogenic organisms that have been inactivated using chemicals, heat, or other treatments but still elicit antibodies against the organism. Vaccines can elicit both prophylactic (preventive or protective) and therapeutic responses. Methods of administration vary depending on the vaccine but may include inoculation, ingestion, inhalation, or other forms of administration. Vaccines can be administered with adjuvants to boost the immune response.

[0066] Vector: An entity containing a DNA or RNA molecule with a promoter(s) operably linked to the coding sequence of an antigen(s) of interest, capable of expressing the coding sequence. Non-limiting examples include naked or packaged (lipid and / or protein) DNA, naked or packaged RNA, viruses or bacteria or other microorganisms, which may be replication-incompetent, or subcomponents of viruses or bacteria or other microorganisms, which may be replication-competent. A vector is sometimes called a construct. A recombinant DNA vector is a vector containing recombinant DNA. A vector may contain a nucleic acid sequence that enables it to replicate in a host cell, such as an origin of replication. A vector may also contain one or more selectable marker genes and other genetic elements known in the art. A viral vector is a recombinant nucleic acid vector that contains at least some nucleic acid sequences derived from one or more viruses.

[0067] II. Recombinant viral vectors Recombinant paramyxoviruses are provided that contain antigens derived from multiple viral pathogens and can be used to induce immune responses against those viral pathogens. The recombinant paramyxoviruses contain a genome encoding a heterologous gene. The recombinant paramyxoviruses contain a genome containing a heterologous gene encoding the ectodomain of a transmembrane protein (e.g., a viral glycoprotein) of a heterologous viral pathogen. The ectodomain can be linked to the CT or TM and CT of the paramyxovirus-derived envelope protein, allowing the paramyxovirus envelope to express the ectodomain of the transmembrane protein from a heterologous virus. For example, the recombinant paramyxovirus can be a recombinant PIV containing a genome containing a heterologous gene encoding the ectodomain of the RSV F protein linked to the TM and CT of the PIV-derived F protein. Further description of recombinant paramyxoviruses and their modifications is provided herein.

[0068] The paramyxovirus genome contains genes encoding the N, P, M, F, HN, and L proteins. The genome also contains a genomic promoter and anti-promoter in the following order: promoter-N, P, M, F, HN, L-anti-promoter. The heterologous gene contained in the genome of the recombinant paramyxovirus can be located anywhere between the genes of the paramyxovirus genome, or between the promoter and the N gene or the L gene and the anti-promoter. The heterologous gene can be flanked by appropriate gene start and gene end sequences to promote expression from the viral genome. In a preferred embodiment, the heterologous gene can be located between the promoter and the N gene or between the N gene and the P gene. do.

[0069] In one embodiment, the heterologous gene contained in the genome of the recombinant paramyxovirus encodes the ectodomain of a type I transmembrane protein (e.g., a glycoprotein of a type I virus) linked to the CT or TM and CT of the F protein of the paramyxovirus. In other embodiments, the heterologous gene contained in the genome of the recombinant paramyxovirus encodes the ectodomain of a type II transmembrane protein (e.g., a glycoprotein of a type II virus) linked to the CT or TM and CT of the HN protein of the paramyxovirus.

[0070] The recombinant paramyxovirus can be, for example, a recombinant HPIV1, HPIV2, HPIV3, BPIV3, PIV5, Sendai virus, or NDV, or chimeras thereof. Further description of such recombinant paramyxoviruses is provided below.

[0071] General methods for generating recombinant paramyxoviruses containing genomes containing heterologous genes are known to those of skill in the art, as are viral sequences and reagents for use in such methods. Non-limiting examples of methods for generating recombinant PIV vectors (such as recombinant HPIV1, HPIV2, HPIV3, or H / BPIV3 vectors) containing heterologous genes, methods for attenuating the vectors (e.g., by recombinant or chemical means), and viral sequences and reagents for use in such methods are described in U.S. Patent Publication No. 2012 / 0045471, each of which is incorporated herein by reference in its entirety; Nos. 2010 / 0119547; 2009 / 0263883; 2009 / 0017517; 8084037; 6,410,023; 8,367,074; 7,951,383; 7,820,182; 7704509; 7632508; 7622123; 7250171; 7208161; 7201907; 7192593 and Newman et al. 2002 Virus genes 24:77-92, Tang et al. 2003 J Virol, 77(20):10819-10828. Non-limiting examples of methods for producing recombinant NDV vectors containing heterologous genes, as well as viral sequences and reagents for use in such methods, are provided in U.S. Patent Publication No. 2012 / 0064112; and Basavarajappa et al., 2014 Vaccine, 32:3555-3563 and McGinnes et al., J. Virol., 85:366-377, 2011, each of which is incorporated by reference in its entirety. Non-limiting examples of methods for producing recombinant Sendai vectors containing heterologous genes, as well as viral sequences and reagents for use in such methods, are provided in U.S. Patent Publication No. 20140186397 and Jones et al., Vaccine, 30:959-968, 2012, each of which is incorporated by reference in its entirety.

[0072] A. HPIV1 Vector In some embodiments, the recombinant paramyxovirus can be a recombinant HPIV1 comprising a viral genome encoding HPIV1 N, P, C, M, F, HN, and L proteins. The nucleic acid sequence of the HPIV1 genome and the genes therein are known in the art, as are the structural and functional genetic elements that control gene expression, such as gene start and gene end sequences and viral genome and antigenome promoters. An exemplary HPIV1 Washington / 1964 strain genome sequence is provided as GenBank Accession No. AF457102.1, which is incorporated herein by reference in its entirety. This exemplary HPIV1 Washington / 1964 strain genome sequence is: HPIV1 N, SEQ ID NO: 24 (GenBank protein number AAL89400.1, incorporated herein by reference); HPIV1 P, SEQ ID NO: 25 (GenBank protein number AAL89402.1, incorporated herein by reference) HPIV1 C, SEQ ID NO: 26 (ORF of P, incorporated herein by reference, GenBank protein number AAL89403.1); HPIV1 M, SEQ ID NO: 27 (GenBank protein number AAL89406.1, incorporated herein by reference) HPIV1 F, SEQ ID NO: 28 (GenBank protein number AAL89407.1, incorporated herein by reference) HPIV1 HN, SEQ ID NO: 29 (GenBank protein number AAL89408.1, incorporated herein by reference) HPIV1 L, SEQ ID NO: 30 (GenBank protein number AAL89409.1, incorporated herein by reference) The N, P, C, M, F, HN and L proteins are encoded by the nucleotides N, P, C, M, F, HN and L.

[0073] The corresponding gene start and gene end sequences for these HPIV1 genes are provided below: [Table 1]

[0074] Additionally, the viral leader / genomic promoter and trailer / antigenome promoter of HPIV2 strain V94, as shown in GenBank accession number AF457102.1 as nucleotides 1 to 96 and 15544 to 15600, respectively.

[0075] The recombinant paramyxovirus can be a recombinant HPIV1 comprising a viral genome encoding the HPIV1 N, P, C, M, F, HN, and L proteins as set forth above, or encoding HPIV1 N, P, C, M, F, HN, and L proteins that individually have at least 90% (such as at least 95%) sequence identity to the HPIV1 N, P, C, M, F, HN, and L proteins set forth above.

[0076] In some embodiments, the recombinant paramyxovirus can be a recombinant HPIV1 comprising a genome comprising a heterologous gene encoding a recombinant viral glycoprotein ectodomain (such as the RSV F ectodomain) derived from a type I membrane protein linked to the HPIV1 F protein TM and CT as shown below, or a recombinant viral glycoprotein ectodomain (such as the RSV F ectodomain) derived from a type I membrane protein linked to the HPIV1 F protein TM and CT as shown below, which has at least 90% (such as at least 95%) sequence identity to the HPIV1 F protein TM and CT as shown below. In some embodiments, the recombinant paramyxovirus can be a recombinant HPIV1 comprising a genome comprising a heterologous gene encoding a recombinant viral glycoprotein ectodomain (such as the RSV F ectodomain) derived from a type I membrane protein linked to the HPIV1 F protein TM and CT as shown below, or a recombinant viral glycoprotein ectodomain (such as the RSV F ectodomain) derived from a type I membrane protein linked to the HPIV1 F protein TM and CT as shown below, which has at least 90% (such as at least 95%) sequence identity to the HPIV1 F protein CT as shown below. The recombinant HPIV1 may comprise a genome containing a heterologous gene encoding a recombinant viral glycoprotein ectodomain (such as the RSV F ectodomain) derived from a type I membrane protein. The HPIV1 F protein TM and CT sequences are known (see, for example, GenBank Accession No. AF457102.1, which is incorporated herein by reference). Exemplary HPIV1 F protein TM and CT sequences are shown below: HPIV1 F TM:QIIMIIIVCILIIIICGILYYLY, residues 1-23 of SEQ ID NO:31 HPIV1 F CT:RVRRLLVMINSTHNSPVNAYTLESRMRNPYMGNNSN, residues 24-59 of SEQ ID NO: 31 HPIV1 F TM+CT:QIIMIIIVCILIIIICGILYYLYRVRRLLVMINSTHNSPVNAYTLESRMRNPYMGNNSN, SEQ ID NO: 31

[0077] B. HPIV2 Vector In some embodiments, the recombinant paramyxovirus vector can be a recombinant HPIV2 comprising a viral genome encoding HPIV2 N, P, V, M, F, HN, and L proteins. The nucleic acid sequences of the genes encoding these HPIV2 proteins are known in the art, as are the structural and functional genetic elements that control gene expression, such as gene start and gene end sequences and viral genome and antigenome promoters. An exemplary HPIV2 V94 strain genome sequence is provided under GenBank accession number AF533010.1, which is incorporated herein by reference in its entirety. This exemplary HPIV2 V94 strain genome sequence is: HPIV2 N, SEQ ID NO: 32 (encoded by GenBank number AF533010.1, incorporated herein by reference) HPIV2 P, SEQ ID NO: 33 (encoded by GenBank number AF533010.1, incorporated herein by reference) HPIV2 V, SEQ ID NO: 34 (ORF of P, encoded by GenBank number AF533010.1, incorporated herein by reference) HPIV2 M, SEQ ID NO: 35 (encoded by GenBank number AF533010.1, incorporated herein by reference) HPIV2 F, SEQ ID NO: 36 (encoded by GenBank number AF533010.1, incorporated herein by reference) HPIV2 HN, SEQ ID NO: 37 (encoded by GenBank number AF533010.1, incorporated herein by reference) HPIV2 L, SEQ ID NO: 38 (encoded by GenBank number AF533010.1, incorporated herein by reference) The N, P, V, M, F, HN and L proteins are encoded by the nucleotides N, P, V, M, F, HN and L.

[0078] The corresponding gene start and gene end sequences for these HPIV2 genes are provided below: [Table 2]

[0079] Additionally, the viral leader / genomic promoter and trailer / antigenomic promoter of HPIV2 strain V94 as shown in GenBank accession number AF533010.1 are shown as nucleotides 1-175 and 15565-15654, respectively.

[0080] The recombinant paramyxovirus can be a recombinant HPIV2 comprising a viral genome encoding HPIV2 N, P, V, M, F, HN, and L proteins as set forth above, or encoding HPIV2 N, P, V, M, F, HN, and L proteins that individually have at least 90% (such as at least 95%) sequence identity to the HPIV2 N, P, V, M, F, HN, and L proteins set forth above.

[0081] In some embodiments, the recombinant paramyxovirus can be a recombinant HPIV2 comprising a genome comprising a heterologous gene encoding a recombinant viral glycoprotein ectodomain (such as the RSV F ectodomain) derived from a type I membrane protein linked to the HPIV2 F protein TM and CT as shown below, or a recombinant viral glycoprotein ectodomain (such as the RSV F ectodomain) derived from a type I membrane protein linked to the HPIV2 F protein TM and CT as shown below that has at least 90% (such as at least 95%) sequence identity to the HPIV2 F protein TM and CT as shown below. In some embodiments, the recombinant paramyxovirus can be a recombinant HPIV2 comprising a genome comprising a heterologous gene encoding a recombinant viral glycoprotein ectodomain (such as the RSV F ectodomain) derived from a type I membrane protein linked to the HPIV2 F protein CT as shown below, or a recombinant viral glycoprotein ectodomain (such as the RSV F ectodomain) derived from a type I membrane protein linked to the HPIV2 F protein CT as shown below that has at least 90% (such as at least 95%) sequence identity to the HPIV2 F protein CT as shown below. HPIV2 F protein TM and CT sequences are known (see, e.g., GenBank Accession No. AF533010.1, incorporated herein by reference). An exemplary HPIV2 F protein TM and CT sequence from the HPIV3 JS strain is shown below: HPIV2 F TM domain: TLYSLSAIALILSVITLVVVGLLIAYII, residues 1-28 of SEQ ID NO: 39 HPIV2 F CT:KLVSQIHQFRALAATTMFHRENPAVFSKNNHGNIYGIS, residues 29-66 of SEQ ID NO:39 HPIV2 F TM+CT:TLYSLSAIALILSVITLVVVGLLIA YIIKLVSQIHQFRALAATTMFHRENPAVFSKNNHGNIYGIS, SEQ ID NO: 39

[0082] C. HPIV3 Vector In some embodiments, the recombinant paramyxovirus can be a recombinant HPIV3 comprising a viral genome encoding HPIV3 N, P, C, M, F, HN, and L proteins. The nucleic acid sequences of the genes encoding these HPIV3 proteins are known in the art, as are the structural and functional genetic elements that control gene expression, such as gene start and gene end sequences and viral genome and antigenome promoters. An exemplary HPIV3 JS strain genome sequence is provided under GenBank accession number Z11575, which is incorporated herein by reference in its entirety. For this exemplary HPIV3 JS strain genome sequence, the nucleic acid sequences encoding the N, P, C, M, F, HN, and L proteins are shown below. HPIV3 N, SEQ ID NO: 40 (encoded by nucleotides 111-1658 of GenBank No. Z11575, incorporated herein by reference) HPIV3 P, SEQ ID NO: 41 (encoded by nucleotides 1784 to 3595 of GenBank No. Z11575, incorporated herein by reference) HPIV3 C, SEQ ID NO: 114 (encoded by nucleotides 1794-2393 of GenBank No. Z11575, incorporated herein by reference) HPIV3 M, SEQ ID NO: 42 (encoded by nucleotides 3753-4814 of GenBank No. Z11575, incorporated herein by reference) HPIV3 F, SEQ ID NO: 43 (encoded by nucleotides 5072 to 6691 of GenBank No. Z11575, incorporated herein by reference) HPIV3 HN, SEQ ID NO: 44 (encoded by nucleotides 6806 to 8524 of GenBank No. Z11575, incorporated herein by reference) HPIV3 L, SEQ ID NO: 45 (encoded by nucleotides 8646 to 15347 of GenBank No. Z11575, incorporated herein by reference)

[0083] In some embodiments, the HN gene in the HPIV3 vector encodes an HPIV3 HN protein comprising the amino acid sequence shown below: (SEQ ID NO: 101)

[0084] An exemplary DNA sequence encoding SEQ ID NO:101 is provided as follows: atggaatactggaagcataccaatcacggaaaggatgctggtaatgagctggagacgtctatggctactcatggcaacaagctcactaataagataatatacatattatggacaataatcctggtgttattatcaatagtcttcatcatagtgctaattaattccatcaaaagtgaaaaggcccacgaatcattgctgcaagacataaataatgagtttatggaaattacagaaaagatccaaatggcatcggataataccaatgatctaatacagtcaggagtgaatacaaggcttcttacaattcagagtcatgtccagaattacataccaatatcattgacacaacagatgtcagatcttaggaaattcattagtgaaattacaattagaaatgata atcaagaagtgctgccacaaagaataacacatgatgtaggtataaaacctttaaatccagatgatttttggagatgcacgtctggtcttccatctttaatgaaaactccaaaaataaggttaatgccagggccgggattattagctatgccaacgactgttgatggctgtgttagaactccgtctttagttataaatgatctgatttatgcttatacctcaaatctaattactcgaggttgtcaggatataggaaaatcatatcaagtcttacagatagggataataactgtaaactcagacttggtacctgacttaaatcctaggatctctcatacctttaacataaatgacaataggaagtcatgttctctagcactcctaaatatagatgtatatca actgtgttcaactcccaaagttgatgaaagatcagattatgcatcatcaggcatagaagatattgtacttgatattgtcaattatgatggttcaatctcaacaacaagatttaagaataataacataagctttgatcaaccatatgctgcactatacccatctgttggaccagggatatactacaaaggcaaaataatatttctcgggtatggaggtcttgaacatccaataaatgagaatgtaatctgcaacacaactgggtgccccgggaaaacacagagagactgtaatcaagcatctcatagtacttggttttcagataggaggatggtcaactccatcattgttgttgacaaaggcttaaactcaattccaaaattgaaagtatggacgatatctatgcgacaaaattactgggggtcagaaggaaggttacttctactaggtaacaagatctatatatatacaagatctacaagttggcatagcaagttacaattaggaataattgatattactgattacagtgatataaggataaaatggacatggcataatgtgctatcaagaccaggaaacaatgaatgtccatggggacattcatgtccagatggatgtataacaggagtatatactgatgcatatccactcaatcccacagggagcattgtgtcatctgtcatattagactcacaaaaatcgagagtgaacccagtcataacttactcaacagcaaccgaaagagtaaacgagctggccatcctaaacagaacactctcagctggatatacaacaacaagctgcattacacactataacaaaggatattgttttcatatagtagaaataaatcataaaagcttaaacacatttcaacccatgttgttcaaaacagagattccaaaaagctgcagttaa(SEQ ID NO: 102)

[0085] The corresponding gene start and gene end sequences for these HPIV3 genes are provided below: [Table 3]

[0086] Additionally, the viral genome and antigenome promoters of HPIV3 strain JS as set forth in GenBank accession number Z11575 are provided as nucleotides 1-96 (genomic promoter) and nucleotides 15367-15462 (antigenome promoter), respectively.

[0087] The recombinant paramyxovirus can be a recombinant HPIV3 comprising a viral genome encoding HPIV3 N, P, C, M, F, HN, and L proteins as set forth above, or encoding HPIV3 N, P, C, M, F, HN, and L proteins that individually have at least 90% (such as at least 95%) sequence identity to the HPIV3 N, P, C, M, F, HN, and L proteins set forth above.

[0088] In some embodiments, the recombinant paramyxovirus can be a recombinant HPIV3 comprising a genome including a heterologous gene encoding a recombinant viral glycoprotein ectodomain (such as the RSV F ectodomain) derived from a type I membrane protein linked to the HPIV3 F protein TM and CT as shown below, or that has at least 90% (such as at least 95%) sequence identity to the HPIV3 F protein TM and CT as shown below. In some embodiments, the recombinant paramyxovirus can be a recombinant HPIV3 comprising a genome including a heterologous gene encoding a recombinant viral glycoprotein ectodomain (such as the RSV F ectodomain) derived from a type I membrane protein linked to the HPIV3 F protein TM and CT as shown below, or that has at least 90% (such as at least 95%) sequence identity to the HPIV3 F protein TM and CT as shown below. The recombinant HPIV3 may comprise a genome containing a heterologous gene encoding a recombinant viral glycoprotein ectodomain (such as the RSV F ectodomain) derived from a type I membrane protein linked to an HPIV3 F protein CT having sequence identity (e.g., at least 95%). HPIV3 F protein TM and CT sequences are known (see, e.g., the protein encoded by nucleotides 5072-6691 of GenBank No. Z11575). Exemplary HPIV3 F protein TM and CT sequences from the HPIV3 JS strain are shown below: HPIV3 F TM domain: IIIILIMIIILFIINITIITIAI, residues 1-23 of SEQ ID NO: 46 HPIV3 F CT:KYYRIQKRNRVDQNDKPYVLTNK, residues 24-46 of SEQ ID NO:46 HPIV3 F TM+CT:IIIILIMIIILFIINITIITIAIKYYRIQKRNRVDQNDKPYVLTNK, SEQ ID NO: 46

[0089] D. Bovine PIV3 and Chimeric Human / Bovine PIV3 Vectors In some embodiments, the recombinant paramyxovirus can be a bovine PIV3 (BPIV3) or chimeric paramyxovirus comprising a viral genome encoding a combination of N, P, C, V, M, F, HN, and L proteins from BPIV3 and HPIV3. For example, the chimeric viral genome can encode HPIV3 F and HN proteins and BPIV3 N, P, C, V, M, and L proteins. The nucleic acid sequences of the genes encoding these HPIV3 and BPIV3 proteins are known in the art, as are the structural and functional genetic elements that control gene expression, such as gene start and end sequences and viral genome and antigenome promoters. An exemplary BPIV3 Kansas genome sequence is provided under GenBank Accession No. AF178654, which is incorporated herein by reference in its entirety. This exemplary BPIV3 Kansas strain genome sequence encodes the N, P, C, V, M, F, HN, and L proteins shown below: BPIV3 N, SEQ ID NO: 47 (GenBank Accession No. AAF28254, each encoded by nucleotides 111 to 1658 of GenBank No. AF178654, which are incorporated herein by reference) BPIV3 P, SEQ ID NO: 48 (GenBank Accession No. AAF28255, each encoded by nucleotides 1784 to 3574 of GenBank No. AF178654, which are incorporated herein by reference) BPIV3 C, SEQ ID NO: 115 (encoded by nucleotides 1794-2399 of GenBank No. AF178654, incorporated herein by reference) BPIV3 V, SEQ ID NO: 116 (gene editing site located at nucleotides 2500-2507, with nucleotide g inserted between nucleotides 2505-2506, encoded by nucleotides 1784-3018 of GenBank number AF178654) BPIV3 M, SEQ ID NO: 49 (GenBank Accession No. AAF28256, each encoded by nucleotides 3735 to 4790 of GenBank No. AF178654, which are incorporated herein by reference) BPIV3 F, SEQ ID NO: 50 (GenBank Accession No. AAF28257, each encoded by nucleotides 5066 to 6688 of GenBank No. AF178654, which are incorporated herein by reference) BPIV3 HN, SEQ ID NO: 51 (GenBank Accession No. AAF28258, each encoded by nucleotides 6800 to 8518 of GenBank No. AF178654, incorporated herein by reference) BPIV3 L, SEQ ID NO: 52 (encoded by nucleotides 8640 to 15341 of GenBank No. AF178654, each of which is incorporated herein by reference) GenBank accession number: AAF28259)

[0090] In some embodiments, the HPIV3 HN gene contained in the chimeric B / HPIV3 vector encodes an HPIV3 HN protein or variant thereof comprising the amino acid sequence set forth as SEQ ID NO: 101 or SEQ ID NO: 44. An exemplary DNA sequence encoding SEQ ID NO: 101 is provided as SEQ ID NO: 102.

[0091] In some embodiments, the chimeric B / HPIV3 vector can include an HPIV3 F gene in place of the BPIV3 F gene, for example, a gene encoding the HPIV3 F amino acid sequence set forth as SEQ ID NO: 43 or a variant thereof.

[0092] The corresponding gene start and gene end sequences of these BPIV3 genes are provided below: [Table 4]

[0093] Additionally, BPIV3 as shown in GenBank accession number AF178654 The viral genome and antigenome promoters of the Kansas strain are provided as nucleotides 1 to 96 (genomic promoter) and nucleotides 15361 to 15456 (antigenome promoter), respectively.

[0094] Recombinant paramyxoviruses comprising viral genomes encoding the N, P, C, V, M, F, HN and L proteins from HPIV3 and BPIV3 viruses may encode a mixture of HPIV3 and BPIV3 N, P, C, V, M, F, HN and L proteins as set forth above, or may encode a mixture of BPIV3 and HPIV3 N, P, C, V, M, F, HN and L proteins that, individually, have at least 90% (such as at least 95%) sequence identity to the BPIV3 or HPIV3 N, P, C, V, M, F, HN and L proteins set forth above.

[0095] In some embodiments, the recombinant paramyxovirus may comprise a viral genome encoding HPIV3 F and HN proteins and BPIV3 N, P, C, V, M, and L proteins as set forth above, or encoding HPIV3 F and HN proteins and BPIV3 N, P, C, V, M, and L proteins that, individually, have at least 90% (such as at least 95%) sequence identity to the corresponding HPIV3 F and HN proteins or BPIV3 N, P, C, V, M, and L proteins set forth above.

[0096] In some embodiments, a recombinant paramyxovirus comprising a genome encoding the N, P, C, V, M, F, HN, and L proteins from BPIV3 is linked to the TM and CT of the BPIV3 F protein as shown below, or In some embodiments, a recombinant paramyxovirus comprising a genome encoding the N, P, C, V, M, F, HN, and L proteins from BPIV3 may further comprise a heterologous gene encoding a recombinant viral glycoprotein ectodomain (such as the RSV F ectodomain) derived from a type I membrane protein linked to a TM and CT having at least 90% (such as at least 95%) sequence identity to the TM and CT of the BPIV3 F protein, as shown below. In some embodiments, a recombinant paramyxovirus comprising a genome encoding the N, P, C, V, M, F, HN, and L proteins from BPIV3 may further comprise a heterologous gene encoding a recombinant viral glycoprotein ectodomain (such as the RSV F ectodomain) derived from a type I membrane protein linked to a CT of the BPIV3 F protein, as shown below, or linked to a CT having at least 90% (such as at least 95%) sequence identity to the CT of the BPIV3 F protein, as shown below.

[0097] In some embodiments, recombinant paramyxoviruses comprising genomes encoding the N, P, C, V, M, F, HN, and L proteins from HPIV3 and BPIV3 viruses (such as HPIV3 F and HN proteins and BPIV3 N, P, C, V, M, and L proteins) may further comprise a heterologous gene encoding a recombinant viral glycoprotein ectodomain (such as the RSV F ectodomain) derived from a type I membrane protein linked to the TM and CT of the BPIV3 F protein as shown below, or linked to a TM and CT that have at least 90% (such as at least 95%) sequence identity to the TM and CT of the BPIV3 F protein as shown below. In some embodiments, recombinant paramyxoviruses comprising genomes encoding the N, P, C, V, M, F, HN, and L proteins from HPIV3 and BPIV3 viruses may further comprise a heterologous gene encoding a recombinant viral glycoprotein ectodomain (such as a RSV F ectodomain) derived from a type I membrane protein linked to the CT of the BPIV3 F protein as shown below, or linked to a CT having at least 90% (such as at least 95%) sequence identity to the CT of the BPIV3 F protein as shown below. An exemplary BPIV3 F protein TM and CT sequence from the BPIV3 Kansas strain is: BPIV3 F TM domain: ITIIIVMIIILVIINITIIVV, residues 1-21 of SEQ ID NO: 53 BPIV3 F CT: IIKFHRIQGKDQNDKNSEPYILTNRQ, residues 22-57 of SEQ ID NO: 53 BPIV3 F TM+CT:ITIIIVMIIILVIINITIIVVIIKFHRIQGKDQNDKNSEPYILTNRQ, SEQ ID NO: 53 is shown as:

[0098] In some embodiments, recombinant paramyxoviruses comprising genomes encoding N, P, C, V, M, F, HN, and L proteins from HPIV3 and BPIV3 viruses (such as HPIV3 F and HN proteins and BPIV3 N, P, C, V, M, and L proteins) may further comprise a heterologous gene encoding a recombinant viral glycoprotein ectodomain (such as the RSV F ectodomain) derived from a type I membrane protein linked to the TM and CT of the HPIV3 F protein as described above, or linked to a TM and CT that have at least 90% (such as at least 95%) sequence identity to the TM and CT of the HPIV3 F protein as described above. In some embodiments, recombinant paramyxoviruses comprising genomes encoding the N, P, C, V, M, F, HN, and L proteins from HPIV3 and BPIV3 viruses may further comprise a heterologous gene encoding a recombinant viral glycoprotein ectodomain (such as the RSV F ectodomain) derived from a type I membrane protein linked to the CT of an HPIV3 F protein as shown below, or linked to a CT having at least 90% (e.g., at least 95%) sequence identity to the CT of an HPIV3 F protein as shown below.

[0099] E. Sendai virus In one embodiment, the recombinant paramyxovirus can be a recombinant Sendai virus comprising a recombinant viral genome encoding the Sendai virus N, P, C, V, M, F, HN, and L proteins containing a heterologous gene encoding a recombinant viral glycoprotein ectodomain derived from a type I membrane protein (such as the RSV F ectodomain), linked to the TM and CT of the Sendai virus F protein, or linked to a TM and CT having at least 90% (e.g., at least 95%) sequence identity to the CT of the Sendai virus F protein. In one embodiment, the recombinant paramyxovirus can be a recombinant Sendai virus comprising a recombinant viral genome encoding the Sendai virus N, P, C, V, M, F, HN, and L proteins containing a heterologous gene encoding a recombinant viral glycoprotein ectodomain derived from a type I membrane protein (such as the RSV F ectodomain), linked to the CT of the Sendai virus F protein, or linked to a CT having at least 90% (e.g., at least 95%) sequence identity to the CT of the Sendai virus F protein. Sendai virus F protein TM and CT sequences are known (see, e.g., GenBank Accession No. BAN84670, incorporated herein by reference). Exemplary Sendai virus F protein TM and CT sequences are: Sendai F TM domain: VITIIVVMVVILVVIIVIIIV (residues 1-21 of SEQ ID NO: 103) Sendai F CT:LYRLRRSMLMGNPDDRIPRDTYTLEPKIRHMYTNGGFDAMAEKR (residues 22 to 65 of SEQ ID NO: 103) Sendai F TM+CT:VITIIVVMVVILVVIIVIIIVLYRLRRSMLMGNPDDRIPRDTYTLEPKIRHMYTNGGFDAMAEKR, SEQ ID NO: 103 is shown as:

[0100] F.NDV In some embodiments, the recombinant paramyxovirus is an NDV, as shown below. The recombinant NDV virus may comprise a recombinant viral genome encoding NDV N, P, V, M, F, HN, and L proteins containing a heterologous gene encoding a recombinant viral glycoprotein ectodomain derived from a type I membrane protein (such as the RSV F ectodomain), linked to the TM and CT of the F protein, or linked to a TM and CT having at least 90% (such as at least 95%) sequence identity to the TM and CT of the NDV F protein as shown below. In some embodiments, the recombinant paramyxovirus may comprise a recombinant NDV virus comprising a recombinant viral genome encoding NDV N, P, V, M, F, HN, and L proteins containing a heterologous gene encoding a recombinant viral glycoprotein ectodomain derived from a type I membrane protein (such as the RSV F ectodomain), linked to the CT of the NDV F protein as shown below, or linked to a CT having at least 90% (such as at least 95%) sequence identity to the CT of the NDV F protein as shown below. NDV viral F protein TM and CT sequences are known (see, e.g., GenBank Accession No. AAC28374, incorporated herein by reference). Exemplary NDV viral F protein TM and CT sequences are: NDV F TM domain: IVLTIISLVFGILSLILACYL (residues 1-21 of SEQ ID NO: 104) NDV F CT:MYKQKAQQKTLLWLGNNTLDQMRATTKM (residues 22-49 of SEQ ID NO: 104) NDV F TM+CT:IVLTIISLVFGILSLILACYLMYKQKAQQKTLLWLGNNTLDQMRATTKM, SEQ ID NO: 104 is shown as:

[0101] G. Heterologous Genes Recombinant paramyxovirus vectors comprise a recombinant genome containing one or more heterologous genes encoding the ectodomain of one or more heterologous envelope proteins (or antigenic fragments thereof) of a heterologous viral pathogen, where the ectodomain is linked to the TM and CT of the envelope protein from the recombinant paramyxovirus. For example, one or more heterologous envelope proteins (or antigenic fragments thereof) from measles virus, subgroup A or subgroup B respiratory syncytial virus, mumps virus, human papillomavirus, human immunodeficiency virus type 1 or type 2, herpes simplex virus, cytomegalovirus, rabies virus, Epstein-Barr virus, filovirus, bunyavirus, flavivirus, alphavirus, human metapneumovirus, Ebola virus (such as Zaire ebola virus), influenza virus, or highly pathogenic coronaviruses (SARS, MERS) can be expressed by the disclosed recombinant paramyxoviruses. Examples of useful envelope proteins include, but are not limited to, measles virus HA and F proteins, subgroup A or subgroup B respiratory syncytial virus F, G, and SH proteins, mumps virus HN and F proteins, human papillomavirus L1 protein, human immunodeficiency virus type 1 or type 2 gp160 protein, herpes simplex virus and cytomegalovirus gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL, and gM proteins, rabies virus G protein, Epstein-Barr virus gp350 protein, filovirus G protein, bunyavirus G protein, flavivirus pre-E and NS1 proteins, human metapneumovirus (HMPV) G and F proteins, Ebola virus GP protein, alphavirus E protein, and SARS and MERS S proteins, and antigenic domains, fragments, and epitopes thereof.Exemplary methods for inserting one or more heterologous genes or transcription units into a paramyxovirus viral genome or antigenome are described in WO04 / 027037 and US2013 / 0052718, each of which is incorporated herein by reference.

[0102] In some embodiments, the heterologous gene contained in the recombinant paramyxovirus genome encodes the ectodomain of a RSV F protein, such as a bovine RSV F protein or a human RSV F protein. Human RSV can be classified into two groups: A and B. Groups A and B include subgroups A1, A2, B1, and B2, primarily based on sequence variability of the joining (G) and fusion (F) proteins. The RSV F ectodomain can be derived from any RSV group (such as group A or group B) or subgroup of RSV, such as subgroups A1, A2, B1, or B2.

[0103] An exemplary human RSV F protein sequence from subgroup A2 and the corresponding GenBank reference (incorporated herein by reference in its entirety) is shown below:

[0104] RSV F A2 HEK protein sequence: (SEQ ID NO: 1) RSV F B1 HEK protein sequence, accession number AAB82436: (SEQ ID NO: 2, incorporated herein by reference in its entirety, GenBank Accession No. AAB82436) RSV F B1 HEK nucleic acid sequence: GenBank Accession No.: AF013254.1, nucleotides 5666 to 7390, incorporated herein.

[0105] As illustrated by the above sequence, the hRSV F protein shows significant sequence conservation, with greater than 85% sequence identity across hRSV subgroups. Given the conservation and breadth of knowledge of the RSV F sequence, those skilled in the art can easily identify the corresponding RSV F amino acid positions between various RSV F chains and subgroups. The numbering of the amino acid substitutions disclosed herein refers to the exemplary hRSV F protein sequence from the A2 strain, set forth as SEQ ID NO: 1, unless otherwise specified.

[0106] For illustrative purposes, the signal peptide, F2 polypeptide, pep27, F1, F1 ectodomain, transmembrane domain, and cytoplasmic domain of the RSV F protein (SEQ ID NO: 1) from the A2 strain are shown as follows: Signal peptide (SEQ ID NO: 1, residues 1-22): MELLILKANAITTILTAVTFCF F2 polypeptide (SEQ ID NO: 1, residues 23-109): ASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKV KLIKQELDKYKNAVTELQLLMQSTPATNNRARR Pep27 (SEQ ID NO: 1, residues 110-136): ELPRFMNYTLNNAKKTNVTLSKKRKRR F1 (SEQ ID NO: 1, residues 137-574): FLGFLLGVGSAIAGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTT PVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQA ETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEG KSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN F1 ectodomain of the mature protein (SEQ ID NO: 1, residues 137-529): FLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNI CLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITT F1 transmembrane domain (SEQ ID NO: 1, residues 530-550): IIIVIIVILLSLIAVGLLLYC F1CT (SEQ ID NO: 1, residues 551-574): KARSTPVTLSKDQLSGINNIAFSN

[0107] In some embodiments, the heterologous gene contained in the recombinant paramyxovirus genome encodes the ectodomain of the human RSF F protein, wherein the RSV F ectodomain is at least 85% (e.g., at least 90% or at least 95%) identical to the RSV ectodomain of one of SEQ ID NOs: 1 (WT RSV FA), 2 (WT RSV FB), 12 (A2 HEK), 14 (A2 HEK + DS) or 19 (A2 HEK + DS-Cav1), or comprises the amino acid sequence of the RSV ectodomain of SEQ ID NOs: 12, 14 or 19.

[0108] In some embodiments, the recombinant paramyxovirus may comprise a genome comprising a heterologous gene encoding a recombinant hRSV F protein that has been codon-optimized for expression in human cells. For example, the gene encoding the recombinant hRSV F protein can be codon-optimized for human expression using the GA, DNA2.0 (D2) or GenScript (GS) optimization algorithm (see Example 1). Non-limiting examples of nucleic acid sequences encoding RSV F proteins that have been codon-optimized for expression in human cells are provided as follows: GeneArt optimized RSV F A2 HEK DNA sequence: cagcgtgtcattctttccacaggccgagacatgcaaggtgcagagcaaccgggtgttctgcgacaccatgaacagcctgaccctgccctccgaagtgaacctgtgcaacgtggacatcttcaaccctaagtacgactgcaagatcatgaccagcaagaccgacgtgtccagctccgtgatcacctccctgggcgccatcgtgtcctgctacggcaagaccaagtgcaccgccagcaacaagaaccggggcatcatcaagaccttcagcaacggctgcgactacgtgtccaacaagggggtggacaccgtgtccgtgggcaacaccctgtactacgtgaacaaacaggaaggcaagagcctgtacgtgaagggcgagcccatcatcaacttctacgaccccctggtgttccccagcgacgagttcgacgccagcatcagccaggtcaacgagaagatcaaccagagcctggccttcatcagaaagagcgacgagctgctgcacaatgtgaatgccggcaagagcaccacaaacatcatgatcaccactatcatcatcgtgatcatcgtcatcctgctgagtctgatcgccgtgggcctgctgctgtactgcaaggccagatccacccctgtgaccctgtccaaggatcagctgtccggcatcaacaatatcgccttctccaactga (SEQ ID NO: 4) GenScript-optimized RSV F A2 HEK DNA sequence:

[0109] Further examples of codon-optimized (for human expression) sequences are provided below.

[0110] The RSV F protein encoded by a heterologous gene may contain one or more amino acid substitutions that improve the expression of the RSV F protein, the availability of the RSV F protein on the virion envelope, or the stability of the RSV F protein, for example, in a pre-fusion conformation. In some embodiments, the RSV F protein may contain a glutamic acid substitution at position 66, a proline substitution at position 101, or both. For example, the RSV F protein may contain a "HEK" substitution of K66E and Q101P. Exemplary DNA and protein sequences of RSV F proteins from the A2 subgroup containing HEK amino acid substitutions are shown below. RSV F A2 protein with HEK substitutions (RSV F_A2_HEK): SEQ ID NO: 1 GeneArt optimized RSV F_A2_HEK DNA sequence: atggaactgctgatcctgaaggccaacgccatcacaacaatcctgaccgccgtgaccttctgcttcgccagcggccagaacatcaccgaggaattctaccagagcacctgtagcgccgtgtccaagggctacctgagcgccctgagaaccggctggtaca ccagcgtgatcaccatcgagctgtccaacatcaaagaaaacaagtgcaacggcaccgacgccaaagtgaagctgatcaagcaggaactggacaagtacaagaacgccgtgaccgagctgcagctgctgatgcagtccacccccgccaccaacaaccgggc GenScript optimized RSV F_A2_HEK DNA sequence:

[0111] In further embodiments, the RSV F protein may contain one or more amino acid substitutions that stabilize the ectodomain of the RSV F protein in its pre-fusion conformation. For example, the RSV F protein may contain a pair of "DS" cysteine ​​substitutions at positions 155 and 290 that form non-native disulfide bonds and stabilize the RSV F protein in its pre-fusion conformation. In some embodiments, the RSV The F protein may contain amino acid substitutions that fill one or more cavities at positions 190 and / or 207 and stabilize the protein in the pre-fusion conformation. For example, the RSV F protein may contain a 190F substitution and / or a 207L substitution. In some embodiments, the RSV F protein may include "Cav1" substitutions of S190F and F207L. In some embodiments, the RSV F protein may include DS-Cav1 substitutions of S155C, S290C, S190F, and V207L ​​to stabilize the protein in the pre-fusion conformation. Exemplary DNA and protein sequences of RSV F proteins from the A2 subgroup (having chimeric TM and / or CT domains) containing DS-Cav1 amino acid substitutions are set forth as SEQ ID NOS: 10-11 and 21-23.

[0112] Additional amino acid substitutions and protein modifications that can be used to stabilize the RSV F ectodomain in the pre-fusion conformation are disclosed, for example, in WO2014160463, the entire contents of which are incorporated herein by reference. The HEK substitution can be combined with any of the amino acid substitutions to stabilize the RSV F protein in the pre-fusion conformation.

[0113] In some embodiments, the heterologous gene contained in the recombinant paramyxovirus genome encodes a recombinant RSV F ectodomain linked to the TM and CT of the F protein of the recombinant paramyxovirus.

[0114] In one embodiment, the recombinant paramyxovirus is a recombinant HPIV1 comprising a recombinant HPIV1 genome containing a heterologous gene encoding a recombinant hRSV F ectodomain. The RSV F ectodomain can be linked to TM and CT from the HPIV1 F protein, e.g., as shown in SEQ ID NO:31, residues 1-23 (TM), SEQ ID NO:31, residues 24-59 (CT), or SEQ ID NO:31 (TM+CT). Exemplary sequences are provided below: hRSV F protein from the A2 strain containing the HEK and DS-Cav1 substitutions and the HPIV1 F CT domain (RSV FA2_HEK_DS-Cav1_H1CT): (SEQ ID NO: 133) GenScript optimized RSV F A2_HEK_DS-Cav1_H1CT DNA sequence: (SEQ ID NO: 134) hRSV F protein from the A2 strain containing HEK and DS-Cav1 substitutions and HPIV1 F TM and CT domains (RSV F A2_HEK_DS-Cav1_H1TMCT): (SEQ ID NO: 135) GenScript optimized RSV F A2_HEK_DS-Cav1_H1TMCT DNA sequence:

[0115] In one embodiment, the recombinant paramyxovirus is a recombinant HPIV2 comprising a recombinant HPIV2 genome containing a heterologous gene encoding a recombinant hRSV F ectodomain. The RSV F ectodomain can be, for example, residues 1-28 of SEQ ID NO: 39 (TM), residues 29-66 of SEQ ID NO: 39 (CT), or residues 39-66 of SEQ ID NO: 39 (TM+CT). It can be linked to the TM and CT from the HPIV2 F protein.

[0116] In one embodiment, the recombinant paramyxovirus can be a recombinant HPIV3 comprising a genome including a heterologous gene encoding a recombinant hRSV F ectodomain. The recombinant RSV F ectodomain can be linked to the TM and CT from the HPIV3 F protein, for example, as shown in SEQ ID NO: 46, residues 1-23 (TM), SEQ ID NO: 46, residues 24-46 (CT), or SEQ ID NO: 46 (TM+CT). Exemplary sequences are provided below: hRSV F protein from the A2 strain containing HEK and DS-Cav1 substitutions and the HPIV3 F CT domain (RSV F_HEK_DS-Cav1_H3CT) protein sequence: (SEQ ID NO: 8) GenScript-optimized RSV F_HEK_DS-Cav1_H3CT DNA sequence: hRSV F protein from strain A2 (RSV F_HEK_DS-Cav1_H3TMCT) protein sequence containing HEK and DS-Cav1 substitutions and HPIV3 F TM and CT domains: MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHL EGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSP LCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIILIMIIILFIINITIITIAIKYYRIQKRNRVDQNDKPYVLTNK (SEQ ID NO: 10) GenScript-optimized RSV F_HEK_DS-Cav1_H3TMCT DNA sequence:

[0117] In one embodiment, the recombinant paramyxovirus is a chimeric PIV comprising a recombinant viral genome encoding HPIV3 F and HN proteins and BPIV3 N, P, C, V, M, and L proteins, wherein the viral genome further comprises a heterologous gene encoding a recombinant hRSV F ectodomain linked to a TM and / or CT from the BPIV3 F protein, e.g., residues 1-21 (TM) of SEQ ID NO: 53, residues 22-57 (CT) of SEQ ID NO: 53, or SEQ ID NO: 53 (TM + CT). Exemplary DNA and protein sequences of recombinant RSV F proteins of the A2 subgroup containing HEK, DS, and / or Cav1 substitutions and heterologous TM and / or CT domains from the BPIV3 F protein that can be used in the disclosed recombinant paramyxoviruses are shown below. hRSV F protein from the A2 strain (RSV F_A2_HEK_B3TMCT) containing HEK substitutions and BPIV3 F TM and CT domains: MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTITIIIVMIIILVIINITIIVVIIKFHRIQGKDQNDKNSEPYILTNRQ(SEQ ID NO:12) GeneArt-optimized RSV F_A2_HEK_B3TMCT DNA sequence: hRSV F protein from the A2 strain (RSV F_A2_HEK_DS_B3CT) containing HEK and DS substitutions, hRSV F TM domain and BPIV3 F CT domain: (SEQ ID NO: 14) gaaccggggcatcatcaagaccttcagcaacggctgcgactacgtgtccaacaagggggtggacaccgtgtctgtgggcaacaccctgtactacgtgaacaaacaggaaggcaa gagcctgtacgtgaagggcgagcccatcatcaacttctacgaccccctggtgttccccagcgacgagttcgatgccagcatctcccaagtgaacgagaagatcaaccagagcctg gccttcatcagaaagtccgatgagctgctgcacaatgtgaacgccggcaagtccaccaccaatatcatgatcaccacaatcatcatcgtgattatcgtgatcctgctgagcctga tcgccgtgggcctgctgctgtactgtatcatcaagttccaccggatccagggcaaggaccagaacgacaagaactccgagccctacatcctgacaaaccggcagtga (SEQ ID NO: 15) hRSV F protein from the A2 strain (RSV F_A2_HEK_DS-Cav1_B3CT) containing HEK and DS-Cav1 substitutions, hRSV FTM domain and BPIV3 FCT domain: MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCIIKFHRIQGKDQNDKNSEPYILTNRQ(SEQ ID NO: 16) GeneArt-optimized RSV F_A2_HEK_DS-Cav1_B3CT DNA sequence: GenScript-optimized RSV F_A2_HEK_DS-Cav1_B3CT DNA sequence: atggaactgctgatcctgaaagccaacgctattactactatcctgaccgccgtgacattttgcttcgcatctggacagaacattactgaggaattctaccagtcaacatgcagcgccgtgtccaaaggatacctgagcgccctgcggaccggctggtatacatcagtgattactatcgagctgtccaacatcaaggaaaacaaatgtaatgggaccgacgcaaaggtgaaactgatcaagcaggagctggataagtacaaaaatgccgtgacagaactgcagctgctgatgcagtccacaccagcaactaacaatcgcgcccggagagagctgccccggttcatgaactataccctgaacaatgctaagaaaaccaatgtgacactgtccaagaaacgcaagaggcgcttcctgggatttctgctgggcgtggggtctgccatcgctagtggagtggccgtctgcaaagtcctgcacctggagggcgaagtgaacaagatcaaaagcgccctgctgtccactaacaaggcagtggtcagtctgtcaaatggcgtgtccgt hRSV F protein from the A2 strain (RSV F_A2_HEK_DS_B3TMCT) containing HEK and DS substitutions and BPIV3 F TM and CT domains: (SEQ ID NO: 19) GeneArt optimized RSV F_A2_HEK_DS_B3TMCT DNA sequence: Genescript optimized RSV F_A2_HEK_DS_B3TMCT DNA sequence: hRSV F protein from the A2 strain containing HEK and DS-Cav1 substitutions and BPIV3 F TM and CT domains (RSV F_A2_HEK_DS-Cav1_B3TMCT) protein sequence: (SEQ ID NO: 21) GeneArt optimized RSV F_A2_HEK_DS-Cav1_B3TMCT DNA sequence: ctccgtctcattctttccacaagccgagacatgcaaggtgcagagcaaccgggtgttctgcgacaccatgaacagcctgaccctgccctccgaagtgaatctgtgcaacgtggacatcttcaaccctaagtacgactgcaagatcatgacctccaagaccgacgtgtccagctccgtgatcacaagcctgggcgccatcgtgtcctgctacggcaagaccaagtgcaccgccagcaacaagaaccggggcatcatcaagaccttcagcaacggctgcgactacgtgtccaacaagggggtggacaccgtgtctgtgggcaacaccctgtactacgtgaacaaacaggaaggcaagagcctgtacgtgaagggcgagcccatcatcaacttctacgaccccctggtgttccccagcgacgagttcgatgccagcatctcccaagtgaacgagaagatcaaccagagcctggccttcatcagaaagtccgatgagctgctgcacaatgtgaacgccggcaagtccaccaccaatatcatgatcaccacaatcaccatcatcattgtgatgattatcatcctcgtgatcatcaacatcacaatcatcgtcgtgattattaagttccaccggatccagggcaaggaccagaacgacaagaactccgagccctacatcctgacaaaccggcagtga (SEQ ID NO: 22) GenScript Optimized RSV F_A2_HEK_DS-Cav1_B3TMCT DNA Sequence:

[0118] In one embodiment, the recombinant paramyxovirus comprises a recombinant Sendai virus genome comprising a heterologous gene encoding a recombinant hRSV F ectodomain. In such embodiments, the TM and CT linked to the RSV F ectodomain can be derived from a Sendai virus F protein, such as residues 1-21 of SEQ ID NO: 103 (TM), residues 22-65 of SEQ ID NO: 103 (CT), or SEQ ID NO: 103 (TM + CT). For example, in some embodiments, the recombinant hRSV F ectodomain linked to the Sendai virus TM and / or CT can comprise an amino acid sequence set forth in one of SEQ ID NOs: 105-108. hRSV F protein from the A2 strain containing the HEK substitution and Sendai virus F CT domain (RSV F_A2_HEK_SeVCT) protein sequence MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLN NAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVN AGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKV QSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDP LVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCLYRLRRSMLMGNPDDRIPRDTYTLEPKIRHMYTNGGFDAMAEKR (SEQ ID NO: 105) hRSV F protein from the A2 strain containing HEK substitutions and the Sendai virus F TM and CT domains (RSV F_A2_HEK_SeVTMCT) protein sequence (SEQ ID NO: 106) hRSV F protein (RSV F_A2_HEK_SeVCT) protein sequence from the A2 strain containing HEK and DS-Cav1 substitutions and the Sendai virus F CT domain (SEQ ID NO: 107) hRSV F protein from the A2 strain (RSV F_A2_HEK_SeVTMCT) protein sequence containing HEK and DS-Cav1 substitutions and the Sendai virus F TM and CT domains (SEQ ID NO: 108)

[0119] In one embodiment, the recombinant paramyxovirus comprises a recombinant NDV genome comprising a heterologous gene encoding a recombinant hRSV F ectodomain. In such embodiments, the TM and CT linked to the RSV F ectodomain can be derived from the NDV viral F protein, cytoplasmic tail, e.g., residues 1-21 of SEQ ID NO: 104 (TM), residues 22-49 of SEQ ID NO: 104 (CT), or SEQ ID NO: 104 (TM + CT). For example, in some embodiments, the recombinant hRSV F ectodomain linked to the NDV TM and / or CT can comprise an amino acid sequence set forth in one of SEQ ID NOs: 109-113. hRSV F protein from the A2 strain (RSV F_A2_HEK_NDVCT) containing the HEK substitution and NDV F CT domains MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYID KQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKT DVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCMYKQKAQQKTLLWLGNNTLDQMRATTKM (SEQ ID NO: 109) hRSV F protein from the A2 strain (RSV F_A2_HEK_NDVTMCT) protein sequence containing the HEK substitution and NDV F TM and CT domains (SEQ ID NO: 110) hRSV F protein (RSV F_A2_HEK_NDVCT) protein sequence from the A2 strain containing HEK and DS-Cav1 substitutions and the NDV F CT domain (SEQ ID NO: 112) hRSV F protein (RSV F_A2_HEK_NDVTMCT) protein sequence from the A2 strain containing HEK and DS-Cav1 substitutions and the NDV F TM and CT domains (SEQ ID NO: 113)

[0120] H. Further description of recombinant paramyxoviruses Specific Embodiments In some embodiments, a recombinant paramyxovirus is provided, comprising a recombinant parainfluenza virus (PIV) comprising a viral genome comprising, from upstream to downstream, a PIV genomic promoter followed by the PIV N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV F ectodomain comprises the 66E, 101P, 155C, 290C, 190F, and 207L substitutions and is linked to the TM and CT of the PIV F protein.

[0121] In some embodiments, a recombinant paramyxovirus is provided that includes a recombinant HPIV1 comprising a viral genome that includes, from upstream to downstream, an HPIV1 genomic promoter followed by the HPIV1 N, P, M, F, HN, and L genes, and further includes a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain, wherein the heterologous gene The gene is located between the genomic promoter and the gene encoding the N protein. The F ectodomain contains the 66E, 101P, 155C, 290C, 190F, and 207L substitutions and is linked to the TM and CT of the HPIV1 F protein.

[0122] In some embodiments, a recombinant paramyxovirus containing a recombinant HPIV1 comprising a viral genome including, from upstream to downstream, an HPIV1 genomic promoter followed by the HPIV1N, P, M, F, HN, and L genes, and further including a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain contains 66E, 101P, 155C, 290C, 190F, and 207L substitutions and is linked to the TM and CT of the HPIV1 F protein.

[0123] In some embodiments, a recombinant paramyxovirus is provided that includes a recombinant HPIV3 comprising a viral genome that includes, from upstream to downstream, an HPIV3 genomic promoter followed by the HPIV3 N, P, M, F, HN, and L genes, and further includes a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and wherein the heterologous gene is located between the genomic promoter and the gene encoding the RSV N protein. The F ectodomain contains the 66E, 101P, 155C, 290C, 190F, and 207L substitutions and is linked to the TM and CT of the HPIV3 F protein.

[0124] In some embodiments, a recombinant paramyxovirus containing a recombinant HPIV3 is provided, comprising a viral genome including, from upstream to downstream, an HPIV3 genomic promoter followed by the HPIV3 N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain contains substitutions 66E, 101P, 155C, 290C, 190F, and 207L and is linked to the TM and CT of the HPIV3 F protein.

[0125] In some embodiments, a recombinant paramyxovirus is provided that includes a recombinant HPIV3 comprising a viral genome that includes, from upstream to downstream, an HPIV3 genomic promoter followed by the HPIV3 N, P, M, F, HN, and L genes, and further includes a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and wherein the heterologous gene is located between the genomic promoter and the gene encoding the RSV N protein. The F ectodomain contains 66E, 101P, 155C, 290C, 190F, and 207L substitutions and is linked to the TM and CT of the BPIV3 F protein.

[0126] In some embodiments, a recombinant paramyxovirus comprising a recombinant HPIV3 comprising a viral genome comprising, from upstream to downstream, an HPIV3 genomic promoter followed by the HPIV3 N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprises substitutions 66E, 101P, 155C, 290C, 190F, and 207L and is linked to the TM and CT of the BPIV3 F protein.

[0127] In some embodiments, a recombinant paramyxovirus containing a recombinant BPIV3 is provided, comprising a viral genome including, from upstream to downstream, a BPIV3 genomic promoter followed by BPIV3 N, P, M, F, HN, and L genes, and further including a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV The F ectodomain contains 66E, 101P, 155C, 290C, 190F and 207 It contains an L substitution and is linked to the TM and CT of the BPIV3 F protein.

[0128] In some embodiments, a recombinant paramyxovirus containing a recombinant BPIV3 comprising a viral genome including, from upstream to downstream, a BPIV3 genomic promoter followed by the BPIV3 N, P, M, F, HN, and L genes, and further including a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain is provided, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain contains 66E, 101P, 155C, 290C, 190F, and 207L substitutions and is linked to the TM and CT of the BPIV3 F protein.

[0129] In some embodiments, a recombinant paramyxovirus comprising a recombinant B / HPIV3 comprising a viral genome including, from upstream to downstream, a BPIV3 genomic promoter followed by the BPIV3 N, P, and M genes, the HPIV3 F and HN genes, and the BPIV3 L gene, and further including a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV F ectodomain contains substitutions 66E, 101P, 155C, 290C, 190F, and 207L and is linked to the TM and CT of the BPIV3 F protein.

[0130] In some embodiments, a recombinant paramyxovirus comprising a recombinant B / HPIV3 comprising a viral genome including, from upstream to downstream, a BPIV3 genomic promoter followed by the BPIV3 N, P, and M genes, the HPIV3 F and HN genes, and the BPIV3 L gene, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprises 66E, 101P, 155C, 290C, 190F, and 207L substitutions and is linked to the TM and CT of the BPIV3 F protein.

[0131] In some embodiments, a recombinant paramyxovirus comprising a recombinant B / HPIV3 comprising a viral genome including, from upstream to downstream, a BPIV3 genomic promoter followed by the BPIV3 N, P, and M genes, the HPIV3 F and HN genes, and the BPIV3 L gene, and further including a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV F ectodomain contains substitutions 66E, 101P, 155C, 290C, 190F, and 207L and is linked to the TM and CT of the HPIV3 F protein.

[0132] In some embodiments, a recombinant paramyxovirus comprising a recombinant B / HPIV3 comprising a viral genome including, from upstream to downstream, a BPIV3 genomic promoter followed by the BPIV3 N, P, and M genes, the HPIV3 F and HN genes, and the BPIV3 L gene, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprises 66E, 101P, 155C, 290C, 190F, and 207L substitutions and is linked to the TM and CT of the HPIV3 F protein.

[0133] In some embodiments, a recombinant paramyxovirus is provided, comprising a recombinant Sendai virus comprising a viral genome comprising, from upstream to downstream, a Sendai virus genomic promoter followed by the Sendai virus N, P, M, F, HN and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain; In this, the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV F ectodomain contains substitutions 66E, 101P, 155C, 290C, 190F, and 207L and is linked to the TM and CT of the Sendai virus F protein.

[0134] In some embodiments, a recombinant paramyxovirus is provided, comprising a recombinant Sendai virus comprising a viral genome comprising, from upstream to downstream, a Sendai virus genomic promoter followed by the Sendai virus N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprises 66E, 101P, 155C, 290C, 190F, and 207L substitutions and is linked to the TM and CT of the Sendai virus F protein.

[0135] In some embodiments, a recombinant paramyxovirus is provided, comprising a recombinant NDV comprising a viral genome comprising, from upstream to downstream, an NDV genomic promoter followed by the NDV N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV F ectodomain comprises 66E, 101P, 155C, 290C, 190F, and 207L substitutions and is linked to the TM and CT of the NDV F protein.

[0136] In some embodiments, a recombinant paramyxovirus is provided, comprising a recombinant NDV comprising a viral genome comprising, from upstream to downstream, an NDV genomic promoter followed by the NDV N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprises 66E, 101P, 155C, 290C, 190F, and 207L substitutions and is linked to the TM and CT of the NDV F protein.

[0137] In some embodiments, a recombinant paramyxovirus containing a recombinant PIV5 comprising a viral genome including, from upstream to downstream, a PIV5 genomic promoter followed by the PIV5 N, P, M, F, HN, and L genes, and further including a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain is provided, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV F ectodomain contains 66E, 101P, 155C, 290C, 190F, and 207L substitutions and is linked to the TM and CT of the PIV5 F protein.

[0138] In some embodiments, a recombinant paramyxovirus containing a recombinant PIV5 comprising a viral genome including, from upstream to downstream, a PIV5 genome promoter followed by the PIV5 N, P, M, F, HN, and L genes, and further including a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain is provided, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain contains 66E, 101P, 155C, 290C, 190F, and 207L substitutions and is linked to the TM and CT of the PIV5 F protein.

[0139] In some embodiments, a recombinant paramyxovirus is provided, including a recombinant parainfluenza virus (PIV) comprising a viral genome comprising, from upstream to downstream, a PIV genome promoter followed by the PIV N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain. In this variant, the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV F ectodomain contains substitutions 66E, 101P, 155C, 290C, 190F, and 207L and is linked to the CT of the PIV F protein.

[0140] In some embodiments, a recombinant paramyxovirus is provided that comprises a recombinant HPIV1 comprising a viral genome that includes, from upstream to downstream, an HPIV1 genomic promoter followed by the HPIV1 N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and The F ectodomain contains 66E, 101P, 155C, 290C, 190F, and 207L substitutions and is linked to the CT of the HPIV1 F protein.

[0141] In some embodiments, a recombinant paramyxovirus comprising a recombinant HPIV1 comprising a viral genome comprising, from upstream to downstream, an HPIV1 genomic promoter followed by the HPIV1N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprises substitutions 66E, 101P, 155C, 290C, 190F, and 207L and is linked to the CT of the HPIV1 F protein.

[0142] In some embodiments, a recombinant paramyxovirus containing a recombinant HPIV3 comprising a viral genome including, from upstream to downstream, an HPIV3 genomic promoter followed by the HPIV3N, P, M, F, HN, and L genes, and further including a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV F ectodomain includes substitutions 66E, 101P, 155C, 290C, 190F, and 207L and is linked to the CT of the HPIV3 F protein.

[0143] In some embodiments, a recombinant paramyxovirus containing a recombinant HPIV3 is provided, comprising a viral genome including, from upstream to downstream, an HPIV3 genomic promoter followed by the HPIV3 N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain contains substitutions 66E, 101P, 155C, 290C, 190F, and 207L and is linked to the CT of the HPIV3 F protein.

[0144] In some embodiments, a recombinant paramyxovirus is provided that includes a recombinant HPIV3 comprising a viral genome that includes, from upstream to downstream, an HPIV3 genomic promoter followed by the HPIV3 N, P, M, F, HN, and L genes, and further includes a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and wherein the heterologous gene is located between the genomic promoter and the gene encoding the RSV N protein. The F ectodomain contains 66E, 101P, 155C, 290C, 190F and 207L substitutions and is linked to the CT of the BPIV3 F protein.

[0145] In some embodiments, a recombinant paramyxovirus is provided that includes a recombinant HPIV3 comprising a viral genome that includes, from upstream to downstream, an HPIV3 genomic promoter followed by the HPIV3 N, P, M, F, HN, and L genes, and further includes a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain is selected from the group consisting of 66E, 101P, 155C, 290C, 190F, and 190C. It contains the 207L and 207L substitutions and is linked to the CT of the BPIV3 F protein.

[0146] In some embodiments, a recombinant paramyxovirus containing a recombinant BPIV3 is provided, comprising a viral genome including, from upstream to downstream, a BPIV3 genomic promoter followed by BPIV3 N, P, M, F, HN, and L genes, and further including a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV The F ectodomain contains 66E, 101P, 155C, 290C, 190F and 207L substitutions and is linked to the CT of the BPIV3 F protein.

[0147] In some embodiments, a recombinant paramyxovirus containing a recombinant BPIV3 comprising a viral genome including, from upstream to downstream, a BPIV3 genomic promoter followed by the BPIV3 N, P, M, F, HN, and L genes, and further including a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain is provided, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain contains substitutions 66E, 101P, 155C, 290C, 190F, and 207L and is linked to the CT of the BPIV3 F protein.

[0148] In some embodiments, a recombinant paramyxovirus comprising a recombinant B / HPIV3 comprising a viral genome including, from upstream to downstream, a BPIV3 genomic promoter followed by the BPIV3 N, P, and M genes, the HPIV3 F and HN genes, and the BPIV3 L gene, and further including a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV F ectodomain contains substitutions 66E, 101P, 155C, 290C, 190F, and 207L and is linked to the CT of the BPIV3 F protein.

[0149] In some embodiments, a recombinant paramyxovirus comprising a recombinant B / HPIV3 comprising a viral genome including, from upstream to downstream, a BPIV3 genomic promoter followed by the BPIV3 N, P, and M genes, the HPIV3 F and HN genes, and the BPIV3 L gene, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprises 66E, 101P, 155C, 290C, 190F, and 207L substitutions and is linked to the CT of the BPIV3 F protein.

[0150] In some embodiments, a recombinant paramyxovirus comprising a recombinant B / HPIV3 comprising a viral genome including, from upstream to downstream, a BPIV3 genomic promoter followed by the BPIV3 N, P, and M genes, the HPIV3 F and HN genes, and the BPIV3 L gene, and further including a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV F ectodomain contains substitutions 66E, 101P, 155C, 290C, 190F, and 207L and is linked to the CT of the HPIV3 F protein.

[0151] In some embodiments, a recombinant paramyxovirus is provided, comprising a recombinant B / HPIV3 comprising a viral genome comprising, from upstream to downstream, a BPIV3 genomic promoter followed by the BPIV3 N, P and M genes, the HPIV3 F and HN genes, and the BPIV3 L gene, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene encodes an N protein. Located between the gene encoding the F protein and the gene encoding the P protein, the RSV F ectodomain contains substitutions 66E, 101P, 155C, 290C, 190F and 207L and is linked to the CT of the HPIV3 F protein.

[0152] In some embodiments, a recombinant paramyxovirus is provided, comprising a recombinant Sendai virus comprising a viral genome comprising, from upstream to downstream, a Sendai virus genomic promoter followed by the Sendai virus N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV F ectodomain comprises substitutions 66E, 101P, 155C, 290C, 190F, and 207L and is linked to the CT of the Sendai virus F protein.

[0153] In some embodiments, a recombinant paramyxovirus is provided, comprising a recombinant Sendai virus comprising a viral genome comprising, from upstream to downstream, a Sendai virus genomic promoter followed by the Sendai virus N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprises 66E, 101P, 155C, 290C, 190F, and 207L substitutions and is linked to the CT of the Sendai virus F protein.

[0154] In some embodiments, a recombinant paramyxovirus is provided, comprising a recombinant NDV comprising a viral genome comprising, from upstream to downstream, an NDV genomic promoter followed by the NDV N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV F ectodomain comprises substitutions 66E, 101P, 155C, 290C, 190F, and 207L and is linked to the CT of the NDV F protein.

[0155] In some embodiments, a recombinant paramyxovirus is provided, comprising a recombinant NDV comprising a viral genome comprising, from upstream to downstream, an NDV genomic promoter followed by the NDV N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprises 66E, 101P, 155C, 290C, 190F, and 207L substitutions and is linked to the CT of the NDV F protein.

[0156] In some embodiments, a recombinant paramyxovirus containing a recombinant PIV5 comprising a viral genome including, from upstream to downstream, a PIV5 genomic promoter followed by the PIV5 N, P, M, F, HN, and L genes, and further including a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain is provided, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV F ectodomain contains 66E, 101P, 155C, 290C, 190F, and 207L substitutions and is linked to the CT of the PIV5 F protein.

[0157] In some embodiments, a recombinant paramyxovirus comprising a recombinant PIV5 is provided, the viral genome comprising, from upstream to downstream, a PIV5 genomic promoter followed by the PIV5 N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the R The SV F ectodomain contains the 66E, 101P, 155C, 290C, 190F and 207L substitutions and is linked to the CT of the PIV5 F protein.

[0158] In some embodiments, a recombinant paramyxovirus is provided, comprising a recombinant parainfluenza virus (PIV) comprising a viral genome comprising, from upstream to downstream, a PIV genomic promoter followed by the PIV N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV F ectodomain comprises 66E, 101P, 155C, and 290C substitutions and is linked to the TM and CT of the PIV F protein.

[0159] In some embodiments, a recombinant paramyxovirus is provided that comprises a recombinant HPIV1 comprising a viral genome that includes, from upstream to downstream, an HPIV1 genomic promoter followed by the HPIV1 N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and The F ectodomain contains 66E, 101P, 155C, and 290C substitutions, and HPIV1 It is linked to the TM and CT of the F protein.

[0160] In some embodiments, a recombinant paramyxovirus containing a recombinant HPIV1 comprising a viral genome including, from upstream to downstream, an HPIV1 genomic promoter followed by the HPIV1 N, P, M, F, HN, and L genes, and further including a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain is provided, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain contains 66E, 101P, 155C, and 290C substitutions and is linked to the TM and CT of the HPIV1 F protein.

[0161] In some embodiments, a recombinant paramyxovirus is provided that includes a recombinant HPIV3 comprising a viral genome that includes, from upstream to downstream, an HPIV3 genomic promoter followed by the HPIV3 N, P, M, F, HN, and L genes, and further includes a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and wherein the heterologous gene is located between the genomic promoter and the gene encoding the RSV N protein. The F ectodomain contains 66E, 101P, 155C, and 290C substitutions, and HPIV3 It is linked to the TM and CT of the F protein.

[0162] In some embodiments, a recombinant paramyxovirus containing a recombinant HPIV3 comprising a viral genome including, from upstream to downstream, an HPIV3 genomic promoter followed by the HPIV3 N, P, M, F, HN, and L genes, and further including a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain is provided, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain contains substitutions 66E, 101P, 155C, and 290C and is linked to the TM and CT of the HPIV3 F protein.

[0163] In some embodiments, a recombinant paramyxovirus is provided that includes a recombinant HPIV3 comprising a viral genome that includes, from upstream to downstream, an HPIV3 genomic promoter followed by the HPIV3 N, P, M, F, HN, and L genes, and further includes a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and wherein the heterologous gene is located between the genomic promoter and the gene encoding the RSV N protein. The F ectodomain contains 66E, 101P, 155C, and 290C substitutions, and BPIV3 It is linked to the TM and CT of the F protein.

[0164] In some embodiments, a recombinant paramyxovirus containing a recombinant HPIV3 is provided, comprising a viral genome including, from upstream to downstream, an HPIV3 genomic promoter followed by the HPIV3 N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain contains substitutions 66E, 101P, 155C, and 290C and is linked to the TM and CT of the BPIV3 F protein.

[0165] In some embodiments, a recombinant paramyxovirus containing a recombinant BPIV3 is provided, comprising a viral genome including, from upstream to downstream, a BPIV3 genomic promoter followed by BPIV3 N, P, M, F, HN, and L genes, and further including a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV The F ectodomain contains 66E, 101P, 155C, and 290C substitutions, and BPIV3 It is linked to the TM and CT of the F protein.

[0166] In some embodiments, a recombinant paramyxovirus containing a recombinant BPIV3 comprising a viral genome including, from upstream to downstream, a BPIV3 genomic promoter followed by the BPIV3 N, P, M, F, HN, and L genes, and further including a heterologous gene encoding a type I membrane protein including a recombinant RSV F ectodomain is provided, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain contains 66E, 101P, 155C, and 290C substitutions and is linked to the TM and CT of the BPIV3 F protein.

[0167] In some embodiments, a recombinant paramyxovirus comprising a recombinant B / HPIV3 comprising a viral genome including, from upstream to downstream, a BPIV3 genomic promoter followed by the BPIV3 N, P, and M genes, the HPIV3 F and HN genes, and the BPIV3 L gene, and further including a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain is provided, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV F ectodomain contains substitutions 66E, 101P, 155C, and 290C and is linked to the TM and CT of the BPIV3 F protein.

[0168] In some embodiments, a recombinant paramyxovirus comprising a recombinant B / HPIV3 comprising a viral genome including, from upstream to downstream, a BPIV3 genomic promoter followed by the BPIV3 N, P, and M genes, the HPIV3 F and HN genes, and the BPIV3 L gene, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprises 66E, 101P, 155C, and 290C substitutions and is linked to the TM and CT of the BPIV3 F protein.

[0169] In some embodiments, a recombinant paramyxovirus is provided that includes a recombinant B / HPIV3 comprising a viral genome that includes, from upstream to downstream, a BPIV3 genomic promoter followed by the BPIV3 N, P, and M genes, the HPIV3 F and HN genes, and the BPIV3 L gene, and further includes a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV F ectodomain includes substitutions 66E, 101P, 155C, and 290C, and the TM and CT of the HPIV3 F protein. It is linked to.

[0170] In some embodiments, a recombinant paramyxovirus comprising a recombinant B / HPIV3 comprising a viral genome including, from upstream to downstream, a BPIV3 genomic promoter followed by the BPIV3 N, P, and M genes, the HPIV3 F and HN genes, and the BPIV3 L gene, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprises 66E, 101P, 155C, and 290C substitutions and is linked to the TM and CT of the HPIV3 F protein.

[0171] In some embodiments, a recombinant paramyxovirus is provided, comprising a recombinant Sendai virus comprising a viral genome comprising, from upstream to downstream, a Sendai virus genomic promoter followed by the Sendai virus N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV F ectodomain comprises 66E, 101P, 155C, and 290C substitutions and is linked to the TM and CT of the Sendai virus F protein.

[0172] In some embodiments, a recombinant paramyxovirus is provided, comprising a recombinant Sendai virus comprising a viral genome comprising, from upstream to downstream, a Sendai virus genomic promoter followed by the Sendai virus N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprises 66E, 101P, 155C, and 290C substitutions and is linked to the TM and CT of the Sendai virus F protein.

[0173] In some embodiments, a recombinant paramyxovirus is provided, comprising a recombinant NDV comprising a viral genome comprising, from upstream to downstream, an NDV genomic promoter followed by the NDV N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV F ectodomain comprises 66E, 101P, 155C, and 290C substitutions and is linked to the TM and CT of the NDV F protein.

[0174] In some embodiments, a recombinant paramyxovirus is provided, comprising a recombinant NDV comprising a viral genome comprising, from upstream to downstream, an NDV genomic promoter followed by the NDV N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprises 66E, 101P, 155C, and 290C substitutions and is linked to the TM and CT of the NDV F protein.

[0175] In some embodiments, a recombinant paramyxovirus containing a recombinant PIV5 comprising a viral genome including, from upstream to downstream, a PIV5 genomic promoter followed by the PIV5 N, P, M, F, HN, and L genes, and further including a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain is provided, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV F ectodomain contains 66E, 101P, 155C, and 290C substitutions and is linked to the TM and CT of the PIV5 F protein.

[0176] In some embodiments, a recombinant paramyxovirus containing a recombinant PIV5 comprising a viral genome including, from upstream to downstream, a PIV5 genome promoter followed by the PIV5 N, P, M, F, HN, and L genes, and further including a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain is provided, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain contains 66E, 101P, 155C, and 290C substitutions and is linked to the TM and CT of the PIV5 F protein.

[0177] In some embodiments, a recombinant paramyxovirus is provided, comprising a recombinant parainfluenza virus (PIV) comprising a viral genome comprising, from upstream to downstream, a PIV genomic promoter followed by the PIV N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV F ectodomain comprises 66E, 101P, 155C, and 290C substitutions and is linked to the CT of the PIV F protein.

[0178] In some embodiments, a recombinant paramyxovirus is provided that comprises a recombinant HPIV1 comprising a viral genome that includes, from upstream to downstream, an HPIV1 genomic promoter followed by the HPIV1 N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and The F ectodomain contains 66E, 101P, 155C, and 290C substitutions, and HPIV1 It is linked to the CT of the F protein.

[0179] In some embodiments, a recombinant paramyxovirus containing a recombinant HPIV1 comprising a viral genome including, from upstream to downstream, an HPIV1 genomic promoter followed by the HPIV1 N, P, M, F, HN, and L genes, and further including a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain is provided, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain contains substitutions 66E, 101P, 155C, and 290C and is linked to the CT of the HPIV1 F protein.

[0180] In some embodiments, a recombinant paramyxovirus is provided that includes a recombinant HPIV3 comprising a viral genome that includes, from upstream to downstream, an HPIV3 genomic promoter followed by the HPIV3 N, P, M, F, HN, and L genes, and further includes a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and wherein the heterologous gene is located between the genomic promoter and the gene encoding the RSV N protein. The F ectodomain contains 66E, 101P, 155C, and 290C substitutions, and HPIV3 It is linked to the CT of the F protein.

[0181] In some embodiments, a recombinant paramyxovirus containing a recombinant HPIV3 is provided, comprising a viral genome including, from upstream to downstream, an HPIV3 genomic promoter followed by the HPIV3 N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain contains substitutions 66E, 101P, 155C, and 290C and is linked to the CT of the HPIV3 F protein.

[0182] In some embodiments, the HPIV3 genome promoter and the The following recombinant paramyxoviruses are provided, including recombinant HPIV3, whose viral genome includes the HPIV3 N, P, M, F, HN, and L genes, and further includes a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and wherein the RSV The F ectodomain contains 66E, 101P, 155C, and 290C substitutions, and BPIV3 It is linked to the CT of the F protein.

[0183] In some embodiments, a recombinant paramyxovirus containing a recombinant HPIV3 is provided, comprising a viral genome including, from upstream to downstream, an HPIV3 genomic promoter followed by the HPIV3 N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain contains substitutions 66E, 101P, 155C, and 290C and is linked to the CT of the BPIV3 F protein.

[0184] In some embodiments, a recombinant paramyxovirus containing a recombinant BPIV3 is provided, comprising a viral genome including, from upstream to downstream, a BPIV3 genomic promoter followed by BPIV3 N, P, M, F, HN, and L genes, and further including a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV The F ectodomain contains 66E, 101P, 155C, and 290C substitutions, and BPIV3 It is linked to the CT of the F protein.

[0185] In some embodiments, a recombinant paramyxovirus containing a recombinant BPIV3 comprising a viral genome including, from upstream to downstream, a BPIV3 genomic promoter followed by the BPIV3 N, P, M, F, HN, and L genes, and further including a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain is provided, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain contains substitutions 66E, 101P, 155C, and 290C and is linked to the CT of the BPIV3 F protein.

[0186] In some embodiments, a recombinant paramyxovirus comprising a recombinant B / HPIV3 comprising a viral genome including, from upstream to downstream, a BPIV3 genomic promoter followed by the BPIV3 N, P, and M genes, the HPIV3 F and HN genes, and the BPIV3 L gene, and further including a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV F ectodomain contains substitutions 66E, 101P, 155C, and 290C and is linked to the CT of the BPIV3 F protein.

[0187] In some embodiments, a recombinant paramyxovirus comprising a recombinant B / HPIV3 comprising a viral genome including, from upstream to downstream, a BPIV3 genomic promoter followed by the BPIV3 N, P, and M genes, the HPIV3 F and HN genes, and the BPIV3 L gene, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprises 66E, 101P, 155C, and 290C substitutions and is linked to the CT of the BPIV3 F protein.

[0188] In some embodiments, from upstream to downstream, a BPIV3 genomic promoter and The following recombinant paramyxoviruses are provided, including recombinant B / HPIV3 containing a viral genome comprising the BPIV3 N, P, and M genes, the HPIV3 F and HN genes, and the BPIV3 L gene, and further comprising a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV F ectodomain contains substitutions 66E, 101P, 155C, and 290C and is linked to the CT of the HPIV3 F protein.

[0189] In some embodiments, a recombinant paramyxovirus comprising a recombinant B / HPIV3 comprising a viral genome including, from upstream to downstream, a BPIV3 genomic promoter followed by the BPIV3 N, P, and M genes, the HPIV3 F and HN genes, and the BPIV3 L gene, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprises 66E, 101P, 155C, and 290C substitutions and is linked to the CT of the HPIV3 F protein.

[0190] In some embodiments, a recombinant paramyxovirus is provided, comprising a recombinant Sendai virus comprising a viral genome comprising, from upstream to downstream, a Sendai virus genomic promoter followed by the Sendai virus N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV F ectodomain comprises 66E, 101P, 155C, and 290C substitutions and is linked to the CT of the Sendai virus F protein.

[0191] In some embodiments, a recombinant paramyxovirus is provided, comprising a recombinant Sendai virus comprising a viral genome comprising, from upstream to downstream, a Sendai virus genomic promoter followed by the Sendai virus N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprises 66E, 101P, 155C, and 290C substitutions and is linked to the CT of the Sendai virus F protein.

[0192] In some embodiments, a recombinant paramyxovirus is provided, comprising a recombinant NDV comprising a viral genome comprising, from upstream to downstream, an NDV genomic promoter followed by the NDV N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the genomic promoter and the gene encoding the N protein, and the RSV F ectodomain comprises substitutions 66E, 101P, 155C, and 290C and is linked to the CT of the NDV F protein.

[0193] In some embodiments, a recombinant paramyxovirus is provided, comprising a recombinant NDV comprising a viral genome comprising, from upstream to downstream, an NDV genomic promoter followed by the NDV N, P, M, F, HN, and L genes, and further comprising a heterologous gene encoding a type I membrane protein comprising a recombinant RSV F ectodomain, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprises 66E, 101P, 155C, and 290C substitutions and is linked to the CT of the NDV F protein.

[0194] In some embodiments, from upstream to downstream, a PIV5 genome promoter and a subsequent A recombinant paramyxovirus containing a recombinant PIV5 comprising a viral genome containing the PIV5 N, P, M, F, HN, and L genes and further containing a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain is provided, wherein the heterologous gene is located between the genome promoter and the gene encoding the N protein, and the RSV F ectodomain contains substitutions 66E, 101P, 155C, and 290C and is linked to the CT of the PIV5 F protein.

[0195] In some embodiments, a recombinant paramyxovirus containing a recombinant PIV5 comprising a viral genome including, from upstream to downstream, a PIV5 genome promoter followed by the PIV5 N, P, M, F, HN, and L genes, and further including a heterologous gene encoding a type I membrane protein containing a recombinant RSV F ectodomain is provided, wherein the heterologous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain contains substitutions 66E, 101P, 155C, and 290C and is linked to the CT of the PIV5 F protein.

[0196] Any of the embodiments of the recombinant paramyxovirus disclosed herein (such as any of the recombinant paramyxoviruses discussed above) comprising a viral genome comprising a heterologous gene encoding an RSV F ectodomain.) In this case, the heterologous gene encoding the recombinant RSV F ectodomain can encode a polypeptide sequence including RSV F positions 1-529.

[0197] Further explanation The disclosed recombinant paramyxoviruses are autonomously replicating, i.e., capable of replicating after infection of a suitable host cell. In some embodiments, the recombinant paramyxoviruses have an attenuated phenotype when administered to, for example, a human subject.

[0198] Attenuation of a recombinant paramyxovirus can be achieved using various methods known in the art, for example, by introducing one or more mutations that alter the biological function of the recombinant paramyxovirus, resulting in an attenuated phenotype. Insertion of a heterologous gene can also result in an attenuated phenotype. Preferably, a paramyxovirus containing a genome encoding a heterologous gene is attenuated by about 100 to 5,000 times or more in cells or mammals compared to wild-type paramyxovirus.

[0199] The disclosed recombinant paramyxoviruses can be tested in well-known in vitro and in vivo models to confirm appropriate attenuation, resistance to phenotypic reversion, and immunogenicity. In in vitro assays, modified paramyxoviruses can be tested for one or more desired phenotypes, such as, for example, temperature-sensitive replication. The disclosed recombinant paramyxoviruses can also be tested in animal models of infection using PIV and / or heterologous viral pathogens contained in the recombinant virus (e.g., RSV). A variety of animal models are known.

[0200] The recombinant attenuated paramyxovirus is preferably attenuated in cells or mammals by about 100-5000 fold compared to wild-type paramyxovirus. In some embodiments, the level of viral replication in vitro is preferably sufficient to provide for the production of viral vaccines for widespread use. In some embodiments, the level of viral replication of the in vitro attenuated paramyxovirus is at least 10 per ml. 6 , more preferably at least 10 7 , most preferably at least 10 8 Preferably, the attenuating mutations are those that are stable. Recombinant paramyxoviruses with at least 2, 3, 4 or even more attenuating mutations are likely to be more stable.

[0201] Ongoing preclinical research has identified several mutations or modifications that attenuate HPIV1, HPIV2, and HPIV3, which can be introduced by reverse genetics to produce attenuated strains as potential vaccine and vector backbones. The inclusion of foreign genes in the HPIV backbone is also attenuating in itself. This can be due to a variety of effects, including increased genome length and gene number, as well as the effect of the foreign protein. Whatever the cause, the attenuating effect of the insert must also be considered when attempting to achieve an appropriate level of attenuation.

[0202] Attenuated strains of HPIV1, 2, and 3 have been or are currently being studied in clinical trials in seronegative infants (Karron et al. 2012 Vaccine 30:3975-3981; Schmidt et al. 2011 Expert Rev. Respir. Med. 5:515-526). These attenuated HPIV1, HPIV2, and HPIV3 strains, or versions thereof, are potential vectors for expressing heterologous RSV F proteins.

[0203] Examples of modifications to the genome of paramyxoviruses that confer an attenuated phenotype are known in the art, e.g., U.S. Patent Publication Nos. 2012 / 0045471; 2010 / 0119547; 2009 / 0263883; 2009 / 0017517; 8084037; 6, No. 410,023; No. 8,367,074; No. 7,951,383; No. 7,820,182; No. 7704509; No. 7632508; No. 7622123; No. 7 No. 250171; No. 7208161; No. 7201907; No. 7192593; No. 2012 / 0064112; No. 20140186397; and Newman et al. 2002 Virus genes 24:77-92; Tang et al., 2003 J Virol, 77(20):10819-10828; Basavarajappa et al., 2014 Vaccine, 32:3555-3563; McGinnes et al., J. Virol., 85:366-377, 2011; and Jones et al., Vaccine, 30:959-968, 2012. For example, attenuation of PIV3 can be achieved by the presence of BPIV3-derived genes that confer host range restriction in primates, including humans, such as B / HPIV3 viruses, which contain BPIV3 genes except for F and HN from HPIV3 (Skiadopoulos MH et al., J Virol, 77:1141-8, 2003). Sendai virus is also restricted in primates due to host range restriction (Jones BG et al. Vaccine 30:959-968 2012). Another means of attenuation is exemplified by missense mutations that can occur in multiple genes, such as in the cp45 HPIV3 virus (Skiadopoulos MH et al. J Virol 73:1374-81 1999). Other examples of attenuating point mutations are provided for HPIV1 in Example 2 below. Deletion of one or several codons, as exemplified by HPIV1 in Example 2, can also confer an attenuated phenotype. Also, as exemplified in Example 1, the presence of vector TM and CT or CT domains linked to a heterologous ectodomain can strongly attenuate the vector. Other examples of attenuating mutations in HPIV1 are described by Bartlett EJ et al. Virol J 4:67 2007) and for HPIV2 by Nolan SM et al. Vaccine 23:4765-4774 2005). Deletion of all or part of one or more accessory genes is also a means of attenuation (Durbin A Virology 261:319-330 1999).

[0204] The immunogenicity of recombinant attenuated paramyxoviruses can be assessed in animal models (non-human primates, such as African green monkeys) by examining the number of animals that form antibodies against the paramyxovirus after one immunization and after a second immunization, as well as the response. The immunogenicity can be assessed by measuring the magnitude of the response. In some embodiments, a recombinant paramyxovirus is sufficiently immunogenic if about 60-80% of the animals develop antibodies after a first immunization and about 80-100% of the animals develop a response after a second immunization. Preferably, the immune response protects against infection by both the source paramyxovirus and the viral pathogen from which the heterologous gene contained in the recombinant paramyxovirus is derived.

[0205] I. Further Vectors It will be appreciated that recombinant RSV F proteins and nucleic acid molecules encoding them can be contained in (or expressed by) vectors other than PIV vectors. For example, plasmid vectors and other viral vectors can be used, for example, to express recombinant RSV F proteins or fragments thereof in host cells, or for immunizing subjects as disclosed herein. In some embodiments, the vector can be administered to a subject as part of a prime-boost vaccination. In some embodiments, the vector is included in a vaccine, such as a primer vaccine or a booster vaccine for use in prime-boost vaccination.

[0206] In some instances, the vector can be a replication-competent and / or attenuated viral vector. The viral vector can also be conditionally replication-competent. In other instances, the viral vector is replication-deficient in the host cell.

[0207] Polyoma, i.e., SV40 (Madzak et al., 1992, J. Gen. Virol., 73:1533-1536), adenovirus (Berkner, 1992, Cur. Top. Microbiol. Immunol., 158:39-6; Berliner et al., 1988, BioTechniques, 6:616-629; Gorziglia et al., 1992, J. Virol., 66:4407-4412; Quantin et al., 1992, Proc. Natl. Acad. Sci. USA, 89:2581-2584; Rosenfeld et al., 1992, Cell, 68:143-155; Wilkinson et al., 1992, Nucl. Acids, 68:145-156). Res., 20:2233-2239; Stratford-Perricaudet et al., 1990, Hum. Gene Ther., 1:241-256), vaccinia virus (Mackett et al., 1992, Biotechnology, 24:495-499), adeno-associated virus (Muzyczka, 1992, Curr. Top. Microbiol. Immunol., 158:91-123; On et al., 1990, Gene, 89:279-282), herpesviruses including HSV and EBV (Margolskee, 1992, Curr. Top. Microbiol. Immunol., 158:67-90; Johnson et al., 1992, J. Virol., 66:295-2965; Fink et al., 1992, Hum. Gene Ther. 3:11-19; Breakfield et al., 1987, Mol. Neurobiol., 1:337-371; Fresse et al., 1990, Biochem. Pharmacol., 40:2189-2199), Sindbis virus (H. Herweijer et al., 1995, Human Gene Therapy 6:1161-1167; U.S. Pat. Nos. 5,091,309 and 5,2217,879), alphavirus (S. Schlesinger, 1993, Trends Biotechnol. 11:18-22; I. Frolov et al., 1996, Proc. Natl. Acad. Sci. USA 93:11371-11377), and retroviruses of avian origin (Brandyopadhyay et al., 1984, Mol. Cell Biol., 4:749–754; Petropouplos et al., 1992, J. Virol., 66:3391–3397), retroviruses of murine origin (Miller, 1992, Curr. Top. Microbiol. Immunol., 158:1–24; Miller et al., 1985, Mol. Cell Biol., 5:431–437; Sorge et al., 1984, Mol. Cell Biol., 4:1730–1737; Mann et al., 1985). Several viral vectors have been constructed that can be used to express recombinant RSV F protein or immunogenic fragments thereof, including retroviruses of human origin (Page et al., 1990, J. Virol., 64:5370-5276; Buchschalcher et al., 1992, J. Virol., 66:2731-2739) and human origin (Page et al., 1990, J. Virol., 64:5370-5276; Buchschalcher et al., 1992, J. Virol., 66:2731-2739). Baculovirus (Autographa californica nuclear polyhedrosis virus; AcMNPV) vectors are also known in the art and can be obtained from commercial sources (PharMingen, San Diego, Calif.; Protein Sciences Corp., Meriden, Conn.; Stratagene, La Jolla, Calif., etc.).

[0208] In some embodiments, the viral vector may comprise an adenoviral vector expressing the disclosed recombinant RSV F protein or an immunogenic fragment thereof (such as the RSV F ectodomain). Adenoviruses from various origins, subtypes, or mixtures of subtypes can be used as the source of the viral genome for the adenoviral vector. Non-human adenoviruses (e.g., monkey, chimpanzee, gorilla, avian, canine, ovine, or bovine adenoviruses) can be used to produce adenoviral vectors. For example, simian adenoviruses can be used as the source of the viral genome for the adenoviral vector. The simian adenovirus can be of serotype 1, 3, 7, 11, 16, 18, 19, 20, 27, 33, 38, 39, 48, 49, 50, or any other simian adenovirus serotype. Simian adenoviruses can be referred to by any suitable abbreviation known in the art, such as SV, SAdV, SAV, or sAV. In some examples, the simian adenovirus vector is a simian adenovirus vector of serotype 3, 7, 11, 16, 18, 19, 20, 27, 33, 38, or 39. In one example, a chimpanzee serotype C Ad3 vector is used (see, for example, Peruzzi et al., Vaccine, 27:1293-1300, 2009). Human adenovirus can be used as the source of the viral genome of the adenovirus vector. Human adenovirus can be of various subgroups or serotypes. For example, the adenovirus can be of subgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, 35, and 50), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 36-39, and 42-48), subgroup E (e.g., serotype 4), subgroup F (e.g., serotypes 40 and 41), an unclassified serogroup (e.g., serotypes 49 and 51), or any other adenovirus serotype.Those skilled in the art are familiar with replication-competent and replication-deficient adenoviral vectors (including singly and multiple replication-deficient adenoviral vectors).Examples of replication-deficient adenoviral vectors, including multiple replication-deficient adenoviral vectors, are disclosed in U.S. Patent No. 5,837,511; U.S. Patent No. 5,851,806; U.S. Patent No. 5,994,106; U.S. Patent No. 6,127,175; U.S. Patent No. 6,482,616; and U.S. Patent No. 7,195,896 and International Patent Application No. WO94 / 28152, WO95 / 02697, WO95 / 16772, WO95 / 34671, WO96 / 22378, WO97 / 12986, WO97 / 21826 and WO03 / 022311.

[0209] III. Recombinant Methods, Vectors and Host Cells The recombinant paramyxoviruses and polynucleotides disclosed herein can be produced by synthetic and recombinant methods. Accordingly, polynucleotides encoding infectious paramyxovirus clones and host cells containing the infectious clones, as well as methods for producing such vectors and host cells by recombinant methods, are also provided.

[0210] Also provided are isolated nucleic acid molecules encoding any of the recombinant RSV F proteins disclosed herein.

[0211] As discussed above, the disclosed paramyxoviruses or polynucleotides can be synthesized or prepared by techniques well known in the art. See, for example, WO94 / 027037 and US20130052718. The nucleotide sequences of wild-type paramyxovirus genomes are known and readily available online, for example, at GenBank (accessible at www-ncbi-nlm-nihgov / entrez). Nucleotide sequences encoding the disclosed recombinant paramyxoviruses can be synthesized or amplified using methods known to those skilled in the art, including the use of DNA polymerase in a cell-free environment. Furthermore, those skilled in the art can readily use the genetic code to construct various functionally equivalent nucleic acids, such as nucleic acids that differ in sequence but encode the same protein components.

[0212] Exemplary nucleic acids can be prepared by cloning techniques. Examples of suitable cloning and sequencing techniques and instructions sufficient to guide the skilled artisan through numerous cloning exercises are known (e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, detailed appendix 104, 2013). Product information from manufacturers of biological reagents and laboratory equipment also provides useful information. Such manufacturers include SIGMA Chemical Company (Saint Louis, MO), R&D Systems (Minneapolis, MN), Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD), Fluka Commercial sources include Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (Carlsbad, Calif.), and Applied Biosystems (Foster City, Calif.), as well as many other commercial sources known to those of skill in the art.

[0213] The genome of a recombinant paramyxovirus can contain one or more variations (e.g., mutations resulting in amino acid deletions, substitutions, or insertions), so long as the resulting recombinant paramyxovirus retains the desired biological function, such as a level of immunogenicity. These variations in sequence can be naturally occurring variations, or they can be engineered through the use of genetic engineering techniques known to those skilled in the art. Examples of such techniques are described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Cloning), both of which are incorporated herein by reference in their entireties. Biology, John Wiley & Sons, New York, through supplement 104, 2013).

[0214] Modifications can be made to the nucleic acids encoding the sequences described herein without diminishing their biological activity. Amino acid substitutions, insertions, and deletions can be made using known recombinant methods such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, PCR mutagenesis, site-specific mutagenesis, cassette mutagenesis, restriction-selection mutagenesis, and the like. (See, for example, Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, detailed appendix 104, 2013). Some modifications can be made to facilitate cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those skilled in the art and include, for example, stop codons, methionines added to the amino terminus to provide initiation sites, and additional nucleotides placed at either end to create conveniently positioned restriction sites.

[0215] A "conservative" amino acid substitution is one that does not substantially affect or reduce the function of a protein, such as the ability of the protein to induce an immune response when administered to a subject. The term conservative variation also includes the use of a substituted amino acid in place of the unsubstituted parent amino acid. Furthermore, one skilled in the art will recognize that individual substitutions, deletions, or additions that alter, add, or delete a single amino acid or a small percentage (e.g., less than 5%, in some embodiments, less than 1%) of amino acids in an encoded sequence are conservative variations in which the change results in the replacement of an amino acid with a chemically similar amino acid.

[0216] Conservative amino acid substitution tables providing functionally similar amino acids are well known to those of skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0217] The disclosed recombinant paramyxovirus can be produced from viruses isolated from biological samples.Polynucleotides and vectors can be produced by standard recombinant methods known in the art, such as polymerase chain reaction (Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, detailed appendix 104, 2013)).Methods for changing or modifying nucleic acid sequences are also known to those skilled in the art.

[0218] Paramyxovirus genomes can be assembled from polymerase chain reaction cassettes sequentially cloned into vectors containing selectable markers for propagation in a host, such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culture in E. coli and other bacteria.

[0219] Using standard recombinant techniques, polynucleotides can be inserted into reproducible vectors for cloning. A variety of vectors are publicly available. Vectors can be in the form of, for example, a plasmid, cosmid, viral particle, or phage. A variety of procedures can be used to insert the appropriate nucleic acid sequence into the vector. Generally, the nucleic acid is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more signal sequences, an origin of replication, one or more marker genes, an enhancer, an endonuclease gene, or a polypeptide. These components include a transcription sensor element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques, which are well known to those skilled in the art.

[0220] Examples of suitable replicable vectors include, but are not limited to, pUC19 or pTM1. The polynucleotide can be operably linked to a suitable promoter, such as a T7 polymerase promoter, a cytomegalovirus promoter, a cellular polymerase II promoter, or an SP1 promoter. The replicable vector can further include a transcription initiation site, a transcription termination site, and a ribosome binding site for translation.

[0221] Introduction of a recombinant vector comprising a polynucleotide encoding a paramyxovirus genome or a paramyxovirus protein into a host cell, such as a bacterial cell or a eukaryotic cell, can be effected by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, nuclear electroporation, chemical transduction, electrotransduction, infection, or other methods. Such methods are described in standard laboratory manuals such as Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, detailed appendix 104, 2013). Commercially available transfection reagents are also available, such as Lipofectamine (Invitrogen, Carlsbad, Calif.) and FuGENE 6™ (Roche Diagnostics, Indianapolis, Ind.). Suitable host cells include, but are not limited to, HEp-2 cells, FRhL-DBS2 cells, LLC-MK2 cells, MRC-5 cells, and Vero cells.

[0222] IV. Immunogenic composition Immunogenic compositions are also provided comprising a recombinant paramyxovirus as described herein (such as a recombinant PIV comprising a genome encoding a heterologous recombinant RSV F protein) and a pharmaceutically acceptable carrier. Such compositions can be administered to a subject by a variety of administration modes known to those skilled in the art, for example, by intranasal administration. Actual methods for preparing administrable compositions are known or apparent to those skilled in the art, and are described in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing. Company, Easton, Pennsylvania, 1995, for example.

[0223] Therefore, the recombinant paramyxoviruses described herein can be formulated with a pharmaceutically acceptable carrier to help maintain biological activity while promoting increased stability during storage within acceptable temperature ranges. Potential carriers include, but are not limited to, physiologically balanced culture media, phosphate buffered saline solutions, water, emulsions (e.g., oil / water or water / oil emulsions), various types of humectants, cryoprotective additives or stabilizers such as proteins, peptides, or hydrolysates (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., monosodium glutamate), or other protective agents. The resulting aqueous solutions can be packaged for immediate use or lyophilized. The lyophilized preparations are combined with a sterile solution prior to administration for either single or multiple doses.

[0224] Formulated compositions, particularly liquid formulations, may contain bacteriostatic agents to prevent or minimize degradation upon storage, including, but not limited to, effective concentrations (usually ≦1% w / v) of benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and / or propylparaben. Bacteriostatic agents may be contraindicated for some patients; therefore, lyophilized formulations are reconstituted with solutions that may or may not contain such ingredients.

[0225] The disclosed pharmaceutical compositions may contain pharmaceutically acceptable vehicle substances as needed to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate.

[0226] Pharmaceutical compositions may optionally contain adjuvants to enhance the host's immune response. Suitable adjuvants include, for example, toll-like receptor agonists, alum, AlPO4, alhydrogel, lipid A and its derivatives or variants, oil emulsions, saponins, neutral liposomes, liposomes containing vaccines and cytokines, nonionic block copolymers, and chemokines. Among many other suitable adjuvants known in the art, nonionic block polymers containing polyoxyethylene (POE) and polyoxypropylene (POP), such as POE-POP-POE block copolymers, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, IN), and IL-12 (Genetics Institute, Cambridge, MA) can be used as adjuvants (Newman et al., 1998, Critical Reviews in Therapeutic Drug Carrier Systems 15:89-142). These adjuvants have the advantage that they stimulate the immune system in a non-specific manner and thus help to enhance the immune response to pharmaceutical preparations.

[0227] In some embodiments, the composition may contain a recombinant paramyxovirus encoding a RSV F ectodomain from a particular RSV subgroup or strain, and a recombinant paramyxovirus encoding a RSV F ectodomain from a different RSV subgroup or strain. For example, the composition may contain a recombinant paramyxovirus containing a recombinant RSV F protein from subtype A and subtype B RSV. The different vectors may be in a mixture and administered simultaneously or separately. Due to the phenomenon of cross-protection between specific strains of RSV, immunization with a certain paramyxovirus encoding a RSV F ectodomain from a first strain may protect against several different strains of the same or different subgroups.

[0228] In some instances, it may be desirable to combine the recombinant viral vector or composition thereof with other pharmaceutical preparations (e.g., vaccines) that induce protective responses against other pathogens, particularly those that cause other childhood diseases. For example, compositions containing a recombinant paramyxovirus as described herein can be administered simultaneously (usually separately) or sequentially with other vaccines recommended by the Advisory Committee on Immunization Practices (ACIP; cdc.gov / vaccines / acip / index.html) for the targeted age group (e.g., infants approximately 1-6 months of age). These additional vaccines include, but are not limited to, vaccines administered intravenously. As such, a recombinant paramyxovirus containing a recombinant RSV F protein described herein can be administered simultaneously or sequentially with vaccines against, for example, hepatitis B (HepB), diphtheria, tetanus, and pertussis (DTaP), pneumococcal virus (PCV), Haemophilus influenzae type b (Hib), polio, influenza, and rotavirus. The next dose can be administered.

[0229] For example, recombinant paramyxoviruses for use in immunogenic compositions such as vaccines are selected based on their attenuation and immunogenicity. These vaccine selection criteria are determined according to well-known methods. Preferably, the candidate virus has a stable attenuation phenotype, exhibits replication in immunized hosts, and effectively induces the generation of an immune response, preferably a protective immune response, in the recipient. Preferably, the candidate virus stimulates and expands the immune response, for example, induces an immune response against different virus strains or subgroups, and / or stimulates an immune response mediated by different immunological bases (e.g., secretory versus serum immunoglobulins, cellular immunity, etc.).

[0230] The pharmaceutical compositions typically contain an effective amount of the disclosed paramyxovirus and can be prepared by conventional techniques. Typically, the amount of recombinant virus in each dose of the immunogenic composition is selected to induce an immune response without significant adverse side effects. In some embodiments, the compositions can be provided in unit dosage forms for use in inducing an immune response in a subject, for example, to prevent PIV and / or RSV infection in a subject. The unit dosage form contains a single preselected dose suitable for administration to a subject, or a suitable significant or measured multiple of two or more preselected unit doses and / or a metering mechanism for administering the unit dose or multiples thereof. In other embodiments, the composition further comprises an adjuvant.

[0231] V. Methods of Inducing an Immune Response Provided herein are methods for eliciting an immune response in a subject by administering one or more of the disclosed recombinant paramyxoviruses to the subject. In certain examples, the subject is a human. The immune response can be a protective immune response, e.g., a response that prevents or reduces subsequent infection with the paramyxovirus or a heterologous virus contained in the recombinant paramyxovirus. The induction of an immune response can be used to treat or inhibit viral infection and diseases associated therewith. In some embodiments, the method comprises administering to a subject a PIV. The immunogenic composition comprises an attenuated recombinant parainfluenza virus comprising a viral genome containing a heterologous gene encoding a recombinant RSV F ectodomain linked to the F protein transmembrane (TM) domain and cytoplasmic tail.

[0232] For example, a subject who has or is at risk of developing a paramyxovirus infection, such as an RSV and / or PIV infection, due to exposure or potential exposure to RSV and / or PIV is selected for treatment. After administration of the disclosed immunogens, the subject can be monitored for paramyxovirus infection or symptoms associated therewith, or both.

[0233] Methods for intranasal administration of a recombinant paramyxovirus to a subject are known to those skilled in the art, as are methods for selecting a subject for administration, preparing an immunogenic composition comprising a recombinant paramyxovirus for intranasal administration, and assessing the subject for an immune response to the recombinant paramyxovirus. An exemplary description of such methods can be found, for example, in Karron et al., 2012 Vaccine, 30(26), 3975-3981, the entire contents of which are incorporated herein by reference.

[0234] Typical subjects intended for treatment with the therapeutic agents and methods of the present disclosure include humans as well as non-human primates and other animals. Because nearly all humans are infected with RSV and PIV by the age of 5, entire birth cohorts are included as relevant populations for immunization. This includes, for example, immunization of pregnant women (or women of childbearing age), newborns, or those still in the womb to protect their infants by passive transfer of antibodies any time from birth to 6 months of age, or from 6 months to 5 years of age. This can be achieved by initiating an immunization program in a family member of a subject over 50 years of age. The scope of this disclosure is intended to include maternal immunization. In some embodiments, the subject is a human subject who is seronegative for RSV or PIV3-specific antibodies. In further embodiments, the subject is under 1 year of age, such as under 6 months, under 3 months, or under 1 month.

[0235] Subjects at greatest risk of developing severe symptoms (e.g., requiring hospitalization) from RSV and / or PIV infection include children with bronchopulmonary dysplasia of prematurity, and congenital heart disease is most susceptible to severe disease. During childhood and adulthood, the disease becomes milder, but may be accompanied by lower respiratory tract disease and is commonly complicated by sinusitis. Disease severity increases in hospitalized elderly people (e.g., people over 65 years of age). Severe disease also occurs in people with severe combined immunodeficiency disease or after bone marrow or lung transplantation. Therefore, these subjects are selected for administration of the disclosed recombinant paramyxoviruses.

[0236] To identify subjects for prevention or treatment according to the methods of the present disclosure, accepted screening methods are used to examine the subject's risk factors associated with the targeted or suspected disease or condition, or to examine the status of an existing disease or condition. These screening methods include, for example, conventional follow-up procedures to determine environmental, familial, occupational, and other such factors that may be associated with the targeted or suspected disease or condition, as well as diagnostic methods such as various ELISA and other immunoassay methods available and well-known in the art for detecting and / or characterizing paramyxovirus infection. These and other conventional methods allow clinicians to select patients who require therapy using the methods and pharmaceutical compositions of the present disclosure. In accordance with these methods and principles, the compositions can be administered as a stand-alone prevention or treatment program, or as a follow-up, adjunctive, or coordinated treatment plan for other treatments, according to the teachings herein or other conventional methods known to those skilled in the art.

[0237] Administration of the disclosed recombinant paramyxoviruses can be prophylactic or therapeutic. When provided prophylactically, the immunogen can be provided prior to any symptoms, e.g., prior to infection. Prophylactic administration serves to induce an immune response that can prevent or ameliorate any subsequent infection. In some embodiments, a method can include selecting a patient at risk of contracting a paramyxovirus infection and administering an effective amount of the disclosed recombinant paramyxovirus to the subject. The recombinant paramyxovirus can be provided prior to anticipated exposure to the paramyxovirus to induce an immune response that attenuates the anticipated severity, duration, or extent of infection and / or associated disease symptoms after exposure or suspected exposure to the virus, or after the actual onset of infection. In some examples, treatment using the methods disclosed herein extends the subject's survival time.

[0238] Administration of the disclosed recombinant paramyxoviruses containing RSV and PIV antigens to a subject can induce the production of an immune response that is protective against severe lower respiratory tract diseases, such as pneumonia and bronchiolitis, or croup if the subject is subsequently infected or reinfected with wild-type RSV or PIV. While naturally circulating viruses can still cause infection, particularly in the upper respiratory tract, the likelihood of rhinitis as a result of vaccination is reduced, and subsequent infection with wild-type viruses can potentially boost resistance. Following vaccination, there are detectable levels of serum and secretory antibodies produced by the host that can neutralize homologous (same subgroup) wild-type viruses in vitro and in vivo. In many instances, host antibodies also neutralize wild-type viruses of different, non-vaccine subgroups. To achieve higher levels of cross-protection, for example, against heterologous strains of different subgroups, at least one major strain of both RSV subgroups A and B can be neutralized. The subject can be vaccinated with a composition comprising a recombinant viral vector containing the RSV F protein derived from

[0239] The recombinant viral vectors and immunogenic compositions thereof disclosed herein are provided to a subject, preferably a human, in an amount effective to induce or enhance an immune response to antigens contained in the virus. An effective amount allows some growth and propagation of the virus to produce the desired immune response, but does not result in virus-related symptoms or disease. Based on the guidance provided herein and knowledge in the art, one of skill in the art can readily determine the appropriate amount of virus to use in a live vaccine. The exact amount will vary depending on several factors, such as the subject's health and weight, the mode of administration, the degree of attenuation of the virus, the nature of the formulation, and whether the subject's immune system is compromised.

[0240] Immunogenic compositions comprising one or more of the disclosed recombinant paramyxoviruses can be used in coordinated (or prime-boost) vaccination protocols or combinatorial formulations. In certain embodiments, the novel combinatorial immunogenic compositions and coordinated immunization protocols each use separate immunogens or formulations directed at eliciting antiviral immune responses, such as immune responses against RSV and PIV proteins. Separate immunogenic compositions that elicit antiviral immune responses can be combined into a multivalent immunogenic composition administered to a subject in a single immunization step, or they can be administered separately (in a monovalent immunogenic composition) in a coordinated (or prime-boost) immunization protocol.

[0241] It is contemplated that there may be several boosts and that each boost may be a different disclosed immunogen. It is also contemplated that in some instances, a boost may be the same immunogen as another boost or prime.

[0242] Upon administration of the disclosed recombinant paramyxoviruses, the subject's immune system typically responds to the immunogenic composition by producing antibodies specific to viral proteins, and such a response indicates that an immunologically effective dose has been delivered to the subject.

[0243] For each individual subject, a specific dosing regimen can be evaluated and adjusted over time according to the individual's needs and the professional judgment of the person administering or supervising the administration of the immunogenic composition. In some embodiments, the subject's antibody response is determined in conjunction with the evaluation of an effective dosage / immunization protocol. In most instances, assessing the antibody titer in serum or plasma obtained from the subject will be sufficient. The decision to administer a booster vaccination and / or modify the amount of therapeutic agent administered to an individual can be based, at least in part, on the antibody titer level. The antibody titer level can be based, for example, on an immunobinding assay that measures the concentration of serum antibodies that bind to an antigen, including the RSV F protein. The actual dosage of the disclosed immunogens will vary according to factors such as disease symptoms and the subject's individual condition (e.g., the subject's age, size, fitness, severity of symptoms, susceptibility factors, etc.), the time and route of administration, other concurrently administered drugs or treatments, and the specific pharmacology of the composition to elicit the desired activity or biological response in the subject. The dosing regimen can be adjusted to provide an optimal prophylactic or therapeutic response.

[0244] Determination of effective dosages is typically based on animal model studies followed by human clinical trials and is guided by an administration protocol that significantly reduces the appearance or severity of the targeted disease symptom or condition in the subject, or induces a desired response (such as a neutralizing immune response) in the subject. Suitable models in this regard include, for example, mice, rats, pigs, cats, ferrets, non-human primates, and other recognized animal model subjects known in the art. Alternatively, effective dosages can be determined using in vitro models (e.g., immunological and histopathological assays). Using such models, only routine calculations and adjustments are required to determine the appropriate concentration and dose for administering a therapeutically effective amount of the composition (e.g., an amount effective to induce a desired immune response or to alleviate one or more symptoms of a targeted disease). In alternative embodiments, an effective amount or dose of a composition may simply inhibit or enhance one or more selected biological activities correlated with a disease or condition as set forth herein, for either therapeutic or diagnostic purposes. In one embodiment, a typical range for viral administration is about 10-15 mg / kg per human subject. 4 ~about 10 5 Approximately 10 per human subject containing PFU virus 3 ~about 10 7 plaque-forming units (PFU) or more of virus.

[0245] Administration of an immunogenic composition that induces an immune response to reduce or prevent infection can eliminate such infection, but not necessarily completely, so long as the infection is measurably reduced by at least about 50%, e.g., at least about 70%, or about 80%, or even about 90%, compared to the absence of the agent or a reference agent. Those in need of treatment include the general population and / or patients infected with or at risk of infection by a paramyxovirus, such as RSV and / or PIV.

[0246] In one example, the desired response is to inhibit, reduce, or prevent RSV and / or PIV infection or reinfection. RSV and / or PIV infection does not need to be completely eliminated, reduced, or prevented for the method to be effective. For example, administering an effective amount of the disclosed recombinant paramyxovirus can reduce subsequent RSV and / or PIV infection by a desired amount, for example, by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (e.g., as measured by infection of cells or by the number or percentage of subjects infected with RSV and / or PIV) (elimination or prevention of detectable RSV and / or PIV infection compared to suitable subjects).

[0247] Dosage and number of doses vary depending on the setting, for example, in adults who have been primed by previous paramyxovirus infection or immunization, or any other, and a single dose may be sufficient booster.In naive subjects, in some cases, at least two doses, for example, at least three doses, can be administered.In some embodiments, for example, annual booster is administered together with annual influenza vaccination.

[0248] After immunization, serum can be collected from the subject at appropriate time points, frozen, and stored for antibody titer assays and / or neutralization testing. Quantification of antibody levels can be performed by subtype-specific neutralization assays or ELISA. Methods for assaying neutralizing activity are known to those skilled in the art and are further described herein, including, but not limited to, plaque reduction neutralization (PRNT) assays, microneutralization assays, flow cytometry-based assays, and single-cycle infection assays. In some embodiments, serum neutralization activity can be assayed using a panel of RSV or PIV pseudoviruses. Virus-neutralizing antibody titers are determined in serum samples by the PRVN assay, as previously described (de Graaf et al., J. Virol. Methods, 143:169-174, 2007). Briefly, serum samples with approximately 50 pfu of NL / 1 / 00 or NL / 1 / 99, which express enhanced green fluorescent protein, can be diluted and incubated at 37°C for 60 minutes. The virus-serum mixture is then added to Vero-118 cells in a 24-well plate and incubated at 37°C. After 2 hours, the supernatant is replaced with an equal volume of a mixture of infection medium and 2% methylcellulose. After 6 days, the cells are imaged using a Typhoon 9410 Variable Mode Imager ( Fluorescent plaques are counted using a GE Healthcare kit. Antibody titers are expressed as the dilution resulting in a 50% reduction in plaque count, calculated according to the method of Reed & Muench, Am. J. Hyg., 27, 493-497, 1938.

[0249] Further embodiments: Item 1. A recombinant paramyxovirus comprising: (a) a viral genome comprising a heterologous gene encoding the ectodomain of a type I transmembrane protein of a heterologous virus linked to the transmembrane domain (TM) and cytoplasmic tail (CT) of the F protein of the paramyxovirus; or (b) a viral genome comprising a heterologous gene encoding the ectodomain of a type II transmembrane protein of a heterologous virus linked to the TM and CT of the HN protein of the paramyxovirus.

[0250] Item 2. The recombinant paramyxovirus of item 1, which is a recombinant human / bovine parainfluenza virus 3 (B / HPIV3), a recombinant human parainfluenza virus 1 (HPIV1), a recombinant human parainfluenza virus 2 (HPIV2), a recombinant human parainfluenza virus 2 (HPIV3), a recombinant parainfluenza virus 5 (PIV5), a recombinant Sendai virus, or a recombinant Newcastle disease virus (NDV).

[0251] Item 3. The recombinant paramyxovirus of item 2, comprising a recombinant parainfluenza virus (PIV) comprising a viral genome comprising a heterologous gene encoding a recombinant respiratory syncytial virus (RSV) F ectodomain linked to PIV F proteins TM and CT; a recombinant NDV comprising a viral genome comprising a heterologous gene encoding a recombinant RSV F ectodomain linked to NDV F proteins TM and CT; or a recombinant Sendai virus comprising a viral genome comprising a heterologous gene encoding a recombinant RSV F ectodomain linked to Sendai virus F proteins TM and CT.

[0252] Item 4. The recombinant paramyxovirus of any one of items 1 to 3, comprising a recombinant PIV comprising a viral genome comprising a heterologous gene encoding a recombinant RSV F ectodomain linked to PIV F proteins TM and CT.

[0253] Item 5. The recombinant paramyxovirus of Item 4, wherein the RSV F ectodomain is derived from a human RSV (hRSV) F protein.

[0254] Clause 6. The recombinant paramyxovirus of clause 4 or 5, wherein the hRSV F protein is derived from a subtype A hRSV or a subtype B hRSV.

[0255] Item 7. The recombinant paramyxovirus of any one of items 4 to 6, wherein the RSV F ectodomain is stabilized in the RSV F prefusion conformation by one or more amino acid substitutions compared to the native RSV F protein sequence.

[0256] Section 8. The RSV F ectodomain comprises the amino acids set forth in any one of clauses 4-7, wherein the amino acid numbering corresponds to the RSV F protein sequence set forth in SEQ ID NO: 1.

[0257] Item 9. The recombinant paramyxovirus of Item 8, wherein the RSV F ectodomain comprises an amino acid substitution set forth as a combination of: (a) K66E; (b) Q101P; (c) S155C and S290C; (d) S190F; (e) V207L; or (f) (a) and (b); (a) and (c); (a) and (d); (a) and (e); (a), (d) and (e); (a), (c), (d) and (e); (a), (b) and (c); (a), (b) and (d); (a), (b) and (e); (a), (b), (e) and (d); (a), (b), (c), (d) and (e); (c) and (d); or (c) and (e); or (c), (d) and (e).

[0258] Paragraph 10. The recombinant paramyxovirus of paragraph 8 or 9, wherein the RSV F ectodomain comprises 66E, 101P, 155C, 290C, 190F and 207L.

[0259] Clause 11. The recombinant paramyxovirus of any one of clauses 4 to 10, wherein the RSV F ectodomain comprises an amino acid sequence at least 85% identical to one of SEQ ID NOs: 1 (WT RSV FA), 2 (WT RSV FB), 12 (A2 HEK), 14 (A2 HEK + DS) or 21 (A2 HEK + DS-Cav1), or the amino acid sequence of the RSV ectodomain of SEQ ID NOs: 12, 14 or 21.

[0260] Paragraph 12. The recombinant paramyxovirus of any one of paragraphs 4 to 11, wherein the PIV is a recombinant PIV1, a recombinant PIV2, or a recombinant PIV3.

[0261] Clause 13. The recombinant PIV of clause 12, wherein the recombinant PIV is a recombinant PIV1, and the TM and CT linked to the RSV F ectodomain are derived from the PIV1 F protein; the recombinant PIV is a recombinant PIV2, and the TM and CT linked to the RSV F ectodomain are derived from the PIV2 F protein; or the recombinant PIV is a recombinant PIV3, and the TM and CT linked to the RSV F ectodomain are derived from the PIV3 F protein.

[0262] Paragraph 14. The recombinant PIV is a recombinant HPIV1, and the PIV F TM and CT linked to the RSV F ectodomain are derived from the HPIV1 F protein; the recombinant PIV is a recombinant HPIV2, and the PIV F TM and CT linked to the RSV F ectodomain are derived from the HPIV1 F protein. The recombinant paramyxovirus of paragraph 12 or 13, wherein the TM and CT are derived from an HPIV2 F protein; the recombinant PIV is a recombinant HPIV3, and the PIV F TM and CT linked to the RSV F ectodomain are derived from an HPIV3 F protein; or the recombinant PIV is a recombinant B / HPIV3, and the PIV F TM and CT linked to the RSV F ectodomain are derived from a BPIV3 F protein.

[0263] Clause 15. The recombinant paramyxovirus of any one of clauses 4 to 14, wherein the RSV F ectodomain is derived from a hRSV F protein, and the TM and CT are derived from a BPIV3 F protein.

[0264] Paragraph 16. The recombinant PIV is a recombinant HPIV1, and the PIV F TM and CT linked to the RSV F ectodomain comprise the amino acid sequence set forth in SEQ ID NO: 31, or an amino acid sequence at least 90% identical to SEQ ID NO: 31; the recombinant PIV is a recombinant HPIV2, and the PIV F TM and CT linked to the RSV F ectodomain comprise the amino acid sequence set forth in SEQ ID NO: 39, or an amino acid sequence at least 90% identical to SEQ ID NO: 39; the recombinant PIV is a recombinant HPIV3, and the PIV F TM and CT linked to the RSV F ectodomain comprise the amino acid sequence set forth in SEQ ID NO: 46. 16. The recombinant paramyxovirus of any one of clauses 4 to 15, wherein the recombinant PIV comprises an amino acid sequence set forth as SEQ ID NO: 53, or an amino acid sequence at least 90% identical to SEQ ID NO: 46; or wherein the recombinant PIV is a recombinant B / HPIV3, and the PIV F TM and CT linked to the RSV F ectodomain comprise an amino acid sequence set forth as SEQ ID NO: 53, or an amino acid sequence at least 90% identical to SEQ ID NO: 53.

[0265] Paragraph 17. The recombinant paramyxovirus of any one of paragraphs 4 to 16, wherein the recombinant PIV is a recombinant HPIV3, and the heterologous gene encodes an hRSV F ectodomain linked to an HPIV3 F TM and CT comprising the amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence at least 90% identical thereto; or the recombinant PIV is a recombinant B / HPIV3, and the heterologous gene encodes an hRSV F ectodomain linked to a BPIV3 F TM and CT comprising the amino acid sequence set forth in SEQ ID NO: 21, or an amino acid sequence at least 90% identical thereto.

[0266] Paragraph 18. The RSV F ectodomain is derived from the hRSV F protein, and the recombinant PIV comprises a viral genome encoding HPIV3 F and HN proteins and BPIV3 N, P, C, V, M, and L proteins, and the TM and CT linked to the RSV F ectodomain are derived from the BPIV3 F protein; the recombinant PIV comprises a viral genome encoding HPIV1 N, P, C, M, F, HN, and L proteins, and the TM and CT linked to the RSV F ectodomain are derived from the HPIV1 F protein; the recombinant PIV comprises a viral genome encoding HPIV2 N, P, V, M, F, HN, and L proteins, and the TM and CT linked to the RSV F ectodomain are derived from the HPIV2 F protein; or the recombinant PIV comprises a viral genome encoding HPIV3 Any one of paragraphs 4 to 17, wherein the recombinant paramyxovirus comprises a viral genome encoding N, P, C, M, F, HN, and L proteins, and the TM and CT linked to the RSV F ectodomain are derived from the HPIV3 F protein.

[0267] Clause 19. The recombinant paramyxovirus of any one of clauses 4 to 18, wherein the recombinant RSV F ectodomain linked to the PIV TM and CT is encoded by a first or second gene downstream of the genomic promoter of the PIV genome.

[0268] Clause 20. The recombinant paramyxovirus of clause 18 or 19, wherein the viral genome comprises, from upstream to downstream, a PIV genome promoter followed by the N, P, C / V, M, F, HN, and L genes; and the gene encoding the recombinant RSV F ectodomain linked to the PIV TM and CT is located between the genome promoter and the gene encoding the N protein or between the gene encoding the N protein and the gene encoding the P protein.

[0269] Paragraph 21. The recombinant paramyxovirus of any one of paragraphs 18 to 19, comprising a viral genome encoding the HPIV3 F and HN genes and the BPIV3 N, P, C, V, M, and L genes, comprising the amino acid sequences set forth as SEQ ID NOs: 21, 101, 47, 48, 49, and 52, respectively, or sequences at least 90% identical thereto.

[0270] Paragraph 22. The recombinant paramyxovirus of any one of the preceding paragraphs, wherein the heterologous gene is codon-optimized for expression in human cells.

[0271] Paragraph 23. A recombinant HPIV3, wherein the heterologous gene encodes the RSV F ectodomain linked to the HPIV3 F TM and CT, and is set forth in SEQ ID NO: 11 (GenScript or a recombinant B / HPIV3, wherein the heterologous gene comprises the nucleotide sequence designated as RSV F_HEK_DS-Cav1_H3TMCT); or a recombinant B / HPIV3, wherein the heterologous gene comprises the nucleotide sequence designated as RSV F_HEK_DS-Cav1_H3TMCT; The recombinant paramyxovirus of claim 22, encoding the RSV F ectodomain linked to the CT and comprising the nucleotide sequence set forth in SEQ ID NO: 22 (GenArt RSV F_HEK_DS-Cav1_B3TMCT) or SEQ ID NO: 23 (GenScript RSV F_HEK_DS-Cav1_B3TMCT).

[0272] Item 24. A recombinant viral vector comprising a viral genome containing a heterologous gene encoding the RSV F ectodomain linked to the TM and CT of the type I membrane protein of the viral genome.

[0273] Clause 25. The viral vector of Clause 24, wherein the RSV F ectodomain comprises K66E and Q101P amino acid substitutions.

[0274] Item 26. A recombinant viral vector comprising a viral genome containing a heterologous gene encoding a RSV F ectodomain containing K66E and Q101P amino acid substitutions.

[0275] Paragraph 27. The viral vector of any one of paragraphs 24 to 26, wherein the RSV F protein is stabilized in a pre-fusion or post-fusion conformation by one or more amino acid substitutions.

[0276] Clause 28. The viral vector of any one of clauses 24 to 27, wherein the RSV F ectodomain is stabilized in a prefusion conformation by S155C, S290C, S190F and V207L ​​amino acid substitutions.

[0277] Paragraph 29. The viral vector of any one of paragraphs 26 to 28, wherein the RSV F ectodomain is soluble and secreted from a host cell containing the viral vector.

[0278] Paragraph 30. The viral vector of any one of paragraphs 24 to 29, which is a recombinant human / bovine parainfluenza virus 3 (B / HPIV3), a recombinant human parainfluenza virus 1 (HPIV1), a recombinant human parainfluenza virus 1 (HPIV2), a recombinant human parainfluenza virus 1 (HPIV3), a recombinant parainfluenza virus 5 (PIV5), a recombinant Sendai virus, or a recombinant Newcastle disease virus (NDV).

[0279] Paragraph 31. The viral vector of any one of paragraphs 24 to 30, wherein the RSV F ectodomain is derived from a human RSV (hRSV) F protein.

[0280] Clause 32. The viral vector of any one of clauses 24 to 31, wherein the heterologous gene encoding the RSV F protein comprises the nucleic acid sequence set forth as nucleotides 1 to 1587 of SEQ ID NO: 18 (the ectodomain encoded by the GenScript-optimized RSV F_A2_HEK_DS-Cav1_B3CT DNA sequence).

[0281] Paragraph 33. The recombinant paramyxovirus or viral vector of any one of the preceding paragraphs, wherein at least 90% of the viral particles produced by a host cell infected with the recombinant paramyxovirus or viral vector comprise a viral envelope that includes an ectodomain encoded by a heterologous gene.

[0282] Paragraph 34. The recombinant paramyxovirus or viral vector of any one of the preceding paragraphs, which is attenuated.

[0283] Paragraph 35. A recombinant paramyxovirus or viral vector of any one of the preceding paragraphs. and a pharmaceutically acceptable carrier.

[0284] Clause 36. The immunogenic composition of Clause 35, further comprising an adjuvant.

[0285] Clause 37. A method of inducing an immune response in a subject against a virus and the heterologous antigen encoded thereby, comprising administering to the subject a therapeutically effective amount of the immunogenic composition of Clause 35 or Clause 36.

[0286] Clause 38. A method of inducing an immune response in a subject to a paramyxovirus and a heterologous antigen encoded thereby, comprising administering to the subject a therapeutically effective amount of the immunogenic composition of Clause 35 or 36, wherein the immunogenic composition comprises a recombinant paramyxovirus comprising a heterologous gene encoding the heterologous antigen.

[0287] Clause 39. A method of inducing an immune response to RSV and PIV in a subject, comprising administering to the subject an immunogenic composition comprising a therapeutically effective amount of the immunogenic composition of Clause 35 or Clause 36, wherein the immunogenic composition comprises a recombinant paramyxovirus comprising a heterologous gene encoding a RSV antigen.

[0288] Clause 40. The method of any one of clauses 37-39, wherein the immune response is a protective immune response.

[0289] Clause 41. The method of any one of clauses 37 to 40, comprising prime-boost administration of the immunogenic composition.

[0290] Clause 42. The method of any one of clauses 37 to 41, comprising intranasal or parenteral administration of the immunogenic composition.

[0291] Paragraph 43. The method of any one of paragraphs 37-42, wherein the subject is a human or veterinary subject.

[0292] Clause 44. The method of any one of clauses 37-43, wherein the subject is at risk for or has a RSV or PIV infection.

[0293] Item 45. The method of any one of items 37 to 44, wherein the subject is under 1 year old.

[0294] Paragraph 46. A nucleic acid molecule comprising the genome of the recombinant paramyxovirus of any one of paragraphs 1 to 25.

[0295] Paragraph 47. A recombinant RSV F protein or immunogenic fragment thereof comprising K66E and Q101P amino acid substitutions.

[0296] Clause 48. The recombinant RSV F protein or immunogenic fragment thereof of clause 47, further comprising: (a) S155C and S290C; (b) S190F; (c) V207L; or (f) (a) and (b); (a) and (c); (b) and (c); Or (a), (b) and (c).

[0297] Clause 49. An immunogenic fragment of the recombinant RSV F protein of clause 47 or clause 48, comprising the RSV F ectodomain.

[0298] Paragraph 50. A nucleic acid molecule encoding the recombinant RSV F protein of any one of paragraphs 47 to 49. [Example]

[0299] The following examples are presented to illustrate certain features of particular embodiments, but the claims should not be limited to the exemplified features. [Example]

[0300] Improved expression and immunogenicity of respiratory syncytial virus (RSV) fusion (F) glycoprotein expressed by an attenuated parainfluenza virus vector This example describes an approach to improve the immunogenicity and stability of expressed RSV F by recombinant B / HPIV3 using RSV F sequences from early passage viruses, by codon optimization, by stable and highly immunogenic pre- and post-fusion forms of RSV F, and by genetically engineering the RSV F proteins TM and CT, thereby allowing it to be more efficiently incorporated into vector particles.

[0301] Introduction. Administering live attenuated RSV strains represents one strategy for RSV vaccines, and these are currently under development (Hurwitz. 2011. Expert. Rev. Vaccines. 10:1415-1433; Collins and Melero. 2011. Virus Res. 162:80-99; Karron et al. 2013. Current Topics Microbiology and Immunology 372:259-284). Live attenuated RSV strains are typically administered via the intranasal (IN) route. However, attenuation generally results in reduced antigen synthesis, which in turn leads to reduced immunogenicity. Achieving the appropriate balance between attenuation and immunogenicity has been challenging for RSV.

[0302] Complete, infectious HPIVs are generated entirely from cloned cDNA in transfected cell cultures (using reverse genetics). A foreign gene designed for expression is modified so that it is flanked by HPIV transcription signals (called gene start and gene end signals, located at the beginning and end of each gene, respectively) and inserted as an additional gene into the HPIV genome by reverse genetics. The foreign gene is then transcribed into a separate mRNA, just like other HPIV genes. HPIVs can accommodate and express several additional foreign genes (Skiadopoulos et al. 2002. Virology 297:136-152). However, multiple genes can be overly attenuated and may collect point mutations (Skiadopoulos et al. 2002. Virology 297:136-152).

[0303] HPIV transcription initiates at a single promoter at the 3' end of the genome and proceeds processively. A fraction of the polymerase leaves the template at each gene junction, creating a negative gradient of gene transcription. Thus, promoter-proximal genes are expressed more frequently than downstream genes. Positioning a foreign gene close to the promoter increases expression but has the potential to affect the expression of downstream vector genes. Other features, such as differences in the efficiency of gene start or gene stop transcription signals or the effects of other structural features in the RNA template that are sometimes present but poorly understood, can also unexpectedly affect the expression of an inserted gene or open reading frame (ORF) (Whelan et al. 2004. Current Topics Microbiology and Immunology 283:61-119). Furthermore, in some cases, the properties of viral constructs are significantly influenced by factors that have yet to be identified; for example, insertion of the RSV F gene into the PM gene junction of a PIV3 vector resulted in a substantially temperature-sensitive and attenuated virus (Liang B et al. 2014. J Virol 88:4237-4250). Thus, although the broad details of expression from the HPIV genome are generally known, specific constructs may produce unpredictable results.

[0304] Previous studies have used the B / HPIV3 vector to express either the RSV G gene and F protein from additional genes in the first and second genomic locations after the promoter, or the RSV F gene from an additional gene in the second genomic location between the N and P genes. The latter virus (called MEDI-534) was evaluated in clinical studies in seronegative children and was found to be attenuated, well-tolerated, and infectious, but less immunogenic to RSV than expected (Bernstein et al. 2012. Pediatric Infectious Disease Journal). 31:109-114). Analysis of vaccine viruses shed from vaccine recipients showed that approximately 50% of specimens contained vaccine viruses with mutations predicted to perturb RSV F expression, likely reducing immunogenicity. Retrospective analysis of clinical trial material (CTM) showed that 2.5% of these viruses did not express RSV F (Yang et al. 2013. Vaccine 31:2822-2827). Furthermore, the observation that RSV F inserts accumulated mutations that inactivated their expression at the protein level and that these mutations were amplified during propagation suggests a selective advantage of silencing RSV F protein expression. This is likely due to the highly fusogenic nature of the RSV F protein, which efficiently mediates syncytium formation. In vitro, this leads to disruption of the cytosol, thereby reducing vector replication. Furthermore, high levels of exogenous glycoprotein synthesis can interfere with the synthesis, processing, and transport of vector glycoproteins through the endoplasmic reticulum and exocytic pathways, and can sterically interfere with virion morphogenesis, among other things. These effects can occur both in vitro and in vivo.

[0305] Expression of early passage (HEK) versions of the RSV F protein and codon-optimized versions of the RSV F open reading frame (ORF). Increased expression of viral antigens typically results in improved immunogenicity. Codon optimization of the vector's antigen-encoding ORF increases its expression, which in turn improves its immunogenicity, as shown, for example, in human immunodeficiency virus antigens expressed from viral or DNA vectors (Gao et al. 2003, AIDS research and human retroviruses 19:817-823; Carnero et al. 2009, J Virol 83:584-597). However, these sequence changes may have effects beyond improving translation, such as effects on mRNA stability and transport, and therefore, the effects of altering the nucleotide sequence of mRNA can be complex and unpredictable. Therefore, a codon-optimized version of the RSV F sequence was designed using the GeneArt (GA) algorithm and evaluated to determine whether it conferred protein expression.

[0306] When designing this codon-optimized ORF, the amino acid sequence of an early passage version of RSV strain A2 from the 1960s was mistakenly used (Connors et al. 1995. Virology 208:478-484; Whitehead et al. 1998. J Virol 72:4467-4471). This early-passage (or low-passage) strain, derived from the 1960s, is called HEK after the human embryonic kidney (HEK) cell culture used for its propagation. This HEK virus differed from the current highly passaged laboratory version of RSV strain A2 by two amino acid assignments (Connors et al. 1995. Virology 208:478-484; Whitehead et al. 1998. J Virol 72:4467-4471). The HEK version had the assignments 66E and 101P, whereas the highly passaged laboratory A2 strain had the assignments 66K and 101Q (hereafter referred to as the "non-HEK" assignments) (Figure 1). However, the appearance of sequence differences between virus strains, or even between stocks of a given strain, is a significant risk for RNA viruses, given their mutation rate. While this is common in RSV vaccine candidates, the HEK difference was not previously known to be significant. Furthermore, the presence of the HEK assignment in an attenuated RSV vaccine candidate, designated RSV NIHΔM2-2, was associated with a slight decrease in replication efficiency in cell culture. Furthermore, the HEK assignment at position 66 was identified to affect syncytium formation during RSV infection. Therefore, given their association with reduced replication, it was intended to avoid the HEK assignment. However, because a version of RSV F containing the HEK assignment was accidentally used in the initial codon optimization, a parallel GA-optimized non-HEK version was constructed to compare the two versions (Figure 1). The two versions of RSV F were inserted into the second position of the rB / HPIV3 vector, under the control of the BPIV3 gene start and gene end transcription signals (Figure 1). The transcription signals and insertion positions were used in all subsequent rB / HPIV3 constructs expressing RSV F, thereby allowing for direct comparison throughout.

[0307] Vero cells were infected with two different vectors (referred to as "HEK / GA-opt" and "non-HEK / GA-opt"). Cell lysates were prepared 48 hours postinfection, and proteins were subjected to gel electrophoresis in the presence of a denaturing detergent and under reducing or non-reducing conditions. Separate proteins were transferred to membranes by Western blotting and analyzed using an antibody specific for RSV F (Figure 2). This showed that the presence of HEK assignment was associated with a small (approximately 2-fold) but consistent increase in RSV F protein expression (Figure 2). One non-limiting explanation for this finding is that HEK assignment may have increased F protein stability, which may have an effect on protein synthesis, although this would be unlikely if the HEK and non-HEK versions of the F ORF were identical except for two codons. Furthermore, when analyzed under non-reducing conditions, the presence of HEK assignment was associated with a decrease in the gel mobility of the RSV F trimer (Figure 2). This suggests that these assignments altered the F protein trimer structure. More strikingly, expression of the HEK version of RSV F was associated with a dramatic reduction in syncytium formation compared to the non-HEK version, as previously noted, even when the HEK version was expressed at slightly increased levels (Fig. 3). While this assay takes advantage of the general lack of obvious syncytia induced in cells infected with the rB / HPIV3 empty vector, expression of the vector-derived RSV F protein resulted in syncytium formation that was generally proportional to the amount of RSV F protein expressed. This provides an assay for the quantity and functionality of the RSV F protein expressed from the PIV vector. These HEK-related observations indicate that the HEK assignment is associated with differences in RSV F synthesis / stability, structure, and fusion activity, and that these effects occurred in the absence of any other RSV proteins and were therefore directly related to expression from a heterologous vector.

[0308] Because HEK assignments originate from low-passage stocks of RSV strain A2 from the 1960s, they are likely representative of the original clinical isolate, whereas non-HEK assignments emerged during extensive in vitro passage over subsequent decades. This suggests that the low-fusogenic phenotype of the HEK version of F is more representative of the original biological virus. Non-HEK versions may represent hyperfusogenic variants selected during passage in cell culture. Hyperfusogenic versions of RSV F may be undesirable in practice because they may destabilize the virus, but may be selected in laboratory settings with rapid growth in cell monolayers. Examination of 226 sequences of RSV F from clinical isolates in the GenBank database revealed that clinical isolates typically contain HEK assignments, consistent with these assignments being representative of circulating RSV. In either case, the HEK assignment resulted in a modest increase in F protein expression, resulting in a form of RSV F that was low-fusogenic. The reduction in syncytium formation suggests that this may be due to cytopathogenicity (which would otherwise prevent HPIV vector replication) and the silencing of the RSV F insert. This is advantageous because it reduces the risk of infection (which can interfere with the preferred selection of infected vectors). HEK assignment therefore has the triple advantage of representing a more native and clinically relevant form of the F protein, resulting in a moderate increase in protein expression and reducing the selection pressure for silencing the RSV F insert.

[0309] The effect of codon optimization on RSV F expression and immunogenicity was also evaluated. The HEK-containing and GA-optimized version (HEK / GA-opt) was used along with two other codon-optimized RSV HEK F sequences generated by two different algorithms. Evaluation of multiple optimized versions is not typical practice, as it increases cost and inconvenience and has not been shown to be useful. Two other sources were the DNA2.0 (D2) and GenScript (GS) algorithms; also included for comparison was a non-HEK, non-codon-optimized version (Figure 4). Codon optimization resulted in significantly improved RSV F protein synthesis, which surprisingly varied in magnitude for the different versions. The highest expression was observed with the HEK-containing GenScript-optimized F protein (HEK / GS-opt), which was significantly higher than unmodified RSV. The expression levels of the more efficient ORFs were 10-fold (Vero cells) and 16-fold (LLC-MK2 cells) higher than those of F (non-HEK / non-opt) (Figure 5). The level of expression with the more efficient ORF was so high that a gradual increase in the level of syncytium formation was evident in association with the increased level of F expression despite the presence of HEK assignment, but syncytium formation was presumably even earlier and more extensive in the absence of HEK assignment.

[0310] Codon pair optimization was also evaluated as a means of increasing F protein expression using a previously described algorithm (Coleman et al. 2008. Science 320:1784-1787). Codon pair optimization increases the frequency of codon pairs associated with high expression. However, this did not result in any increased expression in the case of RSV F.

[0311] Contrary to expectations, the 10- to 16-fold increase in RSV F expression and the concomitant increase in syncytium formation did not have a significant negative impact on vector replication in cell culture (Figure 7). High levels of RSV F expression and syncytium formation, as previously noted, would be expected to interfere with vector replication at any number of steps, including vector glycoprotein synthesis, processing, exocytosis, vector particle formation, and cell survival, but this was not the case. This was particularly surprising because, as previously noted, the accumulation and amplification of mutations that silenced RSV F gene expression in MEDI-534 suggested substantial selective pressure against RSV F protein expression. Compared to the empty vector, all vectors carrying the RSV F insert were moderately attenuated (Figure 7)—presumably including general attenuation effects such as increased genome length and gene number—but replicated with similar kinetics relative to each other and grew to high peak titers slightly lower than those of the empty vector (Figure 7). Small differences in peak titers likely represent experimental variation.

[0312] For in vivo replication, the immunogenicity and protective efficacy of rB / HPIV3 vectors were evaluated in a hamster model. Groups of hamsters were divided into 10 5 Tissue culture infection dose (TCID) 50 units 50 ) and immunized intranasally with the rB / HPIV3 vector at a dose of 10 6 Wild-type (wt) RSV given at a dose of plaque-forming units (pfu) was included as a positive control for the induction of RSV-specific immunity. Although RSV was included with the caveat that it is a non-attenuated virus, this vector was attenuated and may have relatively low immunogenicity for that reason. Six animals per virus per day were euthanized on days 3 and 5 post-infection, and nasal turbinates and lungs were collected for virus titration to measure replication in vivo. This allowed for the development of RSV F. The vectors carrying the inserts were shown to be moderately attenuated in the nasal turbinates (upper respiratory tract) and substantially attenuated in the lungs (lower respiratory tract) compared to the empty vector (Figure 8). The increased attenuation compared to the empty vector was evidenced by lower viral shedding. This was also evidenced by a comparison of titers on days 3 and 5: for the empty vector, titers on days 3 and 5 were comparable, while for the vectors carrying RSV F, the titer on day 3 was lower than the titer on day 5, indicating that these constructs took longer to achieve their maximum titer. Surprisingly, among vectors with RSV F inserts, those with increased RSV F expression were less attenuated than those with lower RSV F expression, i.e., non-HEK / non-opt. Thus, the addition of the RSV F insert to the rB / HPIV3 vector was attenuated in vivo (presumably due to some common feature, such as increased genome length or gene number), although this did not appear to be more substantially affected than the level of RSV F protein synthesis.

[0313] The immunogenicity of the vectors was assessed by measuring serum titers of RSV-neutralizing antibodies using a standard assay, a 60% plaque reduction assay supplemented with guinea pig complement. All vectors expressing RSV F, regardless of HEK assignment or codon optimization, induced similarly high titers of RSV-neutralizing serum antibodies (Figure 9). There was a slight progressive increase in neutralizing titers associated with increasing RSV F expression, but the difference was not statistically significant. wT RSV infected in parallel with the control induced significantly higher titers of RSV-neutralizing antibodies than the vector. However, it is important to note that while the neutralizing antibodies induced by RSV infection contained contributions from both F and G neutralizing antigens, this vector only had F-specific antibodies contributing to its neutralizing titer. Furthermore, the nonattenuated wt RSV control replicated more efficiently than the attenuated vector, particularly in the lungs (Figure 8), which increased its immunogenicity compared to that of the vector.

[0314] To assess the protective efficacy of these vectors, immunized hamsters in groups of six animals from the experiment in Figure 9 were given 10 6 Animals were challenged 30 days after immunization with pfu of wt RSV intranasally. Nasal turbinates and lungs were collected from euthanized animals 3 days postchallenge, and tissue homogenates were prepared and evaluated by plaque assay to measure the level of challenge RSV replication. RSV F-expressing vectors conferred near-complete protection in the lungs and intermediate levels of protection in the nasal turbinates, whereas wt RSV conferred near-complete protection at both anatomical sites (Figure 10). There was no significant difference in protective efficacy against RSV challenge between the RSV F-expressing vectors. It should be noted that protection conferred by RSV includes contributions from neutralizing antibodies against both the F and G proteins, as well as cellular immunity potentially directed against all RSV proteins, whereas protection conferred by the vector includes humoral and cellular immunity directed only against the F protein. Additionally, as noted, the RSV control was an unattenuated wt virus, which replicated to higher titers than the vector during immunization, enhancing its immunogenicity and efficacy, particularly in the lungs (Figure 8).

[0315] These results suggest that RSV resulting from the use of HEK assignments and codon-optimized sequences It has been shown that a 10- to 16-fold increase in F protein expression did not result in a significant increase in the induction of RSV-neutralizing serum antibodies (although a trend toward an increase was observed) or in protection against wt RSV challenge. In contrast, similar levels of increase in human immunodeficiency virus antigen expression resulted in improved protection with other viral vectors and DNA vaccines in different animal models (Gao et al. 2003, AIDS research and human retrovirus 19:817-823; Carnero et al. 2009, J Virol 83:584-597). Previously, the expression of RSV F due to insertions at positions 1 or 2 versus 6 in rB / HPIV3 vectors was not shown to be significant. It has also been observed that a 30- to 69-fold difference in expression induced a significant difference in protective efficacy in hamsters (Liang B et al. 2014. J Virol 88:4237-4250). Therefore, increased antigen synthesis is generally considered to confer increased immunogenicity. However, in some cases, this effect may not be large enough to be detected without question, or a given in vivo model may not be sufficiently sensitive. Therefore, the 10- to 16-fold difference in this study may not be sufficient to induce an effect large enough to be statistically significant in the semi-permissive hamster model. The beneficial effect of higher RSV F expression may be even more pronounced in combination with other characteristics or in permissive hosts, i.e., primates and humans, with larger sample sizes in preclinical and clinical evaluations. Specifically, the 10- to 16-fold increase in F protein expression observed in this study was in Vero (African green monkey) or LLC-MK2 (rhesus macaque) cells, where codon-optimization for human use is likely to be effective given the relatively close phylogenetic relatedness of these primates to humans. In contrast, in vivo immunogenicity assays used hamsters, where codon-optimization for human use may not be effective in increasing expression, and therefore immunogenicity.

[0316] Evaluation of the immunogenicity of pre-fusion and post-fusion forms of RSV F expressed by rB / HPIV3 vectors. Like all paramyxovirus F proteins, the RSV F protein initially assembles into a pre-fusion conformation, which is the version that first accumulates on the surface of infected cells and is incorporated into virions. For example, pre-fusion F undergoes extensive conformational changes that mediate membrane fusion, triggered, for example, by contact with adjacent target cell membranes, resulting in the F protein occupying a post-fusion conformation (Calder et al. 2000. Virology 271:122-131; McLellan et al. 2013. Science 340:1113-1117; McLellan et al. 2011. J Virol 85:7788-7796; Swanson et al. 2011. Proc. Nat'l Acad. Sci. USA 108:9619-9624). The RSV F protein is notable among paramyxoviruses for being highly susceptible to induction and is readily induced before maturation, which may contribute to the significant instability of RSV infectivity. It has also been shown that many of the RSV F proteins that accumulate in infected cells are conformationally heterogeneous, which can function as decoys to reduce the induction of virus-neutralizing antibodies (Sakurai et al. 1999. J Virol 73:2956-2962). Therefore, expressing RSV F in a stabilized conformation is advantageous for more than one reason.

[0317] Recently, stable postfusion forms of RSV F have been described (McLellan et al. 2011, J Virol 85:7788-7796; Swanson et al. 2011, Proc. Nat'l Acad. Sci. USA 108:9619-9624). These stable postfusion forms were recombinantly generated by truncating the hydrophobic fusion peptide and removing the C-terminal transmembrane domain (TM) and cytoplasmic tail (CT) (Ruiz-Arguello et al. 2004, J General Virology 85:3677-3687). Due to the lack of the TM and CT, these postfusion forms are not membrane-anchored and will be secreted. These postfusion forms of RSV F have been shown to be immunogenic and protective in mice (Swanson et al. 2011, Proc. Nat'l Acad. Sci. USA 108:9619-9624).

[0318] However, the prefusion form of RSV F appears to be significantly more immunogenic than the postfusion form (McLellan et al. 2013. Science 340:1113-1117), consistent with the observation that the majority of neutralizing activity in sera from convalescent animals and humans was driven by antibodies that did not bind to the postfusion F protein and were likely specific for the prefusion form. (McLellan et al. 2013, Science 340:1113-1117; Magro et al. 2012, Proc Nat'l Acad. Sci. USA 109:3089-3094). Recently, the structure of the pre-fusion form of RSV F has been determined, making it possible to stabilize this pre-fusion conformation through structure-based mutations: one of these involves the introduction of a disulfide bond (DS), and another involves amino acid substitutions in the predicted cavity of the trimeric structure (Cav1), a combination of which is called DS-Cav1 (McLellan et al. 2013, Science 342:592-598). Recombinant DS and DS-Cav1 RSV F proteins were evaluated as subunit vaccines in mice and monkeys and were shown to induce significantly higher levels of RSV-neutralizing serum antibodies than postfusion versions (DS-Cav1 is more immunogenic than DS) (McLellan et al. 2013. Science 342:592-598).

[0319] The immunogenicity of post-fusion and pre-fusion forms of RSV F was evaluated when expressed from a live attenuated rB / HPIV3 vector. Post-fusion and stabilized pre-fusion forms (DS and DS-Cav1) of RSV F (with HEK assignment) were GA codon-optimized and inserted into the second genomic location of the rB / HPIV3 vector (FIG. 11). These were compared with HEK / GA-opt and a HEK-containing version, the GA-optimized F protein (from which the CT and TM were deleted, leaving the ectodomain (Ecto)) (FIG. 11). These constructs were also compared with non-HEK / non-opt constructs.

[0320] The GA-optimized F ORF was used in the data shown in Figure 11 and subsequent experiments. Parallel constructs with GS-optimized compound ORFs were constructed in some cases (see Figure 35) but are yet to be evaluated. Given the superior expression of the GS-optimized ORFs (Figure 35), the GS-optimized versions may be more immunogenic and protective. Also, the identifiers "HEK" and "GA-opt" are sometimes omitted from the construct names in Figure 11 and subsequent figures, as well as in the subsequent text, for brevity, but the presence of these features is indicated in the figures (i.e., "All above versions of RSV F are HEK, GA-optimized," Figure 11).

[0321] Vectors carrying these various forms of RSV F were rescued and propagated in vitro to similar high titers (Figure 12). They were generally slightly attenuated in terms of growth kinetics, and final yields compared to the empty rB / HPIV3 vector were as previously noted for other vector constructs (see Figure 7).

[0322] The expression efficiency of various forms of RSV F protein was evaluated in Vero and LLC-MK2 cells infected with various constructs (Figures 13A and B). Infected cell cultures were harvested 48 hours post-infection and analyzed by Western blotting. Native RSV F (i.e., HEK / GA-opt) was cell-associated as expected. Post-fusion and Ecto forms were found to be secreted and also cell-associated. Secretion of post-fusion F was consistently more efficient than Ecto; Ecto contains a high content of hydrophobic sequences, so more may remain cell-associated. Unexpectedly, F of DS and DS-Cav1 types was expressed more efficiently (Figure 13B). Because these viruses exhibited similar kinetics and the ORFs were similarly GA-optimized, this increased expression likely reflected increased protein stability of DS and DS-Cav1 types. Genetic manipulation of proteins can substantially affect glycoprotein expression, processing, and stability, often in a negative manner, so the efficient expression of DS and DS-Cav1 by this live vector was an essential property that could not be easily predicted.

[0323] The in vivo replication of these vectors was evaluated in hamsters (Figure 14). In the nasal turbinates, all vectors with RSV F inserts were moderately attenuated compared to the empty vector (Figure 14A). The increased attenuation compared to the empty vector was evidenced by lower values ​​for virus shedding. This was also evidenced by a comparison of titers on days 3 and 5: for the empty vector, these values ​​were comparable, but for vectors carrying RSV F, the titer on day 5 was higher than the titer on day 3, indicating that these constructs took longer to reach their maximum titer. Vectors with post-fusion F replicated to higher titers than vectors with other forms of F, while vectors with pre-fusion F (DS) replicated to lower titers, which may represent experimental variation or a unequal effect on vector replication. In the lungs, all vectors with RSV F inserts were substantially more attenuated than the empty vector (Figure 14B). Consistent with the nasal turbinates, vectors with post-fusion F also replicated somewhat more efficiently in the lungs than the other vectors, whereas vectors expressing the pre-fusion (DS) version appeared somewhat more attenuated. The RSV control, a completely wild-type virus, replicated more efficiently than the attenuated rB / HPIV3 vector expressing RSV F; for example, wild-type RSV replicated to titers 100- and 1000-fold higher in the nasal turbinates and lungs than vectors expressing the pre-fusion (DS) F, respectively. RSV-neutralizing serum antibody titers were determined by a 60% plaque reduction assay. This was performed in two ways: (i) in the presence of added complement (a useful technique, as previously shown in Figure 9), and (ii) without added complement (Figures 15A and 15B, respectively). The presence of added complement provides the most sensitive detection of virus-specific antibodies. This is because complement potentially confers viral lytic potential to all antibodies that bind to virions and may also exert steric effects (Yoder et al. 2004 J Med Virol 72:688-694).In contrast, complement-dependent neutralization assays detect only high-quality neutralizing antibodies that can neutralize RSV without involving the virolytic function or steric effects of complement proteins. It has been suggested that "neutralization assays performed without complement may best reflect the physiological conditions of the respiratory tract" (Yoder et al. 2004 J Med Virol 72:688-694). In complement-containing assays (Figure 15A), vectors with post-fusion F were the least immunogenic among the vectors, even when they replicated to the highest titers in hamsters; on the other hand, vectors with pre-fusion (DS) F, even when they were most attenuated, were the most immunogenic among the vectors tested. In complement-dependent assays (Figure 15B), only vectors expressing pre-fusion (DS) F induced high titers of neutralizing antibodies. None of the other vectors with unmodified post-fusion or EctoF were effective in inducing high-quality neutralizing antibodies. The RSV control was effective in inducing neutralizing antibodies in both complement-containing and complement-dependent assays. Remarkably, vectors bearing the pre-fusion (DS) F were statistically similar to wt RSV in inducing high-quality RSV neutralizing antibodies (Figure 15B). This is noteworthy because this attenuated vector replicated 100-1000-fold less efficiently than nonattenuated wt RSV, and the neutralizing activity conferred by wt RSV was supplemented by a contribution from the RSV G protein. This suggests that vectors expressing the pre-fusion (DS) form of RSV F were highly potent in inducing highly effective neutralizing antibodies and were highly immunogenic.

[0324] To assess the protective efficacy of these vectors, immunized hamsters from the experiment in Figure 15 were administered at 10 6Thirty days after immunization, animals were challenged by intranasal infection with pfu of wt RSV. The animals were sacrificed 3 days after infection, and the nasal turbinates and lungs were harvested and processed into tissue homogenates that were assayed by plaque titration (Figure 16). In the nasal turbinates (Figure 16A), constructs expressing non-HEK / non-opt F, or HEK / GA-opt, or Ecto F conferred modest levels of protection, whereas post-fusion F and especially pre-fusion (DS) F were somewhat more protective. In the lungs (Figure 16B), all The vector constructs provided substantial protection against RSV challenge, except for the postfusion form, which provided the least protection (Fig. 16B). The wt RSV control provided near-complete protection in the nasal turbinates and complete protection in the lungs; however, as noted previously, wt RSV had the advantage of expressing both the F and G neutralization antigens, in addition to expressing all RSV proteins as potential antigens for cellular immunity and replicating up to 1000-fold more efficiently (Fig. 14).

[0325] The addition of Cav-1 mutations to the DS construct resulted in increased immunogenicity as a subunit vaccine (McLellan et al. 2013. Science 342:592-598), which is predicted to further enhance the immunogenicity of pre-fusion RSV F expressed from viral vectors. The DS and DS-Cav1 forms o...

Claims

1. 1. A recombinant paramyxovirus comprising: a viral genome comprising genes encoding the N, P, M, F, HN, and L proteins of human parainfluenza virus 1 (HPIV1), and further comprising a heterologous gene encoding a recombinant respiratory syncytial virus (RSV) F ectodomain linked to the transmembrane domain (TM) and cytoplasmic tail (CT) of the HPIV1 F protein; the recombinant paramyxovirus is a recombinant human parainfluenza virus 1 (HPIV1); The RSV F ectodomain is stabilized in the RSV F prefusion conformation by substitutions S155C, S290C, S190F, and V207L ​​compared to the native RSV F protein sequence; and The recombinant paramyxovirus is an infectious, attenuated, self-replicating virus. The recombinant paramyxovirus.

2. 2. The recombinant paramyxovirus of claim 1, wherein the RSV F ectodomain is derived from human subtype A RSV or human subtype B RSV.

3. The RSV F ectodomain contains glutamic acid and guanine at positions 66 and 101, respectively.

3. The recombinant paramyxovirus of claim 1 or 2, which comprises a nucleotide sequence similar to that of claim 1, and a proline sequence similar to that of claim 2 (66E and 101P).

4. The RSV F ectodomain linked to the TM and CT comprises an amino acid sequence at least 90% identical to SEQ ID NO: 135; The recombinant paramyxovirus of claim 1.

5. The RSV F ectodomain linked to the TM and CT comprises the amino acid sequence set forth as SEQ ID NO:

135. The recombinant paramyxovirus of claim 4.

6. The recombinant paramyxovirus according to any one of claims 1 to 5, wherein the heterologous gene encoding the recombinant RSV F ectodomain is a first or second gene downstream of a genomic promoter in the viral genome.

7. The viral genome comprises, from upstream to downstream, an HPIV1 genome promoter followed by genes encoding HPIV1 N, P, M, F, HN, and L proteins, and a heterologous gene encoding a recombinant RSV F ectodomain is located between the gene encoding the N protein and the gene encoding the P protein. The recombinant paramyxovirus of claim 6.

8. The heterologous gene encoding the recombinant RSV F ectodomain is the third gene downstream of the genomic promoter of the viral genome; The recombinant paramyxovirus according to any one of claims 1 to 7.

9. The viral genome comprises, from upstream to downstream, an HPIV1 genome promoter followed by genes encoding HPIV1 N, P, M, F, HN, and L proteins, and a heterologous gene encoding a recombinant RSV F ectodomain is located between the gene encoding the P protein and the gene encoding the M protein. The recombinant paramyxovirus of claim 8.

10. HPIV1 contains a CΔ170 or LY942A attenuating mutation, The recombinant paramyxovirus according to any one of claims 1 to 9.

11. 11. The recombinant paramyxovirus of any one of claims 1 to 10, wherein the recombinant RSV F ectodomain comprises an amino acid sequence that is at least 90% identical to residues 1 to 529 of SEQ ID NO:

21.

12. The recombinant RSV F ectodomain comprises the amino acid sequence of residues 1 to 529 of SEQ ID NO:

21. The recombinant paramyxovirus of claim 11.

13. The heterologous gene comprises the nucleotide sequence set forth in SEQ ID NO:

136. The recombinant paramyxovirus according to any one of claims 1 to 12.

14. The recombinant paramyxovirus according to any one of claims 1 to 13, wherein at least 90% of the viral particles produced by a host cell infected with the recombinant paramyxovirus or viral vector contain a viral envelope comprising an ectodomain encoded by a heterologous gene.

15. An immunogenic composition comprising the recombinant paramyxovirus according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

16. A medicament for inducing an immune response to an RSV F protein in a subject, the medicament comprising a therapeutically effective amount of the immunogenic composition of claim 15.

17. 16. A medicament for inducing an immune response against respiratory syncytial virus and parainfluenza virus in a subject, the medicament comprising a therapeutically effective amount of the immunogenic composition of claim 15.

18. The method of claim 16 or 17, wherein the immune response is a protective immune response.

19. The medicament according to any one of claims 16 to 18, which is administered as a prime-boost administration of an immunogenic composition.

20. The pharmaceutical composition according to any one of claims 16 to 19, which is administered intranasally.

21. The method of any one of claims 16 to 20, wherein the subject is a human or veterinary subject.

22. The method of any one of claims 16 to 21, wherein the subject is at risk of or has an RSV or PIV infection.

23. The pharmaceutical composition according to any one of claims 16 to 22, wherein the subject is under 1 year old.

24. The method of any one of claims 16 to 23, wherein the subject is immunocompromised or elderly.

25. A nucleic acid molecule comprising the genome of the recombinant paramyxovirus according to any one of claims 1 to 14.

26. A medicament comprising the recombinant paramyxovirus of any one of claims 1 to 14 for inducing an immune response against RSV or RSV and PIV in a subject.

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