Recombinant human / bovine parainfluenza virus 3 (B / HPIV3) expressing chimeric RSV / BPIV3 F protein and its use

Abstract

We provide recombinant paramyxoviruses. [Solution] Recombinant paramyxoviruses containing a viral genome encoding heterologous genes are provided. In some embodiments, the recombinant paramyxovirus is a recombinant parainfluenza virus such as recombinant PIV3, containing a viral genome encoding a heterologous respiratory syncytial virus F ectodomain linked to the transmembrane domain and cytoplasmic tail of the F protein derived from PIV3. Nucleic acid molecules containing the genome of recombinant paramyxoviruses are also provided. Recombinant viruses can be advantageously used in vaccine formulations, such as for vaccines against parainfluenza viruses and respiratory syncytial viruses.

Description

[Technical Field]

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

[0002] This disclosure relates to recombinant paramyxoviruses comprising a viral genome containing heterologous genes encoding antigens of a heterologous virus. For example, a recombinant paramyxovirus may be a recombinant parainfluenza virus (PIV) comprising a genome containing heterologous genes encoding a respiratory syncytial virus (RSV) fusion (F) protein. [Background technology]

[0003] Paramyxoviruses are a family of negative-sense single-stranded RNA viruses that are the leading cause of numerous animal and human deaths worldwide every year. Paramyxoviruses include the subfamilies Paramyxovirinae and Pneumovirinae. Respiratory syncytial virus (RSV) is an enveloped, undivided negative-sense RNA virus in the genus Paramyxoviridae, Pneumovirinae. It is the most common cause of bronchiolitis and pneumonia in infants in the first year of life. RSV also causes recurrent infections, including severe lower respiratory tract illness, which can occur at any age, especially in the elderly or those with immunocompromised heart, lung, or immune systems. Passive immunization is currently used to prevent severe illness 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 virus (PIV), like RSV, is another enveloped, undivided, negative-strand RNA virus within the Paramyxoviridae family. However, PIV belongs to the Paramyxovirinae subfamily. PIV includes members of the respirovirus genus (including PIV1, PIV3, and Sendai virus) and members of the rubravirus genus (including PIV2, PIV4, and PIV5). Furthermore, members of the abrasive virus genus (including Newcastle disease virus NDV) have historically been called PIV and may be considered operationally identical. Human parainfluenza virus (HPIV, serotypes 1, 2, and 3) is the second most common cause of severe respiratory infections in infants and children worldwide, after RSV alone, with HPIV3 being the most significant of the HPIVs in terms of disease impact. The HPIV genome is approximately 15.5 kb and contains the gene sequence 3'-NPMF-HN-L. The main proteins are: N, nucleoprotein; P, phosphorylated protein; M, matrix protein; F, fusion glycoprotein; HN, hemagglutinin-neuraminidase glycoprotein; and L, each gene encoding a separate mRNA that encodes a large polymerase protein. The P gene contains one or more additional open reading frames (ORFs) that encode accessory proteins. Similar to RSV, the development of an effective HPIV vaccine remains elusive. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] Recombinant paramyxoviruses are provided, which include a viral genome encoding heterologous genes. In some embodiments, the recombinant paramyxovirus includes heterologous genes encoding a type I membrane protein, which includes the cytoplasmic tail (CT) or transmembrane domain (TM) of the paramyxovirus F protein and a recombinant RSV F ectodomain linked to the CT. It may be a recombinant parainfluenza virus containing a viral genome. Paramyxoviruses may be, for example, recombinant human / bovine parainfluenza virus 3 (B / HPIV3), recombinant human parainfluenza virus 1 (HPIV1), recombinant human parainfluenza virus 2 (HPIV2), recombinant human parainfluenza virus 3 (HPIV3), or 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 provided a wide variety of increased integration of the RSV F ectodomain into the recombinant paramyxovirus envelope, significantly increasing the induction of an immune response to 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. Therefore, in some embodiments, the disclosed recombinant paramyxovirus can be included in an immunogenic composition to induce a bivalent immune response to paramyxovirus and heterologous RSV F protein.

[0007] The RSV F ectodomain encoded by a heterologous gene may be derived from the human RSV F protein. In some embodiments, the RSV F ectodomain may include 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 may include another amino acid substitution to increase ectodomain expression or integration in the viral envelope (such as "HEK" substitutions, K66E, and Q101P).

[0008] In one, not limited, embodiment, the recombinant paramyxovirus may be recombinant B / HPIV3, and the RSV F ectodomain is ligated to the TM and CT derived from the BPIV3 F protein. In some such embodiments, the RSV F ectodomain ligated to the TM and CT derived from the BPIV3 F protein comprises the amino acid sequence shown as SEQ ID NO: 21 or an amino acid sequence that is at least 90% identical to SEQ ID NO: 21.

[0009] In some embodiments, the recombinant paramyxovirus is a recombinant PIV comprising, from upstream to downstream, a PIV genome promoter followed by a viral genome containing the N, P, M, F, HN, and L genes. In some such embodiments, heterologous genes included in the viral genome may be 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.

[0010] In further embodiments, heterologous genes contained within the viral genome of a recombinant paramyxovirus can be codon-optimized for expression in human cells. In even further embodiments, the recombinant paramyxovirus may be an attenuated virus. In other embodiments, additional genes and the proteins they encode may provide the attenuation required for a vaccine candidate.

[0011] Immunogenic compositions containing recombinant paramyxoviruses are also provided. These compositions may further comprise adjuvants. Methods for inducing an immune response in a subject by administering an effective amount of the disclosed recombinant paramyxovirus to the subject are also disclosed. Isolated nucleic acid molecules containing the viral genome of any of the recombinant paramyxoviruses disclosed herein are also provided.

[0012] Other features and advantages of this disclosure will become more apparent from the detailed description of several embodiments that proceed with reference to the accompanying drawings below. [Brief explanation of the drawing]

[0013] [Figure 1] This is a construct of an rB / HPIV3 vector 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 are referred to as non-HEK / GA-opt and HEK / GA-opt. HEK(66E, 101P) and non-HEK(66K, 101Q) amino acid assignments are indicated by asterisks. Other annotations: S, signal sequence; p27, 27k protein fragment released by cleavage activation; FP, fusion peptide; TM, transmembrane; CT, cytoplasmic tail. The RSV F ORF was inserted into the second genomic position between the N and P genes of the B / HPIV3 vector under the control of BPIV3 gene start and gene terminal transcription signaling. The rB / HPIV3 vector contains the N, P, M, and L genes (derived from BPIV3), as well as the F and NH genes (derived from HPIV3). The same vector genome location and vector transcription signal were used for all other rB / HPIV3 vectors expressing the RSV F protein, as shown in Figures 1-35. [Figure 2] Figures 2A and 2B show that the presence of 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 protein. Vero cells were infected with a vector (derived from GA-optimized ORF, shown in Figure 1) expressing HEK or non-HEK RSV F at an MOI of 10 TCID50 at 32°C. Cell lysates were prepared 48 hours after infection. Equal volumes 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). Natural RSV F trimers were detected with polyclonal antibodies induced in rabbits by repeated immunization using sucrose-purified RSV particles (B). [Figure 3] Figures 3A and 3B are images of syncytial formation in Vero cell monolayers after infection with rB / HPIV3 vectors expressing either non-HEK or HEK RSV F protein. Cells were infected with rB / HPIV3 expressing (A) non-HEK or (B) HEK assignment GA-codon optimized RSV F (see Figure 1) at 32°C with an MOI of 10 TCID50. Images of infected cells were taken 48 hours after infection. Representative syncytials are indicated by dashed outlines. [Figure 4] This diagram 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 with 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 to unoptimized 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 positions and with the same vector signals as in Figure 1. [Figure 5]Figures 5A and 5B show the increased in vitro expression of RSV F protein derived from the rB / HPIV3 vector, attributed to HEK assignment and codon optimization. RSV F expression in (A) Vero and (B) LLC-MK2 cells was evaluated by Western blotting. Cells were infected with the indicated rB / HPIV3 vector at 32°C with an MOI of 10 TCID50, 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. This experiment was performed in a total of three wells per virus. Uncleaved F0 precursors and cleaved F1 subunits were detected using monoclonal antibodies specific to RSV F. RSV F1 band density was quantified and normalized to the band density of the non-HEK / non-opt sample shown as "1". HPIV3 HN protein expression was also determined as an internal regulation of vector protein expression to ensure equivalent MOI and replication; β-actin was used as a loading control. [Figure 6] These images show the effect of codon optimization of HEK and F ORF on syncytial formation in vector-infected Vero cell monolayers. Cells were infected with mock-infection (mock), an empty rB / HPIV3 vector (empty B / H3), or an rB / HPIV3 vector expressing RSV F ORF, which was either 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). Infection was performed at 32°C with an MOI of 10 TCID50, and images were acquired 48 hours post-infection. Representative syncytials are outlined with dashed lines in several panels. [Figure 7]Figures 7A and 7B are graphs showing multicycle in vitro replication of rB / HPIV3 vectors expressing HEK or non-HEK RSV F protein derived from non-optimized or codon-optimized ORFs. (A) LLC-MK2 and (B) Vero cells were triple-infected at 32°C at an MOI of 0.01 TCID50 using either an empty rB / HPIV3 vector (empty B / H3) or a vector expressing an RSV F ORF. These vectors were either 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 the culture medium supernatant were collected at 24-hour intervals for 6 days, and viral titers were determined by limiting dilution assay on LLC-MK2 cells at 32°C and reported as TCID50 / ml. The average titer ± SEM from three independent experiments is shown. [Figure 8]Figures 8A and 8B are graphs showing the replication in hamsters of rB / HPIV3 vectors expressing HEK or non-HEK RSV F protein from unoptimized or codon-optimized ORFs. Golden Syrian hamsters were infected intranasally (IN) with 0.1 ml of inoculation containing either 105 TCID50 rB / HPIV3 vector or 106 PFU of wt RSV (strain A2). The hamsters were sacrificed on days 3 and 5 post-infection (n=6 per virus per day), and (A) turbinates and (B) lungs were removed, homogenized, and viral titers were determined by limiting dilutions against LLC-MK2 (rB / HPIV3 vector) or Vero(RSV) cells at 32°C: open circles and filled circles represent titers from animals sacrificed on days 3 and 5, respectively. Each symbol corresponds to an individual animal, and the average titer for each group is shown by dashed and solid horizontal lines for days 3 and 5, respectively. The limit of detection (LOD) is log10 TCID50 of 1.5 per g of tissue, which is shown by the dashed line. The rB / HPIV3 vector was titrated in LLC-MK2 cells using a limiting dilution assay and reported as TCID50 / g; the RSV was titrated in Vero cells using a plaque assay and reported as PFU / g. [Figure 9]This graph shows serum RSV-neutralizing antibody titers from hamsters infected with rB / HPIV3 vectors expressing unoptimized or codon-optimized ORF-derived HEK or non-HEK RSV F protein. Hamsters (n=6 animals per virus) were inoculated with 0.1 ml of an inoculum containing 10⁵ TCID50 or 10⁶ PFU of wt RSV from the rB / HPIV3 vector shown. Serum samples were collected 28 days after 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 corresponds to an individual animal. The height of each bar corresponds to the average titer of each group. The average titer value is shown above the bar. The standard error of the mean is shown by the horizontal line. The detection limit of the neutralization assay is the reciprocal of 5.3, log2 PRNT60, and is shown by the dashed line. [Figure 10] Figures 10A and 10B are graphs showing the defense of immunized hamsters against RSV challenge. Hamsters immunized using the rB / HPIV3 vector shown or using wt RSV as shown in Figure 9 (n=6 animals per virus) were challenged in IN with 0.1 ml of inoculum containing 106 PFU of wt RSV 31 days after immunization. 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. Each symbol corresponds to an individual animal, and the mean viral titer of the group is shown as the horizontal line. The detection limit of the assay was log10 2.7 PFU per g of tissue, as shown by the dashed line. [Figure 11]This is a diagram of the rB / HPIV3 vector constructs expressing secreted (Ecto), post-fusion, and stabilized pre-fusion RSV F proteins. Each of these modified proteins was expressed from an ORF containing a HEK assignment and optimized for human expression. Annotations: S, signal sequence; p27, 27k 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" type, consists of amino acids 1-513 of the RSV F protein; this lacks the CT and TM anchors and is likely available for secretion. The "post-fusion" type was derived from the ectodomain (1-513aa) by further deleting the first 10aa (FP; 137-146aa) 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 the pre-fusion form by the S155C / S290C mutation (DS) or by the DS and S190F / V207L ​​(Cav1) mutation (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 multicycle 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 constructs shown: HEK / GA-opt;Ecto;Post-fusion; and DS (see Figure 11 for explanation). Viral replication over a 6-day period at 32°C was determined by collecting supernatant samples at 24-hour intervals and by viral titration by ultradilution in LLC-MK2 cells. See Figure 11 for figures of mutant proteins. An asterisk * indicates that all of 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 derived from rB / HPIV3 vectors. Vero cells were infected with either an rB / HPIV3 vector with an MOI of 10 TCID50 or wt RSV with an MOI of 10 PFU. Infected cells were incubated at (A) 32°C or (B) 37°C for 48 hours. Supernatants and lysates of cells infected with (A) an rB / HPIV3 vector expressing post-fusion, Ecto, or HEK / GA-opt, or with wt RSV, and (B) lysates of cells infected with an rB / HPIV3 vector containing non-HEK / non-opt, HEK / GA-opt, DS, or DS-Cav1 type RSV F were collected and analyzed for RSV F expression by Western blotting. Constructs indicated by an asterisk * included the HEK assignment and were GA-optimized. [Figure 14]Figures 14A and 14B are graphs showing the replication of rB / HPIV3 vectors expressing secreted (Ecto), post-fusion, and stabilized pre-fusion RSV F proteins in hamsters. Hamsters were infected with IN using either 105 TCID50 rB / HPIV3 vector or 0.1 ml of inoculum containing 106 PFU of wt RSV. Hamsters were euthanized on post-infection days 3 and 5 (n=6 per virus per day), and (A) nasal turbinates and (B) lungs were removed, homogenized, and viral titers were determined by limiting dilution in LLC-MK2 cells (rB / HPIV3 vector) or Vero(RSV) cells at 32°C: open circles and filled circles represent titers for animals sacrificed on days 3 and 5, respectively. Each symbol corresponds to an individual animal, and the mean titer of each group is shown by a dashed or solid horizontal line for days 3 and 5, respectively. The mean titer on day 5 is shown at the top. The rB / HPIV3 vector was 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, shown by the dotted line. Statistical significance of differences between peak titers was determined by the Tukey-Kramer test and is indicated by an asterisk;*, P≦0.05;**, P≦0.01; or ***, P≦0.001. Constructs indicated by an asterisk* include HEK assignments 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 using either an rB / HPIV3 vector expressing 10⁵ TCID50 or a 0.1 ml inoculum containing 10⁶ PFU of wt RSV. Serum samples were collected 28 days post-immunization, and RSV-neutralizing antibody titers were determined by a 60% plaque reduction neutralization test (PRNT60) performed on Vero cells with and without guinea pig complement at 32°C. 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 shown by the horizontal line. The detection limit of the neutralization assay is shown by the dashed line. The mean neutralizing titer (ND) is below the detection limit. Statistical significance of differences between groups was determined by the Tukey-Kramer test and 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 defense of immunized hamsters against RSV challenge. Hamsters immunized as shown in Figure 15 (n=6 animals per virus) were challenged 31 days after immunization using 0.1 ml of inoculum containing 106 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 viral titer of that group is shown as a horizontal line. The detection limit of the assay was log102.7 PFU per g of tissue, as shown by the dotted line. [Figure 17A]This is a diagram of rB / HPIV3 vectors expressing modified versions of the RSV F protein to increase its integration into vector particles. (A) Structure of the F protein. (B) Cytoplasmic tail (CT), transmembrane (TM) domain, and the ectodomains of the RSV F protein (amino acid assignments in black) and BPIV3 F protein (bold), and the adjacent regions to the indicated amino acid sequence positions. Each of these modified proteins contained a 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 of the RSV F protein (amino acid sequence positions 551-574) replaced by the CT of the BPIV3 F protein (positions 515-540, bold). "B3TMCT" has both the TM and CT of the RSV F protein (positions 530-574) replaced by the TM and CT of the BPIV3 F protein (positions 494-540, bold). "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, along with the same vector signals described in Figures 1, 4, and 11. [Figure 17B]This is a diagram of rB / HPIV3 vectors expressing modified versions of the RSV F protein to increase its integration into vector particles. (A) Structure of the F protein. (B) Cytoplasmic tail (CT), transmembrane (TM) domain, and the ectodomains of the RSV F protein (amino acid assignments in black) and BPIV3 F protein (bold), and the adjacent regions to the indicated amino acid sequence positions. Each of these modified proteins contained a 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 of the RSV F protein (amino acid sequence positions 551-574) replaced by the CT of the BPIV3 F protein (positions 515-540, bold). "B3TMCT" has both the TM and CT of the RSV F protein (positions 530-574) replaced by the TM and CT of the BPIV3 F protein (positions 494-540, bold). "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, along with the same vector signals 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 rB / HPIV3 vector shown at 32°C with an MOI of 0.01 TCID50. The supernatant was collected 6–7 days after infection, cleared by slow centrifugation, and subjected to centrifugation with 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 processed in the same manner. The protein concentration of the sucrose-purified preparations was determined using a standard commercially available kit. (A) Western blot evaluation of the packaging effectiveness of the RSV F protein into rB / HPIV3 particles. To compare the relative amount 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 in the vector particles were quantified for comparison. (B) The packaging efficiency of each form of RSV F into each vector particle was calculated by standardizing its band density against that of the BPIV3 N protein. The order of the lanes is the same as in Part A. The packaging efficiencies of various forms of RSV F are shown relative to the native F protein, which is set to "1". The packaging efficiencies of the B3CT and B3TMCT forms of RSV F into the vector particles were determined to be similar to the efficiency of RSV F into RSV particles. This is because the amount of modified RSV F protein per 0.5 μg of vector particle (lanes 3, 4, 6, 7) was similar to the amount of native RSV F protein per 0.5 μg of RSV particle (lane 5). Constructs indicated by an asterisk * include HEK assignments and were GA-codon-optimized for human expression. [Figure 19-1]Figures 19A–19F are visualizations of the incorporation of B3CT and B3TMCT versions of the RSV F protein into rB / HPIV3 particles by transmission electron microscopy (TEM). Sucrose-purified viruses were labeled with an RSV F-specific mouse monoclonal antibody and a mouse-IgG-specific secondary antibody (labeled with 6 nm gold particles). Virians and gold particles were visualized by TEM. Representative images are shown for (A) RSV, (B) an empty rB / HPIV3 vector (empty B / H3), (C) a vector expressing HEK / GA-opt, (D) a vector expressing B3CT, (E) a vector expressing B3TMCT, and (F) a vector expressing DS / B3TMCT. Arrows point to scattered gold particles in the HEK / GA-opt virion (C). The substantial amount of gold particles associated with the vector particles is evident in D, E, and F. [Figure 19-2] Continuation of Figure 19-1. [Figure 20] Figures 20A and 20B are graphs showing multicycle in vitro replication of rB / HPIV3 vectors expressing B3CT and B3TMCT versions of the RSV F protein. (A) LLC-MK2 and (B) Vero cells were infected at 32°C with a MOI of 0.01 TCID50 using vectors expressing either an empty rB / HPIV3 vector (empty B / H3) or HEK / GA-opt, or B3CT (upper panel), or B3TMCT (upper panel), or DS / B3CT (lower panel), or DS / B3TMCT (lower panel). Aliquots of the culture medium 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 by an asterisk * include the HEK assignment and were 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 the DS or DS-Cav1 mutation that stabilizes the fusion pretype of the RSV F protein. (A) B3CT and B3TMCT expression; and (B) DS and DS-Cav1 (combined with B3CT and B3TMCT) expression. Vero cells were infected with the shown rB / HPIV3 vector at an MOI of 10 TCID50, or with 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 blotting. HPIV3 HN protein was used as a control to show equivalent vector replication; GAPDH was used as a loading control. Constructs indicated by an asterisk * included HEK assignments and were GA-codon-optimized for human expression. [Figure 22] This figure shows syncytial formation in a monolayer of Vero cells infected with an rB / HPIV3 vector expressing the B3CT or B3TMCT version of the RSV F protein, with or without the DS mutation that stabilizes the fusion pre-type of the RSV F protein. Vero cells were infected with an rB / HPIV3 vector expressing the indicated version 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 * include HEK assignments and were GA-codon-optimized for human expression. [Figure 23]Figures 23A and 23B show the replication in hamsters of rB / HPIV3 vectors expressing B3CT or B3TMCT versions of the RSV F protein, with or without the DS mutation that stabilizes the fusion pretype of the RSV F protein. Hamsters were IN-infected using 0.1 ml of inoculation containing 105 TCID50 rB / HPIV3 vector or 106 PFU wt RSV. Hamsters were euthanized on post-infection days 3 and 5 (6 per virus per day), and (A) nasal turbinates and (B) lungs were removed, homogenized, and viral titers were determined by limiting dilution in LLC-MK2 (rB / HPIV3 vector) or Vero(RSV) cells at 32°C: open circles and filled circles indicate titers of animals sacrificed on days 3 and 5, respectively. Each symbol corresponds to an individual animal, and the mean titer of each group is shown by a dashed or solid horizontal line for days 3 and 5, respectively. The mean titer on day 5 is shown at the top. The rB / HPIV3 vector was titrated in LLC-MK2 cells by limiting dilution assay and reported as TCID50 / g; RSV was titrated in Vero cells by plaque assay and reported as PFU / g. The limit of detection (LOD) is shown by a dotted line and is 1.5 log10 TCID50 per g of tissue. Statistical significance of differences between peak titers was determined by the Tukey-Kramer test and is indicated by an asterisk (*, P≦0.05; **, P≦0.01; or ***, P≦0.001). Constructs indicated by an asterisk * along the x-axis contain HEK assignments and were GA-codon-optimized for human expression. Constructions including modifications to DS-Cav1 were not available at the time of this experiment and therefore were not tested. [Figure 24]Figures 24A and 24B are graphs showing serum RSV-neutralizing antibody titers from hamsters infected with rB / HPIV3 vectors expressing B3CT or B3TMCT versions of the RSV F protein, with or without the DS mutation that stabilizes the pre-fusion type of the RSV F protein. Hamsters (n=6 animals per virus) were inoculated with 0.1 ml of an inoculum containing either 10⁵ TCID50 rB / HPIV3 vector or 10⁶ PFU of wt RSV. Serum samples were collected 28 days post-immunization, and antibody titers were determined by the 60% plaque reduction neutralization test (PRNT60) with or without guinea pig complement supplementation (A). The height of each bar corresponds to the average titer shown along the SEM. The average titer value is shown above the bar. The detection limit of the neutralization assay is indicated by the dotted line. Statistical significance of differences in mean titers was determined by the Tukey-Kramer test and indicated by asterisks (*, P ≤ 0.05; **, P ≤ 0.01; ns, P ≥ 0.05). ND, neutralizing titer was below the detection limit. Constructs indicated by asterisks * along the x-axis include HEK assignments and were GA-codon-optimized for human expression. [Figure 25] Figures 25A and 25B are graphs showing the defense of immunized hamsters against RSV challenge. Immunized hamsters (n=6 animals per virus) were challenged 31 days post-immunization using 0.1 ml of inoculum containing 106 PFU of wt RSV, as shown in Figure 24. 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 viral titer of the group is shown as a horizontal line. The detection limit of the assay was log102.7 PFU per g of tissue, shown as a dotted line. [Figure 26]This table shows the stability of RSV F expression by the rB / HPIV3 vector during replication in hamsters. The percentage of recovered vectors expressing RSV F in the nasal turbinates and lungs on days 3 and 5 postimmunization was determined by double-stained plaque assays of vectors recovered directly from tissue homogenates. The results are shown for individual animals. The percentage of rB / HPIV3 expressing the RSV F protein in the tested samples is shown. Samples expressing 100% RSV F protein are colored yellow; samples expressing 90-99% RSV F are colored green; samples expressing 80-89% RSV F are colored orange; samples expressing less than 79% RSV F are colored red. Samples that did not produce plaques due to low titer 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" (where X is equal to the number of plaques). [Figure 27] This table shows the temperature-sensitive phenotypes of B / HPIV3 vectors. The ability of each vector to form plaques in LLC-MK2 cells at the indicated temperatures was evaluated. A decrease in plaque formation of ≥100 times indicates temperature sensitivity. The lowest such limiting temperature for each virus is shown in bold and underlined, and is referred to as the shut-off temperature. [Figure 28] This figure shows rB / HPIV3 constructs evaluated for attenuation and immunogenicity in non-human primates (rhesus macaques). Rhesus macaques were infected with the following constructs, each containing 106 TCID50 per site: non-HEK / non-opt; HEK / GA-opt / DS; and HEK / GA-opt / DS / B3TMCT, in groups of 5, 5, and 4 animals, respectively, via combined intraintracheal and intratracheal routes. [Figure 29]Figures 29A and 29B are graphs showing rB / HPIV3 vector replication in rhesus monkeys. Rhesus monkeys were infected with the rB / HPIV3 vector shown in Figure 28. Vector replication in the respiratory tract was evaluated by collecting (A) nasopharyngeal swabs and (B) tracheal lavage fluid on the indicated days, and by determining viral titers by limiting dilution assays. The limit of detection is 1.2 log10TCID50 / mL, shown as a dotted line. [Figure 30] This graph shows the serum HPIV3-neutralizing antibody titers induced by the rB / HPIV3 vector. Monkey serum was collected at 0, 14, 21, 28, 35, and 56 days post-immunization, and HPIV3-neutralizing antibody titers were determined by a 60% plaque reduction neutralization test (PRNT60) in the presence of supplemental guinea pig complement. The detection limit of the neutralization assay is indicated by the dotted line. The RSV challenge day is also indicated. [Figure 31] This graph shows serum RSV-neutralizing antibody titers induced by the rB / HPIV3 vector. Monkey serum was collected at 0, 14, 21, 28, 35, and 56 days post-immunization (Figure 31). RSV-neutralizing antibody titers were determined at all time points by the 60% plaque reduction neutralization test (PRNT60) in the presence of supplemented guinea pig complement (Figure 32). RSV-neutralizing antibody titers were determined at 28 days post-immunization by the 60% plaque reduction neutralization test (PRNT60) in the absence of supplemented complement. The detection limit of the neutralization assay is indicated by the dotted line. Statistical significance of mean titers was determined by the Tukey-Kramer test and indicated by asterisks (**, P ≤ 0.01; ***, P ≤ 0.001). RSV challenge days are indicated. [Figure 32]This graph shows serum RSV-neutralizing antibody titers induced by the rB / HPIV3 vector. Monkey serum was collected at 0, 14, 21, 28, 35, and 56 days post-immunization (Figure 31). RSV-neutralizing antibody titers were determined at all time points by the 60% plaque reduction neutralization test (PRNT60) in the presence of supplemented guinea pig complement (Figure 32). RSV-neutralizing antibody titers were determined at 28 days post-immunization by the 60% plaque reduction neutralization test (PRNT60) in the absence of supplemented complement. The detection limit of the neutralization assay is indicated by the dotted line. Statistical significance of mean titers was determined by the Tukey-Kramer test and indicated by asterisks (**, P ≤ 0.01; ***, P ≤ 0.001). RSV challenge days are indicated. [Figure 33] This table shows the stability of RSV F expression by rB / HPIV3 vectors during replication in rhesus monkeys. The percentage of recovered vectors expressing RSV F in nasopharyngeal swabs was determined by double-stained plaque assay from 4, 5, and 6 days post-immunization. The percentage of rB / HPIV3 expressing RSV F in the tested samples is shown. Samples with 100% RSV F-expressing virus are colored yellow; samples with 99-90% RSV F-expressing virus are colored green; samples that did not produce plaque due to low titer are marked "NA". [Figure 34]This is a diagram of 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 "foldon" sequence. The RSV F protein expressed from an ORF (containing the DS-Cav1 mutation) with a HEK assignment and GS-codon-optimized (for human expression) was genetically 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 the indicated 27-amino acid foldon sequence derived from a T4 phage (see 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 signal as shown in Figures 1, 4, 11 and 17. [Figure 35] This table summarizes exemplary rB / HPIV3 vectors expressing RSV F, annotated to show constructs evaluated in two different studies in hamsters and two different studies in rhesus monkeys in Example 1. [Figure 36]This is a construct of the antigenomic cDNA for HPIV1 CD170 and LY942A mutations containing the RSV F gene insert at the 1st (F1), 2nd (F2), or 3rd (F3) genome location. The rHPIV1 backbone used for RSV F expression contained either two attenuating mutations: the CD170 mutation in the P / C gene (indicated by *) or the LY942A mutation in the L gene (indicated by ·). For the HPIV1-F1 construct, the RSV F gene was inserted at the 1st genome location prior to the HPIV1 N gene at the 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 the 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 the 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 included HEK amino acid assignments. Copies of the N gene terminus (GE), intergenetic (IG) CTT triplet, and P gene start (GS) sequence were added after (F1, F2) or before (F3) the RSV F insert so that it would be under the control of HPIV1 transcriptional signaling. Sequences 138-140 are shown adjacent to the RSV F insert under HPIV1-F1; sequences 141-143 are shown adjacent to the RSV F insert under HPIV1-F2; and sequences 144-145 are shown adjacent to the RSV F insert under HPIV1-F3. [Figure 37-1]Figures 37A–37D are graphs showing the multi-step replication of HPIV1 / RSV-F virus in Vero (37A and 37C) and LLC-MK2 (37B and 37D) cells. Triple wells of a cell monolayer in a 6-well plate were infected with HPIV1 CΔ170 (A and B) or LY942A (C and D) virus expressing RSV F (F1, F2, or F3) at an MOI of 0.01 TCID50, in parallel with wt HPIV1, HPIV1 LY942A, and HPIV1 CΔ170. The cultures were incubated at 32°C. Aliquots of cell culture medium were collected at 24-hour intervals, and viral titers (log10 TCID50 / ml) were determined at 32°C by serial dilution and erythrocyte adsorption assay in LLC-MK2 cells. The mean titer and the standard error of the mean (SEM) are shown. Statistical significance differences between the titers of each virus against wt HPIV1 were determined using Tukey's multiple comparison test and one-way ANOVA for the first two days after infection, and are indicated by 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 the analysis of RSV F and HPIV1 vector protein expression by Western blotting. Vero cells were infected with the virus indicated by MOI 5. 48 hours post-infection, cells were lysed in SDS sample buffer. All samples were denatured, reduced, and subjected to SDS-PAGE and Western blotting. Proteins were transferred to a PVDF membrane and probed with either an RSV F-specific mouse monoclonal antibody or an HPIV1 N-, P-, HN-, or F-specific polyclonal antibody (induced separately by immunizing rabbits with synthetic peptides corresponding to each protein). The conjugated antibodies were visualized using corresponding anti-mouse (IRDye 680LT) and anti-rabbit (IRDye 800CW) antibodies conjugated with infrared dyes. Images were obtained by scanning the blot using an Odyssey infrared imaging system. The images shown are from a single experiment representative of three independent experiments (B and C). The intensity of protein bands for the rHPIV1 CΔ170(B) and rHPIV1 LY942A(C) constructs was quantified for three independent experiments, with expression shown against the F3 virus set to 1.0. The 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 the expression of their corresponding empty vector scaffolds. *, 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 the cytopathic effect and syncytial formation in a monolayer of LLC-MK2 cells infected with an rHPIV1 vector expressing RSV F. MK2 cells were infected with an MOI of 0.01 TCID50 and incubated for 5 days. Images were obtained at 40× magnification using phase contrast with a light microscope. 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 replication of RSV F-expressing HPIV1 vectors in the nasal turbinates (40A) and lungs (40B) of hamsters. Hamsters were inoculated into the nasal cavity with 105 TCID50 wt HPIV1, rHPIV1 CD170, or rHPIV1 LY942A empty vector, rHPIV1 LY942A expressing RSV F from rHPIV1 CD170 or three genomic locations (F1, F2, or F3), rHPIV1-CR84GCD170HN553ALY942A (the previously described HPIV1 vaccine candidate (Bartlett et al. 2007 Virol J 4:6)), or rB / HPIV3-F2, a chimeric bovine / human PIV3 (also known as HEK / GA-opt, see Figure 1) expressing RSV F from a second genomic location. Viral titers were determined in LLC-MK2 cells by erythrocyte adsorption assay and reported as Log10 TCID 50 / g per gram of tissue. Titers for individual animals (6 animals per group) are shown for day 3 (Δ) and day 5 (·), with each symbol corresponding to an individual animal. Mean values ​​are shown in bold for day 3 and in italics for day 5 for each group. The limit of detection (LOD) is 1.5 log10 TCID 50 / ml and is indicated by a dotted line spanning the bottom of each graph. Statistical significance of the difference between each virus and wt HPIV1 (red asterisk) or rB / HPIV3-F2 (top bar) was confirmed by one-way ANOVA with 95% confidence intervals using Tukey's multiple comparison test for post-infection (pi) days 3 and 5. *, 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 in each group (n=6) were challenged intranasally with 106 PFU of wt RSV A2 30 days post-immunization. Nasal turbinates and lungs were collected from animals euthanized 3 days after challenge, and viral titers were determined for each sample by RSV-specific plaque assay on Vero cells and reported as Log10 PFU per g of tissue. The mean values ​​for each group are shown in 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, no significant difference. [Figure 42] This table illustrates attenuation mutations induced in the HPIV1 skeleton in P / C or L ORFs. Nucleotide changes (deletions or substitutions) in the wt sequence are underlined. [Figure 43] This table illustrates the temperature sensitivity of recombinant viruses to LLC-MK2 cell monolayers. Regarding temperature sensitivity, the underlined values ​​in bold indicate the virus shut-off temperature and the temperature-sensitive phenotype defined as the minimum limit temperature. Here, the mean log10 decrease in viral titer at 32°C compared to the given temperature was 2.0 log10 or greater than that of wt rHPIV1 at the same two temperatures. For the monolayers, serial dilutions of each virus shown were incubated at various temperatures for 7 days in LLC-MK2 cells. Viral titers were determined by erythrocyte adsorption using guinea pig erythrocytes and reported as Log10 TCID50 / ml with a detection limit of 1.2. [Figure 44]This table shows the percentage of the viral population expressing RSV F after in vivo replication. The percentage (stability) of the viral population expressing RSV F after in vivo replication was determined by immunofluorescence double-stained plaque assay. Vero cells were infected with serial dilution tissue homogenates (144 samples in total) of nasal turbinates or lungs from infected hamsters (n=6 per virus) collected on post-infection (pi) days 3 and 5, and incubated for 6 days under a methylcellulose stratified layer. Viral plaques were stained with mouse monoclonal anti-RSV F and goat polyclonal anti-HPIV1 specific antibodies, followed by detection with the corresponding infrared dye conjugate 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, as well as the stability of HPIV1 LY942A-F1, -F2, and F3 in URT and lungs, could not be tested due to the absence of their replication in these tissues. The numbers in parentheses indicate the RSV F expression status for a total of 6 hamsters per virus. ND indicates no plaques were detected. [Figure 45]This table lists results showing that immunization of hamsters with rHPIV1 expressing RSV F induces serum neutralizing antibodies against RSV. A group of 6-week-old hamsters (n=6) were intranasally immunized with 105 TCID50 containing each indicated virus in 0.1 ml of inoculum. Serum samples were collected before immunization and 28 days post-immunization. Antibody titers against RSV and HPIV1 were determined by using the 60% plaque reduction neutralization test (PRNT60) with green fluorescent protein (GFP) or enhanced GFP (eGFP) expressing virus (rRSV-eGFPM or HPIV1-GFP), and neutralizing antibody titers were expressed as mean reciprocal log2 ± SE. Based on the initial serum dilution used in this assay, the PRNT60 assay has titer detection limits 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 significant differences for HPIV1 antibody titers were determined by unpaired t-tests. Mean neutralizing antibody titers were classified into groups (shown in parentheses as A, B, C, and D). Mean antibody titers of treatment groups with different letters are statistically different from each other; titers indicated by two letters are not statistically different from titers indicated by either letter. [Figure 46]This graph shows multicycle in vitro replication of an rB / HPIV3 vector expressing a GA-optimized (GA-opt) pre-fusion RSV F with a DS-Cav1 mutation. (Figure 46) Vero and (Figure 47) LLC-MK2 cells were infected in triplicate at 32°C with an MOI of 0.01 TCID50 using either an empty rB / HPIV3 vector (empty B / H3) or a vector expressing an RSV F ORF. This RSV F ORF is either HEK-containing, GA-opt and contains a DS-Cav1 pre-fusion stabilizing mutation (HEK / GA-opt / DS-Cav1), or HEK-containing, GA-opt and contains the DS-Cav1 mutation and BPIV3-specific TM and CT domains as potential packaging signals (HEK / GA-opt / DS-Cav1 / B3TMCT). Aliquotes of the culture medium supernatant were collected at 24-hour intervals for 6 days. Viral titers were determined by limiting dilution assay in LLC-MK2 cells at 32°C and reported as TCID 50 / ml. The mean titers ± SEM from three independent experiments are shown. [Figure 47] This graph shows multicycle in vitro replication of an rB / HPIV3 vector expressing a GA-optimized (GA-opt) pre-fusion RSV F with a DS-Cav1 mutation. (Figure 46) Vero and (Figure 47) LLC-MK2 cells were infected in triplicate at 32°C with an MOI of 0.01 TCID50 using either an empty rB / HPIV3 vector (empty B / H3) or a vector expressing an RSV F ORF. This RSV F ORF is either HEK-containing, GA-opt and contains a DS-Cav1 pre-fusion stabilizing mutation (HEK / GA-opt / DS-Cav1), or HEK-containing, GA-opt and contains the DS-Cav1 mutation and BPIV3-specific TM and CT domains as potential packaging signals (HEK / GA-opt / DS-Cav1 / B3TMCT). Aliquotes of the culture medium supernatant were collected at 24-hour intervals for 6 days. Viral titers were determined by limiting dilution assay in LLC-MK2 cells at 32°C and reported as TCID 50 / ml. The 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 with a MOI of 0.01 TCID50 using an empty rB / HPIV3 vector (empty B / H3) or a vector expressing an RSV F ORF. This RSV F ORF was either HEK-containing and GS-opt RSV F (HEK / GS-opt), or HEK-containing, GS-opt and possessing a DS-Cav1 pre-fusion stabilization mutation (HEK / GS-opt / DS-Cav1), or HEK-containing, GS-opt and possessing a DS-Cav1 mutation and BPIV3-specific TM and CT domains (HEK / GS-opt / DS-Cav1 / B3TMCT), or a shortened RSV of 1-513 amino acids fused to a 4-amino acid linker and a 27-amino acid oligomerized sequence derived from a T4 phage. This is strain F, which is HEK-containing, GS-opt, and harbors the DS-Cav1 mutation (HEK / GS-opt / DS-Cav1 / (1-513)Foldon). Aliquots of the culture medium 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 TCID 50 / 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 with a MOI of 0.01 TCID50 using either an empty rB / HPIV3 vector (empty B / H3) or a vector expressing an ORF of RSV F. This RSV F ORF was either HEK-containing, GS-opt with the DS-Cav1 mutation (HEK / GS-opt / DS-Cav1), or HEK-containing, GA-opt with the DS-Cav1 mutation (HEK / GA-opt / DS-Cav1). Figures 49C and 49D: (C) Vero and (D) LLC-MK2 cells were infected at 32°C with an empty rB / HPIV3 vector (empty B / H3) or a vector expressing an ORF of RSV F at a MOI of 0.01 TCID50. This RSV F ORF was either HEK-containing, GS-opt and containing DS-Cav1 and B3TMCT variants (HEK / GS-opt / DS-Cav1 / B3TMCT), or HEK-containing, GA-opt and containing DS-Cav1 and B3TMCT variants (HEK / GA-opt / DS-Cav1 / B3TMCT). Aliquots of the culture medium 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. Mean titers ± SEM from three independent experiments are shown. [Figure 50]Figures 50A-50C show the expression of various modified forms of RSV F using 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 with the modified RSV F shown (lanes 2-5 and 8), or with an rB / HPIV3 vector expressing wt RSV (wt RSV, lane 6) at a MOI of 3 PFU / cell, or 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. Cell lysates and supernatants of Vero cells 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 types. 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 the replication of the rB / HPIV3 vector in the upper and lower respiratory tracts of hamsters. Hamsters were infected with IN using 0.1 ml of an inoculation containing either 10⁵ TCID50 rB / HPIV3 vector or 10⁶ PFU of wt RSV. Hamsters were euthanized on days 4 and 5 post-infection (n=6 per virus per day), and (A) turbinates and (B) lungs were removed, homogenized, and viral titers were determined by limiting dilution in LLC-MK2 cells at 32°C and reported as TCID50 / 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 log10 TCID50 per g of tissue, indicated by the dotted line. The open circles and filled circles represent the titers of individual animals slaughtered on day 4 and day 5, respectively. The mean titers for each group are shown by dashed and solid horizontal lines for day 4 and day 5, respectively. The mean titer values ​​for day 4 and day 5 are shown at the top. The mean viral titers on day 5 were assigned to different groups using the Tukey-Kramer test: mean titers of different letters are statistically different (p<0.05), but titers represented by two letters are not significantly different from titers represented by either letter. [Figure 52]This graph shows serum RSV-neutralizing antibody titers from hamsters infected with rB / HPIV3 vectors expressing GA-opt or GS-opt RSV F protein with or without DS, DS-Cav1, or B3TMCT modification. Hamsters (n=6 animals per virus) were inoculated with 0.1 ml of an inoculum containing either 105 TCID50 rB / HPIV3 vector or 106 PFU of wt RSV. Serum samples were collected 28 days post-immunization, and antibody titers were determined by the 60% plaque reduction neutralization test (PRNT60) with and without guinea pig complement supplementation (Figure 52) and (Figure 53). The height of each bar corresponds to the average titer shown along the SEM. The average titer value is shown above the bar. A pairwise Student t-test was used to assess the statistical significance of differences between values: in each of the three horizontal lines above the mean titer, the values ​​shown by vertical bars were compared pairwise with each other and recorded as either statistically significant (*, p<0.05) or not statistically significant (ns). The detection limit of the neutralization assay is shown by the dotted line. ND, the neutralization titer was below the detection limit. [Figure 53]This graph shows serum RSV-neutralizing antibody titers from hamsters infected with rB / HPIV3 vectors expressing GA-opt or GS-opt RSV F protein with or without DS, DS-Cav1, or B3TMCT modification. Hamsters (n=6 animals per virus) were inoculated with 0.1 ml of an inoculum containing either 105 TCID50 rB / HPIV3 vector or 106 PFU of wt RSV. Serum samples were collected 28 days post-immunization, and antibody titers were determined by the 60% plaque reduction neutralization test (PRNT60) with and without guinea pig complement supplementation (Figure 52) and (Figure 53). The height of each bar corresponds to the average titer shown along the SEM. The average titer value is shown above the bar. A pairwise Student t-test was used to assess the statistical significance of differences between values: in each of the three horizontal lines above the mean titer, the values ​​shown by vertical bars were compared pairwise with each other and recorded as either statistically significant (*, p<0.05) or not statistically significant (ns). The detection limit of the neutralization assay is shown by the dotted line. ND, the neutralization titer was below the detection limit. [Figure 54]Figures 54A and 54B are graphs showing the defense against RSV challenge in hamsters immunized with the rB / HPIV3 vector shown. Hamsters immunized as shown in Figure 53 (n=6 animals per immunization group) were IN-challenged 30 days post-immunization with 0.1 ml of inoculum containing 106 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 viral titer of the group is shown above the symbol as a short horizontal line. A pairwise Student's t-test was used to assess the statistical significance of differences between values: for each of the horizontal lines above the mean titer, the values ​​shown by the vertical bars were compared pairwise against each other and recorded as significant (*, p<0.05) or not significantly different (not significantly: ns). The detection limit of the assay was log10 1.7 PFU per g of tissue, shown as the dotted line. [Figure 55] This figure shows rB / HPIV3 constructs evaluated for attenuation and immunogenicity in non-human primates (rhesus macaques). Rhesus macaques were infected in groups of 4, 6, and 6 animals, respectively, using 106 TCID50 on one site of the following constructs via concomitant inhalation and intratracheal routes: 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 monkeys. Rhesus monkeys were infected with the rB / HPIV3 vector shown in Figure 55. Respiratory tract vector replication was evaluated by collecting (A) nasopharyngeal swabs and (B) tracheal lavage fluid on the indicated days, and by determining viral titers by limiting dilution assays. The limit of detection is 1.2 log10TCID50 / mL, shown as a dotted line. [Figure 57]Figures 57A and 57B are graphs showing serum RSV-neutralizing antibody titers induced by the rB / HPIV3 vector. Serum was collected at 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 the 60% plaque reduction neutralization test (PRNT60) in the presence of supplemented guinea pig complement. Statistical significance of the mean titers at each time point was determined by pairwise Student t-tests (ns, P>0.05). Figure 57A: RSV-neutralizing antibody titers 28 days after immunization were determined by PRNT60 in the absence of supplemented complement. The detection limit for the neutralization assay is shown by the dotted line. Statistical significance of the mean titers at each time point was determined by pairwise Student t-tests (ns, P>0.05). [Figure 58] Figures 58A and 58B show constructs of the rB / HPIV3 vector expressing HEK / GS-opt / DS-Cav1 / B3TMCT from the pre-N position, 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 position of rB / HPIV3. Figure 58A: Modification of the HPIV3 HN gene conferring increased phenotypic stability. The HN gene in the original recombinant HPIV3, created by reverse genetics (Durbin et al. Virology 235:323~332 1997), had two genetically engineered nucleotide substitutions in the HN gene at antigenome positions 7913 and 7915 (resulting in amino acid substitution P370T), and a random mutation at antigenome position 7593 (resulting in amino acid substitution T263I). Here, these mutations were reverted to the “wild-type” assignment, i.e., that found in the biologically induced HPIV3 strain JS (Genbank Z11575.1; Stokes et al. Virus Res 25:91~103.1992). [Figure 59]Figures 59A and 59B show the intracellular expression of RSV F and vector proteins by vectors expressing various versions of the RSV F protein at the first gene location (pre-N) or the second gene location (NP). Analysis of rB / HPIV3-wt and HN-HEK / GS-opt / DS-Cav1 / B3TMCT / pre-N, as illustrated in Figure 58A. Vero (Figure 59A) and LLC-MK2 (Figure 59B) cells were infected with an empty rB / HPIV3 vector (empty B / H3, lane 1), or a 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 at a 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 at a pre-N position (lane 6), or RSV (lane 2), or mock-infected (lane 7). The vectors were infected with an MOI of 10 TCID50 / cell, and wt RSV was infected with an MOI of 3 PFU / cell. The 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. The effects on vector protein expression were evaluated using BPIV3 N and P proteins. GAPDH was used as a loading control. [Figure 60] This diagram shows the amino acid sequence positions of the HPIV1 vector, specifically the cytoplasmic tail (CT), transmembrane (TM) domain, and the region adjacent to the ectodomain of the RSV F protein (strain A2, amino acid assignment), as well as the HPIV1 F protein (bold form). RSV-F-TMCT is a chimeric protein consisting of the ectodomain of the RSV F protein bound to the TM and CT domains of the HPIV1 F protein. [Figure 61]This is a construct of an 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 a HEK assignment (HEK) and is codon-optimized by GS for human expression (GS-opt). The RSV F inserts were genetically engineered to be stabilized in the pre-fusion conformation with either the DS and Cav1 mutation (DS-Cav1) alone (upper construct in Figures 61 and 62), or with further modifications by replacing its TMCT domain with a domain derived from HPIV1 F (TMCT, lower construct in Figures 61 and 62). The obtained HEK / GS-opt / DS-Cav1 and HEK / GS-opt / DS-Cav1 / TMCT versions of RSV F were modified by adjacent sequences and inserted into the HPIV1-CΔ170 vector (see Example 2 for a description of the HPIV1 vector and CΔ170 mutations) at the first gene site (MluI site) (Figure 61) or the second gene site (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 is relative to the complete antigenomic RNA sequence of the final construct. The sequences of sequence numbers 146 and 147 are shown adjacent to the RSV F insert below the figure 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]This is a construct of an 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 a HEK assignment (HEK) and is codon-optimized by GS for human expression (GS-opt). The RSV F inserts were genetically engineered to be stabilized in the pre-fusion conformation with either the DS and Cav1 mutation (DS-Cav1) alone (upper construct in Figures 61 and 62), or with further modifications by replacing its TMCT domain with a domain derived from HPIV1 F (TMCT, lower construct in Figures 61 and 62). The obtained HEK / GS-opt / DS-Cav1 and HEK / GS-opt / DS-Cav1 / TMCT versions of RSV F were modified by adjacent sequences and inserted into the HPIV1-CΔ170 vector (see Example 2 for a description of the HPIV1 vector and CΔ170 mutations) at the first gene site (MluI site) (Figure 61) or the second gene site (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 is relative to the complete antigenomic RNA sequence of the final construct. The sequences of sequence numbers 146 and 147 are shown adjacent to the RSV F insert below the figure 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]This graph shows the reaction kinetics of multicycle proliferation in Vero cells of an rHPIV1-CΔ170 vector expressing RSV F stabilized in the pre-fusion conformation (DS-Cav1), with or without TMCT derived from HPIV1 F protein. Vero cells were infected with the construct in triplicate at a MOI of 0.01 and incubated at 32°C for 7 days. A total of 3 mL of culture supernatant was collected at 24-hour intervals over 7 days. After sample collection, 0.5 mL of fresh medium was added to each culture to restore its original volume. Viral titration of the collected samples was performed in LLC-MK2 cells by erythrocyte adsorption assay, and the values ​​are plotted as mean ± SEM. [Figure 64] This figure shows the incorporation of RSV F protein stabilized in the pre-fusion conformation (DS-Cav1) into HPIV1-CΔ170 virion particles, with or without TMCT derived from HPIV1 F protein. The viral constructs shown (HEK / GS-opt names are omitted for simplicity) were grown in LLC-MK2 cells, and the virions were purified by sucrose gradient centrifugation. The protein concentrations of the purified viruses were 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. Binding primary antibodies were detected using infrared-labeled secondary antibodies. The chimeric RSV-F-DS-Cav1 / TMCT protein is visible in lanes 2 and 4 (panel 4). This is because antipeptide serum specific to HPIV1 F protein is induced using a synthetic peptide containing the C-terminal 18 amino acids of the CT domain, and therefore reacts with the RSV F protein possessing the TMCT domain of the HPIV1 F protein. [Figure 65]This figure shows the expression of RSV F protein stabilized in the pre-fusion conformation (DS-Cav1) with and without TMCT derived from HPIV1 F protein in infected Vero cells. Vero cell monolayers in 6-well plates were inoculated with the indicated viruses (named HEK / GS-opt, omitted for brevity) containing wt HPIV1 and rHPIV1-CΔ170 empty vector control at an MOI of 5 and incubated at 32°C for 48 hours. Cell lysates were prepared by dissolving the monolayer in 200 μL of LDS sample buffer. Protein samples were reduced and denatured, and 45 μL of each sample was subjected to electrophoresis, 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] This is a sequence diagram of the cytoplasmic tail (CT), transmembrane (TM) domain, and the adjacent regions of the ectodomains of the RSV F protein (amino acid assignment) and the HPIV3 F protein (bold form), with the indicated amino acid sequence positions. RSV-F-H3TMCT is a chimeric protein consisting of the ectodomain of the RSV F protein bound to the TM and CT domains of the HPIV3 F protein. [Figure 67A]This is a construct of an rHPIV3 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 the rHPIV3 vector particle. The vector is the wild-type rHPIV3 strain JS, which was modified to include amino acid assignments at 263T and 370P in the HN protein, which was found to confer phenotypic stability to the vector (see Figure 58B). Furthermore, the rHPIV3 vector was modified by creating a BlpI site at positions 103–119 for the insertion of RSV F at gene position 1 (or potentially any other insert) (A, upper construct), or by creating an AscI site at positions 1675–1682 for the insertion of RSV F at gene position 2 (B, upper construct). Each modified RSV F insert contained a HEK assignment (HEK) and was codon-optimized by GS for human expression (GS-opt). Furthermore, the RSV F insert was genetically engineered and stabilized in the pre-fusion conformation by further modification, either by the DS and Cav1 mutation (DS Cav1) alone (A and B, second construct) or by the replacement of its TMCT domain with a domain derived from rHPIV3 F (H3TMCT, A and B, third construct). The obtained HEK / GS-opt / DS-Cav1 and HEK / GS-opt / DS-Cav1 / H3TMCT versions of RSV F were modified with adjacent 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 HPIV3 transcriptional signaling for expression as separate mRNAs. Nucleotide numbering is relative to the complete antigenomic 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 sequence numbers 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]This is a construct of an rHPIV3 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 the rHPIV3 vector particle. The vector is the wild-type rHPIV3 strain JS, which was modified to include amino acid assignments at 263T and 370P in the HN protein, which was found to confer phenotypic stability to the vector (see Figure 58B). Furthermore, the rHPIV3 vector was modified by creating a BlpI site at positions 103–119 for the insertion of RSV F at gene position 1 (or potentially any other insert) (A, upper construct), or by creating an AscI site at positions 1675–1682 for the insertion of RSV F at gene position 2 (B, upper construct). Each modified RSV F insert contained a HEK assignment (HEK) and was codon-optimized by GS for human expression (GS-opt). Furthermore, the RSV F insert was genetically engineered and stabilized in the pre-fusion conformation by further modification, either by the DS and Cav1 mutation (DS Cav1) alone (A and B, second construct) or by the replacement of its TMCT domain with a domain derived from rHPIV3 F (H3TMCT, A and B, third construct). The obtained HEK / GS-opt / DS-Cav1 and HEK / GS-opt / DS-Cav1 / H3TMCT versions of RSV F were modified with adjacent 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 HPIV3 transcriptional signaling for expression as separate mRNAs. Nucleotide numbering is relative to the complete antigenomic 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 sequence numbers 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. [Modes for carrying out the invention]

[0014] Sequence List The nucleic acid and amino acid sequences listed in the attached sequence listings are shown using standard letter abbreviations for nucleotide bases and three-letter notations for amino acids, as defined in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but complementary strands are understood to be included by any reference to the shown strand. The sequence listings are incorporated herein by reference. It is included in the form of an ASCII text 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 heterologous genes in Sendai virus, which is the mouse relative of HPIV1 and closely related to HPIV3. These studies showed that only a very small amount of RSV F protein is incorporated into Sendai virus vector particles. The researchers identified the CT or CT and TM of the RSV F protein as corresponding sequences derived from the Sendai F protein, and that this is the foreign RSV The substitutions were made on the premise that they would improve the efficiency of the F protein's interaction with the vector particle. These modifications did indeed increase the integration of genetically 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 the production of infectious attenuated viruses for vaccination, and also removes one of the vector protective antigens that is thought to be necessary for producing a bivalent vaccine.

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

[0017] Efficient packaging of RSV F into vector particles significantly increased the induction of an immune response against the ectodomain (containing all neutralizing epitopes) when recombinant paramyxovirus was administered to subjects. Unexpectedly, the viral neutralizing serum antibody response was qualitatively significantly increased, which was assessed by comparing RSV neutralizing activity in vitro in the absence of complement (measuring strongly neutralizing antibodies) or in its presence (enhancing neutralization by weakly neutralizing or non-neutralizing antibodies). This unexpected qualitative increase in antibodies is particularly significant for RSV, which is known to induce incomplete immunoprotection. Expression of exogenous glycoproteins with the TMCT domain of the vector glycoprotein and effective packaging clearly had the potential to disrupt vector replication and morphogenesis; however, constructs in which this effect was minimal are provided.

[0018] To further enhance immunogenicity, we evaluated the stabilization of the RSV F protein in pre-fusion conformation. Pre-fusion stabilization also naturally resulted in an increase in strongly neutralizing antibodies, suggesting stabilization of the neutralizing epitope. In the hamster model, the effect of pre-fusion stabilization on increased immunogenicity and protection appeared to be additive to that of 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 RSVs, maximum immunogenicity is desirable. Extensive experimental methods allow for other forms of vectors and insert constructs (e.g., use of various insertion sites, codon optimization, and use of early passaged RSV F protein sequences). This was discovered, providing increased RSV F expression and reduced cytopathic effects of syncytial formation mediated by highly membrane-fusionable RSV F proteins.

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

[0021] I. Glossary Unless otherwise specified, technical terms will be used according to their conventional usage. The definitions of general terms in molecular biology are given by Benjamin Lewin, Genes X, Jones & This can be found in Bartlett Publishers, 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, Wiley-VCH, Vol. 16, 2008; and other similar references.

[0022] In this specification, the singular forms “a,” “an,” and “the” refer to both singular and plural unless the context explicitly indicates otherwise. For example, the term “an antigen” includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.” In this specification, the term “comprises” means “includes.” It should be further understood that any base size or amino acid size and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximations and provided for convenience unless otherwise noted. Many methods and materials similar or equivalent to those described herein are used, but specific suitable methods and materials are described herein. In case of any inconsistency, this specification, including the definitions of terms, takes precedence. Furthermore, materials, methods, and examples are merely illustrative and not intended to be limiting. To facilitate an overview of various embodiments, the following definitions of terms are provided:

[0023] Adjuvant: A medium used to enhance antigenicity. Examples of adjuvants include suspensions of minerals (alum, aluminum hydroxide, or aluminum phosphate) on which the antigen is adsorbed; or water-in-oil emulsions, for example, where the antigen solution is emulsified in mineral oil (Freund's incomplete adjuvant); and sometimes containing dead mycobacteria to further enhance antigenicity (inhibiting antigen degradation and / or inducing macrophage influx) (Freund's complete adjuvant). Immunostimulating oligonucleotides (such as those containing CpG motifs) can also be used as adjuvants. Adjuvants also include biomolecules such as costimulatory molecules ("biological adjuvants"). Examples of adjuvants include IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL, immunostimulatory 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 will be 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: Introduction of the composition to the target via the selected route. Administration may be local or systemic. For example, if the selected route is intranasal, the composition (such as the composition containing the disclosed recombinant paramyxovirus) is administered by introducing the composition into the nasal route of the target. Exemplary routes of administration include, but are not limited to, oral, injection (subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous, etc.), sublingual, rectal, percutaneous (e.g., local), 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 zero amino acids. In some cases, an amino acid in a polypeptide is substituted with an amino acid from a homologous polypeptide. For example, an amino acid in recombinant group A RSV F polypeptide is substituted with a corresponding amino acid from group B RSV F polypeptide. A reference to the "66E" amino acid in an RSV F protein refers to an RSV F protein containing a glutamic acid residue at position 66. The amino acid may also be present due to substitution from a reference sequence. A reference to the "K66E" substitution in an RSV F protein refers to an RSV F protein containing a glutamic acid residue at position 66 that has been substituted with a lysine residue in a reference (e.g., native) sequence.

[0026] Attenuated: Paramyxoviruses with an "attenuated" or "attenuated phenotype" refer to paramyxoviruses with reduced virulence compared to a reference wild-type paramyxovirus under similar infection conditions. Attenuation is usually associated with reduced viral replication compared to that of a reference wild-type paramyxovirus under similar infection conditions; therefore, "attenuated" and "restricted replication" are often used as synonyms. In some hosts (typically non-natural hosts, including laboratory animals), the disease is not evident during infection with the reference paramyxovirus in question, and restricted viral replication can be used as a surrogate marker for attenuation. In some embodiments, attenuated recombinant paramyxoviruses (e.g., RSV, PIV3) exhibit at least about 10-fold or greater reductions in viral titers in the upper or lower respiratory tract of mammals, respectively, compared to unattenuated wild-type viral titers in the upper or lower respiratory tract of the same species of mammal under identical infection conditions. Examples of mammals, though not limited to them, include humans, mice, rabbits, rats, hamsters such as the golden hamster (Mesocricetus auratus), and non-human primates such as the savanna monkey (Ceroptihecus aethiops). Attenuated paramyxoviruses may exhibit different phenotypes, including, but not limited to, altered growth, temperature-sensitive growth, and altered growth or plaque size that are restricted by the host range.

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

[0028] Degenerate Variants: In the context of this disclosure, “degenerate variant” refers to a polynucleotide encoding a polypeptide containing a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by two or more codons. Therefore, all degenerate nucleotide sequences encoding a peptide are included, provided that the amino acid sequence of the peptide encoded by the nucleotide sequence is not altered.

[0029] Gene: A nucleic acid sequence, typically a DNA sequence containing regulatory and coding sequences necessary for the transcription of RNA, whether mRNA or not. For example, a gene contains a promoter, one or more enhancers or silencers, and codes for RNA and / or polypeptides. This may include nucleic acid sequences, downstream regulatory sequences, and, if applicable, other nucleic acid sequences involved in the regulation of mRNA expression.

[0030] Heterogeneous: Originating from a different gene source. Heterogeneous genes included in a recombinant viral genome are genes that do not originate from that viral genome. In one specific, non-limiting example, a heterogeneous gene encoding the ectodomain of the RSV F protein is included in the genome of a recombinant PIV vector. Methods for introducing heterogeneous genes into viral vectors are well known in the art and are described herein.

[0031] Host cell: A cell on which a vector can grow and on which its nucleic acid is expressed. The cell may be a prokaryote or a eukaryote. This term also includes any progeny of the target host cell. It is understood that all progeny may not be identical to the parent cell, as mutations can occur during replication. However, such progeny are included when the term “host cell” is used.

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

[0033] Immunogen: A compound, composition, or substance, including a composition injected or absorbed into an animal, that can stimulate antibody production or a T-cell response in an animal. Immunogens react with products of specific humoral or cellular immunity, including those induced by heterologous antigens such as disclosed recombinant paramyxoviruses. Administration of an immunogen to a subject may 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 a measurable B-cell response to an antigen (such as antibody production), encoded by nucleic acid molecules contained on or within the immunogen. For example, an 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 a measurable B-cell response to RSV and / or PIV (such as antibody production). An immunogenic composition may comprise an isolated recombinant paramyxovirus, such as those disclosed herein. For in vivo use, an immunogenic composition typically comprises a recombinant paramyxovirus in a pharmaceutically acceptable carrier and may also comprise other agents, such as adjuvants.

[0035] Isolated: “Isolated” biological components are those that have been substantially separated or purified from other biological components, such as other naturally occurring biological components, including other chromosomes and extrachromosomal DNA, RNA, and proteins. Examples of “isolated” proteins, peptides, nucleic acids, and viruses include those purified by standard purification methods. Isolation does not require absolute purity and may 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 making two molecules into one continuous molecule, for example, linking two polypeptides into one continuous polypeptide by recombination means. References to genes encoding type I membrane proteins containing the TM and CT of heterologous F proteins and the “linked” RSV F ectodomain mean that gene expression is directed from N to C-terminal, with the RSV F ectodomain, TM, and This refers to gene linking by recombinant means between a nucleic acid sequence encoding the RSV F ectodomain in a gene and a nucleic acid sequence encoding the TM and CT of a heterologous F protein, 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 TM (by 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 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 a single continuous molecule. A non-exclusive example of a peptide linker is the glycine-serine linker.

[0038] Native proteins, sequences, or disulfide bonds: e.g., polypeptides, sequences, or disulfide bonds that have not been modified by selective mutation. Selective mutations, e.g., to concentrate the antigenicity of an antigen against a target epitope, or to introduce disulfide bonds into a protein that are not present in the native protein. Native proteins or native sequences are also called wild-type proteins or wild-type sequences. Non-native disulfide bonds are disulfide bonds that are not present in the native protein, e.g., disulfide bonds formed 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 may include both sense and antisense strands of RNA, cDNA, genomic DNA, and the synthetic and mixed polymers described above. A nucleotide refers to a modified form of ribonucleotide, deoxynucleotide, or any of the other types of nucleotides. The term “nucleic acid molecule” is synonymous with “nucleic acid” and “polynucleotide” in this specification. A nucleic acid molecule is usually at least 10 bases long unless otherwise specified. This term includes the single-stranded and double-stranded forms of DNA. A polynucleotide may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide linkages.

[0040] Operatively linked: A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is functionally related to the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence when it affects the transcription or expression of the coding sequence. Generally, operably linked nucleic acid sequences are contiguous and, if the two protein coding regions need to be connected, they are in the same reading frame.

[0041] Paramyxoviruses: A family of enveloped, undivided, minus-stranded single-stranded RNA viruses. Examples of paramyxoviruses, though not limited to them, include human parainfluenza virus (HPIV), including types 1, 2, 3, 4A, and 4B (HPIV1, HPIV2, HPIV3, HPIV4A, and HPIV4B, respectively), mouse parainfluenza virus type 1 (Sendai virus, MPIV1), bovine parainfluenza virus type 3 (BPIV3), parainfluenza virus 5 (PIV5, formerly called monkey virus 5, SV5), monkey virus 41 (SV41), and mumps virus. HPIV1, HPIV3, MPIV1, and BPIV3 belong to the genus Respirovirus. HPIV2, HPIV4, SV5, SV41, and mumps virus belong to the genus Rubravirus. MPIV1, PIV5, and BPIV3 are animal-derived derivatives of HPIV1, HPIV2, and HPIV3, respectively (Chancock et al., Parainfluenza Viruses, Knipe et al. (eds.), 1341-1379, Lippp (Incott Williams & Wilkins, Philadelphia, 2001). HPIV1, HPIV2, and HPIV3 correspond to distinct serotypes and do not induce significant cross-immunity. HPIV is the pathogen of respiratory infections such as croup, pneumonia, or bronchitis.

[0042] Parainfluenza viruses (PIVs): Several enveloped, undivided, minus-stranded single-stranded RNA viruses belonging to the Paramyxoviridae family, which are descriptively grouped together. This includes all members of the Respirovirus genus (e.g., HPIV1, HPIV3) and several members of the Rubravirus genus (e.g., HPIV2, HPIV4, PIV5). Members of the Aburavirus genus (e.g., NDVs) have historically been called PIVs and are considered part of this group. HPIV serotypes 1, 2, and 3 are the second most common, after RSV alone, in causing severe respiratory infections in infants and children worldwide, with HPIV3 being the most significant of the HPIVs in terms of disease impact. PIVs consist of two structural modules: (1) an inner 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 nucleocapsid structural proteins (NP, NC, or N depending on the genus), phosphorylated proteins (P), matrix proteins (M), fusion glycoproteins (F), hemagglutinin-neuraminidase glycoproteins (HN), large polymerase proteins (L), and C and D proteins. The P gene contains one or more further open reading frames (ORFs) encoding accessory proteins. The gene order is 3'-NPMF-HN-L-5', and each gene encodes a distinct protein encoding mRNA. Exemplary PIV strain sequences, such as those of HPIV1, HPIV2, HPIV3, and BPIV3 viruses, are known to those skilled in the art.

[0043] pharmaceutically acceptable carriers: Useful pharmaceutically acceptable carriers are conventional ones. Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 19th edition, 1995, describes compositions and formulations suitable for the pharmacokinetic delivery of the disclosed immunogens.

[0044] Generally, the properties of the carrier vary depending on the specific mode of administration used. For example, parenteral formulations typically contain an injectable fluid, such as water, saline, equilibrium salt solution, aqueous dextrose, or glycerol, as the medium, which is pharmaceutically and physiologically acceptable. For solid compositions (e.g., in the form of powders, pills, tablets, or capsules), conventional non-toxic solid carriers include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to a biologically neutral carrier, the pharmaceutical composition to be administered may contain trace amounts of non-toxic adjuncts, such as humectants or emulsifiers, preservatives, and pH buffers, for example, sodium acetate or sorbitan monolaurate. In specific embodiments suitable for administration to a subject, the carrier is sterile and / or contained in a unit dosage form containing one or more measured doses of a composition suitable for inducing a desired immune response. This is also achieved by drug application for therapeutic purposes. The unit dosage form may be, for example, in a sealed vial containing sterile contents or a syringe for injection into a subject, or lyophilized for subsequent solubilization and administration, or in solid or sustained-release dosage form.

[0045] Polypeptide: Any chain of amino acids that is not related to length or post-translational modifications (e.g., glycosylation or phosphorylation). The term "polypeptide" applies to amino acid polymers, including naturally occurring amino acid polymers and amino acid polymers that are not naturally occurring, and In this case, one or more amino acid residues are non-natural amino acids, for example, artificial chemical mimics of the corresponding naturally occurring amino acids. “Residue” refers to an amino acid or amino acid mimic incorporated into a polypeptide by an amide bond or amide bond mimic. A polypeptide has an amino terminus (N-terminus) and a carboxyl terminus (C-terminus). “Polypeptide” is used synonymously with peptide or protein and, as herein, is used to refer to a polymer of amino acid residues.

[0046] Primer-boost vaccination: Immunotherapy comprising administering a first immunogenic composition (primer vaccine) to a subject, followed by the administration of a second immunogenic composition (booster vaccine), to induce an immune response. The booster vaccine is administered to the subject after the primer vaccine; those skilled in the art will understand a suitable time interval between the administration of the primer vaccine and the booster vaccine, and examples of such time frames are disclosed herein. Further administrations, e.g., a second boost, may be included in the primer-boost protocol.

[0047] Recombinant molecules: Recombinant nucleic acid molecules have sequences that do not exist in nature, for example, including one or more nucleic acid substitutions, deletions, or insertions, and / or sequences created by artificial combinations of two otherwise isolated sequence segments. These artificial combinations can be achieved by chemical synthesis, or more commonly, by artificial manipulation of isolated nucleic acid segments, for example, by genetic engineering techniques.

[0048] Recombinant viruses contain a genome that includes recombinant nucleic acid molecules.

[0049] Recombinant proteins are those that have sequences that do not exist in nature, or sequences that are created by artificially combining two otherwise separate sequence segments. In some embodiments, recombinant proteins are encoded by heterologous (e.g., recombinant) nucleic acids introduced into host cells such as bacteria or eukaryotic cells, or into the genome of a recombinant virus.

[0050] Respiratory syncytial virus (RSV): An enveloped, undivided, minus-stranded single-stranded RNA virus belonging to 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, enabling viral entry into the cytoplasm and also promoting syncytial formation. Based primarily on antigenic differences in the G glycoprotein, two antigenic subgroups of human RSV strains, subgroups A and B, have been described. RSV strains from other species, including bovine RSV, are also known. Exemplary RSV strain sequences are known to those skilled in the art. Furthermore, several models of human RSV infection are available, including model organisms infected with species-specific RSVs, such as the use of model organisms infected with hRSV and bRSV infection in cattle (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 facilitates the fusion of the virus and the cell membrane. In fact, the RSV F protein is initially synthesized as a single polypeptide precursor, designated F0, approximately 574 amino acids long. F0 contains an N-terminal signal peptide that directs its localization to the endoplasmic reticulum, and in this case, the signal peptide (approximately F) The first 22 residues of F0 are cleaved by proteolysis. The remaining F0 residues form an oligomer, which also forms a trimer, which contains two conserved furin consensus cleavage sequences (approximately F0 positions 109 / 110 and 136 / 137; e.g., RARR). 109(Sequence ID 1, residues 106-109) and RKRR 136 (SEQ ID NO: 1, residues 133-136)) is processed by proteolysis by a cellular protease, cleaving the pep27 polypeptide and generating two disulfide-bonded fragments, F1 and F2. The smaller of these fragments, F2, is derived from the N-terminal portion of the F0 precursor and contains approximately 26-109 residues of F0. The larger of these fragments, F1, contains the C-terminal portion of the F0 precursor (approximately residues 137-574), which includes the extracellular / luminal region (approximately residues 137-529), TM (approximately residues 530-550), and CT (approximately residues 551-574) at its C-terminus.

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

[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 consists of three RSV proteins. Contains a protein complex of the F ectodomain.

[0054] The RSV F protein adopts a “pre-fusion” conformation prior to the induction of a membrane fusion event that leads to the transition to the post-fusion conformation of RSV F and subsequent processing to the mature RSV F protein in the secretory system. Exemplary three-dimensional structures of the RSV F protein in the pre-fusion conformation are publicly known and, for example, disclosed 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, as well as “antigen sites” that also contain the epitopes of the D25 and AM22 monoclonal antibodies. Φ The antigenic site is located at the distal apex of the membrane, which is called the "apex." Therefore, recombinant RSV is stabilized in the pre-fusion conformation. The F protein binds to the pre-fusion conformation of the RSV F protein, but not to the post-fusion conformation. For example, antibodies that bind to the antigen site. Φ Antibodies that specifically bind to the epitope within the RSV F fusion, such as D25 or AM22 antibodies, are used for specific binding. Further RSV F pre-fusion specific antibodies include 5C4 and MPE8 antibodies.

[0055] Sequence Identity: The similarity between amino acid sequences is expressed in terms of similarity, also known as sequence identity. Sequence identity is often measured in terms of identity percentage (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologous, orthologue, or variant polypeptides exhibit 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 This is described in *Uter Appls. in the Biosciences* 8, 155-65, 1992; and by Pearson et al., *Meth.Mol.Bio.* 24:307-31, 1994. Altschul et al., *J.Mol.Bio.* 215:403-410, 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 identical nucleotides or amino acid residues exist in both sequences. The sequence identity percentage is determined by dividing the number of matches by either the length of the sequence shown in the identified sequence or the clarified length (such as 100 consecutive nucleotides or amino acid residues derived from the sequence shown in the identified sequence), and then multiplying the resulting value by 100. For example, a peptide sequence with 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 sequence identity percentage value is rounded to one decimal place. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value is always an integer.

[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. Instructions on how to determine sequence identity using this program are available on the NCBI website.

[0059] Homogenetics and variants of polypeptides (such as RSV F ectodomains) are typically characterized by having at least about 75% sequence identity, counted over the full-length alignment with the amino acid sequence of interest, e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Proteins with greater similarity to the reference sequence will show increased identity percentages, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, when evaluated in this manner. For sequence identity, when less than the entire sequence is compared, homologs and variants typically have at least 80% sequence identity over a short window of 10–20 amino acids and may have at least 85% or at least 90% or 95% sequence identity to the reference sequence, depending on their similarity. Methods for determining sequence identity over such a short window are available on the internet from the NCBI website. Those skilled in the art will understand that these sequence identity ranges are provided merely as guidelines; it is entirely possible that strongly significant homologs can be found outside the provided range.

[0060] For sequence comparison of nucleic acid sequences, a sequence typically acts as a reference sequence compared to the test sequence. When the test and reference sequences are input into a computer using a sequence comparison algorithm, subsequence coordinates and sequence algorithm program parameters are specified as needed. Default program parameters are used. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison is described, for example, in Smith & Waterman, Adv.Appl.Mat. According to the local homology algorithm in h.2:482, 1981, Needleman & Alignment can be performed using the homology alignment algorithm of Wunsch, J.Mol.Biol.48:443, 1970, or the similarity search method of Pearson & Lipman, Proc.Nat'l.Acad.Sci.USA 85:2444, 1988, either by computer implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI) or by manual alignment and visual inspection (see, for example, 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, Appendix 104 for details, 2013)). One example of a useful algorithm is PILEUP. PILEUP uses the simplified progressive alignment method described by Feng & Doolittle, J. Mol. Evol.35:351~360, 1987. The method used is similar to that described by Higgins & Sharp, CABIOS5:151~153, 1989. Using PILEUP, the reference sequence is compared to other test sequences, and the sequence identity relation percentage is determined using the following parameters: default gap weighting (3.00), default gap length weighting (0.10), and weighted end gap. PILEUP can be obtained from, for example, the GCG sequencing software package, e.g., version 7.0 (Devereaux et al., Nuc. Acids Res.12:387~395, 1984).

[0061] Another example of an algorithm suitable for determining sequence identity and sequence similarity percentages is the BLAST and BLAST2.0 algorithms, 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 analysis is publicly available from the National Center for Biotechnology Information (ncbi.nlm.nih.gov). The BLASTN program (for nucleotide sequences) uses, by default, 11 word lengths (W), 50 alignments (B), 10 predictions (E), M=5, N=-4, and comparison of both strands. The BLASTP program (for amino acid sequences) uses a word length (W) of 3 and a prediction (E) of 10, along with a BLOSUM62 scoring matrix, by default (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989). Oligonucleotides are linear polynucleotide sequences with a maximum length of approximately 100 nucleotides.

[0062] In this specification, a reference to “at least 90% identity” means “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” of a given reference array.

[0063] Subjects: A category including living multicellular vertebrates, humans, and non-human mammals. In one example, the subject is human. In specific examples, the subject is a newborn. In further examples, subjects requiring inhibition of RSV infection are selected. For example, the subject is either not infected but at risk of RSV infection, or infected and in need of treatment.

[0064] Transmembrane domains (TM): such as the lipid bilayer of cells, viruses, or virus-like particles. An amino acid sequence spanning the lipid bilayer. The transmembrane domain allows the antigen to be immobilized on the membrane. In some examples, the transmembrane domain is the RSV F transmembrane domain.

[0065] Vaccine: Preparation of immunogenic material capable of stimulating an immune response, administered for the prevention, remission, or treatment of infection or other types of disease. Immunogenic material may include attenuated or dead 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 nevertheless retain the ability to induce antibodies and cellular immunity against pathogenic forms. Inactivated (dead) vaccines are previously pathogenic organisms that have been inactivated using chemicals, heat, or other treatments, but still induce antibodies against the organism. Vaccines can induce both prophylactic (preventive or protective) and therapeutic responses. The method of administration varies depending on the vaccine, but may include inoculation, oral ingestion, inhalation, or other forms of administration. Vaccines can be administered with adjuvants to boost the immune response.

[0066] A vector is an entity that contains a DNA or RNA molecule having a promoter(s) operably linked to a coding sequence of an antigen(s) of interest, and is capable of expressing the coding sequence. Examples, but not limited to, include naked or packaged (lipid and / or protein) DNA, naked or packaged RNA, viruses or bacteria or other microorganisms that may or may not be able to replicate, or small components of viruses or bacteria or other microorganisms that may be able to replicate. Vectors are sometimes called constructs. A recombinant DNA vector is a vector that contains recombinant DNA. A vector may contain nucleic acid sequences that enable replication in a host cell, such as an origin of replication. A vector may also contain one or more select marker genes and other genetic elements known in the art. A viral vector is a recombinant nucleic acid vector that contains at least several 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 an immune response to those viral pathogens. The recombinant paramyxoviruses include a genome encoding heterologous genes. The recombinant paramyxoviruses include a genome containing heterologous genes encoding the ectodomain of a transmembrane protein (e.g., a viral glycoprotein) of a heterologous viral pathogen. The ectodomain can be ligated to the CT or TM and CT of a paramyxovirus-derived envelope protein to enable the expression of the ectodomain of the heterologous virus-derived transmembrane protein in the paramyxovirus envelope. For example, a recombinant paramyxovirus may be a recombinant PIV containing a genome containing heterologous genes encoding the ectodomain of an RSV F protein ligated to the TM and CT of a PIV-derived F protein. Further descriptions of recombinant paramyxoviruses and their modifications are provided herein.

[0068] The paramyxovirus genome contains genes encoding N, P, M, F, HN, and L proteins. The genome also contains a genome promoter and anti-promoter in the order promoter-N, P, M, F, HN, L-anti-promoter. Heterogenes contained within the recombinant paramyxovirus genome can be located at any position between genes in the paramyxovirus genome or between the promoter and the N gene, or between the L gene and the anti-promoter. Heterogenes can be flanked by appropriate gene start and termination sequences to promote expression from the viral genome. In preferred embodiments, heterogenes can be positioned between the promoter and the N gene, or between the N gene and the P gene. ru.

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

[0070] Recombinant paramyxoviruses may be, for example, recombinant HPIV1, HPIV2, HPIV3, BPIV3, PIV5, Sendai virus, or NDV, or chimeras thereof. A further description of such recombinant paramyxoviruses is provided below.

[0071] General methods for producing recombinant paramyxoviruses containing heterologous genomes are known to those skilled in the art, as are viral sequences and reagents for use in such methods. Examples, not limited to, of methods for producing recombinant PIV vectors containing heterologous genes (such as recombinant HPIV1, HPIV2, HPIV3, or H / BPIV3 vectors), methods for attenuating vectors (e.g., by recombinant or chemical means), and viral sequences and reagents for use in such methods are, respectively, incorporated herein by reference in their entirety by U.S. Patent Publication No. 2012 / 0045471; These are available in the following publications: 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 in Newman et al. 2002 Virus genes 24:77~92, Tang et al. 2003 J Virol, 77(20):10819~10828. Examples of methods for constructing 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 2012 / 0064112, which is incorporated herein by reference in its entirety; and in Basavarajappa et al., 2014 Vaccine, 32:3555-3563 and in McGinnes et al., J. Virol., 85:366-377, 2011. Examples of methods for constructing 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 20140186397, which is incorporated herein by reference in its entirety; and in Jones et al., Vaccine, 30:959-968, 2012.

[0072] A. HPIV1 vector In some embodiments, recombinant paramyxovirus may be recombinant HPIV1 containing a viral genome encoding HPIV1 N, P, C, M, F, HN, and L proteins. The nucleic acid sequences 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 end sequences, and the viral genome and antigenome promoters. An exemplary HPIV1 Washington / 1964 strain genome sequence is provided as GenBank accession number AF457102.1, the full text of which is incorporated herein by reference. This exemplary HPIV1 Washington / 1964 strain genome sequence is: HPIV1 N, Sequence ID No. 24 (GenBank protein number AAL89400.1, incorporated herein by reference), HPIV1 P, Sequence 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, Sequence ID No. 27 (GenBank protein number AAL89406.1, incorporated herein by reference) HPIV1 F, Sequence ID No. 28 (GenBank protein number AAL89407.1, incorporated herein by reference) HPIV1 HN, Sequence 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) These encode the N, P, C, M, F, HN, and L proteins, which are shown as follows.

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

[0074] Furthermore, the viral leader / genome promoter and trailer / antigenome promoter of the HPIV2 V94 strain, as indicated in GenBank accession number AF457102.1, are represented as nucleotides 1-96 and 15544-15600, respectively.

[0075] Recombinant paramyxoviruses can be recombinant HPIV1s containing viral genomes that encode HPIV1 N, P, C, M, F, HN, and L proteins as shown above, or that individually have at least 90% (e.g., at least 95%) sequence identity to the HPIV1 N, P, C, M, F, HN, and L proteins as shown above.

[0076] In some embodiments, recombinant paramyxovirus may be recombinant HPIV1 comprising a genome containing heterologous genes that encode recombinant viral glycoprotein ectodomains (such as RSV F ectodomains) derived from type I membrane proteins that are linked to HPIV1 F proteins TM and CT as shown below, or that encode recombinant viral glycoprotein ectodomains (such as RSV F ectodomains) derived from type I membrane proteins that have at least 90% (at least 95%) sequence identity to HPIV1 F proteins TM and CT as shown below. Recombinant HPIV1 may include a genome containing heterologous genes encoding recombinant viral glycoprotein ectodomains derived from type I membrane proteins (such as the RSV F ectodomain). The HPIV1 F protein TM and CT sequences are publicly known (see, for example, GenBank accession number AF457102.1, 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, Sequence ID 31

[0077] B.HPIV2 Vector In some embodiments, the recombinant paramyxovirus vector may be recombinant HPIV2 containing 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 end sequences, as well as the viral genome and antigenome promoters. An exemplary HPIV2 V94 strain genome sequence is provided as GenBank accession number AF533010.1, which is incorporated herein by reference in its entirety. This exemplary HPIV2 V94 strain genome sequence is: HPIV2 N, Sequence ID No. 32 (coded by GenBank number AF533010.1, incorporated herein by reference) HPIV2 P, Sequence ID No. 33 (coded by GenBank number AF533010.1, incorporated herein by reference) HPIV2 V, Sequence ID No. 34 (ORF of P, incorporated herein by reference, code by GenBank number AF533010.1) HPIV2 M, Sequence ID No. 35 (coded by GenBank number AF533010.1, incorporated herein by reference) HPIV2 F, Sequence ID No. 36 (coded by GenBank number AF533010.1, incorporated herein by reference) HPIV2 HN, Sequence ID 37 (coded by GenBank number AF533010.1, incorporated herein by reference) HPIV2 L, Sequence ID No. 38 (coded by GenBank number AF533010.1, incorporated herein by reference) These encode the N, P, V, M, F, HN, and L proteins, which are represented as follows.

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

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

[0080] Recombinant paramyxoviruses can be recombinant HPIV2 containing viral genomes that encode HPIV2 N, P, V, M, F, HN, and L proteins as shown above, or that individually have at least 90% (e.g., at least 95%) sequence identity to the HPIV2 N, P, V, M, F, HN, and L proteins as shown above.

[0081] In some embodiments, recombinant paramyxovirus may be recombinant HPIV2 comprising a genome containing heterologous genes that encode recombinant viral glycoprotein ectodomains (such as RSV F ectodomains) derived from type I membrane proteins linked to HPIV2 F proteins TM and CT as shown below, or that encode recombinant viral glycoprotein ectodomains (such as RSV F ectodomains) derived from type I membrane proteins linked to HPIV2 F proteins TM and CT having at least 90% (at least 95%) sequence identity to HPIV2 F proteins TM and CT as shown below. The HPIV2 F protein™ and CT sequences are publicly known (see, for example, GenBank accession number AF533010.1 incorporated herein by reference). Exemplary HPIV2 F protein™ and CT sequences from the HPIV3 JS strain are shown below: HPIV2 F™ 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, Sequence ID 39

[0082] C.HPIV3 vector In some embodiments, recombinant paramyxovirus may be recombinant HPIV3 containing 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 end sequences, and viral genome and antigenome promoters. An exemplary HPIV3 JS strain genome sequence is provided as 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, Sequence ID No. 40 (encoded by nucleotides 111-1658 of GenBank number Z11575, incorporated herein by reference) HPIV3 P, SEQ ID NO: 41 (encoded by nucleotides 1784-3595 of GenBank number Z11575, incorporated herein by reference) HPIV3 C, Sequence ID No. 114 (encoded by nucleotides 1794-2393 of GenBank number Z11575, incorporated herein by reference) HPIV3 M, SEQ ID NO: 42 (encoded by nucleotides 3753-4814 of GenBank number Z11575, incorporated herein by reference) HPIV3 F, Sequence ID No. 43 (encoded by nucleotides 5072-6691 of GenBank number Z11575, incorporated herein by reference) HPIV3 HN, Sequence ID No. 44 (encoded by nucleotides 6806-8524 of GenBank number Z11575, incorporated herein by reference) HPIV3 L, Sequence ID No. 45 (encoded by nucleotides 8646-15347 of GenBank number Z11575, incorporated herein by reference)

[0083] In some embodiments, the HN gene in the HPIV3 vector encodes an HPIV3 HN protein containing the amino acid sequence shown below. (Sequence ID 101)

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

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

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

[0087] Recombinant paramyxoviruses can be recombinant HPIV3s containing viral genomes that encode HPIV3 N, P, C, M, F, HN, and L proteins as shown above, or that individually have at least 90% (e.g., at least 95%) sequence identity to the HPIV3 N, P, C, M, F, HN, and L proteins as shown above.

[0088] In some embodiments, recombinant paramyxovirus may be recombinant HPIV3 comprising a genome containing heterologous genes that encode recombinant viral glycoprotein ectodomains (such as RSV F ectodomains) derived from type I membrane proteins linked to HPIV3 F proteins TM and CT as shown below, or that encode recombinant viral glycoprotein ectodomains (such as RSV F ectodomains) derived from type I membrane proteins linked to HPIV3 F proteins TM and CT as shown below, having at least 90% (at least 95%) sequence identity to HPIV3 F proteins TM and CT as shown below. In some embodiments, recombinant paramyxovirus may be recombinant HPIV3 comprising a genome containing heterologous genes that encode recombinant viral glycoprotein ectodomains (such as RSV F ectodomains) derived from type I membrane proteins linked to HPIV3 F proteins CT as shown below, or that encode at least 90% (at least 95%) sequence identity to HPIV3 F proteins CT as shown below. Recombinant HPIV3 may contain a genome containing heterologous genes that encode recombinant viral glycoprotein ectodomains (such as the RSV F ectodomain) derived from type I membrane proteins, linked to HPIV3 F protein CT, which has sequence identity of at least 95%. The HPIV3 F protein TM and CT sequences are publicly known (see, for example, the protein encoded by nucleotides 5072-6691 of GenBank number Z11575). Exemplary HPIV3 F protein TM and CT sequences from the HPIV3 JS strain are shown below: HPIV3 F™ domain:IIIILIMIIILFIINITIITIAI, residues 1-23 of SEQ ID NO: 46 HPIV3 F CT:KYYRIQKRNRVDQNDKPYVLTNK, residues 24-46 of sequence number 46 HPIV3 F TM+CT:IIIILIMIIILFIINITIITIAIKYYRIQKRNRVDQNDKPYVLTNK, Sequence ID 46

[0089] D. Bovine PIV3 and Chimeric Human / Bovine PIV3 Vectors In some embodiments, recombinant paramyxoviruses may be bovine PIV3 (BPIV3) or chimeric paramyxoviruses containing a viral genome encoding combinations of N, P, C, V, M, F, HN, and L proteins derived from BPIV3 and HPIV3. For example, a chimeric viral genome can encode HPIV3 F and HN proteins as well as 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 as GenBank accession number 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, Sequence ID 47 (GenBank accession number: AAF28254, each encoded by nucleotides 111-1658 of GenBank number AF178654, as incorporated herein by reference) BPIV3 P, Sequence ID 48 (GenBank accession number: AAF28255, each encoded by nucleotides 1784-3574 of GenBank number AF178654, as incorporated herein by reference) BPIV3 C, Sequence ID No. 115 (encoded by nucleotides 1794-2399 of GenBank number AF178654, incorporated herein by reference) BPIV3 V, Sequence ID 116 (a 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, Sequence ID No. 49 (GenBank accession number: AAF28256, each encoded by nucleotides 3735-4790 of GenBank number AF178654, as incorporated herein by reference) BPIV3 F, Sequence ID 50 (each encoded by nucleotides 5066-6688 of GenBank number AF178654, GenBank accession number: AAF28257, as incorporated herein by reference) BPIV3 HN, Sequence ID 51 (GenBank accession number: AAF28258, each encoded by nucleotides 6800-8518 of GenBank number AF178654, as incorporated herein by reference) BPIV3 L, Sequence ID No. 52 (each encoded by nucleotides 8640-15341 of GenBank number AF178654, 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 a variant thereof, comprising an amino acid sequence shown 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 may contain, instead of the BPIV3 F gene, a gene encoding the HPIV3 F amino acid sequence or a variant thereof, for example, the HPIV3 F sequence shown as SEQ ID NO: 43.

[0092] The corresponding start and termination sequences for these BPIV3 genes are provided below: [Table 4]

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

[0094] Recombinant paramyxoviruses containing viral genomes encoding N, P, C, V, M, F, HN, and L proteins derived from HPIV3 and BPIV3 viruses may encode a mixture of HPIV3 and BPIV3 N, P, C, V, M, F, HN, and L proteins as shown above, or individually, a mixture of BPIV3 and HPIV3 N, P, C, V, M, F, HN, and L proteins having at least 90% (e.g., at least 95%) sequence identity to the BPIV3 or HPIV3 N, P, C, V, M, F, HN, and L proteins as shown 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 shown above, or individually, HPIV3 F and HN proteins and BPIV3 N, P, C, V, M, and L proteins having at least 90% (e.g., at least 95%) sequence identity to the corresponding HPIV3 F and HN proteins or BPIV3 N, P, C, V, M, and L proteins shown above.

[0096] In some embodiments, recombinant paramyxoviruses containing genomes encoding N, P, C, V, M, F, HN, and L proteins derived from BPIV3 are linked to the TM and CT of the BPIV3 F protein as shown below, or as shown below The recombinant paramyxovirus, comprising a genome encoding the N, P, C, V, M, F, HN, and L proteins derived from BPIV3, may further include heterogeneous genes encoding recombinant viral glycoprotein ectodomains (such as RSV F ectodomains) derived from type I membrane proteins, which are linked to the TM and CT having at least 90% (e.g., at least 95%) sequence identity to the TM and CT of the BPIV3 F protein, as shown below.

[0097] In some embodiments, recombinant paramyxoviruses comprising genomes encoding N, P, C, V, M, F, HN, and L proteins derived 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 heterologous genes encoding recombinant viral glycoprotein ectodomains (such as RSV F ectodomains) derived from type I membrane proteins, which are linked to the TM and CT of the BPIV3 F protein as shown below, or which have at least 90% (at least 95%, etc.) sequence identity to the TM and CT of the BPIV3 F protein as shown below. In some embodiments, recombinant paramyxoviruses containing genomes encoding N, P, C, V, M, F, HN, and L proteins derived from HPIV3 and BPIV3 viruses may further include heterologous genes encoding recombinant viral glycoprotein ectodomains (such as RSV F ectodomains) derived from type I membrane proteins, which are linked to the CT of the BPIV3 F protein as shown below, or to CTs having at least 90% (e.g., at least 95%) sequence identity to the CT of the BPIV3 F protein as shown below. Exemplary BPIV3 F protein TM and CT sequences derived from the BPIV3 Kansas strain are: BPIV3 F™ domain: ITIIIVMIIILVIINITIIVV, residues 1-21 of SEQ ID NO: 53 BPIV3 F CT: IIKFHRIQGKDQNDKNSEPYILTNRQ, residues 22-57 of sequence number 53 BPIV3 F TM+CT:ITIIIVMIIILVIINITIIVVIIKFHRIQGKDQNDKNSEPYILTNRQ, Sequence ID 53 It is shown as follows.

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

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

[0100] F.NDV In some embodiments, recombinant paramyxoviruses are NDV as shown below. A recombinant NDV virus may be a recombinant NDV virus comprising a recombinant viral genome encoding NDV N, P, V, M, F, HN, and L proteins, comprising a heterogeneous gene encoding a recombinant viral glycoprotein ectodomain (such as the RSV F ectodomain) derived from a type I membrane protein, which is linked to the TM and CT of the F protein, or to a TM and CT having at least 90% (at least 95%) sequence identity to the TM and CT of the NDV F protein as shown below. In some embodiments, a recombinant paramyxovirus may be a recombinant NDV virus comprising a recombinant viral genome encoding NDV N, P, V, M, F, HN, and L proteins, comprising a heterogeneous gene encoding a recombinant viral glycoprotein ectodomain (such as the RSV F ectodomain) derived from a type I membrane protein, which is linked to the TM and CT of the NDV F protein as shown below, or to a CT having at least 90% (at least 95%) sequence identity to the CT of the NDV F protein as shown below. The NDV virus F protein TM and CT sequences are publicly known (see, for example, GenBank accession number AAC28374, incorporated herein by reference). Exemplary NDV virus F protein TM and CT sequences are: NDV F™ domain: IVRTIISLVFGILSLILACYL (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, Sequence ID 104 It is shown as follows.

[0101] G. Heterogenetics A recombinant paramyxovirus vector comprises a recombinant genome containing one or more heterologous genes encoding the ectodomain of one or more heterologous envelope proteins (or antigenic fragments thereof) of heterologous viral pathogens, wherein the ectodomain is linked to the TM and CT of the envelope protein derived from the recombinant paramyxovirus. For example, one or more heterologous envelope proteins (or antigenic fragments thereof) derived from measles virus, subgroup A or subgroup B respiratory syncytial virus, mumps virus, human papillomavirus, type 1 or 2 human immunodeficiency virus, 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 paramyxovirus. Examples of useful envelope proteins, but not limited to them, include 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 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, as well as their antigenic domains, fragments and epitopes.Exemplary methods of inserting one or more heterologous genes or transcription units into a paramyxovirus virus genome or antigenome are described in WO04 / 027037 and US2013 / 0052718, each incorporated herein by reference.

[0102] In some embodiments, the heterologous gene contained in the recombinant paramyxovirus genome encodes an ectodomain of an 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 mainly include subgroups A1, A2, B1, and B2 based on the sequence variability of the attachment (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] Exemplary human RSV F protein sequences derived from subgroup A2 and corresponding GenBank references (incorporated herein by reference in their entirety) are shown below:

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

[0105] As illustrated by the sequences described above, the hRSV F protein exhibits remarkable sequence conservation, with over 85% sequence identity across hRSV subgroups. Given the conservation and breadth of knowledge of RSV F sequences, those skilled in the art can readily identify the corresponding RSV F amino acid positions between various RSV F chains and subgroups. The numbering of amino acid substitutions disclosed herein is performed with reference to the exemplary hRSV F protein sequence from strain A2, shown as Sequence ID No. 1, unless otherwise noted.

[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) derived from strain A2 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 (Sequence ID 1, residues 137-574): FLGFLLGVGSAIAGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTT PVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQA ETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEG KSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN F1 ectodomain of 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, heterologous genes contained in the recombinant paramyxovirus genome encode the ectodomain of the human RSV F protein, where the RSV F ectodomain contains at least 85% (at least 90% or at least 95%) the same amino acid sequence as one of the RSV ectodomains from sequence numbers 1 (WT RSV FA), 2 (WT RSV FB), 12 (A2 HEK), 14 (A2 HEK+DS), or 19 (A2 HEK+DS-Cav1), or contains the amino acid sequence of the RSV ectodomain of sequence numbers 12, 14, or 19.

[0108] In some embodiments, recombinant paramyxoviruses may comprise a genome containing heterologous genes encoding recombinant hRSV F protein that have been codon-optimized for expression in human cells. For example, genes encoding recombinant hRSV F protein can be codon-optimized for human expression using GA, DNA2.0(D2), or GenScript(GS) optimization algorithms (see Example 1). Examples of nucleic acid sequences encoding RSV F protein that have been codon-optimized for expression in human cells are provided below: 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 sequences (for human expression) are provided below.

[0110] RSV F proteins encoded by heterologous genes 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, for example, the stability of the RSV F protein in the pre-fusion conformation. In some embodiments, the RSV F protein may contain a glutamate substitution at position 66, a proline substitution at position 101, or both. For example, the RSV F protein may contain the "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 substitution (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 include 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 include a "DS" substitution of a pair of cysteine ​​substitutions at positions 155 and 290 that forms a non-native disulfide bond and stabilizes the RSV F protein in its pre-fusion conformation. In some embodiments, RSV The F protein may contain amino acid substitutions that fill one or more cavities at positions 190 and / or 207, thereby stabilizing 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 contain the "Cav1" substitutions S190F and F207L. In some embodiments, the RSV F protein may contain the DS-Cav1 substitutions 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™ and / or CT domains) containing the DS-Cav1 amino acid substitution are shown as SEQ ID NOs. 10-11 and 21-23.

[0112] Further 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 full text of which is incorporated herein by reference. HEK substitutions can be combined with any of the amino acid substitutions for stabilizing the RSV F protein in the pre-fusion conformation.

[0113] In some embodiments, heterologous genes contained within the recombinant paramyxovirus genome encode recombinant RSV F ectodomains that are linked to the TM and CT of the recombinant paramyxovirus F protein.

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

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

[0116] In one embodiment, the recombinant paramyxovirus may be recombinant HPIV3 containing a genome with a heterologous gene encoding the recombinant hRSV F ectodomain. The recombinant RSV F ectodomain can be ligated with TM and CT derived from the HPIV3 F protein, for example, as residues 1-23 (TM) of SEQ ID NO: 46, residues 24-46 (CT) of SEQ ID NO: 46, or SEQ ID NO: 46 (TM+CT). Exemplary sequences are provided below: hRSV F protein from strain A2 containing HEK and DS-Cav1 substitutions and the HPIV3 F CT domain (RSV F_HEK_DS-Cav1_H3CT) protein sequence: (Sequence 8) GenScript optimized RSV F_HEK_DS-Cav1_H3CT DNA sequence: The protein sequence of the hRSV F protein (RSV F_HEK_DS-Cav1_H3TMCT) derived from strain A2, containing HEK and DS-Cav1 substitutions and HPIV3 F TM and CT domains: MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHL EGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSP LCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIILIMIIILFIINITIITIAIKYYRIQKRNRVDQNDKPYVLTNK(Sequence ID 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, where the viral genome further comprises heterologous genes encoding recombinant hRSV F ectodomains linked to TM and / or CT from the BPIV3 F protein, for example, as shown as 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, including 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 paramyxovirus, are shown below. The hRSV F protein (RSV F_A2_HEK_B3TMCT) protein sequence from strain A2 containing HEK substitution and BPIV3 F TM and CT domains: MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTITIIIVMIIILVIINITIIVVIIKFHRIQGKDQNDKNSEPYILTNRQ(SEQ ID NO:12) GeneArt optimized RSV F_A2_HEK_B3TMCT DNA sequence: The hRSV F protein (RSV F_A2_HEK_DS_B3CT) protein sequence from strain A2, including HEK and DS substitutions, the hRSV F™ domain, and the BPIV3 F CT domain: (Sequence ID 14) gaaccggggcatcatcaagaccttcagcaacggctgcgactacgtgtccaacaagggggtggacaccgtgtctgtgggcaacaccctgtactacgtgaacaaacaggaaggcaa gagcctgtacgtgaagggcgagcccatcatcaacttctacgaccccctggtgttccccagcgacgagttcgatgccagcatctcccaagtgaacgagaagatcaaccagagcctg gccttcatcagaaagtccgatgagctgctgcacaatgtgaacgccggcaagtccaccaccaatatcatgatcaccacaatcatcatcgtgattatcgtgatcctgctgagcctga tcgccgtgggcctgctgctgtactgtatcatcaagttccaccggatccagggcaaggaccagaacgacaagaactccgagccctacatcctgacaaaccggcagtga (SEQ ID NO: 15) The hRSV F protein (RSV F_A2_HEK_DS-Cav1_B3CT) protein sequence from strain A2, containing HEK and DS-Cav1 substitutions, the hRSV FTM domain, and the BPIV3 F CT 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 The protein sequence of the hRSV F protein (RSV F_A2_HEK_DS_B3TMCT) derived from strain A2, including HEK and DS substitutions and BPIV3 F TM and CT domains: (Sequence ID 19) GeneArt optimized RSV F_A2_HEK_DS_B3TMCT DNA sequence: Genescript-optimized RSV F_A2_HEK_DS_B3TMCT DNA sequence: The hRSV F protein (RSV F_A2_HEK_DS-Cav1_B3TMCT) protein sequence from strain A2 containing HEK and DS-Cav1 substitutions and BPIV3 F TM and CT domains: (Sequence ID 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 containing a heterologous gene encoding a recombinant hRSV F ectodomain. In such embodiments, the TM and CT linked to the RSV F ectodomain may be derived from the Sendai virus F protein, for example, 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 may contain an amino acid sequence shown as one of SEQ ID NOs: 105-108. hRSV F protein (RSV F_A2_HEK_SeVCT) protein sequence from strain A2 containing HEK substitution and Sendai virus F CT domain MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLN NAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVN AGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKV QSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDP LVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCLYRLRRSMLMGNPDDRIPRDTYTLEPKIRHMYTNGGFDAMAEKR (Sequence ID 105) The hRSV F protein (RSV F_A2_HEK_SeVTMCT) protein sequence (SEQ ID NO: 106) derived from strain A2 containing HEK substitution and Sendai virus F TM and CT domains. The hRSV F protein (RSV F_A2_HEK_SeVCT) protein sequence derived from strain A2 containing HEK and DS-Cav1 substitutions and the Sendai virus F CT domain. (Sequence ID 107) The hRSV F protein (RSV F_A2_HEK_SeVTMCT) protein sequence derived from strain A2 containing HEK and DS-Cav1 substitutions and Sendai virus F TM and CT domains. (Sequence ID 108)

[0119] In one embodiment, the recombinant paramyxovirus comprises a recombinant NDV genome containing a heterologous gene encoding the recombinant hRSV F ectodomain. In such embodiments, the TM and CT linked to the RSV F ectodomain may be derived from the NDV viral F protein, cytoplasmic tail, for example, as 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 may contain an amino acid sequence shown as one of SEQ ID NOs: 109-113. hRSV F protein (RSV F_A2_HEK_NDVCT) protein sequence from strain A2 containing HEK substitution and NDV F CT domain MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYID KQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKT DVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCMYKQKAQQKTLLWLGNNTLDQMRATTKM (Sequence ID 109) The hRSV F protein (RSV F_A2_HEK_NDVTMCT) protein sequence from strain A2 containing HEK substitution and NDV F TM and CT domains. (Sequence ID 110) hRSV F protein (RSV F_A2_HEK_NDVCT) protein sequence from strain A2 containing HEK and DS-Cav1 substitutions and NDV F CT domain (Sequence ID 112) The hRSV F protein (RSV F_A2_HEK_NDVTMCT) protein sequence derived from strain A2, containing HEK and DS-Cav1 substitutions and NDV F TM and CT domains. (Sequence ID 113)

[0120] Further explanation of H. recombinant paramyxovirus Specific Embodiments In some embodiments, a recombinant paramyxovirus is provided, comprising a viral genome comprising a PIV genome promoter followed by a heterogeneous gene encoding a type I membrane protein, which includes the PIV N, P, M, F, HN, and L genes, and a recombinant RSV F ectodomain, where the heterogeneous gene is located between the genome promoter and the gene encoding the N protein, and the RSV F ectodomain includes 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 comprising a viral genome comprising, from upstream to downstream, the HPIV1 genome promoter and thereafter, heterologous genes encoding type I membrane proteins, including the HPIV1 N, P, M, F, HN, and L genes and recombinant RSV F ectodomain, in which heterologous genes are provided. The gene is located between the genome promoter and the gene encoding the N protein, RSV The F ectodomain contains 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 is provided comprising a viral genome comprising, from upstream to downstream, an HPIV1 genome promoter and, subsequently, heterologous genes encoding type I membrane proteins, including the HPIV1N, P, M, F, HN, and L genes, and the recombinant RSV F ectodomain, where the heterologous genes are located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain includes 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 comprising a viral genome comprising, from upstream to downstream, an HPIV3 genome promoter, followed by heterologous genes encoding type I membrane proteins, including the HPIV3 N, P, M, F, HN, and L genes, and recombinant RSV F ectodomain, where the heterologous genes are located between the genome promoter and the gene encoding the N protein, and RSV The F ectodomain contains 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 is provided comprising a viral genome comprising, from upstream to downstream, an HPIV3 genome promoter, followed by heterologous genes encoding type I membrane proteins, including the HPIV3 N, P, M, F, HN, and L genes, and the recombinant RSV F ectodomain, where the heterologous genes are located between the genes encoding the N protein and the genes encoding the P protein, and the RSV F ectodomain comprises 66E, 101P, 155C, 290C, 190F, and 207L substitutions, linked to the TM and CT of the HPIV3 F protein.

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

[0127] In some embodiments, a recombinant paramyxovirus is provided comprising a viral genome comprising a BPIV3 genome promoter, followed by heterologous genes encoding type I membrane proteins, including the BPIV3 N, P, M, F, HN, and L genes, and recombinant RSV F ectodomain, arranged from upstream to downstream, wherein the heterologous genes are located between the genome promoter and the gene encoding the N protein, and RSV The F ectodomains are 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 is provided comprising a viral genome comprising a BPIV3 genome promoter, followed by heterologous genes encoding type I membrane proteins, including the BPIV3 N, P, M, F, HN, and L genes, and the recombinant RSV F ectodomain, where the heterologous genes are 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, linked to the TM and CT of the BPIV3 F protein.

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

[0130] In some embodiments, a recombinant paramyxovirus is provided comprising a recombinant B / HPIV3 viral genome comprising, from upstream to downstream, a BPIV3 genome promoter, followed by heterologous genes encoding type I membrane proteins, including the BPIV3 N, P, and M genes, HPIV3 F and HN genes, and the BPIV3 L gene, and further comprising the recombinant RSV F ectodomain, wherein the heterologous genes are 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 is provided comprising a recombinant B / HPIV3 viral genome comprising, from upstream to downstream, a BPIV3 genome promoter, followed by heterologous genes encoding type I membrane proteins, including the BPIV3 N, P, and M genes, HPIV3 F and HN genes, and the BPIV3 L gene, and further comprising the recombinant RSV F ectodomain, wherein the heterologous genes are located between the genome 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 HPIV3 F protein.

[0132] In some embodiments, a recombinant paramyxovirus is provided comprising a recombinant B / HPIV3 viral genome comprising, from upstream to downstream, a BPIV3 genome promoter, followed by heterologous genes encoding type I membrane proteins, including the BPIV3 N, P, and M genes, HPIV3 F and HN genes, and the BPIV3 L gene, and further comprising the recombinant RSV F ectodomain, wherein the heterologous genes are 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 that includes a Sendai virus genome promoter followed by a heterologous gene encoding a type I membrane protein, which includes the Sendai virus N, P, M, F, HN, and L genes, and which comprises recombinant RSV F ectodomain, from upstream to downstream. In this configuration, the heterologous gene is located between the genome 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 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 genome promoter, followed by a heterogeneous gene encoding a type I membrane protein, which comprises the Sendai virus N, P, M, F, HN, and L genes, and which comprises a recombinant RSV F ectodomain, where the heterogeneous 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 genome promoter, followed by heterologous genes encoding type I membrane proteins, including the NDV N, P, M, F, HN, and L genes, and the recombinant RSV F ectodomain, where the heterologous genes are located between the genome promoter and the gene encoding the N protein, and the RSV F ectodomain comprises 66E, 101P, 155C, 290C, 190F, and 207L substitutions, 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 genome promoter and thereafter a heterologous gene encoding a type I membrane protein, comprising the NDV N, P, M, F, HN, and L genes, and the heterologous gene being located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprising 66E, 101P, 155C, 290C, 190F, and 207L substitutions, linked to the TM and CT of the NDV F protein.

[0137] In some embodiments, a recombinant paramyxovirus is provided comprising a viral genome comprising a PIV5 genome promoter, followed by a heterologous gene encoding a type I membrane protein, comprising the PIV5 N, P, M, F, HN, and L genes, and a recombinant RSV F ectodomain, where the heterologous gene is located between the genome 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 PIV5 F protein.

[0138] In some embodiments, a recombinant paramyxovirus containing recombinant PIV5 is provided, comprising a viral genome comprising, from upstream to downstream, a PIV5 genome promoter, followed by heterologous genes encoding type I membrane proteins, including the PIV5 N, P, M, F, HN, and L genes, and the recombinant RSV F ectodomain, where the heterologous genes are 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, linked to the TM and CT of the PIV5 F protein.

[0139] In some embodiments, a recombinant paramyxovirus is provided that includes a recombinant parainfluenza virus (PIV) comprising a viral genome further comprising a PIV genome promoter followed by PIV N, P, M, F, HN, and L genes, and heterologous genes encoding type I membrane proteins including the recombinant RSV F ectodomain. In this configuration, the heterologous gene is located between the genome 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 PIV F protein.

[0140] In some embodiments, a recombinant paramyxovirus is provided comprising a viral genome comprising, from upstream to downstream, the HPIV1 genome promoter and, subsequently, heterologous genes encoding type I membrane proteins, including the HPIV1 N, P, M, F, HN, and L genes, and recombinant RSV F ectodomain, where the heterologous genes are located between the genome promoter and the gene encoding the N protein, and RSV 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 is provided comprising a viral genome comprising, from upstream to downstream, the HPIV1 genome promoter and, thereafter, heterologous genes encoding type I membrane proteins, including the HPIV1N, P, M, F, HN, and L genes, and the heterologous genes located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprising 66E, 101P, 155C, 290C, 190F, and 207L substitutions, linked to the CT of the HPIV1 F protein.

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

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

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

[0146] In some embodiments, a recombinant paramyxovirus is provided comprising a viral genome comprising a BPIV3 genome promoter, followed by heterologous genes encoding type I membrane proteins, including the BPIV3 N, P, M, F, HN, and L genes, and recombinant RSV F ectodomain, arranged from upstream to downstream, wherein the heterologous genes are located between the genome promoter and the gene encoding the N protein, and 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 is provided comprising a viral genome comprising a BPIV3 genome promoter, followed by heterologous genes encoding type I membrane proteins, including the BPIV3 N, P, M, F, HN, and L genes, and the recombinant RSV F ectodomain, where the heterologous genes are 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, linked to the CT of the BPIV3 F protein.

[0148] In some embodiments, a recombinant paramyxovirus is provided comprising a recombinant B / HPIV3 viral genome comprising, from upstream to downstream, a BPIV3 genome promoter, followed by heterologous genes encoding type I membrane proteins, including the BPIV3 N, P, and M genes, HPIV3 F and HN genes, and the BPIV3 L gene, and further comprising the recombinant RSV F ectodomain, wherein the heterologous genes are located between the genome 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 CT of the BPIV3 F protein.

[0149] In some embodiments, a recombinant paramyxovirus is provided comprising a recombinant B / HPIV3 viral genome comprising, from upstream to downstream, a BPIV3 genome promoter, followed by heterologous genes encoding type I membrane proteins, including the BPIV3 N, P, and M genes, HPIV3 F and HN genes, and the BPIV3 L gene, and further comprising the recombinant RSV F ectodomain, wherein the heterologous genes are 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 is provided comprising a recombinant B / HPIV3 viral genome comprising, from upstream to downstream, a BPIV3 genome promoter, followed by heterologous genes encoding type I membrane proteins, including the BPIV3 N, P, and M genes, HPIV3 F and HN genes, and the BPIV3 L gene, and further comprising the recombinant RSV F ectodomain, wherein the heterologous genes are located between the genome 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 CT of the HPIV3 F protein.

[0151] In some embodiments, a recombinant paramyxovirus is provided comprising a recombinant B / HPIV3 viral genome comprising, from upstream to downstream, a BPIV3 genome promoter and thereafter heterologous genes encoding type I membrane proteins, including the BPIV3 N, P, and M genes, HPIV3 F and HN genes, and the BPIV3 L gene, and the recombinant RSV F ectodomain, in which the heterologous genes encode the N protein. Located between the gene encoding the P protein and the RSV F ectodomain, the RSV F ectodomain contains 66E, 101P, 155C, 290C, 190F, and 207L substitutions 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 genome promoter, followed by heterologous genes encoding type I membrane proteins, including the Sendai virus N, P, M, F, HN, and L genes, and the recombinant RSV F ectodomain, where the heterologous genes are located between the genome 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 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 genome promoter and thereafter a heterologous gene encoding a type I membrane protein, comprising the Sendai virus N, P, M, F, HN, and L genes, and the heterologous gene being located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprising 66E, 101P, 155C, 290C, 190F, and 207L substitutions and 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 genome promoter and, subsequently, heterologous genes encoding type I membrane proteins, including the NDV N, P, M, F, HN, and L genes, and the recombinant RSV F ectodomain, where the heterologous genes are located between the genome promoter and the gene encoding the N protein, and the RSV F ectodomain comprises 66E, 101P, 155C, 290C, 190F, and 207L substitutions 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 genome promoter and thereafter a heterologous gene encoding a type I membrane protein, comprising the NDV N, P, M, F, HN, and L genes, and the heterologous gene being located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprising 66E, 101P, 155C, 290C, 190F, and 207L substitutions, linked to the CT of the NDV F protein.

[0156] In some embodiments, a recombinant paramyxovirus is provided comprising a viral genome comprising a PIV5 genome promoter, followed by a heterologous gene encoding a type I membrane protein, comprising the PIV5 N, P, M, F, HN, and L genes, and a recombinant RSV F ectodomain, where the heterologous gene is located between the genome 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 CT of the PIV5 F protein.

[0157] In some embodiments, a recombinant paramyxovirus is provided comprising a viral genome comprising a PIV5 genome promoter, followed by a heterogeneous gene encoding a type I membrane protein, comprising the PIV5 N, P, M, F, HN, and L genes, and the recombinant RSV F ectodomain, in which the heterogeneous gene is located between the gene encoding the N protein and the gene encoding the P protein, and R The SV F ectodomain contains 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 that includes a recombinant parainfluenza virus (PIV) comprising a viral genome further comprising a heterologous gene encoding a type I membrane protein, which includes a PIV genome promoter and, subsequently, PIV N, P, M, F, HN, and L genes, and which includes a recombinant RSV F ectodomain, in which the heterologous gene is located between the genome promoter and the gene encoding the N protein, and the RSV F ectodomain includes a 66E, 101P, 155C, 290C substitution and is linked to the TM and CT of the PIV F protein.

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

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

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

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

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

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

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

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

[0171] In some embodiments, a recombinant paramyxovirus is provided comprising a viral genome comprising a recombinant Sendai virus, which comprises, from upstream to downstream, a Sendai virus genome promoter, followed by heterologous genes encoding type I membrane proteins, including the Sendai virus N, P, M, F, HN, and L genes, and the recombinant RSV F ectodomain, where the heterologous genes are located between the genome promoter and the gene encoding the N protein, and the RSV F ectodomain comprises the 66E, 101P, 155C, 290C substitution 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 viral genome comprising a recombinant Sendai virus, which in turn comprises a Sendai virus genome promoter, followed by a heterogeneous gene encoding a type I membrane protein, which comprises the Sendai virus N, P, M, F, HN, and L genes, and which comprises a recombinant RSV F ectodomain, where the heterogeneous gene is located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprises the 66E, 101P, 155C, 290C substitution 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 an NDV genome promoter, followed by heterologous genes encoding type I membrane proteins, including the NDV N, P, M, F, HN, and L genes, and a recombinant RSV F ectodomain, where the heterologous genes are located between the genome promoter and the gene encoding the N protein, and the RSV F ectodomain comprises the 66E, 101P, 155C, 290C substitution, 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 an NDV genome promoter, followed by heterogeneous genes encoding type I membrane proteins, including the NDV N, P, M, F, HN, and L genes, and a recombinant RSV F ectodomain, where the heterogeneous genes are located between the genes encoding the N protein and the P protein, and the RSV F ectodomain comprises the 66E, 101P, 155C, 290C substitution and is linked to the TM and CT of the NDV F protein.

[0175] In some embodiments, a recombinant paramyxovirus is provided comprising a viral genome comprising a PIV5 genome promoter, followed by a heterologous gene encoding a type I membrane protein, comprising the PIV5 N, P, M, F, HN, and L genes, and a recombinant RSV F ectodomain, where the heterologous gene is located between the genome promoter and the gene encoding the N protein, and the RSV F ectodomain comprises the 66E, 101P, 155C, 290C substitution and is linked to the TM and CT of the PIV5 F protein.

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

[0177] In some embodiments, a recombinant paramyxovirus is provided that includes a recombinant parainfluenza virus (PIV) comprising a viral genome further comprising a heterologous gene encoding a type I membrane protein, which includes a PIV genome promoter and, subsequently, PIV N, P, M, F, HN, and L genes, and which includes a recombinant RSV F ectodomain, where the heterologous gene is located between the genome promoter and the gene encoding the N protein, and the RSV F ectodomain includes a 66E, 101P, 155C, 290C substitution and is linked to the CT of the PIV F protein.

[0178] In some embodiments, a recombinant paramyxovirus is provided comprising a viral genome comprising, from upstream to downstream, the HPIV1 genome promoter and, subsequently, heterologous genes encoding type I membrane proteins, including the HPIV1 N, P, M, F, HN, and L genes, and recombinant RSV F ectodomain, where the heterologous genes are located between the genome promoter and the gene encoding the N protein, and RSV The F ectodomain includes the 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 is provided comprising a viral genome comprising, from upstream to downstream, an HPIV1 genome promoter and thereafter a heterologous gene encoding a type I membrane protein, comprising the HPIV1 N, P, M, F, HN, and L genes, and the recombinant RSV F ectodomain, where 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 the 66E, 101P, 155C, 290C substitution and is linked to the CT of the HPIV1 F protein.

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

[0182] In some embodiments, the HPIV3 genome promoter is routed from upstream to downstream, and to it A recombinant paramyxovirus containing recombinant HPIV3 is provided, which includes a viral genome further comprising heterologous genes encoding type I membrane proteins, including the HPIV3 N, P, M, F, HN, and L genes, and recombinant RSV F ectodomain, where the heterologous genes are located between the genome promoter and the gene encoding the N protein, and RSV The F ectodomain includes the 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 is provided comprising a viral genome comprising, from upstream to downstream, an HPIV3 genome promoter and thereafter a heterologous gene encoding a type I membrane protein, comprising the HPIV3 N, P, M, F, HN, and L genes, and the heterologous gene being located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprising the 66E, 101P, 155C, 290C substitution and linked to the CT of the HPIV3 F protein.

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

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

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

[0188] In some embodiments, the BPIV3 genome promoter is located upstream to downstream, and it is located downstream. A recombinant paramyxovirus containing recombinant B / HPIV3 is provided, which includes a viral genome further comprising heterologous genes encoding type I membrane proteins, including the BPIV3 N, P, and M genes, HPIV3 F and HN genes, and the BPIV3 L gene, and the recombinant RSV F ectodomain, where the heterologous genes are located between the genome promoter and the gene encoding the N protein, and the RSV F ectodomain contains the 66E, 101P, 155C, 290C substitution and is linked to the CT of the HPIV3 F protein.

[0189] In some embodiments, a recombinant paramyxovirus is provided comprising a recombinant B / HPIV3 viral genome comprising, from upstream to downstream, a BPIV3 genome promoter, followed by heterologous genes encoding type I membrane proteins, including the BPIV3 N, P, and M genes, HPIV3 F and HN genes, and the BPIV3 L gene, and further comprising a recombinant RSV F ectodomain, where the heterologous genes are located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprises the 66E, 101P, 155C, 290C substitution 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 genome promoter, followed by heterologous genes encoding type I membrane proteins, including the Sendai virus N, P, M, F, HN, and L genes, and the recombinant RSV F ectodomain, where the heterologous genes are located between the genome promoter and the gene encoding the N protein, and the RSV F ectodomain comprises the 66E, 101P, 155C, 290C substitution 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 genome promoter and thereafter a heterologous gene encoding a type I membrane protein, comprising the Sendai virus N, P, M, F, HN, and L genes, and the heterologous gene being located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprising the 66E, 101P, 155C, 290C substitution and 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 further comprising a heterologous gene encoding a type I membrane protein, which includes an NDV genome promoter and, subsequently, the NDV N, P, M, F, HN, and L genes, and which comprises a recombinant RSV F ectodomain, in which the heterologous gene is located between the genome promoter and the gene encoding the N protein, and the RSV F ectodomain comprises a 66E, 101P, 155C, 290C substitution 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 genome promoter and thereafter heterogeneous genes encoding type I membrane proteins, including the NDV N, P, M, F, HN, and L genes, and the recombinant RSV F ectodomain, where the heterogeneous genes are located between the gene encoding the N protein and the gene encoding the P protein, and the RSV F ectodomain comprises the 66E, 101P, 155C, 290C substitution and is linked to the CT of the NDV F protein.

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

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

[0196] Any embodiment of recombinant paramyxovirus disclosed herein, including a viral genome containing a heterologous gene encoding the RSV ectodomain (such as any of the recombinant paramyxoviruses discussed above).) Therefore, a heterologous gene encoding a recombinant RSV F ectodomain may encode a polypeptide sequence containing RSV F positions 1-529.

[0197] Further explanation The recombinant paramyxoviruses disclosed are self-replicating, i.e., they can replicate after infection with suitable host cells. In some embodiments, the recombinant paramyxoviruses have an attenuated phenotype when administered to, for example, human subjects.

[0198] Attenuation of recombinant paramyxoviruses can be achieved, for example, by introducing one or more mutations that cause alterations in the biological function of the recombinant paramyxovirus and result in an attenuated phenotype, using various methods known in the art. Insertion of heterologous genes can also result in an attenuated phenotype. Preferably, paramyxoviruses containing genomes encoding heterologous genes are attenuated about 100 to 5000 times or more in cells or mammals compared to wild-type paramyxoviruses.

[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 temperature-sensitive replication. The disclosed recombinant paramyxoviruses can also be tested in animal models of infection using heterologous viral pathogens contained in PIV and / or recombinant viruses (e.g., RSV). Various animal models are known.

[0200] Recombinant attenuated paramyxovirus is preferably attenuated about 100 to 5000 times in cells or mammals compared to wild-type paramyxovirus. In some embodiments, it is preferable that the level of viral replication in vitro is sufficient to provide for the production of a viral vaccine for widespread use. In some embodiments, the level of viral replication of the attenuated paramyxovirus in vitro is at least 10 per ml. 6 , more preferably at least 10 7 Most preferably, at least 10 8 It is preferable that the attenuating mutations are stable. Recombinant paramyxoviruses having at least two, three, four or more attenuating mutations are more likely to be stable.

[0201] Ongoing preclinical studies have identified several mutations or modifications that attenuate HPIV1, HPIV2, and HPIV3, which are introduced via reverse genetics to produce attenuated strains as potential vaccine and vector scaffolds. Including foreign genes in the HPIV scaffold is also an attenuation in itself. This is achieved through various effects, including increased genome length and gene number, as well as the effects of foreign proteins. When seeking to achieve an appropriate level of attenuation, the attenuating effect of inserts, whatever the cause, must also be taken into consideration.

[0202] Attenuated strains of HPIV1, 2, and 3 have been or are currently in clinical studies 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 their versions are potential vectors for expressing heterologous RSV F proteins.

[0203] Examples of modifications to the genome of paramyxoviruses that provide attenuated phenotypes are known in the art, for example, U.S. Patent Publications 2012 / 0045471; 2010 / 0119547; 2009 / 0263883; 2009 / 0017517; 8084037; and 6, respectively, whose full texts are incorporated herein by reference. 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. This is described in 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, PIV3 attenuation can be achieved by the presence of BPIV3-derived genes that confer host range restriction in primates, including humans, such as B / HPIV3 viruses containing BPIV3 genes except for F and HN derived 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). Another example of a debilitating point mutation is provided for HPIV1 in Example 2 below. Deletion of one or more codons, as exemplified by HPIV1 in Example 2, can also confer a debilitating phenotype. Furthermore, as exemplified in Example 1, the presence of vector TM and CT or CT domains linked to heterologous ectodomains can potently attenuate the vector. Another example of a debilitating mutation in HPIV1 is 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 determined by examining the number of animals that develop antibodies against paramyxoviruses after one and a second immunization in animal models (non-human primates, e.g., African green monkeys), as well as by assessing the response This can be evaluated by measuring the scale of the response. In some embodiments, the recombinant paramyxovirus is sufficiently immunogenic if about 60–80% of animals develop antibodies after a first immunization and about 80–100% of animals develop a response after a second immunization. Preferably, the immune response protects against infection by both the source paramyxovirus and viral pathogens from heterologous genes contained in the recombinant paramyxovirus.

[0205] I. Further Vectors Recombinant RSV F protein and the nucleic acid molecules encoding it may be found in (or expressed in) vectors other than PIV vectors. For example, plasmid vectors and other viral vectors can be used, for example, for the expression of recombinant RSV F protein or fragments thereof in host cells, or for immunization of 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 booster vaccine, for use in a prime-boost vaccination.

[0206] In some cases, the vector may be a viral vector that is capable of replication and / or attenuated. Viral vectors may also be conditionally capable of replication. In other cases, the viral vector is deficient in replication in host cells.

[0207] Polyomas, i.e., SV40 (Madzak et al., 1992, J. Gen. Virol., 73:1533-1536), adenoviruses (Berkner, 1992, Cur. Top. Microbiol. Immunol., 158:39-6; Berliner et al., 1988, Bio Techniques, 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) 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., 66:29522965; 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), Sindbisvirus (H. Herweijer et al., 1995, Human Gene Therapy 6:1161-1167; U.S. Patent 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), mouse-derived retroviruses (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 its immunogenic fragments, including those from human retroviruses (Page et al., 1990, J. Virol., 64:5370-5276; Buchschalcher et al., 1992, J. Virol., 66:2731-2739). Baculovirus (Autographa californica nuclear polyhedron disease 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 adenovirus vector expressing the disclosed recombinant RSV F protein or its immunogenic fragment (such as the RSV F ectodomain). Adenoviruses from various origins, subtypes, or mixtures of subtypes can be used as sources of the viral genome for the adenovirus vector. Adenovirus vectors can be constructed using non-human adenoviruses (e.g., monkey, chimpanzee, gorilla, bird, dog, sheep, or bovine adenoviruses). For example, monkey adenovirus can be used as a source of the viral genome for the adenovirus vector. The monkey adenovirus may be serotype 1, 3, 7, 11, 16, 18, 19, 20, 27, 33, 38, 39, 48, 49, 50, or any other monkey adenovirus serotype. The monkey adenovirus may be referred to by using any suitable abbreviation known in the art, such as SV, SAdV, SAV, or sAV. In some cases, monkey adenovirus vectors are serotypes 3, 7, 11, 16, 18, 19, 20, 27, 33, 38, or 39. In one example, a chimpanzee serotype C Ad3 vector is used (see, e.g., Peruzzi et al., Vaccine, 27:1293-1300, 2009). Human adenoviruses can be used as a source of viral genome for adenovirus vectors. Human adenoviruses can be from various subgroups or serotypes. For example, an adenovirus could be from 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 adenovirus vectors capable of replication and those lacking replication (including adenovirus vectors that are individually and multipleally lacking replication). Examples of adenovirus vectors lacking replication, including multipleally lacking replication vectors, are disclosed in U.S. Patents 5,837,511; 5,851,806; 5,994,106; 6,127,175; 6,482,616; and 7,195,896, as well as International Patent Application Nos. WO94 / 28152, WO95 / 02697, WO95 / 16772, WO95 / 34671, WO96 / 22378, WO97 / 12986, WO97 / 21826 and WO03 / 022311.

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

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

[0211] As discussed above, the disclosed paramyxoviruses or polynucleotides are 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 publicly known and readily available, for example, on the internet via GenBank (accessible at www-ncbi-nlm-nihgov / entrez). The 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, such as nucleic acids with different sequences but encoding the same protein components, to construct a variety of functionally equivalent nucleic acids.

[0212] Exemplary nucleic acids can be prepared by cloning techniques. Sufficient user manuals are publicly available to guide experienced users through examples of appropriate cloning and sequencing techniques, as well as numerous cloning exercises (e.g., 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). 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), and Chem Genes. Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD), Fluka Examples include Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (Carlsbad, CA), and Applied Biosystems (Foster City, CA), as well as numerous other commercial sources known to those skilled in the art.

[0213] The genome of a recombinant paramyxovirus may contain one or more variations (e.g., mutations causing amino acid deletions, substitutions, or insertions), as long as the resulting recombinant paramyxovirus retains the desired biological function, such as low levels of immunogenicity or other characteristics. These variations in the sequence may be naturally occurring variations, or they may be genetically engineered using genetic engineering techniques known to those skilled in the art. Examples of such techniques are, for example, 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 Cloning), both of which are incorporated herein by reference in their entirety. Seen in Biology, John Wiley & Sons, New York, through supplement 104, 2013.

[0214] The nucleic acid encoding described herein can be modified without reducing its biological activity. Amino acid substitutions, insertions, and deletions can be performed using known recombination methods such as oligonucleotide-mediated (site-specific) mutagenesis, alanine scanning, PCR mutagenesis, site-directed mutagenesis, cassette mutagenesis, and restriction selection mutagenesis. This is possible (see, for example, 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)). Several modifications can be made to facilitate cloning, expression, or incorporation of targeting molecules into fusion proteins. Such modifications are well known to those skilled in the art and include, for example, termination codons, methionine added to the amino terminus to provide an initiation site, and additional nucleotides placed at either end to create conveniently positioned restriction sites.

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

[0216] A table of conserved amino acid substitutions that provide functionally similar amino acids is well known to those skilled in the art. The following six groups are examples of amino acids that are considered to be conserved substitutions of each other: 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 recombinant paramyxoviruses disclosed 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 reactions (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 altering 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 selection markers for replication in a host. Such markers include dihydrofolate reductase or neomycin resistance genes for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culture in Escherichia coli (E. coli) and other bacteria.

[0219] Using standard recombination methods, polynucleotides can be inserted into reproducible vectors for cloning. Various vectors are publicly available. Vectors can be in the form of plasmids, cosmids, viral particles, or phages, for example. Appropriate nucleic acid sequences can be inserted into vectors by various procedures. Generally, nucleic acids are inserted into appropriate restriction endonuclease sites (one or more) using techniques known in the art. Vector components generally include, but are not limited to, one or more signal sequences, origins of replication, one or more marker genes, enhancements, etc. Examples include sensor elements, promoters, and transcription termination sequences. Construction of a suitable vector containing one or more of these components is performed using standard ligation techniques known to those skilled in the art.

[0220] Suitable reproducible vectors include, but are not limited to, pUC19 or pTM1. Polynucleotides can be operably ligated to appropriate promoters, such as the T7 polymerase promoter, cytomegalovirus promoter, cellular polymerase II promoter, or SP1 promoter. Reproducible vectors may further include sites for transcription initiation, transcription termination, and ribosome binding sites for translation.

[0221] The introduction of recombinant vectors, consisting of polynucleotides encoding paramyxovirus genomes or paramyxovirus proteins, into host cells such as bacterial or eukaryotic cells is influenced by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, electrotransport, chemotransduction, electrotransduction, infection, or other methods. Such methods are described in standard laboratory manuals such as 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). Commercially available transfection reagents include Lipofectamine (Invitrogen, Carlsbad, Calif.) and FuGENE 6® (Roche Diagnostics, Indianapolis, Ind.). Suitable host cells, though not limited to these, include HEp-2 cells, FRhL-DBS2 cells, LLC-MK2 cells, MRC-5 cells, and Vero cells.

[0222] IV. Immunogenic composition Immunogenic compositions comprising recombinant paramyxoviruses (such as recombinant PIVs containing genomes encoding heterologous recombinant RSV F protein) and pharmaceutically acceptable carriers, as described herein, are also provided. Such compositions can be administered to subjects by various administration methods known to those skilled in the art, for example, by intranasal routes. Practical methods for preparing administerable compositions are known or obvious to those skilled in the art, as described in Remingtons Pharmaceutical Sciences, 19th edition, Mack Publishing. More detailed information can be found in publications such as Company, Easton, Pennsylvania, 1995.

[0223] Accordingly, recombinant paramyxoviruses described herein can be formulated with pharmaceutically acceptable carriers to help maintain biological activity while promoting increased stability during storage within an acceptable temperature range. Possible carriers include, but are not limited to, physiologically equilibrated culture media, phosphate-buffered saline solution, water, emulsions (e.g., oil / water or water / oil emulsions), various types of wetting agents, 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 during storage, including, but not limited to, benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and / or propylparaben in effective concentrations (usually ≤1% w / v). Bacteriostatic agents may be contraindicated for some patients; therefore, lyophilized formulations may be reconstituted with or without such components.

[0225] The disclosed pharmaceutical compositions may contain, as necessary to approximate physiological conditions, pharmaceutically acceptable mediating substances such as pH adjusters and buffers, tension adjusters, and wetting agents, 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 immune response. Suitable adjuvants include, for example, Toll-like receptor agonists, alum, AlPO4, alpha-hydrogels, lipid A and its derivatives or variants, oil emulsions, saponins, neutral liposomes, vaccine and cytokine-containing liposomes, nonionic block copolymers, and chemokines. Among the 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 of stimulating the immune system in a non-specific way and thus helping to enhance the immune response to pharmaceutical formulations.

[0227] In some embodiments, the composition may comprise recombinant paramyxoviruses encoding the RSV F ectodomain from a particular RSV subgroup or strain, and recombinant paramyxoviruses encoding the RSV F ectodomain from a different RSV subgroup or strain. For example, the composition may comprise recombinant paramyxoviruses containing recombinant RSV F proteins from subtype A and subtype B RSV. Different vectors may be present in the mixture and administered simultaneously or separately. Due to the phenomenon of cross-protection between specific strains of RSV, an immunization using certain paramyxoviruses encoding the RSV F ectodomain from a first strain may provide protection against several different strains of the same or different subgroups.

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

[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 attenuated phenotype, exhibits replication in an immunized host, and effectively induces the generation of an immune response, preferably a protective immune response, in the recipient. Preferably, the candidate virus stimulates and amplifies an immune response, for example, by inducing an immune response to a different virus strain or subgroup, and / or by stimulating an immune response mediated by a different immunological basis (e.g., secretion versus serum immunoglobulin, cellular immunity, etc.).

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

[0231] V. Methods to induce an immune response Methods for inducing an immune response in a subject by administering one or more disclosed recombinant paramyxoviruses are provided herein. In certain examples, the subject is a human. The immune response may be a protective immune response, such as a response that prevents or reduces subsequent infection by a paramyxovirus or a virus with heterologous genes contained in a recombinant paramyxovirus. By inducing an immune response, viral infections and associated diseases can be treated or inhibited. In some embodiments, the method is PIV The treatment involves administration of an immunogenic composition containing an attenuated recombinant parainfluenza virus, which includes a viral genome containing heterologous genes encoding a recombinant RSV F ectodomain linked to the F protein transmembrane (TM) domain and the cytoplasmic tail.

[0232] For example, subjects who have or are at risk of developing paramyxovirus infections, such as RSV and / or PIV infection, due to exposure to or potential exposure to RSV and / or PIV, are selected for treatment. After administration of the disclosed immunogen, subjects can be monitored for paramyxovirus infection or related symptoms, or both.

[0233] Methods for intranasal administration of recombinant paramyxovirus to subjects are known to those skilled in the art, as are methods for selecting subjects for administration, preparing immunogenic compositions containing recombinant paramyxovirus for intranasal administration, and evaluating the subjects for their immune response to recombinant paramyxovirus. Exemplary descriptions of such methods can be found, for example, in Karron et al., 2012 Vaccine, 30(26), 3975-3981, which are incorporated herein by reference in full.

[0234] The usual subjects for which treatment using the therapeutic agents and methods described herein are intended include humans and non-human primates and other animals. Since virtually all humans are infected with RSV and PIV by the age of 5, the entire birth cohort is included as a relevant population for immunization treatment. This includes, for example, pregnant women (or women of childbearing age), newborns or infants still in the womb, at any time from 6 months to 5 years of age, or from birth to 6 months of age, in order to protect their infants by passive transfer of antibodies. This can be done by initiating an immunization program in a family member of a person over 50 years of age. The scope of this disclosure shall include maternal immunization. In some embodiments, the subjects are human subjects who are seronegative for RSV or PIV3-specific antibodies. In further embodiments, the subjects are under 1 year of age, such as under 6 months, under 3 months, or under 1 month.

[0235] Children with premature bronchopulmonary dysplasia and those with congenital heart disease are at highest risk of severe RSV and / or PIV infection (e.g., requiring hospitalization). During childhood and adulthood, the disease is less severe but may be accompanied by lower respiratory tract disease and commonly involves sinusitis. In hospitalized elderly individuals (e.g., those over 65 years of age), disease severity increases. Severe disease also occurs in individuals with severe combined immunodeficiency disease or those who have undergone bone marrow or lung transplantation. Therefore, these subjects are selected for administration of the disclosed recombinant paramyxovirus.

[0236] To identify subjects for prevention or treatment according to the methods of this disclosure, acceptable screening methods are used to examine the subjects for risk factors associated with the targeted or suspected disease or condition, or to examine the status of a pre-existing disease or condition. Examples of such screening methods include conventional post-treatments for determining 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 ELISAs and other immunoassays available and well-known in the art for detecting and / or characterizing paramyxovirus infections. These and other conventional methods enable clinicians to select patients who require therapies using the methods and pharmaceutical compositions of this disclosure. According to these methods and principles, compositions may be administered as an independent prevention or treatment program, or as a follow-up, adjunct, or coordinated treatment plan to other therapies, according to the teachings herein or other conventional methods known to those skilled in the art.

[0237] The administration of the disclosed recombinant paramyxovirus may be for prophylactic or therapeutic purposes. When administered prophylactically, the immunogen may be administered prior to any symptoms, for example, prior to infection. Prophylactic administration works to induce an immune response that can prevent or induce remission of any subsequent infection. In some embodiments, the method may include selecting patients at risk of infection with paramyxovirus and administering an effective amount of the disclosed recombinant paramyxovirus to the subject. By administering the recombinant paramyxovirus prior to anticipated exposure to the paramyxovirus, an immune response can be induced to reduce the anticipated severity, duration, or extent of infection and / or related disease symptoms after exposure or suspected exposure to the virus, or after the actual onset of infection. In some examples, treatment using the method disclosed herein extends the survival time of the subject.

[0238] Administration of the disclosed recombinant paramyxoviruses, including RSV and PIV antigens, to subjects may induce the production of an immune response that is protective against serious lower respiratory tract diseases or croup, such as pneumonia and bronchiolitis, if the subjects subsequently become infected or reinfected with wild-type RSV or PIV. While naturally circulating viruses are still capable of causing infection, particularly in the upper respiratory tract, the likelihood of rhinitis as a result of vaccination is reduced, and there is a possibility of a boost in resistance from subsequent infection with wild-type viruses. After vaccination, there are detectable levels of host-produced serum and secretory antibodies capable of neutralizing homologous (same subgroup) wild-type viruses in vitro and in vivo. In many cases, host antibodies also neutralize wild-type viruses of different non-vaccinated subgroups. To achieve higher levels, for example, against heterologous strains of another subgroup, at least one dominant strain of both RSV subgroups A and B is used. A composition containing a recombinant viral vector with RSV F protein derived from it can be used to vaccinate a target.

[0239] The recombinant viral vectors and their immunogenic compositions disclosed herein are provided to a subject in an amount effective to induce or enhance an immune response to antigens contained in the virus, preferably in humans. The effective amount allows for some growth and replication of the virus to produce the desired immune response, but does not produce virus-related symptoms or disease. Based on the guidelines provided herein and the knowledge of the art, a person skilled in the art can easily determine an appropriate amount of virus for use in a live vaccine. The exact amount varies depending on several factors, such as the subject's health and weight, the mode of administration, the degree of viral attenuation, the nature of the formulation, and whether the subject's immune system is impaired.

[0240] Immunogenic compositions comprising one or more disclosed recombinant paramyxoviruses can be used in coordinated (or prime-boost) vaccination protocols or combinatorial formulations. In certain embodiments, novel combinatorial immunogenic compositions and coordinated immunization protocols each utilize separate immunosources or formulations directed to induce an antiviral immune response, such as an immune response to RSV and PIV proteins. The separate immunogenic compositions that induce an antiviral immune response can be combined with a multivalent immunogenic composition administered to the subject in a single immunization step, or they can be administered separately (as a monovalent immunogenic composition) in a coordinated (or prime-boost) immunization protocol.

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

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

[0243] For each individual subject, a specific dosing plan can be evaluated and adjusted over time, in accordance with the individual's needs and the professional judgment of the person administering or supervising the immunogenic composition. In some embodiments, the subject's antibody response is determined in relation to the evaluation of the effective dose / immunotherapy protocol. In most cases, it is sufficient to evaluate the antibody titer in serum or plasma obtained from the subject. The decision of whether or not to administer a booster dose and / or to change the amount of therapeutic agent administered to the 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 antibodies in serum that bind to an antigen containing the RSV F protein. The actual dose of the disclosed immunogen will vary according to factors such as disease signs and the individual condition of the subject (e.g., age, size, fitness, severity of symptoms, susceptibility factors, etc.), the time and route of administration, other drugs or treatments being administered concurrently, and the specific pharmacology of the composition for inducing the desired activity or biological response in the subject. The dosing plan can be adjusted to provide an optimal prophylactic or therapeutic response.

[0244] The determination of the effective dose is typically guided by a dosage protocol that significantly reduces the appearance or severity of the targeted disease symptoms or condition in the subject, or induces a desired response (such as a neutralizing immune response), based on animal model studies and subsequent human clinical trials. Suitable models in this regard include, for example, mice, rats, pigs, cats, ferrets, non-human primates, and other approved animal models known in the art. can be mentioned. Alternatively, the effective dosage can be determined using in vitro models (e.g., immunological and histopathological assays). Using such models, only ordinary calculations and adjustments are necessary to determine appropriate concentrations and dosages for administering a therapeutically effective amount of the composition (e.g., an amount effective to induce a desired immune response or to reduce one or more symptoms of a targeted disease). In alternative embodiments, the effective amount or effective dosage of the composition can simply inhibit or enhance one or more selected biological activities correlated with a disease or condition as shown herein, for either therapeutic or diagnostic purposes. In one embodiment, the general range of virus administration is from about 10 4 to about 10 5 PFU of virus per human subject, from about 10 3 to about 10 7 or more plaque-forming units (PFU) of virus.

[0245] Administration of an immunogenic composition to induce an immune response to reduce or prevent infection can eliminate such infection, but not necessarily completely, provided that the infection is measurably reduced by at least about 50%, e.g., at least about 70% or about 80% or even about 90% in the absence of the agent or in comparison to a reference agent. Those in need of treatment include the general population and / or patients infected with or at risk of infection with paramyxoviruses 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. For the method to be effective, it is not necessary for RSV and / or PIV infection to be completely eliminated, reduced, or prevented. For example, administration of an effective dose of the disclosed recombinant paramyxovirus can reduce subsequent RSV and / or PIV infection by a desired amount, e.g., 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., by the number or percentage of subjects infected with RSV and / or PIV, compared to a suitable subject) (elimination or prevention of detectable RSV and / or PIV infection).

[0247] The dosage and number of doses vary depending on the setting, for example, in adults primed by prior paramyxovirus infection or immunization, and a single dose may be a sufficient booster. In unsensitized subjects, in some cases at least two doses, for example at least three doses, may be administered. In some embodiments, for example, an annual boost is administered in conjunction with an annual influenza vaccination.

[0248] After immunization of the subject, serum can be collected from the subject at an appropriate time, frozen, and stored for antibody titer assay 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, but are not limited to, plaque reduction neutralization (PRNT) assays, microneutralization assays, flow cytometry-based assays, and single-cycle infection assays. In some embodiments, serum neutralizing activity can be assayed using a panel of RSV or PIV pseudoviruses. In serum samples, virus-neutralizing antibody titers are determined by a PRVN assay, as previously described (de Graaf et al., J. Virol Methods, 143:169-174, 2007). Briefly, serum samples with approximately 50 p. fu of NL / 1 / 00 or NL / 1 / 99 expressing enhanced green fluorescent protein can be diluted and incubated at 37°C for 60 minutes. Next, the virus-serum mixture is 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 infection medium and a mixture of 2% methylcellulose. After 6 days, the cells are scanned using a Typhoon 9410 Variable Mode Imager. Fluorescent plaques were counted using GE Healthcare. Antibody titers are expressed as dilutions 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. Recombinant paramyxovirus comprising (a) a viral genome comprising a heterologous gene encoding the ectodomain of a heterologous virus type I transmembrane protein linked to the transmembrane domain (TM) and cytoplasmic tail (CT) of the paramyxovirus's F protein; or (b) a viral genome comprising a heterologous gene encoding the ectodomain of a heterologous virus type II transmembrane protein linked to the TM and CT of the paramyxovirus's HN protein.

[0250] Item 2. Recombinant paramyxoviruses of Item 1, which are recombinant human / bovine parainfluenza virus 3 (B / HPIV3), recombinant human parainfluenza virus 1 (HPIV1), recombinant human parainfluenza virus 2 (HPIV2), recombinant human parainfluenza virus 2 (HPIV3), recombinant parainfluenza virus 5 (PIV5), recombinant Sendai virus, or recombinant Newcastle disease virus (NDV).

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

[0252] Item 4. A recombinant paramyxovirus according to any of items 1-3, comprising a recombinant PIV containing a viral genome that includes a heterologous gene encoding a recombinant RSV F ectodomain linked to the PIV F protein TM and CT.

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

[0254] Item 6. The hRSV F protein is a recombinant paramyxovirus of item 4 or 5, derived from subtype A hRSV or subtype B hRSV.

[0255] Item 7. A recombinant paramyxovirus from any one of items 4-6, in which the RSV F ectodomain is stabilized in the RSV F pre-fusion conformation by one or more amino acid substitutions compared to the natural RSV F protein sequence.

[0256] Section 8. The RSV F ectodomain contains amino acids represented as (a)66E;(b)101P;(c)155C and 290C;(d)190F;(e)207L; 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) and (e) and (d);(a), (b) and (e) and (d);(a), (b) and (c), (d) and (e);(c) and (d); or (c) and (e); or (c), (d) and (e), where the amino acid numbering corresponds to one recombinant paramyxovirus from items 4 to 7, corresponding to the RSV F protein sequence shown as Sequence ID No. 1.

[0257] Item 9. The RSV F ectodomain is a recombinant paramyxovirus of Item 8, comprising amino acid substitutions indicated as (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), (b) and (c); (a), (b) and (d); (a), (b) and (e); (a), (b) and (e) and (d); (a), (b) and (c), (d) and (e); (c) and (d); or (c) and (e); or a combination of (c), (d) and (e).

[0258] Item 10. The RSV F ectodomain is a recombinant paramyxovirus of item 8 or 9, including 66E, 101P, 155C, 290C, 190F, and 207L.

[0259] Item 11. A recombinant paramyxovirus from any of items 4-10, wherein the RSV F ectodomain contains at least 85% identical amino acid sequence to one of the RSV ectodomains of sequence number 1 (WT RSV FA), 2 (WT RSV FB), 12 (A2 HEK), 14 (A2 HEK+DS), or 21 (A2 HEK+DS-Cav1), or contains the amino acid sequence of the RSV ectodomain of sequence number 12, 14, or 21.

[0260] Item 12. PIV is a recombinant paramyxovirus from any one of items 4-11, which is recombinant PIV1, recombinant PIV2, or recombinant PIV3.

[0261] Item 13. Recombinant paramyxovirus of Item 12, wherein the recombinant PIV is recombinant PIV1, and the TM and CT linked to the RSV F ectodomain are derived from the PIV1 F protein; the recombinant PIV is 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 recombinant PIV3, and the TM and CT linked to the RSV F ectodomain are derived from the PIV3 F protein.

[0262] Section 14. Recombinant PIV is recombinant HPIV1, and PIV F™ and CT are derived from the HPIV1 F protein, with the RSV F ectodomain linked. Recombinant PIV is recombinant HPIV2, and PIV F is linked to the RSV F ectodomain. Recombinant paramyxovirus of item 12 or 13, where TM and CT are derived from HPIV2 F protein; recombinant PIV is recombinant HPIV3 with the RSV F ectodomain linked; or recombinant PIV is recombinant B / HPIV3 with the RSV F ectodomain linked; and PIV F TM and CT are derived from BPIV3 F protein.

[0263] Item 15. A recombinant paramyxovirus from any one of items 4-14, in which the RSV F ectodomain is derived from the hRSV F protein, and TM and CT are derived from the BPIV3 F protein.

[0264] Item 16. Recombinant PIV is recombinant HPIV1, and PIV F™ and CT linked to the RSV F ectodomain contain the amino acid sequence shown as SEQ ID NO: 31, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 31; Recombinant PIV is recombinant HPIV2, and PIV F™ and CT linked to the RSV F ectodomain contain the amino acid sequence shown as SEQ ID NO: 39, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 39; Recombinant PIV is recombinant HPIV3, and PIV F™ and CT linked to the RSV F ectodomain contain the amino acid sequence shown as SEQ ID NO: 46 and A recombinant paramyxovirus from any one of items 4 to 15, comprising the amino acid sequence shown as, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 46; or recombinant PIV is recombinant B / HPIV3 and PIV F TM and CT linked to the RSV F ectodomain comprises the amino acid sequence shown as, or an amino acid sequence that is at least 90% identical to, SEQ ID NO: 53.

[0265] Item 17. Recombinant PIV is recombinant HPIV3, and the heterogene encodes an hRSV F ectodomain linked to HPIV3 F TM and CT containing the amino acid sequence shown as Sequence ID No. 10 or an amino acid sequence at least 90% identical thereto; or recombinant PIV is recombinant B / HPIV3, and the heterogene encodes an hRSV F ectodomain linked to BPIV3 F TM and CT containing the amino acid sequence shown as Sequence ID No. 21 or an amino acid sequence at least 90% identical thereto, one of any recombinant paramyxoviruses from items 4 to 16.

[0266] Item 18. The RSV F ectodomain is derived from the hRSV F protein, and the recombinant PIV contains a viral genome encoding HPIV3 F and HN proteins as well as BPIV3 N, P, C, V, M, and L proteins, with the TM and CT linked to the RSV F ectodomain derived from the BPIV3 F protein; the recombinant PIV contains a viral genome encoding HPIV1 N, P, C, M, F, HN, and L proteins, with the TM and CT linked to the RSV F ectodomain derived from the HPIV1 F protein; the recombinant PIV contains a viral genome encoding HPIV2 N, P, V, M, F, HN, and L proteins, with the TM and CT linked to the RSV F ectodomain derived from the HPIV2 F protein; or the recombinant PIV is derived from HPIV3 A recombinant paramyxovirus comprising a viral genome encoding N, P, C, M, F, HN, and L proteins, with TM and CT linked to the RSV F ectodomain, derived from the HPIV3 F protein, one of items 4-17.

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

[0268] Item 20. The viral genome, from upstream to downstream, includes the PIV genome promoter followed by the N, P, C / V, M, F, HN, and L genes; the gene encoding the recombinant RSV F ectodomain, which is linked to 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, recombinant paramyxovirus as of item 18 or 19.

[0269] Item 21. A recombinant paramyxovirus from any one of items 18-19, comprising a viral genome encoding the HPIV3 F and HN genes and the BPIV3 N, P, C, V, M and L genes, each containing the amino acid sequences shown as sequence numbers 21, 101, 47, 48, 49, and 52, or sequences at least 90% identical thereto.

[0270] Item 22. A heterologous gene is a recombinant paramyxovirus from any one of the previous items, which has been codon-optimized for expression in human cells.

[0271] Item 23. Recombinant HPIV3, the heterologous gene encodes the RSV F ectodomain linked to HPIV3 F TM and CT, and sequence number 11 (GenScript). It contains a nucleotide sequence represented as RSV F_HEK_DS-Cav1_H3TMCT); or recombinant B / HPIV3, and the heterologous gene is BPIV3 F TM A recombinant paramyxovirus according to item 22, comprising a nucleotide sequence that encodes the RSV F ectodomain linked to CT and is shown as sequence number 22 (GenArt RSV F_HEK_DS-Cav1_B3TMCT) or sequence number 23 (GenScript RSV F_HEK_DS-Cav1_B3TMCT).

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

[0273] Item 25. The RSV F ectodomain is the viral vector of item 24, containing the K66E and Q101P amino acid substitutions.

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

[0275] Item 27. Any one of the viral vectors from items 24-26, wherein the RSV F protein is stabilized in the pre-fusion or post-fusion conformation by one or more amino acid substitutions.

[0276] Item 28. A viral vector from any one of items 24-27, in which the RSV F ectodomain is stabilized in the pre-fusion conformation by the S155C, S290C, S190F, and V207L ​​amino acid substitutions.

[0277] Item 29. The RSV F ectodomain is soluble and secreted from a host cell containing a viral vector, one of the viral vectors listed in items 26-28.

[0278] Item 30. A viral vector from any one of items 24-29, which is recombinant human / bovine parainfluenza virus 3 (B / HPIV3), recombinant human parainfluenza virus 1 (HPIV1), recombinant human parainfluenza virus 1 (HPIV2), recombinant human parainfluenza virus 1 (HPIV3), recombinant parainfluenza virus 5 (PIV5), recombinant Sendai virus, or recombinant Newcastle disease virus (NDV).

[0279] Item 31. The RSV F ectodomain is a viral vector derived from the human RSV (hRSV)F protein, one of the viral vectors listed in items 24-30.

[0280] Item 32. A heterogeneous gene encoding the RSV F protein is one of any one of the viral vectors in items 24-31, containing the nucleic acid sequence shown as nucleotides 1-1587 of SEQ ID NO: 18 (the ectodomain encoded by the GenScript-optimized RSV F_A2_HEK_DS-Cav1_B3CT DNA sequence).

[0281] Item 33. At least 90% of the viral particles produced by a host cell infected with a recombinant paramyxovirus or viral vector contain a viral envelope comprising an ectodomain encoded by heterologous genes, which is any one of the recombinant paramyxoviruses or viral vectors described in the preceding items.

[0282] Item 34. A reduced recombinant paramyxovirus or viral vector of any one of the preceding items.

[0283] Item 35. Any one of the recombinant paramyxoviruses or viral vectors from the preceding items An immunogenic composition comprising a pharmaceutically acceptable carrier.

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

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

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

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

[0288] Item 40. The immune response is a protective immune response, one of the methods described in items 37-39.

[0289] Item 41. Any one of the methods described in items 37 to 40, comprising a prime-boost administration of an immunogenic composition.

[0290] Item 42. Any method according to items 37 to 41, comprising intranasal or parenteral administration of an immunogenic composition.

[0291] Item 43. The subject is a human or veterinary subject, and the method is one of items 37-42.

[0292] Section 44. The subject is at risk of RSV or PIV infection, or has RSV or PIV infection, as described in any one of sections 37-43.

[0293] Item 45. The subject is under 1 year of age, and one of the methods described in items 37-44.

[0294] Item 46. A nucleic acid molecule containing the genome of one recombinant paramyxovirus from items 1 through 25.

[0295] Item 47. Recombinant RSV F protein or immunogenic fragment thereof, containing the K66E and Q101P amino acid substitutions.

[0296] Item 48. Recombinant RSV F protein or immunogenic fragments thereof of Item 47, further comprising (a) S155C and S290C; (b) S190F; (c) V207L; or (f) (a) and (b); (a) and (c); (b) and (c); or a combination of (a), (b) and (c).

[0297] Item 49. Immunogenic fragments of recombinant RSV F protein according to item 47 or item 48, comprising the RSV F ectodomain.

[0298] Item 50. A nucleic acid molecule encoding one of the recombinant RSV F proteins listed in items 47-49. [Examples]

[0299] The following examples are provided to illustrate specific features of a particular embodiment, but the claims should not be limited to the illustrated features. [Examples]

[0300] Improved expression and immunogenicity of respiratory syncytial virus (RSV) fusion (F) glycoprotein expressed by attenuated parainfluenza virus vectors. This embodiment describes approaches to improve the immunogenicity and stability of expressed RSV F by recombinant B / HPIV3 using early passage virus-derived RSV F sequences, by codon optimization, by stable and highly immunogenic pre-fusion and post-fusion RSV F, and by genetically engineering RSV F proteins TM and CT, thereby enabling more efficient incorporation into vector particles.

[0301] Introduction. Attenuated live RSV strains represent 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). Attenuated live RSV strains are usually administered via the intranasal (IN) route. However, attenuation generally leads to decreased antigen synthesis, which in turn leads to decreased immunogenicity. Achieving the right balance between attenuation and immunogenicity has been challenging for RSV.

[0302] Complete, infectious HPIV is generated as a whole from cloned cDNA in a transfected cell culture (using reverse genetics). A foreign gene designed for expression is modified by reverse genetics so that it is inserted into the HPIV genome as an additional gene, adjacent to HPIV transcription signals (called gene start and gene end signals, located at the start and end of each gene, respectively). The foreign gene is then transcribed into a different mRNA, like other HPIV genes. HPIV may be expressed in adaptation to several additional foreign genes (Skiadopoulos et al. 2002. Virology 297:136~152). However, multiple genes may be excessively attenuated, leading to the collection of point mutations (Skiadopoulos et al. 2002. Virology 297:136~152).

[0303] HPIV transcription begins at the 3' end of the genome with a single promoter and proceeds sequentially. Polymerase fractions detach from the template at each gene junction, creating a negative gradient of gene transcription. Consequently, genes proximal to the promoter are expressed more frequently than downstream genes. The placement of foreign genes close to the promoter increases their expression but has the ability to influence the expression of downstream vector genes. Other features, such as differences in the efficiency of transcriptional signals for gene initiation or termination, 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 inserted genes or open reading frames (ORFs) (Whelan et al. 2004. Current Topics Microbiology and Immunology 283:61~119). Furthermore, in some cases, the characteristics of viral constructs are significantly influenced by factors that have not yet been identified; for example, insertion of the RSV F gene into the PM gene junction of the PIV3 vector resulted in a substantially temperature-sensitive and attenuated virus (Liang B et al. 2014. J Virol 88:4237~4250). Thus, while the broad details of expression from the HPIV genome are generally known, specific constructs can yield unpredictable results.

[0304] Previous studies have used the B / HPIV3 vector to express the RSV G gene and F protein from additional genes at first and second genomic locations after the promoter, or the RSV F gene from an additional gene at a second genomic location between the N and P genes. When the latter virus (called MEDI-534) was evaluated in a clinical study in seronegative children, it was 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 dropped from vaccine recipients showed that approximately 50% of the samples contained vaccine viruses with mutations predicted to perturb RSV F expression. This is likely to reduce immunogenicity. Retrospective analysis of clinical trial material (CTM) showed that 2.5% of this virus did not express RSV F (Yang et al. 2013. Vaccine 31:2822~2827). Furthermore, observations that the RSV F insert accumulated mutations that inactivated its expression at the protein level, and that these mutations were amplified during proliferation, suggest that it has a selective advantage in silencing RSV F protein expression. This is likely due to the highly fusional nature of the RSV F protein, which efficiently mediates syncytial formation. In vitro, this leads to disruption of the cellular matrix, thereby reducing vector replication. Furthermore, high levels of exogenous glycoprotein synthesis may interfere with the synthesis, processing, and transport of vector glycoproteins through the endoplasmic reticulum and exocytosis pathways, and in particular, may sterically interfere with virion morphogenesis. These effects can occur both in vitro and in vivo.

[0305] Expression of the early passaged (HEK) version of the RSV F protein and a codon-optimized version of the RSV F open reading frame (ORF). Increased expression of the viral antigen typically resulted in improved immunogenicity. Codon optimization of the ORF encoding the vector antigen increased its expression and subsequently improved its immunogenicity, as demonstrated, for example, with human immunodeficiency virus antigen expressed from a viral or DNA vector (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 translational improvements, such as effects on mRNA stability and transport, and therefore the effects of altering the mRNA nucleotide sequence can be complex and unpredictable. Accordingly, codon-optimized versions of the RSV F sequence were designed using the GeneArt (GA) algorithm and evaluated to determine whether they conferred protein expression.

[0306] When designing this codon-optimized ORF, the amino acid sequence of an early passaged 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 passaged (or low passaged) strain, originating in the 1960s, is called HEK after the human embryonic kidney (HEK) cell culture used for its proliferation. This HEK virus differs 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 assignments 66E and 101P, while the highly passaged laboratory A2 strain had assignments 66K and 101Q (hereafter referred to as the "non-HEK" assignment) (Figure 1). However, the emergence of sequence differences between viral strains or between stocks of a given strain is a consequence of the mutation rate of RNA viruses. It is common for HEK assignments to be absent, and the significance of HEK differences was not previously known. Furthermore, the presence of HEK assignments in candidate attenuated RSV vaccines called RSV NIHΔM2-2 was associated with a slight decrease in replication efficacy during cell culture. In addition, HEK assignments at position 66 were identified to affect syncytium formation during RSV infection. Therefore, given their association with reduced replication, avoiding HEK assignments was intended. However, since the version of RSV F containing HEK assignments was used incidentally 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 at position 2 of the rB / HPIV3 vector under the control of BPIV3 gene start and gene terminal transcription signals (Figure 1). Transcription signals and insertion sites were used in all subsequent rB / HPIV3 constructs expressing RSV F, thereby obtaining a direct comparison throughout.

[0307] Vero cells were infected with two different vectors (called "HEK / GA-opt" and "non-HEK / GA-opt"), cell lysates were prepared 48 hours post-infection, and proteins were subjected to gel electrophoresis in the presence of denaturing surfactants and under reducing or non-reducing conditions. Separate proteins were transferred to the membrane by Western blotting and analyzed using antibodies specific to RSV F (Figure 2). This showed that the presence of the HEK assignment was accompanied by a small (approximately 2-fold) but consistent increase in RSV F protein expression (Figure 2). One non-limiting explanation for this finding is that the HEK assignment may have an effect on protein synthesis because it increased F protein stability, although this seems 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 the HEK assignment was accompanied by a decrease in the gel mobility of the RSV F trimer (Figure 2). This suggests that these assignments altered the structure of the RSV F protein trimer. More remarkably, expression of the HEK version of RSV F, as already noted, resulted in a dramatic decrease in syncytiomyogenesis compared to the non-HEK version, even when the HEK version was expressed at only slightly increased levels (Figure 3). This assay utilizes the apparent general absence of syncytiomyogenesis induced in cells infected with the rB / HPIV3 empty vector, while expression of the vector-derived RSV F protein results in syncytiomyogenesis that is generally proportional to the amount of RSV F protein expressed. This provides an assay for the quantity and functionality of RSV F protein expressed from PIV vectors. These observations related to HEK indicate that the HEK assignment is associated with differences in the synthesis / stability, structure, and fusion activity of RSV F, and that these effects occur in the absence of any other RSV protein and are therefore directly related to expression from heterologous vectors.

[0308] Since the HEK assignments originate from low-passage stocks of RSV strain A2 from the 1960s, they are likely representative of the original clinical isolate, while non-HEK assignments emerged during extensive in vitro passaging over the following decades. This suggests that the low-fusion phenotype of the HEK version of F is more representative of the original biological virus. The non-HEK version may correspond to a hyper-fusion variant selected during passaging in cell culture. While the highly-fusion version of RSV F can destabilize the virus and may therefore be undesirable in some cases, it may be chosen in laboratory settings for rapid growth in cell monolayers. Examining 226 sequences of RSV F from clinical isolates in the GenBank database, we found that clinical isolates typically contain HEK assignments. This is consistent with these assignments representing circulating RSV. In any case, the HEK assignments resulted in a moderate increase in F protein expression, leading to the low-fusion form of RSV F. The decrease in syncytium formation is due to cytopathogenicity (otherwise HPIV vector replication and RSV F insert silencing). This is advantageous because it reduces the selective pressure (which may interfere with the preferred selection of the vector). Therefore, the HEK assignment has a triple advantage: it corresponds to a more natural and clinically relevant form of the F protein, it results in a moderate increase in protein expression, and it reduces the selective pressure for silencing the RSV F insert.

[0309] The effects of codon optimization on RSV F expression and immunogenicity were also evaluated. The HEK-containing and GA-optimized versions (HEK / GA-opt) described above were used along with two other codon-optimized RSV HEK F sequences produced by two other different algorithms. Evaluating multiple optimization versions was not a typical implementation as it increased cost and inconvenience and was not shown to be useful. The 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 with respect to the different versions. The highest expression was observed with the HEK-containing GenScript-optimized F protein (HEK / GS-opt), which was superior to the unmodified RSV. Expression levels with more efficient ORF were 10-fold (Vero cells) and 16-fold (LLC-MK2 cells) higher than those with F (non-HEK / non-opt) (Figure 5). The gradual increase in syncytial formation was demonstrated in association with the increase in F expression levels despite the presence of HEK assignment, but it is presumed that syncytial formation was 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 the 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 confer any increase in expression in the case of RSV F.

[0311] Contrary to expectations, the 10- to 16-fold increase in RSV F expression and the accompanying increase in syncytium formation did not have a significant negative impact on vector replication in cell cultures (Figure 7). High levels of RSV F expression and syncytium formation were predicted to interfere with the vector at numerous arbitrary steps, as previously noted, including vector glycoprotein synthesis, processing, exocytosis, vector particle formation, and cell survival; however, this was not the case. This was particularly surprising, as, as previously noted, the accumulation and amplification of mutations that silenced RSV F gene expression in MEDI-534 suggested substantial selective pressure on RSV F protein expression. Compared to empty vectors, all vectors with RSV F inserts were moderately attenuated (Figure 7)—likely including general attenuation effects such as increased genome length and gene number—but replicated with similar kinetics to each other, growing to peak titers slightly lower than those of empty vectors (Figure 7). Slight differences in peak titer are likely to correspond to experimental variability.

[0312] In vivo replication was used to evaluate the immunogenicity and protective efficacy of the rB / HPIV3 vector in a hamster model. The hamster group was divided into groups of 10 animals. 5 Individual tissue culture infection dose (TCID) (50 units) 50 Intranasal immunization was performed using the rB / HPIV3 vector at the following dose: 10 6 Wild-type (wt) RSV administered in the form of plaque-forming units (pfu) was included as a positive control for inducing RSV-specific immunity. The wt RSV control was wt Although RSV was included with a warning that it was a non-attenuated virus, this vector was attenuated and, for that reason, sometimes had relatively low immunogenicity. Six animals per virus per day were euthanized on days 3 and 5 post-infection, and nasal turbinates and lungs were collected for viral titration to measure replication in vivo. This allowed for the determination of RSV F Vectors containing the insert were shown to be moderately attenuated in the nasal turbinates (upper respiratory tract) and substantially attenuated in the lungs (lower respiratory tract) compared to empty vectors (Figure 8). The increased attenuation compared to empty vectors was evidenced by lower values ​​of viral shedding. This was also demonstrated by a comparison of titers on day 3 and day 5: for empty vectors, the titers on day 3 and day 5 were comparable, but for vectors containing RSV F, the titer on day 3 was lower than the titer on day 5, indicating that these constructs took longer to reach their maximum titer. Surprisingly, even among vectors with the RSV F insert, those with increased RSV F expression were not attenuated as much as those with lower RSV F expression, i.e., non-HEK / non-opt. Therefore, the addition of the RSV F insert to the rB / HPIV3 vector was attenuated in vivo (presumably due to some common features such as increased genome length or gene number), however, 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 the serum titer 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 neutralization titer associated with escalating RSV F expression, but the difference was not statistically significant. wt RSV, infected in parallel with controls, induced significantly higher titers of RSV-neutralizing antibodies than the vectors. However, it is important to note that while the neutralizing antibodies induced by RSV infection included contributions from both F and G neutralizing antigens, this vector possessed only F-specific antibodies contributing to the neutralization titer. Furthermore, unattenuated wt RSV controls replicated more efficiently than attenuated vectors, particularly in the lungs (Figure 8), which increased their immunogenicity compared to the vectors.

[0314] To evaluate the protective effectiveness of these vectors, 10 per animal was administered to a group of six immunized hamsters from the experiment shown in Figure 9. 6 PFU-mediated intranasal infection with wt RSV was challenged 30 days after immunization. Nasal turbinates and lungs were collected from animals euthanized 3 days after challenge, and tissue homogenates were prepared and evaluated by plaque assay to measure the level of challenged RSV replication. Vectors expressing RSV F provided nearly complete protection in the lungs and intermediate levels of protection in the nasal turbinates, while wt RSV provided nearly complete protection in both anatomical sites (Figure 10). There was no significant difference in the protective efficacy against RSV challenge between vectors expressing RSV F. It should be noted that protection provided by RSV includes contributions from neutralizing antibodies against both F and G proteins, as well as potentially cellular immunity against all RSV proteins, while protection provided by vectors includes humoral and cellular immunity against F protein only. Furthermore, as noted, the RSV control was an unattenuated wt virus that replicated to higher titers during immunization than the vector, particularly in the lungs, increasing its immunogenicity and control efficiency (Figure 8).

[0315] These results in RSV arising from the use of HEK assignment and codon-optimized sequences. A 10- to 16-fold increase in F protein expression did not result in a significant increase (although a trend toward increase) in the induction of RSV neutralizing serum antibodies, nor in a significant increase 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, RSV F expression was shown to be caused by insertion at positions 1 or 2 vs 6 in the rB / HPIV3 vector. It has been observed that a 30-69 fold difference in current levels can induce a significant difference in protective efficacy in hamsters (Liang B et al. 2014. J Virol 88:4237-4250). Therefore, it is generally thought that increased antigen synthesis confers increased immunogenicity. However, in some cases, this effect may not be large enough to be undoubtedly detected, and given in vivo models may not be sufficiently sensitive. Therefore, the 10-16 fold difference in this study may not be large enough to induce a statistically significant effect in semi-permissive hamster models. The beneficial effects of higher RSV F expression may be even more pronounced in combination with other features, 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 monkey) cells, and here codon optimization for human use is likely to be effective, given the relatively close phylogenetic relationship of these primates to humans. In contrast, in vivo immunogenicity assays using hamsters, here codon optimization for human use may not be effective in increasing expression and consequently immunogenicity.

[0316] Evaluation of pre-fusion and post-fusion immunogenicity of RSV F expressed by the rB / HPIV3 vector. Like all paramyxovirus F proteins, the RSV F protein initially assembles into a pre-fusion conformation, which is the version incorporated into the virion and accumulates on the surface of infected cells. Pre-fusion F undergoes a large number of conformational changes that mediate membrane fusion, induced, for example, by contact with an adjacent target cell membrane, and the F protein ends up in the 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 remarkably sensitive to induction among paramyxoviruses, readily induced before maturity, which may contribute to the significant instability of RSV infectivity. Many RSV F proteins accumulating in infected cells are conformationally heterologous, and this has also been shown to function as a decoy to reduce the induction of virus-neutralizing antibodies (Sakurai et al. 1999. J Virol 73:2956~2962). Therefore, this is an advantage for expressing RSV F in a stabilized conformation for more than two reasons.

[0317] In recent years, stable fusion 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). This stable fusion form was recombinantly generated by shortening 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). The absence of TM and CT would result in this fusion form being unmembrane-free and secreted. The fusion form of RSV F has 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 pre-fusion form of RSV F is considered to be considerably more immunogenic than the post-fusion form (McLellan et al. 2013. Science 340:1113~1117). This is based on the observation that most of the neutralizing activity in the serum of convalescent animals and humans does not bind to the post-fusion F protein and is likely brought about by antibodies that are specific to the pre-fusion form. Based on (McLellan et al. 2013. Science 340:1113~1117; Magro et al. 2012. Proc Nat'l Acad. Sci. USA 109:3089~3094). In recent years, the pre-fusion structure of RSV F has been determined, and it has become 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 lumen of the predicted trimer structure (Cav1), and these combinations are called DS-Cav1 (McLellan et al. 2013. Science 342:592~598). Recombinant DS and DS-Cav1 type 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 the post-fusion type (DS-Cav1 type is more immunogenic than DS type) (McLellan et al. 2013. Science 342:592~598).

[0319] The immunogenicity of post-fusion and pre-fusion RSV F when expressed from attenuated rB / HPIV3 vectors was evaluated. Post-fusion and stabilized pre-fusion (DS and DS-Cav1) RSV F (with HEK assignments) were GA codon-optimized and inserted into the second genomic position of the rB / HPIV3 vector (Figure 11). These were compared with HEK / GA-opt and HEK-containing versions, as well as the GA-optimized F protein (from which CT and TM were deleted, leaving the ectodomain (Ecto)) (Figure 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 still require evaluation. Given the superior expression of the GS-optimized ORF (Figure 35), the GS-optimized version may also be more immunogenic and protective. Furthermore, the identifiers "HEK" and "GA-opt" are sometimes omitted from the construct names for abbreviation in Figure 11 and subsequent figures, as well as in the subsequent text, although the presence of these features is shown in the figures (i.e., "All above versions of RSV F are HEK, GA-optimized," Figure 11).

[0321] These vectors containing various forms of RSV F were rescued and each was grown in vitro to high, similar titers (Figure 12). They were generally slightly attenuated with respect to the growth kinetics, and the final yields compared to empty rB / HPIV3 vectors were as previously noted for the other vector constructs (see Figure 7).

[0322] The effectiveness of expressing various forms of RSV F protein was evaluated in Vero and LLC-MK2 cells infected with various constructs (Figure 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) associated with cells as expected. The post-fusion and ecto forms were found to be secreted and, similarly, associated with cells. Secretion of post-fusion F was consistently more efficient than that of the ecto form: the ecto form contains a high content of hydrophobic sequences, which may result in more remaining associated with cells. Unexpectedly, DS and DS-Cav1 forms of F were expressed more efficiently (Figure 13B). Since these viruses repeated similar reaction kinetics, and the ORF was similarly GA-optimized, this increase in expression likely reflects increased protein stability of the DS and DS-Cav1 forms. Genetic manipulation of proteins can substantially affect glycoprotein expression, processing, and stability, often in a negative way. Therefore, the effective expression of DS and DS-Cav1 by this bio-vector was an inherent characteristic that was not easily predictable.

[0323] The replication of these vectors in vivo was evaluated in hamsters (Figure 14). In the nasal turbinates, all vectors with the RSV F insert were moderately attenuated compared to the empty vector (Figure 14A). The increased attenuation compared to the empty vector was evidenced by lower values ​​for viral shedding. This was also evidenced by a comparison of titers on day 3 and day 5: for the empty vector, these values ​​were comparable, but for vectors containing RSV F, the titer on day 5 was higher than the titer on day 3, indicating that these constructs take 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 be due to experimental variability or the effect of fair anisocoria on vector replication. In the lungs, all vectors with the RSV F insert were substantially more attenuated than the empty vector (Figure 14B). Vectors with post-fusion F, consistent with the nasal turbinates, also replicated to somewhat higher titers in the lungs than other vectors, although vectors expressing the pre-fusion (DS) version appeared to be somewhat further attenuated. Fully wt virus RSV controls replicated more efficiently than attenuated rB / HPIV3 vectors expressing RSV F; for example, wt RSV replicated to titers 100-fold and 1000-fold higher in the nasal turbinates and lungs, respectively, than vectors expressing pre-fusion (DS) F. RSV-neutralizing serum antibody titers were determined by a 60% plaque reduction assay, performed in two ways: (i) in the presence of supplemented complement (a useful technique, as already shown in Figure 9), and (ii) without supplemented complement (Figures 15A and B, respectively). The presence of supplemented complement provides the most sensitive detection of virus-specific antibodies. This is because complement can potentially confer virus-lytic ability to all antibodies that bind to virions and can 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 capable of neutralizing RSV without involvement of complement proteins' viral lytic function or steric effects. It has been suggested that "neutralization assays performed without complement may best reflect the physiological conditions of the respiratory system" (Yoder et al. 2004 J Med Virol 72:688~694). In complement-containing assays (Figure 15A), vectors with post-fusion F were less immunogenic than other vectors, even when replicated to the highest titer in hamsters; conversely, vectors with pre-fusion (DS)F were the most immunogenic of all vectors tested, even when attenuated. In complement-dependent assays (Figure 15B), among the vectors, only those expressing pre-fusion (DS)F induced high-titer neutralizing antibodies. None of the other unmodified post-fusion or EctoF vectors were effective in inducing high-quality neutralizing antibodies. The RSV control was effective in inducing neutralized antibodies in both complement-containing and complement-dependent assays. Notably, vectors containing 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 with 100–1000 times less efficiency than unattenuated wt RSV, and the neutralizing activity conferred by wt RSV had an additional contribution from the RSV G protein. This suggests that vectors expressing the pre-fusion (DS) form of RSV F were highly immunogenic and extremely potent in inducing highly effective neutralizing antibodies.

[0324] To evaluate the protective effectiveness of these vectors, immunized hamsters derived from the experiment shown in Figure 15 were subjected to 10 per animal. 6Animals were challenged 30 days post-immunization by intranasal infection with pfu-containing wt RSV. The animals were sacrificed 3 days after infection, and the turbinates and lungs were collected and treated with tissue homogenates assayed by plaque titration (Figure 16). In the turbinates (Figure 16A), constructs expressing non-HEK / non-opt F, HEK / GA-opt, or Ecto F provided a moderate level of protection, while 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, with the exception of the post-fusion form, which offered the least protection (Figure 16B). The wt RSV control provided near-complete protection in the nasal turbinates and complete protection in the lungs; however, as already noted, wt RSV had the advantage of expressing both F and G neutralizing antigens, in addition to expressing all RSV proteins as potential antigens for cellular immunity and replicating up to 1000 times more efficiently (Figure 14).

[0325] The addition of the Cav-1 mutation to the DS construct resulted in increased immunogenicity as a subunit vaccine (McLellan et al. 2013. Science 342:592~598), which is expected to further enhance the immunogenicity of pre-fusion RSV F expressed from the viral vector. Furthermore, the DS and DS-Cav1 types of RSV F provided the greatest increase in expression, and their immunogenicity and protective efficiency under GS optimization conditions still need to be evaluated (Figures 5 and 6). These further constructs were constructed, recovered, and prepared as working pools (Figure 35).

[0326] The immunogenicity of the RSV F protein is enhanced by facilitating the incorporation of the rB / HPIV3 vector into virion particles. Incorporation of antigens into virus-like particles (VLPs) or adeno-associated virus particles has been shown to increase their immunogenicity (Rybniker et al. 2012. J Virol 86:13800~13804; McGinnes et al. 2011. J Virol 85:366~377). However, it was unclear whether the incorporation of heterologous antigens into the viral envelope of infectious viruses could enhance their immunogenicity. When expressed by rB / HPIV3, the native RSV F protein (i.e., HEK / GA-opt) is incorporated into minute amounts of vector particles (see below).

[0327] In a previous study by Zimmer et al. (Zimmer et al. J Virol 2005 79:10467~77), the expression of the RSV F protein from a gene added to Sendai virus that is mouse-related to HPIV1 and closely related to HPIV3 was evaluated. That study showed that, similar to rB / HPIV3, the extremely small RSV F protein was incorporated into Sendai virus vector particles. The researchers replaced the RSV F protein in CT or CT Plus™ with a corresponding sequence derived from the Sendai F protein, assuming this would improve the effectiveness of the interaction between the vector particles and the exogenous RSV F protein. These modifications did indeed increase the incorporation of genetically engineered RSV F into Sendai particles, but only when the Sendai F protein gene was deleted. The requirement to delete the vector F protein is undesirable in this study because the absence of the vector F protein from rB / HPIV3 has the ability to substantially alter its replication characteristics, particularly in vivo, and also removes one of the HPIV3 protective antigens.

[0328] Despite this clear precedent indicating that this strategy would not be appropriate, we constructed rB / HPIV3 constructs with CT or CT plus TM in which the RSV F protein was replaced with that of the vector (PIV) F protein (resulting in constructs called B3CT and B3TMCT, respectively, Figure 17). The TM and CT regions derived from rB / HPIV3 were 21 and 26 amino acid lengths, respectively. The constructs in this study were also made with a version of the F protein containing HEK assignment and GA optimization in the native F protein (HEK / GA-opt), and also with pre-fusion DS and DS-Cav1 types (Figure 17). (GA-optimized F ORF was used). All chimeric F genes were inserted at the 2nd position of rB / HPIV3 for direct comparison with the constructs described above.

[0329] All viruses were readily recovered by reverse genetics. To quantify the packaging efficiency of RSV F and its modified derivatives, sucrose-purified viruses were prepared for Western blotting analysis to determine the amount of RSV F in the particles (Figure 18). Equal amounts of each sucrose-purified stock (0.5 ug of protein per sample) were subjected to denaturation, reduction gel electrophoresis, and analyzed by Western blotting. This showed that the non-chimeric F protein (derived from HEK / GA-opt) had relatively poor integration in rB / HPIV3 virions (Figure 18, lane 2). However, the B3CT and B3TMCT modifications dramatically improved integration efficiency by 19-20 times (Figure 18, lanes 3 and 4). In fact, compared to wt RSV virions with equal protein amounts (Figure 18, lane 5), the amount of integrated B3CT and B3TMCT F protein in the vector particles appeared to be equal to the amount of native F in the RSV particles. Similarly, improved packaging effectiveness was also observed for the pre-fusion (DS) form of the RSV F chimeric form with B3CT or B3TMCT (Figure 18, lanes 6 and 7). Thus, the efficient packaging of RSV F B3CT and B3TMCT into the rB / HPIV3 vector did not require the deletion of the vector F protein and was therefore dramatically different from Sendai's precedent.

[0330] The packaging of RSV F was also examined by transmission electron microscopy (TEM) using RSV-specific antibodies and immuno-gold labeling (Figures 19A-F). RSV F spikes on the surface of RSV particles were labeled (Figure 19A), but the labeling was not observed on the surface of an empty rB / HPIV3 vector (Figure 19B). The extremely limited labeling of native RSV F was detected in the vector envelope, consistent with the results in Figure 18, which showed that very small amounts of native F were detected in purified rB / HPIV3 virions by Western blot analysis (Figure 19C). In contrast, vectors expressing chimeric F with B3CT or B3TMCT showed enhanced labeling (Figures 19D and 19E), indicating efficient packaging of these chimeric types into the vector envelope. Similarly, pre-fusion (DS) RSV F with B3TMCT was also efficiently packaged into vector particles (Figure 19F). This confirmed that the B3CT and B3TMCT modifications resulted ...

Claims

1. It is a recombinant paramyxovirus, The viral genome includes heterologous genes encoding a type I membrane protein, specifically the cytoplasmic tail (CT) or transmembrane domain (TM) of the paramyxovirus F protein, and the recombinant respiratory syncytial virus RSV F ectodomain linked to the CT. Includes, Recombinant human / bovine parainfluenza virus 3 (B / HPIV3), recombinant human parainfluenza virus 1 (HPIV1), recombinant human parainfluenza virus 2 (HPIV2), recombinant human parainfluenza virus 3 (HPIV3), or recombinant bovine parainfluenza virus 3 (BPIV3). The aforementioned recombinant paramyxovirus.

2. The recombinant paramyxovirus according to claim 1, comprising an RSV F ectodomain linked to the TM and CT of the paramyxovirus F protein.

3. The recombinant paramyxovirus according to claim 1, comprising an RSV F ectodomain linked to the CT of the paramyxovirus F protein, wherein the RSV F ectodomain is linked to the CT of the paramyxovirus F protein via an RSV F transmembrane domain.

4. The recombinant paramyxovirus according to any one of claims 1 to 3, wherein the RSV F ectodomain is derived from human subtype A RSV or human subtype B RSV.

5. The recombinant paramyxovirus according to any one of claims 1 to 4, wherein the RSV F ectodomain is stabilized in the RSV F pre-fusion conformation by one or more amino acid substitutions compared to the natural RSV F protein sequence.

6. The RSV F ectodomain is, (a) 66E; (b) 101P; (c) 155C and 290C; (d) 190F; (e) 207L; 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 a combination of (c), (d) and (e) The recombinant paramyxovirus according to claim 5, comprising amino acids shown as, where the amino acid numbering corresponds to the RSV F protein sequence shown as Sequence ID No.

1.

7. The RSV F ectodomain is, (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 a combination of (c), (d) and (e) Recombinant paramyxovirus according to claim 6, comprising the amino acid substitution shown as.

8. The recombinant paramyxovirus according to claim 6 or 7, wherein the RSV F ectodomain comprises 66E, 101P, 155C, 290C, 190F, and 207L.

9. The RSV F ectodomain is represented by Sequence ID No. 1 (WT RSV F A), No. 2 (WT RSV F B), No. 12 (A2 HEK), No. 14 (A2 HEK DS), or No. 21 (A2 Recombinant paramyxovirus according to any one of claims 1 to 5, comprising or comprising an amino acid sequence that is at least 90% identical to one RSV ectodomain of HEK DS-Cav1 or one RSV ectodomain of Sequence ID No. 1, 2, 12, 14, or 21.

10. (a) Recombinant HPIV1 in which the RSV F ectodomain is linked to the CT of the HPIV1 F protein; (b) Recombinant HPIV2 in which the RSV F ectodomain is linked to the CT of the HPIV2 F protein; (c) Recombinant HPIV3 in which the RSV F ectodomain is linked to the CT of the HPIV3 F protein; (d) Recombinant HPIV3 in which the RSV F ectodomain is linked to the CT of the BPIV3 F protein; (e) Recombinant BPIV3 in which the RSV F ectodomain is linked to the CT of the HPIV3 F protein; (f) Recombinant BPIV3 in which the RSV F ectodomain is linked to the CT of the BPIV3 F protein; (g) Recombinant B / HPIV3 in which the RSV F ectodomain is linked to the CT of the HPIV3 F protein; or (h) Recombinant B / HPIV3 in which the RSV F ectodomain is linked to the CT of the BPIV3 F protein. Recombinant paramyxovirus according to any one of claims 1 to 9, including

11. (a) wherein the CT of the HPIV1 F protein linked to the RSV F ectodomain comprises the amino acid sequence shown as residues 24-59 of SEQ ID NO: 31, or an amino acid sequence that is at least 90% identical to residues 24-59 of SEQ ID NO: 31; (b) wherein the CT of the HPIV2 F protein linked to the RSV F ectodomain comprises the amino acid sequence shown as residues 29-66 of SEQ ID NO: 39, or an amino acid sequence that is at least 90% identical to residues 29-66 of SEQ ID NO: 39; (c) wherein the CT of the HPIV3 F protein linked to the RSV F ectodomain comprises the amino acid sequence shown as residues 24-46 of SEQ ID NO: 46, or an amino acid sequence that is at least 90% identical to residues 24-46 of SEQ ID NO: 46; (d) wherein the CT of the BPIV3 F protein linked to the RSV F ectodomain comprises the amino acid sequence shown as residues 22-57 of SEQ ID NO: 53, or an amino acid sequence that is at least 90% identical to residues 22-57 of SEQ ID NO: 53; (e) the CT of the HPIV3 F protein linked to the RSV F ectodomain comprises the amino acid sequence shown as residues 24-46 of SEQ ID NO: 46, or an amino acid sequence that is at least 90% identical to residues 24-46 of SEQ ID NO: 46; (f) wherein the CT of the BPIV3 F protein linked to the RSV F ectodomain comprises the amino acid sequence shown as residues 22-57 of SEQ ID NO: 53, or an amino acid sequence that is at least 90% identical to residues 22-57 of SEQ ID NO: 53; (g) the CT of the HPIV3 F protein linked to the RSV F ectodomain contains the amino acid sequence shown as residues 24-46 of SEQ ID NO: 46, or an amino acid sequence that is at least 90% identical to residues 24-46 of SEQ ID NO: 46; or (h) The CT of the BPIV3 F protein linked to the RSV F ectodomain contains the amino acid sequence shown as residues 22-57 of SEQ ID NO: 53, or an amino acid sequence that is at least 90% identical to residues 22-57 of SEQ ID NO:

53. Recombinant paramyxovirus according to claim 10, comprising:

12. (a) wherein the RSV F ectodomain linked to HPIV1 F CT comprises the amino acid sequence shown as Sequence ID No. 133, or an amino acid sequence that is at least 90% identical thereto; (c) wherein the RSV F ectodomain linked to HPIV3 F CT comprises the amino acid sequence shown as Sequence ID No. 8, or an amino acid sequence that is at least 90% identical thereto; (d) wherein the RSV F ectodomain linked to BPIV3 F CT comprises the amino acid sequence shown as Sequence ID No. 16, or an amino acid sequence that is at least 90% identical thereto; (e) wherein the RSV F ectodomain linked to HPIV3 F CT comprises the amino acid sequence shown as Sequence ID No. 8, or an amino acid sequence that is at least 90% identical thereto; (f) wherein the RSV F ectodomain linked to BPIV3 F CT comprises the amino acid sequence shown as Sequence ID No. 16, or an amino acid sequence that is at least 90% identical thereto; (g) wherein the RSV F ectodomain linked to HPIV3 F CT contains the amino acid sequence shown as Sequence ID No. 8, or an amino acid sequence that is at least 90% identical thereto; or (h) wherein the RSV F ectodomain linked to BPIV3 F CT contains the amino acid sequence shown as Sequence ID No. 16, or an amino acid sequence that is at least 90% identical thereto. Recombinant paramyxovirus according to claim 10, comprising:

13. (a) Recombinant HPIV1 in which the RSV F ectodomain is linked to the TM and CT of the HPIV1 F protein; (b) Recombinant HPIV2 in which the RSV F ectodomain is linked to the TM and CT of the HPIV2 F protein; (c) Recombinant HPIV3 in which the RSV F ectodomain is linked to the TM and CT of the HPIV3 F protein; (d) Recombinant HPIV3 in which the RSV F ectodomain is linked to the TM and CT of the BPIV3 F protein; (e) Recombinant BPIV3 in which the RSV F ectodomain is linked to the TM and CT of the HPIV3 F protein; (f) Recombinant BPIV3 in which the RSV F ectodomain is linked to the TM and CT of the BPIV3 F protein; (g) Recombinant B / HPIV3 in which the RSV F ectodomain is linked to the TM and CT of the HPIV3 F protein; or (h) Recombinant B / HPIV3 in which the RSV F ectodomain is linked to the TM and CT of the BPIV3 F protein. Recombinant paramyxovirus according to any one of claims 1 to 9, including

14. (a) wherein the TM and CT of the HPIV1 F protein linked to the RSV F ectodomain include the amino acid sequence shown as SEQ ID NO: 31, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 31; (b) wherein the TM and CT of the HPIV2 F protein linked to the RSV F ectodomain contain the amino acid sequence shown as SEQ ID NO: 39, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 39; (c) wherein the TM and CT of the HPIV3 F protein linked to the RSV F ectodomain include the amino acid sequence shown as SEQ ID NO: 46, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 46; (d) wherein the TM and CT of the BPIV3 F protein linked to the RSV F ectodomain contain the amino acid sequence shown as SEQ ID NO: 53, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 53; (e) wherein the TM and CT of the HPIV3 F protein linked to the RSV F ectodomain include the amino acid sequence shown as SEQ ID NO: 46, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 46; (f) wherein the TM and CT of the BPIV3 F protein linked to the RSV F ectodomain contain the amino acid sequence shown as SEQ ID NO: 53, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 53; (g) wherein the TM and CT of the HPIV3 F protein linked to the RSV F ectodomain contain the amino acid sequence shown as SEQ ID NO: 46, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 46; or (h) wherein the TM and CT of the BPIV3 F protein linked to the RSV F ectodomain contain the amino acid sequence shown as SEQ ID NO: 53, or an amino acid sequence that is at least 90% identical to SEQ ID NO:

53. Recombinant paramyxovirus according to claim 13, including the above.

15. (a) wherein the RSV F ectodomain linked to HPIV1 F™ and CT comprises the amino acid sequence shown as Sequence ID No. 135, or an amino acid sequence that is at least 90% identical thereto; (c) wherein the RSV F ectodomain linked to HPIV3 F™ and CT comprises the amino acid sequence shown as Sequence ID No. 10, or an amino acid sequence that is at least 90% identical thereto; (d) wherein the RSV F ectodomain linked to BPIV3 F™ and CT comprises the amino acid sequence shown as Sequence ID No. 21, or an amino acid sequence that is at least 90% identical thereto; (e) wherein the RSV F ectodomain linked to HPIV3 F™ and CT comprises the amino acid sequence shown as Sequence ID No. 10, or an amino acid sequence that is at least 90% identical thereto; (f) wherein the RSV F ectodomain linked to BPIV3 F™ and CT comprises the amino acid sequence shown as Sequence ID No. 21, or an amino acid sequence that is at least 90% identical thereto; (g) wherein the RSV F ectodomain linked to HPIV3 F™ and CT comprises the amino acid sequence shown as Sequence ID No. 10, or an amino acid sequence that is at least 90% identical thereto; or (h) wherein the RSV F ectodomain linked to BPIV3 F™ and CT comprises the amino acid sequence shown as Sequence ID No. 21, or an amino acid sequence that is at least 90% identical thereto. Recombinant paramyxovirus according to claim 13, including the above.

16. Recombinant HPIV1 in which the viral genome contains genes encoding HPIV1 N, P, M, F, HN, and L proteins, and the CT or TM and CT linked to the RSV F ectodomain are derived from the HPIV1 F protein; Recombinant HPIV2 in which the viral genome contains genes encoding HPIV2 N, P, M, F, HN, and L proteins, and the CT or TM and CT linked to the RSV F ectodomain are derived from the HPIV2 F protein; Recombinant HPIV3 in which the viral genome contains genes encoding HPIV3 N, P, M, F, HN, and L proteins, and the CT or TM and CT linked to the RSV F ectodomain are derived from the HPIV3 F protein; Recombinant HPIV3 in which the viral genome contains genes encoding HPIV3 N, P, M, F, HN, and L proteins, and the CT or TM linked to the RSV F ectodomain and the CT are derived from the BPIV3 F protein; Recombinant BPIV3 in which the viral genome contains genes encoding BPIV3 N, P, V, M, F, HN, and L proteins, and the CT or TM linked to the RSV F ectodomain and the CT are derived from the HPIV3 F protein; Recombinant BPIV3 in which the viral genome contains genes encoding BPIV3 N, P, V, M, F, HN, and L proteins, and the CT or TM linked to the RSV F ectodomain and the CT are derived from the BPIV3 F protein; Recombinant B / HPIV3 in which the viral genome contains genes encoding HPIV3 F and HN proteins and BPIV3 N, P, V, M and L proteins, and the CT or TM and CT linked to the RSV F ectodomain are derived from the HPIV3 F protein; or Recombinant paramyxovirus according to any one of claims 1 to 15, comprising recombinant B / HPIV3, wherein the viral genome comprises genes encoding HPIV3 F and HN proteins and BPIV3 N, P, V, M and L proteins, and the TM and CT linked to the RSV F ectodomain are derived from the BPIV3 F protein.

17. The recombinant paramyxovirus according to any one of claims 1 to 16, wherein the heterologous gene encoding the recombinant RSV F ectodomain is a first or second gene downstream of the genome promoter of the viral genome.

18. The recombinant paramyxovirus according to any one of claims 1 to 17, wherein the viral genome includes a gene encoding the parainfluenza virus F protein, and in particular, the parainfluenza virus F protein is the F protein of a recombinant paramyxovirus.

19. Recombinant paramyxoviruses are Recombinant HPIV1, wherein the viral genome, from upstream to downstream, includes the HPIV1 genome promoter and the genes encoding the HPIV1 N, P, M, F, HN, and L proteins, and a heterologous gene encoding the recombinant RSV F ectodomain 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; Recombinant HPIV2, wherein the viral genome, from upstream to downstream, includes the HPIV2 genome promoter and the genes encoding the HPIV2 N, P, M, F, HN, and L proteins, and a heterologous gene encoding the recombinant RSV F ectodomain 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; The viral genome, from upstream to downstream, includes the HPIV3 genome promoter followed by genes encoding the HPIV3 N, P, M, F, HN, and L proteins, and is recombinant. Recombinant HPIV-3 in which a heterologous gene encoding the RSV F ectodomain 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; Recombinant BPIV3, wherein the viral genome comprises, from upstream to downstream, a BPIV3 genome promoter and subsequently genes encoding BPIV3 N, P, M, F, HN, and L proteins, and a heterologous gene encoding the recombinant RSV F ectodomain 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; or The viral genome, from upstream to downstream, includes the BPIV3 genome promoter, followed by genes encoding BPIV3 N, P, and M proteins, HPIV3 F and HN proteins, and BPIV3 L protein, and a heterologous gene encoding the recombinant RSV F ectodomain 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, wherein the recombinant B / HPIV3 Recombinant paramyxovirus according to any one of claims 16 to 18, including

20. Recombinant paramyxovirus according to any one of claims 16 to 19, comprising recombinant B / HPIV3, wherein the HPIV3 F and HN genes and the BPIV3 N, P, M and L proteins each comprise an amino acid sequence shown as SEQ ID NOs. 43, 101, 47, 48, 49, and 52, or a sequence each identical by at least 90% to SEQ ID NOs. 43, 101, 47, 48, 49, and 52.

21. Recombinant B / HPIV3 or recombinant HPIV3, the viral genome is HPIV3 A recombinant paramyxovirus according to any one of claims 1 to 20, comprising a gene encoding an HN protein, wherein the HPIV3 HN protein comprises threonine and proline at residues 263 and 307, respectively.

22. The recombinant paramyxovirus according to any one of claims 1 to 21, wherein the heterologous gene encoding the RSV F ectodomain is codon-optimized for expression in human cells.

23. Recombinant HPIV3 containing the nucleotide sequence (GS RSV F_HEK_DS-Cav1_H3TMCT) in which a heterogene is shown as Sequence ID No. 11; Recombinant B / HPIV3 containing the nucleotide sequence (GS RSV F_HEK_DS-Cav1_H3TMCT) in which a heterogene is shown as Sequence ID No. 11; or Recombinant B / HPIV3 containing a nucleotide sequence represented as SEQ ID NO: 22 (GA RSV F_HEK_DS-Cav1_B3TMCT) or SEQ ID NO: 23 (GS RSV F_HEK_DS-Cav1_B3TMCT), Recombinant B / HPIV3 containing a nucleotide sequence in which the heterologous gene is represented as SEQ ID NO: 20 (GA RSV F_HEK_DS_B3TMCT) or SEQ ID NO: 137 (GS RSV F_HEK_DS_B3TMCT). Recombinant paramyxovirus according to claim 22, comprising:

24. Recombinant HPIV1, wherein the viral genome comprises, from upstream to downstream, the HPIV1 genome promoter and the subsequent HPIV1 N, P, M, F, HN, and L genes, and a heterologous gene encoding the recombinant RSV F ectodomain is located between the genome 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 HPIV1 F protein; The viral genome, from upstream to downstream, consists of the HPIV1 genome promoter and the following: Recombinant HPIV1 comprising HPIV1 N, P, M, F, HN, and L genes, wherein a heterologous gene encoding the recombinant RSV F ectodomain 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; Recombinant HPIV3, wherein the viral genome comprises, from upstream to downstream, the HPIV3 genome promoter and the subsequent HPIV3 N, P, M, F, HN, and L genes, and a heterologous gene encoding the recombinant RSV F ectodomain is located between the genome 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 HPIV3 F protein; Recombinant HPIV3, wherein the viral genome, from upstream to downstream, includes the HPIV3 genome promoter, followed by the HPIV3 N, P, M, F, HN, and L genes, and a heterologous gene encoding the recombinant RSV F ectodomain 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 HPIV3 F protein; Recombinant HPIV3, wherein the viral genome comprises, from upstream to downstream, the HPIV3 genome promoter and the HPIV3 N, P, M, F, HN, and L genes, and a heterologous gene encoding the recombinant RSV F ectodomain is located between the genome 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 BPIV3 F protein; Recombinant HPIV3, wherein the viral genome, from upstream to downstream, includes the HPIV3 genome promoter, followed by the HPIV3 N, P, M, F, HN, and L genes, and a heterologous gene encoding the recombinant RSV F ectodomain 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; Recombinant BPIV3, wherein the viral genome comprises, from upstream to downstream, the BPIV3 genome promoter and the subsequent BPIV3 N, P, M, F, HN, and L genes, and a heterologous gene encoding the recombinant RSV F ectodomain is located between the genome 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 BPIV3 F protein; Recombinant BPIV3, wherein the viral genome, from upstream to downstream, includes the BPIV3 genome promoter followed by the BPIV3 N, P, M, F, HN, and L genes, and a heterologous gene encoding the recombinant RSV F ectodomain 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; Recombinant B / HPIV3, wherein the viral genome comprises, from upstream to downstream, the BPIV3 genome promoter, followed by the BPIV3 N, P, and M genes, the HPIV3 F and HN genes, and the BPIV3 L gene, and a heterologous gene encoding the recombinant RSV F ectodomain is located between the genome 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 BPIV3 F protein; The viral genome, from upstream to downstream, includes the BPIV3 genome promoter, followed by the BPIV3 N, P, and M genes, the HPIV3 F and HN genes, and the BPIV3 L gene, and heterologous genes encoding the recombinant RSV F ectodomain, N-type Located between the gene encoding protein and the gene encoding P protein, RSV Recombinant B / HPIV3 in which 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; Recombinant B / HPIV3, wherein the viral genome comprises, from upstream to downstream, the BPIV3 genome promoter, followed by the BPIV3 N, P, and M genes, the HPIV3 F and HN genes, and the BPIV3 L gene, and a heterologous gene encoding the recombinant RSV F ectodomain is located between the genome 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 HPIV3 F protein; The viral genome, from upstream to downstream, includes the BPIV3 genome promoter, followed by the BPIV3 N, P, and M genes, the HPIV3 F and HN genes, and the BPIV3 L gene, with a heterologous gene encoding the recombinant RSV F ectodomain located between the gene encoding the N protein and the gene encoding the P protein, and RSV Recombinant B / HPIV3 in which the F ectodomain contains 66E, 101P, 155C, 290C, 190F, and 207L substitutions and is linked to the TM and CT of the HPIV3 F protein; Recombinant HPIV1, wherein the viral genome, from upstream to downstream, includes the HPIV1 genome promoter, followed by the HPIV1 N, P, M, F, HN, and L genes, and a heterologous gene encoding the recombinant RSV F ectodomain is located between the genome 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 HPIV1 F protein; Recombinant HPIV1, wherein the viral genome, from upstream to downstream, includes the HPIV1 genome promoter, followed by the HPIV1 N, P, M, F, HN, and L genes, and a heterologous gene encoding the recombinant RSV F ectodomain 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 CT of the HPIV1 F protein; Recombinant HPIV3, wherein the viral genome, from upstream to downstream, includes the HPIV3 genome promoter, followed by the HPIV3 N, P, M, F, HN, and L genes, and a heterologous gene encoding the recombinant RSV F ectodomain is located between the genome 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 HPIV3 F protein; Recombinant HPIV3, wherein the viral genome, from upstream to downstream, includes the HPIV3 genome promoter followed by the HPIV3 N, P, M, F, HN, and L genes, and a heterologous gene encoding the recombinant RSV F ectodomain 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 CT of the HPIV3 F protein; Recombinant HPIV3, wherein the viral genome, from upstream to downstream, includes the HPIV3 genome promoter followed by the HPIV3 N, P, M, F, HN, and L genes, and a heterologous gene encoding the recombinant RSV F ectodomain is located between the genome 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 BPIV3 F protein; The viral genome, from upstream to downstream, includes the HPIV3 genome promoter, followed by the HPIV3 N, P, M, F, HN, and L genes, with heterologous genes encoding the recombinant RSV F ectodomain, and genes encoding the N protein and P protein. Recombinant HPIV3, located between the gene and the RSV F ectodomain, containing 66E, 101P, 155C, 290C, 190F, and 207L substitutions, and linked to the CT of the BPIV3 F protein; Recombinant BPIV3, wherein the viral genome, from upstream to downstream, includes the BPIV3 genome promoter followed by the BPIV3 N, P, M, F, HN, and L genes, and a heterologous gene encoding the recombinant RSV F ectodomain is located between the genome 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 BPIV3 F protein; Recombinant BPIV3, wherein the viral genome, from upstream to downstream, includes the BPIV3 genome promoter followed by the BPIV3 N, P, M, F, HN, and L genes, and a heterologous gene encoding the recombinant RSV F ectodomain 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 CT of the BPIV3 F protein; Recombinant B / HPIV3, wherein the viral genome comprises, from upstream to downstream, the BPIV3 genome promoter, followed by the BPIV3 N, P, and M genes, the HPIV3 F and HN genes, and the BPIV3 L gene, and a heterologous gene encoding the recombinant RSV F ectodomain is located between the genome 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 BPIV3 F protein; The viral genome, from upstream to downstream, includes the BPIV3 genome promoter, followed by the BPIV3 N, P, and M genes, the HPIV3 F and HN genes, and the BPIV3 L gene, with a heterologous gene encoding the recombinant RSV F ectodomain located between the gene encoding the N protein and the gene encoding the P protein, and RSV Recombinant B / HPIV3 in which the F ectodomain contains 66E, 101P, 155C, 290C, 190F, and 207L substitutions and is linked to the CT of the BPIV3 F protein; Recombinant B / HPIV3, wherein the viral genome comprises, from upstream to downstream, the BPIV3 genome promoter, followed by the BPIV3 N, P, and M genes, the HPIV3 F and HN genes, and the BPIV3 L gene, and a heterologous gene encoding the recombinant RSV F ectodomain is located between the genome 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 HPIV3 F protein; or The viral genome, from upstream to downstream, includes the BPIV3 genome promoter, followed by the BPIV3 N, P, and M genes, the HPIV3 F and HN genes, and the BPIV3 L gene, with a heterologous gene encoding the recombinant RSV F ectodomain located between the gene encoding the N protein and the gene encoding the P protein, and RSV The recombinant paramyxovirus according to claim 1, comprising recombinant B / HPIV3, wherein the F ectodomain comprises 66E, 101P, 155C, 290C, 190F, and 207L substitutions and is linked to the CT of the HPIV3 F protein.

25. HPIV1 is C Δ１７０ Or LY ９４２Ａ Including attenuation mutations; HPIV3 is the HPIV3 JS strain; HPIV3 contains I263T and T370P substitutions in the HN protein; or B / HPIV3 contains I263T and T370P substitutions in the HN protein. Recombinant paramyxovirus according to any one of claims 1 to 24.

26. The recombinant RSV F ectodomain includes RSV F positions 1 to 529; In particular, the recombinant RSV F ectodomain consists of as few residues as 1-529 of SEQ ID NO:

21. It also contains an amino acid sequence that is 90% identical; In particular, the recombinant RSV F ectodomain contains an amino acid sequence that is at least 95% identical to residues 1-529 of SEQ ID NO: 21; In particular, the recombinant RSV F ectodomain comprises the amino acid sequence of residues 1 to 529 of SEQ ID NO: 21, according to any one of claims 1 to 25, the recombinant paramyxovirus.

27. The recombinant paramyxovirus according to claim 1, comprising rB / HPIV3-F2-HEK / GS-opt / DS-Cav1 / B3TMCT, further comprising I263T and T370P substitutions in the HN protein.

28. It is a recombinant paramyxovirus, (a) A viral genome containing heterologous genes encoding the transmembrane domain (TM) and ectodomain of a heterologous virus type I transmembrane protein linked to the transmembrane domain (TM) and cytoplasmic tail (CT) of the paramyxovirus; or (b) Viral genome containing heterologous genes encoding the ectodomain of a heterologous virus type II transmembrane protein linked to the TM and CT of the paramyxovirus HN protein. The recombinant paramyxovirus containing the above.

29. The heterologous gene encodes the cytoplasmic tail (CT) of the paramyxovirus F protein, or the recombinant respiratory syncytial virus RSV F ectodomain which is linked to the transmembrane domain (TM) and CT. Recombinant human / bovine parainfluenza virus 3 (B / HPIV3), recombinant human parainfluenza virus 1 (HPIV1), recombinant human parainfluenza virus 2 (HPIV2), recombinant human parainfluenza virus 3 (HPIV3), recombinant bovine parainfluenza virus 3 (BPIV3), recombinant PIV5, recombinant Sendai virus, or recombinant Newcastle disease virus. Recombinant paramyxovirus according to claim 28.

30. The recombinant paramyxovirus according to any one of claims 1 to 29, wherein at least 90% of the viral particles produced by a host cell infected with the recombinant paramyxovirus or viral vector include a viral envelope comprising an ectodomain encoded by heterologous genes.

31. Recombinant paramyxovirus according to any one of claims 1 to 30, which is an infectious virus, an attenuated virus, and a self-replicating virus.

32. The RSV F ectodomain is present on the viral envelope of the paramyxovirus, according to any one of claims 1 to 31, in the recombinant paramyxovirus.

33. An immunogenic composition comprising a recombinant paramyxovirus according to any one of claims 1 to 32 and a pharmaceutically acceptable carrier.

34. The immunogenic composition according to claim 33, further comprising an adjuvant.

35. A method for inducing an immune response to the RSV F protein in a subject, comprising administering a therapeutically effective amount of the immunogenic composition according to claim 33 or 34 to the subject.

36. Immunodeficiency against respiratory syncytial virus and parainfluenza virus in the subjects A method for inducing a response, comprising administering a therapeutically effective amount of the immunogenic composition according to claim 33 or 34 to a subject.

37. The method according to claim 35 or 36, wherein the immune response is a protective immune response.

38. The method according to any one of claims 35 to 37, comprising prime-boost administration of an immunogenic composition.

39. The method according to any one of claims 35 to 38, comprising intranasal or parenteral administration of an immunogenic composition.

40. The method according to any one of claims 35 to 39, wherein the subject is a human or a veterinary subject.

41. The method according to any one of claims 35 to 40, relating to a person at risk of RSV or PIV infection, or who has RSV or PIV infection.

42. The method according to any one of claims 35 to 41, wherein the subject is under one year of age.

43. The method according to any one of claims 35 to 41, wherein the subject is immune-deficient or elderly.

44. A nucleic acid molecule comprising the genome of a recombinant paramyxovirus according to any one of claims 1 to 32.

45. Recombinant RSV F ectodomain comprising K66E and Q101P amino acid substitutions, optionally (a) to (d): (a) S155C and S290C; (b) S190F; (c) V207L; or (d) (a) and (b); (a) and (c); (b) and (c); or a combination of (a), (b) and (c) The recombinant RSV F ectodomain further comprising one of the above.

46. A nucleic acid molecule encoding the recombinant RSV F ectodomain according to claim 44.

47. Use of recombinant paramyxovirus according to any one of claims 1 to 32 for inducing an immune response to RSV or RSV and PIV in a subject.

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