Recombinant RSV vaccines: methods for their production and use.

Abstract

Compositions and methods for inducing an immune response in a subject with RSV, comprising administering a pharmaceutical composition comprising a prophylactic vaccine against RSV infection, the vaccine comprising a live recombinant canine parainfluenza (CPI) vector backbone engineered to express the RSV F protein.

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 382,453, filed November 4, 2022, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Reference This application contains a Sequence Listing that has been submitted electronically in .XML format, which is hereby incorporated by reference in its entirety. The .XML copy created on November 2, 2023 is named "065095.004PCT.xml" and is 42,181 bytes in size. The Sequence Listing contained in this .XML file is made a part of the present specification and is incorporated by reference in its entirety.

[0003] The present invention relates generally to the field of immunomodulation, and more particularly to compositions and methods for modulating immune responses to disease due to respiratory syncytial virus (RSV) infection. [Background technology]

[0004] Human RSV is the leading virus causing lower respiratory tract disease and hospitalization in young children. While the majority of children infected with RSV suffer from mild upper respiratory tract infections, a small subset experience severe RSV-induced lower respiratory tract infections (LRIs) and bronchiolitis, which often require hospitalization and can be life-threatening (Collins et al., Respiratory syncytial virus, In: Fields Virology, Knipe and Howley, eds., Lippincott Williams & Wilcins, New York (1996), pp. 1313-1351). Nearly all children will eventually become infected with RSV, and 20-30% of RSV-infected children will develop significant LRI, resulting in over 130,000 children being hospitalized annually in the United States due to RSV (Shay et al., JAMA, 282(5):1440-1446(1999) and World Health Organization, Initiative for Vaccine Research (IVR), Respiratory Syncytial Virus (RSV)).

[0005] Several risk factors for the development of severe RSV-induced disease have been clearly identified, including preterm birth (Navas et al., J. Pediatr., 121(3):348-54 (1992)), bronchopulmonary dysplasia (Groothuis et al., Pediatrics, 82(2):199-203 (1988)), congenital heart disease (MacDonald et al., N. Engl. J. Med, 307(1):397-400 (1982)), and T-cell immunodeficiency (Mcintosh et al., J. Pediatr., 82(4):578-90 (1973)). However, more than half of children hospitalized with severe RSV-induced disease have no identified risk factors (Boyce et al., J. Pediatr., 137(6):865-70 (2000)). This means that approximately 1-2% of otherwise healthy children without any identifiable risk factors will suffer potentially life-threatening consequences from RSV-induced disease (Collins et al., supra).

[0006] RSV-induced severe disease in children has also been correlated with the development of asthma (e.g., Sigurs et al., Pediatrics, 95(4):500-505 (1995); Welliver et al., Pediatr. Pulmonol, 15(1):19-27 (1993); Cifiientes et al., Pediatr. Pulmonol, 36(4):316-321 (2003); Schauer et al., Eur. Respir. J., 20(5):1277-1283 (2002); Sigurs et al., Am. J. Respir. Crit. Care Med., 161(5):1501-1507 (2000); and Stein et al. (See, e.g., et al., Lancet, 354(9178):541-545 (1999)). The basis for this association is unclear but may be due to underlying genetic factors, immune dysfunction, antigen-specific responses, or structural lesions caused by lung remodeling after severe RSV disease.

[0007] Although RSV infection is nearly universal by age 3, natural RSV infection does not confer complete immunity, and reinfection occurs throughout life (Hall et al., J. Infect. Dis., 163(4):693-698 (1991) and Muelenaer et al., J. Infect. Dis., 164:15-21 (1991)). In the elderly, RSV is a significant cause of morbidity and mortality. In a retrospective cohort study, RSV was responsible for an average of 17 IS hospitalizations and deaths per 1,000 nursing home residents per year, whereas influenza was responsible for an average of 28 hospitalizations and 15 deaths in the same setting (Garofalo et al., Pediatr. Allergy Immunol., 5(2):111-117 (1994)). Thus, RSV was isolated as frequently as influenza A in this population and was associated with a mortality rate comparable to that of influenza A (Ellis et al., J. Am. Geriatr. Soc., 51(6):761-72003 and Falsey et al., J. Infect. Dis., 772(2):389-394 (1995)).

[0008] Currently, there is no FDA-approved vaccine for the prevention of RSV infection or the treatment of RSV-induced disease in children. The only FDA-approved treatment for the prevention of RSV infection is SYNAGIS® (palivizumab) (MedImmune, Gaithersburg, MD), a humanized monoclonal antibody directed against an epitope in the A antigenic site of the RSV F protein administered to high-risk infants. SYNAGIS® has shown significant improvement in preventing acute RSV disease in the lower respiratory tract and reducing lower respiratory tract infections, but has not been shown to be effective against RSV infection in the upper respiratory tract at tolerated doses. In 2023, the FDA approved Bayfortas (nirsevimab), a long-acting RSV monoclonal antibody with an extended half-life, which is recommended for all infants under 8 months of age who are born during the RSV season or entering their first RSV season, as well as infants and children 8 to 19 months of age who are at high risk for severe RSV disease and entering their second RSV season.

[0009] RSV vaccine development has been plagued by a legacy of vaccine-enhanced disease in children following natural RSV infection (Kim et al., Am. J. Epidemiol., 89(4):422-434 (1969), and Kapikian et al., Am. J. Epidemiol., 89(4):405-421 (1969)). For example, a formalin-inactivated, aluminum-precipitated vaccine candidate (FI-RSV) administered to RSV-naive infants in the early 1960s was immunogenic but did not protect the children from natural infection. Furthermore, vaccinees who subsequently became infected with RSV had increased hospitalization rates, more severe illness, and two deaths compared with control children immunized with formalin-inactivated parainfluenza virus (Kapikian et al., supra; Chin et al., Am. J. Epidemiol., 89(4):449-463 (1969); and Polack et al., J. Exp. Med, 196(6):859-65 (2002)). Other approaches to RSV immunization include live-attenuated RSV, RSV subunit proteins, and parainfluenza virus chimeras. Live attenuated RSV vaccines have been tested in clinical trials in RSV-naive infants, but have not demonstrated genetic stability of mutations, an optimal balance of attenuation for safety in infants, or the achievement of a protective immune response (Karron et al., J. Infect. Dis., 191(7):1093-1104 (2005), and Bukreyev et al., J. Virol, 79(15):9515-9526 (2005)). Protein subunit vaccines based on RSV G and F proteins have been safely administered to adults and RSV-seropositive children, but are only moderately immunogenic (Tristram et al., Vaccine, 12(6):551-556 (1994)).

[0010] Thus, there remains a need for compositions and methods for effectively and safely preventing or treating RSV infection.

[0011] Thus, the present invention provides such compositions and methods for effectively and safely preventing or treating RSV infection in mammals, preferably humans. Summary of the Invention

[0012] In accordance with the objective(s) of the invention embodied and broadly described herein, in one aspect, the invention relates to a viral expression vector comprising a parainfluenza virus 5 (PIV5) genome having a heterologous nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 1, wherein the viral expression vector expresses a heterologous polypeptide comprising a live recombinant canine parainfluenza (CPI) vector backbone engineered to express the RSV F protein as a target antigen. In one embodiment, the RSV F protein is encoded by a wild-type or mutant RSV F protein gene. In another embodiment, the RSV F protein gene is codon-optimized for expression in human subjects. In yet another embodiment, the RSV F protein gene is inserted between the SH and HN junctions of the CPI antigenome cDNA, and the CPI antigenome cDNA is sequenced using a primer having a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, or 19. In another embodiment, the parainfluenza (CPI) vector backbone engineered to express the RSV F protein comprises a 22 amino acid extension as part of its cytoplasmic tail. In another aspect, the present invention relates to a pharmaceutical composition comprising a parainfluenza virus 5 (PIV5) viral expression vector having a heterologous nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 1, wherein the viral expression vector expresses a heterologous polypeptide comprising a live recombinant canine parainfluenza (CPI) vector backbone engineered to express the RSV F protein as a target antigen. In one embodiment, the RSV F protein is encoded by a wild-type or mutant RSV F protein gene, wherein the RSV F protein gene is codon-optimized for expression in a human subject. In another embodiment, the RSV F protein gene is inserted between the SH and HN junctions of the CPI antigenome cDNA, and the CPI antigenome cDNA is sequenced using a primer having a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, or 19. In yet another embodiment, the parainfluenza (CPI) vector backbone engineered to express the RSV F protein comprises a 22 amino acid extension as part of its cytoplasmic tail. In another embodiment, the live recombinant canine parainfluenza (CPI) vector backbone engineered to express the RSV F protein is a prophylactic vaccine against RSV infection.

[0013] In yet another aspect, the present invention relates to a method for inducing an immune response in a subject with RSV, the method comprising administering a prophylactic vaccine against RSV infection, wherein the vaccine comprises a live recombinant canine parainfluenza (CPI) vector backbone engineered to express the RSV F protein as described above. In another embodiment, the vaccine induces RSV F protein-specific serum antibodies and cellular responses. In another embodiment, the RSV-F-specific serum antibodies and cellular responses are associated with a reduced incidence of RSV-induced pathological pulmonary responses compared to immune responses obtained by administering formalin-inactivated RSV (FI-RSV). In another embodiment, the pathological pulmonary response is selected from the group consisting of peribronchiolitis, perivasculitis, interstitial pneumonia, and alveolitis. In another embodiment, the vaccine is administered intranasally, intramuscularly, topically, or orally. In another embodiment, the vaccine is administered in a single or multiple dose regimen.

[0014] Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

[0015] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiment(s) of the invention and, together with the description, serve to explain the principles of the invention. [Brief explanation of the drawings]

[0016] [Figure 1]Figures A-B show the RSV F-specific immune response in immunized African green monkeys (AGMs), i.e., the RSV F-specific cellular immune response in immunized AGMs. PBMCs were isolated from immunized AGMs one day before vaccination and 14 and 28 days after immunization. PBMCs were stimulated with an RSV F peptide pool, and the levels of different cytokines in CD4+ cells (A) and CD8+ cells (B) were quantified by ICS and expressed as a percentage of the total cytokines. Group 1 = control, Group 6 = CPI-RSV-F. [Figure 2A] The plasmid map of pSP28 is shown. [Figure 2B] The plasmid map of pAB94 is shown. [Figure 3] The CPI-RSV-F vaccine vector plasmid and map are shown. [Figure 4] 1 shows an exemplary outline of viral rescue. [Figure 5] A-C show representative images of the RSV F protein expression percent assay CPI-RSV-F pre-MVS. [Figure 6] A-C show representative images of the RSV F protein expression percent assay CPI-RSV-F MVS. [Figure 7] An explanatory diagram of the experimental timeline for CHD03 is shown. [Figure 8] An explanatory diagram of the experimental timeline for CHD04 is shown. [Figure 9] A-B show RSV-F serum antibody titers in CHD03 (A) and CHD04 (B). Naive BALB / c mice were treated with PBS or immunized intranasally with 10 PFU of PIV5-RSV-F, PIV5ΔSH-RSV-F, CPI-RSV-F, CPIΔSH-RSV-F, or RSV_rA2. Serum was collected 4 weeks after immunization. RSV-F-specific IgG antibody endpoint titers were determined by ELISA (N=5). The horizontal line represents the geometric mean antibody titer for each group. Statistical significance was determined by ANOVA and Dunnett's multiple comparison test. ****P<0.0001, significance between PBS and vaccine groups. LOD, limit of detection. [Figure 10] Panels A-B show RSV-F ELISPOT titers for CHD03 and CHD04. Naive BALB / c mice were treated with PBS or immunized intranasally with 10 PFU of PIV5-RSV-F, PIV5ΔSH-RSV-F, CPI-RSV-F, CPIΔSH-RSV-F, or RSV_rA2. Splenocytes were harvested and stimulated with RSV F peptide. Results are presented as the number of IFN-γ-secreting cells per 10 splenocytes (N=5). Horizontal lines represent geometric mean ELISPOT titers. Statistical significance was determined by ANOVA and Dunnett's multiple comparison test. *P<0.05, **P<0.01, ***P<0.001, significance between PBS and vaccine groups. [Figure 11] A to B show lung titers for CHD03 (A) and CHDO4 (B). [Figure 12] Pulmonary RSV challenge virus titers after RSV challenge are shown. [Figure 13] 1 shows nasal RSV challenge virus titers following RSV challenge in nasal washes. [Figure 14] RSV neutralizing antibody responses are shown. [Figure 15] Shows anti-RSV F protein IgG antibody responses by ELISA. [Figure 16A] 1 is a bar graph showing RSV NS1 mRNA expression. [Figure 16B] 1 is a bar graph showing IL-4 mRNA expression. [Figure 16C] 1 is a bar graph showing IL-2 mRNA expression. [Figure 16D] 1 is a bar graph showing IFN-γ mRNA expression. [Figure 17]A–F are bar graphs showing serum RSV-specific antibody (Ab) titers before and after vaccination with BLB201 (CPI-RSV-F) in young adults (Group 1) and elderly adults (Group 2). A–B show RSV neutralizing Abs by microneutralization assay. C–D show F-specific RSV IgA Abs, and E–F show F-specific IgG Abs by ELISA. Individual log2 titers and mean log2 titers with their respective standard deviations (error bars) by age group are shown in the left column, and geometric mean fold increases (GMFRs) from baseline by age group at Days 15 and 29 are shown in the right column. *P<0.05, **P<0.01, ***P<0.001 by Wilcoxon test. [Figure 18] A-D are line graphs showing serum RSV neutralizing (nAb) titers and nasal F-specific IgA antibody (Ab) titers (C-D) of individual subjects in Group 1 (A) and Group 2 (B) before and after vaccination with BLB201 (CPI-RSV-F) (days 1, 15, and 29) by age group 1 and group 2. [Figure 19] A–D are bar graphs showing serum PIV5 antibody (Ab) titers before and after vaccination with BLB201 (CPI-RSV-F) for the two vaccination groups. A–B show PIV5-neutralizing Abs by microneutralization assay, and C–D show PIV5-specific RSV IgG Abs by ELISA. Individual log2 titers and mean log2 titers with their respective standard deviations (error bars) by age group at days 1, 15, and 29 are shown in the left column, and geometric mean fold increases (GMFRs) from baseline at days 15 and 29 by age group are shown in the right column. *P<0.05, **P<0.01, ***P<0.001 by Wilcoxon test. [Figure 20](A-C) are bar graphs showing the relationship between response rates to PIV5-RSV vaccination based on baseline (pre-vaccination) PIV5 neutralizing antibody (nAb) levels. (A) Percentage of subjects with a PIV5 neutralizing (nAb) antibody response rate (≥1.5 fold increase in titer) to the PIV5-RSV vaccine, and (B) Percentage of subjects with a RSV neutralizing (nAb) antibody response rate (≥1.5 fold increase in titer) to the PIV5-RSV vaccine, by subgroups based on whether an individual subject's baseline PIV5 nAb titer was below (L) or above (H) the baseline geometric mean titer of PIV5 nAb in each age group (Group 1 and Group 2). C shows the proportion of subjects with a nasal F-specific IgA response (≥2 fold increase in titer) to the PIV5-RSV vaccine by subgroup based on whether the individual baseline nasal F-specific IgA titer was below (L) or above (H) the baseline geometric mean titer of nasal F-specific IgA in each age group (Group 1 and Group 2). [Figure 21] A-B are bar graphs showing nasal IgA antibody titers by ELISA before and after vaccination with F-specific RSV IgA Ab for groups 1 and 2. Individual log2 titers and mean log2 titers with their respective standard deviations (error bars) by age group are shown in the left graph (A), and the geometric mean fold rise (GMFR) from baseline by age group on days 15 and 29 is shown in the left graph (B). [Figure 22-1]Figure 1A shows RSV F-specific CD4+ T cell responses before and after vaccination with BLB201 (CPI-RSV-F). The percentage of CD4+ T cells by age group on days 1, 15, and 29 for each individual (represented by symbols and continuous lines) according to the expression of the indicated immune markers. *P<0.05, **P<0.01, ***P<0.001, ns: not significant. Figure 1B shows RSV F-specific CD4+ T cell responses before and after vaccination with BLB201 (CPI-RSV-F). The percentage of CD4+ T cells by age group on days 1, 15, and 29 for each individual (represented by symbols and continuous lines) according to the expression of the indicated immune markers. *P<0.05, **P<0.01, ***P<0.001, ns: not significant. Figure 1C shows RSV F-specific CD8+ T cell responses before and after vaccination with BLB201 (CPI-RSV-F). Percentage of CD8+ T cells by age group on days 1, 15, and 29 for each individual (represented by symbols and continuous lines) with expression of the indicated immune markers. *P<0.05, **P<0.01, ***P<0.001, ns: not significant. D shows RSV F-specific CD8+ T cell responses before and after vaccination with BLB201 (CPI-RSV-F). Percentage of CD8+ T cells by age group on days 1, 15, and 29 for each individual (represented by symbols and continuous lines) with expression of the indicated immune markers. *P<0.05, **P<0.01, ***P<0.001, ns: not significant. [Figure 22-2]E shows RSV F-specific CD4+ and CD8+ T cell responses before and after vaccination with BLB201 (CPI-RSV-F). The mean percentages of CD4+ T cells (left graph) and CD8+ T cells (right graph) are shown by age group on days 1, 15, and 29, according to the expression of the indicated Th1 / cytotoxicity marker combinations (IFN-γ, TNF-α, CD107a) represented in stacked histograms. *P<0.05, **P<0.01, ***P<0.001; ns: not significant. F shows RSV F-specific CD4+ and CD8+ T cell responses before and after vaccination with BLB201 (CPI-RSV-F). The mean percentages of CD4+ T cells (left graph) and CD8+ T cells (right graph) are shown by age group on days 1, 15, and 29 according to the expression of the indicated combinations of Th1 / cytotoxicity markers (IFN-γ, TNF-α, CD107a) represented as stacked histograms. *P<0.05, **P<0.01, ***P<0.001, ns: not significant. G shows RSV F-specific CD4+ T cell responses before and after vaccination with BLB201 (CPI-RSV-F). The geometric mean fold increase (GMFR) from baseline for CD4+ T cells on days 15 and 29 by age group is shown. The fold change in CD4 T cell responses on days 15 and 29 post-vaccination is shown. *P<0.05, **P<0.01, ***P<0.001, ns: not significant. H shows RSV F-specific CD8+ T cell responses before and after vaccination with BLB201 (CPI-RSV-F). The geometric mean fold increase (GMFR) from baseline for CD8+ T cells by age group on days 15 and 29 is shown. The fold change in CD8 T cell responses on days 15 and 29 after vaccination is shown. *P<0.05, **P<0.01, ***P<0.001, ns: not significant. [Figure 22-3]I shows RSV F-specific CD4+ and CD8+ T cell responses before and after vaccination with BLB201 (CPI-RSV-F). Pie charts show the percentage of F-specific CD4+ T cells and the percentage of CD8+ T cells expressing one, two, or three Th1 / cytotoxicity markers (IFN-γ, TNF-α, CD107a) for Group 1 on days 15 and 29. J shows RSV F-specific CD4+ and CD8+ T cell responses before and after vaccination with BLB201 (CPI-RSV-F). Pie charts show the percentage of F-specific CD4+ T cells and the percentage of CD8+ T cells expressing one, two, or three Th1 / cytotoxicity markers (IFN-γ, TNF-α, CD107a) for Group 2 on days 15 and 29. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention can be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the examples contained therein, as well as the drawings and their preceding and following descriptions.

[0018] I. Definition To facilitate an understanding of the principles and features of various embodiments of the present disclosure, various exemplary embodiments are described herein. While exemplary embodiments of the present disclosure are described in detail, it should be understood that other embodiments are contemplated. Accordingly, it is not intended that the present disclosure be limited in scope to the details of construction and arrangement of components set forth in the description or examples. The present disclosure is capable of other embodiments and of being practiced or carried out in various ways.

[0019] In describing exemplary embodiments, specific terminology is used for the sake of clarity. As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, a reference to a component is intended to include compositions of multiple components. A reference to a composition containing "a" component is intended to include the specified one as well as other components.

[0020] Ranges may be expressed herein as "about," "approximately," or "substantially" from one particular value and / or to another particular value. When such a range is expressed, another exemplary embodiment includes from the one particular value and / or to the other particular value. Unless otherwise indicated, all numbers expressing amounts of ingredients, molecular weights, and the like used in the specification and claims should be understood to be modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of reported significant digits by applying ordinary rounding techniques.

[0021] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0022] For any method disclosed herein that includes separate steps, the steps can be performed in any feasible order, and, where appropriate, any combination of two or more steps can be conducted simultaneously.

[0023] This description illustrates exemplary embodiments. In several places throughout this application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0024] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless specifically stated.

[0025] The present invention is illustrated by the following examples, it being understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention described herein.

[0026] Similarly, as used herein, "substantially free" or "substantially pure" of something, and similar characterizations, can include both being "at least substantially free" or "at least substantially pure" of something, and being "completely free" or "completely pure" of something.

[0027] "Comprising" or "containing" or "including" means that at least the named compounds, elements, particles, or method steps are present in a composition or article or method, but does not exclude the presence of other compounds, elements, particles, or method steps, even if such other compounds, elements, particles, or method steps have the same function as the named ones.

[0028] The terms "preferred" and "preferably" refer to embodiments of the invention that may offer certain advantages, under particular circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0029] The terms "patient," "individual," "subject," and "animal" are used interchangeably herein and refer to mammals, including, but not limited to, humans and veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.), as well as experimental model animals. In particularly preferred embodiments, the subject is a human.

[0030] As used herein, the term "vaccinating" typically refers to the sequential administration of one or more antigens to a subject to generate and / or enhance an immune response to the antigen(s). Sequential administration includes a priming immunization followed by one or several boost immunizations.

[0031] Within the context of the present invention, the term "pathogen" refers to any agent capable of causing a pathological condition. Examples of "pathogens" include, but are not limited to, cells (e.g., bacterial cells, diseased mammalian cells, cancerous mammalian cells), fungi, parasites, viruses, prions, or toxins. A suitable pathogen is an infectious agent. In certain embodiments, the infectious agent is a virus, such as a coronavirus.

[0032] As used herein, "antigen" refers to any molecule capable of eliciting a T-cell or B-cell immune response in a subject. A pathogen-specific antigen is typically an element obtained or derived from the pathogen, containing an epitope and capable of eliciting an immune response against the pathogen. Depending on the pathogenic agent, the antigen may be of various nature, such as a (poly)peptide, protein, nucleic acid, lipid, or cell. Live, attenuated forms of pathogens (e.g., bacteria, viruses), or killed or inactivated forms thereof may also be used, or purified materials therefrom, such as proteins, peptides, or lipids, may be used. The antigen may be naturally occurring or artificially created. It may be foreign to the mammal being treated or endogenous (e.g., tumor antigen). The antigen may be produced by techniques known per se in the art, such as synthetic or recombinant techniques, or enzymatic approaches.

[0033] In certain embodiments, the antigen is a protein, polypeptide, and / or peptide. The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues may be modified, such as an artificial chemical mimic of a corresponding naturally occurring amino acid, or may be non-naturally occurring residues. It should be understood that the term "protein" also includes fragments or variants of different antigens, such as epitope-containing fragments, or proteins obtained from pathogens and subsequently modified enzymatically, chemically, mechanically, or thermally.

[0034] "Therapeutically effective amount" means the amount of a compound (e.g., a PIV5-based composition described herein) that, when administered to a subject for treating a condition, disorder, or pathology, is sufficient to effect such treatment. A "therapeutically effective amount" will vary depending on the composition or bacterium administered, as well as the disease and its severity, and the age, weight, physical condition, and responsiveness of the mammal being treated.

[0035] The phrase "pharmaceutically acceptable," when used in connection with compositions of the present disclosure, refers to molecular entities and other components of such compositions that are physiologically tolerable and typically do not produce an adverse response when administered to a mammal (e.g., a human). Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias for use in mammals, more particularly humans.

[0036] As used herein, the term "pharmaceutically acceptable composition" refers to a composition comprising at least one compound disclosed herein prepared together with one or more pharmaceutically acceptable carriers.

[0037] The term "administration" refers to the introduction of a certain amount of a predetermined substance into a patient by a certain appropriate method. The compositions disclosed herein may be administered via any conventional route, such as, but not limited to, inhalation, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, pulmonary, or rectal administration, as long as they can reach the desired tissue. However, because peptides are digested when administered orally, the active ingredients of compositions for oral administration must be coated or formulated to protect them from degradation in the stomach.

[0038] The term "dose" refers to a single amount of a compound or agent being administered, and / or a "regimen," where a "regimen" refers to multiple predetermined doses given at varying time intervals, which may be of different or similar duration, and which may be of different or similar amount. In some embodiments, a regimen also includes time points for a delivery period (e.g., a drug administration period, or a treatment period). Alternatively, a regimen is multiple predetermined vapor amounts given at predetermined time intervals.

[0039] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or saline solutions and aqueous dextrose and glycerol solutions are preferably used as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including, but not limited to, one or more of binders (for compressed pills), fluidizing agents, encapsulating agents, flavoring agents, and coloring agents. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

[0040] The term "treating" a condition, disorder, or condition or "treatment" thereof includes (1) preventing or delaying the appearance of at least one clinical or subclinical symptom of the condition, disorder, or condition in a subject who may be affected by or susceptible to the condition, disorder, or condition, but who has not yet experienced or exhibited any clinical or subclinical symptoms of the condition, disorder, or condition; or (2) inhibiting the condition, disorder, or condition, i.e., arresting, alleviating, or delaying the onset of the disease or its recurrence (in the case of maintenance treatment) or at least one clinical or subclinical symptom thereof; or (3) palliating the disease, i.e., regressing the condition, disorder, or condition, or at least one clinical or subclinical symptom thereof. The effect on the treated subject is either statistically significant or at least perceptible to the patient or physician.

[0041] Within the context of the present invention, the term "pathogen" refers to any agent capable of causing a pathological condition. Examples of "pathogens" include, but are not limited to, cells (e.g., bacterial cells, diseased mammalian cells, cancerous mammalian cells), fungi, parasites, viruses, prions, or toxins. A suitable pathogen is an infectious agent. In certain embodiments, the infectious agent is a virus, such as a coronavirus.

[0042] "Comprising" or "containing" or "including" means that at least the named compounds, elements, particles, or method steps are present in a composition or article or method, but does not exclude the presence of other compounds, elements, particles, or method steps, even if such other compounds, elements, particles, or method steps have the same function as the named ones.

[0043] As used herein, the term "parainfluenza virus 5" (PIV5) includes, but is not limited to, strains KNU-11, CC-14, D277, 1168-1, and 08-1990. Non-limiting examples of PIV5 genomes are listed in GenBank Accession Nos. NC_006430.1, AF052755.1, KC852177.1, KP893891.1, KC237065.1, KC237064.1, and KC237063.1, which are incorporated herein by reference.

[0044] As used herein, the term "expression" refers to the process by which a polynucleic acid is transcribed into mRNA and translated into a peptide, polypeptide, or protein. If the polynucleic acid is derived from genomic DNA, expression may include splicing of the mRNA, if an appropriate eukaryotic host cell or organism is selected. In the context of the present invention, this term also encompasses the yield of RSV F gene mRNA and RSV F protein achieved after expression.

[0045] As used herein, the term "F protein" or "fusion protein" or "F protein polypeptide" or "fusion protein polypeptide" refers to a polypeptide or protein having all or part of the amino acid sequence of a RSV fusion protein polypeptide. Many RSV fusion proteins and attachment proteins have been described and are known to those skilled in the art. WO / 2008 / 114149 (incorporated herein by reference in its entirety) describes exemplary F and G protein variants (e.g., naturally occurring variants).

[0046] The materials described as comprising various elements of the present disclosure are intended to be exemplary and not limiting. Many suitable materials that would perform the same or similar functions as the materials described herein are intended to be included within the scope of the present disclosure. Such other materials not described herein can include, but are not limited to, materials developed after the time of development of the present disclosure.

[0047] II.CPI-RSV-F composition Respiratory syncytial virus (RSV) is a member of the genus Pneumovirus in the family Paramyxoviridae. Human RSV (HRSV) is a major cause of severe lower respiratory tract disease in young children and is responsible for significant morbidity and mortality in humans. RSV is also recognized as an important vector of disease in immunocompromised adults and the elderly. Following natural infection, RSV can infect multiple times during childhood and adulthood due to incomplete defenses against RSV in infected hosts.

[0048] The virus has a genome consisting of a single negative-stranded RNA, which is tightly associated with viral proteins to form a nucleocapsid. The viral envelope is composed of a lipid bilayer derived from the plasma membrane containing virally encoded structural proteins. Viral polymerase is packaged with the viral particle and transcribes the genomic RNA into mRNA. The RSV genome encodes three transmembrane structural proteins, F, G, and SH, two matrix proteins, M and M2, three nucleocapsid proteins, N, P, and L, and two nonstructural proteins, NS1 and NS2.

[0049] Fusion of HRSV with the infected cell membrane is thought to occur at the cell surface and is a necessary step for the import of viral ribonucleoproteins into the cytoplasm during the early stages of infection. This process is mediated by the fusion (F) protein, which also promotes fusion of the infected cell membrane with that of neighboring cells to form characteristic syncytia, which contribute to both the pronounced cytopathic effect and an additional mechanism for viral spread. Therefore, neutralization of fusion activity is important for blocking viral infection and is crucial for host immunity. Indeed, monoclonal antibodies developed against the F protein have been shown to neutralize viral infectivity and inhibit membrane fusion (Calder et al., 2000, Virology 271:122-131).

[0050] The RSV F protein shares structural features and limited but significant amino acid sequence identity with the F glycoproteins of other paramyxoviruses. It is synthesized as a 574-amino acid inactive (F0) precursor that is cotranslationally glycosylated at asparagine in the endoplasmic reticulum, where it assembles into homo-oligomers. Before reaching the cell surface, the F0 precursor is cleaved by proteases N-terminally to F2 and C-terminally to F1. The F2 and F1 chains remain covalently linked by one or more disulfide bonds.

[0051] CPI-RSV-F is a parainfluenza virus (PIV5)-based RSV vaccine expressing the RSV F protein, and is provided herein as a prophylactic intranasal vaccine for preventing RSV infection and serious complications associated with RSV infection. CPI-RSV-F was designed to induce an immune response against the RSV F protein, a major antigenic protein that is highly conserved between RSV subgroups A and B. Anti-F antibodies inhibit viral entry into host cells, and RSV F is a proven vaccine target based on efficacy data from the commercially available product palivizumab (RSV F monoclonal antibody).

[0052] The present disclosure provides CPI-RSV F compositions and systems and methods for their use in a variety of applications, including functional genomics, drug discovery, target validation, protein production (e.g., therapeutic proteins, vaccines, monoclonal antibodies), gene therapy, and therapeutic treatments such as cancer therapy.

[0053] A. Pharmacological Summary of RSV Studies The present disclosure provides CPI-RSV F compositions and systems and methods for their use in a variety of applications, including functional genomics, drug discovery, target validation, protein production (e.g., therapeutic proteins, vaccines, monoclonal antibodies), gene therapy, and therapeutic treatments such as cancer therapy.

[0054] Previously published studies based on the W3A strain of PIV5 constructs engineered to express the RSV-F protein included immunogenicity and challenge studies in mice, cotton rats, and African green monkeys (1, 2, 3). As part of these studies, permissive PIV5 replication was confirmed in cotton rats and African green monkeys (1, 2). Additionally, the following studies were performed: 1) comparing vector constructs based on the CPI and W3A PIV5 strains, and 2) comparing vaccine constructs expressing the RSV prefusion protein with those expressing the wt F protein inserted in place of the PIV5 SH gene (ΔSH), or between the PIV5 SH and NH (SH-NH) or HN and L (HN-L) sequences. These studies led to the selection of CPI-RSV-F (CPI-RSV-F), which contains the full-length RSV F protein inserted between the SH and HN genes, for use in initial human trials (Figure 1).

[0055] Furthermore, the protective efficacy of the CPI-RSV-F vaccine has been evaluated in RSV challenge studies conducted in mice and cotton rats. Immunization with a single intranasal dose protected animals from RSV infection, based on significantly reduced RSV viral titers observed in lung and nasal washes of immunized animals compared to controls.

[0056] More recent preclinical proof-of-concept studies conducted with the vaccine vector construct CPI-RSV-F included immunogenicity and challenge studies in mice and African green monkeys conducted by Blue Lake Biotechnology Inc. Additionally, we summarize preclinical data from an NIH-sponsored study of the CPI-RSV-F construct in a cotton rat challenge study (for which Blue Lake Biotechnology Inc. provided the vaccine product). The CPI-RSV-F vaccine used in these more recent nonclinical studies used a previous vaccine vector construct (rescued from BHK cells) identical to the vector construct used in the clinical trial material (rescued from 293 / Vero cells), was similarly produced using serum-free Vero cells as a substrate, and formulated in sucrose phosphate glutamate (SPG) buffer. These nonclinical studies included W3AΔSH-RSV-F (a PIV5 W3A strain engineered to express the RSV F protein) as an active control.

[0057] A phase 1 clinical trial of CIP-RSV-F demonstrated the safety of the RSV vaccine in young adults (group 1) and elderly people (group 2). The vaccine induced serum RSV-specific and intranasal IgA antibody responses, as well as strong cellular immune responses (Spearman et al., Sci. Adv. 9, eadj7611 (2023)).

[0058] In summary, preclinical studies in various animal models have demonstrated the ability of CPI-RSV-F to induce RSV F-specific immune responses, as observed by F-specific antibody and cellular responses after a single intranasal administration. CPI-RSV-F was well tolerated in these animal models, and no signs of sensitization were observed after vaccination with CPI-RSV-F. Recent preclinical studies conducted with the CPI-RSV-F vaccine construct (a vaccine based on the CPI PIV5 strain) in mice, cotton rats, and African green monkeys are summarized below. These studies included an active control (a vaccine vector based on the PIV5 W3A strain) with an RSV F protein (ΔSH) replacing the SH gene. For details on the vaccine vector constructs, see Figure 1. For a summary of preclinical studies conducted with various vaccine vector constructs, see Table 1.

[0059] [Table 1]

[0060] i. Testing in mice, cotton rats, and monkeys using the related W3A PIV5 vector expressing the RSV F protein PIV5(W3A)-RSV-F and RSV-G protein study in Balb / c mice (3, Phan et al. 2014): In this study, Balb / c mice received a single intranasal dose (10 6 A single intranasal dose of 50 μl of PFU was administered followed by RSV challenge. The PIV5 W3A vaccine vector construct in this study contained a wild-type RSV F protein inserted into the PIV5 HN and L intergenic region. A single intranasal dose resulted in an IgG2a / IgG1 RSV response 21 days post-immunization similar to that observed after wild-type RSV A2 infection.

[0061] Challenge studies of PIV5(W3A) expressing wild-type or prefusion RSV F proteins in mice and cotton rats (1, Phan et al., 2017): This study evaluated improved PIV5 vector vaccines by altering the F protein insertion site (insertion at the SH-HN junction of PIV5 or replacing SH with the RSV F protein gene). Additionally, this study evaluated both wild-type (wt) F protein and the prefusion conformation F protein (pF).

[0062] Mouse, 1 x 10 6 Mice were immunized intranasally with a single dose of W3AΔSH-RSV-F (RSV F protein gene inserted into the deleted SH region of PIV5) expressing wild-type F protein or a prefusion-stabilized RSV F mutant (W3AΔSH-RSV-pF) or the improved vectors W3A-RSV-F SH-HN or W3A-RSV-pF SH-HN (F protein gene inserted into the SH-HN junction) in PFU. The test groups were as follows (Table 2):

[0063] [Table 2]

[0064] After immunization, both humoral and cellular immune responses were observed. The highest neutralizing antibody responses were detected with vaccine constructs using the wt F protein. Furthermore, vaccine constructs containing wt F inserted with ΔSH before the HN junction appeared to be the most immunogenic. The level of cellular immune responses (based on IFN-gamma using ELISPOT) was similar among the various vaccine constructs.

[0065] Twenty-eight days after immunization, mice were challenged with RSV A2 to determine protective efficacy. Challenge virus was recovered from only one of five mice in the W3AΔSH-RSV-F group on day 4 post-challenge, but not from any mice in the other vaccinated groups. Challenge virus was recovered from all mice in the PBS control group.

[0066] Similar studies were performed in cotton rats, which are more permissive to RSV infection. 3 PFU of modified vectors (W3A-RSV-F(SH-HN), W3A-RSV-pF, W3AΔSH-RSV-F) or 10 2 Rats were immunized with PFU of W3AΔSH-RSV-pF.

[0067] Immune responses, including neutralizing antibody responses against the RSV A Tracy strain (97% identical to the RSV A / A2 strain), were observed in all groups. Similar to the mouse study, groups immunized with vaccine constructs containing the wt F protein had higher antibody levels compared to the pF group, with the highest values ​​(titers of approximately 128) when ΔSH and F were inserted before the HN gene junction. Neutralizing antibody titers against RSV / B / 18537 were significantly lower than those against the RSV / A Tracy strain, with significant antibody levels (titers of approximately 8) detected only in the W3A(SH-HN)-RSV-F and W3AΔSH-RSV-F groups. At day 28, 1.21 × 10 5 After RSV challenge with PFU RSV / A / Tracy, the nasal wash (1.4–1.66 Log 10 in lung lavage fluid (2-3 Log 10 A reduction in RSV viral load of approximately 10 per nasal wash was observed in all vaccine dose groups. RSV challenge virus was recovered from all mice in the PBS control group. 5 PFU, or 10 5 PFU / g of lung wash).

[0068] Overall, these initial studies in mouse and cotton rat models showed no evidence that the pre-fusion F protein was more immunogenic or protective than the wild-type F protein, and no difference in vector performance was observed between the ΔSH and SH-HN insertion vector constructs via the intranasal route.

[0069] A challenge study of Sigmovir Protocol No. XV-131 study report (Phan et al. 2017) in cotton rats using intranasal and subcutaneous routes: A follow-up study (1, Phan et al. 2017) demonstrated a high dose of 10 5 and 10 6 The efficacy, immunogenicity, and safety of PFU W3A(SH-NH)-RSV-F and controls were tested in a cotton rat challenge study evaluating two different routes of administration: intranasal (in) and subcutaneous (sc). The study also included positive controls for disease enhancement (positive control groups consisting of animals immunized with FI-RSV followed by RSV challenge and animals pre-infected with RSV followed by RSV challenge). Animals in this study were challenged with RSV A2 on day 49, followed by necropsy and histology 5 days later (day 54). The test groups in this study were as follows (Table 3):

[0070] [Table 3]

[0071] Immunogenicity: W3A(SH-HN)-RSV-F showed a 10% increase between the intranasal and subcutaneous injection groups. 5 ~10 6 Vaccination with W3AΔSH-RSV-F induced slightly higher neutralizing antibody titers in the intranasal group compared to the subcutaneous group, with a 10 6 The dose resulted in slightly higher titers, but this was not statistically significant.

[0072] Efficacy: W3A(SH-HN)-RSV-F provided complete protection of the lower respiratory tract when administered either intranasally or subcutaneously. Animals also had significantly lower viral loads in the upper respiratory tract.

[0073] W3A(SH-HN)-RSV-F vaccination was significantly higher than control animals sham-immunized with PBS (mean titer 10 5 The reduction in titers observed in lung and nasal washes compared with the mean titer of 103 Subcutaneous administration provided complete protection of the lower respiratory tract, and intranasal administration provided near complete protection, based on PFU.

[0074] Safety: Lung sections from different groups were examined for peribronchiolar inflammation, perivasculitis, interstitial pneumonia, and alveolitis, which are characteristics of pulmonary inflammation, and scored for severity. The greatest lesions were observed in the FI-RSV-immunized RSV-challenged group (positive control group). Lung changes were moderate in the groups immunized with W3A-RSV-F (SH-HN) or W3AΔSH-RSV-F (intranasally or subcutaneously), with levels lower than those observed in the RSV-immunized RSV-challenged positive control group and similar to those in the PBS-sham-immunized RSV-challenged group.

[0075] Cytokine levels measured in lung tissue 5 days after challenge by quantitative real-time PCR (qPCR) did not indicate a potential for enhanced infection. IL-4 mRNA levels were significantly elevated only in the FI-RSV-vaccinated group, consistent with the histopathology results and enhanced disease phenotype. IFNγ mRNA levels were highest in the sham-immunized and FI-RSV-immunized groups. IFN mRNA levels were similarly low between the PIV5-based candidate and RSV-immunized groups. IL-2 mRNA levels were comparable in all groups, but the mean IL-2 levels in the FI-RSV-immunized group were significantly higher than those in the other groups.

[0076] On day 28 after immunization, immunized mice were treated with RSV A2 (10 in 50 μl) 6 After intranasal challenge with 100 PFU of RSV-A2, lung sections obtained 4 days postchallenge showed no progression of lung lesions compared to RSV-A2-immunized mice. Protective immunity, as assessed by viral load in lung tissue, was observed (n = 5 mice per group).

[0077] PIV5(W3A)-expressing RSV F or G protein challenge study in cotton rats and African green monkeys (2, Wang et al., 2017): This study evaluated PIV5-vectored RSV F protein in cotton rats and African green monkeys for replication, immunogenicity, and efficacy in protecting against RSV challenge. In this study, the F protein was inserted into the intergenic region of the PIV5 HN and L genes.

[0078] Replication permissiveness of PIV5 in cotton rats and African green monkeys: 1 x 10 5 PFU of PIV5 was inoculated intranasally in a volume of 10 μl or 100 μl. On day 4, virus titers were measured in nasal homogenates (up to 1 × 10 4 PFU) was observed in all animals inoculated with the larger inoculum volume (100 μl), which had largely disappeared by day 6. With the larger inoculum volume (100 μl), vaccine virus was observed in the lungs of all animals on day 6. This was observed in only one animal inoculated with the smaller volume of 10 μl.

[0079] To assess PIV5 tolerance in African green monkeys, 60 animals were screened for anti-PIV5 antibodies and all were found to be negative. Animals (n = 3 per group) were immunized with 1 × 10 2 ~Max 1×10 8 Mice were infected intranasally with 1 x 10 PFU of PIV5 in a dose volume of 0.25 mL. Nasal washes and bronchoalveolar lavage were assessed for virus shedding on days 3, 5, 7, 10, and 14. Virus was administered at a dose of 1 x 10 2 Doses as low as 1.5 PFU were shedding from both the nose and lungs for up to 10 days with peak replication on day 5. This data demonstrates the permissiveness of African green monkeys to PIV5.

[0080] Immunogenicity in cotton rats and African green monkeys after a single dose control: 1x10 cotton rats 3 , 1x10 4 , 1x10 5 , and 1x10 6Mice were immunized intranasally with full-fledged full-fledged immunization (PFU) of W3A-RSV-F(SH-HN). IgG antibody responses were observed at all dose levels on day 28, with comparable responses across dose groups. Neutralizing antibodies were observed in all dose groups, ranging in titer from 64 to 256 (NT50%). Furthermore, IgA responses in lung homogenates were observed in all dose groups on day 21 postinoculation.

[0081] African green monkeys (PIV5 and RSV seronegative) were cultured at 1 × 10 4 or 1×10 6 A single intranasal immunization with PFU of W3A-RSV-F(SH-HN) was administered. Sera obtained 21 days after inoculation demonstrated high-titer F-specific antibody responses. In addition, low-level neutralizing antibody responses were observed on day 21 (NT50%, 1 × 10 6 In the 1×10 PFU dose group, nasal swabs obtained 21 days after immunization showed significant levels of F protein IgA responses. Cellular responses, as assessed by gamma interferon, were 1×10 6 The PFU dose group showed a low level of response.

[0082] Protection from RSV challenge in cotton rats and African green monkeys: 1 x 10 3 ~1×10 6 Cotton rats immunized intranasally with a single dose ranging from 100 PFU of W3A-RSV-F(SH-HN) were challenged with the RSV A2 strain 28 days after immunization. Protection was assessed by measuring viral load in nasal and lung tissues. Reductions in viral load were greater than 1 x 10 6 A dose-dependent pattern was observed except for the PFU group, which had a concentration of 1 × 10 3 Although the levels were higher compared to the PFU group, they were still reduced compared to the control group. 5 In the 10-dose group, no virus was detected in the lungs and titers were reduced (3 log 10 (of the order of magnitude) was observed in the nose.

[0083] 1×10 4 or 1×10 6African green monkeys immunized with PFU W3A-RSV-F(SH-HN) were challenged with RSV A2 28 days after immunization. Nasal and BAL samples were assessed for RSV viral load 3-14 days after challenge. Control immunization did not shorten viral shedding, but the maximum viral RSV load was reduced 10- to 100-fold in both dose groups, reaching 1 x 10 6 The greatest reduction was observed in the PFU dose group.

[0084] Responses in RSV-exposed African green monkeys relative to controls: Prior exposure to RSV did not interfere with the ability to boost RSV neutralizing antibody titers (50-fold increase) in African green monkeys (seroconverted by intranasal infection with RSV A2).

[0085] Lung pathology of W3A-RSV-F(SH-HN)-immunized cotton rats after RSV challenge: 1 × 10 on day 0 6 Lungs from animals immunized with PFU of W3A-RSV-F and challenged with RSV A2 on day 49 were examined by histopathology in a blinded manner for alveolitis, interstitial pneumonia, perivasculitis, and peribronchiolitis 5 days postchallenge and compared with lungs from control animals inoculated with formalin-inactivated RSV. Scores from positive control animals inoculated with formalin-inactivated RSV were significantly higher than those from PBS control animals, but not as high as those from W3A-RSV-F(SH-HN) animals (ANOVA paired t-test).

[0086] B. Parainfluenza virus 5 (PIV5) Parainfluenza virus 5 (PIV5), a negative-strand RNA virus, is a member of the Rubulavirus genus in the Paramyxoviridae family, which includes many important human and animal pathogens, such as mumps virus, human parainfluenza virus types 2 and 4, Newcastle disease virus, Sendai virus, HPIV3, measles virus, canine distemper virus, rinderpest virus, and respiratory syncytial virus. PIV5 was formerly known as simian virus 5 (SV5). PIV5 infects many animals and humans, but no human symptoms or diseases are known to be associated with PIV5. Unlike most paramyxoviruses, PIV5 infects normal cells and rarely causes cytopathic effects. As a negative-strand RNA virus, the PIV5 genome is highly stable. PIV5 does not have a DNA stage in its life cycle and replicates exclusively in the cytoplasm, meaning that PIV5 cannot integrate into the host genome. Therefore, using PIV5 as a vector avoids potential unintended consequences of genetic modification of host cell DNA. PIV5 can grow to high titers intracellularly, including Vero cells, which have been approved for vaccine production by the WHO and FDA. Thus, PIV5 offers many advantages as a vaccine vector.

[0087] The PIV5-based vaccine vectors of the present invention may be based on any of a variety of wild-type, mutant, or recombinant (rPIV5) strains. Wild-type strains include, but are not limited to, PIV5 strain W3A, WR (ATCC® No. VR-288™), canine parainfluenza virus strain 78-238 (ATCC No. VR-1573) (Evermann et al., 1980, J Am Vet Med Assoc; 177:1132-1134; and Evermann et al., 1981, Arch Virol; 68:165-172), canine parainfluenza virus strain D008 (ATCC No. VR-399) (Binn ​​et al., 1967, Proc Soc Exp Biol Med; 126:140-145), MIL, DEN, LN, MEL, Cryptovirus, CPI+, CPI-, H221, 78524, T1, and SER. See, e.g., Chatziandreou et al., 2004, J Gen Virol; 85(Pt10):3007-16; Choppin, 1964, Virology: 23:224-233, and Baumgartner et al., 1987, Intervirology; 27:218-223. Additionally, PIV5 strains used in commercially available kennel cough vaccines, such as, for example, BI, FD, Merck, and Merial vaccines, may be used.

[0088] C.PIV5 CPI strain vector backbone The PIV5 CPI strain vector backbone differs from that of the PIV5 W3A strain vector as follows. The most notable difference is the PIV5 F protein of the CPI strain, which consists of an additional 22 amino acid extension as part of the cytoplasmic tail. The F protein extension is thought to inhibit the membrane fusion properties of the virus (5, 6). CPI-based viruses are more lytic and produce more progeny virus in infected cells than W3A-based viruses, which do not have the extended PIV5 F protein tail and contain additional amino acids different from CPI (4).

[0089] i.PIV5 CPI-vectored RSV constructs

[0090] The PIV5 vaccine vector of the present invention can be constructed using any of a variety of methods, including, but not limited to, reverse genetics, as described in detail by He et al. (Virology; 237(2):249-60, 1997). PIV5 encodes eight viral proteins. The nucleocapsid protein (NP), phosphoprotein (P), and large RNA polymerase (L) protein are important for transcription and replication of the viral RNA genome. The V protein plays an important role in viral pathogenesis and viral RNA synthesis. The fusion (F) protein, a glycoprotein, mediates both cell-cell fusion and virus-cell fusion in a pH-independent manner, which is essential for viral entry into cells. The structure of the F protein has been determined, and amino acid residues essential for efficient fusion have been identified. The hemagglutinin-neuraminidase (HN) glycoprotein is also involved in viral entry and release from host cells. The matrix (M) protein plays an important role in viral assembly and budding. The hydrophobic (SH) protein is a 44-residue hydrophobic integral membrane protein that is oriented within the membrane with its N-terminus in the cytoplasm. For reviews of the molecular biology of paramyxoviruses, see, e.g., Whelan et al., 2004, Curr Top Microbiol Immunol; 283:61-119, and Lamb & Parks, (2006). Paramyxoviridae: the viruses and their replication. In Fields Virology, 5th edn, pp. 1449-1496. Edited by D.M.K.nipe & P.M. Howley. Philadelphia, PA: Lippincott Williams & Wilkins.

[0091] To date, recombinant PIV5 viruses expressing foreign genes from numerous pathogens, including influenza, rabies, respiratory syncytial virus, tuberculosis, Burkholderia, and MERS-CoV, have been generated and tested as vaccine candidates (Li, Z., et al., J Virol, 87(1):354(2013); Chen, Z., et al., J Virol, 87(6):2986(2013); Wang, D., et al., J Virol, 91(11)(2017); Chen, Z., et al., Vaccin, 33(51):7217(2015); Lafontaine, E.R., et al., Vaccine X., 1:100002(2018); Li, K., et al., mBio, 11(2)(2020)). PIV5-vectored vaccines actively proliferate in the respiratory tract after intranasal immunization and can generate mucosal immunity, including antigen-specific IgA antibodies and long-lived IgA plasma cells (Wang, D., et al., J Virol, 91(11)(2017). Xiao, P., et al., Front Immunol, 12:623996(2021)).

[0092] In one embodiment, the CPI-RSV-F vaccine drug substance (CPI-RSV-F) presented herein comprises a live recombinant PIV5 vectored virus based on the CPI strain of PIV5 expressing the wild-type F protein of RSV (F protein sequence based on GenBank accession FJ614814J, SV A2 strain) codon-optimized for expression in humans (Figure 7). The wild-type RSV F protein is inserted between the SH and NH regions of PIV5 (Figure 1).

[0093] ii. Immunogenicity and challenge studies with CPI-RSV-F vaccine constructs In non-clinical studies, a single dose of CPI-RSV-F was 4 ~10 6It was found to be immunogenic in mice, cotton rats, and AGM after a single intranasal administration at dose levels ranging from 100 PFU to 1000 PFU. Both F-specific serum immune responses and cellular responses were detected, confirming the ability of the PIV5 vector to replicate in cotton rats and AGM.

[0094] A single intranasal dose of CPI-RSV-F or related vaccine constructs was able to protect various challenge model animals (mice, cotton rats, and AGM) from infection using RSV A2 as the challenge. Efficacy was based on the reduction in viral load observed in lung and nasal washes after challenge compared to that observed in control animals.

[0095] In a cotton rat challenge model using the CPI-RSV-F vector construct intended for use in the proposed Phase 1 CPI-RSV-F trial, no enhanced disease / pulmonary pathology was observed. This conclusion was based on the cytokine profile (no increased IL-4 response) observed in lung tissue compared to positive control animals vaccinated with formalin-inactivated RSV (FI-RSV) and challenged with RSV. Similarly, in previously published challenge studies in mice, cotton rats, and AGMs evaluating various W3A-based vector constructs with F protein insertions at different locations within PIV5 W3A, no signs of enhanced disease / pulmonary pathology were observed.

[0096] CPI-RSV-F was chosen over W3A for initial clinical evaluation given its similarity in inducing protective immune responses at similar dose levels as the W3A construct (although the immune response to W3A appeared to be slightly higher). Furthermore, this same CPI PIV5 backbone engineered to express the SARS-CoV-2 S protein is also being evaluated clinically under cross-referenced IND027418. Preclinical and limited clinical data available to date with this related vector construct are based on a 10-day follow-up in healthy adults aged 18-55 years. 6 It has demonstrated a favorable safety profile when administered as a single intranasal dose at PFU.

[0097] III. BLB genome sequence Provided herein is the CPI-RSV-F genome sequence.

[0098] A. CPI-RSV-F genome sequence i.CPI-RSV-F 5' to 3' The CPI-RSV-F 5' to 3' nucleic acid sequence is provided herein.

[0099] IV. Treatment method The present disclosure may be used in gene therapy and / or therapeutic approaches for the treatment of diseases involving the increase or decrease of a nucleotide sequence of interest in a host cell. In these embodiments, the expressible heterologous nucleotide sequence may be derived from a mammalian genome. In some embodiments, it may be particularly useful to have an expressible heterologous nucleotide sequence derived from the human genome, where expression of wild-type RNA and / or protein may result in a therapeutic effect in a patient. For example, the expressible heterologous nucleotide sequence may encode CFTR, NeuroD1, Cas9, and guide RNA, or any other such sequence. In other embodiments, the heterologous nucleotide sequence encodes a secreted protein.

[0100] In other embodiments, the expressible heterologous nucleotide sequence responds to a positive selection stimulus. In other embodiments, the expressible heterologous nucleotide sequence also responds to a negative selection stimulus. In further embodiments, it may be useful for the polynucleotide sequence to further comprise a reporter gene. For example, the reporter gene may be luciferase or green fluorescent protein.

[0101] V. Method of Administration A. Administration by vaccination The present invention includes methods of vaccinating a subject by administering to the subject a viral expression vector, viral particle, or composition described herein.

[0102] The present invention provides the use of a recombinant PIV5-based live vaccine consisting of canine parainfluenza (CPI) engineered to express the RSV F protein as a target antigen to develop a CPI-RSV-F vaccine as a novel prophylactic vaccine against RSV infection.

[0103] i. Intranasal vaccination In one embodiment, the disclosed CPI-RSV-F composition is formulated to allow intranasal administration. Numerous non-clinical studies have been conducted to evaluate the immunogenicity and efficacy of CPI-RSV-F or closely related vaccine constructs (e.g., W3A PIV5-based constructs) expressing the RSV F protein in various animal models using the intranasal route. This included early developmental studies in mice, cotton rats, and African green monkeys (AGMs) evaluating variations in vector constructs (the location of the F protein within the vector) and RSV F protein insertions (wild-type or pre-fusion F protein). Findings from these studies are summarized and the publications are incorporated herein by reference.

[0104] The intranasal composition may include an inhalable dry powder pharmaceutical formulation containing a therapeutic agent, where the therapeutic agent is present as a free base or as a mixture of a salt and the free base. The pharmaceutical formulations disclosed herein may be formulated to be suitable for airway administration, for example, nasal administration, intranasal administration, sinusoidal administration, oral administration, and / or pulmonary administration. Typically, the formulation is manufactured to have a particle size appropriate for the route or target of airway administration. Thus, the formulations disclosed herein may be manufactured to have a defined particle size distribution.

[0105] For example, the particle size distribution of a salt form of a therapeutic agent for intranasal administration may be about 5 μm to about 350 μm. More specifically, the salt form of a therapeutic agent for intranasal administration may have a particle size distribution of about 5 μm to about 250 μm, about 10 μm to about 200 μm, about 15 μm to about 150 μm, about 20 μm to about 100 μm, about 38 μm to about 100 μm, about 53 μm to about 100 μm, about 53 μm to about 150 μm, or about 20 μm to about 53 μm. The salt form of a therapeutic agent in a pharmaceutical composition of the present invention may have a particle size distribution range of less than about 200 μm for intranasal administration. In other embodiments, the salt form of the therapeutic agent in the pharmaceutical composition has a particle size distribution of less than about 150 μm, less than about 100 μm, less than about 53 μm, less than about 38 μm, less than about 20 μm, less than about 10 μm, or less than about 5 μm. The salt form of the therapeutic agent in the pharmaceutical composition of the invention may have a particle size distribution range of more than about 5 μm, more than about 10 μm, more than about 15 μm, more than about 20 μm, more than about 38 μm, less than about 53 μm, less than about 70 μm, more than about 100 μm, or more than about 150 μm for intranasal administration.

[0106] Additionally, the salt form of a therapeutic agent in a pharmaceutical composition of the invention can have a particle size distribution range of about 1 μm to about 10 μm for pulmonary administration. In other embodiments for pulmonary administration, the particle size distribution range is about 1 μm to about 5 μm, or about 2 μm to about 5 μm. In other embodiments, the salt form of a therapeutic agent has an average particle size of at least 1 μm, at least 2 μm, at least 3 μm, at least 4 μm, at least 5 μm, at least 10 μm, at least 20 μm, at least 25 μm, at least 30 μm, at least 40 μm, at least 50 μm, at least 60 μm, at least 70 μm, at least 80 μm, at least 90 μm, or at least 100 μm.

[0107] In some embodiments, the disclosed cannabinoid compositions comprise one or more cannabinoids or pharmaceutically acceptable derivatives or salts thereof, a propellant, an alcohol, and a glycol and / or glycol ether. The alcohol can be a monohydric alcohol or a polyhydric alcohol, preferably a monohydric alcohol. Monohydric alcohols have a lower viscosity than glycols or glycol ethers. Thus, the composition can form droplets with a smaller diameter compared to compositions in which the monohydric alcohol is absent. The inventors surprisingly discovered that a specific ratio of monohydric alcohol to glycol or glycol ether results in a composition with a desirable combination of both long-term stability (e.g., the composition remains a single phase at temperatures between 2 and 40°C for at least one week) and small droplet size.

[0108] a. Vaccine immunogenicity and protection in the lower respiratory tract of mice One embodiment provides a candidate vaccine virus administered intranasally that elicited high levels of anti-F serum antibodies in both CHD03 and CHD04 studies. In W3AASH-RSV-F vaccinated animals, the geometric mean titer of RSV F antibody titers was 3.1 Log at CHD03. 10 / mL and CHD04 3.4Log 10 In CPI-RSV-F vaccinated animals, the geometric mean titer was 3.0 Log 10 / mL and CHD04 3.2Log 10 / mL. These values ​​were not statistically significant from each other and were similar to the values ​​from the RSV_rA2 positive control group. Both vaccine viruses also induced RSV F protein-specific cellular responses as measured by IFN-γ-secreting cells. In CHD03, W3AΔSH-RSV-F had a geometric mean value of 28 IFN-γ-secreting cells, while CPI-RSV-F had a geometric mean value of 10 6 In CHD04, W3AΔSH-RSV-F and CPI-RSV-F had a geometric mean of 23 IFN-γ-secreting cells per splenocyte. 6The CPI-RSV-F group had a geometric mean of 35 and 48 IFN-γ-secreting cells per splenocyte, respectively. These values ​​were statistically significant from the PBS control group but did not differ between the vaccinated groups or from the RSV_rA2 positive control group. IFN-γ-secreting cells in the CPI-RSV-F group in CHD04 were significantly higher than the positive control. Both W3AΔSH-RSV-F and CPI-RSV-F had significantly lower RSV challenge virus titers compared to the PBS control group in both CHD03 and CHD04. The values ​​were 3.21 Log for the PBS control group. 10 PFU / g and 3.33 Log 10 PFU / g for W3AΔSH-RSV-F, whereas for W3AΔSH-RSV-F it was 1.35 Log 10 PFU / g and 1.37Log 10 PFU / g, 1.48 Log for CHD03 and CHD04, respectively 10 PFU / g and 1.40Log 10 The PFU / gCPI-RSV-F was 1.39 Log CFU / gCPI-RSV-F. 10 PFU / g, CHD04 1.32Log 10 The PFU / g values ​​were similar to those of the vaccine group of interest.

[0109] Overall, the results presented here demonstrate that W3AΔSH-RSV-F and CPI-RSV-F, as well as two other candidates (W3A-RSV-F(SH-HN) and CPIΔSH-RSV-F), were significantly more potent than the 10 5 We demonstrated that intranasal administration of 100 PFU of avian flu vaccine induced comparable RSV-specific immune responses and significant protection against RSV challenge virus replication in the lower respiratory tract of BALB / c mice.

[0110] b. Preclinical testing of the PIV5ΔSH-RSV-F vaccine based on the CPI strain of PIV5 in a cotton rat virus challenge model using the RSV A / A2 virus strain, compared with the PIV5 W3A-based PIV5ΔSH-RSV-F vaccine. The efficacy and safety of RSV F protein vaccine candidates based on the CPI or W3A strains of PIV5 (CPI-RSV-F and W3AΔSH-RSV-F, respectively) were evaluated in the cotton rat Sigmodon hispidus model of RSV A / A2 challenge. 4 , 10 5 , or 10 6 Animals were immunized intranasally with 100 μl of PIV5-based vaccine containing 10 PFU of virus, and then 4 weeks later, 10 5 The primary infection control group was mock-immunized with PBS and then infected with RSV A / A2. The secondary infection control group was infected with RSV A / A2 and re-infected 7 weeks later. The vaccine-boosted disease control group was immunized twice with FI-RSV at 4-week intervals and infected with RSV 3 weeks after the second immunization. Five days after RSV challenge, animals were sacrificed and samples were collected. RSV replication in the lungs and nose, lung histopathology, lung cytokine and RSV NS1 mRNA expression, and RSV serum neutralizing antibody (NA) and anti-F protein binding antibody titers were measured. To confirm PIV5 vaccine replication in the respiratory tract of cotton rats, groups of three animals were immunized with 10 6 Cotton rats were inoculated intranasally with PFU of CPI-RSV-F or W3AΔSH-RSV-F and sacrificed 4 days later. These samples were subjected to plaque assay evaluation. Briefly, both candidate vaccine viruses were found to replicate efficiently in the upper and lower respiratory tracts of cotton rats. The W3AΔSH-RSV-F vaccine virus replicated to higher levels in the upper respiratory tract compared to CPI-RSV-F, but replicated to lower levels in the lower respiratory tract compared to CPI-RSV-F.

[0111] Both the CPI-RSV-F and W3AΔSH-RSV-F vaccines were highly effective in protecting the lungs of cotton rats, inducing near-sterilizing immunity at all vaccine doses tested, as demonstrated by plaque assays and qPCR. All doses of both vaccines also induced statistically significant nasal protection, with W3AΔSH-RSV-F immunization being stronger than CPI-RSV-F immunization.

[0112] W3AΔSH-RSV-F reduced nasal viral load to nearly undetectable levels at all three vaccine doses tested. 6 The 10 PFU dose induced the strongest protection. 5 PFU and 10 4 Both the CPI-RSV-F vaccine and the control vaccine induced strong NA responses, with the control vaccine eliciting a strong NA response at the highest dose (10 PFU) at all three doses tested. 6 PFU). The lower the dose of CPI-RSV-F, the weaker the NA response induced. Immunization with both CPI-RSV-F and W3AΔSH-RSV-F induced high levels of binding IgG, although vaccination with W3AΔSH-RSV-F was slightly higher. Both vaccines were significantly more potent than vaccination with the intermediate dose of W3AΔSH-RSV-F (10 5 With the exception of one animal immunized with 10 PFU, none of the W3AΔSH-RSV-F vaccines induced the level of lung histopathology or IL-4 mRNA expression seen in FI-RSV-immunized animals. Overall, the efficacy of the W3AΔSH-RSV-F vaccine appears to exceed that of CPI-RSV-F with respect to nasal protection and neutralizing antibody responses at all doses tested. 4 ~10 6 Increasing the PFU did not provide any clear benefit in terms of improving vaccine efficacy.

[0113] ii. Pulmonary Compositions One embodiment provides formulations and methods for treating SARS-CoV-2 in the pulmonary system by inhalation or pulmonary administration. The diffusion characteristics of a particular formulation through lung tissue are selected to achieve an effective concentration and effective residence time in the treated tissue. Doses may be increased or decreased, or administered more frequently or less frequently, to achieve selected blood levels. Furthermore, the timing and amount of administration of the formulation are preferably controlled to optimize the therapeutic effect of the administered formulation on the treated tissue and / or to titrate to a specific blood level.

[0114] Diffusion through lung tissue can be further modified by various excipients that can be added to the formulation to delay or accelerate the absorption of the drug into lung tissue. For example, the drug can be combined with phospholipids, surfactants such as dimyristoyl phosphatidylcholine, and dimyristoyl phosphatidylglycerol. This drug can also be used in combination with bronchodilators, which can relax the bronchial airways and facilitate the entry of anti-cancer drugs into the lungs. Albuterol is an example of the latter, along with many other examples known in the art. Furthermore, the drug can be complexed with biocompatible polymers, micelle-forming structures, or cyclodextrins.

[0115] The particle size of the aerosolized drug used in this example was measured at about 1.0-5.0 μm with a GSD of less than about 2.0 for deposition in the central and peripheral compartments of the lung. As described elsewhere herein, the particle size is selected depending on the desired deposition site of the drug particles within the respiratory tract.

[0116] Aerosols useful in the present invention include an aqueous vehicle such as water or saline with or without ethanol, and may include preservatives or antimicrobial agents such as benzalkonium chloride, parabens, and / or stabilizers such as polyethylene glycol.

[0117] Powders useful in the present invention include formulations of undiluted drugs or drug formulations in combination with excipients or carriers such as mannitol, lactose, or other sugars. The powders used herein are effectively suspended in a carrier gas for administration. Alternatively, the powder may be dispersed in a chamber containing a gas or gas mixture, which is then inhaled by the patient.

[0118] The agents of the present disclosure may be administered once or in several divided doses at intervals. For example, the agents of the present disclosure may be administered repeatedly, for example, at least 2, 3, 4, 5, 6, 7, 8, or more times, or by continuous infusion. It is understood that the exact dosage and duration of treatment will vary depending on the disease being treated and can be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It should be noted that concentration and dosage values ​​may also vary depending on the severity of the condition to be alleviated. It should be further understood that for any particular subject, the specific dosage regimen should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the composition, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions and methods.

[0119] In some treatment embodiments, an "effective amount" of an agent is an amount that results in a reduction of at least one pathological parameter. Thus, for example, in some aspects of the present disclosure, an effective amount is an amount effective to achieve at least about 10%, at least about 15%, at least about 20%, or at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% reduction in the parameter compared to the expected reduction in the parameter in an individual not treated with the agent.

[0120] In some embodiments, any of the PIV5-based constructs and methods described in WO2013 / 112690 and WO2013 / 112720 (incorporated herein by reference in their entireties) may be used in the present invention.

[0121] As used herein, the term "subject" refers to a living organism, including, for example, a mammal. Mammals include, but are not limited to, humans, non-human primates, and other non-human vertebrates. A subject may be an "individual," a "patient," or a "host." Non-human vertebrates include livestock (such as, but not limited to, cows, horses, goats, and pigs), pet or companion animals such as, but not limited to, dogs or cats, and laboratory animals. Non-human subjects also include non-human primates and rodents, such as, but not limited to, rats or mice. Non-human subjects also include, but are not limited to, poultry, horses, cows, pigs, goats, dogs, cats, guinea pigs, hamsters, mink, and rabbits.

[0122] As used herein, "in vitro" is an intracellular culture, and "in vivo" is within the body of a subject. As used herein, "isolated" refers to material that has been removed from its natural environment (e.g., the natural environment in which it occurs in nature), or that has been produced using recombinant techniques, or that has been chemically or enzymatically synthesized, and thus has been altered "by the hand of man" from its natural state. [Example]

[0123] Example 1: Immunogenicity of CPI-RSV-F in African Green Monkeys (AGM) (Non-GLP Study) Objective: To compare the immunogenicity of CPI-RSV-F and W3AΔSH-RSV-F administered intranasally as a single dose in an African green monkey model.

[0124] method

[0125] On day 0, four monkeys from each dose group were 6 Mice were immunized intranasally with either PFU of CPI-RSV-F (Group 6) or a control (Group 1).

[0126] result

[0127] Antibody responses to the RSV F protein and PIV5 vector on day 28 were determined by ELISA assay (plates coated with recombinant RSV F protein (source: Sino Biologicals: 11049-V088 LC120C 2910) or PIV5 wild-type virus) as shown in Table 4. In summary, all animals developed antibodies against the F protein expressed by the CPI or W3AΔSH vector, and RSV F ELISA titers were comparable between the two groups.

[0128] [Table 4]

[0129] Cellular responses: RSV F protein-specific cellular responses were evaluated by intracellular cytokine staining (ICS) assay. Blood was collected on days -1, 14, and 28 after immunization to quantify RSV F protein-specific CD4 and CD8 cell responses. IFNγ, TNFα, MIP-1b, IL-13, and CD107a-positive cells were quantified (see Figures 1A-1B). PBMCs collected one day before immunization did not respond to RSV F peptide stimulation as expected. After immunization, AGMs immunized with W3AΔSH-RSV-F (group 1) and CPI-RSV-F (group 6) generated RSV F protein-specific CD4 and CD8 cell responses when assessed on days 14 and 28. No F-specific cellular responses were detected in control animals (data not shown), and cellular responses from animals immunized with W3AΔSH-RSV-F and CPI-RSV-F increased from day 14 to day 28 after immunization (Figures 1A-1B). W3AΔSH-RSV-F induced a lower CD4 response than CPI-RSV-F on day 14, but the difference was not statistically significant. On day 28 after immunization, W3AΔSH-RSV-F induced a higher CD4 response than CPI-RSV-F, but this difference was also not statistically significant. The CD8-specific response induced by W3AΔSH-RSV-F was higher than that of CPI-RSV-F, but the difference was not statistically significant on day 14 (P = 0.0883) or day 28.

[0130] Both CPI-RSV-F and W3AΔSH-RSV-F induced an immune response to the RSV F protein. The antibody response to the RSV F protein induced by either CPI-RSV-F or W3AΔSH-RSV-F was 10 6 After a single intranasal dose of PFU, the CD4 and CD8 cell responses induced by CPI-RSV-F were comparable, but were slightly lower than those induced by W3AΔSH-RSV-F, although this difference was not statistically significant.

[0131] Example 2: Manufacturing Process and Process Control Step 1: Construction of vaccine vector Construction of pCVL41 (CPI-RSV-F) antigenome cDNA: The CPI-RSV-Fopt genome was obtained by RsrII and AatII restriction enzyme digestion of the pSP28 plasmid. pSB28 is a low-copy plasmid containing the codon-optimized RSV F protein gene (Fopt, codon-optimized for human, sequence based on GenBank accession M74568, RSV A2 strain) inserted between the SH and HN junctions of the CPI antigenome cDNA (Figure 2A) (3). The pAB94 plasmid, which contains the EGFP gene inserted between the SH and HN junctions of the CPI antigenome cDNA, is a high-copy plasmid (Figure 2B). The RsrII and AatII restriction enzyme-digested Fopt gene from CPI-RSV-Fopt was inserted into RSrII and AatII-digested pAB94, and the EGFP gene was replaced with Fopt using T4 DNA ligase.

[0132] The ligated cDNA was transformed into TOP10 competent cells, and single colonies were grown in LB medium containing chloramphenicol.

[0133] The resulting plasmid DNA (designated pCVL41 or pCPI-RSV-Fopt; Figure 3) was purified using a Qiagen miniprep kit and sequenced. The codon-optimized RSV-F gene inserted between the SH and HN gene junction was confirmed to be correct by Sanger sequencing.

[0134] Step 2: Rescue of vaccine vectors The recombinant vector virus used to generate CPI-RSV-F (CPI-RSV-F) was rescued by transfecting the pCPI-RSV-Fopt plasmid into serum-free 293T suspension cells (obtained from GenHunter Corporation) along with plasmids encoding the PIV5 NP, P, and L proteins and T7 RNA polymerase, allowing rescue of the recombinant virus from the transfected cell culture (Fig. 4).

[0135] The PIV5 plasmid clones encoding the nucleoprotein (N), phosphoprotein (P), or large polymerase protein (L) were previously described (1, 2). Each gene was under the control of the T7 promoter in the pCAGGS vector. Five plasmids were used to generate the CPI-RSV-F RVS:

[0136] 1. pCPI-RSV-Fopt (pCVL41-CPI-RSV-F antigenome cDNA): This plasmid is a high-copy plasmid containing an ampicillin resistance gene. It encodes the RSV F antigen gene inserted between the SH and HN gene junction of canine parainfluenza virus (CPI).

[0137] 2. pCAGGS-NP plasmid: This plasmid contains the PIV5 NP gene under the T7 promoter with the ampicillin resistance gene.

[0138] 3. pCAGGS-P plasmid: This plasmid contains the PIV5 P gene under the T7 promoter with the ampicillin resistance gene.

[0139] 4. pCAGGS-L plasmid: This plasmid contains the PIV5 L gene under the T7 promoter with the ampicillin resistance gene.

[0140] 5. pCAGGS-T7 plasmid (same as pBH437-T7): This plasmid encodes the T7 RNA polymerase gene under the SV40 promoter.

[0141] The medium used for virus rescue was CDM4HEK293 medium (Hyclone) containing 4 mM GlutaMAX (Gibco).

[0142] After 2 days of incubation, 293T cells were co-cultured with serum-free Vero MCB cells (P159) passaged from African green monkey kidney (WHO Vero10-87) CyanVac MCB DOM (19AUG2020-P148). After 4 days of incubation, 2 mL of supernatant containing rescued virus was obtained, mixed with 10X SPG, and stored at -80°C.

[0143] Step 3: Plaque purification and expansion of virus rescue seeds A plaque assay was performed on serum-free Vero cells (P155- derived from WHO Vero10-87 CyanVac MCB DOM:19AUG2020) in a 6-well plate using serial dilutions of an aliquot of the rescued virus-containing frozen stock, aiming to obtain six isolated single plaques. Single plaques were picked using a 1000 μL pipette tip and resuspended in VP-SFM medium containing 4 mM GlutaMAX. The resuspended plaques were then used to infect fresh serum-free Vero cells (P155- derived from WHO Vero10-87 CyanVac MCB DOM:19AUG2020) in a 6-well plate. After 6 days, 2 mL of supernatant from the 6-well plate infected with a single plaque was mixed with 10% 10X SPG and stored at -80°C (to obtain 1X SPG). This material was designated lot number CPI-RSV-F-PP1-PQ10-42621 with a manufacturing date of April 6, 2021. A portion of the supernatant (140 μL) was used for RNA extraction and RT-PCR to verify the viral genome sequence. RT-PCR was performed using the primers listed in Table 5.

[0144] [Table 5]

[0145] Step 4: Production of prMVS (Lot No. 210519MCBCHD-PQ10) in serum-free Vero cells An aliquot from lot number CPI-RSV-F-PP1-PQ10-42621 (plaque-purified virus) was used to infect serum-free Vero cells (P159, derived from WHO Vero10-87 CyanVac MCB DOM:19AUG2020) in a T75 flask and incubated at 37°C for 5 days. The cell culture supernatant was centrifuged at 1,500 rpm for 10 minutes at 4°C to remove cell debris. The clarified supernatant (20 mL) was mixed with 10% 10X SPG containing 10% arginine (Sigma-Aldrich), flash-frozen in 1 mL aliquots, and stored at -80°C as pre-MVS stock. The pre-MVS was designated lot number 210519MCBCHD-PQ10. The pre-MVS was sequenced from the NP gene to the L gene to confirm the viral genomic sequence and the proper insertion of the RSV F protein (a single silent mutation was found at AA54 of NP).

[0146] Prior to use in the cGMP production of MVS, pre-MVS (Lot No. 210519MCBCHD-PQ10) was tested at CRL for sterility (direct method), bacteriostatic and fungal, presence of mycoplasma, mycobacteria, porcine and bovine circoviruses, and apparent viruses.

[0147] The ability of the CPI-RSV-F vaccine vector construct to infect Vero cells and express functional F proteins, including the prefusion F protein, was assessed for both the pre-MVS and MVS stocks using an immunofluorescence assay (IFA), as detailed below. Additionally, the pre-MVS and MVS were sequenced by Sanger sequencing to confirm the presence of the correct F protein gene insertion in the PIV5 viral backbone (sequenced from the leader to the trailer region).

[0148] The following characterization tests were performed on pre-MVS with lot number 210519MCBCHD-PQ10.

[0149] Expression of F protein by CPI-RSV-F pre-MVS in infected Vero cells by IFA: Expression of F protein by CPI-RSV-F was assessed by IFA staining for the PIV5 vector (HN) and RSV F protein using palivizumab. Palivizumab is a humanized murine monoclonal antibody that recognizes antigenic site II on both the pre-fusion (pre-F) and post-fusion (post-F) conformations of the respiratory syncytial virus (RSV) F glycoprotein. The percentage of F expression was determined by calculating the number of cells that stained positive for PIV5 (HN protein) and F protein (Figures 5A-5C and Table 6).

[0150] [Table 6]

[0151] Vero-SF cells were infected with CPI-RSV-F pre-MVS virus at 10x, 100x, and 1000x dilutions. After 1 hour of incubation at 37°C, the medium was replaced, and the cells were incubated at 37°C for 18 hours. Immunostaining was performed using mouse anti-PIV5-HN and human anti-F (palivizumab) antibodies, followed by anti-mouse Cy3 and anti-human FITC secondary antibodies, respectively. Figures 5A-5C show representative images of wells infected with CPI-RSV-F pre-MVS, showing both red and green cells. Table 6 shows the percent expression of RSV F protein in PIV5-infected cells. Images were taken at 10x magnification.

[0152] Expression of F protein by CPI-RSV-F MVS-infected cells by IFA: Expression of F protein by CPI-RSV-F was assessed by IFA staining for PIV-5 vector (HN) and F protein according to CVL-Protocol-034. The percentage of expression was determined by calculating the number of cells that stained positive for PIV5 (HN protein) and RSV F protein (Figures 6A-6C and Table 7).

[0153] [Table 7]

[0154] Vero-SF cells were infected with CPI-RSV-F MVS virus at 1:100, 1:1000, and 1:10,000 dilutions. After 1 hour of incubation at 37°C, the medium was replaced, and the cells were incubated at 37°C for 18 hours. Immunostaining was performed using mouse anti-PIV5-HN and human anti-F (palivizumab) antibodies, followed by anti-mouse Cy3 and anti-human FITC secondary antibodies, respectively. Figures 6A-6C show representative images of wells infected with CPI-RSV-F MVS, showing both green and red cells. Table 7 shows the percent expression of RSV F protein in PIV5-infected cells. Images were taken at 10x magnification.

[0155] Step 5: MVS manufacturing process CPI-RSV-F MVS was manufactured in compliance with current good manufacturing practice (cGMP). Master virus seed stocks (MVS), which also served as Phase 1 clinical lot material, were produced in accordance with current good manufacturing practice (cGMP) at CRL August 2021 (CBR-1862) using serum-free Vero cells, CyanVac MCB (DOM: 19 August 2020, grown at CRL to passage 152).

[0156] Vaccine Bulk Material Preparation: Vero cells grown in T225 flasks in serum-free medium (VP-SFM) were infected with CPI-RSV-F PreMVS Lot No. 210519MCBCHD-PQ10 (two vials used) at an MOI of 0.002 for 7 days at 37°C. The cell culture fluid from the infected cells constituted the crude vaccine bulk material (3.0 liters). Samples of the MVS crude bulk virus fluid were taken for the following tests: sterility (direct method), bacteriostasis and fungistasis, in vitro mycoplasma testing (agar cultivable and non-cultivable), tissue culture safety testing (GP-V810.2), in vitro mycobacterium testing, latent virus, PBERT, and potency.

[0157] The vaccine bulk material was clarified by centrifugation at 1,500 rpm at 2-8°C for 10 minutes to yield 1.2 L and subsequently filtered through a Thermo Fisher 0.45 μM PES filter unit to yield 1.2 L. The clarified and filtered vaccine bulk material was immediately formulated with 10× SPG to stabilize the virus prior to filling. No holding of the vaccine bulk material was performed prior to formulation and filling.

[0158] During the virus production process, control harvest fluids were prepared using uninfected Vero cells from the same production lot. Control fluids were stored at or below -60°C. Production control fluids were collected and tested for bacteriostatic and fungal bulk product (direct method), sterility, detection and quantification of residual Vero DNA, and determination of endotoxin levels (LAL).

[0159] Vaccine Drug Product Manufacturing (Formulation and Filling) / MVS: Filtered virus was stabilized by formulation in 10% 10X SPG buffer and dispensed into 2 mL cryovials using a repeat pipette calibrated to 1 mL volume to create MVS stock / vaccine product (1281 vials total, 1 mL per vial). Aliquoted MVS / vaccine product was flash frozen in a dry ice / methanol bath and stored at -60°C or below.

[0160] Clinical Lot Material: In the proposed initial Phase 1 trial, Master Virus Seed (MVS), Lot Number CPI-RSV-F (CPI-RSV-F-210519PQ10MVS DOM:26AUG2021), will be used as the vaccine formulation.

[0161] MVS characterization: Genetic identity was further confirmed by sequencing the virus antigenomic cDNA spanning the NP gene to L gene region to confirm identity.

[0162] Example 3: Comparative study of PIV5 vector control and CPI-RSV-F vaccine candidate in BALB / c mice Materials and Methods

[0163] The purpose of the two mouse studies in this report (CHD03 and CHD04) was to compare the immunogenicity and protective efficacy of two RSV vaccine candidates, W3AΔSH-RSV-F and CPI-RSV-F, in the BALB / c mouse model. Two other vaccine candidates, W3A-RSV-F(SH-HN) and CPIΔSH-RSV-F, were also included in the study. Antibody titers were measured using an RSV F-specific ELISA assay, and cellular immune responses were measured using an ELISPOT assay. Protection of vaccinated mice against challenge with a wild-type RSV strain in the lower respiratory tract was determined by plaque assay of lung homogenates (Table 8).

[0164] [Table 8]

[0165] Key materials and reagents used are listed in Table 9. Viruses used in this experiment are listed in Table 10.

[0166] [Table 9]

[0167] [Table 10]

[0168] All viruses were diluted to 2 x 10 in 1XPBS prior to vaccination. 6 The RSV_rA2 stock solution was not diluted before challenge.

[0169] The experimental groups applicable to ELISA, ELISPOT, and plaque assay tests are summarized in Tables 11 and 12.

[0170] [Table 11]

[0171] [Table 12]

[0172] Mouse immunization: Six to eight week old female BALB / c mice were anesthetized by intraperitoneal injection of 250 μL of 2,2,2-tribromoethanol in tert-amyl alcohol (Avertin). 5 PFU of each vaccine candidate and the RSV_rA2 positive control were administered intranasally in a volume of 50 μL (25 μL per nostril). Negative controls were administered intranasally with 50 μL (25 μL per nostril) of PBS. The RSV_rA2 positive control was used to mimic natural RSV infection before vaccination. All animal experiments were performed in accordance with protocols approved by the University of Georgia's Institutional Animal Care and Use Committee.

[0173] Blood was collected by buccal sampling for serological analysis on day 27 (mice in groups A, D, G, J, M, P for CHD03 and groups A, E, G, K, M, P for CHD04) or day 28 (mice in groups B, C, E, F, H, I, K, L, N, O, Q, R for CHD03 and groups B, C, D, F, H, I, J, L, N, O, Q, R for CHD04) after immunization. Spleens of mice in cages A, D, G, J, M, P for CHD03 and cages A, E, G, K, M, P for CHD04 were also collected on day 27 after immunization, and ELISPOT assays were performed. Thirty-five days after immunization, mice were anesthetized by intraperitoneal injection of 250 μL of Avertin, and groups B, C, E, F, H, I, K, L, N, O, Q, R for CHD03 and groups B, C, D, F, H, I, J, L, N, O, Q, R for CHD04 were injected with 3.8 × 10 IgG in a volume of 50 μL (25 μL per nostril). 5Mice were challenged intranasally with PFU of RSV A2. After challenge, mice remained housed together within each vaccination group. Four days later, lungs were harvested from 7-10 mice per group, and viral load was assessed by plaque assay on lung homogenates. The experimental timeline is shown in Figures 7 and 8.

[0174] Anti-F ELISA Assay: Anti-F antibody titers were determined by anti-F ELISA assay per CVL SOP-049. Immulon 2HB 96-well plates were coated overnight with 50 μL / well of 12.5 ng / mL RSV F protein. Serum samples were prediluted 10-fold, followed by 3-fold serial dilutions, and added to the F protein-coated plates (50 μL / well) for 1 hour at room temperature. After plate washing, 50 μL / well of goat anti-mouse IgG HRP-conjugated antibody was added at a 1:1250 dilution and incubated for 1 hour at room temperature. After washing, 100 μL / well of SureBlue Reserve TMB Microwell Peroxidase Substrate was added for 3–5 minutes, and the reaction was stopped by adding 100 μL / well of 1N HCl. Immediately after the addition of HCl, the plate was read at 450 nm using a plate reader (SpectraMax iD3 Multimode Microplate Reader). OD 450 Samples above the cutoff value of 0.20 are considered positive for at least two serial dilutions. Antibody titers are defined as the highest reciprocal dilution with a positive signal. The assay was performed at Blue Lake Biotechnology in Los Gatos, CA.

[0175] Enzyme-linked ImmunoSpot Assay (ELISPOT): The level of IFN-γ-secreting cells was measured by ELISPOT assay using the BD™ ELISPOT Mouse IFN-γ Set. BD™ ELISPOT plates were coated with purified anti-mouse IFN-γ antibody 24 hours before the assay. 27 days after immunization, mouse spleens (n=5 per group) were harvested and placed in 15 mL conical tubes containing 5 mL of HBSS. Splenocytes were collected by pressing the spleens through a 70 μM cell strainer, incubating with ACK lysis buffer, washing with HBSS, and resuspending in complete tumor medium (CTM) at 5 × 10 6 The plate was prepared by adjusting the concentration of cells / mL. The capture antibody solution was removed from the plate, and the plate was then washed 5-6 times with PBS. The plate was then blocked with CTM for 90 minutes. The blocking solution was discarded, and 0.1 μg of RSV F peptide (85-93) in 50 μL of CTM was added to the well. Next, 50 μL of splenocytes (2.5 × 10 5 Cells / well) were added to the plate and incubated at 37°C, 5% CO for 48 hours. Spots were immunostained according to the BD™ ELISPOT kit instructions and counted using an ImmunoSpot™ analyzer (Cell Technology Limited, CTL). Results were compared at 10 6 The mean number of IFN-γ secreting cells per splenocyte was expressed. The assay was performed at the University of Georgia in Athens, GA.

[0176] Plaque Assay: RSV virus titer in lung homogenates was measured on Vero cells by plaque assay. Briefly, mouse lungs were collected in gentleMACS M tubes containing 3 mL of Opti-MEM with 1% BSA and kept on ice. Lungs were weighed and then homogenized at 4°C using the Protein_01 program on a gentleMACS Dissociator, followed by centrifugation at 3000 × g for 10 minutes. The supernatant was used to perform three-fold serial dilutions from the original solution to 1:27 in a total volume of 0.6 mL. Vero cells in 24-well plates were infected in triplicate with 100 μL of each dilution. After 1 hour of adsorption at 37°C, the inoculum was removed, the plate was washed once with PBS, and approximately 1 mL of methylcellulose was added to each well. After 7 days of incubation, the methylcellulose was removed, and cells were fixed with approximately 500 μL of 60% acetone / 40% methanol for 20 minutes. The cells were washed and then blocked with 400 μL / well of blot for 30 minutes. After blocking, the cells were incubated with 200 μL / well of human anti-RSV-F antibody (palivizumab, 1 mg / mL) diluted 1:1000 for 1 hour at room temperature. After plate washing, 200 μL / well of goat anti-human IgG HRP-conjugated antibody diluted 1:1000 was added and incubated for 1 hour at room temperature. After washing, 200 μL / well of AEC substrate was added for 30 minutes at room temperature. Virus titers were determined by counting plaques at each dilution, with a plaque count range of 10 to 100. Results were reported as PFU / g of lung. This assay was performed at the University of Georgia in Athens, GA.

[0177] result

[0178] Anti-F Antibody Titers: Anti-F antibody titers were obtained from animals in groups A, D, G, J, M, and P on CHD03 and groups A, E, G, K, M, and P on CHD04 on day 27 post-vaccination. The results are shown in Figures 9A-9B. Overall, anti-F antibody titers were significantly higher in all vaccinated groups compared to the PBS control group. The anti-F antibody titers of groups J (W3AΔSH-RSV-F) and M (CPI-RSV-F) on CHD03 and groups K (W3AΔSH-RSV-F) and M (CPI-RSV-F) on CHD04 were very similar. In CHD03, the geometric mean titer for W3AΔSH-RSV-F was 3.1 Log 10 / mL, and the geometric mean titer of CPI-RSV-F was 3.0 Log 10 In CHD04, the geometric mean titer of W3AΔSH-RSV-F was 3.4 Log 10 / mL, and the geometric mean titer of CPI-RSV-F was 3.2 Log 10 W3A-RSV-F in both CHD03 and CHD04 had the highest anti-F antibody titers, with a geometric mean titer of 3.7 Log 10 The W3AΔSH-RSV-F and CPI-RSV-F groups had similar anti-F antibody titers to the RSV_rA2 positive control group on both CHD03 and CHD04. CPIΔSH-RSV-F also induced similar serum antibody levels in both studies.

[0179] These results demonstrate that both W3AΔSH-RSV-F and CPI-RSV-F, as well as the other two vaccine candidates, were effective against 1 × 10 5 PFU doses induce similar levels of anti-F antibodies in BALB / c mice.

[0180] F-specific cellular immune responses: Cellular immune responses induced by the vaccine candidates were measured by the level of IFN-γ-secreting cells. In CHD03, the levels of IFN-γ-secreting cells were higher in groups J (W3AΔSH-RSV-F) and M (CPI-RSV-F), with group J showing a 10 6 With a geometric mean of 28 IFN-γ-secreting cells per splenocyte, group M was 106 The IFN-γ secreting cells per splenocyte had a geometric mean of 23. These values ​​were not statistically significant from the PBS group or from each other. Group G (W3A-RSV-F) had the highest value compared to the PBS group, with a geometric mean of 10. 6 There were 57 IFN-γ-secreting cells per splenocyte, which was not significantly different from the other groups. The values ​​from the W3AΔSH-RSV-F and CPI-RSV-F groups are similar to the levels of IFN-γ-secreting cells seen in the RSV_rA2 group.

[0181] In CHD04, the levels of IFN-γ-secreting cells were highest in groups K (W3AΔSH-RSV-F) and M (CPI-RSV-F), with group K showing 10 6 With a geometric mean of 35 IFN-γ-secreting cells per splenocyte, group M was 10 6 The mean number of IFN-γ-secreting cells was 48 per splenocyte. These values ​​were statistically significant from the PBS control group but not from each other. Of both vaccine groups of interest, only the CPI-RSV-F group had significantly higher amounts of IFN-γ-secreting cells than the RSV_rA2 positive control group. W3A-RSV-F and CPIΔSH-RSV-F also induced F-specific cellular responses.

[0182] Figures 10A-10B show that both the target vaccine candidates W3AΔSH-RSV-F and CPI-RSV-F, as well as the other two vaccine candidates (W3A-RSV-F and CPIΔSH-RSV-F), induce similar levels of cellular immune responses in BALB / c mice.

[0183] RSV Challenge Virus Lung Titers: Four RSV vaccine candidates were evaluated for protection against RSV_rA2 virus challenge infection in the lower respiratory tract of mice. Overall, all vaccine groups had significantly lower lung virus titers compared to the PBS control on both CHD03 (FIG. 11A) and CHD04 (FIG. 11B), with geometric mean titers of 3.21 Log and 4.21 Log, respectively. 10 PFU / g and 3.33 Log 10In CHD03, the RSV challenge virus titer in groups K and L (W3AΔSH-RSV-F) was 1.35 Log 10 PFU / g, whereas the geometric mean titer for groups N and O (CPI-RSV-F) was 1.48 Log 10 PFU / g. These values ​​are not statistically significant from each other. The other vaccine candidates tested (W3A-RSV-F and CPIΔSH-RSV-F) each had a geometric mean titer of 1.35 Log 10 PFU / g and 1.40Log 10 PFU / g. These values ​​are also significantly lower than the PBS control. In CHD04, the geometric mean titer for groups J and L (W3AASH-RSV-F) was 1.37 Log 10 PFU / g, whereas the geometric mean titer for groups N and O (CPI-RSV-F) was 1.40 Log 10 PFU / g. Similar to CHD03, these values ​​are not statistically significant from each other, but are significantly lower than the PBS control. Vaccine candidates W3A-RSV-F and CPIΔSH-RSV-F each had a geometric mean titer of 1.35 Log 10 PFU / g and 1.40Log 10 PFU / g. These values ​​were the same as in experiment CHD03 and were also significantly lower than the PBS control group. Pulmonary RSV virus titers in all vaccine groups were similar to those seen in the RSV_rA2 positive control group in both CHD03 and CHD04.

[0184] Figures 11A-11B show that both the W3AΔSH-RSV-F and CPI-RSV-F vaccine candidates, as well as the other two vaccine candidates, protected against RSV challenge virus infection in the lower respiratory tract of BALB / c mice.

[0185] Example 4: Virus replication in the upper and lower respiratory tract of cotton rats. Methods and Materials

[0186] Animals: Fifty-five inbred, 6- to 8-week-old Sigmodon hispidus female and male cotton rats (source: Sigmovir Biosystems, Inc., Rockville, MD) were maintained and handled under veterinary supervision in accordance with National Institutes of Health guidelines and a Sigmovir Institutional Animal Care and Use Committee-approved animal study protocol (IACUC protocol number 15). Each group of animals contained three females (the first three animals in each group) and two males (the last two animals in each group). The cotton rats were housed in clear polycarbonate cages and provided with standard rodent chow (Harlan #7004) and tap water ad libitum.

[0187] Virus: Respiratory syncytial virus strain A / A2 (RSV A / A2) (ATCC, Manassas, VA) was propagated in HEp-2 cells after serial plaque purification to reduce defective interfering particles. Approximately 3.0 x 10 8 A pool of virus designated hRSV A / A2 lot number 092215SSM containing pfu / mL was used for this in vivo experiment. This virus stock is stored at -80°C and has been characterized in vivo in the cotton rat model, validating upper and lower respiratory tract replication.

[0188] Procedure(s): Fifty-five young adult female and male cotton rats (6-8 weeks old) were divided into nine groups of five animals each (three females, two males), one group of four animals (two females, two males), and two groups of three animals each (two females and one male). All animals were ear-tagged and pre-bled for serum collection. Animals were immunized or infected with 0.1 ml of the preparation as shown in Table 13 below.

[0189] [Table 13]

[0190] Animals from groups K and L were sacrificed. Nasal tissue was collected and homogenized in 3 ml of HBSS supplemented with 10% SPG for viral titer determination. Lungs were collected en bloc and divided into three equal sections for viral titer determination (left section, homogenized in 3 ml of HBSS supplemented with 10% SPG), histopathology (right section, expanded in 10% neutral buffered formalin), and qPCR (lingual lobe, snap-frozen in liquid nitrogen).

[0191] All animals are eye bled to collect serum. Animals in groups A, B, and C are boosted with 0.1 ml of the preparation as indicated in the table above.

[0192] All animals were eye bled to collect serum. Group A was mock challenged intranasally (IN) with 0.1 ml of PBS (pH 7.4). Groups B to J were challenged with 10 5 Challenge with 0.1 ml of PFU of RSV / A2 (Lot No. 092215SSM). Back titration is performed on the challenge virus to confirm the challenge dose.

[0193] All animals are sacrificed. Nasal tissue is collected and homogenized in 3 ml of HBSS supplemented with 10% SPG for viral titer determination. Lungs are collected en bloc and sectioned into thirds for viral titer determination (left section, homogenized in 3 ml of HBSS supplemented with 10% SPG), histopathology (right section, expanded in 10% neutral buffered formalin), and qPCR (lingual lobe, snap-frozen in liquid nitrogen). Animal sacrifice groups are shown in Table 14, and sample collection is shown in Table 15.

[0194] [Table 14]

[0195] [Table 15]

[0196] Table 16 outlines the endpoint assay protocol.

[0197] [Table 16]

[0198] Lung and nasal RSV virus titer determination: Lung and nasal homogenates are clarified by centrifugation and diluted in EMEM. Confluent HEp-2 monolayers are infected in duplicate with the diluted homogenates in 24-well plates. After 1 hour of incubation at 37°C in a 5% CO2 incubator, the wells are covered with 0.75% methylcellulose medium. After 4 days of incubation, the covers are removed and the cells are fixed with 0.1% crystal violet stain for 1 hour, then rinsed and air-dried. Plaques are counted and virus titers are expressed as plaque-forming units per gram of tissue. Virus titers are calculated as the geometric mean ± standard error for all animals within a group at a given time point.

[0199] Lung histopathology: Lungs were dissected and inflated to normal volume with 10% neutral buffered formalin, then immersed in the same fixative. After fixation, lungs were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Four parameters of pulmonary inflammation were assessed: peribronchiolar inflammation (inflammatory cell infiltration around bronchi), perivasculitis (inflammatory cell infiltration around small blood vessels), interstitial pneumonia (inflammatory cell infiltration and alveolar wall thickening), and alveolitis (cells within the alveolar space). Slides were blindly scored on a severity scale of 0 to 4. The scores were then converted to a histopathological scale of 0 to 100%.

[0200] RSV Neutralizing Antibody Assay (60% Reduction) for Preclinical Studies: Heat-inactivated serum samples were diluted 1:10 in EMEM and serially diluted 1:4. The diluted serum samples were incubated with RSV (25-50 PFU) at room temperature for 1 hour and then seeded in duplicate onto confluent HEp-2 monolayers in 24-well plates. After 1 hour of incubation at 37°C in a 5% CO2 incubator, the wells were covered with 0.75% methylcellulose medium. After 4 days of incubation (6 days for RSV B), the covers were removed and the cells were fixed with 0.1% crystal violet stain for 1 hour, then rinsed and air-dried. The corresponding reciprocal neutralizing antibody titers were determined at the 60% reduction endpoint of the virus control using a statistical program. The geometric mean ± standard error for all animals within a group at a given time point was calculated.

[0201] Preclinical RSV IgG ELISA: UV-inactivated whole RSV or purified F protein extracted from RSV-infected HEp-2 cells was diluted and coated overnight on a 96-well ELISA plate. The coating antigen was decanted, and the plate was incubated in blocking solution for 1 hour at room temperature, followed by washing. Diluted serum (1:500 in duplicate) was added to the wells along with positive and negative controls and incubated for 1 hour at room temperature. After washing the plate, rabbit anti-cotton rat IgG (1:500) was added to all wells and incubated for 1 hour at room temperature. This was followed by incubation with goat anti-rabbit IgG-HRP (1:6,000) for 1 hour at room temperature. Finally, TMB substrate was added to all wells and incubated for 15 minutes at room temperature. TMB stop solution was added to all wells, and the optical density at 450 nm was recorded. The geometric mean of the optical density (OD450) was measured for all triplicate serum samples, and the standard error was determined for all samples within each group at each time point.

[0202] Real-time PCR for preclinical testing: Total RNA is extracted from homogenized tissues or cells using the RNeasy Purification Kit (QIAGEN). 1 μg of total RNA is used to prepare cDNA using SuperScript II RT (Invitrogen) and oligo-dT primer (1 μl, Invitrogen). For real-time PCR reactions, Bio-Rad iQ™ SYBR Green Supermix is ​​used in a final volume of 25 μl with a final primer concentration of 0.5 μM. Reactions are set up in duplicate in a 96-well tray. Amplification is performed in a Bio-Rad iCycler with a 3-minute cycle at 95°C, followed by 40 cycles of 95°C for 10 seconds (s), 60°C for 10 seconds, and 72°C for 15 seconds. Baseline cycles and cycle thresholds (Ct) are calculated using the iQ5 software in PCR baseline subtraction curve fit mode. Relative DNA quantification is applied to all samples. A standard curve is generated using serially diluted cDNA samples with the most abundant transcript of interest (e.g., lungs 6 hours post-RSV infection of FI-RSV immunized animals or PIV5-specific controls). Ct values ​​are expressed as log 10 The Ct values ​​obtained for various samples are plotted against the cDNA dilution factor. These curves are used to convert the Ct values ​​obtained for various samples into relative expression units. These relative expression units are then normalized to the level of β-actin mRNA ("housekeeping gene") expressed in the corresponding sample. In animal studies, mRNA levels are expressed as the geometric mean ± SEM for all animals in a group at a given time point.

[0203] result

[0204] Confirmation of vaccine virus replication in the cotton rat model: To confirm the replication of PIV5-based vaccines in the respiratory tract of cotton rats, groups of 3 animals were divided into 10 groups. 6Cotton rats were inoculated intranasally with PFU of CPI-RSV-F or W3AΔSH-RSV-F, sacrificed 4 days later, and samples were submitted to Blue Lake Biotechnology (BLB) for evaluation. BLB performed plaque assays on the submitted samples. Briefly, both candidate vaccine viruses were found to replicate in the upper and lower respiratory tracts of cotton rats (Table 17). In CPI-RSV-F vaccinated animals, the vaccine virus replicated similarly in the upper respiratory tract (nose) and lower respiratory tract (lungs), with 3.9 and 3.5 Log 10 PFU / mL. W3AΔSH-RSV-F was transmitted to the lower respiratory tract (2.2 Log 10 PFU / mL compared with the upper respiratory tract (5.0 Log 10 The W3AΔSH-RSV-F vaccine virus replicated to higher levels in the upper respiratory tract compared to CPI-RSV-F, but replicated to lower levels in the lower respiratory tract compared to CPI-RSV-F. Overall, replication of both vaccine viruses in the cotton rat model was successfully confirmed (Table 17).

[0205] [Table 17]

[0206] 10 for animals 6 Mice were inoculated intranasally with PFU of CPI-RSV-F or W3AΔSH-RSV-F and sacrificed 4 days later. Lung and nasal samples were collected and sent to the BLB for plaque assay.

[0207] Evaluation of vaccine efficacy and safety in cotton rats, a model of RSV A / A2 infection.

[0208] Pulmonary viral titers: RSV A / A2 loads in the lungs of cotton rats were assessed 5 days after intranasal RSV challenge (Figure 12). RSV-infected animals mock-immunized with PBS (Group B) had a pulmonary viral load of 5.3 Log 10The titers of PFU / g of virus are shown and used for comparison with all other RSV-infected groups (groups C-J). No virus was detectable in the lungs of animals infected twice with RSV (group D). FI-RSV immunization (group C) produced a moderate, but still significant, lung RSV titer of 3.4 Log. 10 The dose of 10 4 , 10 5 , or 10 6 Immunization with CPI-RSV-F at PFU (Groups E, F, and G, respectively), or at a dose of 10 4 , 10 5 , or 10 6 Immunization with W3AΔSH-RSV-F at PFU (Groups H, I, and J, respectively) reduced RSV load to undetectable levels in all vaccinated animals.

[0209] Nasal virus titers: Intranasal RSV A / A2 loads in cotton rats were assessed 5 days after intranasal RSV challenge (Figure 13). RSV-infected animals mock-immunized with PBS (Group B) exhibited intranasal titers of 6.19 Log10 PFU / g of virus and were used for comparison with all other RSV-infected groups (Groups C-J). No virus was detected in the noses of animals infected twice with RSV (Group D). FI-RSV immunization (Group C) did not affect intranasal RSV loads (6.02 Log10 PFU / g). 10 4 , 10 5 , or 10 6 Immunization with CPI-RSV-F at doses of 1.00, 1.00, and 2.44 PFU / g resulted in a dose-dependent reduction in intranasal viral load of 3.65, 3.00, and 2.44 PFU / g in groups E, F, and G, respectively. Immunization with W3AΔSH-RSV-F at all three doses tested (groups H-J) reduced nasal RSV replication to almost undetectable levels, with only single-digit plaque numbers detected in some animals in each group (1, 3, and 1 animal in groups H, I, and J, respectively, had 1-5 plaques).

[0210] Serum RSV A / A2 Neutralizing Antibodies: Serum neutralizing antibodies to RSV A / A2 were measured in all animals prior to the start of the experiment (day 0), 4 weeks (day 28), and 3 weeks (day 49) after the first immunization (Figure 14). Animals infected with RSV A / A2 (Group D) showed high titers of serum RSV A / A2 neutralizing antibodies 4 weeks post-infection (day 28 data, 11.14 Log2) that remained elevated 7 weeks post-infection (day 49 data, 10.07 Log2). 4 , 10 5 , or 10 6 Immunization with CPI-RSV-F at doses of 100 PFU (Groups E, F, and G, respectively) resulted in NA titers of 5.92, 5.87, and 7.55 on day 28.

[0211] The highest vaccine dose tested (Group G, 10 6 PFU) maintained elevated NA titers until day 49 (6.65 Log2), while NA levels in groups E and F had declined to 4.6 and 4.62 Log2, respectively, by that time. 4 , 10 5 , or 10 6 Immunization with PFU of W3AΔSH-RSV-F (groups H-J) resulted in NA titers of 9.24, 10.11, and 9.91 Log2, respectively, on day 28. NA titers in these groups remained significantly elevated (8.79, 8.97, and 8.64 Log2, respectively) on day 49. Animals in other groups did not develop detectable neutralizing antibodies to RSV A / A2.

[0212] Serum RSV A / A2-binding IgG antibodies: Serum-binding IgG antibodies against RSV A / A2 F protein were measured in all animals before the start of the experiment (day 0), 4 weeks (day 28), and 3 weeks (day 49) after the first immunization (Figure 15). A slight increase in binding IgG was observed in animals vaccinated with FI-RSV (group C). Animals infected with RSV A / A2 (group D) and all animals immunized with CPI-RSV-F (groups E-G) or W3AΔSH-RSV-F (groups H-J) showed high levels of binding IgG on days 28 and 49 post-inoculation. No dose-dependence of IgG levels was observed with any dose of CPI-RSV-F (groups E-G) or W3AΔSH-RSV-F (groups H-J). IgG levels were slightly higher in animals immunized with W3AΔSH-RSV-F compared to animals immunized with CPI-RSV-F (groups E-G) on both days 28 and 49.

[0213] Lung histopathology: Lung histopathology was evaluated in all animals 5 days after RSV A / A2 challenge (data not shown). RSV-infected animals mock-immunized with PBS (Group B) or challenged twice with RSV (Group D) had moderate levels of pathology. The highest levels of lung histopathology were detected in animals immunized with FI-RSV (Group C), accompanied by a marked increase in interstitial inflammation and alveolitis. Pathology in animals immunized with CPI-RSV-F at all doses (Groups E-G) did not exceed that seen in animals with secondary RSV infection (Group D). 5 The group of animals immunized with the 1.0 PFU dose of W3AΔSH-RSV-F (Group I) included one animal (number 124872) with elevated interstitial inflammation and alveolitis. The other four animals in that group and all animals immunized with the other two doses of vaccine (Groups H and J) did not exhibit interstitial inflammation or alveolitis.

[0214] qPCR Results: RSV NS1, IL-4, IL-2, and IFN-γ mRNA expression was assessed in lung samples collected 5 days after RSV A / A2 challenge and normalized to the level of β-actin mRNA in each sample (Figures 16A-D). NS-1 mRNA expression was significantly reduced by both vaccines, with W3AΔSH-RSV-F significantly reducing the expression of NS-1 mRNA at the two lower doses (10 4 and 10 5 FI-RSV immunization (Group C) resulted in a moderate reduction in lung RSV NS1 mRNA levels but significantly increased IL-4 mRNA levels. Overall, IL-4, IL-2, and IFN-γ mRNA levels in animals immunized with CPI-RSV-F (Groups E-G) or W3AΔSH-RSV-F (Groups H-J) did not exceed those seen in animals with primary RSV infection (Group B). The group immunized with the intermediate dose of CPI-RSV-F (Group F, 10 5 Several animals in the group immunized with the intermediate dose of W3AΔSH-RSV-F (group I, 10 PFU dose) showed slightly elevated IL-2 and IFN-γ mRNA levels compared to animals with secondary RSV infection (group D). One animal in the group immunized with the intermediate dose of W3AΔSH-RSV-F (group I, 10 PFU dose) showed slightly elevated IL-2 and IFN-γ mRNA levels compared to animals with secondary RSV infection (group D). 5 PFU dose, animal #124872) had elevated IL-4 mRNA. Coincidentally, these same animals also showed elevated alveolitis and interstitial inflammation upon histopathological evaluation.

[0215] Example 5: Phase I Clinical Trial of PIV5-RSV Vaccine - RSV Antibody and CMI Response Rates Materials and Methods

[0216] Participants and Study Conduct: A total of 30 subjects will be enrolled in this Phase 1 study, all of whom will receive 10 7.5A single intranasal dose of PFU PIV5-RSV vaccine was administered. The vaccine was administered as a 0.25 mL spray into each nostril (total volume 0.5 mL) using a MAD Nasal™ intranasal mucosal atomization device (Teleflex MAD300). The mean ages of Group 1 (intended participant age 18-59 years, actual enrollment age 33-59 years) and Group 2 (intended participant age 60-75 years, actual enrollment age 61-75 years) were 45 and 67 years, respectively (Table 18). The majority of participants were female (70%). Most participants in the two groups were Caucasian (73% and 80% in Groups 1 and 2, respectively). All but one participant completed the study. This participant in Group 1 was lost to follow-up after Day 7.

[0217] [Table 18]

[0218] Study Design and Vaccination: A phase 1 clinical trial of the PIV5-RSV vaccine (also known as BLB201; Clinical Trial NCT05281263) was approved by the Advarra central IRB and conducted at two study sites in the United States. Participants were recruited into two study cohorts: healthy young adults (Group 1, 33-59 years old) and healthy older adults (Group 2, 61-75 years old). Participants of childbearing potential were required to use contraception to prevent pregnancy. Exclusion criteria included any live vaccine within 30 days prior to study vaccination, previous administration of any investigational RSV vaccine or any PIV5-based vaccine (CVXGA1) actively enrolled during the study period, and known infection with human immunodeficiency virus, hepatitis B virus, or hepatitis C virus. A complete list of inclusion and exclusion criteria can be found on the clinical trial website. Participants were not prescreened for RSV serum antibody levels. Eligible participants received a 10-day dose of the PIV5-RSV vaccine on Day 1. 7.5A single dose of plaque-forming units (PFU) was administered by spraying 0.25 mL into each nostril (total volume 0.5 mL) using a MAD Nasal™ intranasal mucosal atomization device (Teleflex MAD300) and observed for 30 minutes immediately after administration. Additionally, subjects were asked to maintain a memory aid for solicited systemic AEs and local reactions for one week after vaccination. Four sentinel participants in each group were initially dosed, and their safety data was reviewed by the Safety Monitoring Committee (SMC) before the remaining participants in that group were enrolled.

[0219] Primary outcome measures included (i) solicited AEs (days 1–8) and (ii) unsolicited AEs (days 1–29). Secondary outcome measures included (i) serum IgG titers to RSV proteins (days 15 and 29), (ii) SAEs (days 1–181), and (iii) AEs of special interest (AESIs) and AEs of medical concern, including x-onset chronic medical conditions (days 1–181).

[0220] Immunogenicity Assessment: Blood and nasal samples were collected at baseline (day 1, prevaccination), days 15, and 29 postvaccination. To reduce sample variability, samples from the same participant were run blindly on the same plate for all assays. Serum RSV nAb levels were determined by a qualified RSV A2 microneutralization (MN) assay based on the RSV-A2-rLuc reporter virus (45). Briefly, quadruplicate serially two-fold diluted serum samples from a starting dilution of 1:100 were incubated with 175 ± 75 PFU of RSV-rLuc for 1 hour and then used to infect Vero cells in 96-well white-walled, clear-bottom plates. After 20–24 hours of incubation, cells were lysed using the Renilla-Glo luciferase assay (Promega), and luciferase signals were read on a SpectraMax iD3 multimode microplate reader (Molecular Devices). RSV nAb titers were determined by 5PL curve fitting using Prism (version 9.5.1 for macOS, GraphPad Software) and defined as the reciprocal dilution that inhibited at least 50% of the signal of the virus control. RSV nAb titers were converted to international units based on a standard serum (16 / 284) obtained from NIBSC (London, UK).

[0221] RSV F-specific serum IgG and IgA antibody and nasal IgA antibody levels were determined by ELISA assay using 25 ng / well of purified RSV F protein (SinoBiological, catalog no. 11049-V08B) and two-fold serial dilutions in blocking buffer (1x KPL wash buffer (Seracare) containing 5% milk / 0.5% BSA, added in duplicate). Endpoint titers were calculated by 4PL curve fitting using Prism and reported as reciprocal dilutions. PIV5-specific IgG and nAb titers were determined by ELISA assay using PIV5 virus-coated plates and by a PIV5-rLuc-based MN assay in Vero cells, respectively. PIV5 IgG endpoint titers were calculated by 4PL curve fitting and reported as reciprocal dilutions. The PIV5 MN assay was performed similarly to the RSV-rLuc-based MN assay, except in duplicate instead of quadruplicate. Analysis of PIV5 MN was identical to the RSV-rLuc-based MN assay, with nAb titers defined as the reciprocal dilution that inhibited the signal of the virus control by at least 50%.

[0222] Antigen-specific T cell frequencies were assessed by intracellular cytokine staining assay using cryopreserved peripheral blood mononuclear cells (PBMCs) isolated from whole blood on days 1 (prevaccination), 15, and 29. One million cryopreserved PBMCs were thawed in complete 10% FBS RPMI medium, incubated overnight at 37°C, and then incubated with the RSV F peptide pool from GenScript at a final concentration of 1 μg / mL in the presence of 1 μg / mL anti-CD28 ECD (Beckman Coulter, clone CD28.2), anti-CD107a FITC (BD Biosciences, clone H4A3), and anti-CD49d (BD Biosciences, clone 9F10). Additionally, PBMCs were stimulated with 1 μL of complete medium containing 0.5% dimethyl sulfoxide (DMSO, a negative control corresponding to the DMSO concentration of the RSV-F peptide pool) or 1 μL of PMA / ionomycin (25 ng / mL PMA and 1 μg / mL ionomycin) for negative and positive controls, respectively. After 2 h of incubation at 37°C, 10 μg / mL Brefeldin A (BD Biosciences) was added, and the cells were incubated for an additional 4 h. Cells were washed with PBS and incubated with Aqua-Viability dye (Invitrogen) for 15 min at room temperature. Cells were washed with PBS supplemented with 2% fetal bovine serum (FBS) and surface stained with anti-CD3 Alexa700 (BD Biosciences, clone SP34-2), anti-CD4 BV605 (BD Biosciences, clone L200), anti-CD8 BV450 (BD Biosciences, clone RPA-T8), and anti-CD95 PE-Cy5 (BD Biosciences, clone DX2) for 30 min at 4°C.Cells were washed with PBS containing 2% FBS, fixed with Cytofix / Cytoperm (BD Biosciences), permeabilized with 1x Perm / wash (BD Biosciences), and incubated with anti-IFN-γ PE-Cy7 (BD Biosciences, clone B27), anti-TNF-α APC-Cy7 (BioLegend, clone Mab11), anti-IL-13 PE (Miltenyi Biotec, clone JES10-5A2.2), and anti-MIP-1β APC (eBioscience, clone FL34Z3L) antibodies for 30 minutes at 4°C. Cells were washed with 1x Perm / Wash and PBS containing 2% FBS, then resuspended in PBS / 2% formaldehyde and acquired on a BD FACSAria Fusion cell sorter. CD3+ cells were differentiated into CD4+ cells. + and CD8 + T cells were gated and separated into memory and naive cells using CD28 and CD95. The net percentage of cytokine-secreting cells was determined by subtracting the value obtained with the DMSO-stimulated sample (negative control). Cytokine-positive CD4 + or CD8 + T cell frequencies were considered positive if the detected frequency of T cells was >0.1% after subtracting the baseline. Data were analyzed using FlowJo software (version 10). Boolean combination and SPICE software were used to identify CD4+ cells producing two or more cytokines. + and CD8 + The polyfunctional responses of T cells were determined.

[0223] Statistical analysis: Statistical analysis was performed using GraphPad Prism software (version 9). Given the small sample size, statistical analysis was primarily descriptive and comprehensive. P values ​​were used to indicate differences of potential significance at a significance level of 0.05. Two-group comparisons of RSV-specific CMI and antibody responses were assessed by Wilcoxon matched-pairs signed-rank tests (day 15 or 29 vs. day 1, or day 15 vs. day 29). A 1.5-fold increase in antibody titer after vaccination relative to baseline was considered significant because the assay characteristics demonstrate a significant 1.2- to 1.3-fold change (95% confidence) when samples from a single participant are tested on the same plate.

[0224] result

[0225] Serum antibody titers: All participants were seropositive for RSV-neutralizing Abs (nAbs) at baseline, and titer evolution is presented in Figures 17A-17F and 18A-18B. In Group 1, nAb geometric mean titers (GMTs) were 880 (9.8 log²) at baseline and increased to 1316 (10.4 log²) at 2 weeks and 1312 (10.4 log²) at 4 weeks post-vaccination (P < .05), reflecting a geometric mean fold rise (GMFR) of 1.5. RSV-nAb antibody response rates (≥ 1.5 fold rise) were observed in 7 / 14 (50%) participants. In Group 2, nAb GMTs were 850 (9.7 log²) at baseline and, similar to Group 1, increased to 1103 (10.1 log²) at 2 weeks (P < .05) and 1372 (10.4 log²) at 4 weeks (P < .05), reflecting geometric mean fold increases (GMFRs) of 1.3 and 1.5, respectively. RSV-nAb antibody response rates were identified in 6 / 15 (40%) participants in this group (Table 19) (individual nAb titer changes are shown in Figures 18A-18D). Notably, RSV-nAb antibody response rates were identified in all five participants who were positive for vaccine virus shedding, suggesting a potential correlation between replication and systemic antibody responses.

[0226] [Table 19]

[0227] All participants were also seropositive for RSV-F-specific serum IgA and IgG Abs at baseline (Figures 17A-17F). In Group 1, F-specific serum IgA GMT increased from 530 (9.0 log²) at baseline to 821 (9.7 log²) and 756 (9.6 log²) at 2 and 4 weeks post-vaccination, respectively. F-specific serum IgG GMT increased from 1640 (10.7 log²) at baseline to 2049 (11.0 log²) at 2 weeks and 2055 (11.0 log²) at 4 weeks post-vaccination. F-specific serum IgA and IgG antibody response rates (≥1.5-fold) were identified in 6 / 14 (43%) and 3 / 14 (21%) participants, respectively (Table 19). In Group 2, F-specific serum IgA GMT increased from 445 (8.8 log²) at baseline to 582 (9.2 log²) and 641 (9.3 log²) at 2 and 4 weeks post-vaccination, respectively. F-specific serum IgG GMT increased from 1088 (10.1 log²) at baseline to 1198 (10.2 log²) and 1325 (10.4 log²) at 2 and 4 weeks post-vaccination. F-specific IgA and IgG antibody response rates were identified in 5 / 15 (33%) and 1 / 15 (7%) participants, respectively (Table 19).

[0228] Overall, these results suggest that the PIV5-RSV vaccine boosted RSV-specific serum Ab levels in young adults (33-59 years) and older adults (61-75 years), but to a greater extent in adults than in older adults. Interestingly, the fold increase in RSV-specific Ab titers was inversely correlated with baseline titers. Higher baseline titers may have been closer to maximal levels.

[0229] Next, we investigated vector-specific immune responses. At baseline, 15 / 29 participants (52%) were seropositive for PIV5 nAb, as defined by a titer of 1:10 (Figures 19A-19B). This level of background seropositivity likely reflects passive exposure to PIV5 through exposure to dogs receiving kennel cough vaccination. In Group 1, PIV5-nAb GMT increased from 26 (4.7 log²) at baseline to 48 (5.6 log²) and 51 (5.7 log²) at 2 and 4 weeks postvaccination, respectively, reflecting geometric mean fold increases of 1.8 and 2.0, respectively. PIV5-nAb antibody response rates (≥1.5 fold increase) were identified in 7 / 14 participants (50%) (Table 19). In Group 2, PIV5-nAb GMT increased from 19 (4.2 log²) at baseline to 34 (5.1 log²) and 44 (5.5 log²) at 2 and 4 weeks postvaccination, respectively, reflecting GMFRs of 1.8 and 2.2, respectively. PIV5-nAb antibody response rates (≥1.5 fold increase) were identified in 10 / 15 (67%) participants. Similar kinetics were observed for PIV5-specific IgG titers (Figures 19C-19D), but with higher GMFRs (≤4.6 fold increase in Group 1 and ≤3.4 fold increase in Group 2) and antibody response rates (86% in Group 1 and 87% in Group 2). When participants with low PIV5 nAb titers were compared with participants with high PIV5 nAb titers, baseline PIV5 nAb titers did not clearly affect PIV5-nAb antibody response rates and did not appear to inhibit RSV-nAb antibody response rates (Figures 20A-C).

[0230] RSV F-specific IgA antibody titers in nasal swabs: At baseline, RSV F-specific IgA antibody (Ab) titers in nasal swabs ranged from below the LOD to 514 (9.0 log²) (Figure 21A). In Group 1, the F-specific nasal IgA geometric mean titer (GMT) was 19 (4.2 log²) at baseline and increased to 40 (5.3 log²) at 2 and 4 weeks post-vaccination (P < .05), reflecting a GMFR of 2.1 (Figure 21B). F-specific nasal IgA responses (≥ 2-fold increase) were identified in 9 / 14 (64%) participants (Table 19). In Group 2, the baseline F-specific nasal IgA GMT of 18 (4.2 log2) was similar to that of Group 1, but the GMT was not elevated at 2 and 4 weeks postvaccination (P > .05) (Figures 21A and 21B). However, F-specific nasal IgA responses were identified in 5 / 15 (33%) of Group 2 participants (individual nasal IgA antibody changes are shown in Figures 18C-18D). In Groups 1 and 2, F-specific nasal IgA responses were identified in 4 / 5 participants who tested positive for vaccine virus shedding.

[0231] Overall, these results suggest that the PIV5-RSV vaccine boosted RSV-specific nasal IgA levels in adults and elderly individuals, but to a lesser extent in elderly individuals. Similar to the results seen with systemic antibody responses, the fold increase in F-specific nasal IgA titers in both groups was inversely correlated with baseline titers (Figures 20A-C).

[0232] Baseline cellular immunity, RSV F-specific CD4 + and CD8 + T cells were detected based on the expression of Th1 or cytotoxic T cell cytokines / markers IFN-γ, TNF-α, MIP-1β, and CD107a, and the expression of the Th2 cytokine IL-13 (Figures 22A-22D). In Group 1, F-specific CD4 T cells expressing at least one of the Th1 / cytotoxic markers were detected. +The mean percentage of T cells increased from 0.06% at baseline to a maximum of 0.42% 2 weeks after vaccination (P<0.001, Figures 22E-F), and CD8 + The mean percentage of T cells of the same phenotype increased from 0.08% at baseline to a maximum of 0.38% 4 weeks after vaccination (P<.001). In group 2, CD4 + The mean percentage of T cells of the same phenotype increased from 0.02 to a maximum of 0.26% at 2 weeks postvaccination (P<.001), with CD8 + T cells increased from 0.04 to a maximum of 0.40% at 4 weeks post-vaccination (P<0.001). In Groups 1 and 2, F-specific cellular immune (CMI) responses (defined as a >0.1% increase from baseline in T cells expressing at least two Th1 / cytotoxic biomarkers) to the PIV5-RSV vaccine were identified in 13 / 14 (93%) and 15 / 15 (100%) participants, respectively (Table 19). In addition, F-specific cytotoxic T lymphocyte (CTL) responses (CD8+ cells expressing IFN-γ) to the PIV5-RSV vaccine were also identified. + A ≥0.1% increase in CD4 T cells from baseline was identified in 12 / 14 (86%) and 13 / 15 (87%) participants, respectively, demonstrating that the PIV5-RSV vaccine stimulated F-specific Th1 (CD4 + ) and cytotoxicity (CD8 + ) It has been suggested that it promotes the production of CD4 T cells. + and CD8 + T cell counts were similar in both groups after vaccination, but CD4 + The geometric mean of T cells increased 6.1-fold and 9.1-fold by day 15 in groups 1 and 2, respectively, with CD8 + The geometric mean T cells increased 3.6-fold and 10-fold by day 29 in groups 1 and 2, respectively (Figures 22G-H), reflecting a lower baseline in group 2 than in group 1. This result suggests that, in contrast to nasal IgA responses, similar levels of CMI were induced by the PIV5-RSV vaccine in adults and elderly subjects.

[0233] In both groups 1 and 2, T cells expressing a single Th1 / cytotoxic biomarker were more frequent than T cells expressing at least two Th1 / cytotoxic markers (Figures 22I-J). However, T cells co-expressing at least two Th1 / cytotoxic biomarkers tended to be more frequent in group 1 than in group 2, suggesting that the PIV5-RSV vaccine may induce higher quality antigen-specific effector / memory T cells in younger age groups based on polyfunctional Th1 / cytotoxic responses.

[0234] In contrast to the Th1 and cytotoxic phenotypes, F-specific CD4 expresses IL-13. + T cells and CD8 + The percentage of T cells did not increase after vaccination in either group 1 or group 2 (Figures 22A-22D), indicating the absence of a Th2-biased response to the PIV5-RSV vaccine, which has been identified as a potential risk factor for vaccine-associated enhanced RSV disease (F.P. Polack, et al. J Exp Med 196, 859-865 (2002); B. Bagga, et al., J Infect Dis 212, 1719-1725 (2015)).

[0235] The complete disclosures of all patents, patent applications, and publications cited herein, as well as electronically available materials (including, for example, nucleotide sequence submissions in GenBank and RefSeq, and amino acid sequence submissions in, for example, SwissProt, PIR, PRF, PDB, and translations from the annotated coding regions of GenBank and RefSeq), are incorporated by reference. In the event of a discrepancy between the disclosure of this application and the disclosure(s) of any document incorporated herein by reference, the disclosure of this application shall control. The foregoing detailed description and examples are given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to those skilled in the art are within the scope of the invention as defined by the claims. References 1.Phan SI, Zengel JR, Wei H, et al.Parinfluenza virus 5 expressing wild-type of prefusion respiratory syncytial virus(RSV)fusion protein protects mice and cotton rats from RSV challenge.J of Virology 2017:91(19);e00560-17. 2.Wang D, Phan S, DiStephano DJ, et al.A single-dose recombinant parainfluenza virus 5- vectored vaccine expressing respiratory syncytial virus(RSV)F or G protein protected Cotton rats and African green monkeys from RSV challenge.J of Virology 2017:91(11);e00066-17. 3.Phan SI,Chen Z,Xu P,et al.A respiratory syncytial virus(RSV)vaccine based on parainfluenza virus 5(PIV5).Vaccine 2014:32;3050-3057. 4.Young D,Wignall-Fleming EB,Busse DC,et al.The switch between acute and persistent paramyxovirus infection caused by single amino acid substitutions in the RNA polymerase P subunit.PLoS Pathogens 2019 Feb11;15(2):e1007561.doi:10.1371 / journal.ppat.100756. 5.Seth et al.Mutations in the cytoplasmic domain of a paramyxoviruses fusion glycoprotein rescue syncytium formation and eliminate the HN requirement for membrane fusion.J.of Virology 2003,167-178. 6.Tong et al.Regulation of Fusion Activity by the Cytoplasmic Domain of a Paramyxovirus F Protein.J.of Virology 2002,301:322-333. 7.Schmitt,A.P.,Leser,G.P.,Waning,D.L.& Lamb,R.A.Requirements for Budding of Paramyxovirus Simian Virus 5 Virus-Like Particles.Journal of Virology 76,3952-3964,doi:10.1128 / jvi.76.8.3952-3964.2002(2002). 8.Waning,D.L.,Schmitt,A.P.,Leser,G.P.& Lamb,R.A.Roles for the cytoplasmic tails of the fusion and hemagglutinin-neuraminidase proteins in budding of the paramyxovirus simian virus 5.J Virol 76,9284-9297(2002). 9.F.P.Polack,M.N.Teng,P.L.Collins,G.A.Prince,M.Exner,H.Regele,D.D.Lirman,R.Rabold,S.J.Hoffman,C.L.Karp,S.R.Kleeberger,M.Wills-Karp,R.A.Karron,A role for immune complexes in enhanced respiratory syncytial virus disease.J Exp Med 196,859-865(2002). 10.B.Bagga,J.E.Cehelsky,A.Vaishnaw,T.Wilkinson,R.Meyers,L.M.Harrison,P.L.Roddam,E.E.Walsh,J.P.DeVincenzo,Effect of Preexisting Serum and Mucosal Antibody on Experimental Respiratory Syncytial Virus(RSV)Challenge and Infection of Adults.J Infect Dis212,1719-1725(2015).

Claims

1. 1. A viral expression vector comprising a parainfluenza virus 5 (PIV5) genome having a heterologous nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:1, wherein the viral expression vector expresses a heterologous polypeptide comprising a live recombinant canine parainfluenza (CPI) vector backbone engineered to express the RSV F protein as a target antigen.

2. 2. The viral expression vector of claim 1, wherein the RSV F protein is encoded by a wild-type or mutant RSV F protein gene.

3. 2. The viral expression vector of claim 1, wherein the RSV F protein gene is codon-optimized for expression in a human subject.

4. 2. The viral expression vector of claim 1, wherein the RSV F protein gene is inserted between the SH and HN junctions of the CPI antigenomic cDNA.

5. The viral expression vector of claim 4, wherein the CPI antigenomic cDNA is sequenced using a primer having a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, or 19.

6. 2. The viral expression vector of claim 1, wherein the parainfluenza (CPI) vector backbone engineered to express the RSV F protein comprises a 22 amino acid extension as part of its cytoplasmic tail.

7. 1. A pharmaceutical composition comprising a parainfluenza virus 5 (PIV5) viral expression vector having a heterologous nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:1, wherein the viral expression vector expresses a heterologous polypeptide comprising a live recombinant canine parainfluenza (CPI) vector backbone engineered to express RSV F protein as a target antigen.

8. 8. The pharmaceutical composition of claim 7, wherein the RSV F protein is encoded by a wild-type or mutant RSV F protein gene.

9. 8. The pharmaceutical composition of claim 7, wherein the RSV F protein gene is codon-optimized for expression in a human subject.

10. 8. The pharmaceutical composition of claim 7, wherein the RSV F protein gene is inserted between the SH and HN junctions of the CPI antigenomic cDNA.

11. The pharmaceutical composition of claim 10, wherein the CPI antigenomic cDNA is sequenced using a primer having a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, or 19.

12. 8. The pharmaceutical composition of claim 7, wherein the parainfluenza (CPI) vector backbone engineered to express the RSV F protein comprises a 22 amino acid extension as part of its cytoplasmic tail.

13. 8. The pharmaceutical composition of claim 7, wherein the live recombinant canine parainfluenza (CPI) vector backbone engineered to express the RSV F protein is a prophylactic vaccine against RSV infection.

14. 10. A method for inducing an immune response in a subject with RSV, the method comprising administering a prophylactic vaccine against RSV infection, the vaccine comprising the pharmaceutical composition of claim 7.

15. 15. The method of claim 14, wherein the vaccine induces RSV F protein-specific serum antibody and cellular responses.

16. The RSV-specific cellular response is ＋ T cells and CD8 ＋ 16. The method of claim 15, comprising increasing expression of T cells.

17. The method of claim 15, wherein the RSV-F-specific serum antibodies and the cellular response are associated with a lower incidence of RSV-induced pathological pulmonary responses compared to immune responses obtained by administering formalin-inactivated RSV (FI-RSV).

18. 18. The method of claim 17, wherein the pathological pulmonary response is selected from the group consisting of peribronchiolitis, perivasculitis, interstitial pneumonia, and alveolitis.

19. 15. The method of claim 14, wherein the vaccine is administered intranasally, intramuscularly, topically, or orally.

20. 15. The method of claim 14, wherein the vaccine is administered in a single or multiple dose regimen.

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