Recombinant RSV vaccines: methods of making and using same

Abstract

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

Description

[0001] Citation of Related Applications

[0002] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 382,453, filed November 4, 2022, which is incorporated by reference in its entirety.

[0003] Reference to a sequence listing

[0004] This application contains a sequence listing, which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. A copy of the .XML file, 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 part of this application and is hereby incorporated by reference in its entirety. Technical Field

[0005] The present invention relates generally to the field of immunomodulation, and more particularly to compositions and methods for modulating the immune response to respiratory syncytial virus (RSV) infection. Background Art

[0006] Human RSV is the leading viral cause of lower respiratory tract illness and hospitalization in young children. The vast majority of children infected with RSV suffer from mild upper respiratory tract infections; however, a small number of children 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). Since almost every child will eventually become infected with RSV, and 20-30% of RSV-infected children will develop severe LRIs, RSV causes more than 130,000 childhood hospitalizations in the United States each year (Shay et al., JAMA, 282(5): 1440-1446 (1999), and World Health Organization, Initiative for Vaccine Research (IVR), Respiratory syncytial virus (RSV).

[0007] Several risk factors for developing severe RSV-induced disease have been clearly identified, including premature 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 illness have no identifiable risk factors (Boyce et al., J. Pediatr., 137(6):865-70 (2000)), meaning that approximately 1-2% of healthy children without any identifiable risk factors suffer the potentially life-threatening consequences of RSV-induced illness (Collins et al., supra).

[0008] RSV-induced severe illness in children has also been associated with the development of asthma (see, 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., 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 changes resulting from lung remodeling after severe RSV disease.

[0009] Although RSV infection is almost universal by the age of three, reinfection occurs throughout life because natural RSV infection does not provide complete immunity (Hall et al., J. Infect. Dis., 163(4):693-698 (1991), and Muelenaer et al., J. Infect. Dis., 164:15-21 (1991)). RSV is a significant cause of morbidity and mortality in the elderly. In a retrospective cohort study, RSV caused an average of 15 hospitalizations and 17 deaths per 1,000 nursing home residents per year, whereas influenza caused an average of 28 hospitalizations and 15 deaths in the same setting (Garofalo et al., Pediatr. Allergy Immunol., 5(2): 111-117 (1994)). Thus, in this population, RSV was isolated as frequently as influenza A and had 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)).

[0010] There is currently no FDA-approved vaccine for preventing RSV infection or treating RSV-induced illness in children. The only FDA-approved medication for preventing RSV infection is (palivizumab) (MedImmune, Gaithersburg, MD), a humanized monoclonal antibody directed against an epitope at the A antigenic site of the RSV F protein administered to high-risk infants. It has shown significant progress in preventing acute RSV disease in the lower respiratory tract and in alleviating lower respiratory tract infections, but it has not been shown to be effective against RSV infection in the upper respiratory tract at the approved dose. In 2023, the FDA approved Beyfortus (nirsevimab), a long-acting RSV monoclonal antibody with an extended half-life, which is recommended for use in all infants <8 months of age who were born during or entering their first RSV season, as well as infants and children aged 8-19 months who are at increased risk for severe RSV disease and are entering their second RSV season.

[0011] RSV vaccine development has been influenced by vaccine-enhanced disease following natural RSV infection in children (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, alum-precipitated vaccine candidate (FI-RSV) was administered to RSV-naive infants in the early 1960s and, although immunogenic, failed to protect children from natural infection. Furthermore, vaccine recipients who subsequently became infected with RSV had a higher rate of hospitalization and more severe illness, including two deaths, compared to 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 been shown to achieve an optimal balance between genetic stability of mutations, safe attenuation in infants, or protective immune responses (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 immunogenicity has been modest (Tristram et al. Vaccine, 12(6): 551-556 (1994)).

[0012] Therefore, there remains a need for compositions and methods that effectively and safely prevent or treat RSV infection.

[0013] 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

[0014] In accordance with the purposes of the present invention, as embodied and broadly described herein, the present invention relates in one aspect 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 virus (CPI) vector backbone engineered to express 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 a CPI antigenomic cDNA, wherein the CPI antigenomic cDNA is sequenced using primers 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, a parainfluenza virus (CPI) vector backbone engineered to express RSV F protein comprises a 22 amino acid extension as part of its cytoplasmic tail.

[0015] 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 virus (CPI) vector backbone engineered to express 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 a CPI antigenomic cDNA, wherein the CPI antigenomic cDNA is sequenced using primers 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 virus (CPI) vector backbone engineered to express RSV F protein comprises a 22 amino acid extension as part of its cytoplasmic tail. In another embodiment, the live recombinant canine parainfluenza virus (CPI) vector backbone engineered to express RSV F protein is a prophylactic vaccine for RSV infection.

[0016] In yet another aspect, the present invention relates to a method for inducing an immune response in a subject suffering from RSV, the method comprising administering a prophylactic vaccine for RSV infection, wherein the vaccine comprises a live recombinant canine parainfluenza virus (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 cell-mediated reactions. In another embodiment, RSV-F-specific serum antibodies and cell-mediated reactions are associated with a reduced incidence of pathological lung reactions induced by RSV, compared to the immune response obtained by administering formalin-inactivated RSV (FI-RSV). In another embodiment, the pathological lung reaction 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-dose regimen or a multiple-dose regimen.

[0017] Other advantages of the present invention will be partially listed in the following description and in part will become apparent from the description or can be learned through practice of the present invention. The advantages of the present invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It should be understood that the above summary and the following detailed description are merely exemplary and explanatory and do not limit the invention as claimed. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or several embodiments of the present invention and, together with the description, serve to explain the principles of the invention.

[0019] Figures 1A-1B Figure 3 shows the RSV F specific immune response in the AGM of immunization. RSV F specific cellular immune response in African green monkeys (AGM) of immunization. PBMCs were isolated from the AGM of immunization one day before vaccination and 14 and 28 days after immunization. PBMCs were stimulated with RSV F peptide pools and CD4 + ( Figure 1A ) and CD8 + ( Figure 1B ) The levels of different cytokines in cells are expressed as a % of the sum of all cytokines. Group 1 = control, Group 6 = CPI-RSV-F.

[0020] Figures 2A-2B pSP28 ( Figure 2A ) and pAB94( Figure 2B ) plasmid map.

[0021] Figure 3 The CPI-RSV-F vaccine vector plasmid and map are shown.

[0022] Figure 4 A schematic diagram of virus rescue is shown.

[0023] Figures 5A-5C Representative images of RSV F protein percent expression assay CPI-RSV-F pre-MVs are shown.

[0024] Figures 6A-6C Representative images of the RSV F protein percent expression assay, CPI-RSV-F MVS, are shown.

[0025] Figure 7 A schematic representation of the CHD03 experimental timeline is shown.

[0026] Figure 8 A graphic representation of the CHD04 experimental timeline is shown.

[0027] Figures 9A-9B Shown is CHD03 ( Figure 9A ) and CHD04( Figure 9B ) RSV-F serum antibody titer. Naive BALB / c mice were treated with PBS or 10 5 Naive BALB / c mice were immunized intranasally with 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). Horizontal lines represent the geometric mean antibody titer for each group. Statistical significance was determined by ANOVA and Dunnett's multiple comparison test. ****P < 0.0001, significant between the PBS group and the vaccine group. LOD, limit of detection.

[0028] Figures 10A-10B CHD03 and CHD04 RSV-F ELISPOT titers are shown. Naive BALB / c mice were treated with PBS or with 10 5 Naive BALB / c mice were intranasally immunized with 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 expressed as 10 6 The number of IFN-γ-secreting cells per spleen cell (N=5). The horizontal line represents the geometric mean ELISPOT titer. Statistical significance was determined by ANOVA and Duncan's multiple comparison test. *P<0.05, **P<0.01, ***P<0.001, significant between the PBS group and the vaccine group.

[0029] Figures 11A-11B Shown is CHD03 ( Figure 11A ) and CHDO4( Figure 11B ) lung titer.

[0030] Figure 12 Shown are lung RSV challenge virus titers after RSV challenge.

[0031] Figure 13 Shown are nasal RSV challenge virus titers in nasal washes following RSV challenge.

[0032] Figure 14 RSV neutralizing antibody responses are shown.

[0033] Figure 15 Shown are anti-RSV F protein IgG antibody responses by ELISA.

[0034] Figures 16A-16D is a bar graph showing the expression of RSV NS1, IL-4, IL-2, and IFN-γ mRNA.

[0035] Figures 17A-17F is a bar graph showing serum RSV-specific antibody (Ab) titers in young adults (Group 1) and elderly adults (Group 2) before and after vaccination with BLB201 (CPI-RSV-F). Figures 17A-17B RSV neutralizing antibodies obtained by microneutralization assay are shown; Figures 17C-17D F-specific RSV IgA is shown, and Figures 17E-17F F-specific IgG Abs obtained by ELISA are shown. The left column shows individual log2 titers and mean log2 titers by age group on days 1, 15, and 29, along with their respective standard deviations (error bars), and the right column shows the geometric mean fold increase (GMFR) from baseline by age group on days 15 and 29. *P < .05; **P < .01; ***P < .001, by Wilcoxon test.

[0036] Figures 18A-18D Group 1 ( Figure 18A ) and Group 2 ( Figure 18B ) serum RSV neutralization (nAb) titers and nasal F-specific IgA antibody (Ab) titers ( Figures 18C-18D ) and divided into age groups 1 and 2.

[0037] Figures 19A-19Dis a bar graph showing serum PIV5 antibody (Ab) titers of the two vaccinated groups before and after vaccination with BLB201 (CPI-RSV-F). Figures 19A-19B PIV5 neutralizing antibodies obtained by microneutralization assay are shown; and Figures 19C-19D PIV5-specific RSV IgG Ab obtained by ELISA is shown. The left column shows the individual log2 titers and mean log2 titers and their respective standard deviations (error bars) by age group on days 1, 15, and 29, and the right column shows the geometric mean fold increase (GMFR) relative to baseline by age group on days 15 and 29. *P < .05; **P < .01; ***P < .001, obtained by Wilcoxon test.

[0038] Figures 20A-20C is a bar graph showing the relationship between response rates to PIV5-RSV vaccination based on baseline (pre-vaccination) PIV5 neutralizing antibody (nAb) levels. Patients with PIV5 neutralizing (nAb) serological responses (≥1.5-fold titer increase) to PIV5-RSV vaccination, by subgroup, based on individual subjects' baseline PIV5 nAb titers below (L) or above (H) the baseline geometric mean titer of PIV5 nAb for each age group (Groups 1 and 2). Figure 20A ) and patients with RSV neutralizing (nAb) serological responses (≥1.5-fold titer rise) to PIV5-RSV vaccine ( Figure 20B ) percentage. Figure 20C The percentage of subjects with a nasal F-specific IgA response to PIV5-RSV vaccine (a ≥2-fold increase in titer) is shown, by subgroup, based on individual baseline nasal F-specific IgA titers lower (L) or higher (H) than the baseline geometric mean titer of nasal F-specific IgA for each age group (Group 1 and Group 2).

[0039] Figures 21A-21B is a bar graph showing nasal IgA antibody titers before and after vaccination in Groups 1 and 2. F-specific RSV IgA Ab obtained by ELISA. Left ( Figure 21A ) shows the individual log2 titers and mean log2 titers by age group on days 1, 15, and 29, along with respective standard deviations (error bars), and FIG( Figure 21B ) shows the geometric mean fold increase (GMFR) from baseline by age group on days 15 and 29.

[0040] Figures 22A-22J RSV F-specific CD4 before and after vaccination with BLB201 (CPI-RSV-F) is shown. + and CD8+ T cell responses. CD4 T cells of each individual at days 1, 15, and 29 by age group according to the expression of the indicated immune markers + T cells (top) and CD8 + Percentages of T cells (22A-D) (represented by symbols and continuous lines). Figure 22E and 22F Shown are the CD4 T cells at days 1, 15, and 29 by age group according to the indicated Th1 / cytotoxic marker combinations (IFN-γ, TNF-α, CD107a). + T cells (left) and CD8 + Mean percentages of T cells (right panel) are presented as stacked histograms. Figure 22G and 22H CD4 at days 15 and 29 by age group is shown + and CD8 + Geometric mean fold increase (GMFR) of T cells relative to baseline. Figure 22G and 22H Shown are the fold changes in CD4 and CD8 T cell responses at days 15 and 29 after vaccination. Figure 22I and 22J F-specific CD4 T cells expressing 1, 2, or 3 Th1 / cytotoxicity markers (IFN-γ, TNF-α, CD107a) on days 15 and 29 for groups 1 and 2 + and CD8 + Pie chart showing the proportion of T cells. *P < .05; **P < .01; ***P < .001, ns: not significant. DETAILED DESCRIPTION

[0041] The present invention may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the examples included therein, taken in conjunction with the drawings and its previous and following description.

[0042] I. Definition

[0043] In order to facilitate understanding of the principles and features of the various embodiments of the present disclosure, various exemplary embodiments are explained herein. Although the exemplary embodiments of the present disclosure have been explained in detail, it should be understood that other embodiments may also be considered. Therefore, the scope of the present disclosure is not limited to the details of the construction and arrangement of the components set forth in the description or examples. The present disclosure can have other embodiments and can be implemented or executed in various ways.

[0044] In describing exemplary embodiments, specific terminology will be employed for the sake of clarity. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to a component is intended to include compositions of a plurality of components. Reference to a composition containing "an" component is intended to include additional components in addition to the specified one.

[0045] Ranges may be expressed herein as from "about" or "approximately" or "substantially" one particular value and / or to "about" or "approximately" or "substantially" another particular value. When such a range is expressed, another exemplary embodiment includes from one particular value and / or to another particular value. Unless otherwise indicated, all numerical values used in the specification and claims indicating the amount of a component, molecular weight, etc. are to be understood as being modified in all cases by the term "about". Therefore, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximate values that may vary depending on the desired properties that the present invention seeks to obtain. At a minimum, and without attempting to limit the principle of equivalent ranges to the scope of the claims, each numerical parameter should at least be interpreted in light of the number of reported significant figures and by applying ordinary rounding-up techniques.

[0046] 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.

[0047] For any method disclosed herein comprising discrete steps, the steps may be performed in any feasible order. Also, any combination of two or more steps may be performed simultaneously, if appropriate.

[0048] This description illustrates exemplary embodiments. In several places throughout the application, guidance is provided by lists of examples, which can be used in various combinations. In each case, the enumerated list serves only as a representative group and should not be construed as an exclusive list.

[0049] All headings are for the convenience of the reader and, unless otherwise indicated, should not be used to limit the meaning of the text following the heading.

[0050] The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

[0051] Similarly, as used herein, "substantially free" of a substance or "substantially pure" and similar characterizations can include "at least substantially free" of a substance or "at least substantially pure," as well as "completely free" of a substance or "completely pure."

[0052] “Include” or “contain” or “comprises” means that at least the specified compound, element, particle or method step is present in the composition, article or method, but does not exclude the presence of other compounds, materials, particles, method steps, even if other such compounds, materials, particles, method steps have the same function as the specified one.

[0053] The words "preferred" and "preferably" refer to embodiments of the invention that may provide certain benefits under certain 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 mean that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[0054] 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.) and experimental animal models. In a preferred embodiment, the subject is a human.

[0055] As used herein, the term "vaccination" generally refers to the sequential administration of one or more antigens to a subject to generate and / or enhance an immune response against the antigens. Sequential administration includes a primary immunization followed by one or more booster immunizations.

[0056] In the context of the present invention, the term "pathogen" refers to any agent that can cause 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. Preferred pathogens are infectious pathogens. In specific embodiments, the infectious pathogen is a virus, such as a coronavirus.

[0057] As used herein, antigen refers to any molecule that can cause a T cell or B cell immune response in a subject. Antigens that are specific for pathogens are typically obtained from the pathogen or derived from its elements, which contain epitopes and can cause an immune response against the pathogen. Depending on the pathogen, antigens can have a variety of properties, such as (poly)peptides, proteins, nucleic acids, lipids, cells, etc. Live attenuated forms of pathogens (e.g., bacteria, viruses) or their killing or inactivated forms, or materials purified therefrom, such as proteins, peptides, lipids, etc., can also be used. Antigens can be naturally occurring or artificially produced. For mammals receiving treatment, it may be exogenous or endogenous (e.g., tumor antigens). Antigens can be produced by techniques known in the art per se, such as, for example, synthetic or recombinant techniques, or enzymatic methods.

[0058] In a specific embodiment, 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. The term also applies to amino acid polymers in which one or more amino acid residues are modified or non-naturally occurring residues, such as artificial chemical mimetics of naturally occurring amino acids. It should be understood that the term "protein" also includes fragments or variants of different antigens, such as fragments containing epitopes, or proteins obtained from pathogens and subsequently enzymatically, chemically, mechanically, or thermally modified.

[0059] "Therapeutically effective amount" means an amount sufficient to achieve such treatment when a compound (e.g., a PIV5-based composition as described herein) is administered to a subject for the treatment of a condition, disorder, or illness. The "therapeutically effective amount" will vary depending on the compound or bacterium administered and the disease and its severity, as well as the age, weight, physical condition, and responsiveness of the mammal to be treated.

[0060] The phrase "pharmaceutically acceptable" as used in connection with the compositions of the present disclosure means that the molecular entities and other ingredients of such compositions are physiologically tolerable and do not generally produce adverse reactions when administered to mammals (e.g., humans). Preferably, the term "pharmaceutically acceptable" as used herein means approved by federal regulatory agencies or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly, in humans.

[0061] As used herein, the term "pharmaceutically acceptable composition" refers to a composition comprising at least one compound disclosed herein formulated together with one or more pharmaceutically acceptable carriers.

[0062] The term "administer" refers to the introduction of a predetermined amount of a substance into a patient's body by a suitable method. The compositions disclosed herein can be administered via any common route, as long as they can reach the desired tissue, such as, but not limited to, inhalation, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, or rectal administration. However, since peptides are digested upon oral administration, the active ingredients of compositions for oral administration should be coated or formulated to protect them from degradation in the stomach.

[0063] The term "dose" means a single amount of a compound or agent administered; and / or "regimen" means a plurality of predetermined doses, which may be different or similar in amount, administered at different time intervals, which may be different or similar in duration. In some embodiments, a regimen also encompasses the duration of a delivery period (e.g., an agent administration period or a treatment period). Alternatively, a regimen is a plurality of predetermined vaporized amounts administered at predetermined time intervals.

[0064] 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, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water or aqueous saline solutions and aqueous dextrose solutions and aqueous 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 a binder (for pelleting), a glidant, an encapsulating agent, a flavoring agent and a coloring agent. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin.

[0065] The terms "treat" or "treatment" of a condition, disorder, or condition include: (1) preventing or delaying the onset of at least one clinical or subclinical symptom of the condition, disorder, or condition in a subject who may be at risk for or susceptible to the condition, disorder, or condition but who does not yet experience or display the 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 development of the disease or its recurrence (in the case of maintenance therapy) or at least one clinical or subclinical symptom thereof; or (3) ameliorating the disease, i.e., causing regression of the condition, disorder, or condition or at least one of its clinical or subclinical symptoms. The benefit is either statistically significant for the subject being treated or at least perceptible to the patient or physician.

[0066] In the context of the present invention, the term "pathogen" refers to any agent that can cause 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. Preferred pathogens are infectious pathogens. In specific embodiments, the infectious pathogen is a virus, such as a coronavirus.

[0067] “Include” or “contain” or “comprises” means that at least the specified compound, element, particle or method step is present in the composition, article or method, but does not exclude the presence of other compounds, materials, particles, method steps, even if other such compounds, materials, particles, method steps have the same function as the specified one.

[0068] As used herein, the term "parainfluenza virus 5" (PIV5) includes, for example, but 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 hereby incorporated by reference.

[0069] 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 and an appropriate eukaryotic host cell or organism is selected, expression may include splicing of the mRNA. In the context of the present invention, the term also encompasses the production of RSV F gene mRNA and the RSV F protein obtained after its expression.

[0070] 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 an RSV fusion protein polypeptide. Various RSV fusions and adhesion proteins have been described and are known to those skilled in the art. WO / 2008 / 114149 (which is incorporated herein by reference in its entirety) lists exemplary F protein variants and G protein variants (e.g., naturally occurring variants).

[0071] The materials described as constituting the various elements of the present disclosure are illustrative only and not limiting. It is intended that many suitable materials that perform the same or similar functions as the materials described herein are encompassed within the scope of the present disclosure. Such other materials not described herein may include, but are not limited to, materials developed after the time of development of the present disclosure, for example.

[0072] II. CPI-RSV-F Composition

[0073] Respiratory syncytial virus (RSV) is a member of the genus Pneumovirus of the family Paramyxoviridae. Human RSV (HRSV) is the main cause of severe lower respiratory tract diseases in young children and is also the cause of a large number of morbidity and mortality in people. RSV is also considered to be an important disease pathogen in adults and the elderly with impaired immunity. Due to the incomplete protection of the infected host to RSV after natural infection, RSV may repeatedly infect during childhood and adulthood.

[0074] The genome of this virus is composed of single-stranded negative-sense 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, which contains virally encoded structural proteins. The viral polymerase is packaged together with the virion 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 non-structural proteins NS1 and NS2.

[0075] Fusion of HRSV with the infected cell membrane is thought to occur on the cell surface and is an essential step for the transfer of viral ribonucleoproteins into the cytoplasm in the early stages of infection. This process is mediated by the fusion (F) protein, which also promotes the fusion of the membrane of the infected cell with the membrane of the adjacent cell to form a characteristic syncytium, which is both a significant cytopathic effect and another mechanism of viral transmission. Therefore, neutralizing fusion activity is important for preventing viral infection and is also important for host immunity. In fact, 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).

[0076] 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 an inactive 574-amino acid precursor (F0) that is co-translationally glycosylated on asparagine in the endoplasmic reticulum, where it assembles into homo-oligomers. Before reaching the cell surface, the F0 precursor is cleaved by proteases at the N-terminus into F2 and at the C-terminus into F1. The F2 and F1 chains remain covalently linked by one or more disulfide bonds.

[0077] CPI-RSV-F is a parainfluenza virus (PIV5)-based RSV vaccine that expresses the RSV F protein and is provided herein as a prophylactic intranasal vaccine to prevent RSV infection and serious complications associated with RSV infection. CPI-RSV-F is designed to induce an immune response to the F protein of RSV, which is the major antigenic protein that is highly conserved between RSV subtypes A and B. Anti-F antibodies can inhibit viral entry into host cells, and RSV F is a validated vaccine target based on efficacy data of the commercial product palivizumab (RSV F monoclonal antibody).

[0078] The present disclosure provides CPI-RSV-F compositions, systems, and methods for 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 treatment).

[0079] A. Pharmacological Summary of RSV Studies

[0080] The present disclosure provides CPI-RSV-F compositions, systems, and methods for 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 treatment).

[0081] Previously published studies based on the W3A strain of the PIV5 construct (which was engineered to express the RSV-F protein) included immunogenicity and challenge studies on mice, cotton rats, and African green monkeys (1, 2, 3). As part of these studies, the permissiveness of PIV5 replication was confirmed in cotton rats and African monkeys (1, 2). In addition, the following studies were conducted: 1) a comparison of CPI-based vector constructs with vector constructs based on the W3A PIV5 strain; 2) a comparison of vaccine constructs expressing pre-fusion RSV with vaccine constructs in which the wt F protein replaced the PIV5 SH gene (ΔSH) or was inserted between the PIV5 SH and NH (SH-NH) or was inserted between the HN and L (HN-L). These studies selected CPI-RSV-F (CPI-RSV-F), which contains the full-length RSV F protein inserted between the SH and HN genes (Figure 1), for preliminary human studies.

[0082] In addition, the protective efficacy of the CPI-RSV-F vaccine has been evaluated in RSV challenge studies in mice and cotton rats. Based on the significant reduction in RSV viral titers observed in the lungs and nasal washes of immunized animals compared to controls, a single intranasal dose of immunization protected animals from RSV infection.

[0083] The recent preclinical proof-of-concept studies using the vaccine vector construct CPI-RSV-F include immunogenicity and attack studies performed by Blue Lake Biotechnology Inc. on mice and African green monkeys. In addition, the preclinical data of the CPI-RSV-F construct studies in the cotton rat attack studies sponsored by the NIH were summarized, and the vaccine products were provided by Blue Lake Biotechnology Inc. The CPI-RSV-F vaccine used in these recent non-clinical studies used a previous vaccine vector construct (rescued from BHK cells), which is identical to the vector construct (rescued from 293 / Vero cells) used for clinical trial materials, and is also produced using serum-free Vero cells as substrates and formulated with sucrose phosphate glutamate (SPG) buffer. These non-clinical studies include W3AΔSH-RSV-F (engineered to express the PIV5 W3A strain of RSV F protein) as an active comparator.

[0084] A phase 1 clinical trial of CIP-RSV-F demonstrated the safety of the RSV vaccine in young adults (Group 1) and elderly adults (Group 2). The vaccine elicited serum RSV-specific and nasal IgA antibody responses as well as robust cellular immune responses (Spearman et al., Sci. Adv. 9, eadj7611 (2023)).

[0085] In summary, nonclinical 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 antibodies and cell-mediated responses after a single intranasal dose. CPI-RSV-F is well tolerated in these animal models, and no signs of sensitization were observed after vaccination with CPI-RSV-F. The following summarizes recent nonclinical studies conducted in mice, cotton rats, and African green monkeys using CPI-RSV-F vaccine constructs (vaccines based on the CPI PIV5 strain). These studies included an active comparator (a vaccine vector based on the PIV5 W3A strain) in which the RSV F protein replaced the SH gene (ΔSH). See Figure 1 for more details on the vaccine vector constructs. See Table 1 for an overview of nonclinical studies conducted using various vaccine vector constructs.

[0086] Table 1: Overview of nonclinical studies of previous vaccine construct studies and recent studies using the CPI-RSV-F construct.

[0087]

[0088] i. Studies on related W3A PIV5 vectors expressing RSV F protein in mice, cotton rats, and monkeys

[0089] PIV5(W3A)-RSV-F and RSV-G protein studies in Balb / c mice (3, Phan et al. 2014): In this study, Balb / c mice received a single intranasal dose of PIV5-RSV-F (10 6 PFU dose) followed by RSV challenge. The PIV5 W3A vaccine vector construct in this study consists of the wild-type RSV F protein inserted between the PIV5 HN and L gene regions. A single intranasal dose elicited an IgG2a / IgG1 RSV response similar to that observed following infection with wild-type RSV A2 at day 21 after immunization.

[0090] PIV5 (W3A) challenge study expressing wild-type or prefusion RSV F protein in mice and cotton rats (1, Phan et al. 2017): This study evaluated PIV5 vector vaccines modified by altering the F protein insertion site (insertion into the SH-HN junction of PIV5 or replacement of the SH with the RSV F protein gene). In addition, this study evaluated wild-type (wt) F protein or prefusion conformation F protein (pF).

[0091] Mice were treated with a single dose of 1 x 10 6 PFU intranasal immunization: W3AΔSH-RSV-F (RSV F protein gene inserted into the missing SH region of PIV5) expressing wild-type F protein or prefusion stable RSV F mutant (W3AΔSH-RSV-pF) or improved vectors W3A-RSV-F SH-HN or W3A-RSV-pF SH-HN (F protein gene inserted into the SH-HN junction). The study groups are as follows (Table 2).

[0092] Table 2: Study groups in the mouse challenge study

[0093] Vaccine / route Number of animals Attack virus PBS 10 <![CDATA[1x10 6 PFU RSV A / A2]]> RSV A2 5 <![CDATA[1x10 6 PFU RSV A / A2]]> <![CDATA[W3A(HN-L)-RSV-F,10 6 PFU,i.n.]]> 10 <![CDATA[1x10 6 PFU RSV A / A2]]> <![CDATA[W3A(SH-HN)-RSV-F,10 6 PFU,i.n.]]> 10 <![CDATA[1x10 6 PFU RSV A / A2]]> <![CDATA[W3A(SH-HN)-RSV-pF,10 6 PFU,i.n.]]> 10 <![CDATA[1x10 6 PFU RSV A / A2]]> <![CDATA[W3AΔSH-RSV-F,10 6 PFU,i.n.]]> 10 <![CDATA[1x10 6 PFU RSV A / A2]]> <![CDATA[W3AΔSH-RSV-pF,10 6 PFU,i.n.]]> 10 <![CDATA[1x10 6 PFU RSV A / A2]]>

[0094] Following immunization, both humoral and cell-mediated immune responses were observed. The vaccine construct using the wt F protein detected the highest neutralizing antibody response. Furthermore, the vaccine construct containing the wt F protein inserted before the HN and ΔSH conjugation appeared to have the strongest immunogenicity. The levels of cell-mediated immune responses (based on IFN-γ using ELISPOT) were similar for the various vaccine constructs.

[0095] Mice were challenged with RSV A2 28 days after immunization to determine protective efficacy. On day 4 post-challenge, challenge virus was detected in only one of five mice in the W3AΔSH-RSV-F group, while none of the other vaccinated groups had virus. In the PBS control group, all mice recovered challenge virus.

[0096] Similar studies were conducted in cotton rats, which are more susceptible to RSV infection. 3 PFU of modified vectors (W3A-RSV-F(SH-HN), W3A-RSV-pF, W3AΔSH-RSV-F) containing wild-type or stable F protein (pF) or 10 2 PFU of W3AΔSH-RSV-pF was used for immunization.

[0097] All groups observed immune responses, including neutralizing antibody responses against RSV A Tracy strain (97% identical to RSV A / A2 strain). Similar to the mouse study, the group immunized with the vaccine construct containing the wt F protein had higher antibody levels compared to the pF group, with the F insertion before the HN gene joined to the ΔSH having the highest value (titer of approximately 128). Neutralizing antibody titers against RSV / B / 18537 were significantly lower than those against RSV / A Tracy strain, with only the W3A(SH-HN)-RSV-F and W3AΔSH-RSV-F groups detecting significant antibody levels (titer of approximately 8). At day 28, the group vaccinated with 1.21x10 5 After RSV challenge with PFU of RSV / A / Tracy, a reduction in RSV viral load in nasal washes was observed in all vaccine dose groups (reduction of 1.4-1.66 Log 10 ) and a decrease in RSV viral load in lung lavage fluid (reduced by 2-3 Log 10 RSV challenge virus was recovered from all mice in the PBS control group (approximately 10 5 PFU / nasal wash, or 10 5 PFU / g lung lavage fluid).

[0098] Overall, these preliminary studies in mouse and cotton rat models did not show evidence that the prefusion F protein was more immunogenic and protective than wild-type F. Furthermore, for the intranasal route, no differences in vector performance were observed between the ΔSH and SH-HN insertion vector constructs.

[0099] Sigmovir Protocol #XV-131 Study Report in Cotton Rat Using Intranasal and Subcutaneous Routes (Phan et al. 2017) Challenge Study: In a follow-up study (1, Phan et al. 2017), a higher dose of 10 5 and 10 6The efficacy, immunogenicity, and safety of PFU of W3A(SH-NH)-RSV-F and controls were evaluated by two different routes—intranasal (in) and subcutaneous (sc). This study also included positive controls for enhanced disease (animals immunized with FI-RSV and then challenged with RSV, and a positive control group consisting of animals pre-infected with RSV and then challenged with RSV. Animals in this study were challenged with RSV A2 on day 49, and necropsy and histology were performed 5 days later (day 54). The study groups for this study are as follows (Table 3).

[0100] Table 3: Study Groups in Protocol #XV-131.

[0101] Vaccine / route N = number of animals RSV / A2 attack PBS im 5 PBS PBS im 5 <![CDATA[5Log 10 PFU]]> FI-RSV im 5 <![CDATA[5Log 10 PFU]]> RSV / A2 live, in (positive control) 5 <![CDATA[5Log 10 PFU]]> <![CDATA[W3A(SH-HN)-RSV-F,10 5 PFU i.n.]]> 5 <![CDATA[5Log 10 PFU]]> <![CDATA[W3A(SH-HN)-RSV-F,10 5 PFU s.c]]> 5 <![CDATA[5Log 10 PFU]]> <![CDATA[W3A(SH-HN)-RSV-F,10 6 PFU s.c]]> 5 <![CDATA[5Log 10 PFU]]> <![CDATA[W3AΔSH-RSV-F,10 5 PFU i.n.]]> 5 <![CDATA[5Log 10 PFU]]> <![CDATA[W3AΔSH-RSV-F,10 5 PFU s.c.]]> 5 <![CDATA[5Log 10 PFU]]> <![CDATA[W3AΔSH-RSV-F,10 6 s.c.]]> 5 <![CDATA[5Log 10 PFU]]>

[0102] Immunogenicity: W3A(SH-HN)-RSV-F in 10 5 and 10 6 The neutralizing antibody titers induced by W3AΔSH-RSV-F vaccination were slightly higher in the in group than in the sc group, with 10 6 The titers were slightly higher with the dose, although this was not statistically significant.

[0103] Efficacy: W3A(SH-HN)-RSV-F provided complete protection to the lower respiratory tract by in or sc administration. The viral load in the upper respiratory tract of the animals was also significantly lower.

[0104] Compared with control animals sham-immunized with PBS (mean titer of 10 5 PFU), W3A(SH-HN)-RSV-F vaccination induced complete protection in the lower respiratory tract by sc administration and almost complete protection by in administration, based on the reduction in titers observed in lung and nasal washes (mean titer of 10 3 PFU).

[0105] Safety: Lung sections from different groups were examined for features of lung inflammation: peribronchiolitis, perivasculitis, interstitial pneumonia, and alveolitis, and were scored to determine severity. The largest lesions were observed in the FI-RSV-vaccinated, RSV-challenged group (positive control group). In groups immunized with W3A-RSV-F (SH-HN) or W3AΔSH-RSV-F (intranasal or subcutaneous), lung changes were moderate, with levels lower than those observed in the RSV-vaccinated, RSV-challenged positive control group and similar to those observed in the PBS sham-vaccinated, RSV-challenged group.

[0106] Measurement of cytokine levels in lung tissue 5 days after challenge by quantitative real-time PCR (qPCR) did not indicate the possibility of infection enhancement. IL-4 mRNA levels were significantly elevated only in the FI-RSV vaccination group, which was consistent with the histopathological findings and enhanced disease phenotype. IFNγ-mRNA levels were highest in the sham vaccination group and the FI-RSV vaccination group. IFN-mRNA levels were similarly low in the group vaccinated with PIV5-based candidates and the RSV vaccination group. IL-2 mRNA levels were similar in each group, but the average IL-2 level in the FI-RSV vaccination group was significantly higher than in the other groups.

[0107] On day 28 after immunization, RSV A2 (10 6 After intranasal challenge of immunized mice with 500 PFU (100 mg / kg / day), lung sections obtained on day 4 post-challenge showed no exacerbation of lung lesions compared to RSV-A2-immunized mice. Protective immunity was observed as assessed by viral load in lung tissue (n=5 mice per group).

[0108] PIV5 (W3A) Challenge Study Expressing RSV F or G Protein in Cotton Rats and African Green Monkeys (2, Wang et al. 2017): This study evaluated the replication, immunogenicity, and efficacy of PIV5-vectored RSV F protein in cotton rats and African green monkeys against RSV challenge. In this study, the F protein was inserted into the intergenic region of the PIV5 HN and L genes.

[0109] Replication permissiveness of PIV5 in cotton rats and African green monkeys: 1x10 5 PFU of PIV5 were inoculated intranasally into cotton rats (n=4 per group) in a volume of 10 μl or 100 μl. Virus titers (up 1×10 4 PFU), with viral titers nearly disappearing by day 6. A larger inoculum volume (100 μl) resulted in vaccine virus being observed in the lungs of all animals on day 6, whereas this virus was only observed in one animal vaccinated with the smaller volume of 10 μl.

[0110] To assess the permissivity of PIV5 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 intranasally infected with 1x10 2 Up to 1x10 8 PFU of PIV5. Viral shedding was assessed in nasal and bronchoalveolar washes on days 3, 5, 7, 10, and 14. Virus shedding from the nose and lungs persisted for up to 10 days with doses as low as 1x10 2At 4 PFU, viral replication peaked on day 5. These data indicate the permissiveness of African green monkeys to PIV5.

[0111] Immunogenicity in cotton rats and African green monkeys after single-dose control: 1x10 3 , 1x10 4 , 1x10 5 and 1x10 6 Cotton rats were immunized intranasally with PFU of W3A-RSV-F (SH-HN). IgG antibody responses were observed at day 28 at all dose levels, and the responses were comparable across dose groups. Neutralizing antibodies were observed in all dose groups, with titers ranging from 64 to 256 (NT50%). In addition, IgA responses were observed in lung homogenates from all dose groups on day 21 after vaccination.

[0112] African green monkeys (PIV5 and RSV seronegative) received 1x10 4 or 1x10 6 A single intranasal immunization with PFU W3A-RSV-F (SH-HN) was performed. Serum obtained on day 21 after vaccination showed a high titer of F-specific antibody response. In addition, a low level of neutralizing antibody response (NT50%, 1x10 6 PFU dose group 52). Nasal swabs obtained 21 days after immunization showed a significant level of F protein IgA response. Cell-mediated responses assessed by gamma interferon showed that 1x10 6 The response level was lower in the PFU dose group.

[0113] Protection in cotton rats and African green monkeys challenged with RSV: 1x10 3 to 1x10 6 Cotton rats immunized intranasally with a single dose of PFU W3A-RSV-F(SH-HN) were challenged with RSV A2 strain on day 28 post-immunization. Protection was assessed by measuring viral load in nose and lung tissues. Viral load decreased in a dose-dependent manner, with a 1x10 6 Except for the PFU group, this group was relative to 1x10 3 The PFU group showed a higher level, but still decreased compared with the control group. 5 In the dose group, no virus was detected in the lungs, but a decrease in titer (3log 10 class).

[0114] Use 1x10 4 or 1x10 6African green monkeys immunized with PFU W3A-RSV-F (SH-HN) were challenged with RSV A2 28 days after vaccination. RSV viral loads were assessed in nasal and BAL samples from days 3 to 14 after challenge. Vaccination with the control did not shorten the time to viral shedding; however, peak viral RSV loads were reduced 10- to 100-fold in both dose groups, with a 1x10 6 The highest reduction was observed in the PFU dose group.

[0115] Responses to Controls in RSV-Exposed African Green Monkeys: Prior exposure to RSV did not affect the ability of African green monkeys (seroconverted by intranasal infection with RSV A2) to elevate RSV neutralizing antibody titers (by a 50-fold increase).

[0116] Lung pathology in cotton rats immunized with W3A-RSV-F (SH-HN) after RSV challenge: On day 0, the rats were treated with a dose of 1×10 6 In animals immunized with PFU W3A-RSV-F and challenged with RSV A2 on day 49, lungs obtained five days after challenge were blindly examined for alveolitis, interstitial pneumonia, perivasculitis, and peribronchitis and compared with lungs obtained from control animals vaccinated with formalin-inactivated RSV. Scores in positive control animals vaccinated with formalin-inactivated RSV were significantly higher than those in PBS control animals, but not higher than those in W3A-RSV-F (SH-HN) animals (ANOVA paired t-test).

[0117] B. Parainfluenza virus 5 (PIV5)

[0118] Parainfluenza virus 5 (PIV5) is a negative-strand RNA virus and a member of the Rubulavirus genus of 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 previously known as simian virus-5 (SV5). Although PIV5 is a virus that infects a variety of animals and humans, no known symptoms or diseases associated with PIV5 have been found in humans. Unlike most paramyxoviruses, PIV5 has almost no cytopathic effect when infecting normal cells. As a negative-strand RNA virus, the genome of PIV5 is very stable. Since PIV5 has no DNA stage in its life cycle and replicates only in the cytoplasm, PIV5 cannot be integrated into the host genome. Therefore, using PIV5 as a vector avoids the possible unintended consequences of genetic modification of host cell DNA. PIV5 can be grown to high titers in cells, including Vero cells, which have been approved by the WHO and FDA for vaccine production. Therefore, PIV5 presents many advantages as a vaccine vector.

[0119] The PIV5-based vaccine vectors of the present invention can 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 strains W3A, WR ( 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(Pt 10): 3007-16; Choppin, 1964, Virology: 23: 224-233; and Baumgartner et al., 1987, Intervirology; 27: 218-223. In addition, PIV5 strains used in commercial kennel cough vaccines, such as, e.g., BI, FD, Merck, and Merial vaccines, can be used.

[0120] C. PIV5 CPI strain vector backbone

[0121] The PIV5 CPI strain vector backbone differs from the PIV5 W3A strain vector backbone as follows: The most significant difference is that the PIV5 F protein of the CPI strain consists of an additional 22 amino acid extension as part of its cytoplasmic tail. The extension of the F protein is thought to inhibit the fusogenic properties of the virus (5, 6). Compared with the W3A-based virus, which does not have an extended PIV5 F protein tail and has additional amino acid differences compared to CPI, the CPI-based virus is more soluble and can produce more progeny virus in infected cells (4).

[0122] i. PIV5 CPI vector RSV construct

[0123] The PIV5 vaccine vectors of the present invention can be constructed using any of a variety of methods, including but not limited to the reverse genetics system described in more detail in 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 the 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 is a glycoprotein that mediates cell-to-cell and virus-to-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 the key amino acid residues for efficient fusion have been identified. The hemagglutinin-neuraminidase (HN) glycoprotein is also involved in viral entry into 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 and is located in 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 ed., pp. 1449-1496. DMKnipe & P.M. Howley, eds. Philadelphia, PA: Lippincott Williams & Wilkins.

[0124] Previously, recombinant PIV5 viruses expressing exogenous genes from a variety of 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., Vaccine, 33(51):7217 (2015); Lafontaine, E.R., et al., Vaccine X., 1:100002 (2018); Li, K., et al., mBio, 11(2)(2020)). Because the PIV5 vector vaccine actively replicates in the respiratory tract after intranasal immunization, it can produce 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)).

[0125] In one embodiment, the CPI-RSV-F vaccine drug substance (CPI-RSV-F) presented herein comprises a live recombinant PIV5 vector virus based on the CPI strain of PIV5 that expresses wild-type F protein of RSV (F protein sequence based on GenBank accession number FJ614814J, SV A2 strain) and is codon-optimized for expression in humans ( Figure 7 The wild-type RSV F protein is inserted between the PIV5 SH and NH regions (Figure 1).

[0126] ii. Immunogenicity and challenge studies of CPI-RSV-F vaccine constructs

[0127] In nonclinical studies, a single dose of CPI-RSV-F was found to be immunogenic in mice, cotton rats, and AGMs following a single in dose at dose levels ranging from 10 4 to 10 6 PFU. F-specific serum immune responses and cell-mediated responses were detected. The ability of the PIV5 vector to replicate in cotton rats and AGM was confirmed.

[0128] A single in dose of CPI-RSV-F or related vaccine constructs was able to prevent infection in various animal challenge models (mice, cotton rats, and AGM) using RSV A2 as a challenge. Efficacy was based on a reduction in viral load observed in lung and nasal washes after challenge compared to that observed in control animals.

[0129] No enhanced disease / lung pathology was observed in the cotton rat challenge model of the CPI-RSV-F vector construct used for the proposed Phase 1 study CPI-RSV-F. This conclusion is based on the cytokine profiles in the lung tissue observed (no increase in IL-4 response) 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, various W3A-based vector constructs inserting the F protein at different positions within PIV5W3A were evaluated, and no signs of enhanced disease / lung pathology were observed.

[0130] Given that it was also able to induce protective immune responses at dose levels similar to those of the W3A construct (although the immune response to W3A appeared to be slightly higher), CPI-RSV-F was chosen over W3A for initial clinical evaluation. In addition, the same CPI PIV5 backbone engineered to express the S protein of SARS-CoV-2 is also being clinically evaluated under the cross-referenced IND 027418. Preclinical data and limited clinical data obtained to date using this related vector construct show that when administered at 10 6 A single intranasal dose of PFU has a favorable safety profile when administered to healthy adults aged 18-55 years.

[0131] III. BLB genome sequence

[0132] The CPI-RSV-F genome sequence is provided herein.

[0133] A. CPI-RSV-F genome sequence

[0134] i.CPI-RSV-F 5' to 3'

[0135] The CPI-RSV-F 5'-3' nucleic acid sequence is provided herein.

[0136]

[0137]

[0138]

[0139]

[0140]

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[0151] IV. Treatment Methods

[0152] The present disclosure can be used in gene therapy and / or therapeutic methods for treating diseases involving the increase or decrease of a nucleotide sequence of interest in a host cell. In these embodiments, the expressible heterologous nucleotide sequence can be derived from a mammalian genome. In some embodiments, it may be particularly useful to have an expressible heterologous nucleotide sequence derived from a human genome, where the expression of wild-type RNA and / or protein can produce a therapeutic effect in a patient. For example, the expressible heterologous nucleotide sequence can encode CFTR, NeuroD1, Cas9, and a guide RNA, or any other such sequence. In other embodiments, the heterologous nucleotide sequence encodes a secreted protein.

[0153] In other embodiments, the expressible heterologous nucleotide sequence stimulates response to positive selection. In other embodiments, the expressible heterologous nucleotide sequence also stimulates response to negative selection. In further embodiments, it is useful for the polynucleotide sequence to further comprise a reporter gene. For example, the reporter gene can be luciferase or green fluorescent protein.

[0154] V. Application Methods

[0155] A. Administration through vaccination

[0156] The present invention includes methods of vaccinating a subject by administering to the subject a viral expression vector, viral particle, or composition as described herein.

[0157] The present invention provides a recombinant PIV5-based live vaccine composed of canine parainfluenza virus (CPI) engineered to express RSV F protein as a target antigen to develop CPI-RSV-F vaccine as a novel prophylactic vaccine against RSV infection.

[0158] i. Intranasal vaccination

[0159] In one embodiment, the disclosed CPI-RSV-F composition is formulated to allow intranasal administration. A large number of nonclinical studies have been conducted to evaluate the immunogenicity and efficacy of CPI-RSV-F or very closely related vaccine constructs (e.g., W3A PIV5-based constructs) expressing RSV F protein using the intranasal (in) route in various animal models. This includes early development studies in mice, cotton rats, and African green monkeys (AGMs) that assess changes in vector constructs (position of the F protein in the vector) and RSV F protein inserts (wild-type or pre-fusion F protein). We summarize the findings of these studies and the publications are hereby incorporated by reference.

[0160] Intranasal compositions may include inhalable dry powder pharmaceutical formulations comprising a therapeutic agent, wherein the therapeutic agent is present in the form of a free base or a mixture of a salt and a free base. Pharmaceutical formulations disclosed herein can be formulated to be suitable for airway administration, such as nasal, intranasal, sinusoidal, oral, and / or pulmonary administration. Typically, the formulation is produced in a manner that provides it with an appropriate particle size for an airway administration route or target. Therefore, the formulations disclosed herein can be produced to have a determined particle size distribution.

[0161] For example, the particle size distribution of the salt form of the therapeutic agent for intranasal administration can be between about 5 μm and about 350 μm. More specifically, the salt form of the therapeutic agent can 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, about 53 μm to about 150 μm or about 20 μm to about 53 μm for intranasal administration. The salt form of the therapeutic agent in the pharmaceutical composition of the present invention can have a particle size distribution range of less than about 200 μm for intranasal administration. In other embodiments, the particle size distribution of the salt form of the therapeutic agent in the pharmaceutical composition is 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 present invention may have a particle size distribution range of greater than about 5 μm, greater than about 10 μm, greater than about 15 μm, greater than about 20 μm, greater than about 38 μm, less than about 53 μm, less than about 70 μm, greater than about 100 μm, or greater than about 150 μm for intranasal administration.

[0162] In addition, the salt form of the therapeutic agent in the pharmaceutical composition of the present invention can have a particle size distribution range of between about 1 μm and about 10 μm for pulmonary administration. In other embodiments for pulmonary administration, the particle size distribution range is between about 1 μm and about 5 μm, or between about 2 μm and about 5 μm. In other embodiments, the salt form of the 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.

[0163] In some embodiments, the disclosed cannabinoid compositions include one or more cannabinoids or pharmaceutically acceptable derivatives or salts thereof, a propellant, an alcohol, and a glycol and / or glycol ether. The alcohol may be a monohydric alcohol or a polyhydric alcohol, and is preferably a monohydric alcohol. Monohydric alcohols have a lower viscosity than glycols or glycol ethers. Therefore, the composition is able to form droplets of smaller diameter than a composition in which the monohydric alcohol is not present. The inventors have unexpectedly discovered that a specific ratio of monohydric alcohol to glycol or glycol ether can produce a composition having a desired combination of long-term stability (e.g., the composition remains in a single phase at a temperature of 2-40°C for at least one week) and small droplet size.

[0164] a. Vaccine Immunogenicity and Protection in the Lower Respiratory Tract of Mice

[0165] One embodiment provides intranasally administered candidate vaccine viruses that elicited high levels of anti-F serum antibodies in both the CHD03 and CHD04 studies. The geometric mean titer of RSV F antibody titers in animals vaccinated with W3AΔSH-RSV-F in CHD03 was 3.1 Log 10 / mL, while the geometric mean titer of RSV F antibody titer of CHD04 was 3.4Log 10 / mL. In CPI-RSV-F vaccinated animals, the geometric mean titer of CHD03 was 3.0Log 10 / mL, while the geometric mean titer of CHD04 was 3.2Log 10 / mL. These values were not statistically significant between each other and were similar to those of the RSV_rA2 positive control group. Both vaccine viruses also elicited RSV F protein-specific cellular responses, as measured by IFN-γ-secreting cells. In CHD03, the geometric mean of W3AΔSH-RSV-F was 10 6There were 28 IFN-γ secreting cells per 10 splenocytes, while the geometric mean of CPI-RSV-F was 23 IFN-γ secreting cells. In CHD04, the geometric mean of W3AΔSH-RSV-F and CPI-RSV-F was 23 IFN-γ secreting cells per 10 splenocytes, respectively. 6 There were 35 IFN-γ secreting cells and 48 IFN-γ secreting cells per spleen cell. These values were statistically significant compared with the PBS control group, but were not different from the vaccinated group or the RSV_rA2 positive control group. The IFN-γ secreting cells in the CPI-RSV-F group in CHD04 were significantly higher than the positive control. The RSV challenge virus titers of W3AΔSH-RSV-F and CPI-RSV-F were significantly lower compared with the PBS control group in CHD03 and CHD04. For W3AΔSH-RSV-F, the value was 1.35Log 10 PFU / g and 1.37Log 10 PFU / g, and for CPI-RSV-F, the value was 1.48Log 10 PFU / g and 1.40Log 10 PFU / g (for CHD03 and CHD04, respectively), while the PBS control group was 3.21Log 10 PFU / g and 3.33Log 10 PFU / g. The value of RSV_rA2 positive control group in CHD03 was 1.39Log 10 PFU / g, while the value in CHD04 is 1.32Log 10 PFU / g, similar to the vaccine groups of interest.

[0166] Overall, the results presented here show that W3AΔSH-RSV-F and CPI-RSV-F, as well as two other drug candidates (W3A-RSV-F(SH-HN) and CPIΔSH-RSV-F), have a 10 5 Intranasal administration of PFU dose levels induced a substantial RSV-specific immune response and significantly protected the lower respiratory tract of BALB / c mice from RSV challenge virus replication.

[0167] b. Preclinical testing of the PIV5ΔSH-RSV-F vaccine based on the PIV5 CPI strain in a cotton rat virus challenge model using RSV A / A2 strains, compared with the PIV5ΔSH-RSV-F vaccine based on PIV5 W3A.

[0168] 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 RSV A / A2-challenged cotton rat Sigmodon hispidus model. 4 , 10 5 or 10 6 Animals were immunized intranasally with a PIV5-based vaccine containing PFU virus and then four weeks later with 10 5 PFU RSVA / A2 challenge. 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 reinfected seven weeks later. The vaccine-enhanced disease control group was immunized twice with FI-RSV, four weeks apart, and infected with RSV three weeks after the second immunization. Five days after RSV challenge, the animals were sacrificed to collect samples. RSV replication in the lungs and nose, lung tissue pathology, lung cytokine and RSV NS1 mRNA expression, as well as RSV serum neutralizing antibodies (NA) and anti-F protein binding antibody titers were measured. To confirm the replication of the PIV5 vaccine in the respiratory tract of cotton rats, groups of three animals were inoculated intranasally with 10 6 The rats were given PFU of either CPI-RSV-F or W3AΔSH-RSV-F and sacrificed four days later. These samples were submitted for plaque assay evaluation. In short, 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 at higher levels in the upper respiratory tract than CPI-RSV-F, but at lower levels in the lower respiratory tract than CPI-RSV-F.

[0169] Both CPI-RSV-F and W3AΔSH-RSV-F vaccines were highly effective in protecting the lungs of cotton rats, inducing near-sterile immunity at all vaccine doses tested, as demonstrated by plaque assays and qPCR. All doses of both vaccines also produced statistically significant protection in the nose, with W3AΔSH-RSV-F immunization being more effective than CPI-RSV-F.

[0170] At all three vaccine doses tested, W3AΔSH-RSV-F reduced rhinovirus loads to nearly undetectable levels. 6 The highest protection was induced by the dose of 10 PFU, followed by 5 PFU and 10 4 PFU dose. Both CPI-RSV-F and the control vaccine induced a strong NA response, with the control induced higher NA titers than the highest dose (10 6CPI-RSV-F administered with 100 PFU of CPI-RSV-F. Lower doses of CPI-RSV-F induce weaker NA - Both CPI-RSV-F and W3AΔSH-RSV-F vaccinations induced high levels of binding IgG, with W3AΔSH-RSV-F vaccinations producing slightly higher levels of binding IgG. 5 With the exception of one animal immunized with W3AΔSH-RSV-F at 10 PFU (95% CI, 1.0-2.0), none of the other vaccines induced the lung histopathology or IL-4 mRNA expression levels observed in animals immunized with FI-RSV. Overall, the W3AΔSH-RSV-F vaccine appeared to be more effective than CPI-RSV-F in terms of nasal protection and neutralizing antibody responses at all doses tested. 4PFU Increased to 10 6 PFU has no clear advantage in improving vaccine efficacy.

[0171] ii. Lung Composition

[0172] One embodiment provides a formulation and method for treating SARS-CoV-2 in the pulmonary system by inhalation or pulmonary administration. The diffusion characteristics of a particular pharmaceutical formulation through lung tissue are selected to achieve an effective concentration and effective residence time in the tissue to be treated. The dose can be increased or decreased, or the frequency of administration can be increased or decreased, to achieve a selected blood level. In addition, the administration time and amount of the formulation are preferably controlled to optimize the therapeutic effect of the administered formulation on the tissue to be treated and / or titrate to a specific blood level.

[0173] The diffusion in the lung tissue can also be changed by adding various excipients in the preparation to slow down or accelerate the absorption of the drug to the lung tissue. For example, the drug can be combined with surfactants such as phospholipids, dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol. These drugs can also be used together with bronchodilators to relax the bronchial airways and make it easier for antitumor drugs to enter the lungs. Salbutamol is an example of the latter, and many other known drugs are also available in this area. In addition, the drug can be compounded with biocompatible polymers, micelles, structures or cyclodextrins.

[0174] The particle size of the aerosolized drug used in this example was measured to be approximately 1.0-5.0 μm with a GSD of less than about 2.0 for deposition in the central and peripheral lung compartments. As described elsewhere herein, the choice of particle size depends on the desired site of deposition of the drug particles within the respiratory tract.

[0175] Aerosols useful in the present invention include aqueous vehicles such as water or saline with or without ethanol, and may contain preservatives or antimicrobials such as benzalkonium chloride, parabens, and the like, and / or stabilizers such as polyethylene glycol.

[0176] Powders that can be used in the present invention include preparations of pure drugs or preparations of drugs 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 can be dispersed in a chamber containing a gas or gas mixture and then inhaled by the patient.

[0177] The agent of the present disclosure can be applied once, or can be divided into multiple dosages and applied at intervals. For example, the agent of the present invention can be repeatedly applied, for example at least 2, 3, 4, 5, 6, 7, 8 times or more, or can be applied by continuous infusion. It should be understood that the precise dosage and treatment duration depend on the disease being treated, and can be determined empirically by using known test protocols or by inferring from in vivo or in vitro test data. It should be noted that concentration and dosage values may also vary with the severity of the disease to be alleviated. It is also necessary to understand that for any specific subject, specific dosage regimens should be adjusted over time according to individual needs and the professional judgment of the person administering the composition or the supervisory composition. Any concentration range listed herein is merely exemplary and is not intended to limit the scope or practice of the claimed compositions and methods.

[0178] In some therapeutic embodiments, an "effective amount" of an agent is an amount that results in a reduction in 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 compared to the expected reduction in the parameter in an individual not treated with the agent.

[0179] In some aspects, any of the PIV5-based constructs and methods described in WO 2013 / 112690 and WO 2013 / 112720 (which are hereby incorporated by reference in their entireties) may be used in the present invention.

[0180] As used herein, the term "subject" refers to an 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," "patient," or "host." Non-human vertebrates include livestock animals (such as, but not limited to, cattle, horses, goats, and pigs), domestic pets 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, cattle, pigs, goats, dogs, cats, guinea pigs, hamsters, minks, and rabbits.

[0181] As used herein, "in vitro" means in cell culture, while "in vivo" means within a subject. As used herein, "isolated" refers to material that has been removed from its natural environment (e.g., if naturally occurring, the natural environment), produced using recombinant technology, or chemically or enzymatically synthesized, and thus altered "by the hand of man" from its natural state.

[0182] Example

[0183] Example 1: Immunogenicity of CPI-RSV-F in African Green Monkeys (AGM) (Non-GLP Study)

[0184] Objective: To compare the immunogenicity of single-dose intranasal administration of CPI-RSV-F and W3AΔSH-RSV-F in the African green monkey model.

[0185] method

[0186] On day 0, four monkeys in each dose group were treated with 10 6 PFU of CPI-RSV-F (Group 6) or control (Group 1) were immunized intranasally.

[0187] result

[0188] Antibody responses to RSV F protein and PIV5 vectors on day 28 were determined by ELISA (plates coated with recombinant RSV F protein, source: SinoBiologicals: 11049-V088 LC120C 2910, or PIV5 wild-type virus) as shown in Table 4. In summary, all animals produced antibodies against F protein expressed by CPI or W3AΔSH vectors, and RSV F ELISA titers were comparable between the two groups.

[0189] Table 4: Serum antibody responses to RSV F and PIV5 in the AGM study.

[0190]

[0191] Cell-mediated responses: RSV F protein-specific cellular responses were assessed 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. INFγ, TNFα, MIP-1b, IL-13, and CD107a-positive cells were quantified (see Figures 1A-1B ). PBMCs collected on day -1 before immunization failed to 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 CD4 and CD8 cell responses that were specific for RSV F protein, as assessed on days 14 and 28. No F-specific cellular responses were detected in control animals (data not shown), whereas cellular responses in animals immunized with W3AΔSH-RSV-F and CPI-RSV-F increased from day 14 to day 28 after immunization ( Figures 1A-1B On day 14, W3AΔSH-RSV-F elicited a lower CD4 response than CPI-RSV-F, but the difference was not statistically significant. On day 28 after immunization, W3AΔSH-RSV-F elicited a higher CD4 response than CPI-RSV-F, but this difference was also not statistically significant. W3AΔSH-RSV-F elicited a higher CD8-specific response than CPI-RSV-F, but the difference was not statistically significant on day 14 (P = 0.0883) or day 28.

[0192] Both CPI-RSV-F and W3AΔSH-RSV-F elicited immune responses against RSV F protein. 6 After 100 PFU, CPI-RSV-F and W3AΔSH-RSV-F elicited comparable antibody responses against RSV F protein. CPI-RSV-F elicited slightly lower CD4 and CD8 cell responses than W3AΔSH-RSV-F, but the differences were not statistically significant.

[0193] Example 2: Manufacturing Process and Process Control

[0194] Step 1: Vaccine vector construction

[0195] Construction of pCVL41 (CPI-RSV-F) antigenomic cDNA: The CPI-RSV-Fopt genome was obtained by digesting the pSP28 plasmid with RsrII and AatII restriction endonucleases. pSB28 is a low-copy plasmid containing a codon-optimized RSV F protein gene (Fopt, sequence based on GenBank Accession M74568, RSV A2 strain, codon-optimized for humans) inserted between the SH and HN junctions of the CPI antigenomic cDNA. Figure 2A )(3). pAB94 plasmid is a high copy plasmid in which the EGFP gene is inserted between the SH and HN junctions of the CPI antigenomic cDNA ( Figure 2B ). The Fopt gene in CPI-RSV-Fopt digested with RsrII and AatII restriction endonucleases was inserted into pAB94 digested with RSrII and AatII to replace the EGFP gene with Fopt using T4 DNA ligase.

[0196] The ligated cDNA was transformed into TOP10 competent cells and single colonies were grown in LB medium containing chloramphenicol.

[0197] The resulting plasmid DNA (designated pCVL41 or pCPI-RSV-Fopt; Figure 3 ) was purified by Qiagen miniprep kit and sequenced. Sanger sequencing confirmed that the codon-optimized RSV-F gene inserted into the junction of the SH and HN genes was correct.

[0198] Step 2: Vaccine vector rescue

[0199] The recombinant vector virus used to generate CPI-RSV-F (CPI-RSV-F) was rescued. To rescue the recombinant CPI-RSV-F virus, the pCPI-RSV-Fopt plasmid was transfected into serum-free 293T suspension cells (obtained from GenHunter Corporation) along with plasmids encoding PIV5 NP, P, L proteins, and T7 RNA polymerase, thereby recombinant virus could be rescued from the transfected cell culture ( Figure 4 ).

[0200] Supporting PIV5 plasmid clones encoding the nucleoprotein (N), phosphoprotein (P), or large polymerase protein (L) have been described previously (1, 2), each gene being placed under the T7 promoter in the pCAGGS vector. The following five plasmids were used to generate CPI-RSV-FRVS.

[0201] 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 genes of canine parainfluenza virus (CPI).

[0202] 2. pCAGGS-NP plasmid: This plasmid contains the PIV5 NP gene under the T7 promoter and has the ampicillin resistance gene.

[0203] 3. pCAGGS-P plasmid: This plasmid contains the PIV5 P gene under the T7 promoter and has the ampicillin resistance gene.

[0204] 4. pCAGGS-L plasmid: This plasmid contains the PIV5 L gene under the T7 promoter and has an ampicillin resistance gene.

[0205] 5. pCAGGS-T7 plasmid (same as pBH437-T7): This plasmid encodes the T7 RNA polymerase gene under the SV40 promoter.

[0206] The medium used for virus rescue was CDM4 HEK293 medium (Hyclone) containing 4 mM GlutaMAX (Gibco).

[0207] After two days of incubation, 293T cells were co-cultured with serum-free Vero cells (P159) passaged from Vero MCB cells (obtained from CRL: African green monkey kidney (WHO Vero 10-87) CyanVac MCB DOM: 19AUG2020-P148). After 4 days of incubation, 2 mL of the supernatant containing the rescued virus was taken, mixed with 10XSPG, and stored at -80°C.

[0208] Step 3: Plaque purification and amplification of virus rescue seeds

[0209] Aliquots of the frozen stock containing the rescued virus were serially diluted and plaque assayed on serum-free Vero cells (P155-derived from WHO Vero 10-87 CyanVac MCB DOM: 19AUG2020) in 6-well plates to obtain 6 isolated single plaques. A single plaque was punctured 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 Vero 10-87 CyanVac MCB DOM: 19AUG2020) in 6-well plates. After 6 days, the supernatant (2 mL) of the 6-well plates infected with single plaques was mixed with 10% 10X SPG and stored at -80°C (to produce 1X SPG). This material was designated as Lot# CPI-RSV-F-PP1-PQ10-42621 and manufactured on 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 described in Table 5.

[0210] Table 5: Primers used to amplify viral cDNA for sequencing of CPI-RSV-F antigenomic cDNA

[0211]

[0212]

[0213] Step 4: Production of prMVS in serum-free Vero cells (Lot#210519MCBC HD-PQ10)

[0214] An aliquot from Lot#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 T75 flasks, incubated at 37°C for 5 days, and 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 (Sigma Aldrich) containing 10% arginine, quickly frozen in 1 mL aliquots, and stored at -80°C as a pre-MVS library. The pre-MVS was designated Lot#210519MCBCHD-PQ10. The pre-MVS was sequenced from the NP gene to the L gene to confirm the correct insertion of the viral genomic sequence and the RSV F protein (a single silent mutation was found in AA54 of NP).

[0215] Prior to use in cGMP production of MVS, ex-MVS (Lot# 210519MCBCHD-PQ10) was tested at the CRL for sterility (direct method), bacteriostasis and fungistasis, the presence of mycoplasma, mycobacteria, porcine and bovine circovirus, and overt viruses.

[0216] The ability of the CPI-RSV-F vaccine vector construct to infect Vero cells and express functional F-protein (including pre-fusion F protein) was assessed using immunofluorescence assay (IFA) for pre-MVS and MVS pools, as described below. In addition, pre-MVS and MVS were sequenced by the Sanger method to confirm the presence of the correct F protein gene insert in the PIV5 viral backbone (sequencing from the leader to the trailer).

[0217] The following characteristics were tested on the former MVS of Lot# 210519MCBCHD-PQ10.

[0218] F protein expression by CPI-RSV-F pre-MVs in infected Vero cells obtained by IFA: F protein expression by CPI-RSV-F was assessed by IFA staining of the PIV5 vector (HN) and RSV F protein using palivizumab, a humanized murine monoclonal antibody that recognizes antigenic site II in both the prefusion (pre-F) and postfusion (post-F) conformations of the respiratory syncytial virus (RSV) F glycoprotein. The percentage of F expression was determined by counting the number of cells that stained positive for PIV5 (HN protein) and also stained positive for F protein ( Figures 5A-5C and Table 6 ).

[0219] Table 6: Percent co-expression of CPI-RSV-F pre-MVS.

[0220]

[0221] Vero-SF cells were infected with 10-fold, 100-fold, and 1000-fold diluted CPI-RSV-F pre-MVS virus. After incubation at 37°C for 1 hour, 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 Figure 6 is a representative image of a well infected with CPI-RSV-F pre-MVS, showing red and green cells. Table 6 shows the percentage of RSV F protein expression in PIV5 infected cells. Images were taken at 10X.

[0222] F protein expression by CPI-RSV-F MVS infected cells obtained by IFA: F protein expression 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 counting the number of cells that stained positive for PIV5 (HN protein) and RSV F protein ( Figures 6A-6C and Table 7 ).

[0223] Table 7: Percent co-expression of RSV-F and PIV5 in CPI-RSV-F (MVS).

[0224]

[0225] Vero-SF cells were infected with CPI-RSV-FMVS virus diluted 100-fold, 1000-fold, and 10,000-fold. After incubation at 37°C for 1 hour, 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 Figure 7 is a representative image of a well infected with CPI-RSV-F MVS, showing green and red cells. Table 7 shows the percentage of RSV F protein expression in PIV5-infected cells. Images were taken at 10X.

[0226] Step 5: Manufacturing Process of MVS

[0227] The production of CPI-RSV-F MVS is carried out in accordance with cGMP. The master virus seed stock (MVS), also used as the Phase 1 clinical batch material, is produced using serum-free Vero cells CyanVac MCB (DOM: 19AUG2020, expanded to passage 152 at CRL) in accordance with CRL's cGMP (CBR-1862) of August 2021.

[0228] Vaccine API Manufacturing: Vero cells grown in T225 flasks in serum-free medium (VP-SFM) were infected with CPI-RSV-F pre-MVS Lot# 210519MCBCHD-PQ10 (using 2 vials) at an MOI of 0.002 at 37°C for 7 days. The cell culture fluid of the infected cells constituted the crude vaccine API (3.0 liters). Samples of the MVS crude drug substance were tested for the following: sterility (direct method), bacteriostasis and fungistasis, in vitro mycoplasma test (agar culturable and non-culturable), tissue culture safety test (GP-V810.2), in vitro mycobacterial test, inapparent virus, PBERT, and potency.

[0229] The vaccine drug substance was clarified by centrifugation at 1,500 rpm for 10 min at 2-8°C to obtain 1.2 L, which was then filtered through a ThermoFisher 0.45 μM PES filter unit to obtain 1.2 L. The clarified and filtered vaccine drug substance was immediately formulated with 10XSPG to stabilize the virus before filling. The vaccine drug substance was not stored before formulation and filling.

[0230] During virus production, prepare a control harvest using uninfected Vero cells from the same production batch. Store the control at -60°C or below. Harvest the production control and test for bacteriostatic and fungistatic raw material product (direct method), sterility, detection and quantification of residual Vero DNA, and determination of endotoxin levels (LAL).

[0231] Vaccine Drug Product Manufacturing (Formulation and Filling) / MVS: Filtered virus was stabilized by formulation with 10% 10X SPG buffer and dispensed into 2 mL cryovials at 1 mL volumes using a calibrated repeater pipette to create an MVS library / vaccine product (1281 vials, 1 mL each). The dispensed MVS / vaccine product was flash frozen in a dry ice / methanol bath and stored at or below -60°C.

[0232] Clinical Lot Materials: For the proposed initial Phase 1 trial, the master virus seed (MVS), Lot# CPI-RSV-F (CPI-RSV-F-210519PQ10MVSDOM:26AUG2021) was used as the vaccine drug product.

[0233] MVS characterization: In addition, genetic identity was confirmed by sequencing viral antigenomic cDNA from the NP gene to the L gene region to confirm the identity.

[0234] Example 3: Comparative study between PIV5 vector control and CPI-RSV-F vaccine candidates in BALB / c mice

[0235] Materials and methods

[0236] The purpose of the two mouse studies (CHD03 and CHD04) in this report was to compare the immunogenicity and protective efficacy of two RSV vaccine candidates, W3AΔSH-RSV-F and CPI-RSV-F, in a BALB / c mouse model. In addition, the study included two other vaccine candidates: W3A-RSV-F (SH-HN) and CPIΔSH-RSV-F. Antibody titers were measured by RSV F-specific ELISA, and cellular immune responses were measured by ELISPOT. The protection of vaccinated mice against challenge infection with wild-type RSV strains in the lower respiratory tract was determined by lung homogenate plaque assay (Table 8).

[0237] Table 8. Summary of W3AΔSH-RSV-F and CPI-RSV-F data from the CHD03 and CHD04 studies.

[0238]

[0239]

[0240] dpi = days post-immunization; dpc = days post-challenge

[0241] The key materials and reagents used are listed in Table 9. The viruses used in the experiments are listed in Table 10.

[0242] Table 9. Key Materials and Reagents

[0243]

[0244] Table 10. Viruses

[0245]

[0246]

[0247] Before vaccination, all viruses were diluted to 2X10 6 PFU / mL. RSV_rA2 stock solution was not diluted before challenge.

[0248] The experimental panels suitable for ELISA, ELISPOT and plaque assay testing are summarized in Tables 11 and 12.

[0249] Table 11: CHD03 dosing groups and tests.

[0250]

[0251] Table 12: Dosing groups and tests for CHD04.

[0252]

[0253]

[0254] Immunization of mice: 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). Immunization was performed by intranasal administration of 10 5 Each vaccine candidate was administered with 50 μL of PFU plus an RSV_rA2 positive control. Negative controls were intranasally administered with 50 μL (25 μL per nostril) of PBS. The RSV_rA2 positive control was used to simulate natural RSV infection prior to vaccination. All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of the University of Georgia.

[0255] Twenty-seven days after immunization (mice in groups A, D, G, J, M, P of CHD03 and mice in groups A, E, G, K, M, P of CHD04) or 28 days after immunization (mice in groups B, C, E, F, H, I, K, L, N, O, Q, R of CHD03 and mice in groups B, C, D, F, H, I, J, L, N, O, Q, R of CHD04), blood was collected via buccal bleed for serological analysis. 27 days after immunization, spleens were also collected from mice in cages A, D, G, J, M, P of CHD03 and from mice in cages A, E, G, K, M, P of CHD04 for ELISPOT assay. Thirty-five days after immunization, mice were anesthetized by intraperitoneal injection of 250 μL of Avertin and treated with 50 μL (25 μL per nostril) of 3.8×10 5 Groups B, C, E, F, H, I, K, L, N, O, Q, R of CHD03 and groups B, C, D, F, H, I, J, L, N, O, Q, R of CHD04 were challenged intranasally with PFU of RSV A2. After challenge, mice continued to be housed together according to their vaccination groups. Four days later, lungs of 7-10 mice per group were harvested and viral burden was assessed by plaque assay of lung homogenates. The experimental timeline is as follows: Figure 7 and 8 shown.

[0256] Anti-F ELISA assay: Anti-F antibody titers were determined by anti-F ELISA assay according to CVL SOP-049. 12.5 ng / mL of RSV F protein was coated on Immulon 2HB 96-well plates at 50 μL / well overnight. Serum samples were pre-diluted 10-fold and then serially diluted 3-fold and added to the F protein-coated plates (50 μL / well) for 1 hour at room temperature. After the plates were washed, 50 μL / well of a 1:1250 dilution of goat anti-mouse IgG HRP-conjugated antibody was added and incubated at room temperature for 1 hour. 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 adding HCL, the plate was read at 450 nm using a plate reader (SpectraMax iD3 multi-mode microplate reader). Exceeding OD 450 Samples with a cutoff value of 0.20 were considered positive in at least two serial dilutions. Antibody titer was defined as the highest reciprocal dilution at which a positive signal appeared. The assay was performed by Blue Lake Biotechnology in Los Gatos, California.

[0257] ELISPOT assay: using BD TM ELISPOT Mouse IFN-γ Group The level of IFN-γ secreting cells was measured by ELISPOT assay. BD FACS was coated with purified anti-mouse IFN-γ antibody 24 hours before the assay. TM ELISPOT plate. Mouse spleens (n=5 per group) were harvested 27 days after immunization and placed in a 15 mL conical tube containing 5 mL of HBSS. The spleens were passed through a 70 μM cell strainer, incubated with ACK lysis buffer, washed with HBSS, and then resuspended in complete tumor medium (CTM) to a concentration of 5 × 10 6 Cells / mL were used to prepare splenocytes. The capture antibody solution was removed from the plate and the plate was washed 5-6 times with PBS. The plate was then blocked with CTM for 90 min. The blocking solution was discarded and 50 μL of CTM containing 0.1 μg of RSV F peptide (85-93) was added to the wells. Next, 50 μL of splenocytes (2.5×10 5 Cells / well) were added to the plate and incubated at 37°C, 5% CO2 for 48 h. TM ELISPOT Set instructions were used to immunostain the spots and use The results were counted using a 10 6The mean number of IFN-γ secreting cells per splenocyte is shown. This assay was performed at the University of Georgia, Athens, GA.

[0258] Plaque Assay: RSV viral titers in lung homogenates were measured in Vero cells using a 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 homogenized using the Protein_01 program on the gentleMACS dissociator at 4°C, followed by centrifugation at 3000 × g for 10 min. The supernatant was used to make three-fold serial dilutions in a total volume of 0.6 mL, from undiluted to 1:27. Vero cells in 24-well plates were infected with 100 μL of each dilution in triplicate. After 1 hour of adsorption at 37°C, the inoculum was removed, the plates were washed once with PBS, and approximately 1 mL of methylcellulose was added to each well. After a 7-day incubation, the methylcellulose was removed, and the 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 blotto for 30 minutes. After blocking, the cells were incubated with 200 μL / well of 1:1000 diluted human anti-RSV-F antibody (palivizumab, 1 mg / mL) at room temperature for 1 hour. After the plate was washed, 200 μL / well of 1:1000 diluted goat anti-human IgG HRP conjugated antibody was added and incubated at room temperature for 1 hour. After washing, 200 μL / well of AEC substrate was added at room temperature for 30 minutes. The viral titer was determined by counting plaques at the dilution, where the plaque count range was between 10-100. The results were reported as PFU / g of lung. The assay was performed at the University of Georgia in Athens, Georgia.

[0259] result

[0260] Anti-F antibody titers: Anti-F antibody titers were obtained from animals in groups A, D, G, J, M, and P of CHD03 and animals in groups A, E, G, K, M, and P of CHD04 on day 27 after vaccination, and the results were as follows: Figures 9A-9B As shown. Overall, anti-F antibody titers were significantly higher in all vaccination groups compared to the PBS control group. Anti-F antibody titers were very similar in groups J (W3AΔSH-RSV-F) and M (CPI-RSV-F) in CHD03 and groups K (W3AΔSH-RSV-F) and M (CPI-RSV-F) in CHD04. In CHD03, the geometric mean titer of W3AΔSH-RSV-F was 3.1Log 10 / mL, while the geometric mean titer of CPI-RSV-F was 3.0Log 10 / mL. In CHD04, the geometric mean titer of W3AΔSH-RSV-F was 3.4Log 10 / mL, while the geometric mean titer of CPI-RSV-F was 3.2Log 10 / mL. W3A-RSV-F in both CHD03 and CHD04 had the highest anti-F antibody titer, with a geometric mean titer of 3.7Log in both experiments. 10 In both CHD03 and CHD04, the anti-F antibody titers in the W3AΔSH-RSV-F and CPI-RSV-F groups were similar to those in the RSV_rA2-positive control group. CPIΔSH-RSV-F also elicited comparable serum antibody levels in both studies.

[0261] These results indicate that W3AΔSH-RSV-F and CPI-RSV-F, as well as the other two vaccine candidates, are effective in the treatment of patients with 1×10 5 PFU doses elicited similar levels of anti-F antibodies in BALB / c mice.

[0262] F-specific cellular immune response: The cellular immune response induced by the vaccine candidate was measured by the level of IFN-γ secreting cells. In CHD03, the levels of IFN-γ secreting cells were higher in group J (W3AΔSH-RSV-F) and group M (CPI-RSV-F), with group J having 10 6 The geometric mean of IFN-γ secreting cells was 28 per 10 splenocytes, while the M group had a geometric mean of 10 per 10 6 The geometric mean of 23 IFN-γ secreting cells per 10 splenocytes was 1.04. These values were not statistically significant compared with the PBS group, but they were also not statistically significant between each other. Group G (W3A-RSV-F) had the highest value compared with the PBS group, with 10 6 The geometric mean of 57 IFN-γ-secreting cells per splenocyte was not significantly different from that of the other groups. The values for the W3AΔSH-RSV-F and CPI-RSV-F groups were similar to the levels of IFN-γ-secreting cells observed in the RSV_rA2 group.

[0263] In CHD04, the levels of IFN-γ secreting cells in groups K (W3AΔSH-RSV-F) and M (CPI-RSV-F) were the highest, with group K having 10 6 The geometric mean of IFN-γ secreting cells was 35 per 10 splenocytes, while the M group had a geometric mean of 35 per 10 splenocytes. 6The geometric mean of 48 splenocytes was 48 cells. These values were statistically significant compared with the PBS control group, but they were not statistically significant between each other. Of the two vaccine groups of interest, only the CPI-RSV-F group had significantly higher numbers of IFN-γ-secreting cells than the RSV_rA2-positive control group. W3A-RSV-F and CPIΔSH-RSV-F also elicited F-specific cellular responses.

[0264] Figures 10A-10B It was shown that the interesting W3AΔSH-RSV-F and CPI-RSV-F vaccine candidates, as well as two other vaccine candidates (W3A-RSV-F and CPIΔSH-RSV-F), elicited similar levels of cell-mediated immune responses in BALB / c mice.

[0265] RSV challenge virus lung titers: Evaluation of the protection of four RSV vaccine candidates against RSV_rA2 virus challenge infection in the lower respiratory tract of mice. Overall, Figure 11A ) and CHD04( Figure 11B ), the lung virus titers of all vaccine groups were significantly lower than those of the PBS control, with geometric mean titers of 3.21Log 10 PFU / g and 3.33Log 10 PFU / g. In CHD03, the RSV challenge virus titer of groups K and L (W3AΔSH-RSV-F) was 1.35Log 10 PFU / g, while the geometric mean titer of group N and group O (CPI-RSV-F) was 1.48Log 10 PFU / g. These values were not statistically significant with each other. The geometric mean titers of the other vaccine candidates tested (W3A-RSV-F and CPIΔSH-RSV-F) were 1.35 Log 10 PFU / g and 1.40Log 10 PFU / g. These values were also significantly lower than those of the PBS control. In CHD04, the geometric mean titer of groups J and L (W3AΔSH-RSV-F) was 1.37 Log 10 PFU / g, while the geometric mean titer of group N and group O (CPI-RSV-F) was 1.40Log 10 PFU / g. Similar to CHD03, these values were not statistically significant with each other, but were significantly lower than the PBS control group. The geometric mean titers of the W3A-RSV-F and CPIΔSH-RSV-F vaccine candidates were 1.35 Log 10 PFU / g and 1.40Log 10PFU / g. These values are identical with experiment CHD03, and are also significantly lower than PBS control group. The lung RSV virus titer of all vaccine groups is similar to that observed in the RSV_rA2 positive control group among CHD03 and CHD04.

[0266] Figures 11A-11B It was shown that the W3AΔSH-RSV-F and CPI-RSV-F vaccine candidates, as well as the other two vaccine candidates, protected the lower respiratory tract of BALB / c mice from RSV challenge virus infection.

[0267] Example 4: Virus replication in the upper and lower respiratory tracts of cotton rats.

[0268] Methods and Materials

[0269] Animals: Fifty-five (55) inbred, 6-8 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 the guidelines of the National Institutes of Health and an animal research protocol approved by the Sigmovir Institutional Animal Care and Use Committee (IACUC protocol #15). Each group of animals consisted of 3 females (first three animals per group) and 2 males (last two animals per group). Cotton rats were housed in clear polycarbonate cages and provided ad lib with standard rodent chow (Harlan #7004) and tap water.

[0270] 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. A batch of virus designated hRSV A / A2 Lot# 092215 SSM was used in this in vivo experiment, containing approximately 3.0 x 10 8 pfu / mL. This virus stock was stored at -80°C and has been characterized in vivo in the cotton rat model, where its replication in the upper and lower respiratory tracts has been demonstrated.

[0271] Procedure: Fifty-five young adult male and female cotton rats (6-8 weeks old) were divided into nine groups of five animals (three females, two males), one group of four animals (two females, two males), and two groups of three animals (two females and one male). All animals were pre-bleeded for serum collection and ear-tagged. Animals were immunized or infected with 0.1 ml of the formulations listed in Table 13 below.

[0272] Table 13: Immunization or infection of animals.

[0273]

[0274] Animals in groups K and L were sacrificed. Nasal tissue was harvested and homogenized in 3 ml of HBSS supplemented with 10% SPG for viral titration. Lungs were harvested en bloc and cut into three sections for viral titration (left section, homogenized in 3 ml of HBSS supplemented with 10% SPG), histopathology (right section, aerated with 10% neutral buffered formalin), and qPCR (lingular lobe, snap-frozen in liquid nitrogen).

[0275] All animals were bled to collect serum. Animals in groups A, B, and C were boosted with 0.1 ml of the formulations as shown in the table above.

[0276] All animals were bled to collect serum. Group A was challenged intranasally (IN) with 0.1 ml of PBS (pH 7.4). Groups B to J were challenged with 0.1 ml of RSV / A2 (Lot# 092215SSM), with 10 5 The challenge virus was back titrated to confirm the challenge dose.

[0277] All animals were sacrificed. Nasal tissue was harvested and homogenized in 3 ml of HBSS supplemented with 10% SPG for viral titration. Lungs were harvested en bloc and cut into three sections for viral titration (left section, homogenized in 3 ml of HBSS supplemented with 10% SPG), histopathology (right section, aerated with 10% neutral buffered formalin), and qPCR (lingular lobe, quickly frozen in liquid nitrogen). Animal sacrifice groups are shown in Table 14, and sample collection is shown in Table 15.

[0278] Table 14: Animal sacrifice groups.

[0279] Group Animals A 124831-124834 B 124835-124839 C 124840-124844 D 124845-124849 E 124850-124854 F 124855-124859 G 124860-124864 H 124865-124869 I 124870-124874 J 124875-124879 K 124880-124882 L 124883-124885

[0280] Table 15: Sample collection.

[0281] Sample collection Collection date Number of collections Lung and nose homogenates 4(GrK-L) 3* Lung and nose homogenates 54(GrA-J) 2 Lung histology 4 (GrK-L); 54 (GrA-J) 1 serum 0, 28, and 49 2 Lung total RNA 4 (GrK-L); 54 (GrA-J) 1

[0282] Table 16 shows an overview of the endpoint assay protocol.

[0283] Table 16: Endpoint Assay Protocol

[0284] End point determination sample Storage RSV virus titration (GrA-J) Lung homogenate -80℃ Nose homogenate PIV virus titration (GrK-L) Lung homogenate -80℃ Nose homogenate Lung histopathology (GrA-K) H&E lung sections RT RSV neutralizing Ab (GrA-J) serum -20℃ (RSV / A2) (d0, 28, and 49) RSV / A2 serum IgG ELISA (GrA-J) serum -20℃ (d0, 28, 49) (4-dilution format) qPCR (GrA-J) Lung RNA -80℃ RSV / A2 NS-1 gene; IL-4, IL-2, IFN-γ qPCR PIV(GrK,L) Lung RNA -80℃

[0285] Lung and nasal RSV virus titers: Lung and nasal homogenates were clarified by centrifugation and diluted in EMEM. Confluent HEp-2 monolayers were replicated with the diluted homogenates in 24-well plates. After incubation for one hour in a 37°C, 5% CO2 incubator, the wells were overlaid with 0.75% methylcellulose medium. After four days of incubation, the overlay was removed, and the cells were fixed with 0.1% crystal violet dye for one hour, then rinsed and air-dried. Plaques were counted, and viral titers were expressed as plaque-forming units per gram of tissue. Viral titers were calculated as the geometric mean ± standard deviation of all animals in a group at a given time.

[0286] Lung histopathology: Lungs were dissected and inflated to their normal volume with 10% neutral buffered formalin and then immersed in the same fixative solution. After fixation, the lungs were embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E). Four parameters of lung inflammation were assessed: peribronchiolitis (inflammatory cell infiltration around the bronchioles), perivasculitis (inflammatory cell infiltration around the small blood vessels), interstitial pneumonia (inflammatory cell infiltration and thickening of the alveolar walls) and alveolitis (cells within the alveolar spaces). Slides were blindly scored on a 0-4 severity scale. Scores were subsequently converted to a 0-100% histopathology scale.

[0287] RSV neutralizing antibody assay for preclinical studies (60% reduction): Heat-inactivated serum samples were diluted 1:10 with EMEM and further serially diluted 1:4. The diluted serum samples were incubated with RSV (25-50 PFU) at room temperature for 1 hour and replicated onto confluent HEp - 2 monolayers. After incubation for one hour 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 covering was removed and the cells were fixed with 0.1% crystal violet dye for one hour, then rinsed and air-dried. A statistical program was used to determine the corresponding reciprocal neutralizing antibody titer at the 60% reduction endpoint of the virus control. The geometric mean ± standard deviation of all animals in a group at a given time was calculated.

[0288] RSV IgG ELISA for preclinical studies: Whole RSV inactivated with UV or purified F protein extracted from RSV-infected HEp-2 cells was diluted and coated on a 96-well ELISA plate overnight. The coated antigen was decanted and the plates were incubated in blocking solution for one hour at room temperature and then washed. Diluted serum (1:500, in duplicate) and positive and negative controls were added to the wells and incubated at room temperature for one hour. After washing the plates, rabbit anti-cotton rat IgG (1:500) was added to all wells and incubated at room temperature for one hour. This was followed by incubation with goat anti-rabbit IgG-HRP (1:6,000) at room temperature for one hour. Finally, TMB substrate was added to all wells and incubated at room temperature for 15 minutes. TMB-Stop solution was added to all wells and the optical density at 450 nm was recorded. The optical density (OD 450 ) of the geometric mean + standard error of all samples in the group at each time point.

[0289] Real-time PCR for preclinical studies: Total RNA was extracted from homogenized tissues or cells using the RNeasy purification kit (QIAGEN). One μg of total RNA was used to prepare cDNA using Super Script II RT (Invitrogen) and oligo dT primers (1 μl, Invitrogen). For real-time PCR reactions, Bio-Rad iQ TM The final volume of SYBR Green Supermix was 25 μl, and the final primer concentration was 0.5 μM. Reactions were set up in duplicate in 96-well plates. Amplification was performed on a Bio-Rad iCycler with 1 cycle of 95°C for 3 minutes, followed by 40 cycles of 95°C for 10 seconds, 60°C for 10 seconds, and 72°C for 15 seconds. Baseline cycles and cycle threshold (Ct) values were calculated by iQ5 software in PCR baseline subtraction curve fitting mode. Relative quantification of DNA was performed on all samples. Standard curves were developed using serially diluted cDNA samples that were most enriched in the transcript of interest (e.g., lungs 6 hours after RSV infection from FI-RSV-vaccinated animals or PIV5-specific controls). Ct values were plotted against the log10 cDNA dilution factor. These curves were used to convert the Ct values obtained for the different samples into relative expression units. These relative expression units were then normalized to the level of β-actin mRNA (a “housekeeping gene”) expressed in the corresponding samples. For animal studies, mRNA levels are expressed as the geometric mean ± SEM of all animals in a group at a given time.

[0290] result

[0291] Confirmation of vaccine virus replication in cotton rat model: To confirm the replication of PIV5-based vaccines in the respiratory tract of cotton rats, groups of three animals were treated with 10 6 PFU of CPI-RSV-F or W3AΔSH-RSV-F were inoculated intranasally and sacrificed four days later. The samples were submitted to Blue Lake Biotech (BLB) for evaluation. BLB performed a plaque assay on the submitted samples. In short, it was found that both candidate vaccine viruses could replicate in the upper and lower respiratory tracts of cotton rats (Table 17). In animals vaccinated with CPI-RSV-F vaccine, the vaccine virus replicated similarly in the upper respiratory tract (nose) and lower respiratory tract (lungs), with 3.9 and 3.5 Log, respectively. 10 PFU / mL. The replication titer of W3AΔSH-RSV-F in the upper respiratory tract (5.0Log 10 PFU / mL) was higher than that in the lower respiratory tract (2.2Log 10 PFU / mL). The W3AΔSH-RSV-F vaccine virus replicated at a higher level in the upper respiratory tract than CPI-RSV-F, but at a lower level in the lower respiratory tract than CPI-RSV-F. Overall, the replication of both vaccine viruses in the cotton rat model was successfully confirmed (Table 17).

[0292] Table 17: Evaluation of CPI-RSV-F and W3AΔSH-RSV-F replication in the respiratory tract of cotton rats.

[0293]

[0294] The animals were treated with 10 6 Rats were inoculated intranasally with PFU of CPI-RSV-F or W3AΔSH-RSV-F and sacrificed four days later. Lung and nose samples were collected and transported to BLB for plaque assay.

[0295] To evaluate the efficacy and safety of the vaccine in the cotton rat model of RSV A / A2 infection.

[0296] Lung virus titers: RSV A / A2 loads in cotton rat lungs were assessed 5 days after intranasal RSV challenge ( Figure 12 The virus titer in the lungs of RSV-infected animals (Group B) mock-immunized with PBS was 5.3 Log 10 PFU / g and used for comparison with all other RSV infection groups (Groups C and J). No virus was detected in the lungs of animals infected twice with RSV (Group D). FI-RSV vaccination (Group C) modestly but significantly reduced lung RSV titers to 3.4 Log 10 PFU / g. The dosage is 10 4 , 10 5 or 10 6PFU of CPI-RSV-F (groups E, F and G, respectively) or a dose of 10 4 , 10 5 or 10 6 Immunization with PFU of W3AΔSH-RSV-F (groups H, I, and J, respectively) reduced RSV load to undetectable levels in all vaccinated animals.

[0297] Nasal virus titers: RSV A / A2 loads in cotton rat noses were assessed 5 days after intranasal RSV challenge ( Figure 13 The virus titer in the nose of RSV-infected animals mock-immunized with PBS (Group B) was 6.19 Log10 PFU / g and was used for comparison with all other RSV-infected groups (Groups CJ). No virus was detected in the nose of animals twice infected with RSV (Group D). FI-RSV vaccination (Group C) had no effect on the RSV load in the nose (6.02 Log10 PFU / g). 4 , 10 5 or 10 6 Immunization with CPI-RSV-F at a PFU dose resulted in a dose-dependent reduction in viral load in the nose: 3.65, 3.00, and 2.44 PFU / g in groups E, F, and G, respectively. Immunization with all three doses of W3AΔSH-RSV-F tested (groups HJ) reduced RSV replication in the nose to almost undetectable levels, with some animals in each group having only single-digit plaques (1, 3, and 1 animal in groups H, I, and J, respectively, had 1 to 5 plaques).

[0298] Serum RSV A / A2 neutralizing antibodies: Serum neutralizing antibodies against RSV A / A2 were measured in all animals before the start of the experiment (day 0), 4 weeks after the first immunization (day 28), and 3 weeks after the first immunization (day 49). Figure 14 Animals infected with RSV A / A2 (Group D) showed high serum RSV A / A2 neutralizing antibody titers four weeks after infection (data on day 28, 11.14 Log2) and remained elevated seven weeks after infection (data on day 49, 10.07 Log2). 4 , 10 5 or 10 6 Immunization with CPI-RSV-F at a PFU dose (Groups E, F, and G, respectively) resulted in NA titers of 5.92, 5.87, and 7.55 on day 28.

[0299] Animals immunized with the highest dose of the test vaccine (Group G, 10 6PFU) maintained a high NA titer (6.65Log2) on day 49, while the NA levels in groups E and F decreased to 4.6 and 4.62Log2, respectively, on day 49. 4 , 10 5 or 10 6 Immunization with 100 PFU of W3AΔSH-RSV-F (HJ groups) resulted in NA titers of 9.24, 10.11, and 9.91 Log2, respectively, on day 28. NA titers in these groups remained high on day 49 (8.79, 8.97, and 8.64 Log2, respectively). No other animal groups showed detectable neutralizing antibodies against RSV A / A2.

[0300] 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 after the first immunization (day 28), and 3 weeks after the first immunization (day 49). Figure 15 ). In animals vaccinated with FI-RSV (Group C), a slight increase in binding IgG was seen. Animals infected with RSV A / A2 (Group D) and all animals immunized with CPI-RSV-F (Group EG) or W3AΔSH-RSV-F (Group HJ) had high levels of binding IgG on days 28 and 49 after vaccination. No dose dependence of IgG levels on the dose of CPI-RSV-F (Group EG) or W3AΔSH-RSV-F (Group HJ) was observed. On days 28 and 49, IgG levels in animals vaccinated with W3AΔSH-RSV-F were slightly higher than in animals immunized with CPI-RSV-F (Group EG).

[0301] Lung histopathology: Lung histopathology was assessed 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 intermediate levels of pathology. The highest levels of lung histopathology were detected in animals immunized with FI-RSV (Group C), with significant increases in interstitial inflammation and alveolitis. The pathology of animals immunized with CPI-RSV-F at all doses (Group EG) did not exceed that observed in animals infected twice with RSV (Group D). 5 One animal (#124872) in the animals vaccinated with the PFU dose of W3AΔSH-RSV-F (Group I) developed exacerbated interstitial inflammation and alveolitis. The other four animals in this group, as well as all animals vaccinated with the other two doses of the vaccine (Groups H and J), did not show interstitial inflammation and alveolitis.

[0302] qPCR results: The expression of RSV NS1, IL-4, IL-2, and IFN-γ mRNA was assessed in lung samples collected on day 5 after RSV A / A2 challenge and normalized by the β-actin mRNA level in each sample ( Figures 16A-16D Both vaccines significantly reduced NS-1 mRNA expression, compared to the two lower doses (10 4 and 10 5 Compared with CPI-RSV-F administered with 10 PFU (100 μg / mL), W3AΔSH-RSV-F was slightly more effective in reducing lung NS1 mRNA. FI-RSV vaccination (Group C) resulted in a moderate decrease in lung RSV NS1 mRNA levels, but a significant increase in IL-4 mRNA levels. The IL-4, IL-2, and IFN-γ mRNA levels of animals vaccinated with CPI-RSV-F (Group EG) or W3AΔSH-RSV-F (Group HJ) did not exceed the levels observed in primary RSV-infected animals (Group B) in general. Compared with animals infected with secondary RSV (Group D), several animals in the group vaccinated with medium doses of CPI-RSV-F (Group F, 100 μg / mL) did not show any significant difference in IL-4, IL-2, and IFN-γ mRNA levels. 5 The IL-2 and IFN-γ mRNA levels were slightly increased in one animal in the group immunized with the medium dose of W3AΔSH-RSV-F (Group I, 10 5 PFU dose, animal ##124872) showed elevated IL-4 mRNA. Coincidentally, the same animal demonstrated elevated alveolitis and interstitial inflammation on histopathological evaluation.

[0303] Example 5: Phase I Clinical Trial of PIV5-RSV Vaccine - RSV Antibody and CMI Response Rate

[0304] Materials and methods

[0305] Participants and Study Conduct: A total of 30 subjects were enrolled in this phase 1 trial, and all subjects received 10 7.5 A single intranasal dose of PFU of PIV5-RSV vaccine. TMThe vaccine was administered in the form of a 0.25 mL spray into each nostril (total volume 0.5 mL) using an intranasal mucosal atomization device (Teleflex MAD300). The average ages of Group 1 (planned participants aged 18-59 years and actual participants aged 33-59 years) and Group 2 (planned participants aged 60-75 years and actual participants aged 61-75 years) were 45 and 67 years, respectively (Table 18). Most participants were female (70%). Most participants in both groups were white (73% and 80% in Groups 1 and 2, respectively). All but one participant completed the study; and this participant in Group 1 was lost to follow-up after Day 7.

[0306] Table 18: Trial Cohort Demographics

[0307]

[0308] *One participant withdrew from the study on Day 8 (lost to follow-up).

[0309] ____________________________________________

[0310] 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 was conducted at two study sites in the US. Participants were recruited into two study cohorts: healthy young adults (Group 1, 33-59 years) and healthy older adults (Group 2, 61-75 years). Participants of childbearing potential were required to use contraception to prevent pregnancy. Exclusion criteria included any live vaccine within 30 days before the trial vaccine, any previous vaccination with any investigational RSV vaccine or any PIV5-based vaccine (CVXGA1) being enrolled during the trial, and known infection with human immunodeficiency virus, hepatitis B virus, or hepatitis C virus. A complete list of inclusion and exclusion criteria is available on the clinical trial website. Participants were not prescreened for RSV serum antibody levels. Eligible participants received a single dose of 10 7.5 Plaque forming units (PFU) of PIV5-RSV vaccine using MAD Nasal TM The intranasal mucosal atomization device (Teleflex MAD300) was administered in the form of a 0.25 mL spray to each nostril (total volume 0.5 mL) and observed for 30 minutes immediately after administration. In addition, the subjects were asked to maintain a memory aid for the solicited systemic AEs and local reactions within one week after vaccination. Four sentinel participants in each group were first dosed, and their safety data were reviewed by the Safety Monitoring Committee (SMC) before the remaining participants in the group were enrolled.

[0311] 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), including x episodes of chronic medical conditions and AEs requiring medical attention (Days 1-181).

[0312] Immunogenicity Assessment: Blood and nasal samples were collected at baseline (day 1 before vaccination), day 15, and day 29 after vaccination. To reduce sample variability, samples from the same participant were run on the same plate in a blinded manner in 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, serial 2-fold diluted serum samples (in quadruplicate) with a starting dilution of 1:100 were incubated with 175 ± 75 PFU RSV-rLuc for 1 hour and Vero cells were infected in 96-well, white-walled, clear-bottom plates. After incubation for 20 to 24 hours, cells were lysed using the Renilla-Glo luciferase assay (Promega) and the luciferase signal was read on a SpectraMax iD3 multimode microplate reader (Molecular Devices). RSV nAb titers were defined as the reciprocal dilution that inhibited at least 50% of the signal of the viral control, as determined by 5PL curve fitting using Prism (macOS version 9.5.1, GraphPad software). RSV nAb titers were converted to international units based on standard serum (16 / 284) obtained from NIBSC (London, UK).

[0313] RSV F-specific serum IgG and IgA antibodies and nasal IgA antibody levels were determined by ELISA using 25 ng / well purified RSV F protein (SinoBiological, catalog number 11049-V08B) and 2-fold serial dilutions (in replicates) in blocking buffer (5% milk / 0.5% BSA in 1x KPL wash buffer (Seracare)). 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 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 that two replicates were performed instead of four. The PIV5 MN analysis was identical to the RSV-rLuc-based MN assay, and the nAb titer was defined as the reciprocal dilution that inhibited at least 50% of the signal of the virus control.

[0314] Antigen-specific T cell frequencies were assessed by intracellular cytokine staining assay using cryopreserved peripheral blood mononuclear cells (PBMCs) isolated from whole blood on day 1 (pre-vaccination), day 15, and day 29. One million cryopreserved PBMCs were thawed in complete 10% FBS RPMI medium and incubated overnight at 37°C and incubated with GenScript's RSV F peptide pool 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) at a final concentration of 1 μg / mL. In addition, 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) to serve as negative and positive controls, respectively. After incubation at 37°C for 2 hours, 10 μg / mL brefeldin A (BD Biosciences) was added and the cells were incubated for another 4 hours. The cells were washed with PBS and incubated for 15 min at room temperature with Aqua-Viability dye (Invitrogen). The 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 1× 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 min at 4° C. Cells were washed with 1× Perm / Wash and PBS containing 2% FBS and then resuspended in PBS / 2% formaldehyde for acquisition on a BD FACSAria Fusion cell sorter.CD3+ cells were gated as CD4. + and CD8 + T cells were divided into memory cells and naive cells using CD28 and CD95. The net percentage of cytokine secreting cells was determined by subtracting the value obtained with DMSO-stimulated samples (negative control). If cytokine-positive CD4 + or CD8 + T cell frequency was considered positive if it was >0.1%. Data were analyzed using FlowJo software (version 10). Boolean combinations and SPICE software were used to determine the CD4+ T cells that produced two or more cytokines. + and CD8 + Polyfunctional responses of T cells.

[0315] Statistical analysis: Statistical analyses were performed using GraphPad Prism software (version 9). Due to the small sample size, the statistical analyses were primarily descriptive and summary. P values were used to show potentially significant differences at the 0.05 significance level. Two-group comparisons of RSV-specific CMI responses and antibody responses were assessed by Wilcoxon matched-pairs signed rank test (day 15 or 29 versus day 1, or day 15 versus day 29). A 1.5-fold increase in antibody titers relative to baseline after vaccination was considered significant, as assay characteristics showed a 1.2-1.3-fold (95% confidence level) change to be a significant change when samples from a single participant were tested on the same plate.

[0316] result

[0317] Serum antibody titers: All participants were seropositive for RSV neutralizing antibodies (nAbs) at baseline, and titers changed as Figures 17A-17F and Figures 18A-18BAs shown. In Group 1, the nAb geometric mean titer (GMT) was 880 (9.8 log2) at baseline and increased to 1316 (10.4 log2) 2 weeks after vaccination and to 1312 (10.4 log2) 4 weeks after vaccination (P < .05), reflecting a geometric mean fold increase (GMFR) of 1.5. RSV-nAb seroresponses (≥1.5-fold increase) were identified in 7 / 14 (50%) participants. In Group 2, the nAb GMT at baseline was 850 (9.7 log2), similar to Group 1, and increased to 1103 (10.1 log2) 2 weeks after vaccination (P < .05) and to 1372 (10.4 log2) 4 weeks after vaccination (P < .05), reflecting geometric mean fold increases (GMFRs) of 1.3 and 1.5, respectively. In this group, 6 / 15 (40%) participants were identified as RSV-nAb seroreactive (Table 19) (individual nAb titers varied as shown in Table 19). Figures 18A-18D Notably, RSV-nAb serological responses were identified in all five participants who tested positive for vaccine virus shedding, suggesting a potential correlation between replication and systemic antibody responses.

[0318] Table 19: Summary of RSV Antibody and CMI Response Rates

[0319]

[0320]

[0321] A positive serological response was defined as a ≥1.5-fold increase in RSV F IgG and IgG compared to baseline. A positive nasal IgA antibody response was defined as a ≥2-fold increase compared to baseline. A CMI response was defined as an increase of >0.1% in the total number of single T cells secreting >1 cytokine after subtracting baseline (pre-vaccination). CTL responses were defined as CD8 T cells producing INF-γ. + Abbreviations: CMI, cell-mediated immunity; CTL, cytotoxic T lymphocyte; PBMC, peripheral blood mononuclear cell; PIV5, parainfluenza virus type 5; RSV, respiratory syncytial virus.

[0322] All participants were seropositive for RSV-F-specific serum IgA and IgG Ab at baseline ( Figures 17A-17F). In Group 1, F-specific serum IgA GMTs increased from 530 (9.0 log2) at baseline to 821 (9.7 log2) and 756 (9.6 log2) at weeks 2 and 4 after vaccination, respectively. F-specific serum IgG GMTs increased from 1640 (10.7 log2) at baseline to 2049 (11.0 log2) at weeks 2 and 2055 (11.0 log2) at weeks 4 after vaccination. F-specific serum IgA and IgG seroreactivity (≥1.5-fold) was identified in 6 / 14 (43%) participants and 3 / 14 (21%) participants, respectively (Table 19). In Group 2, F-specific serum IgA GMTs increased from 445 (8.8 log2) at baseline to 582 (9.2 log2) and 641 (9.3 log2) at weeks 2 and 4 after vaccination, respectively. F-specific serum IgG GMTs increased from 1088 (10.1 log2) at baseline to 1198 (10.2 log2) and 1325 (10.4 log2) at weeks 2 and 4 post-vaccination. F-specific IgA and IgG serum responses were identified in 5 / 15 (33%) and 1 / 15 (7%) participants, respectively (Table 19).

[0323] Overall, the results suggest that PIV5-RSV vaccination increases RSV-specific serum Ab levels in both young (33-59 years) and elderly (61-75 years), but the magnitude of the increase is greater in adults than in the elderly. Interestingly, the fold increase in RSV-specific Ab titers was inversely correlated with baseline titers. Higher baseline titers may be closer to peak levels.

[0324] We next examined vector-specific immune responses. For PIV5nAb at baseline, 15 / 29 (52%) participants were seropositive, as defined by a titer of 1:10 ( Figures 19A-19B). This background seropositivity level may reflect passive exposure to PIV5 through exposure to dogs that received kennel cough vaccine. In Group 1, PIV5-nAb GMTs increased from 26 (4.7 log2) at baseline to 48 (5.6 log2) and 51 (5.7 log2) at weeks 2 and 4 after vaccination, respectively, reflecting a geometric mean fold increase of 1.8 and 2.0, respectively. PIV5-nAb seroreactivity (increase ≥1.5-fold) was identified in 7 / 14 (50%) participants (Table 19). In Group 2, PIV5-nAb GMTs increased from 19 (4.2 log2) at baseline to 34 (5.1 log2) and 44 (5.5 log2) at weeks 2 and 4 after vaccination, respectively, reflecting a GMFR of 1.8 and 2.2, respectively. PIV5-nAb seroreactivity (increase ≥1.5-fold) was identified in 10 / 15 (67%) participants. Similar kinetics were observed using PIV5-specific IgG titers ( Figures 19C-19D ), but had higher GMFR (≤4.6-fold in Group 1 and ≤3.4-fold in Group 2) and seroprevalence (86% in Group 1 and 87% in Group 2). Comparing participants with low PIV5 nAb titers with those with high PIV5 nAb titers, baseline PIV5 nAb titers had no significant effect on PIV5-nAb seroprevalence and did not appear to suppress RSV-nAb seroprevalence ( Figures 20A-20C ).

[0325] RSV F-specific IgA antibody titers in nasal swabs: At baseline, RSV F-specific IgA antibody (Ab) titers in nasal swabs varied, ranging 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 weeks 2 and 4 after 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, baseline F-specific nasal IgA GMT was 18 (4.2 log2), similar to Group 1, but GMT did not increase at weeks 2 and 4 after vaccination (P>.05) ( Figure 21A and 21B However, F-specific nasal IgA responses were identified in 5 / 15 (33%) participants in Group 2 (individual nasal IgA antibody changes as Figures 18C-18D In groups 1 and 2, F-specific nasal IgA responses were identified in 4 of 5 participants who were positive for vaccine virus shedding.

[0326] Overall, the results suggest that PIV5-RSV vaccination increases RSV-specific nasal IgA levels in adults and the elderly, but to a lesser extent in the elderly. Similar to the findings observed for systemic antibody responses, the fold increase in F-specific nasal IgA titers in both groups was inversely correlated with baseline titers ( Figures 20A-20C ).

[0327] Baseline cell-mediated immunity, RSV F-specific CD4 T cells, and Th1 or cytotoxic T cell cytokines / markers IFN-γ, TNF-α, MIP-1β, and CD107a, as well as Th2 cytokine IL-13, were detected. + and CD8 + T cells ( Figures 22A-22D In group 1, F-specific CD4 + The mean percentage of T cells increased from 0.06% at baseline to a maximum of 0.42% 2 weeks after vaccination ( P < .001; Figures 22E-22F ), and CD8 + The mean percentage of T cells increased from 0.08% at baseline to a maximum of 0.38% 4 weeks after vaccination (P < .001). + The mean percentage of T cells increased from 0.02 to a maximum of 0.26% 2 weeks after vaccination (P < .001), and CD8 + The mean percentage of T cells increased from 0.04 to a maximum of 0.40% 4 weeks after vaccination (P < .001). In Group 1 and Group 2, F-specific cell-mediated immune (CMI) responses to PIV5-RSV vaccine (defined as an increase of >0.1% from baseline in T cells expressing at least 2 Th1 / cytotoxic biomarkers) were identified in 13 / 14 (93%) participants and 15 / 15 (100%) participants, respectively (Table 19). And F-specific cytotoxic T lymphocyte (CTL) responses to PIV5-RSV vaccine (defined as CD8 expressing IFN-γ) were identified in 12 / 14 (86%) participants and 13 / 15 (87%) participants, respectively. + This suggests that the PIV5-RSV vaccine enhanced F-specific Th1 (CD4 + ) and cytotoxicity (CD8 + )T cell production. Although CD4 + and CD8 + T cell numbers were similar, but by day 15, CD4 +The geometric mean of T cells increased to 6.1-fold and 9.1-fold, respectively, and by day 29, the CD8 + The geometric mean of T cells increased to 3.6 times and 10 times ( Figures 22G-22H ), which reflects that the baseline in Group 2 was lower than that in Group 1. This result indicates that PIV5-RSV vaccine induces similar levels of CMI in adults and the elderly compared with nasal IgA responses.

[0328] In both groups 1 and 2, T cells expressing a single Th1 / cytotoxicity biomarker were more frequent than T cells expressing at least two Th1 / cytotoxicity biomarkers ( Figures 22I-22J However, the frequency of T cells co-expressing at least two Th1 / cytotoxicity biomarkers was higher in group 1 compared with 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.

[0329] Compared with Th1 and cytotoxic phenotypes, F-specific CD4 expressing IL-13 after vaccination in group 1 or 2 + T cells and CD8 + The percentage of T cells did not increase ( Figures 22A-22D ), indicating that there was no Th2-biased response to the PIV5-RSV vaccine, which has been identified as a potential risk factor for vaccine-associated enhancement of RSV disease (FP Polack, et al., J Exp Med 196, 859-865 (2002); B. Bagga, et al., J Infect Dis 212, 1719-1725 (2015)).

[0330] The complete disclosures of all patents, patent applications, and publications cited herein, as well as electronically available materials (including, for example, nucleotide sequences submitted in GenBank and RefSeq, and amino acid sequences submitted in, for example, SwissProt, PIR, PRF, PDB, and translations of annotated coding regions in GenBank and RefSeq) are incorporated by reference. If there is any inconsistency between the disclosure of this application and the disclosure of any document incorporated herein by reference, the disclosure of this application shall prevail. The foregoing detailed description and examples are given for clarity of understanding only. No unnecessary limitations should be understood therefrom. The present invention is not limited to the exact details shown and described, and variations obvious to one skilled in the art are intended to be included in the invention defined by the claims.

[0331] References

[0332] 1.Phan SI.Zengel IR,Wei H,et al.Parainfluenza virus 5 expressingwild-type of prefusion respiratory syncytial virus(RSV)fusion proteinprotects mice and cotten rats from RSV ehallenge.J of Virology 2017:91(19);e00560-17.

[0333] 2.Wang D,Phan S,DiStephano DJ,et al. A singlc-dose reeombinantparainfluenza virus 5-vectored vaccine expressing respiratory syncytial virus(RSV)F or G protein protected Cotton rats and African green monkeys from RSVchallenge.J of Virology 2017:91(11);e00060-17.

[0334] 3.Phan SI,Chen Z,Xu P.et al.A respiratory syncytial virus(RSV)vaccinebased on parainflucnza virus 5(PIV5).Vaccine 2014:32;3050-3057.

[0335] 4.Young D,Wignall-Fleming EB.Busse DC,et al.The switch between acuteand persistent paramyxovirus infection caused by single amino acidsubstitutions in the RNA polymerase P subunit.PLoS Pathogens 2019 Feb 11;15(2):e1007561.doi:10.1371 / journal.ppat.100756.

[0336] 5.Seth et al. Mutations in the cytoplasmic domain of aparamyxoviruses fusion glycoprotein rescue syncytium formation and eliminatethe HN requirement for membrane fusion J.of Virology 2003,167-178.

[0337] 6.Tong et al.Regulation of Fusion Activity by the Cytoplasmic Domainof a Paramyxovirus F Protein.J.of Virology 2002.301:322-333.

[0338] 7.Schmitt.A.P.,Leser,G.P.,Waning,D.L.&Lamb,R.A.Requirements forBudding of Paramyxovirus Simian Vires 5 Virus-Like Particles.Journal ofVirology 76,3952-3964,dor:10.1128 / jvi.76.8.3952-3964.2002(2002).

[0339] 8.Waning,D.L.,Schmitt,A.P.,Leser,G.P.&Lamb,R.A.Roles for thecytoplasmic tails of the fusion and hcmagglutinin-neuraminidase proteins inbudding of the paramyxovirus simian virus 5.JVirol 76,9284-9297(2002).

[0340] 9.FPPolack,MNTeng,PLCollins,GAPrincc,M.ExnerH,Regele,DDLirman,R.Rabold,SJHoffman.CLKarp,SRKleeberger,M.Wills-Karp,RAKarron.A role for immune complexes in cnhanccd respiratory syncytial virus disease.J Exp Med Rev. 196,859–865(2002).

[0341] 10.B.Bagga,JECehelsky,A,Vaishnaw,T.Wilkinson,R.Meyers,LMHarrison,PLRoddam,EEWalsh,JPDeVincenzo,Effect of Preexisting Serum and MueosalAntibody on Experimental Respiratory Syncytial Virus(RSV)Cdifferences and Infection of Adults.J Infect Dis Rev. 212,1719–1725(2015).

Claims

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 virus (CPI) vector backbone engineered to express RSV F protein as a target antigen.

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

4. 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. A viral expression vector as described in claim 4, wherein the CPI antigenomic cDNA is sequenced using primers having a nucleic acid sequence with 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. The viral expression vector of claim 1, wherein the parainfluenza virus (CPI) vector backbone engineered to express the RSV F protein comprises a 22 amino acid extension as part of its cytoplasmic tail.

7. 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 virus (CPI) vector backbone engineered to express RSV F protein as a target antigen.

8. pharmaceutical composition as claimed in claim 7, wherein the RSV F protein is encoded by the RSV F protein gene of wild type or mutation.

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

10. 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. A pharmaceutical composition as described in claim 10, wherein the CPI antigenomic cDNA is sequenced using primers having a nucleic acid sequence with 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. The pharmaceutical composition of claim 7, wherein the parainfluenza virus (CPI) vector backbone engineered to express the RSV F protein comprises a 22 amino acid extension as part of its cytoplasmic tail.

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

14. A method of inducing an immune response in a subject suffering from RSV comprising administering a prophylactic vaccine against RSV infection, wherein the vaccine comprises the pharmaceutical composition of claim 7.

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

16. The method of claim 15, wherein the RSV-specific cell-mediated response comprises CD4 + T cells and CD8 + Increased expression on T cells.

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

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

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

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

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