Trehalose vaccine formulations

Abstract

Stable liquid, lyophilized, and frozen formulations are provided for live attenuated vaccines and / or vector-based vaccines.

Description

[0001] Cross-reference to related applications This application claims priority to U.S. Provisional Application No. 63 / 600,206, filed November 17, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] CRADA Statement This invention was created in the course of a collaborative research and development agreement with the National Institutes of Health of the U.S. Department of Health and Human Services. The U.S. government holds certain rights to this invention.

[0003] sequence list This application contains a sequence list that has been electronically submitted in XML format and incorporated herein by reference in its entirety. The XML copy created on October 2, 2024 is named "01121-0052-00PCT-ST26.xml" and has a size of 42,875 bytes. Background Technology

[0004] Liquid vaccines are typically unstable during storage, and their instability can reduce their safety and efficacy, as well as increase the cost and complexity of manufacturing and storage. This is especially true for live attenuated vaccines that are sensitive to environmental changes. Vaccines consist of proteins and other macromolecules that can change upon exposure to heat, light, oxygen, or radiation, or may interact with container materials or other components of the vaccine mixture. Therefore, identifying these relationships and optimizing stability from production to administration to patients is a crucial part of vaccine development. It is beneficial to produce vaccines that are stable in liquid formulations, which can be maintained at room temperature or 2°C–8°C until they are used. Benefits include, for example, reducing the number of steps required to thaw or reconstitute frozen or lyophilized vaccines, reducing manufacturing costs by allowing all manufacturing steps to be carried out in the liquid phase without requiring changes to the viral form compared to the desired administration form, and allowing for wider distribution without refrigeration. Furthermore, time and cost savings are achieved in manufacturing and storing liquid vaccines that are stable at room temperature or refrigerator temperatures, allowing vaccines to be manufactured, for example, in drug-loaded syringes or other forms, for immediate administration to subjects. Liquid formulations that allow for stability during the maintenance and freeze-thaw stages of the manufacturing process are particularly desirable. Liquid formulations can also be more easily administered immediately to subjects without reconfiguration, thus reducing human error in the administration process. The development of heat-stable vaccines could greatly alleviate this problem and, consequently, improve vaccine accessibility globally.

[0005] One method for making vaccines heat-stable is to add stabilizing additives to the formulation. Many formulations require complex mixtures of multiple additives, which can increase manufacturing costs and complexity. The development of liquid or lyophilized formulations that are long-term stable under refrigeration or no refrigeration will reduce manufacturing, distribution, and storage costs, thereby contributing to wider vaccine use and improved vaccine efficacy. Summary of the Invention

[0006] This document provides, in particular, the following implementation methods: This article discloses liquid, lyophilized, or frozen formulations containing an effective amount of attenuated live virus and / or vector-based virus; about 30% to about 45% (w / v) trehalose; one or more monovalent salts; and buffers and / or antioxidants containing histidine.

[0007] This article discloses liquid, lyophilized, or frozen formulations containing effective amounts of attenuated live virus and / or vector-based virus, as well as approximately 30% to approximately 40% (w / v) trehalose, approximately 100 mM monosodium glutamate (MSG), approximately 160 mM NaCl, and approximately 10 mM histidine.

[0008] This article discloses liquid, lyophilized, or frozen formulations containing an effective amount of attenuated live respiratory syncytial virus (RSV); approximately 30% to approximately 45% (w / v) trehalose; one or more monovalent salts; and buffers and / or antioxidants containing histidine.

[0009] This article discloses liquid, lyophilized, or frozen formulations containing an effective amount of attenuated active RSV; about 30% to about 45% (w / v) trehalose; about 10 to about 300 mM NaCl; about 0.5 to about 300 mM monosodium glutamate (MSG) or potassium glutamate; and about 10 to about 100 mM histidine; wherein the pH of the formulation is about 6 to about 8.

[0010] In some embodiments, the virus is membrane-bound. In some embodiments, the virus is RSV ΔNS2 / Δ1313 / I1314L. In some embodiments, the effective amount of RSV ΔNS2 / Δ1313 / I1314L is about 5 to about 9 log10 plaque-forming units (PFU) / dose. In some embodiments, the effective amount of RSV ΔNS2 / Δ1313 / I1314L is about 5 to about 6 log10 PFU / dose, or about 6.1 to about 7 log10 PFU / dose, or about 7.1 to about 8 log10 PFU / dose, or about 8.1 to about 9 log10 PFU / dose. In some embodiments, the effective amount of RSV ΔNS2 / Δ1313 / I1314L is about 5.6 log10 PFU / dose or about 6.2 log10 PFU / dose.

[0011] In some embodiments, the codon for serine at position 1313 encoding the L protein in the attenuated live RSV is deleted, resulting in an amino acid deletion (Δ1313) in the L protein. In some embodiments, a substitution of the amino acid residue of leucine for isoleucine at position 1314 in the RSV, referring to SEQ ID NO:3, causes a genetically stable mutation in the L gene (I1314L). In some embodiments, the RSV comprises a large polymerase protein (L), a phosphoprotein (P), a nucleocapsid protein (N), an M2-1 protein nonstructural protein 1 (NS1), a glycoprotein (G), a fusion protein (F), a matrix protein (M), an M2-2 protein, and a small hydrophobic protein (SH); and a genome or antigenome comprising the deletion of a codon encoding a serine at position 1313 or the corresponding position of the L protein; a mutation of an amino acid sequence residue 1314 of the L protein or the corresponding position, wherein the mutation of amino acid sequence residue 1314 of the L protein is an amino acid substitution of leucine for isoleucine, wherein leucine is encoded by a codon described as CTG; a deletion of the NS2 gene; and a nucleotide modification at position 14456 of reference SEQ ID NO: 1, which represents a change from thymine (T) to adenine (A).

[0012] In some embodiments, the formulation comprises a liquid formulation. In some embodiments, the formulation comprises a lyophilized formulation. In some embodiments, the liquid formulation is formulated for administration to a human subject. In some embodiments, administration is intranasal. In some embodiments, the formulation does not contain a polyoxyethylene-polyoxypropylene (PEO-PPO) block copolymer. In some embodiments, the formulation is a liquid formulation wherein the liquid formulation is subsequently not dried and reconstituted.

[0013] In some embodiments, the formulation further comprises monosodium glutamate (MSG) or potassium glutamate (PG). In some embodiments, the formulation comprises about 10 to about 100 mM histidine. In some embodiments, the formulation comprises about 30% trehalose. In some embodiments, the formulation comprises about 40% trehalose. In some embodiments, the formulation comprises about 0.5 to about 300 mM monosodium glutamate (MSG) or potassium glutamate (PG). In some embodiments, a monovalent salt, NaCl, is present. In some embodiments, the formulation comprises about 10 to about 300 mM NaCl.

[0014] In some embodiments, the pH is about 6 to about 8. In some embodiments, the pH is about 7 ± 0.5.

[0015] In some embodiments, the attenuated live virus comprises a paramyxovirus. In some embodiments, the attenuated live virus comprises a respiratory syncytial virus (RSV), wherein the RSV is administered to pediatric subjects. In some embodiments, the formulation comprises a vector-based PIV virus, wherein the PIV is PIV3 if desired. In some embodiments, the PIV comprises one or more heterologous RSV or hMPV antigens. In some embodiments, the RSV antigen is RSV F and / or RSV G.

[0016] In some embodiments, the liquid system is prepared, and the titration loss is less than 1 log10 PFU / mL when stored at about 2 to about 8 degrees Celsius for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months. In some embodiments, the titration loss is less than 1 log10 PFU / mL after storage at about 2 to about 8 degrees Celsius for about 12 to about 24 months. In some embodiments, the liquid system is prepared, and the titration loss is less than 1 log10 PFU / mL when stored at about 37 degrees Celsius for at least 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day. In some embodiments, the titration loss is less than 1 log10 PFU / mL when stored at 37°C for about 1 to about 2 weeks. In some embodiments, the system is prepared as a liquid, and the titration loss is less than 1 log10 PFU / mL when stored at room temperature for at least 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, or 5-6 months. In some embodiments, the titration loss is less than 1 log10 PFU / mL when stored at room temperature for 3-4 months. In some embodiments, the system is lyophilized, and the titration loss is less than 1 log10 PFU / mL when stored at 37°C for about 1 to about 2 weeks. In some embodiments, the system is lyophilized, and the titration loss is less than 1 log10 PFU / mL when stored at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. In some implementations, the infectious titration is substantially unaffected by shear stress, repeated freeze-thaw cycles, or remodeling.

[0017] In some embodiments, the vaccine is formulated for intramuscular or subcutaneous administration. In some embodiments, the formulation further comprises an adjuvant. In some embodiments, the formulation further comprises rHSA, a divalent salt, and / or an amino acid.

[0018] This article discloses a method for increasing the stability of liquid or lyophilized vaccines at room temperature or at a temperature of about 2 to about 8 degrees Celsius, comprising formulating attenuated live enveloped virus and / or vector-based virus in a liquid or lyophilized formulation containing about 30% to about 45% (w / v) trehalose, one or more monovalent salts, and buffers and / or antioxidants containing histidine.

[0019] In some embodiments, the concentration of trehalose is about 30% (w / v). In some embodiments, the concentration of trehalose is about 40% (w / v).

[0020] This article discloses a method for immunizing subjects against viral infection, which involves administering the formulation described herein.

[0021] This article discloses liquid, lyophilized, or frozen formulations containing an effective amount of attenuated active RSV comprising RSV ΔNS2 / Δ1313 / I1314L, about 30% to about 40% (w / v) trehalose, about 160 mM NaCl, about 100 mM monosodium glutamate (MSG), and about 10 mM histidine, wherein the pH of the formulation is about 7.

[0022] In some embodiments, the concentration of trehalose is about 30% (w / v). In some embodiments, the concentration of trehalose is about 40% (w / v).

[0023] In some embodiments, the RSV comprises a large polymerase protein (L), a phosphoprotein (P), a nucleocapsid protein (N), an M2-1 protein nonstructural protein 1 (NS1), a glycoprotein (G), a fusion protein (F), a matrix protein (M), an M2-2 protein, and a small hydrophobic protein (SH); and a genome or antigenome comprising the deletion of a codon encoding a serine at position 1313 or the corresponding position of the L protein; a mutation of an amino acid sequence residue 1314 of the L protein or the corresponding position, wherein the mutation of amino acid sequence residue 1314 of the L protein is an amino acid substitution of leucine for isoleucine, wherein leucine is encoded by a codon described as CTG; a deletion of the NS2 gene; and a nucleotide modification at position 14456 of reference SEQ ID NO: 1, which represents a change from thymine (T) to adenine (A).

[0024] The following embodiments are further provided. Embodiment 1 is a liquid, lyophilized, or frozen preparation comprising: a. Effective amounts of attenuated live virus and / or vector-based virus; b. Approximately 30% to approximately 45% (w / v) trehalose; c. One or more monovalent salts; and d. Buffers and / or antioxidants containing histidine.

[0025] Implementation 2 is a liquid, lyophilized or frozen preparation containing an effective amount of attenuated live virus and / or vector-based virus, as well as about 30% to about 40% (w / v) trehalose, about 100 mM monosodium glutamate (MSG), about 160 mM NaCl and about 10 mM histidine.

[0026] Embodiment 3 is a liquid, lyophilized, or frozen preparation comprising: a. An effective amount of attenuated live respiratory syncytial virus (RSV); b. Approximately 30% to approximately 45% (w / v) trehalose; c. One or more monovalent salts; and d. Buffers and / or antioxidants containing histidine.

[0027] Embodiment 4 is a liquid, lyophilized, or frozen preparation comprising: a. An effective amount of attenuated active RSV; b. Approximately 30% to approximately 45% (w / v) trehalose; c. Approximately 10 to approximately 300 mM NaCl; d. Approximately 0.5 to approximately 300 mM sodium glutamate (MSG) or potassium glutamate; and e. Approximately 10 to approximately 100 mM histidine; The pH of this formulation is approximately 6 to approximately 8.

[0028] Embodiment 5 is a formulation as described in Embodiment 1 or Embodiment 2, wherein the virus is membrane-bound.

[0029] Implementation method 6 is a formulation as described in any one of implementation methods 1-4, wherein the virus is RSV ΔNS2 / Δ1313 / I1314L.

[0030] Embodiment 7 is a formulation as described in Embodiment 6, wherein the effective amount of RSV ΔNS2 / Δ1313 / I1314L is about 5 to about 9 log10 plaque-forming units (PFU) / agent.

[0031] Embodiment 8 is a formulation as described in Embodiment 6, wherein the effective amount of RSV ΔNS2 / Δ1313 / I1314L is about 5 to about 6 log10 PFU / dose or about 6.1 to about 7 log10 PFU / dose or about 7.1 to about 8 log10 PFU / dose or about 8.1 to about 9 log10 PFU / dose.

[0032] Embodiment 9 is a formulation as described in Embodiment 6, wherein the effective amount of RSV ΔNS2 / Δ1313 / I1314L is about 5.6 log10 PFU / dose or about 6.2 log10 PFU / dose.

[0033] Embodiment 10 is a formulation as described in any one of Embodiments 3 or 4, wherein the codon for the serine acid at position 1313 encoding the L protein in the attenuated active RSV is deleted, thereby causing a deletion of the amino acid (Δ1313) in the L protein.

[0034] Embodiment 11 is a formulation as described in any one of Embodiments 3, 4 or 10, wherein the substitution of the amino acid residue of leucine for isoleucine at position 1314 in the RSV, with reference to SEQ ID NO: 3, causes a genetically stable mutation (I1314L) in the L gene.

[0035] Implementation method 12 is a formulation as described in any of the foregoing embodiments, wherein the RSV comprises: Large polymerase protein (L), phosphoprotein (P), nucleocapsid protein (N), M2-1 protein, non-structural protein 1 (NS1), glycoprotein (G), fusion protein (F), matrix protein (M), M2-2 protein, and small hydrophobic protein (SH); and A genome or antigenome containing the deletion of a codon encoding a serine acid at position 1313 or the corresponding position of the L protein; a mutation in an amino acid sequence residue 1314 of the L protein or the corresponding position, wherein the mutation in amino acid sequence residue 1314 of the L protein is an amino acid substitution of leucine for isoleucine, wherein the leucine is encoded by a codon described as CTG; a deletion of the NS2 gene; and a nucleotide modification at position 14456 of reference SEQ ID NO: 1, which represents a change from thymine (T) to adenine (A).

[0036] Embodiment 13 is a formulation as described in any of the foregoing embodiments, comprising a liquid formulation.

[0037] Embodiment 14 is a formulation as described in any of the foregoing embodiments, comprising a lyophilized formulation.

[0038] Embodiment 15 is a formulation as described in Embodiment 13, wherein the liquid formulation is formulated for administration to human subjects.

[0039] Embodiment 16 is a formulation as described in Embodiment 15, wherein the administration is intranasal.

[0040] Embodiment 17 is a formulation as described in any of the foregoing embodiments, wherein the formulation does not contain a polyoxyethylene-polyoxypropylene (PEO-PPO) block copolymer.

[0041] Embodiment 18 is a formulation as described in any of the foregoing embodiments, which is a liquid formulation, wherein the liquid formulation is not subsequently dried and reconstituted.

[0042] Embodiment 19 is a formulation as described in any of the foregoing embodiments, further comprising sodium monoglutamate (MSG) or potassium glutamate (PG).

[0043] Embodiment 20 is a formulation as described in any of the foregoing embodiments, comprising about 10 to about 100 mM histidine.

[0044] Embodiment 21 is a formulation as described in any of the foregoing embodiments, which contains about 30% trehalose.

[0045] Implementation method 22 is a formulation as described in any of the foregoing embodiments, which contains about 40% trehalose.

[0046] Embodiment 23 is a formulation as described in any of the foregoing embodiments, comprising about 0.5 to about 300 mM sodium glutamate (MSG) or potassium glutamate (PG).

[0047] Embodiment 24 is a formulation as described in any of the foregoing embodiments, wherein the monovalent salt is NaCl.

[0048] Embodiment 25 is a formulation as described in any of the foregoing embodiments, comprising about 10 to about 300 mM NaCl.

[0049] Implementation 26 is a formulation as described in any of the foregoing implementations, wherein the pH is about 6 to about 8.

[0050] Embodiment 27 is a formulation as described in any of the foregoing embodiments, wherein the pH is about 7 ± 0.5.

[0051] Implementation method 28 is a formulation as described in any of the foregoing embodiments, wherein the attenuated live virus comprises paramyxovirus.

[0052] Implementation 29 is a formulation as described in any of the foregoing implementations, wherein the attenuated live virus comprises respiratory syncytial virus (RSV).

[0053] Implementation 30 is a formulation as described in any of the foregoing implementations, wherein the virus is RSV, and wherein the RSV is administered to pediatric subjects.

[0054] Implementation 31 is a formulation as described in any of the foregoing embodiments, comprising a vector-based PIV virus, wherein the PIV is PIV3 if necessary.

[0055] Embodiment 32 is a formulation as described in Embodiment 31, wherein the PIV contains one or more heterologous RSV or hMPV antigens.

[0056] Embodiment 33 is a formulation as described in Embodiment 32, wherein the RSV antigen is RSV F and / or RSV G.

[0057] Embodiment 34 is a formulation as described in any of the foregoing embodiments, wherein the formulation is a liquid, and wherein the titration loss is less than 1 log10 PFU / mL when stored at about 2 to about 8 degrees Celsius for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 months.

[0058] Embodiment 35 is a formulation as described in any of the foregoing embodiments, wherein after storage at about 2 to about 8 degrees Celsius for about 12 to about 24 months, the titration loss is less than 1 log10 PFU / mL.

[0059] Embodiment 36 is a formulation as described in any of the foregoing embodiments, wherein the formulation is a liquid, and wherein when stored at about 37 degrees Celsius for at least 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day, the titration loss is less than 1 log10 PFU / mL.

[0060] Implementation method 37 is a formulation as described in any of the foregoing implementation methods, wherein at 37°C for about 1 to about 2 weeks, the titration loss is less than 1 log10 PFU / mL.

[0061] Embodiment 38 is a formulation as described in any of the foregoing embodiments, wherein the formulation is a liquid, and wherein the titration loss is less than 1 log10 PFU / mL when stored at room temperature for at least 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5 or 5-6 months.

[0062] Implementation method 39 is a formulation as described in any of the foregoing implementation methods, wherein the titration loss is less than 1 log10 PFU / mL when maintained at room temperature for 3-4 months.

[0063] Embodiment 40 is a formulation as described in any one of Embodiments 1-33, wherein the formulation is lyophilized and wherein, when maintained at 37°C for about 1 to about 2 weeks, the titration loss is less than 1 log10 PFU / mL.

[0064] Embodiment 41 is a formulation as described in any one of Embodiments 1-33, wherein the formulation is lyophilized, and wherein the titration loss is less than 1 log10 PFU / mL when stored at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks.

[0065] Implementation method 42 is a formulation as described in any of the foregoing embodiments, wherein the infectious titration is substantially unaffected by shear stress, repeated freeze-thaw cycles, or remodeling.

[0066] Implementation method 43 is a formulation as described in any of the foregoing embodiments, wherein the vaccine is formulated for intramuscular or subcutaneous administration.

[0067] Embodiment 44 is a formulation as described in any of the foregoing embodiments, which further includes an adjuvant.

[0068] Embodiment 45 is a formulation as described in any of the foregoing embodiments, which further comprises rHSA, divalent salt and / or amino acid.

[0069] Embodiment 46 is a method for increasing the stability of a liquid or lyophilized vaccine at room temperature or at a temperature of about 2 to about 8 degrees Celsius, comprising formulating an attenuated live enveloped virus and / or a vector-based virus in a liquid or lyophilized formulation, the liquid or lyophilized formulation containing about 30% to about 45% (w / v) trehalose, one or more monovalent salts, and a buffer and / or antioxidant containing histidine.

[0070] Implementation method 47 is the method as described in implementation method 46, wherein the concentration of trehalose is about 30% (w / v).

[0071] Implementation method 48 is the method as described in implementation method 46, wherein the concentration of trehalose is about 40% (w / v).

[0072] Implementation method 49 is a method for immunizing a subject against a viral infection, which includes administering a preparation as described in any one of implementation methods 1-48.

[0073] Embodiment 50 is a liquid, lyophilized, or frozen preparation comprising: a. An effective amount of attenuated active RSV containing RSV ΔNS2 / Δ1313 / I1314L; b. Approximately 30% to approximately 40% (w / v) trehalose; c. Approximately 160 mM NaCl; d. Approximately 100 mM monosodium glutamate (MSG); and e. Approximately 10 mM histidine; The pH of this formulation is approximately 7.

[0074] Embodiment 51 is a formulation as described in Embodiment 50, wherein the formulation contains about 30% (w / v) trehalose.

[0075] Embodiment 52 is a formulation as described in Embodiment 50, wherein the formulation contains about 40% (w / v) trehalose.

[0076] Embodiment 53 is a formulation as described in any one of Embodiments 50-52, wherein the attenuated active RSV comprises: Large polymerase protein (L), phosphoprotein (P), nucleocapsid protein (N), M2-1 protein, non-structural protein 1 (NS1), glycoprotein (G), fusion protein (F), matrix protein (M), M2-2 protein, and small hydrophobic protein (SH); and A genome or antigenome containing the deletion of a codon encoding a serine acid at position 1313 or the corresponding position of the L protein; a mutation in an amino acid sequence residue 1314 of the L protein or the corresponding position, wherein the mutation in amino acid sequence residue 1314 of the L protein is an amino acid substitution of leucine for isoleucine, wherein the leucine is encoded by a codon described as CTG; a deletion of the NS2 gene; and a nucleotide modification at position 14456 of reference SEQ ID NO: 1, which represents a change from thymine (T) to adenine (A). Attached Figure Description

[0077] Figure 1A -C illustrates the experimental design for formulation development research ( Figure 1A ); the thermostability of liquid (black bar) and lyo (white bar) formulations with different trehalose concentrations at 37°C for 1 week at titration. Figure 1B The thermal stability of liquid and lyo formulations with the same trehalose concentration (30%) and different salt concentrations at 37°C for 1 week, including titration loss. Figure 1C ).exist Figure 1A In the formula, trehalose (10% = -1, 20% = 0 and 30% = 1), NaCl (0 mM = -1, 80 mM = 0 and 160 mM = 1), monosodium glutamate (MSG) (0 mM = -1, 50 mM = 0 and 100 mM = 1), and histidine (3.33 mM = -1, 10 mM = 0 and 16.67 mM = 1) were present. Figure 1B In the table, the first bar in each column is "Liquid titration at 37°C for 1 week", and the second bar in each column is "Lyo titration at 37°C for 1 week".

[0078] Figure 2A and 2B Different percentages of trehalose liquid formulations were shown at 37°C. Figure 2A ) and 5°C ( Figure 2B The thermal stability curve under ( ).

[0079] Figure 3A and 3B The real-time stability of the liquid formulation containing 30% trehalose, 160 mM NaCl, 100 mM MSG and 10 mM histidine (pH 7.0) was shown at 5°C ± 3°C. Figure 3A It is batch L5 of a high-dose drug product toxicology (Tox) and... Figure 3B This is batch L6 of a GMP-certified clinical high-dose drug product.

[0080] Figures 4A-4C Stability studies of RSV ΔNS2 / Δ1313 / I1314L drug substance (DS) batch L5 are shown in a formulation of 30% trehalose, 160 mM NaCl, 100 mM MSG and 10 mM histidine (pH 7.0) from the 120 L stage I manufacturing process. Figure 4A The results of five freeze-thaw cycles are shown; Figure 4B The stability of DS during thawing at 5°C ± 3°C, and Figure 4C The thaw stability of DS was determined at 25°C ± 2°C.

[0081] Figure 5A and 5B The study compares the real-time stability of 40% trehalose, 160 mM NaCl, 100 mM MSG and 10 mM histidine (pH 7.0) prepared from different DS batches over 36 months at 5°C ± 3°C. Figure 5A The stability curves of high-dose drug products in a 40% trehalose liquid formulation are shown, and Figure 5B The stability curves of low-dose drug products in a 40% trehalose liquid formulation are shown.

[0082] Figures 6A-6D Stability studies of RSV ΔNS2 / Δ1313 / I1314L drug substance (DS) batch L8 are shown in a formulation of 40% trehalose, 160 mM NaCl, 100 mM MSG and 10 mM histidine (pH 7.0) from a 200 L stage III manufacturing process. Figure 6A The results of five freeze-thaw cycles are shown; Figure 6B The stability of DS after thawing at 5°C ± 3°C for up to 90 days. Figure 6C The stability of thawed DS was measured over a period of 130 weeks at 5°C ± 3°C. Figure 6D The thaw stability of DS was determined at 25°C ± 2°C.

[0083] Figures 7A-7GStability curves for infectious titration of liquid pharmaceutical products containing 40% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine (pH 7.0) are shown. Figure 7A The infectious titer was shown for up to 24 weeks at 5°C ± 3°C. Figure 7B The infectious titer was shown to last for up to 24 months at 5°C ± 3°C. Figure 7C The infectious titration at 25°C ±2°C is shown. Figure 7D The infectious titer at 37°C ± 2°C is shown. Figure 7E The infectious titration at 45°C ±2°C is shown. Figure 7F This study investigated the stability of the infectious titration of the prepared bulk product at 5°C for up to 3 months. Figure 7G The system is based on Advanced Dynamics and Technological Solutions (AKTS) modeling and storage period prediction using 3 months of data. The selected best-fit model is Model #53, a competitive 2-step dynamic model, known as the Best Widely Applied Information Criterion (wAIC), which is a generalized version of the Akaike Information Criterion (AIC) on singular statistical models, and the Widely Applied Bayesian Information Criterion (wBIC), which is a generalized version of the Bayesian Information Criterion (BIC) on singular statistical models.

[0084] Figures 8A-8D The results show the drug products made from DS batch L7 and DS batch L9 at 5°C. Figure 8B 240 days and Figure 8C (up to 24 months) and at 37°C ( Figure 8A Stability comparison of liquid formulations containing 40% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine (pH 7.0) at different concentrations. Figure 8D Comparison of AKTS storage period prediction based on titration loss at 5°C.

[0085] Figure 9A -C indicates the thermal stability of the study involving the addition of recombinant human serum albumin (rHSA). Figure 9A Comparison of DS stability with and without rHSA. Figure 9B The stability of DP was compared between the system with and without the addition of a low concentration of rHSA (0.5 mg / mL). Figure 9C The stability of DP was compared between the system with and without the addition of a high concentration of 1.4 mg / mL rHSA.

[0086] Figures 10A-10E Stability curves for the candidate formulations are shown, including accelerated stability tests. Figure 10AThe infectious titer at 5°C ± 3°C is shown. Figure 10B The infectious titer at 25°C ± 2°C is shown. Figure 10C The infectious titer at 37°C ± 2°C is shown. Figure 10D This study predicts the shelf life of AKTS based on titration loss of low-concentration rHSA formulations at 5°C. The selected best-fit model is Model #43, which incorporates one-step kinetics, optimal wAIC, and wBIC. Figure 10E This study aims to predict the shelf life of AKTS based on titration loss of high-concentration rHSA formulations at 5°C. The selected best-fit model is Model #34, which includes one-step kinetics, optimal wAIC, and wBIC.

[0087] Figure 11 A comparison of thermal study curves of 40% trehalose with and without the addition of the new excipient hydroxyethyl starch (HES).

[0088] Figure 12A-12F Candidate formulations with HES stability profiles are shown, including accelerated stability testing. Figure 12A Infectivity titers over 24 weeks (6 months) at 5°C ± 3°C are shown. Figure 12B The infectious titer was shown for up to 104 weeks (24 months) at 5°C ± 3°C. Figure 12C The infectious titer at 25°C ± 2°C is shown. Figure 12D The infectious titer at 37°C ± 2°C is shown. Figure 12E The infectious titer at 45°C ± 2°C is shown. Figure 12F Based on 6 months of data, this study predicts the shelf life of AKTS based on titration loss of a 2.5% HES formulation at 5°C. The selected best-fit model is Model #56, which includes two-step kinetics, optimal wAIC, and wBIC.

[0089] Figure 13 A summary of the thermal stability comparisons from all the dried formulations is presented.

[0090] Figure 14 Results of an exemplary study of the lyo formulation of the herpes simplex virus type 2 (HSV-2) vaccine are shown.

[0091] Figure 15 The genomic structure of RSV ΔNS2 / Δ1313 / I1314L was explained. Detailed Implementation

[0092] This disclosure provides liquid and dry formulations with improved yield and stability, as well as methods for preparing them, wherein the vaccine comprises an attenuated live enveloped virus, greater than about 15% (w / v) trehalose, a monovalent salt, and histidine, wherein the virus is RSV, if desired. These formulations and methods offer at least the following advantages: improved liquid and dry stability profiles, and the ability to integrate liquid vaccines into a drug-loaded syringe or device for immediate administration without reconfiguration. In some embodiments, the device is for intranasal delivery. In some embodiments, the device is pre-filled with a liquid formulation for intranasal delivery. In some embodiments, the formulations provided herein are liquid and are stable when stored at about 2 to about 8 degrees Celsius for at least 12-24 months, with a titer loss of less than 1 log10 PFU / mL. In some embodiments, the formulations provided herein are liquid and, under thermal stress conditions, when stored at about 37 degrees Celsius for at least 12-25 days, maintain a titer loss of less than 1 log10 PFU / mL. In some embodiments, the formulations provided herein are liquids and are stable when stored at room temperature (typically about 25 degrees Celsius) for at least 3-6 months, with a titration loss of less than 1 log10 PFU / mL.

[0093] This invention is partly based on the surprising stability obtained when formulating attenuated live virus vaccines with high concentrations of trehalose.

[0094] I. Definition Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings: Unless the context otherwise requires, "or" is used in an inclusive sense, that is, equivalent to "and / or".

[0095] As used herein, "live attenuated virus" refers to a viable virus that exhibits reduced, weakened, or no clinical symptoms when administered to a subject. "Live attenuated vaccine" contains live attenuated virus.

[0096] As used herein, a “vector” or “vector-based” virus or vaccine comprises a virus modified to express one or more heterologous antigens. For example, vaccine viruses have been used as vaccine vectors expressing influenza genes for immunization against influenza. De Vries and Rimmelzwaan; Human Vaccine Immunother. [Human Vaccine Immunotherology] (2016) Nov; 12(11): 2881-2901. Similarly, parainfluenza viruses (PIVs) have been used as vectors expressing genes from non-PIV viruses, such as, for example, RSV, herpes simplex virus (HSV), and human host pneumonia virus (hMPV), for immunization against, for example, RSV, HSV, and hMPV. Furthermore, a PIV of one species has been used as a vector expressing genes from a PIV virus of another species. In each case, such a vaccine is considered a “vector” or “vector-based” vaccine, and recombinant viruses are considered “vector” or “vector-based” viruses. In some cases, "vector" or "vector-based" vaccines are also live attenuated vaccines, in which the vaccine, when administered to subjects, exhibits reduced, weakened, or no clinical symptoms.

[0097] As used herein, "prevention" means preventing or avoiding disease manifestations, delaying the onset of one or more symptoms of a particular disease, disorder, or condition (e.g., RSV infection), and / or reducing the frequency and / or severity of such one or more symptoms. In some implementations, prevention is assessed on a population basis such that if a statistically significant reduction in the development, frequency, and / or intensity of one or more symptoms of a particular disease, disorder, or condition is observed in a population susceptible to such a disease, disorder, or condition, the agent is considered to have "prevented" the disease, disorder, or condition.

[0098] As used herein, the terms "vaccination" or "vaccinate" refer to the administration of a component intended to elicit an immune response, for example, against a pathogen. Vaccination may be administered before, during, and / or after exposure to a pathogen and / or the appearance of one or more symptoms, and in some embodiments, shortly before, during, and / or after exposure to the pathogen. In some embodiments, vaccination comprises multiple administrations of a vaccine component at appropriate intervals.

[0099] As used herein, "RSV ΔNS2 / Δ1313 / I1314L" refers to RSV ΔNS2 / Δ1313 / I1314L (NIH) or RSV ΔNS2 / Δ1313 / I1314L (Sanofi). Each of ΔNS2 / Δ1313 / I1314L (National Institutes of Health (NIH)) and RSV ΔNS2 / Δ1313 / I1314L (Sanofi) contains an attenuated live RSV having (i) a 523-nucleotide (nt) deletion in the NS2 gene (ΔNS2), (ii) an amino acid deletion in the L protein, and (iii) a genetically stable mutation in the L gene (see [link to original text]). Figure 15 The attenuated live RSV ΔNS2 / Δ1313 / I1314L (Sanofi) also contains a nucleotide modification at position 14456 of reference SEQ ID NO: 1, which represents a change from thymine (T) to adenine (A) in the non-coding region.

[0100] As used herein, "RSV ΔNS2 / Δ1313 / I1314L vaccine" refers to either "RSV ΔNS2 / Δ1313 / I1314L (NIH) vaccine" or "RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine". The RSV ΔNS2 / Δ1313 / I1314L (NIH) vaccine contains an effective amount of RSV ΔNS2 / Δ1313 / I1314L (NIH). The RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine contains an effective amount of RSV ΔNS2 / Δ1313 / I1314L (Sanofi).

[0101] As used herein, the term "immune response" refers to the response of cells of the immune system (such as B cells, T cells, dendritic cells, macrophages, or polymorphonuclear cells) to a stimulus (such as an antigen or vaccine). An immune response can include any cell in the body involved in the host defense response, including, for example, epithelial cells that secrete interferons or cytokines. Immune responses include, but are not limited to, innate and / or adaptive immune responses. As used herein, a "protective immune response" refers to an immune response that protects a subject from infection (e.g., prevents infection or the occurrence of infection-related diseases). Methods for measuring an immune response include measuring, for example, the proliferation and / or activity of lymphocytes (such as B or T cells), the secretion of cytokines or chemokines, inflammation, antibody production, etc. An "antibody response" is an immune response that produces antibodies.

[0102] As used in this article, "immunity" refers to actions that elicit an immune response in a subject or protect the subject from infection.

[0103] As used herein, "adjuvant" refers to a substance or agent that nonspecifically enhances an immune response against an antigen. Adjuvants may include, but are not limited to, suspensions of minerals (e.g., alum, aluminum hydroxide, or phosphate) to which antigens are adsorbed; water-in-oil or oil-in-water emulsions of antigen solutions emulsified in mineral oil or water (e.g., Freund's incomplete adjuvant). Sometimes, killed mycobacteria (e.g., Freund's complete adjuvant) are included to further enhance antigenicity. Immunostimulatory oligonucleotides (e.g., CpG motifs) may also be used as adjuvants (e.g., see U.S. Patent Nos. 6,194,388, 6,207,646, 6,214,806, 6,218,371, 6,239,116, 6,339,068, 6,406,705, and 6,429,199). Adjuvants may also include biomolecules such as Toll-like receptor (TLR) agonists and co-stimulatory molecules. Exemplary biological adjuvants include, but are not limited to, IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL, or combinations thereof.

[0104] As used in this article, "liquid" is given its traditional meaning, and "frozen liquid" is a solid liquid that is distinct from liquid.

[0105] "Formulation" means a composition containing an active pharmaceutical ingredient or biological component and one or more other components. The term "formulation" is used interchangeably herein with the terms "pharmaceutical composition," "vaccine composition," and "vaccine formulation." Depending on the circumstances, other components that may be included include pharmaceutically acceptable excipients, additives, diluents, buffers, sugars, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), chelating agents, surfactants, polyols, fillers, stabilizers, lyophilization protectants, solubilizers, emulsifiers, salts, adjuvants, tonicotinic agents (such as alkali metal halides (e.g., sodium chloride or potassium chloride), mannitol, or sorbitol), delivery mediators, and antimicrobial preservatives.

[0106] As used herein, “stability” or “stabilization” of the active ingredient in a formulation means less than 1 log at a given temperature and time. 10 The loss of PFU / ml. Stability can be measured using the assays described herein as well as assays known in the art for measuring activity or potency or viral infectivity titrations.

[0107] As used herein, “treatment” means any administration or application of a therapeutic agent to a subject’s disease or condition, and includes suppressing the disease, preventing its development, alleviating one or more symptoms of the disease, curing the disease, or preventing the recurrence of one or more symptoms of the disease.

[0108] As used herein, a "therapeutic effective dose" or "effective dose" is the amount of a composition or its active ingredient sufficient to provide a beneficial effect or otherwise reduce harmful or non-beneficial events in an individual to whom the composition is administered. As used herein, reference to "therapeutic effective dose" or "effective dose" means the dose that produces one or more desired or desirable (e.g., beneficial) effects to which it is administered, administered once or more times over a given period of time. The precise dose will depend on the purpose of the treatment and will be determined using known techniques.

[0109] The term "about" or "approximately" is used herein to mean roughly, approximately, or around. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the upper and lower boundaries of the value. Generally, the term "about" can modify a value to be higher or lower (higher or lower) by a variation (e.g., 10%, up or down). In some embodiments, the term indicates a deviation from the indicated value of ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, or ±0.01%. In some embodiments, "about" indicates a deviation from the indicated value of ±10%. In some embodiments, "about" indicates a deviation from the indicated value of ±5%. In some embodiments, "about" indicates a deviation from the indicated value of ±4%. In some embodiments, "about" indicates a deviation from the indicated value of ±3%. In some embodiments, "about" indicates a deviation from the indicated value of ±2%. In some embodiments, "about" means a deviation of ±1% from the indicated value. In some embodiments, "about" means a deviation of ±0.9% from the indicated value. In some embodiments, "about" means a deviation of ±0.8% from the indicated value. In some embodiments, "about" means a deviation of ±0.7% from the indicated value. In some embodiments, "about" means a deviation of ±0.6% from the indicated value. In some embodiments, "about" means a deviation of ±0.5% from the indicated value. In some embodiments, "about" means a deviation of ±0.4% from the indicated value. In some embodiments, "about" means a deviation of ±0.3% from the indicated value. In some embodiments, "about" means a deviation of ±0.2% from the indicated value. In some embodiments, "about" means a deviation of ±0.1% from the indicated value. In some embodiments, "about" means a deviation of ±0.05% from the indicated value. In some embodiments, "about" means a deviation of ±0.01% from the indicated value. All ranges proposed herein are intended to include both the lower and upper limits of the ranges.

[0110] The term "pediatric subject" refers to a subject who is 21 years of age or younger at the time of immunization. Further characteristics of the pediatric subgroups are: (i) neonates – from birth to 28 days after birth; (ii) infants and toddlers – 29 days to under 2 years of age; (iii) children – 2 years to under 12 years of age; and (iv) adolescents – 12 years to 21 years of age. In some respects, pediatric subjects may be 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year of age or younger. In some respects, pediatric subjects may be at least 6 months old. In some respects, pediatric subjects may be 6 months to 18 years of age. In some respects, pediatric subjects may be 6 months to 10 years of age. In some respects, the age of pediatric subjects may be 4 months to 4 years. In some respects, the age of pediatric subjects may be 6 to 18 months. In some respects, the age of pediatric subjects may be 6 months to less than 24 months. The age of pediatric subjects may also include other ranges.

[0111] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying drawings. Although the invention will be described in conjunction with the illustrated embodiments, it should be understood that these embodiments are not intended to limit the invention to those embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the invention as defined in the appended claims and the included embodiments.

[0112] Before describing the contents of this instruction in detail, it should be understood that this disclosure is not limited to specific components or process steps and is therefore subject to variation. It should be noted that, unless the context clearly indicates otherwise, as used in this specification and the appended claims, the singular forms "a / an" and "the" include plural indicators. Thus, for example, a reference to "conjugate" includes multiple conjugates and a reference to "cell" includes multiple cells, etc.

[0113] Numerical ranges include values ​​within defined ranges. Taking into account significant figures and measurement-related errors, measured and measurable values ​​should be understood as approximate values. Furthermore, the use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" is not intended to be restrictive. It should be understood that the foregoing general and detailed descriptions are exemplary and explanatory only, and not intended to limit the content taught.

[0114] Unless otherwise specified in the specification, embodiments in the specification that "comprise" various components are also considered to "consist of" or "substantially consist of" the listed components; embodiments in the specification that "consist of various components" are also considered to "comprise" or "substantially consist of" the listed components; and embodiments in the specification that "substantially consist of various components" are also considered to "consist of" or "include" the listed components (this interchangeability does not apply to the use of such terms in the claims). The term "or" is used in an open sense, that is, equivalent to "and / or," unless the context clearly indicates otherwise.

[0115] The section headings used herein are for organizational purposes only and should not be construed in any way as limiting the intended subject matter. In the event of any conflict between any terminology defined herein or any other express content herein, this specification shall prevail. While this instruction is described in conjunction with various embodiments, it is not intended to limit this instruction to those embodiments. Rather, as will be understood by those skilled in the art, this instruction includes various alternatives, modifications, and equivalents.

[0116] II. Vaccine preparations A. Preparations The formulations described herein may be liquid, lyophilized, or frozen. Liquid formulations may be prepared as liquids (i.e., never lyophilized) and are suitable for long-term storage at 2°C–8°C during the product's shelf life. They may be reconstituted after lyophilization or thawed after freezing and stored at room temperature for hours or days. In some embodiments, the formulations may be lyophilized and stored at 2°C–8°C or room temperature prior to reconstitution. In some embodiments, the formulations do not contain a polyoxyethylene-polyoxypropylene (PEO-PPO) block copolymer or other block copolymers that those skilled in the art consider similar to PEO-PPO.

[0117] In some embodiments, the formulation is liquid and is stable when stored at about 2 to about 8 degrees Celsius (°C) for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months. In some embodiments, "stable" means less than 1 log [value missing] at a given temperature and time. 10Loss of PFU / ml. See also Guidelines on the Stability Evaluation of Vaccines, Geneva: World Health Organization; 2006. p. 28, Report No.: WHO / BS / 06.2049 Final; and WHO Technical Report Series 962: Guidelines on the Stability Evaluation of Vaccines, Geneva: World Health Organization; 2011. 28. Report No.: 57 (incorporated herein by reference), which provide guidance for determining stable formulations. In some such embodiments, after storage at about 2 to about 8 degrees Celsius for about 12-24 months, titration loss is less than 1 log. 10 PFU / mL.

[0118] In some embodiments, the formulation is a liquid, and under thermal stress conditions, when stored at approximately 37 degrees Celsius for at least 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, the formulation can maintain 1 log 10 Titration loss below PFU / mL. In some such embodiments, titration loss is less than 1 log at 37°C for 1-2 weeks. 10 PFU / mL.

[0119] In some embodiments, the formulation is a liquid system, and when stored at room temperature for at least 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, or 5-6 months, the titration loss is less than 1 log. 10 PFU / mL. In some embodiments, the formulation is liquid and is stable when stored at room temperature for at least 3 months. In some embodiments, the formulation is liquid and is stable when stored at room temperature for at least 4 months. In some embodiments, the formulation is lyophilized, and wherein titration loss is less than 1 log when stored at 37°C for about 1 to about 2 weeks. 10 PFU / mL. In some embodiments, the formulation is lyophilized, and wherein the titration loss is less than 1 log when stored at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. 10 PFU / mL.

[0120] In some implementations, the infectious titration of the formulation is substantially unaffected by shear stress, repeated freeze-thaw cycles, or remodeling.

[0121] Trehalose In some embodiments, a liquid, lyophilized, or frozen formulation is provided, comprising attenuated live enveloped virus and trehalose. In some embodiments, a liquid or lyophilized formulation is provided, comprising attenuated live enveloped virus and trehalose. In some embodiments, the concentration of trehalose is between about 15% and 60% w / v. In some embodiments, the concentration of trehalose is between about 20% and 55% w / v. In some embodiments, the concentration of trehalose is between about 25% and 50% w / v. In some embodiments, the concentration of trehalose is between about 30% and 45% w / v. In some embodiments, the concentration of trehalose is between about 35% and 40% w / v. In some embodiments, the concentration of trehalose is between about 30% and 40% w / v. In some embodiments, the concentration of trehalose is between about 30% and 35% w / v. In some embodiments, the concentration of trehalose is between about 15% and 20%, 15% and 25%, 15% and 30%, 15% and 35%, 15% and 40%, or 15% and 45% w / v. In some embodiments, the concentration of trehalose is greater than about 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45%. In some embodiments, the concentration of trehalose is between about 20% and 25%, 20% and 30%, 20% and 35%, 20% and 40%, or 20% and 45% w / v. In some embodiments, the concentration of trehalose is between about 25% and 30%, 25% and 35%, 25% and 40%, or 25% and 45% w / v. In some embodiments, the concentration of trehalose is between about 30% and 35%, 30% and 40%, or 30% and 45% w / v. In some embodiments, the concentration of trehalose is about 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45% w / v. In some embodiments, the concentration of trehalose is about 20% w / v. In some embodiments, the concentration of trehalose is about 25% w / v. In some embodiments, the concentration of trehalose is about 30% w / v. In some embodiments, the concentration of trehalose is about 35% w / v. In some embodiments, the concentration of trehalose is about 40% w / v. In some embodiments, the concentration of trehalose is about 45% w / v. In some embodiments, trehalose can act as a stabilizer.In some embodiments, trehalose can act as a freeze-drying protectant. In various embodiments, trehalose can act as both a stabilizer and a freeze-drying protectant.

[0122] Salt In some embodiments, the formulation further comprises a salt. In some embodiments, the salt is a monovalent salt. In some embodiments, the salt is a divalent salt.

[0123] a. Monovalent salts (e.g., NaCl) In some embodiments, a liquid, lyophilized, or frozen formulation is provided, comprising attenuated live enveloped virus, trehalose, and a monovalent salt. In some embodiments, a liquid or lyophilized formulation is provided, comprising attenuated live enveloped virus, trehalose, and a monovalent salt. In some embodiments, the monovalent salt is sodium chloride (NaCl). In some embodiments, the monovalent salt is sodium monoglutamate (MSG) or potassium monoglutamate (PG; for example, potassium monoglutamate (MPG)).

[0124] In some embodiments, the concentration of the monovalent salt is between about 10 and 300 mM. In some embodiments, the concentration is between about 10 and 290 mM, 20 and 180 mM, 30 and 170 mM, 40 and 160 mM, 50 and 150 mM, 60 and 140 mM, 70 and 130 mM, 80 and 120 mM, or 90 and 110 mM. In some embodiments, the concentration is between about 100 and 200 mM, 110 and 200 mM, 120 and 200 mM, 130 and 200 mM, 140 and 200 mM, 150 and 200 mM, or 160 and 200 mM. In some embodiments, the concentration is between about 100 and 200 mM, 100 and 190 mM, 100 and 180 mM, 100 and 170 mM, 100 and 160 mM, or 100 and 150 mM. In some embodiments, the concentration is about 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, or 200 mM. In some embodiments, the concentration is about 150 mM. In some embodiments, the concentration is about 160 mM. In some embodiments, the concentration is about 170 mM. In some embodiments, the salt is NaCl, and the concentration of the monovalent salt is about 160 mM.

[0125] In some embodiments, a liquid, lyophilized, or frozen formulation is provided, comprising: attenuated live enveloped virus; about 30%-45% w / v trehalose; and about 160 mM monovalent salt, wherein the monovalent salt is NaCl, if desired. In some embodiments, a liquid or lyophilized formulation is provided, comprising: attenuated live enveloped virus; about 30% w / v trehalose; and about 160 mM monovalent salt, wherein the monovalent salt is NaCl, if desired. In some embodiments, a liquid, lyophilized, or frozen formulation is provided, comprising: attenuated live enveloped virus; about 30% w / v trehalose; and about 160 mM monovalent salt, wherein the monovalent salt is NaCl, if desired. In some embodiments, a liquid or lyophilized formulation is provided, comprising: attenuated live enveloped virus; about 30% w / v trehalose; and about 160 mM monovalent salt, wherein the monovalent salt is NaCl, if desired. In some embodiments, a liquid, lyophilized, or frozen formulation is provided, comprising: attenuated live enveloped virus; approximately 40% w / v trehalose; and approximately 160 mM monovalent salt, wherein the monovalent salt is NaCl, if desired. In some embodiments, a liquid or lyophilized formulation is provided, comprising: attenuated live enveloped virus; approximately 40% w / v trehalose; and approximately 160 mM monovalent salt, wherein the monovalent salt is NaCl, if desired. In some embodiments, the virus is RSV. In some embodiments, the vaccine is an RSV ΔNS2 / Δ1313 / I1314L vaccine.

[0126] b. Divalent salts (e.g., MgCl₂) 2 or CaCl 2 ） In some embodiments, the formulation further comprises a divalent salt, wherein the divalent salt is MgCl2 or CaCl2, if desired. In some embodiments, a liquid, lyophilized, or frozen formulation is provided, comprising: attenuated live enveloped virus; trehalose; a monovalent salt, wherein the monovalent salt is NaCl, if desired; a stabilizer, wherein the stabilizer is MSG or PG, if desired; histidine; and a divalent salt, wherein the divalent salt is MgCl2 or CaCl2, if desired. In some embodiments, a liquid or lyophilized formulation is provided, comprising: attenuated live enveloped virus; trehalose; a monovalent salt, wherein the monovalent salt is NaCl, if desired; a stabilizer, wherein the stabilizer is MSG or PG, if desired; histidine; and a divalent salt, wherein the divalent salt is MgCl2 or CaCl2, if desired.

[0127] In some embodiments, the concentration of the divalent salt (e.g., MgCl2 or CaCl2) is between about 0 and 50 mM. In some embodiments, the concentration of the divalent salt (e.g., MgCl2 or CaCl2) is between about 0 and 10 mM, 0 and 20 mM, 0 and 30 mM, 0 and 40 mM, or 0 and 50 mM. In some embodiments, the concentration of the divalent salt (e.g., MgCl2 or CaCl2) is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, or 55 mM. In some embodiments, the concentration of the divalent salt (e.g., MgCl2 or CaCl2) is about 10 mM.

[0128] In some embodiments, a liquid, lyophilized, or frozen formulation is provided, comprising: attenuated live enveloped virus; about 30%-45% w / v trehalose; about 160 mM monovalent salt, optionally NaCl; about 100 mM stabilizer, optionally MSG or PG; about 10 mM histidine; and about 0-50 mM divalent salt, optionally MgCl2 or CaCl2. In some embodiments, a liquid or lyophilized formulation is provided, comprising: attenuated live enveloped virus; about 30%-45% w / v trehalose; about 160 mM monovalent salt, optionally NaCl; about 100 mM stabilizer, optionally MSG or PG; about 10 mM histidine; and about 0-50 mM divalent salt, optionally MgCl2 or CaCl2. In some embodiments, a liquid, lyophilized, or frozen formulation is provided, comprising: attenuated live enveloped virus; about 30% w / v trehalose; about 160 mM monovalent salt, optionally NaCl; about 100 mM stabilizer, optionally MSG or PG; about 10 mM histidine; and about 0-50 mM divalent salt, optionally MgCl2 or CaCl2. In some embodiments, a liquid or lyophilized formulation is provided, comprising: attenuated live enveloped virus; about 30% w / v trehalose; about 160 mM monovalent salt, optionally NaCl; about 100 mM stabilizer, optionally MSG or PG; about 10 mM histidine; and about 0-50 mM divalent salt, optionally MgCl2 or CaCl2. In some embodiments, a liquid, lyophilized, or frozen formulation is provided, comprising: attenuated live enveloped virus; about 40% w / v trehalose; about 160 mM monovalent salt, optionally NaCl; about 100 mM stabilizer, optionally MSG or PG; about 10 mM histidine; and about 0-50 mM divalent salt, optionally MgCl2 or CaCl2. In some embodiments, a liquid or lyophilized formulation is provided, comprising: attenuated live enveloped virus; about 40% w / v trehalose; about 160 mM monovalent salt, optionally NaCl; about 100 mM stabilizer, optionally MSG or PG; about 10 mM histidine; and about 0-50 mM divalent salt, optionally MgCl2 or CaCl2. In some embodiments, the virus is RSV. In some implementations, the vaccine is the RSV ΔNS2 / Δ1313 / I1314L vaccine.

[0129] stabilizer In some embodiments, the formulation further comprises a stabilizer. In some embodiments, the stabilizer is sodium monoglutamate (MSG) or potassium glutamate (PG). In some embodiments, a liquid, lyophilized, or frozen formulation is provided, comprising: attenuated live enveloped virus; trehalose; a salt, if desired, wherein the salt is NaCl; and a stabilizer, if desired, wherein the stabilizer is MSG or PG. In some embodiments, a liquid or lyophilized formulation is provided, comprising: attenuated live enveloped virus; trehalose; a salt, if desired, wherein the salt is NaCl; and a stabilizer, if desired, wherein the stabilizer is MSG or PG.

[0130] In some embodiments, the concentration of the stabilizer is between about 0.5 and 300 mM. In some embodiments, the concentration of the stabilizer is between about 10 and 290 mM, 20 and 180 mM, 30 and 170 mM, 40 and 160 mM, 50 and 150 mM, 60 and 140 mM, 70 and 130 mM, 80 and 120 mM, or 90 and 110 mM. In some embodiments, the concentration of the stabilizer is between about 100 and 200 mM, 110 and 200 mM, 120 and 200 mM, 130 and 200 mM, 140 and 200 mM, 150 and 200 mM, or 160 and 200 mM. In some embodiments, the concentration of the stabilizer is between about 100 and 200 mM, 100 and 190 mM, 100 and 180 mM, 100 and 180 mM, 100 and 170 mM, 100 and 160 mM, or 100 and 150 mM. In some embodiments, the concentration of the stabilizer is between about 50 and 100 mM. In some embodiments, the concentration of the stabilizer is about 80, 85, 90, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 mM. In some embodiments, the concentration of the stabilizer is about 100 mM.

[0131] In some embodiments, a liquid, lyophilized, or frozen formulation is provided, comprising: attenuated live enveloped virus; about 30%-45% w / v trehalose; about 160 mM salt, optionally NaCl; and about 100 mM stabilizer, optionally MSG or PG. In some embodiments, a liquid or lyophilized formulation is provided, comprising: attenuated live enveloped virus; about 30% w / v trehalose; about 160 mM salt, optionally NaCl; and about 100 mM stabilizer, optionally MSG or PG. In some embodiments, a liquid, lyophilized, or frozen formulation is provided, comprising: attenuated live enveloped virus; about 30% w / v trehalose; about 160 mM salt, optionally NaCl; and about 100 mM stabilizer, optionally MSG or PG. In some embodiments, a liquid or lyophilized formulation is provided, comprising: attenuated live enveloped virus; about 30% w / v trehalose; about 160 mM salt, optionally NaCl; and about 100 mM stabilizer, optionally MSG or PG. In some embodiments, a liquid, lyophilized, or frozen formulation is provided, comprising: attenuated live enveloped virus; about 40% w / v trehalose; about 160 mM salt, optionally NaCl; and about 100 mM stabilizer, optionally MSG or PG. In some embodiments, a liquid or lyophilized formulation is provided, comprising: attenuated live enveloped virus; about 40% w / v trehalose; about 160 mM salt, optionally NaCl; and about 100 mM stabilizer, optionally MSG or PG. In some embodiments, the virus is RSV. In some implementations, the vaccine is the RSV ΔNS2 / Δ1313 / I1314L vaccine.

[0132] Buffers and / or antioxidants, histidine In some embodiments, the formulation further comprises a buffer and / or antioxidant containing histidine. In some embodiments, a liquid, lyophilized, or frozen formulation is provided comprising: attenuated live enveloped virus; trehalose; a salt, if desired, wherein the salt is NaCl; a stabilizer, if desired, wherein the stabilizer is MSG or PG; and a buffer and / or antioxidant containing histidine. In some embodiments, a liquid or lyophilized formulation is provided comprising: attenuated live enveloped virus; trehalose; a salt, if desired, wherein the salt is NaCl; a stabilizer, if desired, wherein the stabilizer is MSG or PG; and a buffer and / or antioxidant containing histidine. In some embodiments, histidine has a buffering function. In some embodiments, histidine has an antioxidant function. In various embodiments, histidine has both buffering and antioxidant functions. In other words, histidine can have both buffering and antioxidant functions.

[0133] In some embodiments, the concentration of histidine is between about 10 and 100 mM. In some embodiments, the concentration of histidine is between about 10 and 90 mM, 20 and 80 mM, 30 and 70 mM, or 40 and 60 mM. In some embodiments, the concentration of histidine is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 mM. In some embodiments, the concentration of histidine is about 10 mM.

[0134] In some embodiments, a liquid, lyophilized, or frozen formulation is provided, comprising: attenuated live enveloped virus; about 30%-45% w / v trehalose; about 160 mM salt, optionally NaCl; about 100 mM stabilizer, optionally MSG or PG; and about 10 mM histidine. In some embodiments, a liquid or lyophilized formulation is provided, comprising: attenuated live enveloped virus; about 30%-45% w / v trehalose; about 160 mM salt, optionally NaCl; about 100 mM stabilizer, optionally MSG or PG; and about 10 mM histidine. In some embodiments, a liquid, lyophilized, or frozen formulation is provided, comprising: attenuated live enveloped virus; about 30% w / v trehalose; about 160 mM salt, optionally NaCl; about 100 mM stabilizer, optionally MSG or PG; and about 10 mM histidine. In some embodiments, a liquid or lyophilized formulation is provided, comprising: attenuated live enveloped virus; about 30% w / v trehalose; about 160 mM salt, optionally NaCl; about 100 mM stabilizer, optionally MSG or PG; and about 10 mM histidine. In some embodiments, a liquid, lyophilized, or frozen formulation is provided, comprising: attenuated live enveloped virus; about 40% w / v trehalose; about 160 mM salt, optionally NaCl; about 100 mM stabilizer, optionally MSG or PG; and about 10 mM histidine. In some embodiments, a liquid or lyophilized formulation is provided, comprising: attenuated live enveloped virus; about 40% w / v trehalose; about 160 mM salt, optionally NaCl; about 100 mM stabilizer, optionally MSG or PG; and about 10 mM histidine. In some embodiments, the virus is RSV. In some embodiments, the vaccine is an RSV ΔNS2 / Δ1313 / I1314L vaccine.

[0135] amino acids In some embodiments, a liquid, lyophilized, or frozen formulation is provided, comprising attenuated live enveloped virus, trehalose, and one or more amino acids as desired.

[0136] The amino acids may be alanine, arginine, aspartic acid, asparagine, cysteine, leucine, isoleucine, methionine, proline, serine, lysine, histamine, glycine, glutamic acid, phenylalanine, threonine, tryptophan, tyrosine, valine, and combinations thereof. In some embodiments, the amino acids or combinations thereof are present at concentrations ranging from about 0.1% to about 10% (w / v). The amino acids may also be provided by enzymatic digestion of the protein. For example, NZ-Amine™ A (an enzymatic digest of casein) can be used to provide the combination of amino acids. In some embodiments, the virus strain is RSV. In some embodiments, the vaccine strain is RSV ΔNS2 / Δ1313 / I1314L vaccine.

[0137] rHSA In some embodiments, the formulation further comprises albumin, wherein the albumin is human serum albumin (HSA) if desired, and recombinant human serum albumin (rHSA) if desired. In some embodiments, a liquid, lyophilized, or frozen formulation is provided, comprising: attenuated live enveloped virus; trehalose; a salt, wherein the salt is NaCl if desired; a stabilizer, wherein the stabilizer is MSG or PG if desired; histidine, wherein MgCl2 or CaCl2 if desired; and HSA if desired. In some embodiments, a liquid or lyophilized formulation is provided, comprising: attenuated live enveloped virus; trehalose; a salt, wherein the salt is NaCl if desired; a stabilizer, wherein the stabilizer is MSG or PG if desired; histidine, wherein MgCl2 or CaCl2 if desired; and HSA if desired.

[0138] In some embodiments, the concentration of HSA is between about 0 and 20 mg / mL. In some embodiments, the concentration of HSA is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / mL. In some embodiments, the concentration of HSA is about 5, 10, 15, or 20 mg / mL.

[0139] In some embodiments, a liquid, lyophilized, or frozen formulation is provided, comprising: attenuated live enveloped virus; about 30%-40% w / v trehalose; about 160 mM salt, optionally NaCl; about 100 mM stabilizer, optionally MSG or PG; about 10 mM histidine; about 0-50 mM MgCl2 or CaCl2; and about 0-20 mg / mL rHSA. In some embodiments, a liquid or lyophilized formulation is provided, comprising: attenuated live enveloped virus; about 30%-40% w / v trehalose; about 160 mM salt, optionally NaCl; about 100 mM stabilizer, optionally MSG or PG; about 10 mM histidine; about 0-50 mM MgCl2 or CaCl2; and about 0-20 mg / mL rHSA. In some embodiments, a liquid, lyophilized, or frozen formulation is provided, comprising: attenuated live enveloped virus; about 30% w / v trehalose; about 160 mM salt, optionally NaCl; about 100 mM stabilizer, optionally MSG or PG; about 10 mM histidine; about 0-50 mM MgCl2 or CaCl2; and about 0-20 mg / mL rHSA. In some embodiments, a liquid or lyophilized formulation is provided, comprising: attenuated live enveloped virus; about 30% w / v trehalose; about 160 mM salt, optionally NaCl; about 100 mM stabilizer, optionally MSG or PG; about 10 mM histidine; about 0-50 mM MgCl2 or CaCl2; and about 0-20 mg / mL rHSA. In some embodiments, a liquid, lyophilized, or frozen formulation is provided, comprising: attenuated live enveloped virus; about 40% w / v trehalose; about 160 mM salt, optionally NaCl; about 100 mM stabilizer, optionally MSG or PG; about 10 mM histidine; about 0-50 mM MgCl2 or CaCl2; and about 0-20 mg / mL rHSA. In some embodiments, a liquid or lyophilized formulation is provided, comprising: attenuated live enveloped virus; about 40% w / v trehalose; about 160 mM salt, optionally NaCl; about 100 mM stabilizer, optionally MSG or PG; about 10 mM histidine; about 0-50 mM MgCl2 or CaCl2; and about 0-20 mg / mL rHSA. In some embodiments, the virus is RSV. In some implementations, the vaccine is the RSV ΔNS2 / Δ1313 / I1314L vaccine.

[0140] B. Virus Any live attenuated vaccine can be formulated as described herein. Vector-based vaccines are also covered, which in some cases can also be considered live attenuated vaccines. In some cases, live attenuated vaccines use a weakened strain of the virus that causes disease. In some cases, live attenuated vaccines are considered attenuated because they are administered to a host species that does not produce a significant immune response to the virus, but the virus can produce an immune response in another host species. Each virus discussed herein can be present in a live attenuated formulation or a vector-based formulation. In embodiments comprising a vector-based formulation, the virus may be a vector virus containing heterologous viral antigens, or may be a source of heterologous viral antigens.

[0141] In some embodiments, the virus is a enveloped virus. In some embodiments, the virus is an attenuated live enveloped virus. In some embodiments, the virus is an RNA virus. In some embodiments, the virus is a DNA virus. In some embodiments, the virus is an enveloped DNA virus. In some embodiments, the virus is an enveloped RNA virus.

[0142] In some embodiments, the virus belongs to the Paramyxoviridae family, which is an antisense, enveloped, single-stranded RNA virus family with a genome of approximately 13-19 kb. In some embodiments, the paramyxovirus is a respiratory syncytial virus (RSV). In some embodiments, the RSV is human RSV (also known as human orthopneumovirus).

[0143] In some embodiments, the viral lineage is RSV and includes a formulation for administration to pediatric subjects (RSV ΔNS2 / Δ1313 / I1314L), as disclosed, for example, in WO 2017100756 A1, which is incorporated herein by reference in its entirety. In some embodiments, the viral lineage is RSV and includes an attenuated RSV with point mutations, as described, for example, in WO 2013154728 A1, which is incorporated herein by reference in its entirety. In some embodiments, the viral lineage is RSV and includes mutations that interfere with RSV protein expression within RSV open reading frames, as described, for example, in WO 2017100759 A1, which is incorporated herein by reference in its entirety. In some vector-based formulation embodiments, the heterologous antigen is derived from RSV virus, wherein, if desired, the antigen is RSV F and / or RSV G. In some embodiments, the virus is RSV and includes a formulation (RSVΔNS2 / Δ1313 / I1314L) for administration to pediatric subjects. This is a recombinant infectious respiratory fusion cell virus comprising a large polymerase protein (L), a phosphoprotein (P), a nucleocapsid protein (N), an M2-1 protein, nonstructural protein 1 (NS1), a glycoprotein (G), a fusion protein (F), a matrix protein (M), an M2-2 protein, and a small hydrophobic protein (SH), and a genome or antigenome having: a deletion of the codon encoding the serine at position 1313 or the corresponding position of the L protein; a mutation at amino acid sequence residue 1314 or the corresponding position of the L protein, wherein the mutation at amino acid sequence residue 1314 of the L protein is an amino acid substitution of leucine for isoleucine (wherein leucine is encoded by a codon described as CTG); and a deletion of the NS2 gene, as described, for example, in WO 2013154728 A1.

[0144] In some implementations, when used with any of the formulations described herein, the effective amount of RSV comprises approximately 5.6 log. 10 Plaque-forming units (PFU) / agent. In some embodiments, when used with any of the formulations described herein, the effective amount of RSV comprises approximately 6.2 log... 10 Plaque-forming units (PFU) / agent. In some embodiments, when used with any of the formulations described herein, the effective amount of RSV comprises approximately 5.6 log... 10 PFU to 6.2 log 10 PFU / dosage. In some embodiments, when used with any of the formulations described herein, the effective amount of RSV comprises about 5 log [units missing]. 10 PFU to 9 log 10PFU / dosage. In some embodiments, when used with any of the formulations described herein, the effective amount of RSV comprises about 5 log [units missing]. 10、 6 log 10 7 log 10 8 log 10 or 9 logs 10 PFU / dosage. In some embodiments, when used with any of the formulations described herein, the effective amount of RSV comprises about 5 log [units missing]. 10 Approximately 8 log 10 PFU / dosage. In some embodiments, when used with any of the formulations described herein, the effective amount of RSV comprises about 5 to about 6 log. 10 PFU / dose or approximately 6.1 to approximately 7 log 10 PFU / dose or approximately 7.1 to approximately 8 log 10 PFU / dose or approximately 8.1 to approximately 9 log 10 PFU / dosage.

[0145] In some embodiments, the RSV vaccine is delivered intranasally as a liquid formulation. In some embodiments, the RSV vaccine dose is delivered intranasally in a volume of about 0.2 mL. In some embodiments, the 0.2 mL dose is delivered intranasally, with about 0.1 mL delivered to each nostril. In some embodiments, an intranasal nebulizer is used to deliver the RSV vaccine dose intranasally in volumes of about 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, or 1.0 mL. As described above, the dose can be evenly distributed between the two nostrils, substantially evenly distributed between the two nostrils, unevenly distributed between the two nostrils, or delivered entirely to one nostril in one or more deliveries.

[0146] In some embodiments, the virus (e.g., paramyxovirus) is a parainfluenza virus (PIV), including human (HPIV), wherein the HPIV is HPIV-1, HPIV-2, HPIV-3, or HPIV-4, as needed. In some embodiments, the virus or paramyxovirus causes respiratory infection. In some embodiments, the PIV is bovine PIV (BPIV). In some embodiments, the PIV is mouse PIV (MPIV). In some embodiments, the PIV and / or PIV vaccine are disclosed in US 20180312544 A1, WO9853078 A1, WO 0104320 A1, WO 0103744 A3, WO 0142445 A3, or WO 0202605 A3, each of which is incorporated herein by reference in its entirety. In some vector-based formulation embodiments, the heterologous antigen is derived from the PIV virus. In some vector-based formulation embodiments, the virus containing the heterologous antigen is derived from the PIV virus.

[0147] In some embodiments, the PIV is recombinant. In some embodiments, the PIV contains one or more phenotype-specific mutations, which are introduced in a selected combination into the genome or antigenome of an infectious strain to produce desired properties, including attenuation, temperature sensitivity, cold adaptation, small plaque size, host range restriction, etc. In some embodiments, infectious PIV particles (e.g., viral or subviral particles) are generated from one or more separate polynucleotide molecules encoding the PIV genome or antigenome. Infectious PIV particles can be generated by co-expressing in a cellular or cell-free system an expression vector containing a separate polynucleotide molecule encoding the PIV genome or antigenome with an expression vector containing one or more separate polynucleotide molecules encoding the N, P, and L proteins of PIV. In some embodiments, the PIV or recombinant PIV is used as a live attenuated vaccine (i.e., a non-vector-based vaccine).

[0148] In some embodiments, the vaccine is a PIV-based vaccine. In one such embodiment, the formulation comprises a recombinant human, bovine, or murine parainfluenza virus vector, wherein the PIV is bovine or human PIV3, if desired. In some embodiments, the PIV is used as a vector for expressing heterologous antigens (e.g., RSV, hMPV, measles virus, or mumps virus). In some embodiments, the formulation comprises a PIV vector and RSV F antigen. In some embodiments, the formulation comprises a PIV vector and hMPV antigen. In some embodiments, the formulation comprises a PIV vector and human metastatic pneumonia virus (hMPV), RSV, HSV, coronavirus, Newcastle disease virus, yellow fever virus, dengue virus, or other enveloped virus antigens.

[0149] In some embodiments, the vaccine is a PIV-based vector vaccine containing PIV3. In some embodiments, the vaccine further contains at least one antigen from a non-PIV virus. In some embodiments, additional non-PIV antigens include human host pneumonia virus (hMPV), RSV, HSV, coronavirus, Newcastle disease virus, yellow fever virus, dengue virus, or other enveloped viruses.

[0150] In some embodiments, the virus is a member of the Herpesviridae family. In some embodiments, the virus is herpes simplex virus (HSV). In some embodiments, HSV is herpes simplex virus 1 (HSV-1), human alpha herpesvirus 1, herpes simplex virus 2 (HSV-2), or human alpha herpesvirus 2. In some embodiments, HSV causes blisters, small ulcers, and / or fever.

[0151] In some embodiments, the virus is a member of the Pneumoviridae family. In some embodiments, the virus is human host pneumonia virus (hMPV). In some embodiments, hMPV causes respiratory infection.

[0152] In some embodiments, the virus is an influenza (flu) virus. In some embodiments, the influenza virus is influenza A, B, C, or D. In various embodiments, the influenza A virus belongs to subtypes H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H7N9, or H6N1. In some embodiments, the virus belongs to the Yamagata lineage. In some embodiments, the virus belongs to the Victoria lineage. In some embodiments, the influenza antigen is incorporated into a vector-based vaccine, wherein the influenza antigen is, as needed, a hemagglutinin (HA) or neuraminidase (NA) protein. In some embodiments, the influenza virus causes fever, runny nose, sore throat, muscle and joint pain, headache, cough, and / or malaise.

[0153] In some embodiments, the virus is a flavivirus. In various embodiments, the flavivirus is one of approximately 70 viruses belonging to the genus *Flavaviridae* of the family Flaviviridae. In some embodiments, the flavivirus includes yellow fever virus, dengue virus, Japanese encephalitis virus, West Nile virus, Zika virus, and tick-borne encephalitis virus. In some embodiments, yellow fever virus causes fever, chills, muscle pain, headache, or jaundice. In some embodiments, dengue virus causes fever, headache, muscle and joint pain, or rash. In some embodiments, Japanese encephalitis virus causes headache, fever, vomiting, confusion, and / or seizures. In some embodiments, West Nile virus causes fever, headache, vomiting, neck stiffness, confusion, seizures, and / or rash. In some embodiments, Zika virus causes fever, red eyes, joint pain, headache, and / or maculopapular rash. In some embodiments, tick-borne encephalitis virus causes fever, malaise, headache, nausea, vomiting, and / or myalgia.

[0154] In some embodiments, the virus is measles virus. In some embodiments, the measles virus belongs to the genus Measlesvirus within the family Paramyxoviridae. In some embodiments, the measles virus is measles virus (MV), erythrocyte virus, or rubella virus. In some embodiments, the measles virus causes fever, cough, runny nose, eye inflammation, and / or rash.

[0155] In some embodiments, the virus is mumps virus. In some embodiments, the mumps virus belongs to the genus Orthopaedicvirus and the family Paramyxoviridae. In some embodiments, the mumps virus is orthopaedicvirus (… Mumps orthorubulavirus ) or classic mumps virus ( Mumps rubulavirus In some implementations, the mumps virus causes fever, muscle pain, headache, and / or painful swelling of the parotid glands.

[0156] In some embodiments, the virus is rubella virus. In some embodiments, the rubella virus is chlamydia virus. In some embodiments, the rubella virus causes fever, swollen lymph nodes, malaise, and / or rash.

[0157] In some embodiments, the virus is the varicella-zoster virus. In some embodiments, the varicella-zoster virus is the varicella-zoster virus. In some embodiments, the varicella-zoster virus causes small itchy blisters, fever, fatigue, and / or headache.

[0158] In some embodiments, the virus is a smallpox virus. In some embodiments, the smallpox virus is a severe form of smallpox (Severe smallpox). Variola major or lightweight ceiling ( Variola minorIn some embodiments, the smallpox virus causes fever, vomiting, oral ulcers, and / or a rash that develops into a characteristic fluid-filled mass with a central depression, which crusts over and falls off, leaving a scar.

[0159] In some embodiments, the virus is a poliovirus. In some embodiments, the poliovirus belongs to enterovirus C of the Picornaviridae family. In some embodiments, the poliovirus causes abortive poliomyelitis. In various embodiments, the poliovirus causes paralytic or nonparalytic poliomyelitis.

[0160] In some embodiments, the virus is rotavirus. In some embodiments, the rotavirus belongs to the Reoviridae family. In some embodiments, the rotavirus is rotavirus A, B, C, D, E, F, G, H, I, or J. In some embodiments, the rotavirus causes nausea, vomiting, watery diarrhea, and / or low-grade fever.

[0161] C. Adjuvant In some embodiments, the vaccine contains an adjuvant. This includes any adjuvant known to those skilled in the art. Non-limiting exemplary adjuvants include incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, alum, Stimulon™ QS-21 (Aquila Biopharmaceuticals, Inc., Worchester, Massachusetts), MPL™ (3-O-deacylated monophospholipid A; RIBI ImmunoChem Research, Inc., Hamilton, Montana), and interleukin-12 (Genetics Institute, Cambridge, Massachusetts).

[0162] In some embodiments, the formulation is a liquid. In some embodiments, the formulation is dry, for example, lyophilized.

[0163] III. Products In some embodiments, the formulation is provided in a container, device, or kit. In one embodiment, the device is for parenteral delivery, such as intranasal, intramuscular, subcutaneous, or by inhalation. In some embodiments, the formulation is provided in a syringe (such as a drug-loaded syringe), an autoinjector, an intranasal device (such as a syringe), a nebulizer delivery device (e.g., the formulation is nebulized), a dropper, or an injection device. Such a drug-loaded syringe or autoinjector device contains a formulation that can maintain a titer loss of 1 log at room temperature. 10For at least 3-6 months, the PFU / ml level should remain below 2-8 degrees Celsius, and for at least 12-24 months.

[0164] In some embodiments, the formulation is provided in a container, including but not limited to syringes (including drug-loaded syringes), autoinjectors, intranasal nebulizers, or bottles. This includes pre-filled devices (e.g., spray or nebulizer containers) for intranasal administration containing the formulation. In some embodiments, the pre-filled intranasal device is formulated for direct administration to a subject, as needed, a pediatric subject. Such a pre-filled device for intranasal administration contains a formulation that can maintain a titer loss of 1 log at room temperature. 10 For at least 3, 4, 5 or 6 months, the PFU / ml level must be below 2-8 degrees Celsius, and the level must be maintained for at least 12-24 months.

[0165] IV. Usage and Applications In some embodiments, a method is provided to prevent or reduce the likelihood of a subject being infected with a membrane-bound RNA or DNA virus, comprising administering a formulation as disclosed herein. In some embodiments, a method is provided to vaccinate a subject against membrane-bound RNA or DNA virus infection, comprising administering a formulation as disclosed herein. In some embodiments, a method is provided to trigger or generate an immune response in a subject, comprising administering a formulation as disclosed herein.

[0166] In some embodiments, the formulation is used to prevent or reduce the likelihood of a subject being infected with a membrane-bound RNA or DNA virus. In some embodiments, the formulation is used in a method of vaccinating a subject against membrane-bound RNA or DNA virus infection. In some embodiments, the formulation is used to trigger or generate an immune response in a subject.

[0167] In some embodiments, the formulation is provided for use in the preparation of a medicament for preventing or reducing the likelihood of a subject being infected with a membrane-bound RNA or DNA virus. In some embodiments, the formulation is provided for use in the preparation of a medicament for vaccinating a subject against membrane-bound RNA or DNA virus infection. In some embodiments, the formulation is provided for use in the preparation of a medicament for triggering or generating an immune response in a subject.

[0168] In each of the methods and uses described herein, infection may be caused by any of the viruses described above.

[0169] In some embodiments, the method and its use are for treating infections caused by viruses of the Paramyxoviridae family. In some embodiments, the paramyxovirus is a respiratory syncytial virus (RSV). In some embodiments, the RSV is human RSV (also known as human orthopneumovirus). In some embodiments, the virus is an RSV and includes a formulation for administration to pediatric subjects (RSV ΔNS2 / Δ1313 / I1314L), as disclosed, for example, in WO 2017100756 A1, which is incorporated herein by reference in its entirety. In some embodiments, the virus is an RSV and includes an attenuated RSV with point mutations, as described, for example, in WO 2013154728 A1, which is incorporated herein by reference in its entirety. In some embodiments, the virus is an RSV and includes mutations that interfere with RSV protein expression within an RSV open reading frame, as described, for example, in WO 2017100759 A1, which is incorporated herein by reference in its entirety. In some carrier-based formulation implementations, the heterologous antigen is derived from RSV virus, wherein, if necessary, the antigen is RSV F and / or RSV G. In some embodiments, the virus is RSV and includes a formulation (RSV ΔNS2 / Δ1313 / I1314L) for administration to pediatric subjects. This is a recombinant infectious respiratory fusion cell virus comprising a large polymerase protein (L), a phosphoprotein (P), a nucleocapsid protein (N), an M2-1 protein, nonstructural protein 1 (NS1), a glycoprotein (G), a fusion protein (F), a matrix protein (M), an M2-2 protein, and a small hydrophobic protein (SH), and a genome or antigenome having: a deletion of the codon encoding the serine at position 1313 or the corresponding position of the L protein; a mutation at amino acid sequence residue 1314 or the corresponding position of the L protein, wherein the mutation at amino acid sequence residue 1314 of the L protein is an amino acid substitution of leucine for isoleucine (wherein leucine is encoded by a codon described as CTG); and a deletion of the NS2 gene, as described, for example, in WO 2013 / 154728 A1.

[0170] In some embodiments, the method and use are for treating infections caused by parainfluenza viruses (PIVs), including human PIV (HPIV) as needed, wherein the HPIV is HPIV-1, HPIV-2, HPIV-3, or HPIV-4. In some embodiments, the PIV causes a respiratory infection. In some embodiments, the PIV is bovine PIV (BPIV). In some embodiments, the PIV is mouse PIV (MPIV). In some embodiments, the PIV and / or PIV vaccines are disclosed in US 20180312544 A1, WO9853078 A1, WO 0104320 A1, WO 0103744 A3, WO 0142445 A3, or WO 0202605 A3, each of which is incorporated herein by reference in its entirety.

[0171] In some embodiments, the method and its use are for treating infections caused by viruses of the Herpesviridae family. In some embodiments, the virus is herpes simplex virus (HSV). In some embodiments, the HSV is herpes simplex virus 1 (HSV-1), human alpha herpesvirus 1, herpes simplex virus 2 (HSV-2), or human alpha herpesvirus 2. In some embodiments, HSV causes blisters, small ulcers, and / or fever.

[0172] In some embodiments, the method and its use are for treating infections caused by viruses of the Pneumoviridae family. In some embodiments, the virus is human host pneumonia virus (hMPV). In some embodiments, hMPV causes respiratory infections.

[0173] In some embodiments, the method and its use are for treating infections caused by influenza (flu) viruses. In some embodiments, the influenza virus is influenza A, B, C, or D. In various embodiments, the influenza A virus belongs to subtypes H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H7N9, or H6N1. In some embodiments, the virus belongs to the Yamagata lineage. In some embodiments, the virus belongs to the Victoria lineage. In some embodiments, the influenza virus causes fever, runny nose, sore throat, muscle and joint pain, headache, cough, and / or malaise.

[0174] In some embodiments, the method and its use are for treating infections caused by flaviviruses. In various embodiments, flaviviruses are one of approximately 70 viruses belonging to the genus *Flavaviridae* of the family Flaviviridae. In some embodiments, flaviviruses include yellow fever virus, dengue virus, Japanese encephalitis virus, West Nile virus, Zika virus, and tick-borne encephalitis virus. In some embodiments, yellow fever virus causes fever, chills, muscle pain, headache, or jaundice. In some embodiments, dengue virus causes fever, headache, muscle and joint pain, or rash. In some embodiments, Japanese encephalitis virus causes headache, fever, vomiting, confusion, and / or seizures. In some embodiments, West Nile virus causes fever, headache, vomiting, neck stiffness, confusion, seizures, and / or rash. In some embodiments, Zika virus causes fever, red eyes, joint pain, headache, and / or maculopapular rash. In some embodiments, tick-borne encephalitis virus causes fever, malaise, headache, nausea, vomiting, and / or myalgia.

[0175] In some embodiments, the method and its use are for treating infections caused by measles virus. In some embodiments, the measles virus belongs to the genus Measlesvirus within the family Paramyxoviridae. In some embodiments, the measles virus is measles virus (MV), erythrocyte virus, or rubella virus. In some embodiments, the measles virus causes fever, cough, runny nose, eye inflammation, and / or rash.

[0176] In some embodiments, the method and its use are for treating infections caused by the mumps virus. In some embodiments, the mumps virus belongs to the genus Orthopaedicvirus and the family Paramyxoviridae. In some embodiments, the mumps virus is orthopaedic virus or classical mumps virus. In some embodiments, the mumps virus causes fever, muscle pain, headache, and / or painful swelling of the parotid gland.

[0177] In some embodiments, the method and its use are for treating infections caused by rubella virus. In some embodiments, the rubella virus is a chlamydia virus. In some embodiments, the rubella virus causes fever, swollen lymph nodes, malaise, and / or rash.

[0178] In some embodiments, the method and its use are for treating infections caused by the varicella-zoster virus. In some embodiments, the varicella-zoster virus is the varicella-zoster virus. In some embodiments, the varicella-zoster virus causes small, itchy blisters, fever, fatigue, and / or headache.

[0179] In some embodiments, the method and its use are for treating infections caused by the smallpox virus. In some embodiments, the smallpox virus is severe or mild smallpox. In some embodiments, the smallpox virus causes fever, vomiting, oral ulcers, and / or a rash that develops into a characteristic fluid-filled mass with a central depression, which crusts over and falls off, leaving a scar.

[0180] In some embodiments, the method and its use are for treating infections caused by poliovirus. In some embodiments, the poliovirus belongs to enterovirus C of the Picornaviridae family. In some embodiments, the poliovirus causes abortive poliomyelitis. In various embodiments, the poliovirus causes paralytic or nonparalytic poliomyelitis.

[0181] In some embodiments, the method and its use are for treating infections caused by rotavirus. In some embodiments, the rotavirus belongs to the Reoviridae family. In some embodiments, the rotavirus is rotavirus A, B, C, D, E, F, G, H, I, or J. In some embodiments, the rotavirus causes nausea, vomiting, watery diarrhea, and / or low-grade fever.

[0182] The formulations disclosed herein can be used alone at appropriate doses that allow for optimal inhibition of viral infection while minimizing potential toxicity. Furthermore, co-administration or sequential administration of other agents may be desirable.

[0183] The formulations and compositions of the present invention can be administered to a patient intranasally, intramuscularly, subcutaneously, intradermally, or via skin imprint. Other administration methods are also contemplated, such as intraperitoneal, intravenous, or inhalation delivery. In some embodiments, the formulations and compositions disclosed herein can be delivered by intranasal nebulization. In some embodiments, the formulation is administered directly to the subject, for example, without reconstitution or dilution.

[0184] In some embodiments, the pharmaceutical components and formulations disclosed herein are administered to a patient in various prime / booster combinations to induce an enhanced and durable immune response. In this case, the two pharmaceutical components are administered in a "primer and booster" regimen. For example, the first component is administered once or more, followed by the second component once or more after a predetermined time interval (e.g., 2 weeks, 1 month, 2 months, 6 months, or other appropriate interval).

[0185] In some embodiments, the live attenuated virus vaccine is administered by injection. In some embodiments, the live attenuated virus vaccine is in the form of a nasal spray. In some embodiments, the live attenuated virus vaccine is administered as an intranasal aerosol spray. In some embodiments, the live attenuated virus vaccine is an oral vaccine. In various embodiments, the vaccine is administered in one or more doses.

[0186] In any of the foregoing embodiments involving a subject, the subject may be a mammal. In any of the foregoing embodiments involving a subject, the subject may be a human. In any of the foregoing embodiments involving a subject, the subject may be a cow, pig, monkey, sheep, dog, cat, fish, or poultry.

[0187] Example The following examples are provided to illustrate certain implementation methods of the disclosure and should not be construed in any way as limiting the scope of this disclosure.

[0188] Example 1. Materials and Methods 1.1. Materials RSV vaccine strain RSV ΔNS2 / Δ1313 / I1314L is an attenuated live virus vaccine. It is genetically engineered to contain a full gene deletion (ΔNS2) and point mutations (Δ1313 and I1314L) to achieve an appropriate level of attenuation. At the same time, it maintains an effective level of immunogenicity by intranasal administration, thereby inducing innate, humoral and cellular responses on the mucosal surface.

[0189] Table 1: List of Active Materials *The pharmaceutical product formulations tested in the following examples are made from the active materials listed in Table 1.

[0190] Table 2: List of Chemical Materials Example 1.2. Method 1.2.1. Stability Assessment Accelerated temperature stability studies were designed to evaluate sample formulations stored at 37°C ± 2°C for up to 2 or 3 weeks. Additionally, data were collected from samples stored at 25°C ± 2°C for up to 8 or 12 weeks and at 45°C ± 2°C for up to 5 or 8 days. In this context, "accelerated" means operating above storage temperatures of approximately 2°C–8°C, such that reactions or degradation may be accelerated compared to the storage temperature.

[0191] Real-time stability studies were conducted using storage conditions of 2°C to 8°C for up to 12 months, 24 months, 36 months or longer.

[0192] Freeze-thaw (F / T) stress: A freeze-thaw stress study was designed, and five freeze-thaw cycles were performed between -60°C and room temperature. All five samples were evaluated together with a frozen control at time zero (T0).

[0193] The bioactivity of samples in each stability assessment study was evaluated using plaque assays (stability indicator assays that can directly measure the viral infectivity titer at each stability time point), as described below.

[0194] 1.2.2. Freeze-drying (FD) Freeze-drying (FD) (sometimes referred to herein as “lyo”, “freeze-drying”, etc.) is a process used to remove water from formulations at low temperatures and prevent thermal degradation through sublimation. This FD process involves three stages: freezing, primary drying, and secondary drying. All samples underwent a pre-cooling step at 4°C to ensure a uniform starting temperature before freeze-drying. In this study, the formulations were freeze-dried (lyo) in an FTS LyoStar II freeze dryer. Drying parameters are provided in Table 3.

[0195] Table 3: Examples of FD loop parameters 1.2.3 Spray drying Spray drying (SD) is a process that transforms a liquid formulation into a dry powder by removing moisture. In SD, the liquid product is sprayed through nozzles into a chamber containing hot air. When the droplets of the liquid formulation come into contact with the hot air, the hot air evaporates the solvent of the liquid formulation into powder form. The liquid formulation described in this article was processed using a BUCHI MiniSpray Dryer B-290 according to the operating procedures. All SD components were autoclaved before use to allow for aseptic operation. The drying parameters in Table 4 were used for testing.

[0196] Table 4: Examples of SD process parameters 1.2.4 Foam Drying Foam drying is an alternative to freeze drying. Foam drying is performed in an FTS LyoStar II freeze dryer without a freezing stage. The dried product produced by foam drying does not form a cake, but rather produces a bubbly, glassy foam. Exemplary foam drying cycle parameters are included in Table 5.

[0197] Table 5: Examples of foam drying cycle parameters 1.2.5. Infectivity titer measurement (plaque assay) Viral infectivity titers are determined by plaque assays, which are indicators of viral infectivity used to monitor the biological stability of samples. Higher titers indicate better sample stability, or lower titer loss indicates better sample stability. No other stability indicators have been identified for measuring the biological stability of respiratory syncytial virus (RSV). Plaque assays are used for formulations and stability evaluation as described herein.

[0198] The infectivity titer of RSV samples was assessed by titration on complementary Vero cell lines. 48-well plates were seeded one day prior to infection. Samples were serially diluted, plated, and incubated at 34°C and 5% CO2 for 4 days. RSV plaques were visualized by immunostaining. Immunostaining was performed by adding conjugated primary anti-RSV antibody (5353C75.1) (1:1000 dilution) to each well and incubating the 48-well plates at 36°C and 5% CO2 for 20 minutes with a 1-step TMB blot solution (Thermo Fisher Scientific, USA). After washing, plaques were automatically counted using a Viruscope (MicroVision Instruments, EVRY Cedex France). The infectivity titer was then calculated by averaging triplicate wells and expressed as plaque-forming units per milliliter (PFU / mL) or log 10 PFU / mL. The measurement deviation is within ±0.2 log₂. 10 Within PFU / mL.

[0199] 1.2.6. Data Analysis 1) Lyo loss – The Lyo loss value is calculated as the difference between the average log titration amount before lyo and the average log titration amount after lyo.

[0200] 2) Storage loss – The storage loss value is calculated as the difference between the average logarithmic titration value at time zero and the average logarithmic titration value after different time points and storage conditions.

[0201] 3) Data analysis of viral degradation rates – Commercial plotting software was used to plot the trend of the degradation curves and perform regression analysis. The slope or constant of the regression equation was considered the degradation rate. The degradation rate (i.e., the slope obtained from linear regression analysis) was used to compare formulation stability and extrapolate the predicted degradation time at 37°C for 14 days. The degradation rate and time were used to guide the selection of stable formulations.

[0202] 4) Analysis of Covariance (ANOVA) – Perform ANOVA analysis of the degradation slope of the drug product (DP) formulation across multiple batches in development studies of high- and low-dose formulations. Measure representative drug substance (DS) batches formulated at high and low doses in validation studies to investigate the significance of differences between batches and formulations, thereby aiding decision-making. p A value <0.05 is considered statistically significant.

[0203] 5) Advanced Kinetics and Technical Solutions (AKTS) Analysis – AKTS is software capable of analyzing kinetic models for reaction complexity, independent of reactions ranging from one-step to multi-step. AKTS uses a combination of two Sestak-Berggren (SB) models, including the Arrhenius equation. Arrhenius's contribution to the model allows analysis of degradation data obtained at various storage temperatures (e.g., 5°C, 25°C, 37°C, 45°C, etc.), in contrast to traditional linear ICH modeling methods, which are based solely on degradation data under normal storage conditions at 5°C (ICH Guideline Q1E). The simultaneous combination of the SB equations in AKTS enables consideration of all kinetic models used in the literature and ranking of models applied to autocatalytic reactions. AKTS provides Akaike Information Criterion and Bayesian Information Criterion (AIC and BIC) to automatically help prevent overfitting and distinguish the best kinetic reaction model among all mathematically fitted models. AKTS utilizes a bootstrap method to calculate the prediction confidence interval (CI) (e.g., 95% CI), thereby allowing for reliable estimation of long-term stability or shelf-life predictions (see, for example, D. Clenet et al., Advanced Kinetic Analysis as a Tool for Formulation Development and Prediction of Vaccine Stability], J. PHARM. SCI. 103:3055–3064, 2014). Here, a 3-month data analysis was used for formulation selection purposes.

[0204] 1.2.7. Research Design Stabilizing attenuated live enveloped viruses can be challenging. Therefore, many attenuated live virus vaccine candidates (e.g., herpes simplex virus-2 (HSV-2), ALVAC, etc.) have been developed as cryopreserved liquids, and there are currently few successful cases in developing thermostable liquid live enveloped virus vaccines. The industry gold standard is often freeze-dried susceptible viruses such as RSV, measles, yellow fever, and dengue fever. Formulating enveloped viruses into liquids that can be stored for extended periods can be advantageous (e.g., commercially advantageous). Extending the shelf life of enveloped viruses as freeze-dried formulations is also advantageous.

[0205] One reason for developing more stable liquid and lyo formulations is the complexity and cost of cold chain logistics, as well as the challenges of industrializing frozen liquid formulations. RSV ΔNS2 / Δ1313 / I1314L, sometimes referred to as RSVΔNS2 (as described in WO 2013154728), was evaluated as an improved storage option for both liquid and lyophilized forms. RSV ΔNS2 / Δ1313 / I1314L with different stabilizers was first evaluated in liquid and lyo formulations at an accelerating temperature of 37°C ± 2°C. Potentially stable formulations were then further investigated under real-time storage conditions of 5°C ± 3°C for durations of 3, 6, 12, 24, or 36 months.

[0206] 1.2.8. Selection Criteria for Formulations The selection criterion for formulations is infectious titration loss. Generally, the lower the titration loss during processing and storage, the better the stability of the formulation.

[0207] The World Health Organization (WHO) has established guidelines for the evaluation of vaccine stability and recommends conducting basic and accelerated stability studies (Guidelines on the Stability Evaluation of Vaccines, Geneva: World Health Organization [Geneva Guidelines on the Stability Evaluation of Vaccines: World Health Organization]; 2006. Page 28, Report No.: WHO / BS / 06.2049 Final (WHO Technical Report Series No. 962: Guidelines on the Stability Evaluation of Vaccines, Geneva: World Health Organization [Geneva Guidelines for the Evaluation of Vaccine Stability: World Health Organization]; 2011. 28. Report No.: 57). However, different criteria have been used in the literature, and different titer losses have been reported for different attenuated live virus formulations. For example, for influenza vaccines, less than 1 log [amount missing] was observed after use at 25°C for up to 6 weeks. 10 The loss of PFU / mL (White JA et al., Vaccine [Vaccines], 2016;34(32):3676-83), this influenza vaccine provides storage stability for up to one year at 2°C to 8°C. In another instance, for measles preparations, less than 1 log [unclear] was used for 8 hours at 40°C. 10 PFU / mL (Schlehuber et al.) Vaccine [Vaccines], 2011;29(31):5031-9). Typically, the general standard described in the literature is less than 1 log [value] at 37°C for one week. 10 Titration loss of PFU / mL (Ohtake S et al., Vaccine [vaccine]; 2010;28(5):1275-84; Wiggan et al., Vaccine [Vaccines], 2011;29(43):7456-62; and Bhambhabi A et al., U.S. Patent Application Publication No. 2016 / 0250319).

[0208] For these studies, our goal is to find a formulation that will allow titration loss to remain less than 1 log for 1–2 weeks. 10 PFU / mL, for example, less than 1 log at 37°C for 10 days or 2 weeks. 10 PFU / mL (Ohtake S et al., Vaccine [vaccine]; 2010;28(5):1275-84).

[0209] Accelerated stability data at 37°C were used for formulation selection, with fewer real-time stability time points because accelerated stability testing is convenient and time-saving. Viral stability was also monitored at different accelerated storage temperatures of 25°C and 45°C in the validation study. Test samples exhibited significant and easily detectable degradation over relatively short time periods. The degradation rate of each formulation at 37°C was compared for formulation selection. A freeze-thaw stress study was designed, and five freeze-thaw cycles were performed between ≤ -60°C and room temperature.

[0210] Example 2. Screening study of excipients for liquid and Lyo formulations A group of stabilizers and buffers with different pH values ​​were scanned to understand their function and role during formulation. Both liquid and LYO formulations were examined after screening excipients.

[0211] 2.1. Effects of Trehalose and Salt Concentration The formulation studies in Study 1 were conducted at different trehalose and salt concentrations, starting with drug substance (DS) L1 and adjusted by dilution or addition of components as needed. Four excipients and their combinations were investigated in both liquid and lyo formulations. Figure 1A The experimental design described in the figure evaluates liquid and lyo formulations (legend: trehalose (10% = -1, 20% = 0 and 30% = 1), NaCl (0 mM = -1, 80 mM = 0 and 160 mM = 1), monosodium glutamate (MSG) (0 mM = -1, 50 mM = 0 and 100 mM = 1), histidine (His) (3.33 mM = -1, 10 mM = 0 and 16.67 mM = 1). Figure 1A The plaque determination results of all formulation components are shown in Table 6 and Figure 1B and 1C Table 6 includes a comparison of accelerated thermal stability performance based on different formulations.

[0212] Table 6: Summary of the effect of excipient concentration on stability Note: T = trehalose; N = NaCl; M = MSG; H = histidine Figure 1B The observed dose-response effect is depicted, and it is shown that RSV ΔNS2 / Δ1313 / I1314L is a key stabilizer in trehalose-based liquid formulations (see black bars). Notably, except for F5 (30% trehalose, MSG and NaCl-free) and F10 (30% trehalose, NaCl-free) in liquids, the titration loss observed in the 10% trehalose formulation (see horizontal arrows marked 10%, 20%, and 30%) is greater than that observed in the 20% and 30% trehalose formulations. In most 30% trehalose formulations containing salt, the storage loss over 1 week at 37°C is approximately or <1 log. 10 PFU / mL. For example... Figure 1B As shown in Table 6, the 10% trehalose formulation also demonstrates that, compared to the 20% and 30% trehalose formulations, the 10% trehalose formulation has a reduced protective effect in liquid formulations.

[0213] in addition, Figure 1B The trehalose concentration is also a key stabilizer in the LYO formulation (see white bar). Compared to the 20% and 10% trehalose formulations, the 30% trehalose formulation exhibits the least titration loss during lyophilization (e.g., manufacturing) and storage (e.g., shelf life) (see arrows labeled 10%, 20%, and 30%). The 20% trehalose LYO formulation also shows reduced titration loss compared to the 10% trehalose LYO formulation.

[0214] like Figure 1C As shown, NaCl can act as a stabilizer for 30% trehalose liquid formulations. For example, formulations F1 and F8 containing NaCl exhibit better stability compared to formulations F10 and F5, which lack NaCl. Furthermore, formulation F5, lacking both monovalent salts NaCl and MSG, showed an infectious titration loss of 3 log10 PFU / mL after one week at 37°C, even when containing 30% trehalose and histidine.

[0215] It was determined that both the liquid and lyo 30% trehalose formulations maintained biological activity after incubation at 37°C for 2 weeks. Furthermore, samples containing NaCl, in addition to 30% trehalose, exhibited better liquid stability curves, indicating that NaCl enhances liquid stability. Samples containing 30% trehalose and MSG, with or without NaCl, also showed improved liquid stability. Therefore, 30% trehalose, NaCl, and / or MSG effectively preserve the virus and improve the thermal stability of both the liquid and lyo formulations. Among the excipients tested, trehalose was the only excipient assessed to significantly affect the stability curves. Moreover, monovalent salts have a synergistic effect with trehalose.

[0216] Formulations with low (10%) trehalose content are unstable in both liquid and lyo.

[0217] 2.2. Study on the concentration of 40% trehalose liquid formulation To determine whether a higher percentage of trehalose could further improve the stability of the liquid formulation, a study (Study 2) was conducted to compare a 30% trehalose control (F1 control) with a 40% trehalose control (F2). This study used development batch L2 of RSV ΔNS2 / Δ1313 / I1314L (in 30% or 40% trehalose, 10 mM histidine, 160 mM NaCl, and 100 mM MSG (pH 7)). Table 7 summarizes the stability data at different storage temperatures. The results showed that the 30% trehalose formulation was stable for up to 12 months at 5°C with a loss of approximately 0.6 log [missing value]. 10PFU / mL. The 40% trehalose formulation (F2) offers even better stability after 12 months of storage at 5°C, with less titration loss (only about 0.5 log). 10 (PFU / mL). Therefore, under these conditions, the 40% trehalose formulation exhibits better stability compared to the 30% trehalose formulation.

[0218] Table 7: Summary of stability data from Study 2 2.3. Study on the concentration of 15% trehalose liquid formulation A study (Study 3) was designed to evaluate the short-term stability performance of the 15% trehalose formulation compared to the 30% trehalose formulation, both of which included 160 mM NaCl, 100 mM MSG, and 10 mM histidine. This study was intended to determine the mixing or dilution strategy for the drug product (DP) formulations (L3 and L4) and adjuvants, or for co-dosing with other antigens (if necessary). After a 14-day period, the titer loss exceeded 1.0 log [value missing] discussed above. 10 The PFU / mL limit is set, and a 15% trehalose concentration is not used for further formulations.

[0219] Figure 2A Thermostability profiles of 15%, 20%, 30%, and 40% trehalose formulations with the same salt / buffer composition in each formulation were summarized, depicting a comparison of the decreasing trend of infectious titer over a period of up to 21 days at 37°C.

[0220] Figure 2B Stability curves for 15%, 30%, and 40% trehalose formulations were summarized, which depicted a comparison of the decreasing trend of infectious titer over a period of up to 12 months at 5°C.

[0221] Based on these comparisons, it was concluded that the formulations with 30% and 40% trehalose exhibited better stability than those with 15% and 20% trehalose.

[0222] Example 3. Stable 30% trehalose liquid formulation of RSV ΔNS2 / Δ1313 / I1314L Based on the above data, a formulation comprising 30% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine (pH 7.0) was selected for testing. Stability studies were conducted on RSV ΔNS2 / Δ1313 / I1314L. 3 mL clear glass vials with rubber stoppers and aluminum caps were filled with 0.6 mL of the vaccine product. The following studies demonstrate the long-term real-time stability of RSV ΔNS2 / Δ1313 / I1314L in this liquid formulation at 5°C.

[0223] 3.1 Real-time stability studies of two formulations of RSV ΔNS2 / Δ1313 / I1314L For RSV ΔNS2 / Δ1313 / I1314L (L5) (in 30% trehalose, 160 mM NaCl, 100 mM MSG and 10 mM histidine (pH 7.0)), stability results from the development stability study (Study 4) confirmed a shelf life of 36 months at ≤ -60°C from the date of vial filling. Furthermore, Figure 3A This indicates that after 24 months of storage at 5°C in the liquid formulation, the titration loss was only 0.79 log. 10 PFU / mL.

[0224] For the clinical DPRSV ΔNS2 / Δ1313 / I1314L (L6) in 30% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine (pH 7.0), stability results from the developmental stability study (Study 5) confirmed a shelf life of 24 months at ≤ -60°C from the date of vial filling. Furthermore, Figure 3B This indicates that after 24 months of storage at 5°C in the liquid formulation, the titration loss is only 0.87 log [value missing]. 10 PFU / mL.

[0225] The evidence provided by these studies suggests that, as described in this article, 30% and 40% trehalose formulations with a combination of salt and histidine (pH 7.0) are stable liquid virus formulations.

[0226] 3.2. Stability Study of RSV ΔNS2 / Δ1313 / I1314L (DS L5) The purified drug substance (DS) of RSV ΔNS2 / Δ1313 / I1314L (L5) was stable in the same formulation composition as described above, consisting of 30% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine (pH 7.0). Stability data for this DS L5 obtained from 120 LDS fermentation and purification batches were obtained after 5 freeze-thaw cycles (see [link to relevant documentation]). Figure 4A The results of Study 6, using RSV ΔNS2 / Δ1313 / I1314L(L5) thawed from -60°C and stored at 5°C for up to 3 months, are shown in [the table / incomplete]. Figure 4B The results of Study 6, conducted using RSV ΔNS2 / Δ1313 / I1314L (L5) thawed from -60°C and stored at 25°C for up to 5 weeks, are shown in [reference needed]. Figure 4C These results indicate that RSV ΔNS2 / Δ1313 / I1314L in 30% trehalose, 160 mM NaCl, 100 mM MSG and 10 mM histidine (pH 7.0) is a stable formulation and suitable for industrial manufacturing.

[0227] Example 4. Stable liquid formulation in 40% trehalose As further described below, Example 4 demonstrates that formulations with higher trehalose concentrations (specifically, 40% trehalose, 100 mM MSG, 160 mM NaCl, and 10 mM histidine, pH 7.0) can further minimize titration loss in liquid DP formulations during both short-term and long-term storage. This supports the use of the formulation, for example, in commercial applications as a stable liquid product stored at 5°C in classic glass vials or drug-loaded syringes.

[0228] 4.1 Stability study of 40% trehalose liquid formulation with RSV ΔNS2 / Δ1313 / I1314L (Study 7) This study confirms that the 40% trehalose liquid formulation at high doses (HD, 6.7 log) of RSV ΔNS2 / Δ1313 / I1314L 10 PFU / mL) and low dose (LD, 5.7 log) 10 Stability at various incubation temperatures (PFU / mL). Two DS batches, L5 and L7, were used in this study. Each RSV ΔNS2 / Δ1313 / I1314L DS batch had the same composition: 40% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine (pH 7.0).

[0229] RSVΔNS2 / Δ1313 / I1314L in a liquid formulation (DP formulation) containing 40% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine (pH 7.0) was monitored at four different temperatures, including a real-time storage condition of 5°C ± 3°C for up to 24 months. Accelerated stability testing was performed at incubation temperatures of 25°C ± 2°C, 37°C ± 2°C, and 45°C ± 2°C. Infectivity titration results for the 40% trehalose formulation under real-time storage conditions of 5°C ± 3°C are shown below. Figure 5A (High dose, for example, about 6 log) 10 PFU / dose) and 5B (low dose, e.g., about 5 log) 10 The stability data are shown in Table 8-11. (PFU / dose).

[0230] Table 8: Accelerated stability of development batches of 40% trehalose liquid formulation (high dose of RSV ΔNS2 / Δ1313 / I1314L) at 37°C Table 9: Accelerated stability of development batches of 40% trehalose liquid formulation (low dose (LD) of RSV ΔNS2 / Δ1313 / I1314L) at 37°C Table 10: Real-time stability of development batches of 40% trehalose liquid formulation (RSV ΔNS2 / Δ1313 / I1314L High Dose (HD)) at 2°C to 8°C Table 11: Real-time stability of development batches of 40% trehalose liquid formulation (low dose (LD) of RSV ΔNS2 / Δ1313 / I1314L) at 2°C to 8°C The above results indicate that the 40% trehalose-based stable liquid formulation maintains a stability level of no more than 1 log1 after 36 months of storage at 5°C in the long-term stability study. 10 Titration loss of PFU / mL was observed. Furthermore, no more than 1 log [value missing] was observed after storage at 37°C for 7 days. 10 Titration loss of PFU / mL.

[0231] 4.2. Stability study of DS batch 8 (Study 8) In view of the above stability results, the purified DS RSV ΔNS2 / Δ1313 / I1314L L8 was stabilized in a formulated composition comprising 40% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine (pH 7.0) for further scale-up of industrial production and an attempt was made to obtain a more stable liquid product. The DS batch 8 of RSV ΔNS2 / Δ1313 / I1314L was freeze-thawed five times and stability data were measured as Figure 6A shown. The results of Study 8 using RSVΔNS2 / Δ1313 / I1314L (L8) thawed from -60°C and stored at 5°C for up to 3 months are shown in Figure 6B , the results for up to 24 months are shown in Figure 6C , where the titer loss was 1.31 log, and the results for storage at 25°C for up to 5 weeks are shown in Figure 6D . The results indicate that 40% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine (pH 7.0) is a stable formulation suitable for commercial manufacturing implementation.

[0232] 4.5. Stability study of DP batches and AKTS prediction (Study 9) Development studies were conducted to document the stability performance of a 2 L scale-up DP batch of RSV ΔNS2 / Δ1313 / I1314L (L9) in a formulation of 40% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine (pH 7.0), and to predict the DP shelf life of the liquid product stored at 5°C ± 3°C and under accelerated conditions (25°C ± 2°C, 37°C ± 2°C, and 45°C ± 2°C) at the 3-month time point by AKTS. The 2 L DP batch was made from 200 L of processed DS batch 9. 3 mL clear glass vials with rubber stoppers and aluminum caps were filled with 0.6 mL samples of the DP batch. The above plaque assay was used to measure the sample infectious titer at all stability time points.

[0233] The development stability study data and product shelf life predictions are summarized in Figure 7A -F. Figure 7A shows the stability curve for up to 24 weeks under storage conditions of 2°C - 8°C; Figure 7B shows the stability curve for up to 24 months under storage conditions of 2°C - 8°C; Figure 7C shows the stability curve for up to 9 weeks under accelerated storage conditions of 25°C; and Figure 7D shows the stability curve for up to 21 days under forced degradation conditions of 37°C. Figure 7EStability curves for up to 5 days under forced degradation conditions at 45°C are shown. Figure 7F The stability profile of the prepared bulk product (FBP) is shown for up to 3 months at 5°C. The product is stable without titration loss. Further details of the FBP stability study are described in Section 4.6. The 3-month stability data were analyzed using the AKTS program to predict the 24-month shelf life (SL) at 5°C. Figure 7G The predicted storage period of AKTS is summarized in Table 12. Figure 7B The actual titration loss at 5°C for 24 months was 0.785 log.

[0234] 4.6. Study on the shelf life of prepared bulk products (FBP) FBP formulation is a stage in the vaccine manufacturing process that uses calculations based on known values, variables, and formulas of formulation parameters to generate component volume requirements (i.e., no serial dilution is required). Component amounts are calculated based on the expected batch size of the FBP. The amount of each component (or fraction) to be allocated is based on weight. The specific gravity (SG) value of the DS is required when preparing the formulation to convert the volume of active ingredient added into a weight that can be measured on a scale. If necessary, samples of formulation buffer (40% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine (pH 7.0) and DS can be taken. Here, the FBP is transferred to a 250 mL Nalgene bottle. 200 mL of FBP is maintained at 2°C–8°C, and samples are taken at 1 day, 2 days, 3 days, 1 month, 2 months, and 3 months. Figure 7E As shown, the results indicate that there was no significant loss of titration over the 3-month period.

[0235] 4.7. Comparability between DP batches from different DS batches The stability of the formulation, comprising 40% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine (pH 7.0), was further evaluated. Stability was compared between two DP batches to assess stability profiles at 37°C and 5°C. One DP batch was prepared from a 120L process RSV ΔNS2 / Δ1313 / I1314L (L7), and the other DP batch was prepared from a 200L process DS batch RSV ΔNS2 / Δ1313 / I1314L (L9). Figure 8A The thermal stability of DS L7 and DS L9 at 37°C was compared (at 21 days). p The value is 0.2682. Figure 8BThe thermal stability of DS L7 and DS L9 at 5°C was compared (at 6 months). p The value is 0.8850. Figure 8C The thermal stability of DS L7 and DS L9 at 5°C was compared (at 24 months). Figure 8D Comparison of AKTS predictions (see also Table 12).

[0236] Table 12: AKTS SL - Predictions based on 3 months of stability data *Due to insufficient data, the estimated values ​​output by the AKTS model are for reference only. The above data and comparisons show that the formulation with 40% trehalose, 160 mM NaCl, 100 mM MSG and 10 mM histidine (pH 7.0) is a stable liquid formulation, with comparable stability data and shelf-life predictions.

[0237] Example 5. Stable liquid formulation with added recombinant human serum albumin (rHSA) rHSA can be used as a supplement in vaccine manufacturing to ensure optimal cell growth and has been shown to be safe in a variety of approved biological products (see Peters T. All About Albumin: Biochemistry, Genetics and Medical Applications. Academic Press Inc, California, USA (1996)). However, as a product excipient for stabilizing enveloped viruses, rHSA has not been fully studied, but some licensed vaccine products (i.e., MMR and varicella vaccines) include rHSA as a residual product from the manufacturing process (see Richard T. Wiedmann et al.). Vaccine [Vaccines] April 27, 2015; 33(18); Roman Prymul et al., BMC Pediatr [BMC Pediatrics] 2016; 16).

[0238] Generally, rHSA is used as a stabilizer in three main ways: to protect against shear stress, to prevent adsorption to the container surface, and to enhance overall thermal stability (see O'Neil Wiggan et al., Vaccine. 6 October 2011; 29(43)). However, in the study described herein, the use of rHSA for stabilizing RSV liquid vaccine products has not been identified. The role of rHSA in the stabilization of RSV ΔNS2 / Δ1313 / I1314L (e.g., as an excipient stabilizer) is investigated below.

[0239] 5.1 rHSA stabilizing effect - Study 11 To better understand the role of rHSA and the minimum concentration level required for it to function, studies were conducted using RSV ΔNS2 / Δ1313 / I1314L containing non-rHSA and incorporating rHSA. Two RSV ΔNS2 / Δ1313 / I1314L DS batches were used, the first without rHSA (L10) and the second incorporating 5 mg / mL rHSA (L11), both prepared from fresh, small-scale developed RSV ΔNS2 / Δ1313 / I1314L DS batches that were never frozen. The DP formulation was prepared from the aforementioned DS L11 with two levels of rHSA concentration: 0.05 mg / mL and 1.4 mg / mL. This was used to investigate the stabilizing effect of rHSA and the influence of rHSA concentration on the stabilizing effect in the formulated DP.

[0240] The thermal stability of DP at 37°C for up to 14 days was compared after preparation in samples containing 0 mg / mL rHSA, 0.05 mg / mL rHSA and 1.4 mg / mL rHSA, 160 mM NaCl and 10 mM histidine (pH 7.0).

[0241] The thermal stability of rHSA and its effect on RSV ΔNS2 / Δ1313 / I1314L L10 and L11 (containing 0 or 5 mg / mL rHSA) indicate that rHSA is an effective stabilizer for DS. p (Value < 0.0001).

[0242] Titration results for DS and DP batches (see) Figures 9A-9C The results showed that formulations without rHSA exhibited significantly higher titration loss compared to DS and DP batches containing rHSA. Adding a small amount of rHSA (0.05 mg / mL) proved effective. pValue < 0.0001). No effect of freeze-thaw titration loss was observed when the formulation contained rHSA. Therefore, this study concludes that rHSA concentrations from 0.05 mg / mL (low-dose (LD) rHSA) to 1.4 mg / mL (high-dose (HD) rHSA) exhibit relatively good DP thermal stability (compared to the control without rHSA). p Value < 0.0001). rHSA levels below 0.05 mg / mL may result in lower stability. DS and DP without rHSA exhibit lower thermal stability.

[0243] 5.2 Stability studies of stable formulations with different concentrations of rHSA (Study 12) rHSA was added as a stabilizer at two concentrations to the first 200 L RSV ΔNS2 / Δ1313 / I1314L DS batch L9 of a formulation containing 40% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine (pH 7.0), with a titration target DP of 8.0 log. 10 PFU / mL. The concentrations of rHSA were 0.05 mg / mL (F2, low concentration rHSA) and 1.4 mg / mL (F3, high concentration rHSA).

[0244] Plaque assay results from high-dose RSV ΔNS2 / Δ1313 / I1314L DP with low or high concentrations of rHSA at different incubation temperatures are shown in the figure. Figures 10A-10E In samples subjected to heat stress at 37°C, 1.4 mg / mL rHSA significantly reduced DP degradation compared to 0.05 mg / mL rHSA. p Value = 0.0004, see [link / reference] Figure 10C ).

[0245] AKTS modeling for predicting SL based on 3-month stability data is listed in Tables 13 and 14. Figure 10D-10E middle.

[0246] Table 13: AKTS SL predictions for F2 formulations with 0.05 mg / mL rHSA (high-dose DP) based on 3-month stability data. *Due to insufficient data, the estimated values ​​based on the model output are for reference only. Table 14: AKTS SL predictions for F3 formulations with 1.4 mg / mL rHSA (high-dose DP) based on 3-month stability data. *Due to insufficient data, the estimated values ​​based on the model output are for reference only. Example 6. Stable liquid formulation with added hydroxyethyl starch (HES) Many polymers were screened to examine their ability to stabilize attenuated live RSV virus, including but not limited to PVP, PEG, polydextrose, and HES. Surprisingly, HES was observed to stabilize attenuated live RSV virus in some cases.

[0247] 6.1 Study on the stabilizing effect and concentration of HES (Study 13) The efficacy of the MSG and HES combination in improving RSV thermal stability was evaluated compared to a 40% trehalose liquid formulation (10 L DS RSV ΔNS2 / Δ1313 / I1314L L12, in 40% trehalose, 160 mM NaCl, and 10 mM histidine (pH 7.0)). All samples were incubated at 37°C under accelerated conditions for up to 14 days. To incorporate HES into the DP formulation buffer, HES powder was directly dissolved at twice the target excipient concentration in a base DP buffer (40% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine, pH 7). Histidine was added until the final buffer concentration was 30 mM, and the formulation was then mixed with DS at a 1:1 ratio to achieve the final target excipient concentration. It is noteworthy that when preparing a DP buffer with a 5% HES concentration, the HES exhibited viscous, gel-like properties in solution upon dissolution. After dissolution, the 5% HES DP buffer passed through a 0.2-µm filter unit without issue, and the viscosity of the formulation showed no significant difference during the filling process.

[0248] The thermal stability of the three formulations is summarized in Figure 11 The results showed that MSG (F2, 40% trehalose, 10 mM histidine, 160 mM NaCl and 100 mM MSG) and HES (F3, 40% trehalose, 10 mM histidine, 160 mM NaCl + 2.5% HES and 100 mM MSG) were significantly superior to the control (F1, 40% trehalose, 10 mM histidine, 160 mM NaCl) (with stability at 37°C for up to 14 days of [missing information - likely related to stability]. p Value = 0.04 and p Value = 0.0002). It also indicates that when comparing stability at 37°C for up to 14 days, HES formulation F3 was slightly superior to MSG formulation F2, but the difference was not statistically significant. p Value = 0.22).

[0249] 6.2 Stability studies of stable formulations with added HES (Study 14) The stability performance of candidate liquid formulations for commercial use at 3 months was evaluated using AKTS modeling for shelf-life prediction. F2 is a potential formulation: 40% trehalose, 160 mM NaCl, 100 mM MSG, 20 mM histidine, and 2.5% HES, pH = 7.0. F2 was tested using RSV ΔNS2 / Δ1313 / I1314L L12. The infectious titer of stable samples at specified time points was determined using the plaque assay described above. The raw data from this study used for AKTS analysis are summarized in Table 15.

[0250] Table 15: Raw data of plaque determination titration of F2 formulation A kinetic model was selected based on 6 months of data from all temperatures (5°C, 25°C, 37°C, and 45°C). Figure 12E Table 16 uses kinetic model #56 (a two-step kinetic model with optimal AIC and optimal BIC) to demonstrate the predictive model and the predicted titration loss at 5°C.

[0251] Table 16: Prediction of AKTS storage period based on 24-week stability data *Due to insufficient data, the estimated values ​​based on the model output are for reference only. The data provided by these studies support the use of a combination of rHSA and / or HES with formulations containing 40% trehalose, 160 mM NaCl, 100 mM MSG, and 20 mM histidine (pH 7.0) to further improve the stability of liquid products.

[0252] Example 7. Other Studies 7.1. Excipient screening and pH study A study (Study 15) was conducted to confirm and optimize the RSV ΔNS2 / Δ1313 / I1314L liquid formulation based on 40% trehalose, which was prepared in 40% trehalose with 160 mM NaCl and 10 mM histidine (pH 7) by adding different concentrations of screening excipients (such as rHSA, PEG-400 and CaCl2) and screening with 10 L RSV ΔNS2 / Δ1313 / I1314L DS L13.

[0253] The effect of pH on the processed 10 L-scale RSV ΔNS2 / Δ1313 / I1314L DS L14 was investigated (Study 16). All formulation samples were incubated at 37°C for 1 and 2 weeks.

[0254] The composition and thermal stability properties of the formulation are summarized in Table 17.

[0255] Table 17: Summary of Studies 15 and 16 In all groups containing rHSA (F5, F6, and F7), higher rHSA content resulted in higher titration losses in the 40% trehalose formulation (see composition concentrations in Table 17). Notably, the optimal formulation was F5, with 0.5 mg / mL rHSA. However, compared to the F2 control, 0.5 mg / mL rHSA did not appear to provide a significant benefit. p = 0.3275). The addition of CaCl2 to F8 showed improved thermal stability at 37°C at the 1-week time point, with an observed improvement of only 0.68 log [value missing]. 10 The titration loss was measured in PFU / mL. However, the thermal stability of F9 in combination with PEG400 was not improved, with a titration loss of 0.97 log at 37°C at the 1-week time point. 10 PFU / mL.

[0256] The screened potential stabilizers have shown effectiveness in formulations with lower trehalose and / or lower salt concentrations in other studies.

[0257] 7.2. Headspace Nitrogen Purging Study A headspace nitrogen purge study (Study 2) was conducted to determine whether nitrogen headspace could improve the stability of RSV ΔNS2 / Δ1313 / I1314L in different formulations. Side-by-side comparisons of the formulations at selected time points are listed in Table 18 below: Table 18: Results of Plaque Measurement in Nitrogen Purge Test This result indicates that headspace oxygen had no effect on any of the formulations tested, suggesting that the 10 mM histidine in each formulation provided sufficient antioxidant activity. Compared to 30% trehalose, 40% trehalose did not provide additional protection against infectious titration loss in the tested formulations (A. Michael Wade et al., J. Nutritional Biochemistry, 1998). In other words, nitrogen headspace purging did not increase the benefits of preventing oxidation and improving the thermal stability of liquid DP.

[0258] Example 8. Different drying methods Different drying methods were evaluated to compare the feasibility and stability of dried formulations with liquid formulations. The drying methods evaluated included freeze drying, spray drying, and foam drying.

[0259] 8.1 Research on freeze-drying Studies (Studies 1 and 18) were conducted to examine the effect of freeze-drying on the infectious titration loss of RSV ΔNS2 / Δ1313 / I1314L in different formulations. The freeze-drying parameters used are summarized in Table 3. Examples of formulation components and their properties before and after drying are shown in Table 19 below. The data in Table 19 show that the drying process loss and storage loss of the three (3) formulations were very similar. Furthermore, the three (3) formulations prepared by freeze-drying met the predetermined criteria that the loss did not exceed 1 log during 7 days of incubation at 37°C. 10 PFU / mL, they are considered to be heat-stable dry formulations.

[0260] Table 19: Summary of titration results from Lyo Development Research 18 8.2 Spray Drying Proof-of-Concept (POC) Study A point-of-concept (POC) study (Study 19) was conducted to evaluate whether spray drying is an effective method for stabilizing RSV ΔNS2 / Δ1313 / I1314L. The spray drying method was evaluated using a 1:6 dilution of RSV ΔNS2 / Δ1313 / I1314L DS L15 (30% trehalose, 160 mM NaCl, 100 mM potassium glutamate, and 10 mM histidine, pH 7.0) and a benchtop spray dryer (Buchi 290). Drying parameters are listed in Table 4. The results of plaque assays performed on samples before and after drying are summarized in Table 20 below. Table 20: Summary of titrant loss during spray drying and titrant loss during storage at 37°C The results in Table 20 indicate that the spray drying process causes 0.64 log 10 The PFU / mL viral titer loss was higher than that observed during freeze-drying. Storage loss at 37°C was less than 1 log [missing value]. 10 PFU / mL for up to 4 weeks, and it is very stable as a dried preparation.

[0261] 8.3 Foam Drying POC Study 1 A proof-of-concept (POC) study (Study 20) was designed to demonstrate that the foam drying process can be used to stabilize RSV ΔNS2 / Δ1313 / I1314L. DP was prepared using RSVΔNS2 / Δ1313 / I1314L DS L4 in 30% trehalose, 10 mM histidine, 160 mM NaCl, and 100 mM sodium glutamate (pH 7.0), and the foam was dried using the parameters shown in Table 5.

[0262] The stability of the foam-dried formulation was monitored for 12 weeks at 5°C ± 3°C and for 2 weeks at 37°C ± 2°C. The infectious titer was used as a stability indicator in this study. The results of the infectious titer are summarized in... Figure 13 And in Table 21.

[0263] Table 21: Summary of stability data from the first foam drying study 20 This study provides 12 months of data on foam-dried samples generated at 2°C–8°C, with Table 21 showing a 0.34 log loss in infectious titer during the 12 months at 2°C–8°C. 10 PFU / mL. The infectious titer loss during the drying process was 0.7 log. 10 PFU / mL.

[0264] 8.4 Foam Drying POC Study 2 Because a high loss of infectious titer was observed during the drying process in the first study, the cycle parameters were adjusted to a lower temperature for each stage of the second foam drying study to observe whether the loss of infectious titer (RSV ΔNS2 / Δ1313 / I1314L) could be reduced. The adjusted process cycle parameters are shown in Table 22. The results are shown in Table 23 and... Figure 13 middle.

[0265] In this study, 6-month data on foam-dried samples at 2°C–8°C showed that the infectious titer loss during 6 months at 2°C–8°C (Table 23) was 0.37 log [data missing]. 10 PFU / mL. The infectious titer loss during the drying process was 0.5 log [value missing]. 10 PFU mL.

[0266] Table 22: Second example of foam drying process parameters Table 23: Plaque Measurement Data from Foam Drying Study 2 8.5 Comparison and Summary of Drying Methods The acceleration stability curves are compared in Table 24. From this information, we can see that: (1) Compared to the first run (0.7 log) 10 Compared to the second foam drying process (PFU / mL), the second foam drying process had a 0.52 log 10 The decrease in PFU / mL resulted in a greater titration loss than the loss during the freeze-drying process (0.3 log). 10 (PFU / mL), but slightly better than the loss during spray drying (0.6 log). 10 PFU / mL).

[0267] (2) Compared with the foam-dried product from the first run, the foam-dried product from the second run exhibited greater titration loss at all time points at 37°C. Storage loss was comparable to that of the freeze-dried and spray-dried products.

[0268] (3) POC data show that all drying methods have similar total losses regardless of the drying process losses. This formulation is suitable for all different drying methods and produces well-stable dried products.

[0269] (4) During the two weeks of accelerated conditions, the liquid formulations showed relatively lower losses compared to the dry form, especially for the 40% trehalose formulation with the best stability.

[0270] Figure 13 A comparison of the stability of the above-mentioned different formulations and drying conditions or liquid formulations is summarized.

[0271] Table 24: Summary of the stability performance of drying methods for formulations containing 30% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine (pH 7.0) The data in Table 24 provide evidence that the 30% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine (pH 7.0) formulation with the combination of salt and histidine is a stable dried virus formulation. The study also showed that the 40% trehalose, 160 mM NaCl, 100 mM MSG, and 10 mM histidine (pH 7.0) formulation with the combination of salt and histidine (pH 7.0) is also a stable dried virus formulation.

[0272] In summary, the RSV liquid formulation exemplified herein (i.e., having greater than 15% trehalose, monovalent salts, and histidine) is stable as a liquid formulation at room temperature and over extended periods at 2°C–8°C. This liquid formulation can be dispensed directly from shelf storage or storage at 2°C–8°C.

[0273] In contrast, US 2016 / 0250319 describes the liquid formulation only as a pre-treatment step for the bulk formulation before drying. The liquid formulation is not suitable for direct administration to human subjects; instead, it is rapidly frozen to -180°C before drying to form ice particles, thereby maintaining viral stability (see paragraph

[038] of US Patent Application Publication No. 2016 / 0250319, which states that "preferably, the vaccine is frozen by a quick-freezing or rapid freezing method, especially for formulations with high disaccharide content"). No normal freezing rate and F / T data are provided. Furthermore, the formulation described herein exhibits improved stability parameters, as shown in Table 25 below.

[0274] Table 25: Described Liquid Formulations ( Figure 3A , 7A A comparison of the examples in 10A and 12A and the dried formulations (examples in Table 19) with the formulations described in US 2016 / 0250319 (Table 5). Stable formulation composition Table 26: Stable trehalose formulation composition and possible optimal concentrations, with an optimal pH range of 6–8. RSV has been shown to be unstable even under freezing conditions and difficult to freeze-dry (Gupta CK et al., Vaccine, 1996;14(15):1417-20 and Liljeroos L et al., Proc Natl Acad SciUSA, 2013;110(27):1133-8), and is therefore an exemplary vaccine for formulation studies. Furthermore, live attenuated RSV has been shown to be unstable. The RSV ΔNS2 / Δ1313 / I1314L DP formulation development studies disclosed in this paper cover many aspects of the formulation, including pH, excipients, trehalose concentration (15% to 40%), buffer / antioxidant histidine and monovalent salts, and real-time storage stability. rHSA, HES, and MgCl2 can be added as potential stabilizers. Multiple batches of 30% trehalose formulations in liquid and freeze-dried forms were also compared. The formulations disclosed in this paper achieve minimal loss of infectivity under stress and exhibit extremely low titration loss rates at 2°C–8°C.

[0275] Compared to existing viral vaccines in terms of acceleration and real-time stability, the liquid and lyo formulations of RSV ΔNS2 / Δ1313 / I1314L disclosed in this study exhibited suitable stability profiles across multiple batches.

[0276] Stable DP formulations with 30%-40% trehalose were developed. Both liquid and LYO formulations exhibited excellent thermal stability, outperforming other viral vaccine benchmarks when compared at 37°C. The 30%-40% trehalose formulations are robust to many process parameters, such as dilution, mixing shear stress, headspace, etc. Furthermore, based on the stage and needs of early and late development phases, the 30%-40% trehalose formulations can be flexibly prepared as cryolipids, liquids, and lyophilized formulations.

[0277] The results also indicate that the 40% trehalose formulation is a stable liquid formulation of attenuated live RSV ΔNS2 / Δ1313 / I1314L. Typically, the 40% trehalose liquid formulation provides stability for at least 12–24 months. Three potential stabilizers, MgCl2, rHSA, and HES, all showed evidence of stabilizing RSV ΔNS2 / Δ1313 / I1314L.

[0278] Example 9. HSV-2 Trehalose Formulation To investigate the effects of trehalose and MSG on the stability of attenuated HSV-2 virus (HSV 529, as described in Da Costa, X.; Kramer, MF; Zhu, J.; Brockman, MA; Knipe, DM Construction, phenotypic analysis, and immunogenicity of a UL5 / UL29 double deletion mutant of herpes simplex virus 2, J. Virol., 2000, 74, 7963-7971), a full-factor study of the lyophilization process was conducted. The study design included two variables at three levels: 10%, 20%, and 30% trehalose dehydration product, and 0 mM, 50 mM, and 100 mM MSG. The results are summarized in Table 27 below. All preparations were compared with controls of 10% sucrose, 160 mM NaCl, 50 mM MSG, and 10 mM histidine buffer (pH 7.0).

[0279] Table 27: Effects of Trehalose and MSG on titration loss during freeze-drying and storage The results of this study (Table 27) show that increasing the concentration of trehalose can significantly reduce lyophilization loss. Interestingly, the formulation containing 30% trehalose and 100 mM MSG was the only one that showed significantly better results compared to the control and met the stability criteria. Although trehalose was the only component that significantly affected lyophilization loss, it exhibited a clear interaction with MSG levels.

[0280] The titration amount used was 7.99 log. 10 The confirmatory stability study of PFU / mL HSV-2 DS in HSV-2 lyo buffer (30% trehalose, 160 mM NaCl, 100 mM MSG and 10 mM histidine, pH 7.0) was performed by directly filling 0.5 mL to 3 mL glass vials. The lyo cycling parameters used were the same as those in the optimization process in Table 3.

[0281] The results of the confirmation run (Table 28) Figure 14 This indicates that the 30% trehalose formulation significantly stabilizes HSV-2 with lyo presentation. For the lyo process, the titration loss was 0.32 log [value missing]. 10 PFU / mL; when stored at 37°C for 4 weeks, the titration loss was 1 log [value missing]. 10 PFU / mL. However, it is completely ineffective in stabilizing HSV-2 in liquid presentation, as the titer is completely lost after storage at 25°C for one week.

[0282] Table 28: Summary of infectious titration of HSV-2 30% trehalose formulation Example 10. Trehalose formulation for yellow fever Data were also generated to evaluate the formulations described herein and to test their suitability for other attenuated live viruses (e.g., yellow fever attenuated live virus) on RSV. Stability data are summarized in Table 29 below.

[0283] Table 29: Stability of Yellow Fever Lyophilized Products in 30% Trehalose Formulation In comparison, the 30% trehalose formulation (YF F2: trehalose 331.6 mg / mL; NaCl 9.35 mg / mL; MSG 18.713 mg / mL; histidine 9.35 mg / mL, pH 7.0) showed slightly better stabilization performance in terms of lyo loss and storage loss than the original yellow fever formulation (YF F1: trehalose 150 mg / mL; PVP10 20 mg / mL; CaCl2·H2O 1.325 mg / mL; proline 3.2 mg / mL; urea 2.5 mg / mL; P407 5 mg / mL; lysine·H2O 0.337 mg / mL; sorbitol 30 mg / mL, in 20 mM Tris buffer, pH 8.0). The 30% trehalose formulation was able to stabilize yellow fever virus at 37°C for up to 2 weeks, with a stability of approximately 1.06 log... 10 Cell culture infection dose, 50% endpoint (CCID) 50 Titration loss.

[0284] Example 11. PIV Trehalose Formulation Liquid and lyophilized vector-based formulations containing PIV were prepared according to the methods described herein, and their stability was tested at 2–8°C, room temperature, and 37°C. A PIV3 vector-based vaccine was prepared, comprising a PIV3-derived vector virus and one or more heterologous RSV or hMPV antigens. In some embodiments, the RSV antigen is RSV F and / or RSVG. A PIV3 vector-based vaccine comprising a PIV3-derived vector virus and non-PIV heterologous antigens was also prepared.

[0285] 15% and higher trehalose concentrations (e.g., 30% and 40% trehalose) provide stabilization for liquid formulations containing PIV similar to that of the RSV and HSV-2-based formulations described above.

[0286] Appendix A: Sequence Summary Sequence Description Appendix A provides a list of some of the sequences cited in this article. The amino acid sequences provided are from the N-terminus to the C-terminus. The nucleic acid sequences are 5' to 3'.

Claims

1. A liquid, lyophilized, or frozen preparation comprising: a. Effective amounts of attenuated live virus and / or vector-based virus; b. Approximately 30% to approximately 45% (w / v) trehalose; c. One or more monovalent salts; and d. Buffers and / or antioxidants containing histidine.

2. A liquid, lyophilized, or frozen formulation comprising an effective amount of attenuated live virus and / or vector-based virus, and about 30% to about 40% (w / v) trehalose, about 100 mM monosodium glutamate (MSG), about 160 mM NaCl, and about 10 mM histidine.

3. A liquid, lyophilized, or frozen preparation comprising: a. An effective amount of attenuated live respiratory syncytial virus (RSV); b. Approximately 30% to approximately 45% (w / v) trehalose; c. One or more monovalent salts; and d. Buffers and / or antioxidants containing histidine.

4. A liquid, lyophilized, or frozen preparation comprising: a. An effective amount of attenuated active RSV; b. Approximately 30% to approximately 45% (w / v) trehalose; c. Approximately 10 to approximately 300 mM NaCl; d. Approximately 0.5 to approximately 300 mM sodium glutamate (MSG) or potassium glutamate; and e. Approximately 10 to approximately 100 mM histidine; The pH of this formulation is approximately 6 to approximately 8.

5. The formulation of claim 1 or claim 2, wherein the virus is membrane-bound.

6. The formulation according to any one of claims 1-4, wherein the virus is RSV ΔNS2 / Δ1313 / I1314L.

7. The formulation of claim 6, wherein the effective amount of RSV ΔNS2 / Δ1313 / I1314L is about 5 to about 9 log. 10 Plaque-forming units (PFU) / agent.

8. The formulation of claim 6, wherein the effective amount of RSV ΔNS2 / Δ1313 / I1314L is about 5 to about 6 log. 10 PFU / dose or approximately 6.1 to approximately 7 log 10 PFU / dose or approximately 7.1 to approximately 8 log 10 PFU / dose or approximately 8.1 to approximately 9 log 10 PFU / dosage.

9. The formulation of claim 6, wherein the effective amount of RSV ΔNS2 / Δ1313 / I1314L is approximately 5.6 log₂S₂ / Δ1313 / I1314L. 10 PFU / dose or approximately 6.2 log 10 PFU / dosage.

10. The formulation of any one of claims 3 or 4, wherein the codon for the serine acid at position 1313 encoding the L protein in the attenuated active RSV is deleted, thereby causing a deletion of the amino acid (Δ1313) in the L protein.

11. The formulation according to any one of claims 3, 4 or 10, wherein the substitution of the amino acid residue of leucine for isoleucine at position 1314 in the RSV, referring to SEQ ID NO: 3, causes a genetically stable mutation (I1314L) in the L gene.

12. The formulation according to any one of claims 3, 4, or 10-11, wherein the RSV comprises: Large polymerase protein (L), phosphoprotein (P), nucleocapsid protein (N), M2-1 protein, non-structural protein 1 (NS1), glycoprotein (G), fusion protein (F), matrix protein (M), M2-2 protein, and small hydrophobic protein (SH); and A genome or antigenome containing the deletion of a codon encoding a serine acid at position 1313 or the corresponding position of the L protein; a mutation in an amino acid sequence residue 1314 of the L protein or the corresponding position, wherein the mutation in amino acid sequence residue 1314 of the L protein is an amino acid substitution of leucine for isoleucine, wherein the leucine is encoded by a codon described as CTG; a deletion of the NS2 gene; and a nucleotide modification at position 14456 of reference SEQ ID NO: 1, which represents a change from thymine (T) to adenine (A).

13. The formulation as described in any of the preceding claims, comprising: a liquid formulation.

14. The formulation as described in any of the preceding claims, comprising: a lyophilized formulation.

15. The formulation of claim 13, wherein the liquid formulation is formulated for administration to a human subject.

16. The formulation of claim 15, wherein the administration is intranasal.

17. The formulation as described in any of the preceding claims, wherein the formulation does not contain a polyoxyethylene-polyoxypropylene (PEO-PPO) block copolymer.

18. The formulation as described in any of the preceding claims is a liquid formulation, wherein the liquid formulation is not subsequently dried and reconstituted.

19. The formulation according to any one of claims 1 and 3, further comprising sodium monoglutamate (MSG) or potassium glutamate (PG).

20. The formulation according to any one of claims 1 and 3, comprising about 10 to about 100 mM histidine.

21. The formulation as described in any of the preceding claims, comprising about 30% trehalose.

22. The formulation as described in any of the preceding claims, comprising about 40% trehalose.

23. The formulation as described in any of the preceding claims comprises about 0.5 to about 300 mM sodium glutamate (MSG) or potassium glutamate (PG).

24. The formulation as described in any one of claims 1 or 3, wherein the monovalent salt is NaCl.

25. The formulation according to any one of claims 1, 3, 5 or 20-22, comprising about 10 to about 300 mM NaCl.

26. The formulation as described in any of the preceding claims, wherein the pH is about 6 to about 8.

27. The formulation as described in any of the preceding claims, wherein the pH is about 7 ± 0.

5.

28. The formulation of any one of claims 1 or 2, wherein the attenuated live virus comprises paramyxovirus.

29. The formulation according to any one of claims 1, 2, 5 or 28, wherein the attenuated live virus comprises respiratory syncytial virus (RSV).

30. The formulation as described in any of the preceding claims, wherein the virus is RSV, and wherein the RSV is administered to a pediatric subject.

31. The formulation according to any one of claims 1, 2, 5 or 20-22, comprising a vector-based PIV virus, wherein the PIV is PIV3 if desired.

32. The formulation of claim 31, wherein the PIV comprises one or more heterologous RSV or hMPV antigens.

33. The formulation of claim 32, wherein the RSV antigen is RSV F and / or RSV G.

34. The formulation as described in any of the preceding claims, wherein the formulation is a liquid, and wherein the titration loss is less than 1 log when stored at about 2 to about 8 degrees Celsius for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 months. 10 PFU / mL.

35. The formulation of claim 34, wherein after storage at about 2 to about 8 degrees Celsius for about 12 to about 24 months, the titration loss is less than 1 log. 10 PFU / mL.

36. The formulation as described in any of the preceding claims, wherein the formulation is a liquid, and wherein the titration loss is less than 1 log when stored at about 37 degrees Celsius for at least 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day. 10 PFU / mL.

37. The formulation of claim 34, wherein the titration loss is less than 1 log when maintained at 37°C for about 1 to about 2 weeks. 10 PFU / mL.

38. The formulation as described in any of the preceding claims, wherein the formulation is a liquid, and wherein the titration loss is less than 1 log when stored at room temperature for at least 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, or 5-6 months. 10 PFU / mL.

39. The formulation of claim 38, wherein the titration loss is less than 1 log during 3-4 months at room temperature. 10 PFU / mL.

40. The formulation according to any one of claims 1-33, wherein the formulation is lyophilized, and wherein the titration loss is less than 1 log when maintained at 37°C for about 1 to about 2 weeks. 10 PFU / mL.

41. The formulation according to any one of claims 1-33, wherein the formulation is lyophilized, and wherein the titration loss is less than 1 log when stored at room temperature for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. 10 PFU / mL.

42. The formulation as described in any of the preceding claims, wherein the infectious titration is substantially unaffected by shear stress, repeated freeze-thaw cycles, or remodeling.

43. The formulation as described in any of the preceding claims, wherein the vaccine is formulated for intramuscular or subcutaneous administration.

44. The formulation as described in any of the preceding claims further comprises an adjuvant.

45. The formulation as described in any of the preceding claims, further comprising rHSA, a divalent salt and / or an amino acid.

46. ​​A method for increasing the stability of a liquid or lyophilized vaccine at room temperature or at a temperature of about 2 to about 8 degrees Celsius, comprising formulating an attenuated live enveloped virus and / or a vector-based virus in a liquid or lyophilized formulation, the liquid or lyophilized formulation comprising about 30% to about 45% (w / v) trehalose, one or more monovalent salts, and a buffer and / or antioxidant containing histidine.

47. The method of claim 46, wherein the concentration of trehalose is about 30% (w / v).

48. The method of claim 46, wherein the concentration of trehalose is about 40% (w / v).

49. A method for immunizing a subject against a viral infection, comprising administering a formulation as described in any one of claims 1-48.

50. A liquid, lyophilized, or frozen preparation comprising: a. An effective amount of attenuated active RSV containing RSV ΔNS2 / Δ1313 / I1314L; b. Approximately 30% to approximately 40% (w / v) trehalose; c. Approximately 160 mM NaCl; d. Approximately 100 mM monosodium glutamate (MSG); and e. Approximately 10 mM histidine; The pH of this formulation is approximately 7.

51. The formulation of claim 50, wherein the formulation comprises about 30% (w / v) trehalose.

52. The formulation of claim 50, wherein the formulation comprises about 40% (w / v) trehalose.

53. The formulation according to any one of claims 50-52, wherein the attenuated active RSV comprises: Large polymerase protein (L), phosphoprotein (P), nucleocapsid protein (N), M2-1 protein, non-structural protein 1 (NS1), glycoprotein (G), fusion protein (F), matrix protein (M), M2-2 protein, and small hydrophobic protein (SH); and A genome or antigenome containing the deletion of a codon encoding a serine acid at position 1313 or the corresponding position of the L protein; a mutation in an amino acid sequence residue 1314 of the L protein or the corresponding position, wherein the mutation in amino acid sequence residue 1314 of the L protein is an amino acid substitution of leucine for isoleucine, wherein the leucine is encoded by a codon described as CTG; a deletion of the NS2 gene; and a nucleotide modification at position 14456 of reference SEQ ID NO: 1, which represents a change from thymine (T) to adenine (A).

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