Live attenuated influenza vaccine compositions and methods for making the same

文档序号：245826 发布日期：2021-11-12 浏览：38次中文

阅读说明：本技术 减毒活流感疫苗组合物和用于制备其的方法 (Live attenuated influenza vaccine compositions and methods for making the same ) 是由拉耶夫·姆哈尔萨坎特·德雷莱纳·拉温德拉·耶了卡米兰·肖梅纳特·甘古利帕里克西特·达拉姆于 2020-02-07 设计创作，主要内容包括：本公开提供了用于制造和获得可鼻内递送以提供针对流感病毒感染的保护的减毒活流感疫苗(LAIV)组合物的组合物和方法。所述LAIV毒株基于包含野生型大流行性或季节性流感毒株的表面糖蛋白基因的主供体病毒(MDV)的冷适应、温度敏感和减毒表型。而且,所述LAIV毒株进一步适于在MDCK细胞(马丁达比狗肾细胞)中生长。在大规模疫苗生产中避免使用蛋。纯化过程没有色谱步骤。所述LAIV组合物包括一种或多种减毒活流感疫苗病毒,并且不含聚合物和表面活性剂。(The present disclosure provides compositions and methods for making and obtaining Live Attenuated Influenza Vaccine (LAIV) compositions that can be delivered intranasally to provide protection against influenza virus infection. The LAIV strain is based on the cold-adapted, temperature sensitive and attenuated phenotype of a Master Donor Virus (MDV) comprising the surface glycoprotein genes of wild-type pandemic or seasonal influenza strains. Moreover, the LAIV strain is further suitable for growth in MDCK cells (madin dabigatran etes kidney cells). The use of eggs is avoided in large scale vaccine production. The purification process has no chromatographic step. The LAIV composition comprises one or more live attenuated influenza vaccine viruses and is free of polymers and surfactants.)

1. An immunogenic composition comprising:

a) one or more influenza viruses;

b) one or more carbohydrates;

c) one or more amino acids; and

d) gelatin.

2. The immunogenic composition of claim 1, wherein the influenza virus comprises a pandemic or seasonal influenza virus, a Live Attenuated Influenza Vaccine (LAIV) virus, an inactivated influenza virus, a chimeric influenza virus, or a recombinant influenza virus.

3. The immunogenic composition of claim 1, wherein the composition is monovalent for influenza viruses derived from influenza a or influenza b or influenza c or subtypes thereof.

4. The immunogenic composition of claim 1, wherein the composition is multivalent with respect to influenza viruses derived from influenza a or influenza b or influenza c or subtypes thereof.

5. The immunogenic composition of claim 1, wherein the influenza virus is a rearranged LAIV virus comprising cold-adapted, temperature sensitive and/or attenuated phenotype gene segments of a Master Donor Virus (MDV) and Hemagglutinin (HA) gene segments and/or Neuraminidase (NA) gene segments of a wild-type pandemic or seasonal influenza a or influenza b or influenza c strain in a ratio of 1:7, 2:6, 3:5, 4:4, 5:3, 6:2 or 7: 1.

6. The immunogenic composition of claim 5, wherein the Master Donor Virus (MDV) is selected from the group comprising A/Leninggler/134/17/57 (H2N2) influenza A strain, B/USSR/60/69 influenza B strain.

7. The immunogenic composition of claim 5, wherein the rearranged LAIV virus comprises a Hemagglutinin (HA) gene and/or a Neuraminidase (NA) gene from influenza A virus or H1 to H18 and N1 to N11 and subtypes thereof H1N1, H2N2, H3N2, H5N1, H5N3, H9N2, H7N1, H7N3, H7N7, H6N1, H7N9 or H10N 8.

8. The immunogenic composition of claim 5, wherein the rearranged LAIV virus comprises a Hemagglutinin (HA) gene and/or a Neuraminidase (NA) gene from influenza A strain pdmH1N1 strain A/California/07/2009 (referred to as A/Cal) -like strain or A/Cal, A/Michigan/45/2015-like strain, A/south Africa/3626/2013-like strain or H3N 2-A/hong Kong/4801/2014-like strain.

9. The immunogenic composition of claim 5, wherein the rearranged LAIV virus comprises a Hemagglutinin (HA) gene and/or a Neuraminidase (NA) gene from influenza B strain Victoria lineage B/Britisb/60/2008-like strain or Yamagata lineage B/Pregiisland/3073/2013-like strain.

10. The immunogenic composition of claim 1, wherein the one or more carbohydrates are selected from the group comprising natural carbohydrates, synthetic carbohydrates, monosaccharides, disaccharides, trisaccharides, oligosaccharides, reducing sugars, non-reducing sugars, sugar alcohols, polyols, chemically modified carbohydrates, glass transition promoters, wherein the glass transition promoters are selected from the group comprising sucrose, mannitol, mannose, raffinose, lactitol, lactobionic acid, glucose, maltulose, isomaltulose, maltose, lactose, dextrose, fucose or combinations thereof.

11. The immunogenic composition of claim 10, wherein the one or more carbohydrates comprise sucrose present in an amount of 1% to 10% (w/v).

12. The immunogenic composition of claim 1, wherein the one or more amino acids are selected from the group comprising tris (hydroxymethyl) methylglycine, leucine, isoleucine, histidine, glycine, glutamine, arginine, lysine, alanine, or combinations thereof.

13. The immunogenic composition of claim 10, wherein the one or more amino acids comprise tris (hydroxymethyl) methylglycine present in an amount of 0.1% to 2% (w/v), histidine present in an amount of 0.1% to 2% (w/v), alanine present in an amount of 0.01% to 1% (w/v) and arginine present in an amount of 0.1% to 5% (w/v).

14. The immunogenic composition according to claim 1, wherein gelatin is present in an amount of 0.1% to 5% (w/v).

15. The immunogenic composition of claim 1, wherein the composition further comprises an adjuvant selected from the group of aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate, and aluminum potassium sulfate, or mixtures thereof.

16. The immunogenic composition of claim 1, wherein the composition further comprises an immunostimulatory component selected from the group consisting of: oil and water emulsions, MF-59, liposomes, lipopolysaccharides, saponins, lipid A derivatives, monophosphoryl lipid A, 3-deacylated monophosphoryl lipid A, AS01, AS03, oligonucleotides comprising at least one unmethylated CpG and/or liposomes, Freund's adjuvant, Freund's complete adjuvant, Freund's incomplete adjuvant, CRL-8300 adjuvant, muramyl dipeptide, TLR-4 agonists, flagellin derived from gram-negative bacteria, TLR-5 agonists, fragments of flagellin capable of binding TLR-5 receptors, QS-21, ISCOMS, deacetylated chitin, saponins in combination with sterols and lipids.

17. The immunogenic composition of claim 1, wherein single dose composition is preservative-free and multiple dose composition comprises one or more preservatives selected from the group consisting of: 2-phenoxyethanol, benzethonium chloride (phemol), phenol, thimerosal, formaldehyde, methylparaben, propylparaben, benzyl alcohol, or combinations thereof.

18. The immunogenic composition of claim 1, wherein the composition comprises a buffer selected from the group consisting of: sodium chloride, carbonate, citrate, lactate, gluconate, tartrate, phosphate buffered saline, HEPES, citrate-phosphate or TRIS.

19. The immunogenic composition of claim 1, wherein the composition comprises a pharmaceutically acceptable transporter, excipient, binder, carrier, isotonicity agent, emulsifying agent, or wetting agent.

20. The immunogenic composition of claim 19, wherein the composition comprises a pharmaceutically acceptable excipient selected from the group consisting of: a sugar; a polyol; comprises NaCl, KCl and KH₂PO₄、Na₂HPO₄.2H₂O、CaC1₂Or MgCl₂A salt; amino acids or pH modifiers.

21. The immunogenic composition of claim 1, wherein the composition is formulated as a single dose vial or multiple dose kit or pre-filled syringe or nasal spray for use in a method of reducing or preventing a health condition comprising an influenza a virus infection or subtype thereof, an influenza b virus infection or subtype thereof or an influenza c virus infection or subtype thereof.

22. The immunogenic composition of claim 1, wherein the final pH of the immunogenic composition comprises pH 6.5 to 8.

23. The immunogenic composition of claim 1, wherein the influenza virus is propagated in a madding-bitch dog kidney (MDCK) cell selected from, but not limited to, ATCC CCL-34, MDCK33016 cell line (DSMACC 2219), MDCK (ATCC CCL34MDCK (NBL2)), MDCK33016(DSMACC 2219), DSMACC3309, ATCC CRL-12042, ATCC PTA-7909, ATCC PTA-7910, ATCC PTA-6500, ATCC PTA-6501, ATCC PTA-6502, ATCC PTA-6503, 'MDCK-S', 'MDCK-SF 101', 'MDCK-SF 102', 'MDCK-SF 103', and FERM BP 7449.

24. The immunogenic composition of claim 1, wherein the influenza virus is propagated in madin bitch dog kidney (MDCK) cells (ATCC CCL-34).

25. The immunogenic composition of claim 1, wherein the influenza b virus has a Log EID of 6 to 7₅₀0.5ml, more preferably not less than 6.5Log EID₅₀In a dose of 0.5 ml.

26. The immunogenic composition of claim 1, wherein the influenza a virus has a Log EID of 6 to 7₅₀0.5ml, more preferably not less than 7Log EID₅₀In a dose of 0.5 ml.

27. A method of making an immunogenic composition comprising:

a) infecting a MDCK cell culture host with an influenza virus at a MOI between 1:100 and 1: 10000;

b) harvesting the supernatant comprising influenza virus after 40 to 70 hours incubation in MEM containing trypsin in the range of 5 to 25U/ml;

c) filtering the viral harvest by Direct Flow Filtration (DFF) through at least one clarification filter having a pore size between about 6 microns to about 0.45 microns;

d) treating the CVP with a non-specific endonuclease at a temperature ranging from 30 ℃ to 34 ℃ for 2 hours to 6 hours, and subsequently at a temperature ranging from 2 ℃ to 8 ℃ for 5 hours to 15 hours;

e) concentrating the endonuclease-treated CVP by Tangential Flow Filtration (TFF) using a membrane having a molecular weight cut-off (MWCO) of 100KDa to 500 KDa;

f) stabilizing the TFF concentrate with a stabilizer composition comprising one or more carbohydrates, one or more amino acids, and gelatin to form a stabilized viral harvest;

g) sterilizing the stabilized TFF concentrate by DFF through at least one sterilization grade filter having a pore size between about 0.8 microns to about 0.2 microns to form sterilized CMVP;

wherein the total recovery of purified virus is greater than or equal to 40%.

28. The method of making an immunogenic composition according to claim 27, wherein step (d) comprises the presence of Ca in an amount between 0.1mM and 100mM selected from the group consisting of²⁺、Mg²⁺、Mn²⁺And Cu²⁺In the case of divalent cations in the group, the viral harvest is treated with a non-specific endonuclease, more particularly a benzoate, at a concentration in the range of 0.5 units/ml to 5 units/ml.

29. The method of manufacturing an immunogenic composition according to claim 27, wherein step (d) comprises the presence of divalent cation Mg at a concentration of 1mM to 3mM²⁺In the case of salt, the viral harvest is treated with a non-specific endonuclease, more particularly a benzoate, at a concentration in the range of 0.5 units/ml to 5 units/ml.

30. The method of making an immunogenic composition according to claim 27, wherein step (e) comprises concentrating the viral harvest by Tangential Flow Filtration (TFF) to produce at least a 4-fold concentrated viral harvest.

31. The method of making an immunogenic composition of claim 27, wherein step (f) comprises stabilizing the virus harvest with a stabilizer composition comprising sucrose at a concentration of 1% to 10% (w/v), histidine at a concentration of 0.1% to 2% (w/v), alanine at a concentration of 0.01% to 1% (w/v), tris (hydroxymethyl) methylglycine at a concentration of 0.1% to 2% (w/v), arginine at a concentration of 0.1% to 5% (w/v), and gelatin at a concentration of 0.1% to 5% (w/v).

32. The method of making an immunogenic composition of claim 27, wherein step (f) comprises stabilizing the virus harvest with a stabilizer composition comprising sucrose at a concentration of 3% to 6% (w/v), histidine at a concentration of 0.1% to 1% (w/v), alanine at a concentration of 0.05% to 0.5% (w/v), tris (hydroxymethyl) methylglycine at a concentration of 0.1% to 0.5% (w/v), arginine at a concentration of 0.1% to 3% (w/v), and gelatin at a concentration of 0.1% to 3% (w/v).

33. The method of making an immunogenic composition according to claim 27, wherein step (f) comprises stabilizing the virus harvest with a stabilizer composition comprising 4% (w/v) sucrose, 0.21% (w/v) histidine, 0.1% (w/v) alanine, 0.3% (w/v) tris (hydroxymethyl) methylglycine, 2.1% (w/v) arginine and 0.85% (w/v) gelatin.

34. The method of making an immunogenic composition according to claim 27, wherein step (f) comprises stabilizing the virus harvest with a stabilizer composition comprising 4% (w/v) sucrose, 0.21% (w/v) histidine, 0.1% (w/v) alanine, 0.3% (w/v) tris (hydroxymethyl) methylglycine, 2.1% (w/v) arginine and 1.0% (w/v) gelatin.

35. The method of making an immunogenic composition according to claim 27, wherein step (f) comprises stabilizing the virus harvest with a stabilizer composition comprising 4% (w/v) sucrose, 0.21% (w/v) histidine, 0.1% (w/v) alanine, 0.3% (w/v) tris (hydroxymethyl) methylglycine, 1.6% (w/v) arginine and 1.0% (w/v) gelatin.

36. The immunogenic composition of claim 1, wherein the method of administering the immunogenic composition to a human subject comprises an intranasal, intramuscular, intravenous, subcutaneous, transdermal or intradermal route.

37. An immunogenic composition comprising:

a) one or more Live Attenuated Influenza Vaccine (LAIV) viruses at a dose of 6 to 7log EID₅₀/0.5ml；

b) 1% to 10% (w/v) sucrose;

c) 0.1% to 2% (w/v) histidine;

d) 0.01% to 1% (w/v) alanine;

e) 0.1% to 2% (w/v) tris (hydroxymethyl) methylglycine;

f) 0.1% to 5% (w/v) arginine;

g) 0.1% to 5% (w/v) gelatin.

38. The immunogenic composition of claim 37, wherein the composition comprises:

a) one or more Live Attenuated Influenza Vaccine (LAIV) viruses at a dose of 6 to 7log EID₅₀/0.5ml；

b) 3% to 6% (w/v) sucrose;

c) 0.1% to 1% (w/v) histidine;

d) 0.05% to 0.5% (w/v) alanine;

e) 0.1% to 0.5% (w/v) tris (hydroxymethyl) methylglycine;

f) 0.1% to 3% (w/v) arginine;

g) 0.1% to 3% (w/v) gelatin.

39. The immunogenic composition of claim 37, wherein the composition comprises:

a) one or more Live Attenuated Influenza Vaccine (LAIV) viruses at a dose of 6 to 7log EID₅₀/0.5ml；

b) 4% (w/v) sucrose;

c) 0.21% (w/v) histidine;

d) 0.1% (w/v) alanine;

e) 0.3% (w/v) tris (hydroxymethyl) methylglycine;

f) 2.1% (w/v) arginine;

g) 0.85% (w/v) gelatin.

40. The immunogenic composition of claim 37, wherein the composition comprises:

a) one or more Live Attenuated Influenza Vaccine (LAIV) viruses at a dose of not less than 6 to 7log EID₅₀/0.5ml；

b) 4% (w/v) sucrose;

c) 0.21% (w/v) histidine;

d) 0.1% (w/v) alanine;

e) 0.3% (w/v) tris (hydroxymethyl) methylglycine;

f) 2.1% (w/v) arginine;

g) 1% (w/v) gelatin.

41. The immunogenic composition of claim 37, wherein the composition comprises:

a) one or more Live Attenuated Influenza Vaccine (LAIV) viruses at a dose of 6 to 7log EID₅₀/0.5ml；

b) 4% (w/v) sucrose;

c) 0.21% (w/v) histidine;

d) 0.1% (w/v) alanine;

e) 0.3% (w/v) tris (hydroxymethyl) methylglycine;

f) 1.6% (w/v) arginine;

g) 1% (w/v) gelatin.

Technical Field

The present disclosure relates to the field of viral vaccine manufacture, and more particularly, to live attenuated influenza vaccine compositions and methods of making the same. The present disclosure relates to methods for producing a virus or virus antigen produced by cell culture, viruses or virus antigens obtained by the methods, and vaccines comprising such viruses or virus antigens.

Background

The following background information herein is relevant to the present disclosure and is not necessarily prior art.

Epidemics and pandemics caused by infectious pathogens have occurred for centuries, causing significant damage, varying in morbidity and mortality. Influenza viruses have become one of the major participants in the history of pandemics. Four influenza pandemics occurred over the last century, and at least 15 influenza pandemics were recorded to date, with an estimated 5000 million deaths in 1918 to 1919 alone.

Vaccines play an important role in the control of viral transmission, and Inactivated Influenza Vaccines (IIV) as well as Live Attenuated Influenza Vaccines (LAIV) have been used for many years. When circulating strains are antigenically matched to vaccines, the widely used parenterally administered inactivated influenza vaccines induce serum antibody responses, effectively preventing influenza disease. In contrast, Live Attenuated Influenza Vaccines (LAIVs) are administered intranasally, mimicking natural infection, inducing local and systemic humoral and cellular immune responses, providing protection for matched strains as well as drifted strains. In addition, needleless application of LAIV can lower the acceptance threshold and thereby improve coverage of influenza vaccine.

To date, LAIV has gained license in the united states (since 2003), europe (since 2010), russia (since the 1980 s), and india (since 2010). LAIV was based on attenuated influenza a and influenza b Major Donor Viruses (MDV) developed independently but in essentially the same way in the united states and russia in the 60 th century. Med immune seasonal and epidemic LAIV viruses are currently produced by Reverse Genetics (RG). In contrast, russian LAIV was produced by traditional genetic recombination in chicken embryos. Russian LAIV is recently being registered for use in china and thailand.

Russian LAIV consists of a recombinant virus containing Hemagglutinin (HA) gene segments and in most cases Neuraminidase (NA) gene segments from the circulating wild-type (WT) virus of interest on the backbone of the remaining 6 internal protein genes (PB1, PB2, PA, NP, M and NS) from (MDV). A/Leninggler/134/17/57 (H2N2) (Len-MDV) and B/USSR/60/69MDV are currently used in Russia as MDVs for LAIV. MDVs contain mutations in multiple gene segments, making them cold-adapted (ca), temperature-sensitive (ts), and attenuated (att). The surface glycoproteins Hemagglutinin (HA) and Neuraminidase (NA) of contemporary strains are incorporated into these MDVs by rearrangement. Their ca, ts and att phenotypes indicate that LAIV replicates at low temperatures and ceases to replicate at higher temperatures (>38 ℃), limiting replication to the upper respiratory tract. Russian MDVs contain fewer mutations present at different sites in the gene segments than MDVs used in the united states, suggesting that they may be attenuated differently. Direct comparison in animals and humans suggests that russian MDV and its derived single strain rearrangements are more immunogenic than the equivalent.

Over the years, russian LAIV has been safely administered to 7500 more than ten thousand people and has proven safe in terms of attenuation (genetic stability) and dissemination. Reversal of virulence (loss of the attenuated phenotype) was never observed and is highly unlikely to occur because it requires reversal of multiple mutations in multiple gene segments. Neurotoxicity of the russian LAIV virus has never been reported, and both MDV and its derived rearrangements show no neurotoxic properties. Following LAIV administration, no serious adverse events associated with immunity were reported except for self-limiting flu-like symptoms (runny nose, nasal congestion, sore throat, cough, headache, and low fever) reported in a few cases. In addition to being safe, LAIV has been shown to be effective in preventing diseases caused by influenza virus infection. LAIV-induced immunity is extensive and has been shown to protect against drifting strains. LAIV is more effective than inactivated influenza vaccines in clinical protection and/or protection against culture-confirmed influenza, particularly in children, and has also been shown to have a community immunity effect.

As with inactivated influenza vaccines, russian LAIV is produced using chicken embryos, its own inherent disadvantages limit vaccine quality suppliers, and eggs that do not contain specific pathogens need to be ordered at least 4 months in advance before they can be used for large-scale vaccine production. Specialized facilities for egg incubation, harvesting, etc. in turn limit the ability to scale up quickly. The extended time required to produce an egg-dependent vaccine may result in too few doses being available to cope with pandemic situations (such as those occurring in 2009) or to prevent a pandemic from highly pathogenic influenza viruses. Therefore, current vaccine production systems are not sufficient to cope with influenza pandemics, and a new type of rapid emergency vaccine production process is required for this purpose. Theoretically, the production of influenza vaccines in cell culture offers important advantages in pandemic situations. Large scale production of vaccines can be easily achieved using pre-existing tissue culture manufacturing units for other viral vaccines.

The availability of control substrate consistency and production flexibility, which is important in case of a pandemic, is rapidly upscaled, and independence from egg supply and chicken flock maintenance is a major advantage of tissue culture methods. The eggshell is a porous structure and the exterior of the egg is not sterile. The manufacturing process of culturing influenza virus in eggs requires puncturing the eggshell for inoculation and open processing for harvesting, which has an inherent risk of contamination. Furthermore, cell culture is a more controlled system with defined cell culture media and validated cell banks that meet Good Manufacturing Practices (GMP), and therefore attempts have been made to transfer production from eggs to cell culture.

Several cell lines are currently being investigated for the production of influenza virus on a cell culture basis, and MRC-5 cells (ref: de Ona et al (1995) J Clin Microbiol 33: 1948-49), HepG2 cells (ref: Ollier et al (2004) J Clin Microbiol 42(12):5861-5), LLC-MK2 cells (ref: Schepetiuk and Kok (1993) J Virol Methods 42(2-3):241-50), Madin Darby dog kidney (MDCK) cells (ref: Tobita et al (1975) Med Microbiol Immunol (Berl) 162(1):9-14 and 23-27), African green monkey Vero cells (ref: Monto et al (1981) J Clin Microbiol 13 (1): 233-235 and vorkova et al (1995) J implant Dis (1):250-3) and Gorkova et al (1995) J Impect J1889: 1887, 2009R 9-9, 2009). Previously, for MRC-5, WI-38 and FRhL cells, it has been reported to have a log equal to or lower than 5.0₁₀ TCID₅₀Low to moderate titers of virus per ml, whereas for MDCK cells up to 6.7log has been reported₁₀ TCID₅₀Viral titer per mL. Although cell culture derived influenza vaccines (egg isolated influenza viruses adapted/optimized for cell culture growth) have gained approval in europe (Optaflu Novartis; 2007) and the united states (Flucelvax Novartis; 2012), only a very small fraction of influenza vaccines on the market are cell culture derived. This may be due to the use of cellsThe culture production system combines the conventional purification method and the complicated stable practice, and the yield of the influenza vaccine is inconsistent. Theoretical safety issues associated with the use of continuous cell lines, such as MDCK cells, have been addressed in connection with their use in vaccine production (VRBPAC, 2008). These problems are mainly associated with residual cellular components (DNA and proteins) in vaccine drug products, especially Live Attenuated Influenza Vaccine (LAIV) product vaccines that are neither inactivated nor extensively biochemically purified as in traditional inactivated influenza vaccines. Two strategies have been taken to minimize these risks: cell line characterization and vaccine purification processing steps. One particular aspect of the purification process is to reduce the amount and size of residual host cell DNA in the vaccine product. The manufacture of Optaflu Novartis (2007) involves multiple steps to eliminate residual host cell DNA. These include cellulose sulfate ion exchange chromatography, which binds influenza virus and allows DNA to pass through, and subsequent CTAB precipitation steps, particularly to precipitate DNA.

Previously reported purification processes are costly and time consuming because they employ one or more chromatography methods from hydroxyapatite, affinity, anion exchange and size exclusion. It has been reported that the overall recovery of virus is not ideal for conventional vaccine production using chromatographic methods. In addition, circulating influenza viruses undergo changes in the antigenic properties of the viral particles due to the antigenic drift and antigenic shift observed in the virus. These changes affect the physicochemical properties of the virus and, in turn, the binding capacity of the virus to the chromatographic matrix. This can result in high variability in yield of drifted or drifted strains, making chromatography unsuitable for routine production. Another method is used to remove/reduce host cell DNA.

Higher concentrations of the benzoic acid enzyme (e.g., 50U/ml, 100U/ml) are used, which is expensive.

Typical nonionic surfactants used in pharmaceutical formulations include Triton^TMX-100、F-68, F-88 and F-127 (poloxamers),Brij 35 (polyoxyethylene alkyl ether), polyoxyethylene stearate 40, EL and alpha-tocopherol TPGS. Each of these surfactants shares a common fact that they contain polyoxyethylene moieties and therefore exhibit more or less similar problems in that the polyoxyethylene moieties auto-oxidize to produce reactive peroxides, which can lead to an increase in the immunogenicity of the unwanted protein (see Edward T. Maggio et al; Polysorbates, peroxides, protein aggregation, immunogenicity-a growth control; Journal of Excipients and Food Chemicals 3(2): 46-53; 2012).

Various stabilizers are used to stabilize vaccine formulations to achieve a desired shelf life. Stabilizers such as polyvinylpyrrolidone (PVP), trehalose, and sorbitol are also used in virus preparations. However, PVP has been reported to destabilize live attenuated virus preparations. (see: JA White et al; Development of a stable liquid formation of a liquid attached underfluorza Vaccine; Vaccine Volume 34, Issue 32, 12July 2016, Pages 3676-3683; 2016).

Trehalose is high in cost; it must be combined with other carbohydrate and protein additives (gelatin) to achieve stability. Moreover, other stabilizers are superior to trehalose in improving the shelf-life stability of lyophilized vaccines.

Sorbitol has a low glass transition temperature (Tg) (-1.6 ℃) and therefore cannot be used as a major formulation component. The low Tg of sorbitol limits its use. Sorbitol must be combined with other carbohydrate and protein additives (gelatin) to achieve stability.

It has been suggested that for the route of administration of the vaccine, the theoretical impact of host residual DNA on product safety should be considered, as tissue distribution and clearance can vary based on the mode of administration. Studies have shown that the uptake and clearance of MDCK DNA from tissues varies depending on the route of administration. When DNA was administered intranasally, detectable levels of DNA were lower at all time points compared to intramuscular injections. Thus, the intranasal route of administration of the vaccine appears to reduce the potential risks associated with residual host cell DNA that may be present in the final vaccine product produced in cell culture. (see: D.E.Tabor et al; Biologicals 41(2013) 247-.

It is an object of the present disclosure to overcome the above limitations and also to provide compositions and methods for manufacturing MDCK (madin bitch dog kidney) cells based on an intranasal delivered live attenuated influenza virus vaccine (LAIV) to prevent influenza virus. The present disclosure further provides improved manufacturing methods that utilize low concentrations of endonucleases, more particularly benzoxygenases, and do not have chromatographic steps suitable for large scale cell culture production. Still further, the present disclosure provides a polymer and surfactant free LAIV formulation comprising one or more live attenuated influenza vaccine viruses.

Disclosure of Invention

The present disclosure provides MDCK cell-based intranasal delivery of a Live Attenuated Influenza Vaccine (LAIV) composition comprising:

a) one or more live attenuated influenza vaccine viruses;

wherein the live attenuated influenza vaccine strain is derived by a rearranged "classical" or "reverse genetics" method, consisting essentially of the Hemagglutinin (HA) gene and/or the Neuraminidase (NA) gene from wild-type pandemic or seasonal influenza virus and the genes expressing PB1, PB2, PA, NP, M and NS proteins, and in some cases the NA proteins are derived from the donor virus (MDV) A/Lianggelle/134/17/57 (H2N2) and/or B/USSR/60/69 strains,

b) one or more kinds of amino acids selected from the group consisting of,

c) one or more carbohydrates, and

d) gelatin.

The present disclosure further provides methods for making such vaccine compositions/formulations.

Purpose(s) to

Some of the objects of the present disclosure that are met by at least one embodiment herein are as follows:

it is an object of the present disclosure to alleviate one or more problems of the prior art or to at least provide a useful alternative.

It is another object of the present disclosure to provide vaccine compositions and methods of making Live Attenuated Influenza Vaccines (LAIVs) that can be delivered intranasally.

It is yet another object of the present disclosure to provide a Live Attenuated Influenza Vaccine (LAIV) composition based on madin bitch dog kidney (MDCK) cells, wherein the use of eggs is completely avoided.

It is yet another object of the present disclosure to provide a LAIV composition comprising one or more live attenuated influenza vaccine viruses and being free of polymers and surfactants.

It is a further object of the present disclosure to provide a LAIV composition comprising one or more live attenuated influenza vaccine viruses, wherein the LAIV strain is based on the cold-adapted, temperature sensitive and attenuated phenotype of a Master Donor Virus (MDV) comprising the surface glycoproteins of one or two wild-type pandemic or seasonal influenza strains.

It is yet another object of the present disclosure to provide live attenuated MDCK cell-based influenza vaccine (LAIV) compositions for intranasal delivery, wherein the compositions retain desirable characteristics of the virus, including immunogenicity and stability.

It is yet another object of the present disclosure to provide Live Attenuated Influenza Vaccine (LAIV) compositions/formulations based on intranasal delivery of MDCK cells, which are suitable for treating or preventing influenza virus infection, or preventing, alleviating or delaying the onset or progression of its clinical manifestations.

It is yet another object of the present disclosure to provide an improved process suitable for large scale cell culture production in the field of MDCK cell based attenuated live influenza vaccine production.

Other objects and advantages of the present disclosure will become more apparent in the following description which is not intended to limit the scope of the present disclosure.

Drawings

The disclosure will now be described with the aid of the figures listed below:

FIG. 1 illustrates a schematic representation of rearrangement of wild-type pandemic or seasonal influenza virus and attenuated MDV to produce rearranged vaccine strains, wherein (A) represents a wild-type viral infectious pathogen, (B) represents a main donor virus comprising temperature sensitive (ts), cold-adapted (ca) and attenuated (at) phenotypic gene segments, and (C) represents a rearranged vaccine strain.

FIG. 2: the graph illustrates the virus log yield titer (EID) for influenza A strains (A/17/Turkey/05/133-A/H5N 2)₅₀0.5ml) with stabilizer composition 1 disclosed in table 3B at 37 ℃.

FIG. 3: the graph illustrates the virus log yield titer (EID) of influenza A strains (A/17/Turkey/05/133-A/H5N 2 and A/17/Anhui/2013/61-A/H7N 9)₅₀0.5ml) with the stabilizer composition 1 disclosed in table 3A at 2 ℃ to 8 ℃.

FIG. 4: the graph illustrates the virus log yield titer (EID) of influenza A strain (A/17/California/2009/38-A/H1N 1) and influenza B strain (B/Texas/02/13-CDC)₅₀0.5ml) with stabilizer composition 3 disclosed in table 3B at 37 ℃.

FIG. 5: the graph illustrates the virus log yield titer (EID) of influenza A strain (A/17/California/2009/38-A/H1N 1) and influenza B strain (B/Texas/02/13-CDC)₅₀0.5ml) with the stabilizer composition 3 disclosed in table 3A at 2 ℃ to 8 ℃.

FIG. 6: the graph illustrates the virus log yield titer (EID) of influenza A strain (A/17/California/2009/38-A/H1N 1) and influenza B strain (B/Texas/02/13-CDC)₅₀0.5ml) with stabilizer composition 4 disclosed in table 3B at 37 ℃.

FIG. 7: the graph illustrates the virus log yield titer (EID) of influenza A strain (A/17/California/2009/38-A/H1N 1) and influenza B strain (B/Texas/02/13-CDC)₅₀0.5ml) with the stabilizer composition 4 disclosed in table 3A at 2 ℃ to 8 ℃.

FIG. 8: the graph illustrates the virus log yield titer (EID) of influenza A strain (A/south Africa/3626/13-H1N 1) and influenza B strain (B/Texas/02/13-CDC)₅₀0.5ml) with stabilizer composition 2 disclosed in table 3B at 37 ℃.

FIG. 9: the graph illustrates the virus log yield titer (EID) of influenza A strain (A/south Africa/3626/13-H1N 1) and influenza B strain (B/Texas/02/13-CDC)₅₀0.5ml) with the stabilizer composition 2 disclosed in table 3A at 2 ℃ to 8 ℃.

FIG. 10: the graph illustrates the virus log yield titer (EID) of influenza A strain (A/south Africa/3626/13-H1N 1) and influenza B strain (B/Texas/02/13-CDC)₅₀0.5ml) with stabilizer composition 1 disclosed in table 3B at 37 ℃.

FIG. 11: the graph illustrates the virus log yield titer (EID) of influenza A strain (A/south Africa/3626/13-H1N 1) and influenza B strain (B/Texas/02/13-CDC)₅₀0.5ml) with the stabilizer composition 1 disclosed in table 3A at 2 ℃ to 8 ℃.

FIG. 12: infectious virus testing in turbinate and lung samples. Animals were vaccinated with one or two doses of H5 LAIV, H7 LAIV, or placebo. Groups 1 through 4 were challenged with H5/Tk/Tk, groups 5 and 6 with H5/Vt, and groups 7 through 10 with H7/An. Turbinate samples (a) and lung samples (b) collected on day 4 post-infection were titrated to determine if replication-competent viral particles were present. Individual titers are shown as group means indicated by the solid black line.

FIG. 13: HI and VN antibody responses after immunization. At day 28 after final immunization (day 28 of the one-dose regimen study, day 56 of the two-dose regimen study), geometric mean antibody responses of H7/An in H5/Tk/Tk and H7 LAIV immunized animals against the cognate challenge virus of H5 LAIV immunized animals. (a) HI antibody titer, (b) VN antibody titer. Each set of N-6, error bars represent standard error of the mean. Statistical significance was determined by the U test of Mann-Whitney. P <0.05 and p < 0.01.

Detailed Description

While the present disclosure may be susceptible to various embodiments, there is shown in the drawings and will hereinafter be described in detail certain embodiments with the understanding that the present disclosure is to be considered an exemplification of the principles of the disclosure and is not intended to limit the scope of the disclosure to that illustrated and disclosed in the specification.

Embodiments of the present disclosure will now be described with reference to the accompanying drawings.

The embodiments are provided to repeatedly and fully convey the scope of the disclosure to those skilled in the art. Numerous details are set forth regarding specific components and methods in order to provide a thorough understanding of embodiments of the present disclosure. It will be apparent to those skilled in the art that the details provided in the embodiments should not be construed as limiting the scope of the disclosure. In some embodiments, well-known processes, well-known device structures, and well-known technologies are not described in detail.

The terminology used in the present disclosure is for the purpose of describing particular embodiments only, and such terminology is not to be taken as limiting the scope of the present disclosure. As used in this disclosure, the forms "a", "an" and "the" are also intended to include the plural forms as well, unless the context clearly indicates otherwise.

The terms first, second, third and the like should not be construed as limiting the scope of the present disclosure, as the foregoing terms may be used solely to distinguish one element, component, region, layer or section from another component, region, layer or section. Unless the disclosure expressly states otherwise, as used herein, terms such as first, second, third, etc. do not denote a particular sequence or order.

As used herein, the term "influenza virus" refers to an RNA virus that includes influenza a, b, c and d viruses representing the orthomyxoviridae family. The influenza virus may be a live wild-type pandemic or seasonal influenza virus, a live attenuated influenza vaccine virus, an inactivated influenza virus, a chimeric influenza virus or a recombinant influenza virus.

The present disclosure provides compositions comprising Live Attenuated Influenza Vaccine (LAIV) viruses for protection against influenza virus infection. The present disclosure further provides methods for manufacturing a composition comprising one or more influenza vaccine viruses.

According to a first embodiment of the present disclosure, the LAIV composition may comprise one or more live attenuated influenza vaccine viruses, one or more amino acids, one or more carbohydrates, and gelatin.

The term "live" is used in its conventional sense, a live virus being a virus that has not been inactivated, i.e. a virus that is capable of replicating on permissive cells. Live attenuated influenza vaccine viruses are viruses that do not induce disease in animals or humans caused by the corresponding wild-type virus and are capable of eliciting a specific immune response.

According to a second embodiment of the present disclosure, one or more live attenuated influenza vaccine viruses may be derived by a "classical" or "reverse genetics" rearrangement process that includes gene segments from one or more influenza strains.

According to a first aspect of the second embodiment, the live attenuated rearranged influenza vaccine virus is a rearranged LAIV virus comprising cold-adapted, temperature sensitive and/or attenuated phenotype gene segments (PB1, PB2, PA, NP, M and/or NS proteins) of a Main Donor Virus (MDV) strain and Hemagglutinin (HA) gene segments and/or Neuraminidase (NA) gene segments of a wild-type pandemic or seasonal influenza a or b or c virus strain in a ratio of 1:7, 2:6, 3:5, 4:4, 5:3, 6:2 or 7: 1.

Still preferably, the rearranged live attenuated influenza vaccine virus may comprise gene segments from a Main Donor Virus (MDV) strain and the wild-type pandemic or seasonal influenza virus strain in a ratio of 6:2 (as illustrated in fig. 1).

According to a second aspect of the second embodiment, the live, rearranged attenuated influenza vaccine virus may comprise gene segments derived from influenza a virus of any subtype or may be derived from the Main Donor Virus (MDV) of influenza b virus of any subtype.

The live, rearranged attenuated influenza vaccine virus may comprise gene segments from a Main Donor Virus (MDV) selected from the group comprising influenza a/liennegure/134/17/57 (H2N2) and influenza B/USSR/60/69.

Still preferably, the rearranged attenuated influenza a vaccine virus may comprise gene segments from a Main Donor Virus (MDV) comprising an influenza a/lienninggler/134/17/57 (H2N2) strain.

Still preferably, the rearranged live attenuated influenza vaccine type a strain a/17/california/2009/38 may comprise gene segments from a Main Donor Virus (MDV) comprising a/lienggler/134/17/57 (H2N2) influenza a strain.

Still preferably, the rearranged live attenuated influenza vaccine type a strain a/17/turkish/05/133 may comprise gene segments from a Main Donor Virus (MDV) comprising influenza a/lienninggler/134/17/57 (H2N 2).

Still preferably, the rearranged live attenuated influenza vaccine strain a/17/anhui/2013/61 may comprise gene segments from a Main Donor Virus (MDV) comprising influenza a/lienggler/134/17/57 (H2N 2).

Still preferably, the rearranged live attenuated influenza vaccine strain a/17/new york/15/5364 may comprise gene segments from a Main Donor Virus (MDV) comprising influenza a/lienninggler/134/17/57 (H2N 2).

Still preferably, the rearranged live attenuated influenza vaccine strain a/17/hong kong/2014/8296 may comprise gene segments from a Main Donor Virus (MDV) comprising influenza a/lieng gler/134/17/57 (H2N 2).

Still preferably, the rearranged live attenuated influenza vaccine strain a/south africa/3626/2013-CDC-LV 14A may comprise gene segments from a Main Donor Virus (MDV) comprising influenza a/lienninggler/134/17/57 (H2N 2).

Still preferably, the rearranged attenuated influenza B vaccine virus may comprise gene segments from a Main Donor Virus (MDV) comprising the influenza B/USSR/60/69B strain.

Still preferably, the rearranged live attenuated vaccine influenza B strain B/texas/02/2013-CDC-LV 8B may comprise gene segments from a Main Donor Virus (MDV) comprising the influenza B/USSR/60/69 strain B.

Still preferably, the rearranged live attenuated vaccine influenza B strain B/pregabalin/3073/2013 may comprise gene segments from a Main Donor Virus (MDV) comprising the influenza B/USSR/60/69 strain.

Still preferably, the rearranged live attenuated vaccine influenza B strain B/56/brisban/60/08 may comprise gene segments from a Master Donor Virus (MDV) comprising an influenza B/USSR/60/69 strain.

According to a third aspect of the second embodiment, the rearranged, attenuated, live influenza vaccine virus may comprise a Hemagglutinin (HA) gene and/or a Neuraminidase (NA) gene from an influenza a virus or an influenza b virus or an influenza c virus.

Still preferably, the rearranged live attenuated influenza a vaccine strain may comprise Hemagglutinin (HA) genes from any other reported HA subtype of influenza a virus H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17 or H18 and/or Neuraminidase (NA) genes from the NA subtype of influenza a virus N1, N2, N3, N4, N5, N6, N7, N8, N9, N10 or N11 or any other reported NA subtype.

Still preferably, the rearranged live attenuated influenza vaccine virus may comprise a Hemagglutinin (HA) gene and/or a Neuraminidase (NA) gene from a pandemic influenza strain or a potential pandemic influenza strain.

Still preferably, the rearranged live attenuated influenza vaccine virus may comprise a Hemagglutinin (HA) gene and/or a Neuraminidase (NA) gene from a seasonal influenza strain.

Still preferably, the rearranged live attenuated influenza vaccine strain may comprise Hemagglutinin (HA) genes and/or Neuraminidase (NA) genes from influenza a virus subtypes H1N1, H2N2, H3N2, H5N1, H5N2, H9N2, H7N1, H7N3, H7N7, H6N1, H7N9 and H10N8 or any other previously reported or newly detected strain.

Still preferably, the rearranged live attenuated influenza vaccine strain may comprise the Hemagglutinin (HA) gene and/or the Neuraminidase (NA) gene from the pdmH1N1 strain of influenza a virus.

Still preferably, the rearranged live attenuated influenza vaccine virus strain may comprise the Hemagglutinin (HA) gene and/or the Neuraminidase (NA) gene from an influenza a virus a/california/07/2009 (referred to as a/Cal) like strain.

Still preferably, the rearranged live attenuated influenza vaccine virus strain may comprise a Hemagglutinin (HA) gene and/or a Neuraminidase (NA) gene from an influenza a virus a/michigan/45/2015-like strain.

Still preferably, the rearranged live attenuated influenza vaccine virus strain may comprise Hemagglutinin (HA) genes and/or Neuraminidase (NA) genes from influenza b viruses belonging to two different lineages Yamagata-like or victoria-like.

Still preferably, the rearranged live attenuated influenza vaccine virus strain may comprise a Hemagglutinin (HA) gene and/or a Neuraminidase (NA) gene from a victoria lineage B/brisbane/60/2008-like strain of influenza B virus.

Still preferably, the rearranged live attenuated influenza vaccine virus strain may comprise a Hemagglutinin (HA) gene and/or a Neuraminidase (NA) gene from a Yamagata lineage B/pregabalin/3073/2013-like strain of influenza B virus.

There are two methods for generating rearrangements

1. Typical rearrangement Process

The process of shuffling both wild-type pandemic or seasonal influenza vaccine viruses of rearranged somatic strains and attenuated MDVs involves co-infection of a culture host (typically an egg) with an MDV strain and a wild-type virus strain. The reassortant virus is selected by adding an antibody specific for the HA and/or NA protein of the MDV, so that a reassortant virus comprising the HA and/or NA protein of a wild-type virus strain is selected. After several generations of such treatments, rapidly growing reassortant viruses can be selected that contain wild-type pandemic or seasonal influenza vaccine virus strains HA and/or NA fragments as well as internal genes of MDV.

2. Reverse genetics methods for rearrangements

Reverse genetics is a method of producing infectious viral particles from DNA replication. The six internal genes of MDV, as well as the HA and NA genes from the wild-type strains recommended for inclusion in the vaccine, were cloned in plasmids that, when transfected in cultured cells, produced full-length viral RNA. These plasmids are transfected into cultured cells along with four plasmids expressing viral polymerase subunits. Expression of the polymerase subunit and production of full-length viral RNA results in viral assembly and release of infectious viral particles in the supernatant. This recovered virus exhibited the antigenic characteristics of the recommended strain as well as the ca, ts, att phenotypes of MDV.

Rearranged LAIV strains were purchased from russia st petderburgh laboratory medicine Institute (IEM) or WHO cooperative centers, such as the atlanta disease control and prevention center (CDC).

According to a third embodiment of the present disclosure, the LAIV composition may comprise one or more carbohydrates selected from, but not limited to, the group of: natural carbohydrates, synthetic carbohydrates, polyols, glass transition promoters, monosaccharides, disaccharides, trisaccharides, oligosaccharides and their corresponding sugar alcohols, polyols, such as carbohydrate derivatives and chemically modified carbohydrates, hydroxyethyl starch and sugar copolymers. Both natural and synthetic carbohydrates are suitable for use. Synthetic carbohydrates include, but are not limited to, those in which the glycosidic bond is replaced by a thiol or carbon bond. Carbohydrates in the D and L forms may be used. The carbohydrate may be non-reducing or reducing. When a reducing carbohydrate is used, a Maillard reaction inhibitor is preferably added. Reducing carbohydrates suitable for use in the composition are those known in the art and include, but are not limited to, glucose, sucrose, maltose, lactose, fructose, galactose, mannose, maltulose, and lactulose. Non-reducing carbohydrates include, but are not limited to, non-reducing glycosides of polyhydroxy compounds selected from sugar alcohols and other linear polyols. Other useful carbohydrates include raffinose, stachyose, melezitose, dextran, cellobiose, mannose and sugar alcohols. The sugar alcohol glycoside is preferably a monoglycoside, in particular a compound obtained by reducing disaccharides such as lactose, maltose, lactulose and maltulose. The glass former is selected from the group consisting of: sucrose, mannitol, trehalose, mannose, raffinose, lactitol, lactobionic acid, glucose, maltulose, isomaltulose, maltose, lactose sorbitol, glucose, fructose, glycerol or combinations thereof.

However, according to a preferred aspect of the third embodiment, the LAIV composition may comprise sucrose as a suitable carbohydrate stabilizer in the range between 1% and 20% weight/volume, preferably between 1% and 10%, more preferably between 3% and 6%, most preferably equal to 4% (w/v).

According to a fourth embodiment of the present disclosure, the LAIV composition may comprise one or more amino acids selected from the group consisting of, but not limited to: tris (hydroxymethyl) methylglycine, arginine, leucine, isoleucine, histidine, glycine, glutamine, lysine, alanine, a peptide, a hydrolysed protein or a protein, such as serum albumin.

According to still a preferred aspect of the fourth embodiment, the LAIV composition may comprise tris (hydroxymethyl) methylglycine, arginine, histidine and alanine, alone or in combination, as suitable amino acids.

According to still a preferred aspect of the fourth embodiment, the one or more amino acids may comprise tris (hydroxymethyl) methylglycine in the range between 0.1% and 2% weight/volume (w/v), preferably between 0.1% and 1%, more preferably between 0.1% and 0.5%, most preferably equal to 0.3% (w/v).

According to still a preferred aspect of the fourth embodiment, the one or more amino acids may comprise histidine in the range of between 0.1% and 2% (w/v), preferably between 0.1% -1%, more preferably between 0.1% -0.5%, most preferably equal to 0.21% (w/v).

According to still a preferred aspect of the fourth embodiment, the one or more amino acids may comprise alanine in the range between 0.01% and 1% weight/volume, preferably between 0.05% and 0.5%, more preferably between 0.08% and 0.2%, most preferably equal to 0.1% (w/v).

Also, according to a preferred aspect of the fourth embodiment, the one or more amino acids may comprise arginine in a range between 0.1% and 10% weight/volume, preferably between 0.1-5%, more preferably between 0.1-3%, most preferably equal to 2.1% (w/v).

According to a fifth embodiment of the present disclosure, the LAIV composition may comprise gelatin in a range between 0.1% and 10% weight/volume, preferably between 0.1% -5%, more preferably between 0.1% -3%, most preferably equal to 0.85% (w/v).

As used herein, the term "gelatin" refers to a sterile, pyrogen-free protein preparation (e.g., fraction) produced by partial acid hydrolysis (type a gelatin) or partial base hydrolysis (type B gelatin) of animal collagen, the most common sources being bovine, porcine, and fish. Gelatin of different molecular weight ranges can be obtained. Recombinant gelatin sources may also be used.

According to a sixth embodiment of the present disclosure, the LAIV composition may further comprise a buffering agent selected from the group consisting of: HEPES, citrate-phosphate, carbonate, phosphate, citrate, lactate, gluconate, and tartrate buffers, as well as more complex organic buffers including phosphate buffers containing sodium and/or potassium phosphate in selected proportions to achieve a desired pH. In another embodiment, the buffer comprises Tris (hydroxymethyl) aminomethane or "Tris" formulated to achieve a desired pH value. In yet another embodiment, the buffer can be minimal medium with hanks salts.

According to a seventh embodiment of the present disclosure, wherein the single dose composition is preservative-free and the multi-dose composition may additionally comprise a preservative selected from the group comprising: 2-phenoxyethanol, benzethonium chloride (Phemerol), phenol, m-cresol, thimerosal, formaldehyde, a paraben (e.g., methyl-, ethyl-, propyl-, or butyl paraben), benzalkonium chloride, benzyl alcohol, chlorobutanol, p-chloro-m-cresol, or benzyl alcohol, or a combination thereof. Vaccine compositions may include materials for a single immunization, or may include materials for multiple immunizations (i.e., "multi-dose" kits). Preservatives are preferably included in the multi-dose arrangement. As an alternative to including a preservative (or in addition to a preservative) in a multi-dose composition, the composition may be contained in a container having a sterile adapter for removal of material.

According to an eighth embodiment of the present disclosure, the LAIV composition may further comprise a pharmaceutically acceptable transporter, an excipient, a binder, a carrier, an isotonicity agent, an emulsifier, or a wetting agent, wherein the pharmaceutically acceptable excipient is selected from the group consisting of: surfactants, polymers and salts. Examples of surfactants may include nonionic surfactants such as polysorbate 20, polysorbate 80, and the like. Examples of polymers may include dextran, carboxymethyl cellulose, hyaluronic acid, cyclodextrin, and the like. Examples of salts may include NaCl, KCl, KH₂PO₄、Na₂HPO₄.2H₂O、CaCl₂、MgCl₂And the like.

According to a ninth embodiment of the present disclosure, the LAIV composition may further comprise an adjuvant selected from the group consisting of: aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate, and aluminum potassium sulfate, or a mixture thereof.

According to a tenth embodiment of the present disclosure, the LAIV composition further comprises an immunostimulatory component selected from the group consisting of: oil and water emulsions, MF-59, liposomes, lipopolysaccharides, saponins, lipid A derivatives, monophosphoryl lipid A, 3-deacylated monophosphoryl lipid A, AS01, AS03, oligonucleotides comprising at least one unmethylated CpG and/or liposomes, Freund's adjuvant, Freund's complete adjuvant, Freund's incomplete adjuvant, polymers, copolymers, such AS polyoxyethylene-polyoxypropylene copolymers, including block copolymers, polymer p 1005, CRL-8300 adjuvant, muramyl dipeptide, TLR-4 agonists, flagellin derived from gram-negative bacteria, TLR-5 agonists, fragments of flagellin capable of binding to TLR-5 receptors, alpha-C-galactosyl ceramide, deacetylated chitin, interleukin-2, QS-21, QS-5, and pharmaceutically acceptable salts thereof, ISCOMS, saponins in combination with sterols and lipids.

According to an eleventh embodiment of the present disclosure, the method of preparing a LAIV composition based on MDCK cell cultures may comprise any subset or all of the following steps:

a) the LAIV candidate vaccine virus was initially passaged in SPF chick embryos to produce an egg-based Master Seed Virus (MSV).

b) The egg-based master seed virus is suitable for growth on cell culture hosts to produce cell-based Working Seed Viruses (WSV). The cell-based WSV is in a different cell culture vessel/system, e.g., 175cm in surface area²The surface area of the Tissue Culture Flasks (TCFs) of (1) is 850cm²Roller Bottles (RBs) of 6320cm in surface area²Cell Facilities (CFs) and fixed bed bioreactors (e.g., fromOf Life sciences Co., Ltd (Washington harbor, N.Y.)Bioreactors such as Nano bioreactor and 500/100 bioreactor).

c) The cultured virus was harvested.

d) The virus harvest is filtered by Direct Flow Filtration (DFF) through at least one clarification filter to obtain a Clarified Virus Pool (CVP).

e) The CVP is treated with a non-specific endonuclease to degrade cellular DNA.

f) The treated endonuclease-treated CVP was subjected to tangential flow filtration.

g) The TFF concentrate is stabilized with a stabilizer composition comprising one or more carbohydrates, one or more amino acids, and gelatin to form a stabilized viral harvest.

h) The stable TFF concentrate is sterilized by DFF through at least one sterilization grade filter to obtain a sterilized Clear Monovalent Virus Pool (CMVP).

i) The sterilized CMVP is stored in a polycarbonate bottle at-60 ℃ or below.

j) The sterilized formulations were filled into vials and stored at 2 ℃ to 8 ℃.

According to the first aspect of the eleventh embodiment, the egg-based LAIV virus candidate suitable for growth in a cell culture host may be any eukaryotic cell. Still preferably, the cell culture host may be a mammalian or avian cell. Suitable mammalian cells include, but are not limited to, hamster, bovine, primate (including human and monkey) and dog cells. Various cell types include, but are not limited to, kidney cells, fibroblasts, retinal cells, and lung cells. An example of a suitable hamster cell is the cell line named BHK21 or HKCC. Suitable monkey cells are, for example, african green monkey cells, such as kidney cells in the Vero cell line. Suitable dog cells are, for example, kidney cells in CLDK and MDCK cell lines.

Further suitable cells include, but are not limited to: CHO; 293T; BHK; MRC 5; c6, PER; FRhl.2; WI-38, etc. Suitable cells are widely available from, for example, the American Type Cell Culture (ATCC) collection, the Coriell cell bank, or the European cell culture Collection (ECACC). For example, ATCC provides various Vero cells having catalog numbers CCL 81, CCL 81.2, CRL 1586, and CRL-1587, and it provides MDCK cells having catalog number CCL 34. C6 is obtainable from ECACC under accession number 96022940.

Still preferably, the cell culture host may be a Madin Darby dog kidney (MDCK) cell selected from, but not limited to, ATCC CCL-34, the MDCK33016 cell line (DSM ACC 2219), MDCK (ATCC CCL34MDCK (NBL2)), MDCK33016(DSM ACC 2219), DSM ACC3309, ATCC CRL-12042, ATCC PTA-7909, ATCC PTA-7910, ATCC PTA-6500, ATCC PTA-6501, ATCC PTA-6502, ATCC PTA-6503, "MDCK-S", "MDCK-SF 101", "MDCK-SF 102", "MDCK-SF 103", and FERM BP-7449.

Still preferably, the cell culture host may be a madin bitch dog kidney (MDCK) cell ATCC CCL34 (NBL 2).

According to a second aspect of the eleventh embodiment, the MDCK cells may be cultured in a Minimal Essential Medium (MEM) comprising 10% Fetal Bovine Serum (FBS). The culturing of the cells may be carried out at 37 ℃. + -. 1 ℃. The pH of the medium during propagation of the cells prior to infection may be in the range of pH6.8 and pH7.6, and more preferably between pH 7.0 and pH 7.4.

Still, MDCK cells can be cultured in serum-free or protein-free media.

According to a third aspect of the eleventh embodiment, MDCK cells may be washed with MEM prior to infection, and subsequently washed with MEM containing a protease in the range of 5U/ml to 25U/ml.

However, the protease may be selected from, but is not limited to, trypsin, chymotrypsin, fungal protease, pepsin, papain, bromelain, and subtilisin.

Still preferably, the protease may be trypsin obtained from porcine or bovine origin or from fungal or bacterial origin.

Still preferably, the protease may be a recombinant trypsin expressed in a yeast or plant or bacterial host cell selected from the group consisting of, but not limited to, aspergillus, streptomyces griseus, maize, escherichia coli, pichia pastoris. Preferably, the recombinant trypsin is selected from Biogenomics (e.coli as host), d.k.bio Pharma pvt.ltd (e.coli as host), Richcore (pichia pastoris as host) and Gibco (fungi).

Still, the preferred trypsin concentration is 12.5U/ml.

According to a fourth aspect of the eleventh embodiment, prior to infection, the working seed virus may be obtained by diluting the virus with MEM containing a protease in the range of 5U/ml to 25U/ml and incubating at a temperature of 31 ℃ to 33 ℃ for 10 minutes to 60 minutes.

The protease may be selected from, but is not limited to, trypsin, chymotrypsin, fungal protease, pepsin, papain, bromelain, and subtilisin.

Still preferably, the protease may be trypsin obtained from porcine or bovine origin or from fungal or bacterial origin.

Still, the preferred trypsin concentration is 12.5U/ml.

Still, a preferred trypsin concentration is 2000 to 3000 units of trypsin per roller bottle.

According to a fifth aspect of the eleventh embodiment, infection of MDCK cells with LAIV candidate virus may occur in a bioreactor (4 m)²) Has a preferred TCF of about 40-60X 10⁶TCF, RB about 150-⁶/RB, and 7000-10000 × 10⁶/BR(4m²) MDCK cell density of (a).

According to a sixth aspect of the eleventh embodiment, the LAIV candidate virus may be grown in adherent culture or suspension culture mode on MDCK cells.

According to a seventh aspect of the eleventh embodiment, infecting MDCK cells with an egg-based LAIV candidate virus may occur at an MOI between 1:100 and 1: 10000.

According to an eighth aspect of the eleventh embodiment, the post-infection MDCK cells may be cultured in a Minimum Essential Medium (MEM) containing trypsin in the range of 5U/ml to 25U/ml and at a temperature of 32 ℃ ± 1 ℃. The pH of the medium after infection may be in the range of pH6.8 and pH7.6, and most preferably in the range of 7.2 to 7.6.

According to a ninth aspect of the eleventh embodiment, the time may be 40 to 70 hours after infection; more preferably, the cell supernatant may be harvested after an incubation period of 54 ± 8 hours.

Still alternatively, multiple harvests may be performed at appropriate time intervals for about 4 to 5 times before input material is discarded and processed separately to obtain a Clarified Monovalent Virus Pool (CMVP).

After harvest, EID of at least 7.0 to 9.2Log can be achieved₅₀Viral yield of 0.5 ml.

According to a tenth aspect of the eleventh embodiment, the virus-containing medium can be clarified, typically through a filter of reduced pore size (e.g., 6 μ, 5 μ, 0.8 μ, 0.65 μ, 0.45 μ, 0.2 μ). Suitable commercially available filters and filtration devices are well known in the art and can be selected by the skilled artisan. Exemplary filtration devices can be made from polypropylene or cellulose acetate or polyethersulfone, and commercially available filters can be millipak (millipore), kleenpak (pall), and sartobran (sartorius) filtration devices.

According to an eleventh aspect of the eleventh embodiment, the filtered harvest can be treated with a non-specific endonuclease, most preferably a benzoate enzyme, at a concentration varying between 0.5 units/ml and 2 units/ml at a temperature ranging between 30 ℃ and 34 ℃ for 2 hours to 6 hours, and then at a temperature of 2 ℃ to 8 ℃ for 5 hours to 15 hours.

Still alternatively, the filtered harvest may be treated with a non-specific endonuclease, most preferably a benzoate enzyme, in the presence of an amount of divalent cations between 0.1mM and 100mM, selected from the group consisting of Ca²⁺、Mg²⁺、Mn²⁺And Cu²⁺In the group consisting of.

Alternatively still, divalent cation Mg may be present at a concentration of 1mM to 3mM²⁺In the case of salt, the filtered harvest is treated with a non-specific endonuclease, most preferably a benzoate enzyme.

According to a twelfth aspect of the eleventh embodiment, the benzoate enzyme treated harvest may be further subjected to Tangential Flow Filtration (TFF), typically by a Molecular Weight Cut Off (MWCO) filter between 100KDa and 500KDa, to obtain a 2-fold to 10-fold concentrated viral harvest, and further to remove residual impurities.

Still preferably, the residual impurities may include residual DNA, residual Bovine Serum Albumin (BSA), and residual host cell proteins.

According to a thirteenth aspect of the eleventh embodiment, the above method can produce a purified and concentrated LAIV virus harvest comprising trace amounts of residual cellular DNA (<10 ng/dose), residual BSA (<50 ng/dose), and residual cellular proteins. Furthermore, according to the above method, the total recovery of purified virus may be at least 40%.

According to a fourteenth aspect of the eleventh embodiment, the concentrated monovalent virus stock solution (TFF concentrate) may be stabilized with a stabilizer composition to obtain a final LAIV composition comprising one or more carbohydrates, one or more amino acids and gelatin.

Still preferably, the concentrated virus bulk (TFF concentrate) may be stabilized with a stabilizer composition comprising any combination of sucrose, histidine, alanine, tris (hydroxymethyl) methylglycine, arginine and gelatin.

Still preferably, the concentrated virus bulk (TFF concentrate) may be stabilized with a stabilizer composition comprising sucrose at a concentration of 1% to 10% (w/v), histidine at a concentration of 0.1% to 2% (w/v), alanine at a concentration of 0.01% to 1% (w/v), tris (hydroxymethyl) methylglycine at a concentration of 0.1% to 1% (w/v), arginine at a concentration of 0.1% to 5% (w/v), gelatin at a concentration of 0.1% to 5% (w/v).

Still preferably, the concentrated virus bulk (TFF concentrate) may be stabilized with a stabilizer composition comprising sucrose at a concentration of 3 to 6% (w/v), histidine at a concentration of 0.1% to 1% (w/v), alanine at a concentration of 0.05% to 0.5% (w/v), tris (hydroxymethyl) methylglycine at a concentration of 0.1% to 0.5% (w/v), arginine at a concentration of 0.1% to 3% (w/v) and gelatin at a concentration of 0.1% to 3% (w/v).

Still preferably, the concentrated virus bulk (TFF concentrate) may be stabilized with a stabilizer composition comprising 4% (w/v) sucrose, 0.21% (w/v) histidine, 0.1% (w/v) alanine, 0.3% (w/v) tris (hydroxymethyl) methylglycine, 2.1% (w/v) arginine and 0.85% (w/v) gelatin.

According to a fifteenth aspect of the eleventh embodiment, the stabilized viral harvest is sterilizable by Direct Flow Filtration (DFF) through at least one sterilization grade filter, preferably 0.2 μ. Suitable commercially available filters and filtration devices are well known in the art and can be selected by the skilled artisan. Exemplary filtration devices can be made from polypropylene or cellulose acetate or polyethersulfone or polyvinylidene fluoride, and commercially available filters can be Millipak (Millipore), Kleenpak (pall), and Sartobran^TMP (sartorius) filtration device.

According to a sixteenth aspect of the eleventh embodiment, the LAIV composition may be multivalent, comprising more than one LAIV strain or subtype as disclosed in the previous embodiments. The LAIV composition may be divalent or trivalent or tetravalent.

Still alternatively, the LAIV composition may be monovalent, including any one of the LAIV strains or subtypes as disclosed in the previous embodiments.

According to a seventeenth aspect of the eleventh embodiment, the LAIV composition may comprise a dose of 6 to 7LogEID₅₀0.5ml of influenza virus.

Still preferably, the LAIV composition may comprise influenza a virus or any subtype at a dose of 6 to 7LogEID₅₀0.5 ml; more preferably not less than 7LogEID₅₀/0.5ml。

Still preferably, the LAIV composition may comprise influenza b virus or any subtype at a dose of 6 to 7LogEID₅₀0.5 ml; more preferably not less than 6.5LogEID₅₀/0.5ml。

According to a twelfth embodiment, a method of making an immunogenic composition can comprise the steps of:

a) infecting a MDCK cell culture host with an influenza virus at a MOI between 1:100 and 1: 10000;

b) harvesting the supernatant comprising influenza virus after 40 to 70 hours incubation in MEM containing trypsin in the range of 5 to 25U/ml;

c) filtering the viral harvest by Direct Flow Filtration (DFF) through at least one clarification filter having a pore size between about 6 microns to about 0.45 microns;

d) treating the CVP with a non-specific endonuclease at a temperature in the range of 30 ℃ to 34 ℃ for 2 hours to 6 hours, and then at a temperature of 2 ℃ to 8 ℃ for 5 hours to 15 hours;

e) concentrating the endonuclease-treated CVP by Tangential Flow Filtration (TFF) using a membrane having a molecular weight cut-off (MWCO) of 100KDa to 500 KDa;

f) stabilizing the TFF concentrate with a stabilizer composition comprising one or more carbohydrates, one or more amino acids, and gelatin to form a stabilized viral harvest;

g) sterilizing the stabilized TFF concentrate by DFF through at least one sterilization grade filter having a pore size between about 0.8 microns to about 0.2 microns to form sterilized CMVP;

wherein the total recovery of purified virus is greater than or equal to 40%.

According to a first aspect of the twelfth embodiment, the method of preparing an immunogenic composition, wherein step (d) may comprise adding a pharmaceutically acceptable carrier selected from the group consisting of calcium, magnesium, calcium, magnesium²⁺、Mg²⁺、Mn²⁺And Cu²⁺(ii) treating the viral harvest with a non-specific endonuclease, more particularly a benzoate enzyme, in a concentration range of 0.5 units/ml to 5 units/ml in the case of divalent cations in a group and in an amount of between 0.1mM and 100 mM.

According to a second aspect of the twelfth embodiment, a method of preparing an immunogenic composition, wherein step (d) may comprise the presence of divalent cation Mg in a concentration of 1mM to 3mM²⁺In the case of salt, the viral harvest is treated with a non-specific endonuclease, more particularly a benzoate, at a concentration in the range of 0.5 units/ml to 5 units/ml.

According to a third aspect of the twelfth embodiment, a method of making an immunogenic composition, wherein step (e) can comprise concentrating a viral harvest by Tangential Flow Filtration (TFF) to obtain a viral harvest that is at least 4-fold concentrated.

According to a fourth aspect of the twelfth embodiment, the method of preparing an immunogenic composition, wherein step (f) may comprise stabilizing the viral harvest with a stabilizer composition comprising sucrose at a concentration of 1% (w/v), histidine at a concentration of 0.1% to 2% (w/v), alanine at a concentration of 0.01% to 1% (w/v), tris (hydroxymethyl) methylglycine at a concentration of 0.1% to 2% (w/v), arginine at a concentration of 0.1% to 5% (w/v) and gelatin at a concentration of 0.11% to 5% (w/v).

According to a fifth aspect of the twelfth embodiment, the method of preparing an immunogenic composition, wherein step (f) may comprise stabilizing the viral harvest with a stabilizer composition comprising sucrose at a concentration of 3% to 6% (w/v), histidine at a concentration of 0.1% to 1% (w/v), alanine at a concentration of 0.05% to 0.5% (w/v), tris (hydroxymethyl) methylglycine at a concentration of 0.1% to 0.5% (w/v), arginine at a concentration of 0.1% to 3% (w/v) and gelatin at a concentration of 0.1% to 3% (w/v).

According to a sixth aspect of the twelfth embodiment, the method of making an immunogenic composition, wherein step (f) can comprise stabilizing the viral harvest with a stabilizer composition comprising 4% (w/v) sucrose, 0.21% (w/v) histidine, 0.1% (w/v) alanine, 0.3% (w/v) tris (hydroxymethyl) methylglycine, 2.1% (w/v) arginine and 0.85% (w/v) gelatin.

According to a seventh aspect of the twelfth embodiment, the method of making an immunogenic composition, wherein step (f) can comprise stabilizing the viral harvest with a stabilizer composition comprising 4% (w/v) sucrose, 0.21% (w/v) histidine, 0.1% (w/v) alanine, 0.3% (w/v) tris (hydroxymethyl) methylglycine, 1.6% (w/v) arginine and 1.0% (w/v) gelatin.

According to a thirteenth embodiment of the disclosure, an immunogenic composition may comprise a) one or more Live Attenuated Influenza Vaccine (LAIV) viruses at a dose of 6 to 7LogEID₅₀0.5 ml; b) 1% to 10% (w/v) sucrose; c) 0.1% to 2% (w/v) histidine; d) 0.01% to 1% (w/v) alanine; e) 0.1% to 2% (w/v) tris (hydroxymethyl) methylglycine; f) 0.1% to 5% (w/v) arginine; g) 0.1% to 5% (w/v) gelatin.

Still preferably, the immunogenic composition may comprise a) one or more Live Attenuated Influenza Vaccine (LAIV) viruses at a dose of 6 to 7LogEID₅₀0.5 ml; b) 3% to 6% (w/v) sucrose; c) 0.1% to 1% (w/v) histidine; d) 0.05% to 0.5% (w/v) alanine; e) 0.1% to 0.5% (w-v) tris (hydroxymethyl) methylglycine; f) 0.1% to 3% (w/v) arginine; g) 0.1% to 3% (w/v) gelatin.

Still preferably, the immunogenic composition may comprise a) one or more Live Attenuated Influenza Vaccine (LAIV) viruses at a dose of 6 to 7LogEID₅₀0.5 ml; b) 4% (w/v) sucrose; c) 0.21% (w/v) histidine; d) 0.1% (w/v) alanine; e) 0.3% (w/v) tris (hydroxymethyl) methylglycine; f) 2.1% (w/v) arginine; g) 0.85% (w/v) gelatin.

Still preferably, the immunogenic composition may comprise a) one or more Live Attenuated Influenza Vaccine (LAIV) viruses at a dose of not less than 6 to 7LogEID₅₀0.5 ml; b) 4% (w/v) sucrose; c) 0.21% (w/v) histidine; d) 0.1% (w/v) alanine; e) 0.3% (w/v) tris (hydroxymethyl) methylglycine; f) 2.1% (w/v) arginine; g) 1% (w/v) gelatin.

According to a fourteenth embodiment of the present disclosure, the LAIV composition may be liquid-full.

Still alternatively, the LAIV composition may be a lyophilized or freeze-dried composition.

As used herein, the term "freeze-drying" or "lyophilization" relates to lyophilization and refers to the process in which a suspension is frozen, followed by removal of water by sublimation at low pressure. As used herein, the term "sublimation" refers to a change in a physical property of a composition, wherein the composition changes directly from a solid state to a gas state without changing to a liquid.

According to a fifteenth embodiment of the present disclosure, the LAIV composition may be formulated as a method for reducing the onset of or preventing a health condition comprising immunization of a human subject by intranasal or other route with an effective amount of the LAIV composition.

According to a preferred aspect of the embodiments, the LAIV composition may be administered to the human subject by an intranasal route. In one embodiment, it is an intranasal dispersion device, such as a device in the form of an aerosol (intranasal spray) or a droplet delivery system. Liquid nasal formulations can be delivered by nasal spray, drip and nasal catheter, compressed air nebulizer, squeeze bottle, metered pump spray (e.g., multi-dose metered spray pump or single/dual dose spray pump, spray device connected to syringe). Other dosage forms may be selected from nasal powders (insufflators, dry powder inhalers), nasal gels, nasal drops, solutions, suspensions, co-solvent systems, microspheres, nanoparticles, microemulsions, nasal inserts.

The intranasal delivery device may be selected from, but is not limited to, Becton Dickinson (BD) Accuspray^TMDelivery device, Bi-Directional Optinose device, Teleflex's MAD intranasal mucosal atomization device, AeroLife^TMAnd AeroVax (Aerovacx, Inc., Atlanta, GA), jet injector-A needleless injector; mutjis multi-purpose nozzle jet injector: an Aquapuncture device, Disposable syringe jet injector:Vitajet^TM、LectraJet HS、ZetaJet^TM、Aktiv-DryPuffHaler^TMand nasal spray influenza vaccine devices.

According to a sixteenth embodiment of the present invention, the LAIV composition may be formulated as a method for reducing the onset of or preventing a health condition comprising an influenza a virus infection or a subtype thereof as disclosed in a previous embodiment of the present disclosure, an influenza b virus infection or a subtype thereof as disclosed in a previous embodiment of the present disclosure or an influenza c virus infection or a subtype thereof as disclosed in a previous embodiment of the present disclosure.

According to a seventeenth embodiment of the present disclosure, the LAIV composition described above may be administered intranasally at a dose that is effective for protection. The vaccine is administered in a manner compatible with the dosage form and in a prophylactically effective amount. The immunogenic compositions of the present disclosure may be administered as a primary prophylactic in adults or children at risk of infection. For example, the live attenuated influenza vaccine compositions disclosed herein can be used in adults or children at risk of infection with influenza virus.

More preferably, the LAIV composition may be administered intranasally in a dosage volume of about 0.1ml to 0.5 ml.

According to an eighteenth embodiment of the present disclosure, the LAIV composition may be formulated as a single dose vial or multiple dose kit or pre-filled syringe or nasal spray, wherein the LAIV composition may be administered in a single dose regimen, or preferably a multiple dose regimen, wherein the main course of vaccination is followed by 1 to 2 individual doses administered at subsequent time intervals required to maintain and/or boost the immune response, e.g., a second dose administered within 1-4 months if needed, followed by subsequent vaccinations within months or years or annually. The dosage regimen will also depend, at least in part, on the need for a booster dose needed to confer protective immunity.

According to a nineteenth embodiment of the present disclosure, the final pH of the immunogenic composition can comprise 6.5 to 8.

Other embodiments disclosed herein also include a vaccine kit comprising a first container containing a lyophilized (freeze-dried) immunogenic composition and a second container containing an aqueous solution, optionally saline or WFI (water for injection), for reconstitution of the lyophilized (freeze-dried) LAIV composition.

Throughout the specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps, and the word "comprising", will be understood to be open-ended, allowing for the presence of more than the stated, provided that the stated essential or novel features are not changed by the presence of the stated, but not excluding prior art embodiments.

Throughout this specification, the word "immunogenic composition" encompasses any composition that elicits an immune response against an antigen or immunogen of interest expressed from a vector; for example, following administration to a subject, an immune response is elicited against a targeted immunogen or antigen of interest. The terms "vaccine composition" and "vaccine" encompass any composition that induces a protective immune response against an antigen of interest or effectively provides protection against an antigen; for example, following administration or injection to a subject, a protective immune response is elicited against a targeted antigen or immunogen or effective protection is provided against an antigen or immunogen expressed from a vector.

The use of the expression "one or more" or "at least one" suggests the use of one or more elements or ingredients or quantities, as such use may be in embodiments of the invention to achieve one or more desired objectives or results. While certain embodiments of the present invention have been described, these embodiments have been presented by way of example only, and are not intended to limit the scope of the invention. Variations or modifications of the compositions of the present invention that are within the scope of the invention may occur to those of skill in the art upon reading the disclosure herein. Such changes or modifications are also within the spirit of the present disclosure.

Unless stated to the contrary in the specification, the numerical values given for the various physical parameters, dimensions, and quantities are only approximate, and values higher than the numerical values specified for the physical parameters, dimensions, and quantities are contemplated to be within the scope of the invention.

Similarly, the components used in the purification, e.g., filters, chromatography columns, are in no way intended to be limiting or exclusive, and may be replaced with other components at the discretion of the practitioner to achieve the same purpose.

While considerable emphasis has been placed herein on specific features of the preferred embodiments, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other changes in the preferred embodiments of the present disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be clearly understood that the foregoing description is to be interpreted merely as illustrative of the present disclosure and not as a limitation.

Technical advantages

1. Almost all influenza vaccines produced today use eggs as hosts to prepare virus pools. The use of eggs to make LAIV has certain disadvantages that can be overcome using cell culture as a substrate. Limitations in using eggs as substrates are:

limited suppliers of vaccine quality eggs and eggs free of specific pathogens.

Ordering at least 4 months in advance before eggs are available.

Some candidate pandemic strains can cause poultry to become fatally infected, resulting in the unavailability of substrates (eggs) for vaccine production.

Egg-based manufacturing requires specialized egg incubation, harvesting, etc. facilities, thereby limiting the ability to scale up quickly.

2. Tissue culture based manufacturing has the advantage of a fully controlled system, easy to scale up.

3. In the case of a pandemic, large scale production of vaccines can be easily achieved using pre-existing tissue culture production units for other viral vaccines.

4. Cell culture derived viruses have greater similarity to circulating strains, whereas viruses produced in eggs may have antigenic modifications.

5. The components involved in the vaccine composition are minimal.

6. No preservative, polymer and surfactant.

7. The purification process uses a low concentration of endonuclease (benzoate).

8. Without the costly and cumbersome purification of chromatographic steps.

9. The process for producing such stable compositions/formulations is improved, thereby increasing the yield.

10. Intranasal administration is the simplest route of immunization because it does not require a high level of expertise, is suitable for multiple dose administration,

11. it is not reported to be associated with Guillain Barre syndrome and provides better protection due to delivery at the site of infection.

12. Liquid vaccines are not readily available, helping to overcome the problems of limited lyophilization capacity, the need to supply diluent for reconstitution, and the additional reconstitution step required prior to delivery of the vaccine.

13. The MDV backbone used to generate the LAIV recombination has a complete safety profile and is reported to provide a high level of protection.

Examples

The following examples are included to illustrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the compositions and techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Rearranged LAIV strains were purchased from russia st petderburgh laboratory medicine Institute (IEM) or WHO cooperative centers, such as the atlanta disease control and prevention center (CDC).

Example 1: rearranged LAIV Virus immunogenic composition stability data

Component 1-live attenuated influenza vaccine Virus (LAIV)

Influenza vaccine viruses are rearranged LAIV viruses derived by a typical rearrangement process comprising cold-adapted, temperature sensitive and/or attenuated phenotype gene segments of a Main Donor Virus (MDV) and Hemagglutinin (HA) gene segments and/or Neuraminidase (NA) gene segments of wild-type pandemic or seasonal influenza a or b or c virus strains in a ratio of 6:2 or 7: 1.

For the LAIV immunogenic composition:

in the dose range of 6 to 7log EID50/0.5 ml; more preferably 7log EID50/0.5ml of the dose of type A virus

In the dose range of 6 to 7log EID50/0.5 ml; more preferably 6.5log EID50/0.5ml of the dose of the virus B

For the rearranged LAIV strains used in the immunogenic compositions disclosed in any combination in table 1, the LAIV compositions are monovalent or multivalent (bivalent; trivalent; tetravalent).

Explanation:

stabilizer composition 3 (1% w/v gelatin + 5% w/v sorbitol + 0.1% w/v L-alanine + 0.21% w/v L-histidine + 0.3% w/v tris (hydroxymethyl) methylglycine + 1.6% w/v L-arginine hydrochloride):

unacceptable degradation rates were observed for both influenza a/H1N1 and b vaccine strains, both stress stability at 37 ℃ and real-time stability at temperatures from 2 ℃ to 8 ℃. (refer to FIGS. 4 and 5)

Stabilizer composition 4 (1% w/v gelatin + 5% w/v sorbitol + 0.1% w/v L-alanine + 0.21% w/v L-histidine + 0.9% w/v tris (hydroxymethyl) methylglycine + 1.6% w/v L-arginine hydrochloride):

Stabilizer composition 2 (0.85% w/v gelatin + 3% w/v sucrose + 0.1% w/v L-alanine + 0.21% w/v L-histidine + 0.3% w/v tris (hydroxymethyl) methylglycine + 2.1% w/v L-arginine hydrochloride):

unacceptable degradation rates were observed for both stress stability at 37 ℃ and real-time stability at temperatures from 2 ℃ to 8 ℃. (refer to FIGS. 8 and 9)

Stabilizer composition 1 (0.85% w/v gelatin + 4% w/v sucrose + 0.1% w/v L-alanine + 0.21% w/v L-histidine + 0.3% w/v tris (hydroxymethyl) methylglycine + 2.1% w/v L-arginine hydrochloride):

acceptable degradation rates (values within acceptable ranges) were observed for both stress stability at 37 ℃ and real-time stability at temperatures from 2 ℃ to 8 ℃. (refer to FIGS. 2, 3, 10 and 11)

Example 2: MDCK cell-based LAIV virus manufacturing process

The method of preparing a LAIV composition based on MDCK cell cultures may include any subset or all of the following steps:

k) the LAIV vaccine candidate virus was initially passaged in SPF chick embryos producing egg-based Master Seed Virus (MSV).

l) egg-based master seed virus was grown on a MDCK cell culture (ATCC CCL-34) host to produce cell-based Working Seed Virus (WSV). This is achieved byCell-based WSVs were used in different cell culture vessels/systems (e.g., 175cm surface area)²The surface area of the Tissue Culture Flasks (TCFs) of (1) is 850cm²Roller Bottles (RBs) of 6320cm in surface area²Cell Factories (CFs) and fixed-bed bioreactors (e.g., fromOf Life sciences Co., Ltd (Washington harbor, N.Y.)Bioreactors such as Nano bioreactor and 500/100 bioreactor)) infected MDCK cell cultures at a MOI of 1:100 to 1: 10000. (MDCK cells are cultured in MEM containing FBS; rinsed with MEM containing trypsin 5U/ml to 25U/ml before inoculation; WSV is inoculated into cells at an MOI of 1:10 to 1:10000, incubated at 31 ℃ to 33 ℃ for 48 hours to 72 hours)

m) harvesting the cultured virus.

n) filtering the virus harvest through at least one clarification filter by Direct Flow Filtration (DFF) to obtain a Clarified Virus Pool (CVP).

o) treating the CVP with a non-specific endonuclease (e.g. a benzoate enzyme) at a temperature in the range of 30 ℃ to 34 ℃ for 2 hours to 6 hours, and then at a temperature of 2 ℃ to 8 ℃ for 5 hours to 15 hours;

p) concentrating the endonuclease-treated CVP by Tangential Flow Filtration (TFF) using a membrane with a molecular weight cut-off (MWCO) of 100KDa to 500 KDa;

q) stabilizing the TFF concentrate with a stabilizer composition comprising one or more carbohydrates, one or more amino acids, and gelatin to form a stabilized viral harvest.

r) sterilizing the stabilized TFF concentrate by DFF through at least one sterilization grade filter having a pore size of about 0.2 microns to obtain sterilized CMVP (clear monovalent virus pool).

s) storing the sterilized CMVP in a polycarbonate bottle at a temperature of-60 ℃ or less.

t) filling the sterilized formulation into vials and storing at 2 ℃ to 8 ℃.

Cell culture medium: MEM with 10% FBS (pH adjusted to 7.0 to 7.4 with 1N HCl)

(additional Glutamine 350mg/L)

Virus culture medium: MEM without FBS (pH adjusted to 7.2 to 7.6 with 1N HCl)

(glucose 500mg/L, Glutamine 350mg/L otherwise)

Additional 0.4% glucose was added to the CM and VM for the bioreactor system

Example 3: effect of viral input (MOI) and post-inoculation incubation period on yield

Infection:

A. infection time:

on the basis of the optimization study, the upper limit of the number of cells infected with MDCK cell-derived influenza working seed virus was selected to be 1.2 to 1.8 billion cells per roller bottle and 70 to 100 billion cells per bioreactor system. Monolayer confluent microscopic observations were made on roller bottles with MDCK cells prior to the infection procedure.

Moi and post-inoculation incubation period:

according to all observations of the MOI optimization study, the MOI selection range for influenza a (H1N1), a (H3N2) and b viruses is between 1:100 and 1:10000, and the post-inoculation incubation period is between 48 and 72 hours.

Example 4: effect of varying concentrations of Trypsin on yield

And (3) deducing: trypsin is required to activate influenza virus to inoculate MDCK cells. From the above results, it can be seen that using 2000 to 3000 units of trypsin per roller bottle produces the greatest viral potency.

Example 5: effect of the concentration and temperature of the benzoic acid enzyme on cellular DNA content and viral titer

Different concentrations of benzoate enzyme were tested to degrade host cell DNA at different temperatures. The virus pool (CVP) was clarified to 500 (containing 2mM MgCl)₂) 500, 1000, 2500 and 5000U/L were treated with the enzyme benzoate and the treated CVP was held at 32 ℃ for 3 hours and further treatment was continued overnight at 2 ℃ to 8 ℃. Samples were taken at each stage, and the following is the DNA content results for each stage.

And (3) deducing: as can be seen from the results, in contrast to the absence of 2mM MgCl₂In the presence of 2mM MgCl compared with 500U/L concentration of benzoylase-treated CVP₂In the case of (2), CVP treated with 500U/L of benzoylase showed higher DNA degradation. It can also be seen that the higher concentrations of benzoylase 1000U/L, 2500U/L and 5000U/L show comparable to 500U/L (with 2mM MgCl)₂) The level of benzoic acid enzyme of (a) is equivalent to the degree of DNA degradation.

Example 6: viral yield at different stages of manufacture

CVP: the virus pool was clarified (harvested after filtration),

BCVP: CVP treated with a benzoylase enzyme,

CMVP: clarification of monovalent virus pools (after TFF, addition of stabilizer and 0.2. mu. filtration)

And (3) deducing: the stage virus concentration of each of the seasonal influenza viruses a (H1N1), a (H3N2) and b was examined, and it was observed that the initial virus concentration at the harvest level remained throughout the process until the final stage, i.e. the preparation of the vaccine batch (CMVP).

And (3) deducing: virus recovery can be calculated as the percentage of virus retained during manufacturing, where harvest is the starting point and CMVP is the end point of manufacturing. From the results, it can be concluded that the average virus recovery was 44.67%, which corresponds to 0.34Log EID₅₀Titer of 0.5mlAnd (4) loss. It was also observed that the final virus recovery (virus titer) at CMVP level was within an acceptable range and CMVP could be used to produce a final product batch of MDCK-based LAIV virus.

Example 7: comparative data on DNA concentrations of staged host cells during CMVP production

Clarified Virus Pools (CVPs) of different strains were treated with benzoate enzyme and DNA content was sampled in stages. The following are the results of the DNA content at each stage.

And (3) deducing:

as can be seen from the results, CVP showed a significant reduction in DNA when treated with benzoate enzyme. And it was further observed that residual DNA was again efficiently removed during the TFF process (diafiltration/concentration) and CMVP preparation. The DNA content at the final CMVP level is within desired and acceptable limits.

Example 8: various attempts of TFF experiments

Various TFF experiments were performed taking into account parameters such as dilution media (virus media and PBS) for the TFF process, diafiltration and concentration procedures. TFF concentrate samples were tested for virus concentration.

A/Cal: A/17/California/2009/38;

B/Tex ═ B/texas/02/2013-CDC-LV 8B;

a/HK ═ a/17/hong kong/2014/8296;

VM: virus culture medium

Explanation:

from the previous set of experiments, the TFF process was developed and then 2X DF and 4X concentration stages were chosen to obtain the desired TFF concentrate with the best virus yield. PBS has better stability to virus than VM.

Example 9: immunogenic results

Studies were performed to evaluate the immune response and vaccine efficacy of trivalent and tetravalent seasonal influenza vaccines based on egg and MDCK cell cultures in the ferret model. All animals were immunized intranasally on day 0 using trivalent or tetravalent formulations based on egg and MDCK cell cultures containing strains similar to a/michigan/45/2015 (H1N1), a/hong kong/4801/2015 (H3N2), B/brisban/60/2008 and B/pregabalin/3073/2013 and challenged four weeks later (day 28).

Table 19: geometric mean hemagglutination inhibition (HAI) and Neutralization Titers (NT) of sera collected on day 28

And (3) deducing: (refer to FIGS. 12 and 13)

It was concluded that egg-based and MDCK-based trivalent and tetravalent vaccines containing type a-H1N 1 protect animals from type a-H1N 1 infection when challenged with homologous type a-H1N 1 viruses.

Another study aimed to evaluate the immune response and efficacy of egg and MDCK cell-based monovalent LAIVs (type a-H5N 2 and type a-H7N 9) in ferret models. Different groups of animals were immunized with type a-H5N 1 and type a-H7N 9 monovalent LAIV-based eggs and MDCK cell cultures and challenged with homologous type a-H5N 1 and type a-H7N 9 viruses, respectively. The results show that animals immunized with monovalent H5N2 LAIV are protected from the homologous challenge of a-H5N 1 virus. Animals immunized with monovalent a-H7N 9 LAIV were protected from the homologous challenge of a-H7N 9 virus.

39页详细技术资料下载

上一篇：一种医用注射器针头装配设备

下一篇：抗癌组合疗法

Live attenuated influenza vaccine compositions and methods for making the same

相关技术

网友询问留言