Yellow fever virus (YFV) compositions and production thereof

Abstract

The present invention relates to a process for producing high yields of yellow fever virus (YFV) particles for vaccine manufacture. Also provided are liquid frozen or lyophilized YFV or chikungunya virus (CHIKV) formulations, and their use in the prevention of a yellow fever and chikungunya virus infections. Further provided are combination liquid or lyophilized formulations of YFV and CHIKV, and their use in the prevention of yellow fever virus and chikungunya virus infections.

Description

[0001] YELLOW FEVER VIRUS (YFV) COMPOSITIONS AND PRODUCTION THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a high yield process of producing yellow fever virus (YFV). The invention also encompasses formulations that allow stability of immunogenic YFV compositions, and uses of these formulations in preventing yellow fever infection. Additionally, the invention involves a high yield process of producing chikungunya virus (CHIKV), as well as producing a combined YFV and CHIKV formulation, the formulations thereof, and uses of these formulations in preventing yellow fever and chikungunya infection.

[0004] BACKGROUND

[0005] Yellow Fever (YF) is an acute viral infectious disease that is transmitted to humans through the bite of infected mosquitoes. As of 2023, 34 countries in Africa and 13 countries in Central and South America are either endemic for, or have regions that are endemic for, yellow fever. A modelling study based on African data sources estimated the burden of yellow fever during 2013 was 84,000-170,000 severe cases, and 29,000-60,000 deaths. Because of large explosive outbreaks in the recent years, YF has reemerged as a major international public health threat.

[0006] Whilst YF can be prevented by a single dose vaccine, supply cannot keep up with outbreaks. Traditional commercially available YF vaccines (such as Stamaril®) are produced in chicken embryo fibroblast (CEF) primary cultures which are limited in scale and harvest titer (~6.5 - 7 logw PFU / mL). Consequently, in 2016, an explosive outbreak occurred in Angola, spreading to neighboring areas in the Democratic Republic of Congo and infecting expatriate workers. Thirty million doses of YF vaccine were needed to stop the outbreak, which outstripped the available global vaccine supply and lack of supply led to the unprecedented use of fractional doses of the vaccine to prevent further disease spread (Situation report: yellow fever, 28 October 2016. Geneva: World Health Organization. http: / / apps.who. int / iris / bitstream / 10665 / 250661 / 1 / yellowfeversitrep28Oct16-eng.pdf?ua=1).

[0007] The YF outbreaks and resulting public health crises in Angola, the Democratic Republic of Congo, and Brazil underscore the need for a comprehensive, updated and intensified strategy to eliminate YF epidemics. Whilst WHO aims to increase YF vaccine manufacturing to distribute 1.3 billion YF vaccine doses to endemic countries by 2026, there have only been approximately 1 billion YF doses distributed in the last 80 years (refer to Hansen and Barret 2021 , The Present and Future of Yellow Fever Vaccines). Indeed, the current six commercial YF vaccine manufacturers combined can only produce a maximum of about 80-120 million Valneva Austria GmbH PAT070-PCT doses I year, though the real number of annually produced vaccine doses is usually in the range of 33-80 million, with production capabilities varying greatly from year to year.

[0008] To that end, YF vaccines that are similarly efficacious and safe as the existing ones, but which can be manufactured more easily, more quickly, at lower cost, or in larger quantities are needed.

[0009] SUMMARY OF THE INVENTION

[0010] The YF vaccine manufacturing method described herein surprisingly significantly increases YF vaccine manufacturing capacities. It is expected that with the described process in a commercial-scale 500m2cell growth surface bioreactor system (e.g., iCELLis 500+), over 100 million doses of YFV vaccine can be produced per lot. Assuming 10 lots per year, the output would be over 1 billion YF vaccine doses per year.

[0011] This is the first time that attenuated YF has been produced at high titer (>8 log-io TCIDso / mL).

[0012] Therefore, there is provided a method of preparing a composition comprising yellow fever virus (YFV), wherein the method comprises: a) Culturing host cells in a serum-free culture media in a fix-bed bioreactor; b) Infecting the host cells with YFV; and c) Collecting the culture medium 60-84 hours post-infection to produce a (first) YFV harvest (i).

[0013] The fixed-bed bioreactor may use a falling film for medium oxygenation. The shear stress and / or medium foaming may be minimized by maintaining the Falling Film Height (FFH) at the lowest possible level while still maintaining required dissolved oxygen (DO) levels. This may minimise damage to virus particles, particularly compared with more turbulent incubation conditions.

[0014] The dissolved oxygen (DO) level for the first 3 hours post-infection may be not less than 10%, preferably not less than 15%. The DO level from 3 hours post-infection to collection of culture medium may be between 40-60%

[0015] The host cells may be Vero cells.

[0016] The culture media may be replaced 1 day post infection with fresh culture media.

[0017] The culture media may be collected by perfusion between hours 60-84 hours post-infection.

[0018] The method may further comprise: d) adding fresh culture medium to the bioreactor, and collecting culture medium 7-9 days post-infection to produce a second YFV harvest (ii). Valneva Austria GmbH PAT070-PCT

[0019] The serum free culture medium in a) used for culturing the host cells is OptiPRO-SFM or Virus Production SFM (VP-SFM), optionally OptiPRO-SFM. The culture medium may be replaced in b) with VP-SFM.

[0020] The method may further comprise dialyzing the virus harvest (i) and / or (ii) to form a liquid formulation (iii); wherein said liquid formulation (iii) comprises a) at least one strain of YFV; b) 2% to 8% (w / w) sugar; c) 1 mmol / kg to 20 mmol / kg phosphate; d) 1 mmol / kg to 50 mmol / kg of at least one carboxylate buffering agent; e) 1 mmol / kg to 10 mmol / kg MgCh; f) 0.1% to 5% (w / w) D-sorbitol; g) 1 to 20 mmol / kg L-methionine; and h) 0.001 % to 0.1% (w / v) human albumin (HA), wherein the formulation is at a pH of 7.0 to 7.5.

[0021] The sugar may be sucrose is 5% (w / w), the phosphate may be 5 mmol / kg, the at least one carboxylate buffering agent may be 25 mmol / kg, the MgCh may be 5 mmol / kg, the D-sorbitol may be 0.5% (w / w), the L-methionine may be 10 mmol / kg , and the HA may be 0.01% (w / v). The formulation may be at a pH of 7.3.

[0022] The sugar may be sucrose at 5% (w / w), the phosphate may be potassium phosphate at 5 mmol / kg , the at least one carboxylate buffering agent may be sodium citrate at 25 mmol / kg , the MgCh may be 5 mmol / kg , the D-sorbitol may be 0.5% (w / w), the L-methionine may be 10 mmol / kg , and the HA may be 0.01%. (w / v). The formulation may be at a pH of 7.3.

[0023] The HA may be a recombinant human albumin (rHA); and / or may be a serum derived human albumin (HSA). HSA and rHA are structurally the same but may indicate a different source (recombinant versus plasma derived) but may also indicate exactly the same (i.e. are interchangeable). rHA may be prepared w / v or w / w dependent on the scale of formulation buffer.

[0024] The formulation may further comprise of at least one diluent selected from the group consisting of tissue culture medium, saline and water.

[0025] The method may further comprise lyophilizing the liquid formulation (iii) to produce a lyophilized formulation (iv). Therefore, there is also provided a method of producing a lyophilized formulation.

[0026] The method according to the invention, wherein b) further comprises dividing the composition (ii) after dialysis to produce 102TCIDso to 106TCIDso per dose of at least one strain of YFV, preferably about 104TCIDso dose.

[0027] The YFV may comprise or consist of a polynucleotide sequence with at least 80% identity to SEQ ID NO: 1. The YFV polynucleotide sequence may comprise one or more mutations Valneva Austria GmbH PAT070-PCT selected from: A2608C, A5641G, A7225G, C8212T, A10243G, G10559GT, and / or A10722G mutations in SEQ ID NO: 1.

[0028] The YFV may be a live, attenuated virus.

[0029] Also provided is a method of preparing a composition comprising yellow fever virus (YFV), wherein the method comprises: a) Culturing host cells in a serum-free culture media in roller bottles; b) Infecting the host cells with YFV; and c) Collecting the culture medium 90 ± 12 hours post-infection to produce a YFV harvest. YFV particles obtained from said method are suitable for all purification and formulation methods set forth herein.

[0030] Also provided is an immunogenic composition of YFV obtained or obtainable by the methods described herein. Also provided is a pharmaceutical composition of YFV (e.g., comprising YFV) obtained or obtainable by the methods described herein.

[0031] Provided is a liquid or lyophilized yellow fever virus (YFV) formulation comprising: a) an effective amount of YFV particles such as YFV particles with 102TCID50 to 106TCID50, preferably about 104TCIDso / dose of YFV particles; b) 2% to 8% (w / w) sucrose; c) 1 mmol / kg to 20 mmol / kg phosphate; d) 1 mmol / kg to 50 mmol / kg of at least one carboxylate buffering agent; e) 1 mmol / kg to 10 mmol / kg MgCh; f) 0.1% to 5% (w / w) D-sorbitol; g) 1 to 20 mmol / kg L-methionine; and h) 0.001 % to 0.1% (w / v) human albumin (HA), wherein the formulation is at a pH of 7.0 to 7.5.

[0032] The lyophilized YFV formulation may be lyophilized from a composition comprising: a) YFV particles; b) 2% to 8% (w / w) sucrose; c) 1 mmol / kg to 20 mmol / kg phosphate; d) 1 mmol / kg to 50 mmol / kg of at least one carboxylate buffering agent; e) 1 mmol / kg to 10 mmol / kg MgCh; f) 0.1% to 5% (w / w) D-sorbitol; g) 1 to 20 mmol / kg L-methionine; and h) 0.001 % to 0.1 % (w / v) human albumin (HA), at a pH of 7.0 to 7.5.

[0033] The YFV may comprise or consist of a polynucleotide sequence with at least 80% sequence identity to SEQ ID NO: 1. The YFV polynucleotide sequence may comprise one or more mutations selected from: A2608C, A5641G, A7225G, C8212T, A10243G, G10559GT, and / or A10722G mutations in SEQ ID NO: 1.

[0034] The sugar may be sucrose at 5% (w / w), the phosphate may be 5 mmol / kg , the at least one carboxylate buffering agent may be 25 mmol / kg , the MgCh may be 5 mmol / kg , the D-sorbitol may be 0.5% (w / w), the L-methionine may be 10 mmol / kg , and the HA may be 0.01% (w / v), optionally wherein the formulation is at a pH of 7.3.

[0035] The sugar may be sucrose at 5% (w / w), the phosphate may be potassium phosphate at 5 mmol / kg , the at least one carboxylate buffering agent may be sodium citrate at 25 mmol / kg , Valneva Austria GmbH PAT070-PCT the MgCh may be 5 mmol / kg , the D-sorbitol may be 0.5% (w / w), the L-methionine may be 10 mmol / kg , and the HA may be 0.01% (w / v), optionally wherein the formulation may be at a pH of 7.3.

[0036] The HA may be a recombinant human albumin (rHA). HA may be prepared w / v or w / w dependent on the scale of formulation buffer.

[0037] The YFV formulation may further comprise at least one diluent selected from the group consisting of tissue culture medium, saline and water.

[0038] The YFV formulation may comprise an effective amount of at least one strain of the virus. The effective amount may be 102TCIDso / dose to 106TCIDso / dose, preferably about 104TCIDso / dose of YFV particles.

[0039] The YFV may be a live, attenuated virus.

[0040] The YFV formulation may further comprise one or more enveloped viruses. The enveloped viruses may be selected from Flaviviridae, Alphaviridae, Togaviridae, Coronavirus, Orthomyxoviruses, Paramyxoviridae, and Rhabdoviruses.

[0041] The YF vaccine manufacturing method described above may also be used for any Chikungunya (CH IK) pharmaceutical composition as described in WO2019057793.

[0042] Also provided is a method of preparing a CHIKV composition, wherein the method comprises: c) Producing a CHIKV virus harvest (i); d) Dialyzing the CHIKV virus harvest (i) to form a liquid formulation (ii); wherein said liquid formulation (ii) comprises a) at least one strain of CHIKV; b) 2% to 8% (w / w) sucrose; c) 1 mmol / kg to 20 mmol / kg phosphate; d) 1 mmol / kg to 50 mmol / kg of at least one carboxylate buffering agent; e) 1 mmol / kg to 10 mmol / kg MgCI2; f) 0.1% to 5% (w / w) D-sorbitol; g) 1 to 20 mmol / kg L-methionine; and h) 0.001 % to 0.1 % (w / v) human albumin (HA), wherein the formulation is at a pH of 7.0 to 7.5;

[0043] The CHIKV may comprise or consist of a polynucleotide sequence with at least 80%, 90%, 95%, 99% identity to SEQ ID NO: 2, i.e. having a 60 amino acid deletion in the nsP3 protein of a complete CHIKV strain as set out in SEQ ID NO: 2.

[0044] The sucrose may be at 5% (w / w), the phosphate may be 5 mmol / kg, the at least one carboxylate buffering agent may be 25 mmol / kg, the MgCh may be 5 mmol / kg, the D-sorbitol may be 0.5% (w / w), the L-methionine may be 10 mmol / kg, and the HA may be 0.01% (w / v), optionally wherein the formulation may be at a pH of 7.3. Valneva Austria GmbH PAT070-PCT

[0045] The sucrose may be at 5% (w / w), the phosphate may be potassium phosphate at 5 mmol / kg, the at least one carboxylate buffering agent may be sodium citrate at 25 mmol / kg , the MgCh may be 5 mmol / kg, the D-sorbitol may be 0.5%(w / w), the L-methionine may be 10 mmol / kg, and the HA may be 0.01% (w / v), optionally wherein the formulation may be at a pH of 7.3.

[0046] The HA may be a recombinant human albumin (rHA). HA may be prepared w / v or w / w dependent on scale of formulation buffer.

[0047] The CHIKV formulation may further comprise at least one diluent selected from the group consisting of tissue culture medium, saline and water.

[0048] The CHIKV formulation may comprise an effective amount of at least one strain of the virus. The effective amount may be 102TCIDso / dose to 106TCIDso / dose, preferably about 104TCIDso / dose of CHIKV particles.

[0049] The CHIKV may be a live, attenuated virus.

[0050] The CHIKV formulation may further comprise one or more enveloped viruses. The enveloped viruses may be selected from Flaviviridae, Alphaviridae, Togaviridae, Coronavirus, Orthomyxoviruses, Paramyxoviridae, and Rhabdoviruses.

[0051] Also provided is a method of preparing a YFV or CHIKV composition, wherein the method comprises: a) Producing a virus harvest (YFV or CHIKV) (i); b) Dialyzing the virus harvest (i) to form a liquid formulation (ii); wherein said liquid formulation (ii) comprises a) at least one strain of virus; b) 2% to 8% sucrose; c) 1 mmol / kg to 20 mmol / kg phosphate; d) 1 mmol / kg to 50 mmol / kg of at least one carboxylate buffering agent; e) 1 mmol / kg to 10 mmol / kg MgCh; f) 0.1% to 5% D-sorbitol; g) 1 to 20 mmol / kg L-methionine; and h) 0.001% to 0.1% human albumin (HA), wherein the formulation is at a pH of 7.0 to 7.5.

[0052] The method may optionally further provide a step c) Mixing the YFV liquid formulation (ii) and / or CHIKV liquid formulation (ii) to produce a combined liquid formulation (iii).

[0053] The method may further comprise a step d) Lyophilizing the combined YFV and CHIKV liquid formulation (iii) to produce a combined YFV and CHIKV lyophilized formulation (vi). Therefore, there is also provided a method of producing a lyophilized combined YFV and CHIKV formulation. Valneva Austria GmbH PAT070-PCT

[0054] The method may comprise diluting the composition (ii) after dialysis to produce 102TCIDso to 106TCID50 per dose of YFV, optionally about 104TCI D50 dose, and / or

[0055] The YFV and / or CHIKV viruses may be live, attenuated viruses.

[0056] Also provided is an immunogenic combined YFV and CHIKV composition obtained or obtainable by the method described herein. Also provided is a pharmaceutical composition of YFV and CHIKV (e.g., combined pharmaceutical composition) obtained or obtainable by the method described herein.

[0057] Also provided is a liquid or lyophilized combination formulation comprising: a) an effective amount of YFV particles such as 102TCIDso / dose to 106TCIDso / dose b) an effective amount of CHIKV particles such as 102TCIDso / dose to 106TCIDso / dose c) 2% to 8% (w / w) sugar; d) 1 mmol / kg to 20 mmol / kg phosphate; e) 1 mmol / kg to 50 mmol / kg of at least one carboxylate buffering agent; f) 1 mmol / kg to 10 mmol / kg MgCh; g) 0.1% to 5% (w / w) D-sorbitol; h) 1 to 20 mmol / kg L-methionine; and i) 0.001% to 0.1% (w / v) human albumin (HA). Optionally, the formulation is at a pH of 7.0 to 7.5. The combined YFV and CHIKV formulation may be lyophilized from a composition comprising a) YFV particles; b) CHIKV particles; c) 2% to 8% (w / w) sugar; d) 1 mmol / kg to 20 mmol / kg phosphate; e) 1 mmol / kg to 50 mmol / kg of at least one carboxylate buffering agent; f) 1 mmol / kg to 10 mmol / kg MgCh; g) 0.1% to 5% (w / w) D- sorbitol; h) 1 to 20 mmol / kg L-methionine; and i) 0.001 % to 0.01 % (w / v or w / w) human albumin (HA), at a pH of 7.0 to 7.5.

[0058] The CHIKV may comprise or consist of a polynucleotide sequence with at least 80%, 90%, 95%, 99% identity to SEQ ID NO: 2, i.e. having a 60 amino acid deletion in the nsP3 protein of a complete CHIKV strain and also as set out in SEQ ID NO: 2.

[0059] The YFV may comprise or consist of a polynucleotide sequence with at least 80% sequence identity to SEQ ID NO: 1 , suitably the YFV polynucleotide sequence may comprise one or more mutations selected from: A2608C, A5641G, A7225G, C8212T, A10243G, G10559GT, and / or A10722G mutations in SEQ ID NO: 1.

[0060] The sugar may be sucrose at 5% (w / v), the phosphate may be 5 mM, the at least one carboxylate buffering agent may be 25 mM, the MgCh may be 5 mM, the D-sorbitol may be 0.5% (w / v), the L-methionine may be 10 mM, and the HA may be 0.01% (w / v orw / w), optionally wherein the formulation is at a pH of 7.3.

[0061] The sugar may be sucrose at 5% (w / w), the phosphate may be potassium phosphate at 5 mmol / kg, the at least one carboxylate buffering agent may be sodium citrate at 25 mmol / kg, Valneva Austria GmbH PAT070-PCT the MgCh may be 5 mmol / kg, the D-sorbitol may be 0.5% (w / w), the L-methionine may be 10 mmol / kg, and the HA may be 0.01 %, optionally the formulation is at a pH of 7.3.

[0062] The HA may be a recombinant human albumin (rHA).

[0063] The YFV and CHIKV combination formulation may further comprise at least one diluent selected from the group consisting of tissue culture medium, saline and water.

[0064] The YFV and CHIKV combination formulation may comprise an effective amount of at least one strain of each virus, optionally wherein the effective amount for each virus is 103TCIDso / dose to 106TCIDso / dose, preferably about 104TCIDso / dose. In other words, the effective amount may be 104TCIDso / dose for YFV and 104TCIDso / dose for CHIKV.

[0065] The degradation kinetic of the YFV and CHIKV combination formulation may be between - 0.25 log-io loss / year and 0.5 log™ loss / year.

[0066] The viruses may be live, attenuated viruses.

[0067] The YFV and CHIKV combination formulation may further comprise one or more enveloped viruses, optionally wherein the enveloped viruses are selected from Flaviviridae, Alphaviridae, Togaviridae, Coronavirus, Orthomyxoviruses, Paramyxoviridae, and Rhabdoviruses.

[0068] Also provided is use of the YFV formulations described herein, or any of the combined YFV and CHIKV formulations described herein, in preventing YFV infection.

[0069] Also provided is use of the combined YFV and CHIKV formulations described herein in preventing YFV and / or CHIKV infections.

[0070] The formulations may be used as a vaccine. The vaccine may be a one-shot vaccine.

[0071] Also provided is a method of preventing YFV infection comprising administering a YFV formulation described herein to a subject, such as a liquid frozen formulation or lyophilized formulation.

[0072] Also provided is a method of preventing CHIKV infection comprising administering a CHIKV formulation described herein to a subject, such as a liquid frozen formulation or lyophilized formulation.

[0073] Also provided is a method of preventing YFV and CHIKV infection comprising administering a combined CHIKV and YFV formulation described herein to a subject, such as a liquid formulation or lyophilized formulation. Valneva Austria GmbH PAT070-PCT

[0074] Also provided is a method of preventing YFV and CHIKV infection comprising administering a combined CHIKV and YFV formulation described herein to a subject, such as a liquid formulation or lyophilized formulation.

[0075] Also provided is a method of preventing YFV and / or CHIKV infection comprising administering a CHIKV and / or YFV Liquid Frozen formulation described herein to a subject, using a prefilled syringe.

[0076] BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1. Examples of bioreactors using a falling film component for Circulation / oxygenation of cell culture medium.

[0078] A) A perspective view of a bioreactor for culturing cells, wherein the bioreactor includes an external casing or housing 112 forming an interior compartment and a removable cover 114 for covering the interior compartment, which may include various openings or ports P with removable covers or caps C for allowing for the selective introduction or removal of fluid, gas (including by way of a sparger), probes, sensors, samplers, or the like. The bioreactor may be used in connection with an external reservoir and conduits (e.g., forward and return) to form a continuous loop for circulating fluid to the bioreactor (adapted from Figure 1 of WO2019122239A1).

[0079] B) The bioreactor according to A) above, showing the medium recirculation loop (adapted from Figure 20 of WO2019122239A1).

[0080] C) A bioreactor arranged such that an upper plate 330 is provided with peripheral openings 330c to allow fluid to flow directly along the inner wall formed by tube 334. In this manner, a thin layer or film of fluid may be created, which flows downwardly while passing through the fifth chamber 328. This may serve to increase the volume of the fluid exposed to gas (air) within the fifth chamber 328, prior to it being returned to the first chamber 316. This implementation can allow for more oxygen transfer which may be needed for larger sizes or otherwise to increase cell growth rates or adjust process parameters based on the biologic being produced (adapted from Figures 11 and 12 of WO2019122239A1).

[0081] D) A structured bag bioreactor 100, which is placed in fluid communication with a circulation tube 410. A first end of the circulation tube 410 is fluidly connected to a dip tube 110 in the structured bag 100 and a second end of the circulation tube 410 is fluidly connected to a circulation port 420 disposed in the fitment of the structured bag 100. The circulation port 420 fluidly connects the circulation tube 410 to the interior compartment of the structured bag 100. Valneva Austria GmbH PAT070-PCT

[0082] The circulation system also includes a pump configured to move fluid and / or other components out of the structured bag 100, into the circulation system and back into the structured bag 100. Fluid and / or other components are circulated using the pump 400 to increase oxygenation. Media in the interior compartment of the structured bag 100 is drawn into the circulation tube 410 via the dip tube 110. Media is then returned to the structured bag 100 through the circulation port 420. As the media flows into the bag though the circulation port 420, oxygen diffuses into the media while the media cascades down the bag, for example, down the interior of the sidewall to minimize splashing or disruption of cells in the structured bag 100. The cascading of the media functions like a "falling film oxygenator" (adapted from Figure 4 of WO2017197154A1).

[0083] E) An exemplary fixed-bed bioreactor (iCELLis 500), showing the falling film component (sidecuts). The cell culture medium flows through the fixed bed from the bottom to the top. At the top, the medium falls as a thin film down the outer wall where it takes up 02 to maintain high a Volumetric Oxygen Transfer Coefficient (kLa) in the bioreactor. The arrows represent the direction of the liquid 10 circulation (adapted from PALL Biotech USD3262d “Productivity and Reduced Footprint for Industrialization of Animal Cell Culture Processes in a Fixed-Bed Bioreactor”, 2021).

[0084] Figure 2. Production of recombinant YFV plasmid. A) pVVS01828 plasmid comprising a YFV ASLE genome flanked by a SP6 promoter at the 5’ terminus and an Aflll run-off restriction site at the 3’ terminus. The backbone of this plasmid is pACNR (Silva PA, et al., 2010). B) recombinant YFV sequence with alignment to FGT2, FGT3 and FGT4 fragments and the respective restriction sites in the recombinant YFV sequence. C) pVVS02030 plasmid comprising a full length recombinant YFV sequence in a pACNR backbone.

[0085] Figure 3. YFV yield with changed parameters. A) TCIDso at days 3, 4 (harvest 1) and 8 (harvest 2) post infection when OptiPRO-SFM is used for host cell expansion and virus infection, propagation and harvest (OptiPRO-all) or when OptiPRO-SFM is used for host cell expansion and media is switched to VP-SFM for viral infection, propagation and harvest (Opti PRO- VP- switch). Graphical representation of mean log™ virus titers (infective virus particles) analysed by TCIDso assay. The results shown in text boxes represent the titers. B) TCIDso at days 3 and 4 (harvest 1) post infection when OptiPRO-SFM is used for host cell expansion and media is switched to VP-SFM for viral infection, propagation and harvest (Opti PRO- VP- switch). In the first condition, a recirculation mode was used during the virus propagation phase, whereas in the second condition, the virus is harvested by perfusion from day 3 until final harvest. C) YFV virus yields obtained for in-process and harvest samples over time for different conditions. Dotted lines represent the polynomial (second order) trendlines. Valneva Austria GmbH PAT070-PCT

[0086] D) Turbidity (NTU) measurements of post-infection in-process samples of YFV taken from the recirculation bottle. The dotted lines represent the exponential trendlines. E) summary of TCID50 at day 4 post infection (harvest 1) for different conditions.

[0087] Figure 4. YFV purification Process Overview (iCellis). Primary recovery in this process consists of host cell DNA (hcDNA) reduction by precipitation with Protamine Sulphate (PS) in the crude harvest. Precipitated hcDNA is removed together with cell debris by clarification using a membrane filter. Clarified harvest is then concentrated by an ultrafiltration step (UF1). Further purification of the concentrated harvest material is performed by a flowthrough chromatography step using the multi-modal Capto™Core 400 (CC400) chromatography resin. CC400 pool is then again concentrated by a final ultrafiltration step (UF2) followed by a buffer exchange by diafiltration to remove residual media components. A final 0.2 pm filtration is performed to prepare the drug substance (DS).

[0088] Figure 5. SE-HPLC of UF2 pool sample of YFV from OptiPRO-VP-switch + media change 24h PI Condition. The chromatogram depicts peak integration as well as allocation of rHA and LMW impurities I Buffer salts present in the chromatogram.

[0089] Figure 6. Yield and infectious titer of YFV during recovery and downstream processing. A) Process yields of YFV of OptiPRO-VP-switch + media change 24h PI Condition during downstream processing steps, calculated from TCI D50 results. B) Total infectious titer during recovery and downstream process steps (YFV from OptiPRO-VP-switch + media change 24h PI Condition).

[0090] Figure 7. Stability of lyophilized YFV formulation over time at different temperatures. Stability data for YFV Lyophilized DP at lower infectious virus concentration (6 log-10 TCIDso / mL) plotted against time (month). Data with circles represents DP stored at 2-8 °C and data with squares represents DP stored at 37 °C. Each data point is a geometric mean of three individual measurements of three separate vials (N=3). Dashed lines represent linear fits along with their equations and fit parameter (R2). B) Comparison of lyophilized YFV DP and Stamaril® at 37°C.

[0091] Figure 8. Stability of liquid YFV formulation over time at different temperatures. A) Stability of liquid frozen formulation against time (months). Data with circles represents DP stored as frozen liquid in vials at -70 °C and data with squares represents DP stored as frozen liquid in vials at -20 °C. Each data point is a geometric mean of three individual measurements of three separate vials (N=3). Dashed lines represent linear fits along with their equations and fit parameter (R2). B) Stability of liquid formulation against time (month) in the graph. Data Valneva Austria GmbH PAT070-PCT with circles represents DP from a first batch and data in squares represents DP from a second batch. Dashed lines represent linear fits along with their equations and fit parameter (R2). Data points up to 2 months for both batches are a geometric mean of the three individual measurement of three separate vials (N=3). 3-month and 4-month data points are geometric means of two such measurements while the 6-month data point is a single data point.

[0092] Figure 9. Stability of liquid YFV formulation stored in vials or pre-filled syringes. A) Stability of YFV liquid formulation (2-8°C and 25°C) and liquid frozen formulation (-20°C) stored in vials. B) Stability of YFV liquid formulation (2-8°C and 25°C) and liquid frozen formulation (-20°C) stored in pre-filled syringes. C) Side-by-side stability comparison of YFV liquid formulation (2-8°C and 25°C) stored in vials and prefilled syringes. D) Side-by-side stability comparison of YFV liquid formulation stored in vials or prefilled syringes and reconstituted lyophilized Stamaril® stored in vials at 2-8°C. For all experiments, each data point is a geometric mean of three individual measurements of three separate vials (N=3).

[0093] Figure 10. YFV purification process overview (Roller Bottles). Part 1 : Process flow for virus recovery from crude harvest, hcDNA reduction and initial concentration. Part 2: Process flow for purification and DS formulation.

[0094] Figure 11. Growth kinetics for YFV in roller bottles following vero cell infection with an MOI of 0.01. Virus titers were measured at the indicated timepoints after infection in two experiments run in duplicate (#25-0092 and #25-0093). The groups labeled “-01” were harvested on d3; the groups labelled “-02” were monitored longer as indicated. An optimal harvest window of 90 ± 12 hours post infection was identified.

[0095] DETAILED DESCRIPTION OF THE INVENTION

[0096] Definitions

[0097] Any reference to ‘or’ herein is intended to encompass ‘and / or’ unless otherwise stated.

[0098] As used herein, the singular forms ‘a’, ‘an’, and ‘the’ include both singular and plural referents unless the context clearly dictates otherwise.

[0099] The terms ‘comprising’, ‘comprises’ and ‘comprised of’ may be synonymous with including, includes or containing, contains, and may be inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The term also encompasses ‘consisting of’. Valneva Austria GmbH PAT070-PCT

[0100] ‘Consisting’ may be understood to mean that only the specified items are present. In other words, this term may be exclusive and closed, and may exclude additional, non-recited members, elements or method steps.

[0101] Whereas the term ‘one or more’ or ‘at least one’, such as one or more members of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.

[0102] ‘Around’ and ‘about’ may be used to indicate that the stated value includes values within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or less in either direction (greater than or less than) of the stated value unless otherwise stated or otherwise evident from the context. For example, ‘about’ or ‘around’ may indicate + / - 10% of the stated value.

[0103] ‘Up to’ may be used to indicate a maximum value. ‘Up to’ may include the value indicated. For example, ‘up to 10’ may include all possible values up to and including the number 10.

[0104] ‘At least’ may be used to indicate a minimum value. ‘At least’ may include the value indicated. For example, ‘at least 10’ may include all possible values above 10, and including the number 10.

[0105] ‘Improve’, ‘increase’, or ‘reduce’, or grammatical equivalents, may be used to indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or control population or control value) who have not received the treatment.

[0106] ‘Nucleotide’, ‘nucleic acid’ or ‘nucleic acid residue’ may refer to any nucleoside having one or more phosphate groups joined in ester linkages to a sugar moiety. Exemplary nucleotides include nucleoside monophosphates, diphosphates and triphosphates. In ‘ribonucleic acid’ (RNA), the sugar is ribose. In deoxyribonucleic acid’ (DNA) the sugar is deoxyribose. The term nucleotide is used generally to refer to nucleotides found in DNA and RNA; adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U), and any analogues thereof. The term encompasses modified nucleotides, and nucleotides with modified backbone residues or linkages, including modifications which are synthetic, naturally occurring, and non-naturally occurring, and may have similar binding properties as the reference polynucleotide.

[0107] ‘Polynucleotide’ may refer to a polymer of nucleotides or nucleic acid residues joined together by a phosphodiester or phosphorothioate linkage between 5' and 3' carbon atoms, including Valneva Austria GmbH PAT070-PCT

[0108] DNA and RNA. In preferred examples, ‘polynucleotide’ may refer to a messenger RNA (mRNA). A polynucleotide may be any length, including at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 50, 100, 1000, 10,000 or more nucleotides in length.

[0109] ‘Amino acid’ may be used to refer to molecules which have an amine and carboxyl functional group. Amino acids can be divided into the following four groups: (1) acidic amino acids, (2) basic amino acids, (3) neutral polar amino acids, and (4) neutral, nonpolar amino acids. Representative amino acids within these various groups include, but are not limited to, (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine.

[0110] ‘Polypeptide’ or ‘protein’ may be used to refer to a polymer of amino acids or amino acid residues, e.g. at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100 amino acid residues, or a variant, fragment or isoform thereof. One or more amino acid residues in a polypeptide may be an artificial chemical analogue of a corresponding naturally occurring amino acid. The terms are also inclusive of modifications amino acids including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. A polypeptide sequence may include conservative modifications in the encoded polypeptide. A conservative substitution may be where one or more amino acids are replaced by one or more amino acids with the same properties as the original amino acids, such that the properties (e.g., chemical or secondary or tertiary structure) of the encoded polypeptide do not change or do not substantially change. Conservative substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and the amphipathic nature of the resulting amino acid residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine. Similarly, conservative substitutions may be made within the amino acid groups discussed above.

[0111] ‘Variant’ may include amino acid modifications of the polypeptide but still retain the intended function of the polypeptide. Valneva Austria GmbH PAT070-PCT

[0112] ‘Sequence’ may refer to any contiguous string of nucleotides or amino acids. A ‘sequence’ may be at least 2, 5, 10, 15, 20, 30, 40, 50, 100, 200, 500, 1000 nucleotides or amino acids in length. Unless otherwise specified, all sequences are by convention written from 5’ to 3’ end for nucleotide sequences, or from the N-terminal to C-terminal end for amino acid sequences.

[0113] ‘Homology’, ‘sequence identity’ or ‘sequence similarity’ in the context of two or more polynucleotides may refer to the extent to which the sequence of nucleotides are the same over a specified region. The extent of homology may also be reported as a ‘percentage sequence similarity’, ‘percentage sequence identity’ or ‘percentage homology’, which may be calculated by aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which identical nucleotides occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. The similarity between nucleotide sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologues or variants of the nucleotide sequence will possess a relatively high degree of sequence identity when aligned using standard methods. Methods of aligning sequences for comparison are well known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet. Identity may be determined manually or by using a computer sequence algorithm such as ClustalW, ClustalX, BLAST, FASTA or Smith-Waterman. Percentage sequence identity may be defined across a whole sequence, or across regions of a sequence, such as at least 5, 10, 20, 30, 40, 50, 75, 100, or 150 amino acids in a polypeptide sequence or at least 5, 10, 25, 50, 75, 100, 250, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250 or 2500 nucleic acid residues in a polynucleotide sequence.

[0114] ‘Mutations’ may refer to nucleotide substitutions, deletions or insertions with reference to a specific sequence. For example, A2608C in SEQ ID NO: 1 may refer to a nucleotide change at position 2608 from an adenine nucleic acid residue to a cytosine nucleic acid residue.

[0115] ‘Enveloped virus’ refers to viruses with an envelope surrounding the capsid (protein shell of the virus). The envelope may be a lipid bilayer. Valneva Austria GmbH PAT070-PCT

[0116] ‘Yellow Fever Virus’ (YFV) belongs to the Flaviviridae family, and Flavivirus genus. There are several strains of YFV; West Africa I, West Africa II, East and Central Africa, South America I and South America II. The live attenuated strain YF17D (full genome at GenBank: X03700.1) is widely used for vaccines.

[0117] ‘Chikungunya virus’ (CHIKV) belongs to the Togaviridae family and alphavirus genus. There are several strains of CHIKV.

[0118] ‘Strain’ may be used to distinguish genetically distinct lineages of the same virus, i.e. , genetic variants or subtypes of the same virus.

[0119] ‘Live attenuated’ may describe a virus or antigenic composition which demonstrates reduced virulence (compared to a wild-type virus) and is not inactivated.

[0120] ‘Yield’ refer to the number of virus particles obtained from a process. ‘High yield’ is by comparison to existing processes.

[0121] ‘Composition’ may refer to a mixture two or more constituents, suitably wherein one of the constituents is YFV and / or CHIKV particles.

[0122] ‘Formulation’ may also refer to a composition but may refer particularly to a composition that facilitates storage of the composition. The formulation may therefore include solubilizers, stabilizers, buffers, tonicity modifiers, bulking agents, viscosity enhancers / reducers, surfactants, chelating agents, and adjuvants.

[0123] A ‘pharmaceutical composition’ or ‘pharmaceutical formulation’ may refer to a composition suitable for administration to a human subject. ‘Immunogenic composition’ may refer to a composition which is able to produce an immune response in a subject when administered to the subject.

[0124] ‘Vaccine’ may refer to a material that contains or encodes an antigen which will provide active immunity to material comprising the antigen but will not cause the disease. ‘Vaccine’ may refer to a composition comprising viral particles that is suitable for administration to humans to prevent a specific disease or infection.

[0125] However, pharmaceutical composition, formulation or vaccine may be used interchangeably. Valneva Austria GmbH PAT070-PCT

[0126] ‘Combined composition’, ‘combined YFV and CHIKV composition’, ‘combined formulation’, ‘combined YFV and CHIKV formulation’, ‘combined vaccine’, ‘combined YFV and CHIKV vaccine’, ‘combination composition’, ‘combination formulation’, or ‘combination vaccine’, may refer to a composition, formulation or vaccine which contains both YFV and CHIKV particles. Suitably the particles are immunogenic. Suitably the combined composition, formulation or vaccine comprises an effective amount of each of YFV and CHIKV such that the composition, formulation or vaccine can prevent both YFV and CHIKV infections.

[0127] ‘Liquid formulation’ may refer to a composition which is in liquid form, suitably the virus particles in a composition which is in liquid form. Therefore ‘liquid frozen formulation’ may refer to a liquid formulation which has been frozen, for example by storing at temperatures below 0, -5, -10, -15, -20 or -70°C.

[0128] ‘Lyophilization’ may refer to freeze-drying a substance. Lyophilization may remove water from a composition under vacuum.

[0129] ‘Lyophilized formulation’ may refer to a composition which has been freeze-dried. A lyophilized formulation may not contain water. A lyophilized formulation may be in powder form. The lyophilized formulation may be reconstituted in a diluent such as water to produce a liquid formulation.

[0130] ‘Effective amount’ may refer to an amount of virus particles which produces a protective effect against later infection.

[0131] ‘TCID50’ may refer to the amount of viral particles that infects 50% of a given cell culture. TCID50 is a measure used to indicate the infectious viral titer, i.e. a level of replication competent infectious virus. TCID50 may be determined using an assay as described in the examples below. TCID50 may be determined on Vero cells, e.g. using an endpoint dilution assay. TCID50 may be defined as an amount of virus required to infect 50% of tissue culture cells, e.g. Vero cells. Infection may be determined by the detection of a cytopathic effect by microscopic evaluation, or by use of a cell dye to detect cell health.

[0132] The process of producing viral particles may include a host cell expansion phase, a virus infection phase, a virus propagation phase and viral harvest. Valneva Austria GmbH PAT070-PCT

[0133] ‘Virus production’ or ‘virus production process’ may refer to the process of virus infection, virus propagation and virus harvest. ‘Virus production’ or ‘virus production process’ may include the cell expansion phase.

[0134] ‘Host cell’ may refer to cells which are or are intended to be infected with a virus to allow amplification of the virus.

[0135] ‘Cell culturing’ may refer to the process of growing and maintaining cells using standard techniques. Culturing may include splitting or passaging cells when cells reach around 60- 100% confluency to increase the stock of cells.

[0136] ‘Culture media’ or ‘cell culture media’ may refer to the liquid that cells are cultured in. The media used may include various reagents that support the growth of the host cells, including amino acids, vitamins, organic and inorganic salts, carbohydrates.

[0137] ‘Serum-free media’ (SFM) may refer to a culture media formulated without any serum, such as e.g. animal or human serum.

[0138] ‘Fresh culture media’ culture media that has not been exposed to host cells.

[0139] ‘Host cell propagation’, ‘host cell propagation phase’, ‘host cell expansion’ or ‘host cell expansion phase’ may refer to culturing of the host cells to allow the host cells to reach a specified cell density or confluency. The host cell propagation phase may be 1 to 10, 2 to 10, 3 to 10, 4 to 10, 5 to 10, 1 to 9, 2 to 9, 3 to 9, 4 to 9, 5 to 9, 1 to 8, 2 to 8, 3 to 8, 4 to 8, 5 to 8, or 6 to 8 days, or around 7 days. The specified density may be in cells / cm2or may be measured as the percentage of the growth surface covered by cells (referred to as confluency). The specified density at the end of host cell propagation may be between 0.1 to 4.0, 0.1 to 3.5, 0.1 to 3.0, 0.1 to 2.5, 0.5 to 4.0, 0.5 to 3.5, 0.5 to 3.0, 0.5 to 2.5, 1.0 to 4.0, 1.0 to 3.5, 1.0 to 3.0, 1.0 to 2.5, or 1.0 to 2.0 x 105cells / cm2, or around 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 or 1.9 x 105cells / cm2. The percentage of the growth surface covered by cells at the end of host cell propagation may be between 30-100%, 30-95%, 30-90%, 30-85%, 30-80%, 30-75%, 30-70%, 30-60%, 30-50%, 40-100%, 40-95%, 40-90%, 40-85%, 40-80%, 40-75%, 40-70%, 40-60%, 40-50%, 50-100%, 50-95%, 50-90%, 50-85%, 50-80%, 50-75%, 60-100%, 60-95%, 60-90%, 60-85%, 60-80%, 60-75%, 70-100%, 70-95%, 70-90%, 70-85%, or 70-80%. The percentage of the growth surface covered by cells at the end of host cell propagation may be at least 40, 50, 60, 70, 80, or 90%. The percentage of the growth surface covered by cells at the end of host cell propagation may be around 40, 50, 60, 70, 80, or 90%. Valneva Austria GmbH PAT070-PCT

[0140] ‘Virus infection’ or ‘virus infection phase’ may refer to the introduction of a virus to the cell culture. This may be achieved by adding the viral particles to the cultured host cells e.g. in the medium. In this phase, the viral particles may enter the host cells. The virus infection phase may last between 0.5 to 10, 1 to 10, 0.5 to 9, 1 to 9, 0.5 to 8, 1 to 8, 2 to 8, 0.5 to 7, 1 to 7, 2 to 7, 0.5 to 6, 1 to 6, 2 to 6, 0.5 to 5, 1 to 5, 2 to 5, 0.5 to 4, 1 to 4, 1.5 to 4, 2 to 4, 3 to 4, 0.5 to 3.5, 1 to 3.5, 1.5 to 3.5, 2 to 3.5, or 2.5 to 3.5 hours, or around 3 hours post-infection.

[0141] ‘Multiplicity of infection’ may refer to the average number of virus particles infecting each cell. The MOI may refer to the approximate number of virus particles introduced in the virus infection phase per number of host cells to be infected. The multiplicity of infection (MOI) used to infect the host cells may be between 0.001 to 1 , 0.01 to 0.1 , 0.01 to 0.5, 0.03 to 0.5, 0.03 to 0.1 or around 0.01 or 0.03.

[0142] ‘Virus propagation’, ‘virus production’, ‘virus propagation phase’ or ‘virus production phase’ may refer to replication of the virus within the host cells. Early virus production may require active host cell metabolism, requiring relatively more nutrients and oxygen. Early virus production is generally considered the time directly following infection of the host cells up to about 26 hours post infection (PI), especially to 18 to 30 hours PI, to about 20 to 28 hours PI, to about 22 to 26 hours PI, especially to about 24 or 26 hours PI. The later part of the virus propagation phase is characterized by a slowing down of cell metabolism and increasing cytopathic effect (CPE) as virus production and cell density are both higher. The later part of the virus propagation phase is broadly defined as greater than 26 hours post infection (PI) until virus harvest.

[0143] ‘Collection of culture media’ may refer to ‘viral harvest’. In this step, virus particles are retrieved from the bioreactor by removing and keeping culture media from the host cells that were infected with the virus. Harvesting of the virus may be performed 24-120, 24-108, 24-96, 24- 84, 24-72, 36-120, 36-108, 36-96, 36-84, 36-72, 48-120, 48-108, 48-96, 48-84, 48-72, 60-120, 60-108, 60-96, 60-84, 60-80, 60-78, 60-76, 60-74, 60-72, 62-80, 62-78, 62-76, 62-74, 62-72, 64-80, 60-78, 64-76, 64-74, 64-72, 66-80, 66-78, 66-76, 66-74, 66-72, 68-80, 68-78, 68-76, 68-74, 68-72, 70-80, 70-78, 70-76, 70-74, 70-72, or around 72 hours post-infection. Harvesting of the virus may be performed 2-4, 2.5-3.5 or around 3 days post-infection.

[0144] After this harvest, the culture media may be replaced with fresh culture media, and the host cells left to incubate in the bioreactor. A ‘second viral harvest’ may then be performed at a later time period to remove and keep culture media from the host cells infected with the virus after the first viral harvest was performed. The second viral harvest may be performed 144-216, 144-204, 144-196, 156-216, 156-204, 156-196, 168-216, 168-204, 168-196, 180-216, 180- Valneva Austria GmbH PAT070-PCT

[0145] 204, 180-196, 190-194 or around 192 hours post-infection. The second viral harvest may be performed 7-9, 7.5-8.5 or around 8 days post-infection.

[0146] ‘Perfusion’ may refer to the continual removal of ‘spent’ culture media from the bioreactor at a specified rate and for a specified period of time. Perfusion for the first virus harvest may occur between 48-96, 48-92, 48-90, 48-87, 48-84, 48- 61 , 48-78, 48-75, 48-72, 54-96, 54-92, 54-90, 54-87, 54-84, 54-81 , 54-78, 54-75, 54-72, 60-96, 60-92, 60-90, 60-87, 60-84, 60-81 , 60-78, 60-75, 60-72, 66-96, 66-92, 66-90, 66-87, 66-84, 66-81 , 66-78, 66-75, 66-72, 69-96, 69-92, 69-90, 69-87, 69-84, 69-81 , 69-78, 69-75, 69-72, 72-96, 72-94, 72-92, 72-90, or 72-84 hours post-infection. Perfusion for the second virus harvest may occur between 144-216, 144-204, 144-196, 156-216, 156-204, 156-196, 168-216, 168-204, 168-196, 180-216, 180-204, ISO- 196 hours post infection. These ranges (e.g., 72-84) indicate that viral harvest using perfusion may start at the first time point in the range (e.g., 72 hours post infection for 72-84) and may finish at the second time point in the range (e.g., 84 hours post infection for 72-84).

[0147] ‘Bioreactor’ may refer to a device that supports a biologically active environment in which a biological process such as propagation of virus and vectors under controlled conditions may be carried out. A bioreactor may be used to propagate both suspended and adherent cells. The bioreactor may be a controlled environment wherein the dissolved oxygen (DO), nitrogen, carbon dioxide, and pH levels may be adjusted. Parameters such as DO, pH, temperature, and biomass may be measured at periodic intervals.

[0148] A ‘fixed-bed bioreactor’ (FBBR) may refer to a type of bioreactor which includes a fixed-bed of packing material that promotes cell adhesion and growth.

[0149] ‘Falling film’ may refer to use of cascading or falling cell culture medium to oxygenate the cell culture medium. This also be referred to as a ‘falling film component’ or ‘falling film oxygenator’. Therefore ‘falling film height’ (FFH) may refer to the height that the media falls from and where it falls to. FFH may be measured in cm.

[0150] ‘Dissolved oxygen’ (DO) may refer to the oxygen levels in the culture media (in the bioreactor).

[0151] ‘Post infection’ may refer to the time elapsed since the time that a virus was added to the host cells in the bioreactor.

[0152] ‘Dialyzing’ or ‘dialysis’ may refer to a process of separating molecules in a solution using a semipermeable membrane to allow certain molecules through whilst blocking others. This may be used to purify or concentrate virus particles. Valneva Austria GmbH PAT070-PCT

[0153] ‘Disease’, ‘disorder’, ‘condition’ may be used to indicate a pathology, injury or abnormal physiology in a subject, which may be associated with negative symptoms. ‘Disease’ may be used to define a pathology which be generally known to have a clear cause and / or diagnostic criteria, ‘disorder’ may be used to define a pathology where normal function is disrupted, but a cause and / or diagnostic criteria is not defined, and ‘condition’ may be used more broadly to refer to any abnormal physiology, including injuries.

[0154] ‘Pharmaceutically acceptable’ may be used herein to refer to substances, compounds, materials, compositions, and / or dosage forms which are suitable for use in human beings in a clinical environment, e.g., without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0155] ‘Prevent’ may be used to refer to use of the formulations described herein as a vaccine, e.g., to reduce the severity, or delay the onset of, the manifestation of clinical or subclinical symptoms, complications, pathologies or biochemical indicia of a specific disease or infection, or to reduce or inhibit the spread / transmission of a specific virus. As used

[0156] ‘Administer’ may refer to application of a composition or formulation to a subject.

[0157] ‘Unit dose’ may be used to define a discrete amount of a composition (optionally a pharmaceutical composition). As such, a unit dose may comprise or consist of a specified amount of viral particles. In embodiments, the unit dose may be configured to deliver a single dosage to a subject.

[0158] ‘“Millimolal” or “mmol / kg” may refer to a concentration unit representing millimoles of a solute per kilogram of solvent. Essentially, 1 millimolal solution contains 1 millimole of solute dissolved in 1 kilogram of solvent.

[0159] Viruses

[0160] Yellow fever virus (YFV) is a single-stranded RNA alphavirus with a diameter of approximately 50-70 nm. YFV may have a gene order of structural and non-structural proteins of: 5’-C-prM- NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3’ (Chambers et al., 1990, Virology, 177: 159-174). C may refer to capsid, prM may refer to precursor to M, and E may refer to envelope protein.

[0161] The YFV may be any YFV strain. The YFV may be an inactivated or live-attenuated YFV. Suitably the YFV is YFV17D. The ‘17D strain’ may refer to the yellow fever strain that was isolated from a mild human case, in Ghana in 1927 and was passaged 18 times in minced murine embryo tissue and then passaged 58 times in minced chicken embryo tissue as Valneva Austria GmbH PAT070-PCT described in Monath (2005). The RNA sequence of the 17D strain may be found as Genbank accession number KF769016. YFV may be from any virus bank.

[0162] The YFV may comprise or consist of a polynucleotide sequence with at least 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 1. The polynucleotide sequence of the YFV may comprise one or more mutations selected from: A2608C, A5641G, A7225G, C8212T, A10243G, G10559GT, and / or A10722G mutations in SEQ ID NO: 1. A2608C is a missense mutation. A2608C is a mutation in the ns1 protein of YFV. The corresponding amino acid mutation for A2608C is Glu52Asp in SEQ ID NO: 5. A5641G is a silent mutation. A5641G is a mutation in the ns3 protein of YFV. The corresponding amino acid mutation for A5641G is Leu357Leu in SEQ ID NO: 5. A7225G is a missense mutation. A7225G is a mutation in the ns4b protein of YFV. The corresponding amino acid mutation forA7225G is lle113Met in SEQ ID NO: 5. C8212T is a silent mutation. C8212T is a mutation in the ns5 protein of YFV. The corresponding amino acid mutation for C8212T is Leu192Leu in SEQ ID NO: 5. A10243G is a silent mutation. A10243G is a mutation in the ns5 protein of YFV. The corresponding amino acid mutation for A10243G is Leu869Leu in SEQ ID NO: 5. G10559GT is a downstream (frameshift) mutation. The G10559GT mutation is in the ns3 protein of YFV. A10722G is a downstream (frameshift) mutation. The A10722G mutation is in the ns3 protein of YFV. A2608C, A7225G, C8212T, and / or G10559GT mutations may occur in only 10-30, IQ- 25, 10-20, 10-18, 10-15, 11-13, 11.5-12.5 or around 12% of the YFV particles (e.g., total YFV particles obtained or in a composition). A5614G, A10243G, and A10722G may be present in at least 95, 96, 97, 98, 99, or 99.5, or in 100%, of the YFV particles. Less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 % of the YFV particles may have one or more mutations in the E protein of YFV (SEQ ID NO: 4). The E protein may not be mutated in the YFV particles.

[0163] The YFV may be adapted to grow on Vero cells.

[0164] The Chikungunya (CHIKV) virus genome encodes two polyproteins: non-structural proteins 1- 4 (nsP1-4) and structural proteins (C, E3, E2, 6K, E1). The CHIKV may be a CHIKV-A5nsP3. Compared to the wild type CHIKV genome, CHIKV-A5nsP3 sequence contains a 183-bp deletion in the 3' part of the sequence encoding nsP3 (amino acids 1656 to 1717 in the nsP1- 4 polyprotein) which results in a 60 amino acid deletion in the nsP3 replicase protein (indicated by A60aa, see Hallengard et al.). Therefore, ‘CHIKV’ may refer to a virus with at least 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99%, or 100%, sequence identity SEQ ID NO: 3. The E2 structural protein of CHIKV-A5nsP3 is set out in SEQ ID NO: 3. Variants of CHIKV-A5nsP3 include variants in the E2 structural protein which have one or more mutations, such as E168K, G55R, E247K, G82R, and / or H232Y. Suitably, mutations in the E2 structural protein are present at a Valneva Austria GmbH PAT070-PCT frequency of less than 70%, 60, 50, 40, 30 or 25% compared to the total number of CHIKV- A5nsP3 particles.

[0165] The CHIKV particles may comprise or consist of CHIKV particles expressing an E2 structural protein as defined by the amino acid sequence of SEQ ID NO: 3. The CHIKV particles may comprise or consist of (i) CHIKV particles expressing an E2 structural protein as defined by the amino acid sequence of SEQ ID NO: 3 and (ii) CHIKV particles expressing an E2 structural protein having the mutation G55R or E168K in the amino acid sequence of SEQ ID NO: 3. CHIKV (i) may be between 30-100, 30-90, 30-80, 40-100, 40-90, 50-100, 50-90 of the CHIKV particles in the composition, formulation or vaccine. CHIKV (ii) may be 1-70, 1-60, 1-50, IQ- 70, 10-60, 10-50, 15-70, 15-60, 15-50, 15-40, 20-70, 20-50, 20-40, or 20-30% of the CHIKV particles in the composition, formulation or vaccine. In an example, the CHIKV particles may comprise or consist of (i) 30-90% of CHIKV particles expressing an E2 structural protein as defined by the amino acid sequence of SEQ ID NO: 3 and (ii) 10-50% of CHIKV particles expressing an E2 structural protein having the mutation G55R or E168K in the amino acid sequence of SEQ ID NO: 3.

[0166] Viral sequences and / or mutations may be identified using standard sequencing techniques, such as next generation sequencing (NGS).

[0167] YFV virus banks may be produced using standard techniques in the art. The YFV may be encoded by a plasmid. The plasmid may comprise a sequence encoding YFV. The plasmid may comprise a sequence with at least 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 1. The YFV sequence in the plasmid may be preceding by a promoter, such as an SP6 promoter. The plasmid may comprise a pACNR backbone (Silva PA, et al., 2010). / n vitro transcription can then be performed to produce RNA. The RNA can then be used to transfect host cells, such as Vero cells. The Vero cells may then be cultured for at least 2, 3, 4 or 5 days, or between 1-10, 1-9, 1-8, 1-7, 1-6, 2-10, 2-8, 2-7, 2-6, 2-5 or 3- 5 days. The culture medium containing viral particles may then be collected. The culture medium may be filtered, such as through a 0.2 pm filter. The culture medium may be stabilised, for example in sucrose, such as 5-15%, 7-12%, 8-12%, 9-11%, 9.5-10.5% (w / v) sucrose. Optionally, the culture medium may be stabilised in 10% w / w sucrose / 25mM TRIS, pH 7.3 and stored at -70 °C.

[0168] Virus production process

[0169] The virus production process may comprise host cell expansion, host cell infection with the virus, virus propagation and virus harvest. Valneva Austria GmbH PAT070-PCT

[0170] The virus production process may use a bioreactor. Several types of bioreactors are available and generally include an impeller (stirring mechanism) for circulation of medium, a head space for provision of oxygen and a pump for circulation and refreshing of medium.

[0171] Suitably, the bioreactor is a fixed-bed bioreactor. An example of fixed-bed bioreactors are shown in Figure 1. The fixed-bed bioreactor may be an iCELLis Nano (Pall Corporation), iCELLis 500 bioreactor (Pall Corporation, described in LISTR 3473 “iCELLis® 500+ Generation R Bioreactors”, published by Pall, May 2022), or a Univercells fixed-bed bioreactor (Univercells SA) such as a Scale-X™ bioreactor. The fixed-bed bioreactor may comprise carriers composed of woven medical-grade polyethylene terephthalate (PET) fibres in a robust, single, closed system that does not require any aseptic handling. The fixed-bed bioreactor may be used at small-scale (such as in the iCELLis Nano) e.g., where the growing area is between 0.5 to 4 m2. The fixed-bed bioreactor may be used at manufacturing scale (such as in iCELLis 500) e.g., where the growing area ranges from 66 to 500 m2. The fixed- bed height may range from 10 to 20 mm, providing a growth area of 5300 to 40,000 cm2in an 800 mL fixed-bed bioreactor and 660,000 to 5,000,000 cm2in a 25L fixed-bed bioreactor. Processes developed in the small-scale system may be scaled up to that of the manufacturing scale. A fixed-bed bioreactor may have sensors that measure and monitor the pH, temperature, dissolved oxygen, and the biomass, which indicates adherent cell density. A fixed-bed bioreactor may also have different ports that enable the addition of oxygen or nitrogen, a media exchange port, ports for the addition of sodium hydroxide (NaOH) and / or CO2 to adjust the pH.

[0172] The bioreactor may comprise a fixed bed in a spiral shape wherein the medium recirculates through the bed, such as in Figure 1A and 1B. The bioreactor may comprise a fixed bed embedded in a chamber through which the medium circulates such as the bioreactor shown in Figure 1C. The bioreactor may comprise a flask, wherein the medium is pumped out through a dip tube and returned to the chamber in a waterfall such as shown in Figure 1D. The bioreactor may allow the FFH to be adjusted by the weight of the medium introduced into the system such as shown in Figure 1E.

[0173] The fixed bed bioreactor may use cascading cell culture medium to oxygenate the cell culture medium. This may be referred to as a ‘falling film component’ or ‘falling film oxygenator’. The cascading media may be directed along a surface, such as an interior sidewall of the bioreactor, to minimize splashing or disruption of cells. The falling film may be characterized in that shear stress and / or medium foaming are minimized by maintaining the Falling Film Height (FFH) at the lowest possible level while still maintaining required dissolved oxygen (DO) levels. The required DO may refer to DO levels at each stage of the virus production Valneva Austria GmbH PAT070-PCT process, thereby minimizing damage to virus particles compared with more turbulent incubation condition.

[0174] The host cells used for the virus production process may be any eukaryotic cell that is suitable for the production of virus antigen, viral vector, or virus production. The host cell may be an ‘adherent cell’ that adheres to a surface in culture. Suitable host cells include Vero cells, MBCK cells, MDBK cells, MRC-5 cells, BSC-1 cells, LLC-MK cells, CV-1 cells, CHO cells, COS cells, murine cells, human cells, avian cells, insect cells, HeLa cells, HEK-293 cells, MDOK cells, CRFK cells, RAF cells, TCMK cells, LLC-PK cells, PK 15 cells, WI-38 cells, T-FLY cells, BHK cells, SP2 / 0 cells, NSO cells, NTCT cells, and PerC6 cells, 3T3 cells, or a combination, derivative or modification thereof. Suitably, the host cells are Vero cells or a derivative or modification thereof. The Vero cells may be a Vero (WHO) cell line, obtained from the Health Protection Agency (currently the UK Health Security Agency) general cell collection under catalogue number 88020401.

[0175] Vero cell stocks can be thawed and seeded onto adherent cell culture dishes and incubated at 37 °C / 5%CO2. Vero cells can then be passaged using standard techniques in the art. For example, by the addition of a proteolytic enzyme such as trypsin-EDTA for 2-3 minutes at 37 °C, adding cell culture media, centrifuging the cells, resuspending the cell pellet in media and re-plating a portion of the Vero cells.

[0176] The Vero cells may be cultured in a serum-free medium selected from DM EM, DMEM / F12, Medium 199, MEM, RPMI, OptiPRO-SFM, VP-SFM, VP-SFM AGT, HyQ PF-Vero, MP-Vero, suitably OptiPRO-SFM and VP-SFM, more suitably OptiPRO-SFM for culturing the host cells. The culture media may also be animal-free media; that is, not containing any product of animal origin. The culture media may also be protein-free media; that is, media is formulated without added proteins. The serum-free or protein-free media may be formulated without serum or protein but may contain cellular protein derived from the host cells, and optionally proteins specifically added to the serum-free or the protein-free media. The serum free medium may be supplemented with glucose, sodium bicarbonate and / or L-glutamine. The glucose may be between 1-10, 2-9, 3-8, 4-8, 5-7, 5.5-6.5 or around 6 g / L. The sodium bicarbonate may be between 1-5, 2-5, 2-4.5, 3-5, 3-4.5, 3-4, or around 3.7 g / L. The L-glutamine may be between 1-10, 1-9, 1-8, 1-7, 2-10, 2-9, 2-8, 2-7, 2-6, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3.5-4.5 or around 3 mM, particularly 4 mM.

[0177] The pH for culturing can be between 6.5-7.5, depending on the pH stability of the host cells.

[0178] Preferably the cells are cultured at a pH of 7.4. The host cells may be cultured at a temperature Valneva Austria GmbH PAT070-PCT of between 20 and 40°C, specifically between 30 and 40°C, and preferably between 35 and 37°C for mammalian cells, especially at 36.5°C to 37.5°C.

[0179] The bioreactor may be prepared and incubated overnight with cell culture medium.

[0180] Vero cells may then be passaged for transfer to the bioreactor. Passaged Vero cells may be counted and tested for cell viability (including the presence of cell (%) of cells in aggregates) manually or automatically (for example, using a NucleoCounter NC-200 cell counter).

[0181] Host cells may be seeded in the bioreactor at a density between 500-20000, 500-15000, 500- 10000, 500-7000, 500-5000, 1000-20000, 1000-15000, 1000-10,000, 1000-7000, 1000-5000, 2000-20000, 2000-15000, 2000-10000, 2000-7000, 2000-5000, 2000-4000, 2500 to 3500, or around 3000 cells per cm2. The seeding density may be adjusted based on the type of host cell, the volume of the bioreactor, the height of fixed-bed in a fixed-bed bioreactor, etc. It is within the knowledge of one skilled in the art to select the optimum seeding density for the process. Cells may be placed into the bioreactor by placing in the inoculation bottle.

[0182] The transfer of cells may be checked by taking a sample from the medium of the bioreactor and determining the concentration of cells that had not attached yet by performing a cell count. Agitation speed can be reduced after 3 hours, with the agitation speed corresponding to a linear speed of medium through the fixed-bed of 1 cm / sec.

[0183] Recirculation of medium between the bioreactor and the recirculation bottle can be initiated one day after seeding. Recirculation rate can be fixed to 4 mL / min / m2, i.e. 16 mL / min during cell expansion. pH of the bioreactor may be maintained at pH 7-7.5, or around pH 7.4 + / - 0.2. pC>2 may be maintained at between 40-60, 45-55 or around 50% air saturation. Agitation speed may be maintained at a speed corresponding to fluid circulation at 1 cm / sec (with the exception of cell attachment and virus infection phases, during which the linear speed of medium through the fixed- bed was adjusted to 2 cm / sec).

[0184] Host cells may be incubated in the bioreactor for between 2-10, 3-10, 3-9, 4-10, 4-9, 4-8, 5-8, 5-7, 5.5-8.5 or around 6 days. Host cells may be infected by the virus particles when they reach a cell density between 0.1 to 4.0, 0.1 to 3.5, 0.1 to 3.0, 0.1 to 2.5, 0.5 to 4.0, 0.5 to 3.5, 0.5 to 3.0, 0.5 to 2.5, 1.0 to 4.0, 1.0 to 3.5, 1.0 to 3.0, 1.0 to 2.5, or 1.0 to 2.0 x 105cells / cm2, or around 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 or 1.9 x 105cells / cm2. Alternatively, host cells may be infected by the virus particles when they reach a confluency of 30-100%, 30-95%, 30- 90%, 30-85% 30-80%, 30-75%, 30-70%, 30-60%, 30-50%, 40-100%, 40-95%, 40-90%, 40- Valneva Austria GmbH PAT070-PCT

[0185] 85%, 40-80%, 40-75%, 40-70%, 40-60%, 40-50%, 50-100%, 50-95%, 50-90%, 50-85%, 50- 80%, 50-75%, 60-100%, 60-95%, 60-90%, 60-85%, 60-80%, 60-75%, 70-100%, 70-95%, 70- 90%, 70-85%, or 70-80%.

[0186] Before infecting host cells with virus particles, the media may be changed. The media may be changed to a serum-free media. The media may be OptiPRO-SFM or VP-SFM, suitably VP- SFM. The serum free medium may be supplemented with glucose, sodium bicarbonate and / or L-glutamine. The glucose may be between 1-10, 2-9, 3-8, 4-8, 5-7, 5.5-6.5 or around 6 g / L. The sodium bicarbonate may be between 1-5, 2-5, 2-4.5, 3-5, 3-4.5, 3-4, or around 3.7 g / L. The L-glutamine may be between 1-10, 1-9, 1-8, 1-7, 2-10, 2-9, 2-8, 2-7, 2-6, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3.5-4.5 or around 3 mM, particularly 4 mM.

[0187] The host cells can then be infected with the virus (e.g., YFV or CHIKV). To do this, the virus particles can be added to the medium in the bioreactor. The host cells may be infected at a multiplicity of infection (MOI) between 0.001 to 1 , 0.01 to 0.1 , 0.01 to 0.5, 0.03 to 0.5, 0.03 to 0.1 or around 0.03. The virus and cells may be incubated in the bioreactor with the impeller speed increased to a value corresponding to a linear speed of medium through the fixed-bed between 1-4, 1-3, 1.5-2.5 or around 2 cm / sec. The impeller speed may be increased for 0.5 to 10, 1 to 10, 0.5 to 9, 1 to 9, 0.5 to 8, 1 to 8, 2 to 8, 0.5 to 7, 1 to 7, 2 to 7, 0.5 to 6, 1 to 6, 2 to 6, 0.5 to 5, 1 to 5, 2 to 5, 0.5 to 4, 1 to 4, 1 .5 to 4, 2 to 4, 3 to 4, 0.5 to 3.5, 1 to 3.5, 1 .5 to 3.5, 2 to 3.5, or 2.5 to 3.5 hours, or around 3 hours post-infection of the host cells.

[0188] The virus infection phase may last between 0.5 to 10, 1 to 10, 0.5 to 9, 1 to 9, 0.5 to 8, 1 to 8, 2 to 8, 0.5 to 7, 1 to 7, 2 to 7, 0.5 to 6, 1 to 6, 2 to 6, 0.5 to 5, 1 to 5, 2 to 5, 0.5 to 4, 1 to 4, 1.5 to 4, 2 to 4, 3 to 4, 0.5 to 3.5, 1 to 3.5, 1.5 to 3.5, 2 to 3.5, or 2.5 to 3.5 hours, or around 3 hours post-infection of the host cells.

[0189] The medium may be changed between 6-30, 12-30, 18-30, 20-30, or 24 hours post-infection.

[0190] Suitably, the virus infection phase may have a DO of at least 10, 12, 15, 20, 25, or 30%, suitably between 5-20%, 10-20%, 10-15%, more suitably around 10% or 15%. Suitably, in the virus infection phase the FFH may be at 0-5cm, 0-4cm, 0-3cm, 0-2cm, 0-1cm, 0-0.5cm, 0- 0.05cm, 0-0.01 cm or around 0cm. Suitably, in the virus infection phase the FFH may be maintained for between 50-100, 60-100, 70-100, 80-100, 90-100, or 100% of the duration of the virus infection phase e.g., within the first 5, 4.5, 4, 3.5, 3, 2.5 or 2 hours post infection. If the DO falls below 10% during the virus infection phase, an FFH of 1 cm may be set and increased by increments up to a maximum of 4cm, and / or until a DO of at least 15% can be maintained. During virus infection the DO may not exceed 4cm. The increments may be 0.5cm. Valneva Austria GmbH PAT070-PCT

[0191] Suitably, in the virus infection phase the FFH is maintained for at least 50, 60, 70, 80, 90, or 100% of the duration of the virus infection phase e.g., within the first 5, 4.5, 4, 3.5, 3, 2.5 or 2 hours post infection. In the virus infection phase, the stirrer speed of the bioreactor may remain constant for the entire duration of the virus infection phase e.g., within the first 5, 4.5, 4, 3.5, 3, 2.5 or 2 hours post infection.

[0192] The host cells may then undergo virus propagation phase. This may last from virus infection phase to viral harvest. In this phase, recirculation rate may be reduced to 1-5, 1-4, 1-3, 1.5- 2.5 or around 2 mL / min / m2. Suitably, the recirculation rate may be 8 mL / min.

[0193] Suitably, the virus propagation phase may have a DO of at least 10, 15, 20, 25, 30, 35, 40, 45, or 50%, suitably between 5-60%, 10-60%, 20-60%, 30-60%, 40-60%, 45-55%, or around 50%. In the virus production phase, the FFH may be between 1-5, 1-4.5, 1-4, 1-3.5, 1-3, 1-2.5, 1.5- 5, 1.5-4.5, 1.5-4, 1.5-3.5, 1.5-3, 1.5-2.5, 2-4, 2-3, or around 2 cm. The FFH may be increased incrementally up to a maximum of 4 cm. The increments may be 0.5 cm. During virus production the FFH may not exceed 4 cm.

[0194] The virus may then be harvested by collecting the culture media from the bioreactor. Harvesting of the virus may be performed 24-120, 24-108, 24-96, 24-84, 24-72, 36-120, 36- 108, 36-96, 36-84, 36-72, 48-120, 48-108, 48-96, 48-84, 48-72, 60-120, 60-108, 60-96, 60- 84, 60-80, 60-78, 60-76, 60-74, 60-72, 62-80, 62-78, 62-76, 62-74, 62-72, 64-80, 60-78, 64- 76, 64-74, 64-72, 66-80, 66-78, 66-76, 66-74, 66-72, 68-80, 68-78, 68-76, 68-74, 68-72, 70- 80, 70-78, 70-76, 70-74, 70-72, or around 72 hours post-infection. Harvesting of the virus may be performed 2-4, 2.5-3.5 or around 3 days post-infection.

[0195] After this harvest, the culture media may be replaced with fresh culture media, and the host cells left to incubate in the bioreactor. A ‘second viral harvest’ may then be performed at a later time period to remove and keep culture media from the host cells infected with the virus after the first viral harvest was performed. The second viral harvest may be performed 144-216, 144-204, 144-196, 156-216, 156-204, 156-196, 168-216, 168-204, 168-196, 180-216, 180- 204, 180-196, 190-194 or around 192 hours post-infection. The second viral harvest may be performed 7-9, 7.5-8.5 or around 8 days post-infection.

[0196] The media may collected at a single time point, or by perfusion using standard techniques in the art. Perfusion for the first virus harvest may occur between 48-96, 48-92, 48-90, 48-87,

[0197] 48-84, 48-61 , 48-78, 48-75, 48-72, 54-96, 54-92, 54-90, 54-87, 54-84, 54-81 , 54-78, 54-75,

[0198] 54-72, 60-96, 60-92, 60-90, 60-87, 60-84, 60-81 , 60-78, 60-75, 60-72, 66-96, 66-92, 66-90,

[0199] 66-87, 66-84, 66-81 , 66-78, 66-75, 66-72, 69-96, 69-92, 69-90, 69-87, 69-84, 69-81 , 69-78, Valneva Austria GmbH PAT070-PCT

[0200] 69-75, 69-72, 72-96, 72-94, 72-92, 72-90, or 72-84 hours post-infection. Perfusion for the second virus harvest may occur between 144-216, 144-204, 144-196, 156-216, 156-204, 156- 196, 168-216, 168-204, 168-196, 180-216, 180-204, or 180-196 hours post infection. These ranges (e.g., 72-84) indicate that viral harvest using perfusion may start at the first time point in the range (e.g., 72 hours post infection for 72-84) and may finish at the second time point in the range (e.g., 84 hours post infection for 72-84).

[0201] Downstream processing

[0202] The harvested culture medium may be precipitated to produce a virus supernatant. An example downstream processing method is shown in Figure 4. The downstream process may include clarification, concentration, and diafiltration using standard techniques in the art. The process may also include precipitation before clarification.

[0203] The harvested culture medium may be precipitated to remove Vero cell DNA and other undesired material, such as Vero cell debris, from the harvested culture medium before concentration. This may be performed by adding a protamine salt or benzonase, suitably protamine sulphate, to the harvested culture medium. The protamine sulphate may be added at concentrations between to 0.1 to 5, 0.1 to 2, 0.1 to 1 , 0.1 to 0.5, 0.1 to 0.3, 0.15 to 0.25 or around 0.2 mg / mL. The mixture with protamine sulphate may be incubated at room temperature or on ice. The mixture with protamine sulphate may be incubated for between 15- 90, 15-60, 20-90, 20-60, 20-50, or 30-45 minutes.

[0204] Debris from infected host cells may be removed from the harvested viral particles, referred to as a "clarification" of the harvest. The harvested particles may be clarified by common methods known in the art, such as such as low-speed centrifugation, for example, at 1500 g for 10 min, and / or by filtration through a filter, for example with a pore size of 0.45 pm. The clarified harvest may then be concentrated. Clarification may be performed using Sartopore 2 membrane filter capsules (Sartorius Sartopore 2, 5445306G9-S-A, 0.8 / 0.45 pm, 2000 cm2filter area).

[0205] To concentrate the titer of the virus in the harvested culture medium, it may be subjected to concentration by any method known in the art, including ultrafiltration, ultracentrifugation, centrifugal concentrator, vacuum centrifugation, and lyophilization. The harvested culture medium may be concentrated by ultrafiltration and the retentate containing the virus is collected. The harvested culture medium may be concentrated by precipitation in which polyethylene glycol (PEG) 8000 dissolved in the culture medium (up to 10%) and the precipitate dissolved in a buffer, for example phosphate-buffered saline (PBS, pH 7.0). An example of concentration may be a sucrose density gradient centrifugation step which also Valneva Austria GmbH PAT070-PCT removes excess protamine sulphate. The sucrose density gradient centrifugation step may be adding 10% + / - 10% (w / w) sucrose comprising the protamine-sulphate treated particles to a simple three layer sucrose density gradient ( e.g. a gradient comprising a 15% + / - 10% (w / w) sucrose solution, a 35% + / - 10% (w / w) sucrose solution, and a 50% + / - 10% (w / w) sucrose solution) as described in WO 2017 / 109225 (incorporated by reference in its entirety). Alternatively, the concentrated harvest may be concentrated using chromatography, such as flowthrough chromatography, such as multi-modal Capto™Core 400 (CC400) or Capto™Core (CC700) chromatography resin following manufacturer protocol.

[0206] Concentrated harvest material was stabilized for freezing by addition of sucrose (final sucrose concentration 10% w / w using a 50% w / w stock dissolved in 25 mM Tris pH 7.4). Stabilized concentrated harvest can be frozen at -65°C. Alternatively, concentrated harvest material may be further purified and diafiltrated immediately.

[0207] Purification of the concentrated harvest material may be by any method known in the art, including ultrafiltration, ultracentrifugation, centrifugal concentrator, vacuum centrifugation, and lyophilization. The harvested culture medium may be concentrated by ultrafiltration and the retentate containing the virus is collected. Ultrafiltration and tangential flow filtration (TFF) as used herein can be interchangeable. The purification may be through chromatography such as flowthrough chromatography, preferably multi-modal Capto™Core 400 (CC400) or Capto™Core (CC700) chromatography resin following manufacturer protocol.

[0208] The composition may then undergo diafiltration to further remove trace amounts of residual media components. The diafiltration buffer may comprise or consist of sugar, phosphate, a carboxylate buffering agent, MgCh, D-sorbitol, and an amino acid. The diafiltration buffer may further comprise human albumin. The diafiltration buffer may comprise or consist of sugar, phosphate, a carboxylate buffering agent, MgCh, D-sorbitol, an amino acid, and human albumin (HA) in a diluent. Diafiltration is optional and may be performed as part of the primary harvest recovery steps and / or as part of the downstream purification steps.

[0209] The sugar may be sucrose, mannitol, lactose, sorbitol, dextrose, fucose and trehalose, suitably sucrose or trehalose, more suitably sucrose. The sugar (e.g., sucrose) may be at concentrations between 1 to 50%, 1 to 40%, 1 to 30%, 1 to 20%, 1 to 10%, 2 to 8%, 3 to 7%, 4 to 6%, 4.5 to 5.5% w / v, or around 5% w / v, in the diafiltration buffer. The sugar may protect the viral particles during freeze / thaw stress. In instances where reduced viscosity is desired, it is known in the art to be preferable to use lower concentrations of sugar, e.g., sucrose. The upper limit for the concentration of sugar can be dictated by the ability of a formulation to undergo required filtration or processing steps. Valneva Austria GmbH PAT070-PCT

[0210] The phosphate may be a monophosphate, polyphosphate or phosphorylated compound. The phosphate may be sodium phosphate or potassium phosphate, suitably potassium phosphate. The phosphate may be provided as di-Potassium Hydrogen Phosphate and / or Potassium diHydrogen Phosphate, suitably di-Potassium Hydrogen Phosphate and Potassium diHydrogen Phosphate combined. The phosphate (e.g., potassium phosphate) may be at concentrations between 1-30, 1-25, 1-20, 1-15, 1-12, 1-10, 2-25, 2-20, 2-15, 2-12, 2-10, 2-8, 3-20, 3-15, 3-12, 3-10, 3-7, 4-20, 4-15, 4-12, 4-10, 4-8, 4-6, 4.5-5.5 mM, or around 5 mM, in the diafiltration buffer. Lower amounts of phosphate are preferable to avoid precipitation of viral particles.

[0211] The carboxylate buffering agent may be a succinate, citrate, fumarate, tartarate, maleate or lactate, suitably sodium citrate, more suitably provided as trisodium citrate dihydrate, carboxylate buffering agent (e.g., sodium citrate) may be at concentrations between 1-50, 1- 40, 5-50, 5-40, 10-50, 10-40, 10-30, 15-50, 15-40, 15-35, 15-30, 20-50, 20-40, 20-35, 20-30, 21-29, 22-28, 23-27, 24-26 mM, or around 25 mM, in the diafiltration buffer. The carboxylate buffering agent may help to inhibit aggregation.

[0212] The magnesium chloride (MgCh) may be provided as magnesium chloride hexahydrate. The MgCh may be at concentrations between 1-15, 1-12, 1-10, 2-9, 3-8, 3-7, 4-6, 4.5-5.5 mM, or around 5 mM, in the diafiltration buffer. The magnesium chloride may stabilize RNA structure of the viral particles and exhibit a positive effect on infectivity after storage.

[0213] The D-sorbitol may be at concentrations between 0.01-5%, 0.01-3%, 0.01-2%, 0.01-1%, 0.1- 5%, 0.1-3%, 0.1-2%, 0.1-1%, 0.1-0.9%, 0.2-0.8%, 0.3-0.7%, 0.4-0.6%, 0.45-0.55% w / v, or around 0.5% w / v, in the diafiltration buffer. D-sorbitol can result in retention of protein native structure and improved stability.

[0214] The amino acid may be L-methionine, arginine or glutamine, suitably L-methionine. The amino acid (e.g., L-methionine) may be at concentrations between 1-20, 1-15, 3-20, 3-15, 5-20, 5- 15, 7-13, 8-12, 9-11 , 9.5-10.5 mM, or around 10 mM, in the diafiltration buffer. The amino acids may improve the stability of the lyophilized vaccine.

[0215] The human albumin (HA) may be recombinant human albumin (rHA). The HA may be at concentrations between 0.001 to 2, 0.001 to 1.5, 0.001 to 1 , 0.005 to 2%, 0.005 to 1.5%, 0.005 to 1%, 0.005 to 0.5%, 0.005 to 0.02%, 0.005 to 0.015%, 0.09-1%, 0.09-0.5%, 0.09-0.1% w / v, or around 0.01% w / v, in the diafiltration buffer. The HA may be at concentrations between 0.001-1 , 0.01-1 , 0.01-0.5, 0.05-1 , 0.05-0.5, 0.05-0.2, 0.05-0.15, 0.08-0.12 mg / mL, or around 0.1 mg / mL, in the diafiltration buffer. Valneva Austria GmbH PAT070-PCT

[0216] The pH of the diafiltration buffer may be between pH 7-8, pH 7.0-7.5, pH 7.0-7.8, pH 7.1-7.5, pH 7.2-7.4, pH 7.25-7.35, or around pH 7.3 + / - 0.2, or around pH 7.3 + / - 0.1.

[0217] The diluent may be tissue culture medium, saline or water, such as water for injection (WFI) or milliQ water, preferably WFI.

[0218] The diafiltration buffer may comprise or consist of sucrose, potassium phosphate, sodium citrate, MgCh, D-sorbitol, L-methionine, and human albumin (HA) in water, suitably wherein the buffer comprises or consists of sucrose at 5% w / v, potassium phosphate at 5 mM, sodium citrate at 25 mM, MgCh at 5 mM, D-sorbitol at 0.5% w / v, L-methionine at 10 mM, and human albumin (HA) at 0.1 mg / mL, in water, suitably at pH 7.3. These values may be deviate by + / - 10%.

[0219] A final filtration (e.g., 0.2 pm filtration) may be performed to prepare the drug substance (DS).

[0220] Preparation of formulation

[0221] Liquid formulations can be prepared using standard techniques in the art by either (1) dilution of bulk vaccine into the stabilizer, (2) dialysis / diafiltration into the stabilizer, or (3) concentration of bulk vaccine and dilution or diafiltration into the stabilizer. A lyophilized drug product can be prepared from the liquid formulation by lyophilization.

[0222] The drug substance may be further diluted to provide the formulation, vaccine or unit dose. The diluent may be the diafiltration buffer, or another diluent described herein. The dilution factor of drug substance (DS) to drug product (DP) bulk depends on the viral concentration of DS (determined by TCIDso assay). Formulation buffer for DS and DP formulation are identical and are as described above. The target concentrations after dilution to DP is between 102TCIDso / dose to 106TCID50 / dose, 102TCID50 / dose to 105TCID50 / dose, 102TCID50 / dose to 104TCIDso / dose, or between 103TCIDso / dose to 104TCIDso / dose. The dose may be in 0.5mL. The unit dose of the liquid frozen or lyophilized formulation may comprise between 1 to 20, 1 to 15, 1 to 10, 3 to 20, 3 to 15, 3 to 10, 3 to 9, 4 to 15, 4 to 10, 4 to 8, 5 to 15, 5 to 7, 5.5 to 6.5 or around 6 logw TCIDso per mL.

[0223] Preferably the total volume of the liquid formulation per individual vial before lyophilization is 0.3 to 1.0 mL, more preferably 0.4 to 0.6 mL, most preferably about 0.5 mL.

[0224] The liquid formulation may be lyophilized. Any general method of lyophilization known in the art may be used. Particularly preferred is a lyophilization method providing an adequate cake structure, i.e. , a cake without cracks and with minimal shrinkage. Valneva Austria GmbH PAT070-PCT

[0225] As a general example of a lyophilization process, freezing may be performed followed by primary drying and secondary drying. The freezing may be performed at temperatures between -30 to -50, -35 to -45 or around -40°C. The freezing may be performed for between 15-180, 15-120, 15-90, 15-60, 30-180, 30-120, 30-90, 30-60, 45-180, 45-120, 45-90, 45-60, 60-180, 60-150, 60-120, 50-70 or around 60 minutes. The freezing may be performed in steps that gradually reduce the temperature to the freezing temperature. In one example, freezing may be in 2, 3, 4 or 5 steps. In an example, freezing may be performed in three steps. The first step may be at 3-10, 3-7, 4-6, 4.5 to 5.5 or around 5 °C. The next step may be at -10 to 3, -10 to 0, -7 to 1 , -7 to 0, -6 to 0, -5 to -1 , -4 to -2, -2.5 to -3.5 or around -3 °C. The final freezing step may be at -30 to -50, -35 to -45 or around -40°C. The first freezing step may be between 10-90, 15-90, 10-60, 15-60, 15-45, 20-40, 25-35 or around 30 minutes. The second freezing step may be between 10-90, 10-60, 15-90, 15-60, 20-60, 30-60, 35-55, 40-50, or around 45 minutes. The third freezing step may be between 15-180, 15-120, 15-90, 15-60, 30-180, 30-120, 30-90, 30-60, 45-180, 45-120, 45-90, 45-60, 50-70 or around 60 minutes. The freezing step(s) may not be performed under vacuum.

[0226] The primary drying may be performed in 1 , 2, 3, 4 or 5 steps. The primary drying may be performed under vacuum, such as under 20-1000, 20-500, 20-200, 20-150, 20-100, 25-100, 25-80, 25-75, 25-60, 25-50, 30-50, 30-40, or around 35 pbar. The primary drying may be for 1-96, 1-72, 1-48, 1-36, 1-31 , 1-28, 1-26, 1-24, 5-96, 5-72, 5-48, 5-36, 5-30, 5-28, 5-26, 5-24, 10-96, 10-72, 10-48, 10-36, 10-30, 10-28, 10-26, 10-24, 18-96, 18-72, 18-48, 18-36, 18-30, 18-28, 18-26, 18-24, 20-30, 22-26, 25-35, 27-35, or around 31 hours. Where there are two or more primary drying steps, the steps may be at different temperatures. The first temperature may be between -30 to -50, -35 to -45 or around -40 °C. The second temperature may be between -35 to -15, -35 to -20, -30 to -20, -30 to -25, -26 to -28 or around -27 °C. In an example where primary drying is over three steps, the first step may be at a temperature between -30 to -50, -35 to -45 or around -40 °C. The first step may be performed for 15-180, 15-120, 15- 90, 30-180, 30-120, 30-90, 45-180, 45-120, 45-90, 45-75, 55-65 or around 60 minutes. The second primary drying step may be performed at 35 to -15, -35 to -20, -30 to -20, -30 to -25, - 26 to -28 or around -27 °C. The second step may be performed for 1-96, 1-72, 1-48, 1-36, 1- 31 , 1-28, 1-26, 1-24, 5-96, 5-72, 5-48, 5-36, 5-30, 5-28, 5-26, 5-24, 10-96, 10-72, 10-48, IQ- 36, 10-30, 10-28, 10-26, 10-24, 18-96, 18-72, 18-48, 18-36, 18-30, 18-28, 18-26, 18-24, 20- 30, 22-26, 25-35, 27-33, or around 30 hours.

[0227] Secondary drying may be performed at between 10-40, 15-30, 10-30, 15-30, 15-25, or around 20 °C. The secondary drying step may be performed under vacuum, such as under 20-1000, 20-500, 20-200, 20-150, 20-100, 25-100, 25-80, 25-75, 25-60, 25-50, 30-50, 30-40, or around Valneva Austria GmbH PAT070-PCT

[0228] 35 pbar. Secondary drying may be for 1-24, 1-18, 1-12, 1-10, 3-24, 3-18, 3-12, 3-10, 3-8, 3-7, 4-10, 4-8, 4-6, 4.5-5.5 or around 5 hours.

[0229] There are also specific chemical compounds that interact with and stabilize biological molecules and / or general pharmaceutical excipients that can be added to the vaccine and used in conjunction with either lower temperature storage or lyophilization methods. However, use and effectiveness of chemicals may vary between each virus.

[0230] The liquid formulation may be frozen, for example at -20, or -70 °C.

[0231] Formulations

[0232] Any of the pharmaceutical compositions, formulations, or vaccines provided in the current invention may further comprise an adjuvant and / or a pharmaceutically acceptable excipient.

[0233] Excipients include substances such as buffers, stabilizers or further active ingredients, especially ingredients known in connection with pharmaceutical compositions and / or vaccine production.

[0234] The formulations, compositions or vaccines may be administered to a subject with, prior to, or after administration of one or more adjuvants. An adjuvant may refer to a molecule or formulation that enhances a response in a subject, such as an immune response, to an antigen or other molecule. Therefore, administering any of the formulations, vaccines or compositions prior to, or after administration of an adjuvant, may enhance the production of virus neutralizing antibodies. An adjuvant may stabilize an antigen or other molecule. Determining whether a formulation, vaccine or composition is administered with an adjuvant depends on various factors (e.g., type and extent of response desired) and will be evident to one of skill in the art. Examples of adjuvants may include aluminium salt (aluminium hydroxide or aluminium phosphate), calcium phosphate hydroxide, paraffin oil, killed bacteria, bacterial toxins, toxoids, subunits of bacteria, squalene, thimerosal, detergents, IL-1, IL-2, IL-12, 2-component adjuvants, such as 2-component adjuvants containing an antibacterial peptide and a TLR9 agonist (e.g., IC31®), and combinations such as Freund's complete adjuvant and Freund's incomplete adjuvant. Said IC31 ® adjuvant comprises a combination of KLKLLLLLKLK peptide (SEQ ID NO: 6) and oligo(dldC)i3 (a.k.a. ODN1a; SEQ ID NO: 7), suitable aluminium hydroxide or aluminium phosphate salt. An aluminium salt may be an aluminium hydroxide with reduced Cu content, e.g. lower than 1.25 ppb based on the weight of the final pharmaceutical composition or vaccine, an adjuvant described in detail in WO2013 / 083726A1 and Schlegl et al., Vaccine (2015) 33:5989-5996. Valneva Austria GmbH PAT070-PCT

[0235] The amounts and concentrations of the components of the formulations described herein may refer to the weight / volume percentage when referring to lyophilized or liquid formulations. For example, a 10% concentration in a liquid formulation is 10 grams per 100 milliliters and a 10% concentration of a lyophilized formulation refers to 10 grams per 100 milliliters in the liquid form before lyophilization. The lyophilized formulations can be reconstituted in a diluent to approximately the same volume as before lyophilization, i.e., the concentrations in the lyophilized formulations as used herein generally refer to concentrations both before lyophilization and after reconstitution in the same volume. Other measures such as the molarity of a compound refer to a liquid formulation or to a lyophilized formulation both before lyophilization and after reconstitution.

[0236] Therefore, reference to concentrations and components of a lyophilized formulation may be understood to refer to the initial liquid formulation (i.e., before lyophilization), or the reconstituted product after lyophilization. If the lyophilized formulation is reconstituted in the same volume of diluent to the original volume of the liquid formulation before lyophilization, then the concentrations and components can be understood to be the same in the reconstituted formulation as in the original liquid formulation.

[0237] In a previous application (WO 2021 / 028406, herein incorporated by reference in its entirety), it was found that certain buffer systems, such as e.g. histidine, under certain conditions, induced aggregation of CHIKV vaccine candidates. A low (e.g. 5 mM) phosphate-citrate buffer system was shown to be amenable to lyophilization and promoted the stability and desired size of CHIKV particles, also containing other stabilizing components, was developed for the lyophilized and liquid (frozen) CHIKV vaccine. In the present examples, we surprisingly found that the same formulation provided a stable lyophilized and liquid frozen formulation of YFV at different temperatures over time.

[0238] Therefore, the lyophilized or liquid frozen formulation may comprise YFV. The lyophilized or liquid frozen formulation may further comprise one or more further viruses. The further virus may be chikungunya virus (CHIKV), Zika virus (ZIKV), Japanese encephalitis virus (JEV), Dengue virus (DENV), suitably CHIKV. In embodiments, the viral particles of the lyophilized or liquid frozen formulation may comprise or consist of YFV and CHIKV particles.

[0239] The lyophilized or liquid frozen formulation may comprise sugar, phosphate, a carboxylate buffering agent, MgCh, D-sorbitol, and an amino acid. The lyophilized or liquid frozen formulation may further comprise human albumin. In some embodiments, the lyophilized or liquid frozen formulation may comprise or consist of viral particles (e.g., YFV, or YFV and Valneva Austria GmbH PAT070-PCT

[0240] CHIKV, described herein), sugar, phosphate, a carboxylate buffering agent, MgCh, D-sorbitol, an amino acid, and human albumin (HA) in a diluent.

[0241] The sugar may be sucrose, mannitol, lactose, sorbitol, dextrose, fucose and trehalose, suitably sucrose or trehalose, more suitably sucrose. The sugar (e.g., sucrose) may be at concentrations between 1 to 50%, 1 to 40%, 1 to 30%, 1 to 20%, 1 to 10%, 2 to 8%, 3 to 7%, 4 to 6%, 4.5 to 5.5% w / v, or around 5% w / v, in the lyophilized or liquid frozen formulation. The sugar may protect the viral particles during freeze / thaw stress. In instances where reduced viscosity is desired, it is known in the art to be preferable to use lower concentrations of sugar, e.g., sucrose. The upper limit for the concentration of sugar can be dictated by the ability of a formulation to undergo required filtration or processing steps.

[0242] The phosphate may be a monophosphate, polyphosphate or phosphorylated compound. The phosphate may be sodium phosphate or potassium phosphate, suitably potassium phosphate. The phosphate may be provided as di-Potassium Hydrogen Phosphate and / or Potassium diHydrogen Phosphate, suitably di-Potassium Hydrogen Phosphate and Potassium diHydrogen Phosphate combined. The phosphate (e.g., potassium phosphate) may be at concentrations between 1-30, 1-25, 1-20, 1-15, 1-12, 1-10, 2-25, 2-20, 2-15, 2-12, 2-10, 2-8, 3-20, 3-15, 3-12, 3-10, 3-7, 4-20, 4-15, 4-12, 4-10, 4-8, 4-6, 4.5-5.5 mM, or around 5 mM, in the lyophilized or liquid frozen formulation. Lower amounts of phosphate are preferable to avoid precipitation of viral particles.

[0243] The carboxylate buffering agent may be a succinate, citrate, fumarate, tartarate, maleate or lactate, suitably sodium citrate, more suitably provided as trisodium citrate dihydrate, carboxylate buffering agent (e.g., sodium citrate) may be at concentrations between 1-50, 1- 40, 5-50, 5-40, 10-50, 10-40, 10-30, 15-50, 15-40, 15-35, 15-30, 20-50, 20-40, 20-35, 20-30, 21-29, 22-28, 23-27, 24-26 mM, or around 25 mM, in the lyophilized or liquid frozen formulation. The carboxylate buffering agent may help to inhibit aggregation.

[0244] The magnesium chloride (MgCh) may be provided as magnesium chloride hexahydrate. The MgCh may be at concentrations between 1-15, 1-12, 1-10, 2-9, 3-8, 3-7, 4-6, 4.5-5.5 mM, or around 5mM, in the lyophilized or liquid frozen formulation. The magnesium chloride may stabilize RNA structure of the viral particles and exhibit a positive effect on infectivity after storage.

[0245] The D-sorbitol may be at concentrations between 0.01-5%, 0.01-3%, 0.01-2%, 0.01-1%, 0.1- 5%, 0.1-3%, 0.1-2%, 0.1-1%, 0.1-0.9%, 0.2-0.8%, 0.3-0.7%, 0.4-0.6%, 0.45-0.55% w / v, or Valneva Austria GmbH PAT070-PCT around 0.5% w / v, in the lyophilized or liquid frozen formulation. D-sorbitol can result in retention of protein native structure and improved stability.

[0246] The amino acid may be L-methionine, arginine or glutamine, suitably L-methionine. The amino acid (e.g., L-methionine) may be at concentrations between 1-20, 1-15, 3-20, 3-15, 5-20, 5- 15, 7-13, 8-12, 9-11, 9.5-10.5 mM, or around 10 mM, in the lyophilized or liquid frozen formulation. The amino acids may improve the stability of the lyophilized vaccine.

[0247] The human albumin (HA) may be recombinant human albumin (rHA). The HA may be at concentrations between 0.001 to 2, 0.001 to 1.5, 0.001 to 1 , 0.005 to 2%, 0.005 to 1.5%, 0.005 to 1%, 0.005 to 0.5%, 0.005 to 0.02%, 0.005 to 0.015%, 0.09-1%, 0.09-0.5%, 0.09-0.1% w / v, or around 0.01% w / v, in the lyophilized or liquid frozen formulation. The HAS may be at concentrations between 0.001-1, 0.01-1, 0.01-0.5, 0.05-1 , 0.05-0.5, 0.05-0.2, 0.05-0.15, 0.08- 0.12 mg / mL, or around or exactly 0.1 mg / mL, in the lyophilized or liquid frozen formulation.

[0248] The pH of the lyophilized or liquid frozen formulation may be between pH 7-8, pH 7.0-7.5, pH 7.0-7.8, pH 7.1-7.5, pH 7.2-7.4, pH 7.25-7.35, or around pH 7.3 + / - 0.2, or around pH 7.3 + / - 0.1.

[0249] The diluent may be tissue culture medium, saline or water, such as water for injection (WFI) or milliQ water, preferably WFI. The amount of diluent used to reconstitute the lyophilized cake may be from 0.25 to 2.5 times the volume of the formulation before lyophilization, or 0.5 to 1.75 times, 0.75 to 1.25 times; however, there is a clear preference for reconstituting the lyophilized cake to about the same volume as before lyophilization.

[0250] In some embodiments, the lyophilized or liquid frozen formulation may comprise or consist of viral particles (e.g., YFV, or YFV and CHIKV, described herein), sucrose, potassium phosphate, sodium citrate, MgCh, D-sorbitol, L-methionine, and human albumin (HA) in water, suitably wherein the formulation comprises or consists of viral particles (e.g., YFV, or YFV and CHIKV, described herein), sucrose at 5% w / v, potassium phosphate at 5mM, sodium citrate at 25 mM, MgCh at 5 mM, D-sorbitol at 0.5% w / v, L-methionine at 10 mM, and human albumin (HA) at 0.1 mg / mL, in water, suitably at pH 7.3. These values may deviate by + / - 10%.

[0251] In some embodiments, the lyophilized or liquid frozen formulation may comprise or consist of more than one effective dose (e.g., two or more effective doses) of viral particles (e.g., YFV, or YFV and CHIKV), sucrose, potassium phosphate, sodium citrate, MgCh, D-sorbitol, L- methionine, and human albumin (HA) in water, suitably wherein the formulation comprises or consists of more than one effective dose (e.g., two or more effective doses) of viral particles (e.g., YFV, or YFV and CHIKV, described herein), sucrose at 5% w / v, potassium phosphate at Valneva Austria GmbH PAT070-PCT

[0252] 5mM, sodium citrate at 25 mM, MgChat 5 mM, D-sorbitol at 0.5% w / v, L-methionine at 10 mM, and human albumin (HA) at 0.1 mg / mL, in water, suitably at pH 7.3. These values may deviate by + / - 10%.

[0253] Any general method of lyophilization known in the art may be used. As a general example of a lyophilization process, freezing may be performed at -40°C, followed by primary drying in 2 steps (e.g., -35°C for 3 hours, followed by -25°C for 30 hours, both at 100 bar measured by Pirani). Secondary drying may be performed at 20°C followed by a second step at 25°C (both at 70 bar vacuum). Particularly preferred is a lyophilization method providing an adequate cake structure, i.e. , a cake without cracks and with minimal shrinkage.

[0254] A unit dose of the liquid frozen or lyophilized formulation may comprise between 102TCIDso / dose to 106TCID50 / dose, 102TCID50 / dose to 105TCID50 / dose, 102TCID50 / dose to 104TCIDso / dose, or between 103TCIDso / dose to 104TCIDso / dose. The dose may be in 0.5mL. The unit dose of the liquid frozen or lyophilized formulation may comprise between 1 to 20, 1 to 15, 1 to 10, 3 to 20, 3 to 15, 3 to 10, 3 to 9, 4 to 15, 4 to 10, 4 to 8, 5 to 15, 5 to 7, 5.5 to 6.5 or around 6 log TCIDso per mL.

[0255] The total number of YFV or CHIKV particles in the formulation may be determined using standard techniques, e.g. reverse transcription quantitative polymerase chain reaction (RT- qPCR). Amplification of viral sequences may be performed.

[0256] The lyophilized formulation may be provided as a kit with a diluent. The kit may include instructions. The kit may further comprise one or more additional formulations, such as one or more further formulations for use as a vaccine against one or more other viruses. The further formulation may be for use as a vaccine against CHIKV, JEV, Dengue virus, or Zika virus infections.

[0257] The liquid frozen formulation may be provided in pre-filled syringes. This form ensures ease of administration and precise dosing, while maintaining the stability and efficacy of the vaccine. The liquid frozen pre-filled syringes are designed to be stored at low temperatures to preserve the live, attenuated viruses and other components until use for at least 12 months and longer, preferably for at least 6, 12, 24 or 48 months.

[0258] The liquid frozen formulation may be provided in a pre-filled syringe assembly featuring crosslinked silicone, ensuring the exclusion of extraneous components and to withstand impacts during transportation, particularly under low-temperature conditions. The pre-filled syringe should not crack, deform, or break even when exposed to rough handling or extreme temperatures during shipping. Additionally, the pre-filled syringe assembly includes a barrier Valneva Austria GmbH PAT070-PCT layer that prevents the diffusion of water vapor or oxygen into the formulation during freezing and thawing cycles. This barrier is critical for maintaining the stability of the liquid frozen vaccine formulation, ensuring that the vaccine does not degrade or lose its potency due to exposure to moisture or air. The pre-filled syringe assembly is designed to be compatible with automated syringe filling equipment, ensuring that the syringe assembly can be filled in a sterile environment, maintaining the vaccine’s sterility and quality throughout the production process.

[0259] TCID50 is the measure to indicate the level of replication competent infectious virus from culture supernatant. This endpoint dilution assay quantifies the amount of virus required to infect 50% of tissue culture cells through the detection of cytopathic effect by microscopic evaluation or by use of a cell dye to detect cell health. Methods of performing TCID50 assays are known in the art. Briefly, cells can be seeded in microplates and infected with 10-fold serially diluted virus samples in media. After a one-week incubation, virus-induced cytopathic effects can be assessed and viral titers calculated according to the Reed and Muench method (Reed, L.J. and Muench, H. A simple method of estimating fifty percent endpoints (1938) The American Journal of Hygiene 27:493-497).

[0260] Multidose

[0261] There is also provided a multi-dose lyophilized formulation of YFV, as well as liquid formulations reconstituted from such lyophilized compositions. There is also provided a multidose formulation of YFV and CHIKV, as well as liquid formulations reconstituted from such lyophilized compositions.

[0262] The formulation may be lyophilized from a single dose formulation (i.e., with the same volume and excipient composition), but when reconstituted, can provide more than one effective dose. The main changes for a multidose formulation are that a higher amount of YFV or CHIKV particles are added to the same amount of excipients used for one dose. A higher volume of diluent is then used for reconstitution to achieve multiple doses.

[0263] The lyophilized formulation may be for multidose administration, comprising a) two or more effective doses of a YFV, or YFV and CHIKV; b) sucrose at a concentration of 3-10% (w / v); c) potassium phosphate at a concentration of 3-10 mM; d) sodium citrate at a concentration of 15-50 mM; e) MgCh at a concentration of 3-10 mM; f) D-sorbitol at a concentration of 0.3- 1.0% (w / v), g) L-methionine at a concentration of 3-20 mM; and h) recombinant human albumin at a concentration of 0.003-0.02% (w / v), and wherein the pH of the formulation is Valneva Austria GmbH PAT070-PCT

[0264] 7.3±0.2; optionally wherein the multidose pharmaceutical composition is present in an individual vial that allows the withdrawal of two or more effective doses after reconstitution.

[0265] The lyophilized formulation may be lyophilized from a liquid formulation comprising i) more than one effective dose (e.g. two or more effective doses) of a YFV, or YFV and CHIKV, viral particles, ii) 1 .02 mg trisodium citrate dihydrate, iii) 0.09 mg di-potassium hydrogen phosphate, iv) 6.94 mg sucrose, v) 0.14 mg magnesium chloride hexahydrate, vi) 0.69 mg D-sorbitol, vii) 0.21 mg L-methionine, and viii) 0.14 mg recombinant human albumin (rHA), wherein the pH of the formulation is 7.3±0.2, and wherein the total volume of the formulation per individual vial is 0.5 mL. In case of a higher or lower total volume of the formulation per individual vial, the amount of excipients may be increased or reduced accordingly.

[0266] The total number of YFV or CHIKV particles in an effective dose may comprise or consist of at least 6.0 log™ particles / dose, e.g. 6.0 to 9.0, 6.0 to 8.0 or 6.0 to 7.0 logic CHIKV-A5nsP3 particles. In some embodiments, an effective dose comprises 6.1 to 6.7, 6.2 to 6.6, 6.3 to 6.5, or about 6.4 log™ total YFV or CHIKV particles.

[0267] Methods of preventing infection

[0268] The formulation may increase serum antibody titers in a vaccinated human subject by at least 1 log, relative to a control, within about 5 to 28 days. The formulation may increase serum antibody titers in a vaccinated human subject by at least 1 log, relative to a control, within about 14, or 7 days. The control may be pre-immune sera from the same human subject or sera from a placebo-treated or non-vaccinated subject or subjects.

[0269] The formulation may stimulate seroconversion in at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, up to 100%, of vaccinated subjects within 14 or 7 days of a single vaccination. Seroconversion may be defined as reaching a virus-specific antibody titer, i.e. , a neutralizing antibody titer, of at least 10. The neutralization of the virus may be assessed in an in vitro assay, such as a TCIDso assay and / or a neutralizing assay such as a PRNT, i.e. PRNT50 or microtiter assay, i.e., pNTso wherein a range of serum dilutions are tested for neutralization of virus growth and calculating the dilution that neutralizes 50% of growth compared with a negative control. A PRNT50 assay may give a readout in countable plaques and a pNTso assay may give a colorimetric readout which is proportional to cytopathic effect. The 50% reduction of virus growth in a TCID50 assay (or a PRNT50 or pNTso assay) by a 1 :10 or 1 :20, preferably 1 :20, or higher dilution of immune sera may be defined as seroconversion. Valneva Austria GmbH PAT070-PCT

[0270] The value may be reported as the reciprocal of the dilution factor, e.g., 50% virus neutralization at a 1 :10 immune serum dilution is referred to as a neutralizing titer of 10, or 50% virus neutralization at a 1 :20 immune serum dilution is referred to as a neutralizing titer of 20. Neutralizing titer values of 10 or higher may be defined as seroconversion, with 10 being the minimum and 20 or higher being preferred. A threshold of neutralizing antibodies of 1 :10 dilution of serum in a PRNT50 is generally accepted as evidence of protection (Rombach et. al. Vaccine (2005) 23:5205-5211).

[0271] The formulation may confer a protective immune response against at least YFV infection and / or CHIKV infection or YFV infection and a further viral infection when using a combined vaccine (e.g., against YFV and CHIKV). Protective response is expected against all strains of the virus vaccinated against, i.e. , all YFV strains. It is expected that the protective immune response is long-lasting. The formulation may confer lifelong protection. Alternatively, the formulation may confer a protective immune response for at least 6, 12, 24 or 48 months, or at least 5, 10, 15, 20 years. A protective immune response is an immune response in which neutralizing antibodies are produced which are sufficient for reducing or preventing signs or symptoms of the viral infection (i.e., YFV and / or CHIKV).

[0272] The formulations described herein may be suitable for use in a method of treating or preventing a YFV and / or CHIKV infection. In the combined formulation, the formulation may be used in a method of treating or preventing a YFV infection and a further infection. In a YFV / CHIKV combined formulation, the formulation may be used in a method of treating or preventing a YFV and a CHIKV infection. The method may include administering an effective amount of the formulation to a subject in need thereof. Where using lyophilized formulations, the formulation may be reconstituted in a diluent as described herein.

[0273] The formulations described herein may be for use in preventing a YFV and / or CHIKV infection when using a combined formulation, in a subject in need thereof. The formulations described herein may also be for use in vaccinating a human subject and stimulating a protective immune response in said subject against YFV and / or CHIKV in the combined formulation.

[0274] The formulations described herein may be used in the manufacture of a medicament for use in preventing a YFV and / or CHIKV infection when using a combined formulation, in a subject in need thereof.

[0275] A subject in need of vaccination against YFV and / or CHIKV, can be any mammal, including humans (infants, children, adolescents and adults) in danger of exposure to the virus, such as Valneva Austria GmbH PAT070-PCT a traveller to an endemic or outbreak country or an inhabitant of an endemic or outbreak country or a country in danger of an outbreak.

[0276] The formulation may be administered by the intravenous, intradermal, intracutaneous, intraarticular, intraperitoneal, intrathecal, intramuscular or subcutaneous route, suitably intramuscular or subcutaneous routes.

[0277] The formulation may be administered once. Therefore, the formulation may be used as a one- shot vaccine. Alternatively, the formulation may be administered on repeat occasions. For example, 7 days, 14 days, 1 month, 3 months, 6 months, 1 , 2, 3, 5 or 10 years after the first administration.

[0278] EXAMPLES

[0279] Example 1 - Preparation of YFV plasmids and virus bank

[0280] YFV plasmid production

[0281] To construct a plasmid with YFV full length genome, the starting material was the pVVS01828 (Figure 2A) comprising a YFV ASLE genome is flanked by a SP6 promoter at the 5’ terminus and an Afll I run-off restriction site at the 3’ terminus. The backbone of this plasmid is pACNR (Silva PA, et al., 2010).

[0282] Segments of the YFV ASLE genome that were different to YFV sequence (SEQ ID NO: 1) were replaced. This involved the fragment from 4000bp to 10600 bp of the YFV sequence, of which was obtained by synthesis from GeneArt and split into 3 fragments; FGT2, FGT3, FGT4 (Figure 2B) in 3 separate plasmids. Three mutations were introduced; A5614G, A10243G, and A10722G to mimic current YFV vaccine strains (Barban et al., 2007). The construction of the recombinant YFV genome was therefore performed in 3 steps;

[0283] Step 1), the FGT4 fragment was inserted into pVVS01828 between the Zral and Xbal site (Figure 2B).

[0284] Step 2), The concatenation of FGT2 and FGT3 in a plasmid from Mlul to Sail restriction site (Figure 2B). This was performed in parallel to step 1.

[0285] Step 3), The substitution of the FGT2 / FGT3 concatenated sequence into the recombinant produced by step 1. Valneva Austria GmbH PAT070-PCT

[0286] The result of the step 3 was a recombinant YFV complete genome sequence flanked by a SP6 promoter at the 5’ terminus and a run-off restriction site at the 3’ terminus in a pACRN backbone (referred to as pVVS02030 plasmid, Figure 2C).

[0287] The pVVS02030 plasmid was amplified with lllustra TempliPhi D NA Amplification kit, purified and then linearized by Aflll to be used for in vitro transcription and cell transfection.

[0288] YFV viral rescue and generation of virus banks

[0289] In vitro transcription was performed using 1 pg of linearized pVVS01828 plasmid using MEGAscript SP6 transcription kit. The single band for genomic DNA was used to transfect EB66 and Vero cells; 2 and 4 pg RNA for Vero cells, and 10 pg for EB66. Virus titration for both rescues confirmed that YFV was rescued in both cell lines with titers around 8.5 Log TCIDso / mL, with only a mild cytopathic effect 5 days post infection.

[0290] As a positive control for the transfection and virus rescue, we used YFV ASLE plasmid and virus, respectively, using the same steps described for the recombinant YFV.

[0291] YFV virus banks were then produced on vero cells using either serum-free culture medium (OptiPRO SFM) or Minimum essential medium (MEM) supplemented with FBS. Virus material was harvested, filtered and stabilized in 10% w / w sucrose / 25mM TRIS, pH 7.3 and stored at -70 °C.

[0292] Mutations

[0293] YFV RNA was extracted using QIAmp virus RNA mini kit (Qiagen) following the manufacturer protocol. Next generation sequencing (NGS) was performed by GATC Genomics (Eurofins). Briefly, strand specific cDNA was synthesised. The library was then prepared using NEBNext Ultra II DNA Library Kit (NEB) and sequenced on a NovaSeq 6000 S4 instrument in paired- end mode with 150 bp read length.

[0294] The generated fastq-files were quality checked with FASTQC (version 0.11.9), and raw reads were quality trimmed using CUTADAPT (version 3.5, Martin, 2011) by removing base cycles. The quality trimmed reads were mapped to the reference genome (YFV strain 17D-204, NCBI accession number X15062.1) using BOWTIE2 (version 2.3.5.1 , Langmead & Salzberg, 2012). The computed alignments were coordinate sorted and indexed with SAMTOOLS (version 1.14, Danecek et al., 2021). The reads were then re-aligned using a Viterbi algorithm (LOFREQ VITERBI, version 2.1.5, Wilm et al., 2012) and indel quality scores were inserted using the Dindel-algorithm (LOFREQ INDELQUAL DINDEL, Albers et al., 2011). After sorting and indexing, alignments were visually checked with the Integrated Genomics Viewer (IGV, Valneva Austria GmbH PAT070-PCT version 2.10.0). Variants and indels were then called using LOFREQ CALL-INDELS. Next, base quality scores were recalibrated using GATK BASERECALIBRATOR followed by GATK APPLYBQSR implemented in the GATK4 (version 4.2.1.) program suite (https: / / gatk.broadinstitute.org / hc / en-us) whereby the output of the first variant call round served as input to mark known polymorphic sites. After base recalibration, indel quality scores were re-inserted as before (LOFREQ INDELQUAL DINDEL). After sorting the alignments with SAMTOOLS, variants were called for a second time as above (LOFREQ CALL-INDELS), annotated with SNPEFF (version 5.0.1 , Cingolani et al., 2012) and subsequently filtered at a 10% allele frequency level. Mapping statistics for the alignments were pulled-out with SAMTOOLS.

[0295] NGS showed no insertions or deletions in the recombinant YFV particle genome. Coverage analysis with SAMTOOLS confirmed this finding. Variant calling and annotation with LOFREQ and SNPEFF revealed that in the whole YFV genome a total of seven single nucleotide polymorphisms (SNP) occur that pass the 10% prevalence threshold (see Table 1).

[0296] Table 1. Variants detected by LOFREQ passing the 10 % prevalence threshold.

[0297] ‘mutations in bold font were introduced into the plasmid to mimic current YFV vaccine strains.

[0298] Of the seven mutations, three (A5614G, A10243G, and A10722G) were introduced to mimic current YFV vaccine strains (Barban et al., 2007) and are present in 100% of particles. Of the remaining four mutations, C8212T is a silent SNP not leading to any amino acid substitution. A2608C and A7225G are missense SNPs (ns1 : E52D and ns4b: I113M, respectively), while the G10559GT) is a frameshift inducing-UTR of the virus genome. However, these SNPs are present in only 12% of the viral particles, and none of them are located in the viral E-protein, the major antigen of YFV and so are not expected to negatively impact immunogenicity of the YFV particles. Valneva Austria GmbH PAT070-PCT

[0299] Example 2 - Increasing yield of YFV in manufacture

[0300] Background

[0301] YFV development was initially in roller bottles. However, this was identified as potentially limiting scale-up for manufacture. We therefore attempted to provide a process for a fixed-bed bioreactor system (iCELLis - system; PALL / Cytiva) for virus production. The iCELLis bioreactor system can be scaled-up from a 0.5 to 4 m2development-scale system (iCELLis Nano) to a maximum of 500 m2manufacturing system (iCELLis 500+).

[0302] Methods

[0303] Bioreactor preparation

[0304] For the development of a fixed-bed bioreactor process, we used the iCELLis Nano system equipped with a 4 m2cell growth area bioreactor (100 mm fixed-bed height, 0.2 L fixed-bed volume, high compaction (C1.5)). iCELLis Nano bioreactors, mPath controllers and iCELLis Nano docking stations and bioreactor probes (temperature, pH (AppliSens sensor PH+ L=120mm [ref# Z001012051]), DO (optical DO sensor Hamilton VisiFerm DO ECS 120 H2 [ref# 243666-212]), biomass) were assembled, prepared and calibrated according to manufacturer instructions with iCELLis Nano stainless steel lid and octopus tubing (PALL “lid tubing” manifold) attached). All connections to and from the iCELLis Nano systems were done aseptically inside of a class II biological safety cabinet. Preparation included autoclaving of the assembled bioreactor at 121 °C for 1 hour. An inoculation bottle (PALL “sampling manifold 1 L”) was aseptically connected.

[0305] Complete cell culture growth medium, (media (Opti or VP) including additional 4mM L- Glutamine, 6g Glucose, 3.7g Sodium Bicarbonate, (700 mL) was subsequently added to the bioreactor and equilibrated overnight (temperature regulation, gas supply and bioreactor stirrer were turned on; temperature SP: 37.0 °C, CO2 flow SP: 20 mL / min, AIR flow SP: 180 mL / min, stirrer rpm SP: 1120 rpm (corresponding to 2 cm / sec linear speed of medium through fixed-bed)).

[0306] The next morning a sample was taken from the bioreactor and the pH-sensor recalibrated (if necessary) following a pH measurement on a Nova Biomedical pHOx analyzer, the AIR flow was switched to 30 mL / min in the gas overlay and the DO-sensor recalibrated to 90 % air saturation. In addition, the pH regulation (set point pH = 7.40) was started.

[0307] Transfer of host cells to bioreactor and host cell expansion Valneva Austria GmbH PAT070-PCT

[0308] When temperature and pH were stable at their set points, vero cells were passaged and counted, and tested for cell viability (including the presence of cell (%) of cells in aggregates with five or more cells ) using a NucleoCounter NC-200 cell counter. Agitation was turned off prior transferring cells to the bioreactor to prevent air bubbles being introduced into the bioreactor fixed-bed.

[0309] Vero cells were transferred to the iCELLis Nano bioreactor system (bioreactor inoculation) at a seeding density of 3.0x103cells / cm2and cultured in serum- and animal / human origin- free media. Two serum- and animal / human origin- free media were tested for cell expansion; OptiPRO SFM and VP-SFM, which were supplemented to final concentrations of 6g / L Glucose, 3.7 g / L Sodium Bicarbonate and 4 mM L-Glutamine.

[0310] For cell transfer, the required volume of cell suspension was pooled into 75 mL complete cell culture growth medium in the inoculation bottle and the cells were transferred into the bioreactor by applying gentle air pressure to the inoculation bottle. The inoculation bottle and the inoculation line were subsequently rinsed with 75 mL cell culture growth medium and bioreactor agitation was turned on using a stirrer speed corresponding to 2 cm / sec linear speed through the fixed-bed. After around 3 hours (range: 2.5-3.5 hours) of cell attachment to the PET (polyethylene terephthalate) macrocarrier fixed-bed, the proportion of cells attached to the fixed-bed was checked by taking a sample from the bioreactor and determining the concentration of cells that had not attached yet by performing a cell count on the NC-200 cell counter. Agitation speed was subsequently reduced, corresponding to a linear speed of medium through the fixed-bed of 1 cm / sec.

[0311] Recirculation of medium between the bioreactor and the recirculation bottle was initiated one day after seeding (day 1 post inoculation). Briefly, recirculation rate was fixed to 4 mL / min / m2, i.e. 16 mL / min during cell expansion, whereas during the virus production phase, recirculation rate was reduced to 2 mL / min / m2, i.e. 8 mL / min. Volumes of medium in recirculation bottles / loops and thereby total system volume (VTOT) were chosen to be representative of the large- scale iCELLis 500+ manufacturing process.

[0312] Process set points were as follows: pH was maintained at 7.4, pCh at 50% air saturation and agitation speed corresponding to fluid circulation at 1 cm / sec (with the exception of cell attachment and virus infection phases, during which the linear speed of medium through the fixed- bed was adjusted to 2 cm / sec).

[0313] Virus infection and propagation phases Valneva Austria GmbH PAT070-PCT

[0314] Host cells were allowed to grow for around 6 days to a cell density between 1 and 2 x105cells / cm2.

[0315] The host cells were then infected on day 6 by adding the YFV virus bank to the bioreactor (in the media) via a top screw cap using a multiplicity of infection (MOI) of 0.03.

[0316] Two serum- and animal / human origin- free media were tested for virus infection and propagation; OptiPRO-SFM and VP-SFM, which were supplemented to final concentrations of 6g / L Glucose, 3.7 g / L Sodium Bicarbonate and 4 mM L-Glutamine. In the first experimental condition, OptiPRO-SFM was used for both cell expansion and the infection and propagation phase. In the second experimental condition, OptiPRO-SFM was used for cell expansion and switched to VP-SFM for virus infection and propagation.

[0317] Virus was incubated with bioreactor impeller speed increased to a value corresponding to a linear speed of medium through the fixed-bed of 2 cm / sec for 3 hours.

[0318] Viral harvest

[0319] The effects of applying different harvest strategies on product quality and virus yield were investigated.

[0320] Virus was harvested on days 3, 4 and 8 post infection by collecting culture media from the bioreactor.

[0321] Samples

[0322] Samples of medium containing released virus particles were obtained from the iCELLis Nano recirculation bottles daily after infection.

[0323] Cell counting

[0324] Cell counts were obtained from microcarrier strips from the fixed-bed bioreactor. Briefly, the microcarrier strips were sampled aseptically using sterile forceps and incubated in cell lysis buffer (1 mL PBS without Ca / Mg + 1 mL ChemoMetec Lysis Buffer A100) for 5 minutes at room temperature (static), followed by vortexing for 2 minutes and addition of 1 mL ChemoMetec Stabilizing Buffer B. The released nuclei were counted on a NucleoCounter NC-200 cell counter (ChemoMetec), following manufacturer protocol.

[0325] Dissolved oxygen and pH Valneva Austria GmbH PAT070-PCT

[0326] Dissolved oxygen concentration and pH were measured by sampling cell culture medium from the bioreactor and promptly analyzing the sample on a pHOx analyzer (Nova Biomedical) according to the manufacturer’s instructions.

[0327] SE-HPLC

[0328] Protamine sulfate (PS) treatment was performed by adding PS to a final concentration of 200 pg / mL to the samples. Addition of protamine sulfate precipitates host cell DNA (hcDNA) and proteins resulting in a significant reduction in turbidity and viscosity. Sample tubes were immediately inverted several times, incubated for 15 minutes at room temperature, followed by centrifugation at 3000 ref for 10 minutes. The sample supernatant was mixed with sucrose / TRIS-stabilizer (S / T) [50 % sucrose / 125 mM TRIS] buffer and transferred into an HPLC vial containing 2 mg / mL BSA stock solution (to prevent unspecific adsorption of virus particles).

[0329] A Superose 6 Increase 10 / 300 GL size exclusion chromatography column (Cytiva) was equilibrated with 1 x PBS w / o Ca / Mg containing additional 250 mM NaCI (pH range 7.2 ± 0.3) at a flow rate of 1 mL / min and operated at 25 °C on a Thermo Fisher Scientific Vanquish HPLC system running Chromeleon software v. 7.2.10.

[0330] An SE-HPLC method with UV 214 nm detection wavelength was used for the detection, characterization and quantification of YFV particles in pre-inactivation process stages (e.g. virus growth kinetics and harvest samples).

[0331] TCIDso plaque assay

[0332] Titers / concentrations of active virus in pre-inactivation samples were determined by TCID50 assay. In brief, Vero cells were seeded into 96-well plates at 2.0x104cells per well on day -1 and incubated at 35.0°C, 5% CO2. On day 0, serial dilution series of the medium samples were prepared (3 independent dilution series / replicates per sample) and 100 pL (for high titer / control samples) or 500 pL (for low titer) of each sample dilution pipetted into the corresponding well. The plates were incubated for 7-8 days at 35°C, 5% CO2. On day 8 (on some occasions on day 7) post infection plates were read out microscopically and subsequently TCI D50 titers calculated using the Reed-Muench method.

[0333] Sequencing analysis

[0334] Viral RNA was extracted using Qiagen’s QIAmp virus RNA mini kit according to manufacturer instructions and sequencing performed on cDNA as described in Example 1.

[0335] Results Valneva Austria GmbH PAT070-PCT

[0336] Effect of media and harvest time

[0337] Where OptiPRO-SFM was used for all stages of virus production (host cell expansion, infection, propagation and harvest), herein referred to as the ‘OptiPRO-all condition’, cell density at infection was 1 .6 x105cells / cm2and a MOI of 0.03 used. Where OptiPRO-SFM was used for host cell expansion and then media switched to VP-SFM for virus infection, propagation and harvest (referred to herein as ‘OptiPRO-VP-switch), cell density at infection was 1 .3 x105cells / cm2and a MOI of 0.03 used.

[0338] For both conditions, relatively low virus titers were detected on day 1 post infection, after which titers steadily increased until day 3 post infection when maximum virus titers were observed (Figure 3A). Titers slightly decreased from day 3 to day 4 post infection when the first harvest was performed (Figure 3A).

[0339] Surprisingly, the peak virus titers were actually achieved on day 3 post infection regardless of the media used, with titers of log TCIDso 7.14 for OptiPRO-all and 8.15 for OptiPRO-VP- switch. In contrast, the titers of the harvests performed on day 4 post infection were lower, namely 6.9 and 7.8 log TCID50 for OptiPRO-all and OptiPRO-VP-switch, respectively. Therefore, a higher yield can be obtained by harvesting viral particles sooner. This also avoids the costs and resources required for an additional day of virus propagation, which can escalate when manufacturing on a large scale.

[0340] With regard to media, OptiPRO-VP-switch surprisingly showed a higher titer at 3 and 4 days post infection. However, more surprising was that, where OptiPRO SFM was the propagation medium, the viral particle titer did not recover after the harvest at day 4 and instead remained stable until day 8 post infection. In contrast, where VP-SFM was used as the propagation medium, the first harvest on day 4 post infection resulted in a slight drop in virus concentration in the bioreactors (96 hpinf sample), but titers then increased until the maximum titer was reached again on day 8 post infection (second harvest). This means that a further virus harvest can be obtained when using VP-SFM for virus infection, propagation and harvest, reducing the costs and time required to set up new host cell cultures and infections. However, a late second harvest may increase host cell protein and host cell DNA which may reduce yield or increase impurities.

[0341] We then compared the effect of switching from OptiPRO-SFM for host cell expansion to VP- SFM for virus infection, propagation and harvest (‘OptiPRO-VP-switch’), compared to using VP-SFM for all stages of virus production (host cell expansion, infection, propagation and Valneva Austria GmbH PAT070-PCT harvest), referred to as ‘VP-all’. The same methods were used as in the experiments above. However, for OptiPRO-VP-switch, the VP-SFM was replaced 24 hours post infection.

[0342] For OptiPRO-VP-switch, cell density at infection was 1.4 x105cells / cm2and MOI was 0.03. For VP-all, cell density at infection was 1.6 x 105cells / cm2and MOI was 0.03. As with the conditions above, in the VP-all condition relatively low virus titers were detected on day 1 post infection, after which titers steadily increased until day 3 post infection (data not shown). A higher titer was achieved with the OptiPRO-VP-switch condition than the VP-all condition (titers of log™ TCIDso of 8.50 for OptiPRO-VP-switch and 8.25 for VP-all). The increase in titer of OptiPRO-VP-switch to 8.50 log™ TCI Dso compared to 8.15 log™ TCIDso in the previous experiment may be due to the media change post infection.

[0343] Harvest strategy

[0344] In this experiment, experiments were performed as described for the OptiPRO-VP-switch condition, with an MOI of 0.03. However, in these experiments, VP-SFM medium as replaced 21 hours post infection. This was done to ensure that levels of glucose and L-glutamine were not significantly decreased by harvest, and that the levels of lactate and ammonia were not significantly increased by harvest.

[0345] In addition, the virus harvest was either as a single harvest on day 4 (93 hours) post infection (cell density at infection 1.9 x105cells / cm2) or using a perfusion harvest between 69-93 hours post infection (cell density at infection 1.3 x105cells / cm2). The total perfusion volume was 4.35 L with a perfusion-out rate of 1 .2 mL / min.

[0346] A summary of the obtained TCIDso results is shown in Figure 3B. The number of infectious particles steadily increased over time with peak productivity on day 3 (68 hours) post infection of more than 8.4 logw TCIDso on day 3 post infection in both runs. In both runs, again, a decline was observed after day 3 post infection, and harvest at day 4 post infection was 8.1 and 8.3 logw TCIDso for single harvest and perfusion harvest, respectively (Figure 3B).

[0347] This suggests that a perfusion harvest may be advantageous in increasing YFV yield. It is hypothesised that this may be due to reducing shear stress on viral particles. However, a perfusion harvest strategy may complicate the process and increase costs.

[0348] Summary data for media and harvest time

[0349] The effect of media and harvest time on yield is summarised in Figure 3C. As can be seen from Figure 3C, virus yield peaks around 68-74 hours post infection and drops between days Valneva Austria GmbH PAT070-PCT

[0350] 74 and 94 hours post infection. This is corroborated by the fact that a significant increase in turbidity from day 3 post infection onwards was detected (Figure 3D).

[0351] A summary of virus yield of harvest at day 4 post infection is shown in Figure 3E. As can be seen from Figure 3E, the virus yield at day 4 was improved by changing media after infection, and perfusion harvest. The best yield at day 4 was obtained using the OptiPRO-VP-switch media followed by the VP-all media and then OptiPRO-all media.

[0352] Mutations

[0353] Sequencing confirmed that seven mutations were identified in the viral particles produced using the processes discussed above (see Tables 2 and 2a). These seven mutations are those identified in Example 1. Additional mutations were not detected.

[0354] Table 2. Variants detected by LOFREQ passing the 10% prevalence threshold.

[0355] ‘mutations in bold font were introduced into the plasmid to mimic current YFV vaccine strains.

[0356] Table 2a. Variants detected by LOFREQ passing the 5% (0.05) prevalence threshold. Time in left column refers to time of sample taken for sequencing. WVB = working virus bank, VSB = virus seed bank, h = hours, pi = post infection, OptiPro-VP-switch + media change at 24h post infection - sample taken at 91 h PI, mutations in bold were introduced into the plasmid to mimic current YFV vaccine strains; pos.: nucleotide position in genome; ref.: nucleotide in reference sequence; alt.: detected alternative nucleotide; n.d.: not detected I below 5% (0.05) threshold. Valneva Austria GmbH PAT070-PCT

[0357] *mutations in bold font were introduced into the plasmid to mimic current YFV vaccine strains.

[0358] This data confirms that the frequency of mutations did not significantly change over time, nor as a result of the experimental conditions. Importantly, the E protein is preserved, and attenuation of the virus is not affected by the process.

[0359] Discussion

[0360] The yields discussed in this example are surprising as, when previously attempting to produce YFV in roller bottles, it was noted that a lower YFV yield was obtained using reduced-serum medium. In particular, a yield of 1.45 E+07 TCID50 per cm2cell growth surface area was obtained when using MEM media supplemented with 3% v / v FBS and 2 mM L-glutamine. In contrast, the yield decreased to 2.67 E+06 TCID50 per cm2was obtained when MEM was supplemented with 0.03% FBS and 2 mM L-glutamine.

[0361] Indeed, the data above indicate that OptiPRO-VP switch condition is preferred for obtaining the highest YFV yield from fixed-bed bioreactors and vero cells. The data shows that a higher YFV yield can surprisingly be obtained at 3 days post infection. Harvesting at day 3 may Valneva Austria GmbH PAT070-PCT increase the quality of the harvest (e.g., by reducing the amount of process-related impurities) compared to harvest at day 4. An earlier harvest would also be associated with reduced production costs.

[0362] The data also shows that a single harvest is preferred for increased yield and quality, although a dual harvest strategy may be utilised to further increase yield.

[0363] A perfusion harvest may be advantageous in increasing YFV yield. It is hypothesised that this may be due to reducing shear stress on viral particles. However, a perfusion harvest strategy may complicate the process and increase costs.

[0364] In addition, our data shows that virus yield may be increased by changing the media one day post infection.

[0365] The data supports that the process producing an increased YFV yield does not result in additional mutations, nor a significant change in the prevalence of mutations in the YFV particles. Importantly, the E protein is preserved, and attenuation of the virus is not affected by the process. Therefore, immunogenicity and safety of the produced YFV is not affected by the processes described here.

[0366] In conclusion, the serum-free fixed bed bioreactor YFV process can increase the total specific productivity per growth surface area by 1 .4- and 7.5- fold compared to the FBS-supplemented and serum free media containing roller bottle YFV processes, respectively.

[0367] Based on these results obtained in the scale-down 4 m2development bioreactor, the total batch-yield for a 500 m2manufacturing-scale bioreactor process is expected to be approximately 14 log TCIDso (pre DSP and formulation / lyophilization losses).

[0368] Alternative MOIs and cell densities may be used to further increase the yield.

[0369] Example 4 - Downstream processing of YFV

[0370] Background

[0371] Using YFV obtained as described in Example 3 in the OptiPRO-VP-switch + media change 24h PI condition was harvested and tested for primary recovery and downstream purification. YFV obtained from the VP-all condition described in Example 3 was harvested and tested for primary recovery only.

[0372] In brief, primary recovery consists of host cell (hcDNA) reduction by precipitation with Protamine Sulphate in the crude harvest. Precipitated hcDNA is removed together with cell Valneva Austria GmbH PAT070-PCT debris by clarification using a membrane filter. Clarified harvest is then concentrated by an ultrafiltration step (UF1). At this stage, the harvest pool may be stabilised and stored at -65 °C.

[0373] The next stage is downstream purification of the concentrated harvest material is performed by a flowthrough chromatography step using the multi-modal Capto™Core 400 (CC400) or Capto™Core (CC700) chromatography resin. CC400 or CC700 pool is then again concentrated by a final ultrafiltration step (UF2). The final product then undergoes buffer exchange by diafiltration to remove residual media components. A final 0.2 pm filtration is performed to prepare the drug substance (DS).

[0374] As the YFV virus is attenuated, viral inactivation is not part of the downstream process.

[0375] An overview of the overall downstream process is shown in Figure 4.

[0376] Methods

[0377] Protamine sulphate precipitation (primary recovery)

[0378] Primary recovery in this process consists of host cell (hcDNA) reduction by precipitation with Protamine Sulphate in the crude harvest. In brief, protamine sulphate is added directly to the crude harvest at a final concentration of 0.2 mg / mL and the mixture agitated for around 2 minutes. The mixture was then incubated at room temperature for 30 to 45 minutes. Precipitation was performed inside a biosafety cabinet.

[0379] Clarification (primary recovery)

[0380] Insolubles (cell debris and precipitated hcDNA) were removed from the crude harvest by clarification using Sartopore 2 membrane filter capsules (Sartorius Sartopore 2, 5445306G9- -S-A, 0.8 / 0.45 pm, 2000 cm2filter area). Clarified harvest was collected in a 5 L Flexboy bag. Clarification was performed inside a biosafety cabinet.

[0381] Concentration (primary recovery)

[0382] The YFV purification process includes a concentration of the Clarified Harvest (UF1). UF1 was performed using two Pall Centramate 100 kDa membrane modules of 1000 cm2membrane area resulting in 2000 cm2total membrane area.

[0383] In brief, membrane modules were installed in the holders and flushed with MilliQ water according to the manufacturer instructions. After the flush the modules were stored in 0.1 M NaOH until use. Prior to a run the storage solution was flushed out with 2 L MilliQ water Valneva Austria GmbH PAT070-PCT followed by equilibration with 1 L of 25 mM Tris / 125 mM NaCI, pH 7.5. Post run, the membranes were first flushed with 1 L 1 M NaCI solution followed by sanitation using 0.5 M NaOH. First, 500 mL NaOH were flushed through the system followed by recirculation for 30 minutes. After recirculation the system was flushed with another 500 mL 0.5 M NaOH followed by 1 L MilliQ water. Finally, the system was stored in 0.1 M NaOH until the next run.

[0384] Concentration was then performed using a feed flowrate of 400 mL / min. During concentration the transmembrane pressure (TMP) was kept in the range of 0.2-0.4 bar. If necessary TMP was adjusted by restricting the retentate line of the system. After concentration a wash of approximately one system dead volume was performed with 25 mM Tris / 125 mM NaCI, pH 7.5.

[0385] Stabilising and storage (primary recovery)

[0386] Concentrated harvest material was stabilized for freezing by addition of Sucrose (final sucrose concentration 10% w / w using a 50% w / w stock dissolved in 25 mM Tris pH 7.4).

[0387] Stabilized concentrated harvest was frozen at -65 °C.

[0388] Flowthrough chromatography (downstream purification)

[0389] Further purification of the concentrated harvest material is performed by a flowthrough chromatography step using the multi-modal Capto™Core 400 (CC400) chromatography resin following manufacturer protocol. In brief, CC400 slurry (approx. 80 % slurry in 20 % ethanol) was washed five times with 400 mM NaCI / 20 % ethanol. A 50 % slurry in 400 mM NaCI / 20% ethanol was prepared and approximately 43 mL dispensed into a Omnifit 10x250 column housing. The column plug was inserted, and the column packed at 300 cm / h. The plug was then lowered approximately 3 mm into the packed bed. The resulting bed height and column volume was 250 mm and 20 mL respectively. The column was sanitized with 2 CV of 1 M NaOH and then stored in 20% ethanol until use. Prior to equilibration the CC400 column was flushed with MilliQ water for at least 5 CV at a flowrate of 150 cm / h followed by a preequilibration with 250 mM NaPO4 1 250 mM NaCI, pH 7.5. Final equilibration of the column was performed with DPBS for 5 CV.

[0390] Chromatography was then performed on an AKTA Pure 25 system. The chromatography method consisted of 2 CV equilibration with DPBS via line A1 at 150 cm / h followed by loading of the CC400 Load via A2 at a flowrate of 150 cm / h. After an absorbance increase to 20 mAU (UV280) collection of the flowthrough (CC400 Pool) was started by switching to Outlet 2. CC400 Pool was collected in a PETG bottle containing rHA. The amount of rHA added to the Valneva Austria GmbH PAT070-PCT collection bottle was calculated prior to the run based on the expected final CC400 Pool volume (final rHA concentration target 50 pg / mL). Completion of the load step was detected by the system via the air sensor of pump block A. After air was detected in Line A2, the system switched to line B1 to perform a post load wash step with DPBS. Collection of the CC400 Pool was stopped by switching to Outlet 1 after UV280 absorbance decreased to 20 mAU at the descending edge of the flowthrough peak. The wash step was continued for an additional 1 CV until end of the method.

[0391] Sanitation and CIP of the column was performed after the method finished. After completion of the run, the column was flushed with CaptoCore CIP solution (30% Isopropanol, 1 M NaOH) for 2 CV at 150 cm / h at reverse flow followed by a hold time of 60 minutes. After the hold time the CIP solution was flushed out using MilliQ water for 5 CV at a flowrate of 150 cm / h. The column was then stored in 20 % EtOH.

[0392] Concentration / diafiltration (downstream purification)

[0393] Concentration / diafiltration of the CC400 Pool (UF2) was performed as a simple concentration step using Sartorius Vivaspin centrifugal concentrator tubes of 100 kDa cutoff following manufacturer instructions.

[0394] Preparation of drug substance (downstream purification)

[0395] The final product then undergoes buffer exchange by diafiltration (explained above) to remove residual media components. A final 0.2 pm filtration is performed to prepare the drug substance (DS).

[0396] Sample preparation

[0397] During primary recovery, samples were drawn after clarification and after concentration and stabilization (UF1). The sample drawn after clarification was first stabilized for freezing by addition of 125 mM TRIS I 50% Sucrose sample stabilization buffer (4 parts sample, 1 part stabilization buffer). Sample aliquots for TCIDso and SEC analysis were prepared from the stabilized sample. TCID50 sample aliquots were prepared in triplicate in cryovials. For SE- HPLC analysis 300 pL stabilized sample were transferred to a HPLC vial containing 7.5 pL of a 2 mg / mL BSA stock solution. All samples were stored frozen at -65 °C until analysis.

[0398] During DSP samples were drawn from CC400 Load, CC400 pool and concentrated CC400 pool. CC400 Load samples for SE-HPLC were prepared by transferring 300 pL sample were to HPLC vials containing 7.5 pL of 2 mg / mL BSA. CC400 pool and concentrated CC400 pool Valneva Austria GmbH PAT070-PCT

[0399] I PC samples did not require additional sample preparation as they already contained 25 mM TRIS / 10% Sucrose and 50 pg / mL rHA.

[0400] SE-HPLC and TCI Dso were performed as described in Example 1.

[0401] SEC analysis

[0402] Brief description

[0403] A SE-HPLC (size exclusion high performance liquid chromatography) method is used for the detection and characterization of viral particles in various process samples (e.g. harvest, CC400 load and CC400 pol etc) and purified Durg Substance samples utilizing a Superose 6 Increase 10 / 300 SEC column coupled to a Dionex Ultimate 3000 HPLC system. The column is equilibrated with PBS + 250 mM NaCI, pH 7.4 at a flow rate of 1 mL / min and operated at 25°C. UV monitoring is done at 214 nm detection wavelength.

[0404] The coupled online MALS (multi angle light scatter) analysis is used for continuative characterization of viral particles. MALS enables the determination of the radius of gyration (Rz), rms radius (root mean square radius) and Cumulative Number Distribution and Differential Weight Fraction analyses of particles eluting from the SEC column. This method is used to characterize viral particles and to demonstrate viral integrity of viral particles (e.g. monomer vs. aggregated particles). Like other flaviviruses, the YF particles can be described as uniform in size and shape with diameters expected in the range of approximately 40nm.

[0405] Results

[0406] SE-HPLC

[0407] Step yield for the CaptoCore 400 chromatography process step (primary recovery) was determined as 84% according to SE-HPLC (see Table 3).

[0408] Table 3. Overview of CC400 chromatography yield Valneva Austria GmbH PAT070-PCT

[0409] Final volume of the UF2 pool was 70 mL (2 fold concentration compared to the CC400 pool). Step yield for the UF2 process step was determined as 77.5 % according to SE-HPLC (see Table 4).

[0410] Table 4. Overview of UF2 yield

[0411] Analytical SEC was used to assess the relative amount of total virus particles and aggregation status. Figure 5 depicts integration of the peaks for YFV, rHA / BSA and low MW impurities on the chromatogram.

[0412] Results of SE-HPLC analysis as well as yield calculations are summarized in Table 5. SE- HPLC of YFV particles from OptiPRO-VP-switch + media change 24h PI condition shows a high virus particle recovery of >70 % until UF2 pool. Aggregates were effectively reduced from 12% in the Clarified Harvest to 5% in the UF2 pool. Surprisingly, YFV from the VP-all condition showed a higher presence of aggregates (-20%) compared to corresponding samples of YFV from the OptiPRO-VP-switch + media change 24h PI condition.

[0413] Table 5. SE-HPLC results of YFV downstream processing

[0414] *#23-0050 is the OptiPRO-VP-switch + media change 24h PI condition and #23-0051 is the VP-all condition.

[0415] TCID50 Results Valneva Austria GmbH PAT070-PCT

[0416] Infectious virus titer was determined by TCID50 assay. A summary of the TCID50 results for samples drawn during Primary recovery and downstream purification is shown in Table 6. Process yields for recovery and downstream purification were calculated based on TCID50 results and are shown in Figure 6A. Note that the variability of the TCID50 assay is approximately ± 0.5 logw TCID50. A comparison of the total infectious titers for the process steps of primary recovery and downstream processing showed that the number of infectious particles remains stable throughout these process steps (Figure 6B). The final titer of concentrated purified bulk from OptiPRO-VP-switch + media change 24h PI condition was 9.5 logw TCIDso / mL.

[0417] Table 6. Summary of TCID50 results

[0418] *#23-0050 is the OptiPRO-VP-switch + media change 24h PI condition and #23-0051 is the

[0419] VP-all condition.

[0420] Discussion

[0421] Analysis of the IPC samples showed a virus particle yield > 70% based on SE-HPLC until the final concentration step (UF2). The total number of infectious particles as determined by TCI D50 remained stable throughout the assessed downstream purification process steps. The final titer of concentrated purified bulk from downstream processing of OptiPRO-VP-switch + media change 24h PI YFV particles was determined as 9.5 logw TCIDso / mL.

[0422] Considering the total processing of the harvest material (4 m2bioreactor) to a final pool (4x70 mL UF2 pool), a total amount of 11.95 logw TCI D50 infectious YFV particles could have been recovered from the OptiPRO-VP-switch + media change 24h PI condition. A preliminary yield estimate for larger scale (iCellis 500+) could result in up to 14 log TCID50 infectious particles per batch. Assuming that the minimum dose at end of shelf life is > 1000 active particles per dose and assuming a drug product starting point of 5 log TCIDso / dose at t=0 and a maximum loss in infectious titer / stability of ~0.5 logw loss / year to ensure at least 3 years shelf life, the estimated batch-yield of a manufacturing-scale iCELLis 500+ YFV - process is >100 million Valneva Austria GmbH PAT070-PCT up to 600 million doses per iCellis 500 YF lot. Assuming 10 lots per year, the output would match >1 billion YF vaccine doses per year suggested by WHO as being required by 2026.

[0423] Example 5 - formulation of YFV vaccines

[0424] Background

[0425] Purified YFV material (as described in Example 4) was used to test the stability of yellow fever live-attenuated virus vaccine in lyophilized (2-8°C), liquid-frozen (-20°C ± 5°C and -70°C ± 5°C) and liquid form (2-8°C), in different formulations, over 12 months. The methods below used a formulation and freezing cycle that we developed for CHIKV (a Togaviridae) as a starting point. However, as YFV is a different virus, and particularly a virus from a different family (Flaviviridae), the expectation was that the formulation and methods would need to be optimised for YFV.

[0426] Methods

[0427] Preparing formulation

[0428] All liquid formulations were prepared by diluting the YFV particles produced in Example 4 (Opti PRO- VP- switch + media change 24h PI with downstream processing) into a formulation buffer in a 100 mL volumetric flask.

[0429] The formulation buffer composition was: Trisodium citrate dihydrate (25 mmol / kg), di- Potassium Hydrogen Phosphate and Potassium di-Hydrogen Phosphate (5 mmol / kg, combined), Sucrose (5% w / w), Magnesium Chloride hexahydrate (5 mmol / kg ), D-Sorbitol (0.5% w / w), L-Methionine (10 mmol / kg ), 20% Recombinant Human Albumin (0.1 mg / mL final cone., stock of 200mg / ml (20%) was diluted 2000 times), Water for Injection, final pH 7.3.

[0430] The target infectious virus concentration in the formulation was 6 log TCIDso per mL in 0.5 mL in the drug product based on our experience with previous drug products. However, one lyophilized sample was prepared at a concentration of 8 log TCIDso per mL in 0.5 mL to see if there was any effect of virus concentration on stability.

[0431] Lyophilization

[0432] Details of the lyophilization cycle are shown in Table 7.

[0433] Table 7. Lyophilization cycle details.

[0434] | Step | Temperature (°C) | Ramp (hours) | Hold (hours) | Pressure | Valneva Austria GmbH PAT070-PCT

[0435] TCID50

[0436] Determination of infectious virus concentration in the lyophilized, liquid, and liquid-frozen drug products after preparation (TO) and after storage for various times (2 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 18 months and 24 months) and at various temperatures (-70°C, -20°C, 2-8°C, 25°C, 37°C) was performed by TCI Dso assay as described in Example 1 or 2 (assays are the same). For each time point, three vials were stored. Therefore, the results obtained provide a geometric mean of three values obtained from the analysis of each vial.

[0437] Results

[0438] Stability of lyophilized formulation

[0439] The formulation batch was prepared at a target infectious virus concentration of 6 log™ TCIDso / mL. Lyophilized DP resulted in producing a DP (stored at 2-8 °C) with infectious virus concentration of 8.25 logw TCIDso / mL when measured on day 7 (Figure 7A). Compared to this, the DP stored at 37°C was measured with an infectious virus concentration of 4.95 logw TCIDso / mL on day 12 (Figure 7A). Data collected at various time points of stability study indicates that DP stored at 2-8 °C is relatively stable with a slope value of negligible -0.0094 logs / month. Compared to this, the DP stored at 37 °C indicates a much higher loss of infectious virus particles with a slope of -1.24 logs / month.

[0440] Direct comparison of our lyophilized DP with an existing YFV vaccine Stamaril® at 37°C over two weeks indicated that our lyophilized product had similar stability to Stamaril® (Figure 7B).

[0441] Stability of liquid formulation

[0442] To check the feasibility of a liquid DP, liquid formulations prepared at a target infectious virus concentration of 6 logw TCIDso / mL were filled in vials (0.5 mL) and stored at 2-8 °C, -20 °C or -80 °C.

[0443] Results from the analysis of respective samples is plotted in Figure 8A that confirms comparable stability of frozen liquid DP to the lyophilized DP. This figure shows that the Liquid Valneva Austria GmbH PAT070-PCT frozen formulation stored at -20°C and -70°C is comparatively stable compared to the lyophilized drug product stored at 2-8°C. Linear fits indicate slope values of 0.01 and 0.00007, which can arguably be considered as 0, suggesting minimal loss of infectious virus concentration under these storage conditions. The positive slope values observed may be artifacts of analytical error. The stability of the frozen liquid formulation at these temperatures ensures that the vaccine remains potent and effective, even during extended storage periods.

[0444] The data from storing liquid DP at 2-8°C is shown in Figure 8B. This figure shows that the liquid formulation is not as stable as the lyophilized DP at 2-8°C. Linear fits indicate much higher negative slope values of around -0.5 logs / month for both batches indicating a higher loss of infectious virus concentration even at refrigerated storage condition. Due to the observed high degradation rate at 2-8°C, samples stored at higher temperatures were not analysed for liquid formulations.

[0445] Stability of liquid formulations stored in vials or pre-filled syringes The stability of YFV liquid formulation (2-8°C and 25°C) and liquid frozen formulation (-20°C) stored in vials or pre-filled syringes was assessed over several weeks (Figure 9A and B). It was observed that syringes stored at 2-8°C and 25°C showed a trend of higher stability than vials. A further experiment of side-by-side comparison of vials and syringes stored at 2-8°C and 25°C showed the same effect (Figure 9C) In addition, it was observed that reconstituted Stamaril stored in vials was not as stable as the YFV liquid formulation at 2-8°C (Figure 9D). For all experiments, each data point is a geometric mean of three individual measurements of three separate vials (N=3).

[0446] Discussion

[0447] Liquid Yellow Fever DP was either lyophilized, frozen as a liquid at -20°C and -70°C or stored in the liquid form at 2-8°C.

[0448] WHO, in their technical report (Requirements for yellow fever vaccine, World Health Organization (WHO), Technical Report Series, No 872, 1998) highlighted that freeze-dried yellow fever vaccines should undergo a thermal stability test. The test requires the freeze- dried product to be incubated at 37°C for two weeks and at or below the recommended storage condition (which was 2-8°C in our case). At the end of the 2-week incubation period, the geometric mean of infectious virus concentration of the freeze-dried final lot should not have decreased by more than 1.0 log unit and it should be at least equal to the required minimum number of infectious units per human dose.

[0449] Accordingly, the lyophilized DPs were evaluated at 2-8°C and 37°C. Therefore, considering the slope value of -1.24 logs / month (Figure 7) over 14 days at 37°C months, we confirm that Valneva Austria GmbH PAT070-PCT within 2 weeks of incubation at 37°C, the drop in infectious virus concentration was less than 1 log units, i.e. 0.6 log TCIDso after 2 weeks at 37°C.

[0450] Furthermore, the lyophilized DPs appear very stable at 2-8°C, with negligible loss of infectious virus concentration over 12 months. Liquid drug product, as expected, showed relatively poor stability due to the inherent nature of live viruses to degrade naturally, but frozen liquid drug products appeared to be very stable and thus provide an alternative for the lyophilized drug product. The comparative stability to the existing YFV vaccine shows that the high yield process described herein does not have a detrimental effect on the stability of the final drug product.

[0451] Furthermore, it was expected that the initial formulation buffer tested (a buffer we had previously optimised for Chikungunya virus, CHIKV) would show poor stability and need to be optimised. This is because formulations have to be adapted for each virus, even for viruses within the same virus family. Therefore, the fact that the YFV virus particles were stable in a formulation we have previously used for CHIKV (WO 2021 / 028406) was entirely surprising. This means that, because we are able to use the same CHIKV and YFV formulation buffer, a combination vaccine against CHIKV and YFV can be produced without concern of reducing the stability of one or both of the viral particles over time.

[0452] Example 6 - Immunogenicity studies

[0453] In a further example, it is expected that clinical trials of a single vaccination of YFV particles produced with the process described herein in humans will result in a protective immune response. This can be measured by levels of CHlKV-specific neutralizing antibodies in sera (e.g., by measuring neutralizing antibodies in a microneutralization assay, pNT), assessment of immunogenicity of CHIKV-A5nsP3 after challenge and assessment of antibody persistence (e.g. up to month 12) after a single vaccination (e.g., by comparing geometric mean titers (GMTs) and seroconversion rates (SCRs) in the per-protocol population between dose groups and untreated individuals). It is expected that 14 days after a single vaccination, 100% of subjects will be seroconverted (Seroconversion defined as subjects achieving a YFV-specific neutralizing antibody titer of at least 20 [ NTsoS2O]) and that this will be sustained for at least 12 months. A lack of an anamnestic response, i.e., a booster effect of the challenge dose, is expected in the majority of patients following challenge, indicating sterilizing immunity as characterized by a less than or equal to a four-fold rise in antibody titers as compared to prechallenge titers. Immunization is not expected to be specific to one YFV strain, but to provide protection against multiple YFV strains. Valneva Austria GmbH PAT070-PCT

[0454] It is expected that the vaccine will be tolerable with few adverse events.

[0455] As the present invention relates to increased yield of YFV, has shown no immunogenicityimpacting mutations appearing during the process, and relates to improved stability of the YFV formulation, it is expected that immunogenicity will be similar, if not improved by comparison to other YFV formulations.

[0456] Example 7 Drug Substance (DS) manufacturing process in Roller Bottles (RB) including Upstream and Downstream processes and Drug Substance formulation

[0457] This yellow fever virus candidate, which is based on the approved Stamaril vaccine strain 17D- 204, was rescued from Vero cells transfected with an in vitro synthesized recombinant viral RNA genome based on the published sequence for the Stamaril vaccine strain 17D-204, followed by production of YFV virus banks as described in Example 1. The attenuated sequence was confirmed by NGS (next generation sequencing) and the infectious virus titer was determined by TCID50 analysis.

[0458] GMP master (passage P2) and working (passage P3) virus seed banks will be manufactured, enabling the manufacture of Yellow Fever vaccine drug substance (passage P4) based on internal processes.

[0459] Production of YFV in roller bottles provides a smaller scale process in comparison with production in large bioreactors. The production process for DS described in this example utilizes an animal component - free roller bottle upstream process (RB850CB), using serum- free cell culture media for Vero cell expansion (OptiPRO-SFM) and virus production (VP-SFM). The final scale for infection is 60* RB850CB, resulting in -12L harvest with a single harvest on day 4 post infection. hcDNA reduction is done by protamine sulphate (PS) treatment, followed by clarification (filtration) and concentration / diafiltration (100 kDa MWCO TFF). Removal of host cell proteins is achieved by Capto™Core 700 batch chromatography. After removal of the CC700 beads by filtration, the material is 7-fold diluted to DS and 0.2pm sterile-filtered. Notably, the DS formulation buffer has the same composition as used for IXCHIQ® (licensed live - attenuated vaccine, used to prevent infection caused by chikungunya virus). Approximately 6 liters of DS are expected, aliquoted in PETG bottles and stored frozen at < -65°C. For each process step, preliminary targets and ranges are provided, all based on lab-scale experiments.

[0460] The main component of the YFV vaccine candidate is a purified live-attenuated yellow fever virus based on the approved yellow fever vaccine strain 17D-204 (active component of yellow Valneva Austria GmbH PAT070-PCT fever vaccine ‘Stamaril’). Yellow fever virus strain 17D is an attenuated vaccine strain derived from a wild-type YF virus strain (Asibi) isolated in 1927 and attenuated by several passages in chicken embryo tissue culture. Two sub-strains of the 17D vaccine virus are currently used for vaccine production in embryonated chicken eggs, namely 17D-204 and 17DD.

[0461] As mentioned above, the yellow fever virus strain described herein, is based on the licensed 17D-204 strain used in the 'Stamaril' vaccine. Unlike Stamaril, which is manufactured using embryonated chicken eggs, the current YFV vaccine candidate is produced in Vero cells. This cell substrate represents a World Health Organization (WHO) approved continuous cell line for human vaccine production.

[0462] The YFV described herein was generated by reverse genetics, whose sequence is identical to that of the commercially available 'Stamaril' vaccine. The yellow fever virus (YFV) sequence used for this project was based on the originally published Stamaril sequence (Dupuy et al. 1989; GeneBank-ID: X15062). In addition, this sequence was modified with 3 nucleotide substitutions (at positions 5641 [ns3; silent, Leu357Leu], 10243 [ns5; silent, Leu869Leu], and 10722 [3'UTR] nt; see SEQ ID NO: 1), which were identified in Stamaril vaccine batches (Barban et al., 2007). The sequence was thus modified to match the vaccine candidate to the published commercial Stamaril vaccine strain sequence. No foreign sequence was introduced into the viral genome.

[0463] The YFV generated herein therefore exactly corresponds to the attenuated 17D-204 yellow fever virus (YFV) strain established through multiple passages in cell culture, which is used for the commercially available yellow fever vaccine 'Stamaril'.

[0464] Description of Manufacturing Process and Process Controls

[0465] The developed manufacturing process of the YFV described herein is based on several principles of the upstream and downstream principles of Valneva’s approved IXIARO® and IXCHIQ® manufacturing processes for commercial vaccine production of the Japanese encephalitis Virus (JEV) and Chikungunya virus (CHIKV).

[0466] USP (Upstream Process) The upstream process (USP) utilizes an animal component - free process for Vero host cell expansion in T-flasks and roller bottles (RB850CB). The USP includes host (vero) cell expansion and infection, followed by harvesting of virus-containing cell culture supernatant. Key reagents, biological material, consumables and equipment needed for the USP are summarized in Table 8. Valneva Austria GmbH PAT070-PCT

[0467] Table 8. Key reagents, biological material, consumables and equipment needed for the

[0468] USP. key reagents, biological material, consumables and equipment

[0469] Host cell expansion Vero host cell expansion is performed using an SFM - adapted Vero cell bank in an animal component - free process in T-flasks and roller bottles (RB850CB) according to laboratory biosafety and good laboratory practices including aseptic techniques based on established procedures.

[0470] Briefly, Vero cells of a SFM - adapted cell bank are expanded in OptiPRO-SFM cell growth medium (supplemented to final concentration of 4mM L-GIn) in T-flasks (Corning TC treated polystyrene with vent cap) and subsequently roller bottles (Corning CellBIND 850 cm2polystyrene roller bottle with easy grip vent cap). For virus production VP-SFM medium (supplemented to 4mM L-GIn) is utilized.

[0471] The passaging procedure is comprised of carefully washing the cell monolayer once with 1 * PBS (w / o Ca2+ & Mg2+), followed by the addition of TrypLE Select 1 * for cell detachment. After a short (approximately 2 minutes) incubation at room temperature, the TrypLE 1 * is removed and discarded to waste and the vessels transferred into an incubator. After approximately 15 minutes (set points: 37.0°C, 5.0% CO2, (0.3 rpm for roller bottles)), growth / cell expansion medium is added to the vessel and the cells are fully detached from the growth surface. The cell suspension is gently triturated to disperse any cell clumps. Cell concentration and viability are measured using an automated cell counter (e.g. NC-200) and the volume of cells required to target a defined seeding density of viable Vero cells per cm2are transferred into new cell culture vessels already containing fresh growth / cell expansion medium. Valneva Austria GmbH PAT070-PCT

[0472] Media and key process parameters for the Vero cell expansion phase are summarized in Table 9 and Table 10.

[0473] Table 9. Media used in USP.

[0474] Table 10. Key set-point process parameters for the Vero cell expansion phase.

[0475] Vero cells are seeded based on a defined seeding density (viable cells per cm2), which varies with the planned duration for the respective passage as summarized in Table 11 and Table 12 below.

[0476] Table 11. Seeding densities for Vero cell expansion phase in T-flasks (Coming TC- treated polystyrene with vent cap).

[0477] 1for initiation 3.0E+04 cells / cm2for 4 days

[0478] Table 12. Seeding densities for Vero cell expansion phase in roller bottles (Coming 850cm2CellBIND-surface polystyrene roller bottle with vent cap).

[0479] 1last passage prior infection 4 days

[0480] Host cell infection with YFV working virus seed bank (WVSB) After 4 days of incubation (set points: 37.0°C, 5.0% CO2; 0.3 rpm) some (e.g. five (5)) of the roller bottles are selected at random for cell counting, some (e.g. five (5)) of the roller bottles are used for culturing negative control cells for negative control testing and sixty (60) of the available roller bottles are infected with yellow fever virus.

[0481] The host cells are counted and the MOI is calculated. Briefly, the cell monolayer of each RB850CB is washed once with 1 * DPBS w / o Ca2+ & Mg2+ and TrypLE Select 1 * added to detach the cells. After a short (approximately 2 minutes) incubation at room temperature the Valneva Austria GmbH PAT070-PCT

[0482] TrypLE is removed and discarded to waste and the vessels transferred into an incubator. After approximately 15 minutes (set points: 37.0°C, 5.0% CO2, 0.3 rpm), growth I cell expansion medium is added to the roller bottles and the cells fully detached and the cell suspension gently triturated to disperse any cell clumps. Cell concentration and viability are measured using an automated cell counter (e.g. NC-200) for calculation of the MOI.

[0483] Negative Control (5* RB850) On the day of infection, the growth medium is removed from each of the randomly selected roller bottles, 200 mL pre-warmed virus production medium (VP-SFM + 4mM L Gin) are added (but no virus inoculum) and the roller bottles are incubated at 35.0°C, 5.0% CO2, 0.3 rpm. On virus harvest day the roller bottles are checked for CPE, the production medium is removed, 2000 mL pre-warmed virus production medium is added to each RB and the roller bottles are further incubated at 35.0°C, 5.0% CO2 until testing.

[0484] Infection with YFV WVSB (60* RB850CB) The total amount of cells available for infection in sixty (60) roller bottles will be calculated based on the cell count obtained. The required number of working virus seed bank vials is calculated and thawed (at room temperature). The total amount of virus needed is calculated and the required volume is added to virus production medium (VP-SFM + 4mM L-GIn) in order to prepare the virus infection inoculum (with excess). The MOI is set to 0.01.

[0485] For Vero host cell infection, the used cell culture medium is carefully removed from the roller bottles by aspirating the used medium into a waste bag (or other suitable vessel) without disturbing the cell monolayer, followed by the addition of 190 mL fresh virus production medium (VP-SFM + 4mM L-GIn) to each of the 60* roller bottles (Corning RB850CB) that will be infected.

[0486] Subsequently 10 mL of the prepared virus infection inoculum are added to each of the roller bottles and the roller bottles transferred to a roller bottle incubator at set points 35.0°C, 5.0% CO2, 0.3 rpm. Key process parameters for the virus production phase are summarized in Table 13.

[0487] Table 13. Key process parameters for virus production phase.

[0488] Harvest of YFV Based on data using a non-GMP YF virus bank, a favorable harvest window of 90 ± 12 hours post infection has been identified (refer to Figure 11). It should be noted that infection of Vero host cells with yellow fever virus generally results in a low level of cytopathic Valneva Austria GmbH PAT070-PCT effect (CPE), which may be subtle under microscopic evaluation. The yellow fever virus containing medium / cell culture supernatant is transferred / pumped from the infected roller bottles into a suitable vessel (e.g. Flexboy bag, 20L).

[0489] DSP (Downstream Process) The downstream process is divided into two parts; Part 1 : Recovery, hcDNA reduction and concentration, and Part 2: Purification and DS formulation (see Figure 10). All process steps are described for a 60 RB850CB - scale, equivalent to 12L harvest volume.

[0490] DSP Part 1 Initial process steps include recovery of the virus, harvest stabilization, hcDNA reduction and initial concentration and diafiltration to reduce the harvest volume and exchange the cell culture medium to a defined buffer for intermediate storage and further purification. These process steps are summarized in Figure 10.

[0491] A single harvest is collected in a 20L Flexboy bag (~12L crude volume). After addition of a 1 M Tris stock pH 7.4 to a final concentration of 25 mM, the crude harvest is stored at 2-8°C overnight before further processing.

[0492] On the next day, Protamine Sulphate (PS) stock solution is added to the cold harvest to a final concentration of 0.2 mg / mL. The harvest bag is incubated for 30 min (range 25-35 min) at room temperature (18-25°C) with manual mixing by hand every 5 minutes. Cell debris and hcDNA-PS precipitate are subsequently removed by filtration using a 5” EKV filter (0.2 pm; PALL, 2200 cm2filter area). Concentration and diafiltration is done immediately after filtration using a 100 kDa TFF PES membrane with 2000 cm2membrane size. Concentration is complete when the volume is reduced to L (resulting in a — 12-fold concentration).

[0493] Diafiltration is done using 5 volumes of DF buffer to remove growth media components and the phenol red present in VP-SFM production media. Recovery of diafiltered product is done in a 2 L PETG bottle including a wash-out of one system volume (final total volume for diafiltrate of ~1 L). After diafiltration, the virus is stabilized by the addition of sucrose to a final concentration of 10% w / w using a 50% w / w stock dissolved in 25 mM Tris pH 7.4.

[0494] The stabilized harvest solution can be stored up to three days at 2-8°C in the 2 L PETG bottle until further processing. Optionally, and for long term storage, the material can be sub- aliquoted and frozen at < -65°C (e.g. 5 x 250 mL).

[0495] DSP Part 2 Purification and DS formulation

[0496] The process steps are summarized in Figure 10. Depending on the storage of the stabilized diafiltered virus material the purification step is done: Valneva Austria GmbH PAT070-PCT

[0497] For concentrated diafiltered harvest material stored at 2-8 °C The material can be immediately processed by addition of Capto™ Core 700 slurry. The mixture is incubated under gentle mixing via a magnetic stirrer (no vortex, e.g. 100 rpm) for 60 min (50-70 min) at room temperature (15-25°C). The Capto™ Core resin is removed by filtration using a bottle top filter (e.g. Nalgene rapid flow 0.2 pm) into a fresh 2 L PETG bottle.

[0498] For concentrated diafiltered harvest material stored at < -65 °C: The calculated number of aliquots is transferred to a fridge (2-8°C) and thawed overnight. Prior to purification the completely thawed aliquots are pooled in a PETG bottle of appropriate size (e.g. 1-2 L PETG). The pooled material is purified by addition of Capto™Core 700 slurry. The mixture is incubated under gentle mixing via a magnetic stirrer (no vortex, e.g. 100 rpm) for 60 min (range 50-70 min) at room temperature (15-25 °C). The Capto™Core resin is removed by filtration using a bottle top filter into a fresh 2L PETG bottle.

[0499] After Capto™ Core purification, the material is diluted 7-fold with DS buffer (see Table 15) in a PC bottle of appropriate size (e.g. 10 L) where 6 volumes of DS buffer have been predispensed into the DS mixing bottle followed by the purified virus solution. The pre-filtration DS is mixed gently (no vortex, e.g. 100 rpm; ~10 min) followed by 0.2 pm filtration using an EKV filter (220 cm2filter area) into a fresh 10 L Flexboy bag.

[0500] Finally, the filtered DS is aliquoted into 125 mL PETG bottles (100mL per bottle) using a peristaltic pump and the individual aliquots are frozen at < -65 °C.

[0501] Table 14. Buffers and solutions.

[0502] Table 15. Recipe for the preparation of 16 L formulation buffer (DS buffer) with a final pH of 7.3 (range 7.1 - 7.5). Valneva Austria GmbH PAT070-PCT

[0503] *WFI may also be added up to a final weight of 16 kg, in which case the concentrations would be w / w or mmol / kg. This recipe may be scaled up or down according to need.

[0504] QTPP and CQAs

[0505] QTPP The Quality Target Product Profile (QTPP) is a strategic element of pharmaceutical development that outlines the desired quality attributes of a drug product to ensure its safety, efficacy, and regulatory compliance. As a core component of the Quality by Design (QbD) framework defined in ICH Q8(R2), the QTPP guides formulation and process development, supports risk management, and facilitates alignment with global regulatory standards.

[0506] Key elements typically include dosage form, route of administration, strength, purity, stability, and manufacturing considerations. The QTPP is a dynamic document, evolving throughout the development lifecycle based on emerging data and regulatory expectations.

[0507] Table 16. Quality Target Product Profile (QTPP) for yellow fever vaccine. Valneva Austria GmbH PAT070-PCT

[0508] CQAs Critical Quality Attributes (CQAs) for live virus vaccines are specific properties or characteristics that must be carefully controlled to ensure the safety, efficacy, and consistency of the final product. Regulatory agencies (e.g., FDA, EMA, WHO) require thorough characterization and control of CQAs for live virus vaccines. CQAs are linked to Critical Process Parameters (CPPs) during manufacturing to ensure consistent quality. Stability studies must demonstrate that CQAs are maintained throughout the product's shelf life. These attributes are particularly important due to the biological complexity and variability of live virus vaccines. The CQAs for live virus vaccines are specific to their biological nature and the need to balance potency, safety, and stability. Below are the key CQAs for live virus vaccines:

[0509] • Potency (Viral Titer)

[0510] Definition: The concentration of live, infectious virus particles in the vaccine.

[0511] Importance: Potency directly correlates with the vaccine's ability to induce a protective immune response.

[0512] Measurement: Plaque-forming units (PFU), tissue culture infectious dose (TCID50), or other infectivity assays.

[0513] YF control strategy: TCID50 (DS, DP, I PC)

[0514] • Identity

[0515] Definition: Confirmation that the vaccine contains the correct viral strain.

[0516] Importance: Ensures the vaccine targets the intended pathogen.

[0517] Measurement: Genetic sequencing, PCR, or immunological assays to verify the viral strain.

[0518] YF control strategy: qPCR (DS)

[0519] • Purity

[0520] Definition: Freedom from process-related impurities (e.g., host cell proteins, DNA, media components) and product-related impurities (e.g. defective viral particles).

[0521] Importance: Ensures safety and reduces the risk of adverse reactions.

[0522] Measurement: Chromatography, electrophoresis or mass spectrometry.

[0523] YF control strategy: DS

[0524] Residual Host Cell DNA (qPCR),

[0525] Residual Host cell Protein (ELISA), Valneva Austria GmbH PAT070-PCT

[0526] Residual BSA (ELISA)

[0527] • Safety

[0528] Definition: Absence of harmful contaminants, such as adventitious agents (e.g. bacteria, mycoplasma, other viruses) and endotoxins.

[0529] Importance: Prevents contamination that could harm recipients.

[0530] Measurement: Sterility testing, mycoplasma testing, and adventitious agent testing.

[0531] YF control strategy: DS

[0532] Bioburden (membrane filtration)

[0533] Test for detection of Mycoplasmas (culture method)

[0534] Rapid detection of Mycoplasma by PCR

[0535] Test for Mycobacteria

[0536] • Stability

[0537] Definition: The ability of the vaccine to maintain its potency, purity, and safety over time under specified storage conditions.

[0538] Importance: Ensures the vaccine remains effective throughout its shelf life.

[0539] Measurement: Real-time and accelerated stability studies to monitor viral titer, degradation, and physical changes.

[0540] YF control strategy: DS and DP stability studies

[0541] • Genetic Stability

[0542] Definition: Consistency of the viral genome during manufacturing and storage.

[0543] Importance: Ensures the virus does not revert to a virulent form or loses its attenuated properties.

[0544] Measurement: Genetic sequencing or PCR to detect mutations.

[0545] YF control strategy: DS and virus banks

[0546] RNA sequencing by Sanger under GLP conditions or NGS

[0547] • Viability

[0548] Definition: The proportion of live, infectious virus particles relative to non-infectious or defective particles.

[0549] Importance: Ensures the vaccine can elicit a robust immune response.

[0550] Measurement: total viral particles by qPCR and total infectious particles by infectivity assays (e.g. TCID50, PFU).

[0551] Immunogenicity Valneva Austria GmbH PAT070-PCT

[0552] Definition: The ability of the vaccine to induce a protective immune response in the target population.

[0553] Importance: Determines the vaccine's efficacy.

[0554] Measurement: Animal models, serological assays (e.g., neutralizing antibody titers), or clinical trials.

[0555] • Physical Attributes

[0556] Definition: Characteristics such as appearance, pH, osmolality, particle size distribution and particulate matter.

[0557] Importance: Ensures the vaccine meets physical standards for administration.

[0558] Measurement: Visual inspection, pH meters, and particle counters.

[0559] YF control strategy: pH, osmolality, visual control, particle size distribution (characterization)

[0560] • Attenuation

[0561] Definition: The degree to which the virus has been weakened to prevent disease while retaining immunogenicity.

[0562] Importance: Ensures the vaccine is safe and effective.

[0563] Measurement: In vitro and in vivo assays to confirm attenuation.

[0564] YF control strategy: Sequencing

[0565] • Dose Uniformity

[0566] Definition: Consistency of viral titer across vaccine batches and individual doses.

[0567] Importance: Ensures each dose delivers the intended level of protection.

[0568] Measurement: Statistical analysis of viral titer data from multiple batches.

[0569] YF control strategy: DP filling homogeneity (TCID50)

[0570] • Residual Moisture (for Lyophilized Vaccines)

[0571] Definition: The amount of moisture remaining in lyophilized (freeze-dried) vaccines.

[0572] Importance: Excessive moisture can destabilize the virus and reduce shelf life.

[0573] Measurement: Karl Fischer titration or other moisture analysis techniques.

[0574] • Reconstitution Time (for Lyophilized Vaccines)

[0575] Definition: The time required for the vaccine to dissolve completely when reconstituted with a diluent.

[0576] Importance: Ensures ease of administration and consistent dosing.

[0577] Measurement: Visual inspection and timing during reconstitution. Valneva Austria GmbH PAT070-PCT

[0578] PREFERRED ASPECTS

[0579] Aspect 1A. A high-yield method of preparing a composition comprising yellow fever virus (YFV), wherein the method comprises: a. Culturing host cells in a serum-free culture media in a fix-bed bioreactor; b. Infecting the host cells with YFV; and c. Collecting the culture medium 60-84 hours post-infection to produce a first YFV harvest (i).

[0580] Aspect 2A. The method according to aspect 1A, wherein the fixed-bed bioreactor uses a falling film for medium oxygenation, suitably wherein the shear stress and / or medium foaming are minimized by maintaining the Falling Film Height (FFH) at the lowest possible level while still maintaining required dissolved oxygen (DO) levels.

[0581] Aspect 3A. The method according to aspect 2A, wherein, the dissolved oxygen (DO) level for the first 3 hours post- infection is not less than 10%, suitably not less than 15%, and / or wherein the DO level from 3 hours post-infection to collection of culture medium is between 40-60%.

[0582] Aspect 4A. The method according to any preceding aspect, wherein the host cells are Vero cells.

[0583] Aspect 5A. The method according to any preceding aspect, wherein the culture media is replaced 1-day post-infection with fresh culture media.

[0584] Aspect 6A. The method according to any preceding aspect, wherein the culture media is collected by perfusion between 60-84 hours post-infection.

[0585] Aspect 7A. The method according to any preceding aspect, wherein the method further comprises: d) adding culture medium to the bioreactor and collecting culture medium 7-9 days post-infection to produce a second YFV harvest (ii). Valneva Austria GmbH PAT070-PCT

[0586] Aspect 8A. The method according to any preceding aspect, wherein the serum free culture media in (a) for culturing the host cells is OptiPRO-SFM or VP-SFM, suitably OptiPRO- SFM, and / or wherein the culture medium is replaced in (b) with VP-SFM.

[0587] Aspect 9A. The method according to any preceding aspect, wherein the method further comprises: dialyzing the virus harvest (i) and / or (ii) to form a liquid formulation (iii); wherein said liquid formulation (iii) comprises a) at least one strain of YFV; b) 2% to 8% (w / v) sugar; c) 1 mM to 20 mM phosphate; d) 1 mM to 50 mM of at least one carboxylate buffering agent; e) 1 mM to 10 mM MgCh; f) 0.1 % to 5% (w / v) D-sorbitol; g) 1 to 20 mM L- methionine; and h) 0.01 to 1 mg / mL human albumin (HA), wherein the formulation is at a pH of 7.0 to 7.5.

[0588] Aspect 10A. The method according to aspect 9A, wherein the sugar is sucrose is 5% (w / v), the phosphate is 5 mM, the at least one carboxylate buffering agent is 25 mM, the MgCh is 5 mM, the D-sorbitol is 0.5%, the L-methionine is 10 mM, and the HA is 0.01% (w / v), optionally wherein the formulation is at a pH of 7.3, and / or wherein the sugar is sucrose is 5% (w / v), the phosphate is potassium phosphate at 5 mM, the at least one carboxylate buffering agent is sodium citrate at 25 mM, the MgCh is 5 mM, the D-sorbitol is 0.5%, the L-methionine is 10 mM, and the HA is 0.01% (w / v), optionally wherein the formulation is at a pH of 7.3, and / or wherein the HA is a recombinant human albumin (rHA); and / or the formulation further comprises at least one diluent selected from the group consisting of tissue culture medium, saline and water.

[0589] Aspect 11 A. The method of aspect 9A or 10A, wherein the method further comprises lyophilizing the liquid formulation (ii) to produce a lyophilized YFV formulation (iii).

[0590] Aspect 12A. The method according to any of aspects 9A to 11 A, wherein (b) further comprises dividing the formulation (ii) after dialysis to produce 102TCIDsoto 106TCID50 per dose of YFV. Valneva Austria GmbH PAT070-PCT

[0591] Aspect 13A. The method according to any preceding aspect, wherein the YFV comprises or consists of a polynucleotide sequence with at least 80% identity to SEQ ID NO: 1, suitably wherein the YFV polynucleotide sequence comprises one or more mutations selected from: A2608C, A5641G, A7225G, C8212T, A10243G, G10559GT, and / or A10722G mutations in SEQ ID NO: 1, and / or wherein the YFV is a live, attenuated virus.

[0592] Aspect 14A. A pharmaceutical composition of YFV obtained or obtainable by the method of aspect any of aspects 9A to 13A.

[0593] Aspect 15A. A liquid yellow fever virus (YFV) formulation comprising: a) YFV particles; b) 2% to 8% (w / v) sucrose; c) 1 mM to 20 mM phosphate; d) 1 mM to 50 mM of at least one carboxylate buffering agent; e) 1 mM to 10 mM MgCh; f) 0.1% to 5% (w / v) D- sorbitol; g) 1 to 20 mM L-methionine; and h) 0.001% to 0.1% (w / v) human albumin (HA), wherein the formulation is at a pH of 7.0 to 7.5, wherein the formulation is for preparation of a liquid frozen or lyophilized formulation.

[0594] Aspect 16A. A lyophilized yellow fever virus (YFV) formulation comprising: a) YFV particles; b) 2% to 8% sucrose; c) 1 mmol / kg to 20 mmol / kg phosphate; d) 1 mmol / kg to 50 mmol / kg of at least one carboxylate buffering agent; e) 1 mmol / kg to 10 mmol / kg MgCh; f) 0.1% to 5% D-sorbitol; g) 1 to 20 mmol / kg L-methionine; and h) 0.001% to 0.1% human albumin (HA), wherein the formulation is at a pH of 7.0 to 7.5.

[0595] Aspect 17A. The formulation according to aspect 15A and 16A, wherein the YFV comprises or consists of a polynucleotide sequence with at least 80% sequence identity to SEQ ID NO: 1, suitably wherein the YFV polynucleotide sequence comprises one or more mutations selected from: A2608C, A5641G, A7225G, C8212T, A10243G, G10559GT, and / or A10722G mutations in SEQ ID NO: 1, and / or wherein the sugar is sucrose at 5% (w / v), the phosphate is 5 mM, the at least one carboxylate buffering agent is 25 mM, the MgCh is 5 mM, the D-sorbitol is 0.5%, the L-methionine is 10 mM, and the HA is 0.01% (w / v), optionally wherein the formulation is at a pH of 7.3, and / or Valneva Austria GmbH PAT070-PCT wherein the sugar is sucrose at 5% (w / v), the phosphate is potassium phosphate at 5 mM, the at least one carboxylate buffering agent is sodium citrate at 25 mM, the MgCh is 5 mM, the D-sorbitol is 0.5%, the L-methionine is 10 mM, and the HA is 0.01% (w / v), optionally wherein the formulation is at a pH of 7.3, and / or wherein the HA is a recombinant human albumin (rHA); and / or the YFV formulation further comprises at least one diluent selected from the group consisting of tissue culture medium, saline and water, and / or wherein the liquid or lyophilized formulation comprises an effective amount of at least one strain of YFV, optionally wherein the effective amount is 102TCIDso / dose to 106TCIDso / dose of YFV and / or wherein the YFV is a live attenuated virus, and / or wherein the formulation further comprises one or more enveloped viruses, optionally wherein the enveloped viruses are selected from Flaviviridae, Alphaviridae, Togaviridae, Coronavirus, Orthomyxoviruses, Paramyxoviridae, and Rhabdoviruses.

[0596] Aspect 18A. A method of preparing a YFV or CHIKV composition, wherein the method comprises: a. Producing a virus harvest (i); b. Dialyzing the virus harvest (i) to form a liquid formulation (ii); wherein said liquid formulation (ii) comprises a) at least one strain of virus; b) 2% to 8% sucrose; c) 1 mmol / kg to 20 mmol / kg phosphate; d) 1 mmol / kg to 50 mmol / kg of at least one carboxylate buffering agent; e) 1 mmol / kg to 10 mmol / kg MgCI2; f) 0.1% to 5% D- sorbitol; g) 1 to 20 mmol / kg L-methionine; and h) 0.001 % to 0.1% human albumin (HA), wherein the formulation is at a pH of 7.0 to 7.5.

[0597] Aspect 19A. The method according to aspect 18A, further comprising mixing the YFV liquid formulation (ii) and / or CHIKV liquid formulation (ii) to produce a combined liquid formulation (iii).

[0598] Aspect 20A. The method of aspect 18A or 19A, further comprising lyophilizing the liquid formulation (ii) to produce a lyophilized formulation (iv). Valneva Austria GmbH PAT070-PCT

[0599] Aspect 21A. A method of preparing a combined YFV and CHIKV composition, wherein the method comprises: a. Producing a YFV virus harvest (i); b. Dialyzing the YFV virus harvest (i) to form a liquid formulation (ii); wherein said liquid formulation (ii) comprises a) at least one strain of YFV; b) 2% to 8% (w / v) sucrose; c) 1 mM to 20 mM phosphate; d) 1 mM to 50 mM of at least one carboxylate buffering agent; e) 1 mM to 10 mM MgCh; f) 0.1% to 5% (w / v) D- sorbitol; g) 1 to 20 mM L-methionine; and h) 0.001% to 0.1% (w / v) human albumin (HA), wherein the formulation is at a pH of 7.0 to 7.5; c. producing a Chikungunya virus (CHIKV) harvest (iii); d. dialyzing the CHIKV virus harvest (iii) to form a liquid formulation (iv); wherein said liquid formulation (iv) comprises a) at least one strain of CHIKV; b) 2% to 8% (w / v) sucrose; c) 1 mM to 20 mM phosphate; d) 1 mM to 50 mM of at least one carboxylate buffering agent; e) 1 mM to 10 mM MgCI2; f) 0.1% to 5% (w / v) D- sorbitol; g) 1 to 20 mM L-methionine; and h) 0.001% to 0.1% (w / v) human albumin (HA), wherein the liquid formulation is at a pH of 7.0 to 7.5; e. mixing the YFV liquid formulation (ii) and CHIKV liquid formulation (iv) to produce a combined YFV and CHIKV liquid formulation (v).

[0600] Aspect 22A. The method according to aspect 21A, further comprising mixing the YFV liquid formulation (ii) and / or CHIKV liquid formulation (ii) to produce a combined liquid formulation (iii).

[0601] Aspect 23A. The method of aspect 21 A or 22A, the method further comprising (f) lyophilizing the combined YFV and CHIKV liquid formulation (v) to produce a combined YFV and CHIKV lyophilized formulation (vi).

[0602] Aspect 24A. The method of aspect 18A to 23A, wherein the CHIKV comprises or consists of a polynucleotide sequence with at least 80%, 90%, 95%, 99% identity to SEQ ID NO: 2, and / or wherein the sucrose is 5% (w / v), the phosphate is 5 mM, the at least one carboxylate buffering agent is 25 mM, the MgCh is 5 mM, the D-sorbitol is 0.5%, the L-methionine is 10 mM, and the HA is 0.01% (w / v), optionally wherein the formulation is at a pH of 7.3, and / or Valneva Austria GmbH PAT070-PCT wherein the sucrose is 5% (w / v), the phosphate is potassium phosphate at 5 mM, the at least one carboxylate buffering agent is sodium citrate at 25 mM, the MgCh is 5 mM, the D-sorbitol is 0.5%, the L-methionine is 10 mM, and the HA is 0.01% (w / v), optionally wherein the formulation is at a pH of 7.3, and / or wherein the HA is a recombinant human albumin (rHA); and / or the formulation further comprises at least one diluent selected from the group consisting of tissue culture medium, saline and water, and / or wherein (b) and (d) further comprise diluting the composition (ii) and (iv) after dialysis to produce 102TCIDso to 106TCIDso per dose of YFV and 102TCIDso to 106TCIDso per dose of CHIKV, and / or wherein the YFV and / or CHIKV viruses are live, attenuated viruses.

[0603] Aspect 25A. The method of aspect 18A to 23A, wherein the YFV comprises or consists of a polynucleotide sequence with at least 80% sequence identity to SEQ ID NO: 1 , suitably wherein the YFV polynucleotide sequence comprises one or more mutations selected from: A2608C, A5641G, A7225G, C8212T, A10243G, G10559GT, and / or A 10722G mutations in SEQ ID NO: 1 , and / or wherein the CHIKV comprises or consists of a polynucleotide sequence with at least 80%, 90%, 95%, 99% identity to SEQ ID NO: 2, and / or wherein the sucrose is 5%, the phosphate is 5 mmol / kg, the at least one carboxylate buffering agent is 25 mmol / kg, the MgCh is 5 mmol / kg, the D-sorbitol is 0.5%, the L- methionine is 10 mmol / kg, and the HA is 0.01 %, optionally wherein the formulation is at a pH of 7.3, and / or wherein the sucrose is 5%, the phosphate is potassium phosphate at 5 mmol / kg, the at least one carboxylate buffering agent is sodium citrate at 25 mmol / kg, the MgCh is 5 mmol / kg, the D-sorbitol is 0.5%, the L-methionine is 10 mmol / kg, and the HA is 0.01 %, optionally wherein the formulation is at a pH of 7.3, and / or wherein the HA is a recombinant human albumin (rHA); and / or Valneva Austria GmbH PAT070-PCT the formulation further comprises at least one diluent selected from the group consisting of tissue culture medium, saline and water, and / or wherein step (b) and (d) further comprise diluting the composition (ii) and (iv) after dialysis to produce 102TCI D50 to 106TCI D50 per dose of YFV and / or 102TCI D50 to 106TCID50 per dose of CHIKV and / or wherein the YFV and / or CHIKV viruses are live, attenuated viruses.

[0604] Aspect 26A. A pharmaceutical composition of YFV and CHIKV obtained or obtainable by the method of aspects 17A to 25A.

[0605] Aspect 27A. A liquid or lyophilized combination formulation comprising: a) YFV particles; b) CHIKV particles; c) 2% to 8% (w / v) sugar; d) 1 mM to 20 mM phosphate; e) 1 mM to 50 mM of at least one carboxylate buffering agent; f) 1 mM to 10 mM MgCh; g) 0.1 % to 5% (w / v) D-sorbitol; h) 1 to 20 mM L-methionine; and i) 0.001 % to 0.1% (w / v) human albumin (HA), wherein the formulation is at a pH of 7.0 to 7.5.

[0606] Aspect 28A. The formulation according to aspect 27A, wherein the CHIKV comprises or consists of a polynucleotide sequence with at least 80%, 90%, 95%, 99% identity to SEQ ID NO: 2, and / or wherein the YFV comprises or consists of a polynucleotide sequence with at least 80% sequence identity to SEQ ID NO: 1 , suitably wherein the YFV polynucleotide sequence comprises one or more mutations selected from: A2608C, A5641G, A7225G, C8212T, A10243G, G10559GT, and / or A10722G mutations in SEQ ID NO: 1, and / or wherein the sugar is sucrose is 5% (w / v), the phosphate is 5 mM, the at least one carboxylate buffering agent is 25 mM, the MgCh is 5 mM, the D-sorbitol is 0.5% (w / v), the L-methionine is 10 mM, and the HA is 0.01% (w / v), optionally wherein the formulation is at a pH of 7.3, and / or wherein the sugar is sucrose is 5% (w / v), the phosphate is potassium phosphate at 5 mM, the at least one carboxylate buffering agent is sodium citrate at 25 mM, the MgCh is 5 mM, the D-sorbitol is 0.5%, the L-methionine is 10 mM, and the HA is 0.01% (w / v), optionally wherein the formulation is at a pH of 7.3, and / or wherein the HA is a recombinant human albumin (rHA); and / or Valneva Austria GmbH PAT070-PCT the formulation further comprises at least one diluent selected from the group consisting of tissue culture medium, saline and water, and / or wherein the liquid or lyophilized formulation comprises an effective amount of at least one strain of YFV and CHIKV, optionally wherein the effective amount is 102TCIDso / dose to 106TCID50 / dose of YFV and 102TCID50 / dose to 106TCID50 / dose, of CHIKV, and / or wherein the viruses are live, attenuated viruses, and / or wherein the formulation further comprises one or more enveloped viruses, optionally wherein the enveloped viruses are selected from Flaviviridae, Alphaviridae, Togaviridae, Coronavirus, Orthomyxoviruses, Paramyxoviridae, and Rhabdoviruses.

[0607] Aspect 29A. The formulation according to any of aspects 14A to 17A and 27A to 28A, for use in preventing YFV infection.

[0608] Aspect 30A. The formulation according to any of aspects 27A to 28A, for use in preventing YFV and / or CHIKV infection.

[0609] Aspect 31 A. A method of preventing YFV infection comprising administering the formulation according to any of aspects 14A to 17A and 27A to 28A to a subject.

[0610] Aspect 32A. A method of preventing YFV and / or CHIKV infection comprising administering the formulation according to aspects 27A or 28A to a subject.

[0611] Aspect 33A. The method according to aspect 18A, wherein the YFV and / or CHIKV liquid formulation is provided in a single-dose pre-filled syringe.

[0612] Aspect 34A. The method according to aspect 19A, wherein the YFV and / or CHIKV liquid formulation is provided in a single-dose pre-filled syringe for simultaneous administration.

[0613] Aspect 35A. The pre-filled syringe assembly of aspects 33A and 34A, wherein the YFV and / or CHIKV liquid formulation is stable after being stored at temperatures ranging from -15°C to -80°C for at least 12 months. Valneva Austria GmbH PAT070-PCT

[0614] Aspect 36A. A lyophilized composition in unit dosage form comprising or consisting of a) an effective amount of YFV and / or CHIKV live attenuated virus; b) 22,5 to 27,5 mg, preferably about 25 mg sucrose; c) 0,25 to 0,35, preferably 0,3128 mg di-potassium hydrogen phosphate; d) 0,075 to 0,125 mg, preferably 0,0925 mg potassium dihydrogen phosphate; e) 3,35 to 4,05 mg, preferably 3,7 mg trisodium citrate dihydrate; f) 0,45 to 0,55 mg, preferably 0,563 mg magnesium chloride hexahydrate; g) 2 to 3 mg, preferably 2,5 mg D-sorbitol; h) 0,5 to 1,0 mg, preferably 0,746 mg L- methionine; and i) 0,2 to 0,3 mg, preferably 0,2515 mg human albumin (HA), wherein said composition is for preparation of a reconstituted lyophilized composition; for which said lyophilized composition is reconstituted in water for injection to end up with a pharmaceutical composition of 0,5 g total weight or of 0,5 ml total volume.

[0615] Aspect 37A. A pharmaceutical composition comprising YFV and optionally CHIKV, wherein the formulation is a liquid frozen formulation.

[0616] Aspect 38A. A pharmaceutical composition comprising YFV and optionally CHIKV, wherein the formulation is lyophilized formulation.

[0617] Aspect 39A. The pharmaceutical composition of aspect 37A or 38A, wherein the formulation is a single dose or multi-dose formulation.

[0618] Aspect 40A. The composition of aspect 38A or 39A wherein the lyophilized formulation is reconstitution with water for Injection (WFI).

[0619] Aspect 41A. The pharmaceutical composition of aspect 39A, wherein the single dose formulation is reconstituted in 0.5 mL of WFI.

[0620] Aspect 1B. A high-yield method of preparing a composition comprising yellow fever virus (YFV), wherein the method comprises: a. Culturing host cells in a serum-free culture medium in roller bottles; b. Infecting the host cells with YFV; and c. Collecting the culture medium 78-102 hours post-infection to produce a YFV harvest (i). Valneva Austria GmbH PAT070-PCT

[0621] Aspect 2B. The method according to aspect 1 B, wherein the serum free culture media in (a) for culturing the host cells is OptiPRO-SFM or VP-SFM, preferably OptiPRO-SFM, and / or wherein the culture medium is replaced in (b) with VP-SFM.

[0622] Aspect 3B. The method according to aspect 1 B or 2B, wherein the method further comprises: dialyzing the virus harvest (i) to form a liquid formulation (iii); wherein said liquid formulation (iii) comprises a) at least one strain of YFV; b) 2% to 8% sugar; c) 1 mmol / kg to 20 mmol / kg phosphate; d) 1 mmol / kg to 50 mmol / kg of at least one carboxylate buffering agent; e) 1 mmol / kg to 10 mmol / kg MgCh; f) 0.1% to 5% D- sorbitol; g) 1 to 20 mmol / kg L-methionine; and h) 0.01 to 1 mg / mL human albumin (HA), wherein the formulation is at a pH of 7.0 to 7.5.

[0623] Aspect 4B. The method according to any of aspects 1 B to 3B, wherein the sugar is sucrose at 5%, the phosphate is 5 mmol / kg, the at least one carboxylate buffering agent is 25 mmol / kg, the MgCh is 5 mmol / kg, the D-sorbitol is 0.5%, the L-methionine is 10 mmol / kg, and the HA is 0.01%, optionally wherein the formulation is at a pH of 7.3, and / or wherein the sugar is sucrose at 5%, the phosphate is potassium phosphate at 5 mM, the at least one carboxylate buffering agent is sodium citrate at 25 mmol / kg, the MgCh is 5 mmol / kg, the D-sorbitol is 0.5%, the L-methionine is 10 mmol / kg, and the HA is 0.01 %, optionally wherein the formulation is at a pH of 7.3, and / or wherein the HA is a recombinant human albumin (rHA); and / or the formulation further comprises at least one diluent selected from the group consisting of tissue culture medium, saline and water.

[0624] Aspect 5B. The method of aspect 3B or 4B, wherein the method further comprises lyophilizing the liquid formulation (iii) to produce a lyophilized YFV formulation (iv).

[0625] Aspect 6B. The method according to any of aspects 3B to 5B, wherein (b) further comprises dividing the formulation (ii) after dialysis to produce 102TCIDsoto 106TCID50 per dose of YFV. Valneva Austria GmbH PAT070-PCT

[0626] Aspect 7B. The method according to any preceding aspect, wherein the YFV comprises or consists of a polynucleotide sequence with at least 80% identity to SEQ ID NO: 1 , suitably wherein the YFV polynucleotide sequence comprises one or more mutations selected from: A2608C, A5641G, A7225G, C8212T, A10243G, G10559GT, and / or A10722G mutations in SEQ ID NO: 1 , and / or wherein the YFV is a live, attenuated virus.

[0627] Aspect 8B. A pharmaceutical composition of YFV obtained or obtainable by the method of any preceding aspect.

[0628] Aspect 9B. A liquid yellow fever virus (YFV) formulation comprising: a) YFV particles; b) 2% to 8% sucrose; c) 1 mmol / kg to 20 mmol / kg phosphate; d) 1 mmol / kg to 50 mmol / kg of at least one carboxylate buffering agent; e) 1 mmol / kg to 10 mmol / kg MgCh; f) 0.1 % to 5% D-sorbitol; g) 1 to 20 mmol / kg L-methionine; and h) 0.001% to 0.1% human albumin (HA), wherein the formulation is at a pH of 7.0 to 7.5, wherein the formulation is for preparation of a liquid frozen or lyophilized formulation.

[0629] Aspect 10B. A reconstituted lyophilized yellow fever virus (YFV) formulation comprising: a) YFV particles; b) 2% to 8% sucrose; c) 1 mmol / kg to 20 mmol / kg phosphate; d) 1 mmol / kg to 50 mmol / kg of at least one carboxylate buffering agent; e) 1 mmol / kg to 10 mmol / kg MgCh; f) 0.1% to 5% D-sorbitol; g) 1 to 20 mmol / kg L-methionine; and h) 0.001% to 0.1% human albumin (HA), wherein the formulation is at a pH of 7.0 to 7.5.

[0630] Aspect 11 B. The formulation according to aspect 9B or 10B, wherein the YFV comprises or consists of a polynucleotide sequence with at least 80% sequence identity to SEQ ID NO: 1 , suitably wherein the YFV polynucleotide sequence comprises one or more mutations selected from: A2608C, A5641G, A7225G, C8212T, A10243G, G10559GT, and / or A10722G mutations in SEQ ID NO: 1 , and / or wherein the sugar is sucrose at 5%, the phosphate is 5 mmol / kg, the at least one carboxylate buffering agent is 25 mmol / kg, the MgCh is 5 mmol / kg, the D-sorbitol is 0.5%, the L-methionine is 10 mmol / kg, and the HA is 0.01%, optionally wherein the formulation is at a pH of 7.3, and / or wherein the sugar is sucrose at 5%, the phosphate is potassium phosphate at 5 mmol / kg, the at least one carboxylate buffering agent is sodium citrate at 25 mmol / kg, Valneva Austria GmbH PAT070-PCT the MgCh is 5 mmol / kg, the D-sorbitol is 0.5%, the L-methionine is 10 mmol / kg, and the HA is 0.01%, optionally wherein the formulation is at a pH of 7.3, and / or wherein the HA is a recombinant human albumin (rHA); and / or the YFV formulation further comprises at least one diluent selected from the group consisting of tissue culture medium, saline and water, and / or wherein the liquid or lyophilized formulation comprises an effective amount of at least one strain of YFV, optionally wherein the effective amount is 102TCIDso / dose to 106TCIDso / dose of YFV and / or wherein the YFV is a live attenuated virus, and / or wherein the formulation further comprises one or more enveloped viruses, optionally wherein the enveloped viruses are selected from Flaviviridae, Alphaviridae, Togaviridae, Coronavirus, Orthomyxoviruses, Paramyxoviridae, and Rhabdoviruses.

[0631] Aspect 12B. A method of preparing a YFV or CHIKV composition, wherein the method comprises: a. Producing a virus harvest (i); b. Dialyzing the virus harvest (i) to form a liquid formulation (ii); wherein said liquid formulation (ii) comprises a) at least one strain of virus; b) 2% to 8% sucrose; c) 1 mmol / kg to 20 mmol / kg phosphate; d) 1 mmol / kg to 50 mmol / kg of at least one carboxylate buffering agent; e) 1 mmol / kg to 10 mmol / kg MgCh; f) 0.1% to 5% D- sorbitol; g) 1 to 20 mmol / kg L-methionine; and h) 0.001 % to 0.1% human albumin (HA), wherein the formulation is at a pH of 7.0 to 7.5.

[0632] Aspect 13B. The method according to aspect 12B, further comprising mixing the YFV liquid formulation (ii) and / or CHIKV liquid formulation (ii) to produce a combined liquid formulation (iii).

[0633] Aspect 14B. The method of aspect 12B or 13B, further comprising lyophilizing the liquid formulation (iii) to produce a lyophilized formulation (iv).

[0634] Aspect 15B. The method of aspects 12B to 14B, wherein the YFV comprises or consists of a polynucleotide sequence with at least 80% sequence identity to SEQ ID NO: 1 , suitably wherein the YFV polynucleotide sequence comprises one or more mutations Valneva Austria GmbH PAT070-PCT selected from: A2608C, A5641G, A7225G, C8212T, A10243G, G10559GT, and / or

[0635] A10722G mutations in SEQ ID NO: 1 , and / or wherein the CHIKV comprises or consists of a polynucleotide sequence with at least 80%, 90%, 95%, 99% identity to SEQ ID NO: 2, and / or wherein the sucrose is 5%, the phosphate is 5 mmol / kg, the at least one carboxylate buffering agent is 25 mmol / kg, the MgCh is 5 mmol / kg, the D-sorbitol is 0.5%, the L- methionine is 10 mmol / kg, and the HA is 0.01 %, optionally wherein the formulation is at a pH of 7.3, and / or wherein the sucrose is 5%, the phosphate is potassium phosphate at 5 mmol / kg, the at least one carboxylate buffering agent is sodium citrate at 25 mmol / kg, the MgCh is 5 mmol / kg, the D-sorbitol is 0.5%, the L-methionine is 10 mmol / kg, and the HA is 0.01 %, optionally wherein the formulation is at a pH of 7.3, and / or wherein the HA is a recombinant human albumin (rHA); and / or the formulation further comprises at least one diluent selected from the group consisting of tissue culture medium, saline and water, and / or wherein the (b) further comprises dividing the composition (ii) after dialysis to produce 102TCI D50to 106TCI D50per dose of YFV and / or 102TCI D50to 106TCI D50per dose of CHIKV and / or wherein the YFV and / or CHIKV viruses are live, attenuated viruses.

[0636] Aspect 15B. A pharmaceutical composition of YFV and / or CHIKV obtained or obtainable by the method of aspects 12B to 14B.

[0637] Aspect 16B. A liquid or reconstituted lyophilized combination formulation comprising: a) YFV particles; b) CHIKV particles; c) 2% to 8% sugar; d) 1 mmol / kg to 20 mmol / kg phosphate; e) 1 mmol / kg to 50 mmol / kg of at least one carboxylate buffering agent; f) 1 mmol / kg to 10 mmol / kg MgCh; g) 0.1 % to 5% D-sorbitol; h) 1 to 20 mmol / kg L- methionine; and i) 0.001 % to 0.1 % human albumin (HA), wherein the formulation is at a pH of 7.0 to 7.5. Valneva Austria GmbH PAT070-PCT

[0638] Aspect 17B. The formulation according to aspect 16B, wherein the CHIKV comprises or consists of a polynucleotide sequence with at least 80%, 90%, 95%, 99% identity to SEQ ID NO: 2, and / or wherein the YFV comprises or consists of a polynucleotide sequence with at least 80% sequence identity to SEQ ID NO: 1 , suitably wherein the YFV polynucleotide sequence comprises one or more mutations selected from: A2608C, A5641G, A7225G, C8212T, A10243G, G10559GT, and / or A10722G mutations in SEQ ID NO: 1, and / or wherein the sugar is sucrose at 5%, the phosphate is 5 mmol / kg, the at least one carboxylate buffering agent is 25 mmol / kg, the MgCh is 5 mmol / kg, the D-sorbitol is 0.5%, the L-methionine is 10 mmol / kg, and the HA is 0.01%, optionally wherein the formulation is at a pH of 7.3, and / or wherein the sugar is sucrose at 5%, the phosphate is potassium phosphate at 5 mmol / kg, the at least one carboxylate buffering agent is sodium citrate at 25 mmol / kg, the MgCh is 5 mmol / kg, the D-sorbitol is 0.5%, the L-methionine is 10 mmol / kg, and the HA is 0.01%, optionally wherein the formulation is at a pH of 7.3, and / or wherein the HA is a recombinant human albumin (rHA); and / or the formulation further comprises at least one diluent selected from the group consisting of tissue culture medium, saline and water, and / or wherein the liquid or lyophilized formulation comprises an effective amount of at least one strain of YFV, optionally wherein the effective amount is 102TCIDso / dose to 106TCIDso / dose of YFV and 102TCID50 / dose to 106TCID50 / dose, of CHIKV, and / or wherein the viruses are live, attenuated viruses, and / or wherein the formulation further comprises one or more enveloped viruses, optionally wherein the enveloped viruses are selected from Flaviviridae, Alphaviridae, Togaviridae, Coronavirus, Orthomyxoviruses, Paramyxoviridae, and Rhabdoviruses.

[0639] Aspect 18B. The formulation according to aspects 8B to 11 B and 15B to 17B, for use in preventing YFV infection. Valneva Austria GmbH PAT070-PCT

[0640] Aspect 19B. The formulation according to any of aspects 15B to 17B, for use in preventing YFV and / or CHIKV infection.

[0641] Aspect 20B. A method of preventing YFV infection comprising administering the formulation according to any of aspects 8B to 11 B and 15B to 17B to a subject.

[0642] Aspect 21 B. A method of preventing YFV and / or CHIKV infection comprising administering the formulation according to any of aspects 15B to 17B to a subject.

[0643] Aspect 22B. The method according to aspect 21 B, wherein the YFV and / or CHIKV liquid formulation is provided in a single-dose pre-filled syringe.

[0644] Aspect 23B. The method according to aspect 22B, wherein the YFV and / or CHIKV liquid formulation is provided in a single-dose pre-filled syringe for simultaneous administration.

[0645] Aspect 24B. The pre-filled syringe assembly of aspects 22B and 23B, wherein the YFV and / or CHIKV liquid formulation is stable after being stored at temperatures ranging from -15°C to -80°C for at least 12 months.

[0646] Aspect 25B. A pharmaceutical composition comprising YFV and optionally CHIKV, wherein the formulation is liquid frozen formulation.

[0647] Aspect 26B. A pharmaceutical composition comprising YFV and optionally CHIKV, wherein the formulation is lyophilized formulation.

[0648] Aspect 27B. The pharmaceutical composition of aspect 25B or 26B, wherein the formulation is a single dose or multi-dose formulation.

[0649] Aspect 28B. The composition of aspect 26B or 27B, wherein the lyophilized formulation is reconstitution with water for Injection (WFI).

[0650] Aspect 29B. The pharmaceutical composition according to aspect 27B, wherein the single dose formulation is reconstituted in 0.5mL of WFI.

[0651] SEQUENCE LISTING Valneva Austria GmbH PAT070-PCT

[0652] SEQ ID NO: 1 YFV (At position 8,212 there is a 50% heterogeneity C / T which does not affect the amino acid. In the provided sequence, position 8,212 is a C.)

[0653] AGTAAATCCTGTGTGCTAATTG AG GTG CATTG GTCTG CAAATCG AGTTG CTAG G CAATAA AC AC ATTTG G ATTAATTTTAATCG TTCGTTG AG CG ATTAG CAG AG A ACTG ACCAG AACATGTCTG GTCGTAAAG CTCAG G G AAAAACCCTGG G CGTC AATATG GT

[0654] ACGACGAGGAGTTCGCTCCTTGTCAAACAAAATAAAACAAAAAACAAAACAAATTGGAAACAGACCTGGACCTTCAAGAG GTGTTCAAGGATTTATCTTTTTCTTTTTGTTCAACATTTTGACTGGAAAAAAGATCACAGCCCACCTAAAGAGGTTGTGGAAA ATGCTGGACCCAAGACAAGGCTTGGCTGTTCTAAGGAAAGTCAAGAGAGTGGTGGCCAGTTTGATGAGAGGATTGTCCTC

[0655] AAGGAAACGCCGTTCCCATGATGTTCTGACTGTGCAATTCCTAATTTTGGGAATGCTGTTGATGACGGGTGGAGTGACCTTG GTGCGGAAAAACAGATGGTTGCTCCTAAATGTGACATCTGAGGACCTCGGGAAAACATTCTCTGTGGGCACAGGCAACTGC ACAACAAACATTTTGGAAGCCAAGTACTGGTGCCCAGACTCAATGGAATACAACTGTCCCAATCTCAGTCCAAGAGAGGAG

[0656] CCAGATGACATTGATTGCTGGTGCTATGGGGTGGAAAACGTTAGAGTCGCATATGGTAAGTGTGACTCAGCAGGCAGGTCTA

[0657] GGAGGTCAAGAAGGGCCATTGACTTGCCTACGCATGAAAACCATGGTTTGAAGACCCGGCAAGAAAAATGGATGACTGGA AGAATGGGTGAAAGGCAACTCCAAAAGATTGAGAGATGGTTCGTGAGGAACCCCTTTTTTGCAGTGACGGCTCTGACCATT

[0658] GCCTACCTTGTGGGAAGCAACATGACGCAACGAGTCGTGATTGCCCTACTGGTCTTGGCTGTTGGTCCGGCCTACTCAGCTC ACTGCATTGGAATTACTGACAGGGATTTCATTGAGGGGGTGCATGGAGGAACTTGGGTTTCAGCTACCCTGGAGCAAGACA

[0659] AGTGTGTC ACTGTTATG G CCCCTG ACAAG CCTTCATTG G ACATCTCACTAG AG AC AGTAG CCATTG ATAG ACCTG CTG AG GTG AGGAAAGTGTGTTACAATGCAGTTCTCACTCATGTGAAGATTAATGACAAGTGCCCCAGCACTGGAGAGGCCCACCTAGCTG AAGAGAACGAAGGGGACAATGCGTGCAAGCGCACTTATTCTGATAGAGGCTGGGGCAATGGCTGTGGCCTATTTGGGAAA G G G AG CATTGTG GCATGCG CC AAATTCACTTGTG CCAAATCCATG AGTTTGTTTG AG GTTG ATCAG ACCAAAATTCAGTATGT CATCAGAGCACAATTGCATGTAGGGGCCAAGCAGGAAAATTGGAATACCGACATTAAGACTCTCAAGTTTGATGCCCTGTCA

[0660] GGCTCCCAGGAAGTCGAGTTCATTGGGTATGGAAAAGCTACACTGGAATGCCAGGTGCAAACTGCGGTGGACTTTGGTAAC AGTTACATCGCTGAGATGGAAACAGAGAGCTGGATAGTGGACAGACAGTGGGCCCAGGACTTGACCCTGCCATGGCAGAG

[0661] TGGAAGTGGCGGGGTGTGGAGAGAGATGCATCATCTTGTCGAATTTGAACCTCCGCATGCCGCCACTATCAGAGTACTGGC CCTGGGAAACCAGGAAGGCTCCTTGAAAACAGCTCTTACTGGCGCAATGAGGGTTACAAAGGACACAAATGACAACAACC

[0662] TTTACAAACTACATGGTGGACATGTTTCTTGCAGAGTGAAATTGTCAGCTTTGACACTCAAGGGGACATCCTACAAAATATGC ACTGACAAAATGTTTTTTGTCAAGAACCCAACTGACACTGGCCATGGCACTGTTGTGATGCAGGTGAAAGTGTCAAAAGGA GCCCCCTGCAGGATTCCAGTGATAGTAGCTGATGATCTTACAGCGGCAATCAATAAAGGCATTTTGGTTACAGTTAACCCCAT CGCCTCAACCAATGATGATGAAGTGCTGATTGAGGTGAACCCACCTTTTGGAGACAGCTACATTATCGTTGGGAGAGGAGAT

[0663] TCACGTCTCACTTACCAGTG G CACAA AG AG G G A AG CTCAATAG G AAAGTTGTTC ACTCAG ACCATG AAAGGCGTGGAACGC CTGGCCGTCATGGGAGACACCGCCTGGGATTTCAGCTCCGCTGGAGGGTTCTTCACTTCGGTTGGGAAAGGAATTCATACG GTGTTTG G CTCTG CCTTTCAG G G G CTATTTG G CG GCTTG A ACTG G ATAACAAAG GTCATCATG G G G G CG GTACTTATATG G G TTGGCATCAACACAAGAAACATGACAATGTCCATGAGCATGATCTTGGTAGGAGTGATCATGATGTTTTTGTCTCTAGGAGTT GGGGCGGATCAAGGATGCGCCATCAACTTTGGCAAGAGAGAGCTCAAGTGCGGAGATGGTATCTTCATATTTAGAGACTCT GATGACTGGCTGAACAAGTACTCATACTATCCAGAAGATCCTGTGAAGCTTGCATCAATAGTGAAAGCCTCTTTTGAAGAAG GGAAGTGTGGCCTAAATTCAGTTGACTCCCTTGAGCATGAGATGTGGAGAAGCAGGGCAGATGAGATCAATGCCATTTTTG

[0664] AGGAAAACGAGGTGGACATTTCTGTTGTCGTGCAGGATCCAAAGAATGTTTACCAGAGAGGAACTCATCCATTTTCCAGAAT TCGGGATGGTCTGCAGTATGGTTGGAAGACTTGGGGTAAGAACCTTGTGTTCTCCCCAGGGAGGAAGAATGGAAGCTTCAT CATAGATGGAAAGTCCAGGAAAGAATGCCCGTTTTCAAACCGGGTCTGGAATTCTTTCCAGATAGAGGAGTTTGGGACGGG

[0665] AGTGTTCACCACACGCGTGTACATGGACGCAGTCTTTGAATACACCATAGACTGCGATGGATCTATCTTGGGTGCAGCGGTG AACGGAAAAAAGAGTGCCCATGGCTCTCCAACATTTTGGATGGGAAGTCATGAAGTAAATGGGACATGGATGATCCACACC

[0666] TTGGAGGCATTAGATTACAAGGAGTGTGAGTGGCCACTGACACATACGATTGGAACATCAGTTGAAGAGAGTGAAATGTTC ATGCCGAGATCAATCGGAGGCCCAGTTAGCTCTCACAATCATATCCCTGGATACAAGGTTCAGACGAACGGACCTTGGATGC AGGTACCACTAGAAGTGAAGAGAGAAGCTTGCCCAGGGACTAGCGTGATCATTGATGGCAACTGTGATGGACGGGGAAAA

[0667] TCAACCAGATCCACCACGGATAGCGGGAAAGTTATTCCTGAATGGTGTTGCCGCTCCTGCACAATGCCGCCTGTGAGCTTCC

[0668] ATGGTAGTGATGGGTGTTGGTATCCCATGGAAATTAGGCCAAGGAAAACGCATGAAAGCCATCTGGTGCGCTCCTGGGTTAC AGCTGGAGAAATACATGCTGTCCCTTTTGGTTTGGTGAGCATGATGATAGCAATGGAAGTGGTCCTAAGGAAAAGACAGGG ACCAAAGCAAATGTTGGTTGGAGGAGTAGTGCTCTTGGGAGCAATGCTGGTCGGGCAAGTAACTCTCCTTGATTTGCTGAA ACTCACAGTGGCTGTGGGATTGCATTTCCATGAGATGAACAATGGAGGAGACGCCATGTATATGGCGTTGATTGCTGCCTTTT

[0669] CAATCAGACCAGGGCTGCTCATCGGCTTTGGGCTCAGGACCCTATGGAGCCCTCGGGAACGCCTTGTGCTGACCCTAGGAG CAG CCATG GTG G AG ATTG CCTTG G GTG G CGTG ATG G G CG G CCTGTG G AAGTATCTAAATG CAGTTTCTCTCTG CATCCTG AC AATAAATGCTGTTGCTTCTAGGAAAGCATCAAATACCATCTTGCCCCTCATGGCTCTGTTGACACCTGTCACTATGGCTGAGGT GAGACTTGCCGCAATGTTCTTTTGTGCCGTGGTTATCATAGGGGTCCTTCACCAGAATTTCAAGGACACCTCCATGCAGAAG ACTATAC CTCTG GTG G CCCTCAC ACTCAC ATCTTACCTG G G CTTG ACACAACCTTTTTTG G G CCTGTGTGCATTTCTG G CAACC CGCATATTTGGGCGAAGGAGTATCCCAGTGAATGAGGCACTCGCAGCAGCTGGTCTAGTGGGAGTGCTGGCAGGACTGGC TTTTCAGGAGATGGAGAACTTCCTTGGTCCGATTGCAGTTGGAGGACTCCTGATGATGCTGGTTAGCGTGGCTGGGAGGGT

[0670] GGATGGGCTAGAGCTCAAGAAGCTTGGTGAAGTTTCATGGGAAGAGGAGGCGGAGATCAGCGGGAGTTCCGCCCGCTAT Valneva Austria GmbH PAT070-PCT

[0671] GATGTGGCACTCAGTGAACAAGGGGAGTTCAAGCTGCTTTCTGAAGAGAAAGTGCCATGGGACCAGGTTGTGATGACCTC GCTGGCCTTGGTTGGGGCTGCCCTCCATCCATTTGCTCTTCTGCTGGTCCTTGCTGGGTGGCTGTTTCATGTCAGGGGAGCT AGGAGAAGTGGGGATGTCTTGTGGGATATTCCCACTCCTAAGATCATCGAGGAATGTGAACATCTGGAGGATGGGATTTATG GCATATTCCAGTCAACCTTCTTGGGGGCCTCCCAGCGAGGAGTGGGAGTGGCACAGGGAGGGGTGTTCCACACAATGTGG

[0672] CATGTCACAAGAGGAGCTTTCCTTGTCAGGAATGGCAAGAAGTTGATTCCATCTTGGGCTTCAGTAAAGGAAGACCTTGTC GCCTATGGTGGCTCATGGAAGTTGGAAGGCAGATGGGATGGAGAGGAAGAGGTCCAGTTGATCGCGGCTGTTCCAGGAAA

[0673] GAACGTGGTCAACGTCCAGACAAAACCGAGCTTGTTCAAAGTGAGGAATGGGGGAGAAATCGGGGCTGTCGCTCTTGACT ATCCG AGTG G C ACTTCAG G ATCTCCTATTGTTAAC AG G AACG G AG AG GTG ATTG G G CTGTACG G C AATG G CATCCTTGTCG G

[0674] TGACAACTCCTTCGTGTCCGCCATATCCCAGACTGAGGTGAAGGAAGAAGGAAAGGAGGAGCTCCAAGAGATCCCGACAA TGCTAAAGAAAGGAATGACAACTGTCCTTGATTTTCATCCTGGAGCTGGGAAGACAAGACGTTTCCTCCCACAGATCTTGGC CGAGTGCGCACGGAGACGCTTGCGCACTCTTGTGTTGGCCCCCACCAGGGTTGTTCTTTCTGAAATGAAGGAGGCTTTTCA CGGCCTGGACGTGAAATTCCACACACAGGCTTTTTCCGCTCACGGCAGCGGGAGAGAAGTCATTGATGCCATGTGCCATGC CACCCTAACTTACAGGATGTTGGAACCAACTAGGGTTGTTAACTGGGAAGTGATCATTATGGATGAAGCCCATTTTTTGGATC

[0675] CAGCTAGCATAGCCGCTAGAGGTTGGGCAGCGCACAGAGCTAGGGCAAATGAAAGTGCAACAATCTTGATGACAGCCACA CCGCCTGGGACTAGTGATGAATTTCCACATTCAAATGGTGAAATAGAAGATGTTCAAACGGACATACCCAGTGAGCCCTGGA ACACAGGGCATGACTGGATCCTGGCTGACAAAAGGCCCACGGCATGGTTCCTTCCATCCATCAGAGCTGCAAATGTCATGGC TGCCTCTTTGCGTAAGGCTGGAAAGAGTGTGGTGGTCCTGAACAGGAAAACCTTTGAGAGAGAATACCCCACGATAAAGCA

[0676] GAAGAAACCTGACTTTATATTGGCCACTGACATAGCTGAAATGGGAGCCAACCTTTGCGTGGAGCGAGTGCTGGATTGCAG GACGGCTTTTAAGCCTGTGCTTGTGGATGAAGGGAGGAAGGTGGCAATAAAAGGGCCACTTCGTATCTCCGCATCCTCTGC

[0677] TGCTCAAAGGAGGGGGCGCATTGGGAGAAATCCCAACAGAGATGGAGACTCATACTACTATTCTGAGCCTACAAGTGAAAA TAATGCCCACCACGTCTGCTGGTTGGAGGCCTCAATGCTCTTGGACAACATGGAGGTGAGGGGTGGAATGGTCGCCCCACT CTATGGCGTTGAAGGAACTAAAACACCAGTTTCCCCTGGTGAAATGAGACTGAGGGATGACCAGAGGAAAGTCTTCAGAG

[0678] AACTAGTGAGGAATTGTGACCTGCCCGTTTGGCTTTCGTGGCAAGTGGCCAAGGCTGGTTTGAAGACGAATGATCGTAAGT GGTGTTTTGAAGGCCCTGAGGAACATGAGATCTTGAATGACAGCGGTGAAACAGTGAAGTGCAGGGCTCCTGGAGGAGC AAAGAAGCCTCTGCGCCCAAGGTGGTGTGATGAAAGGGTGTCATCTGACCAGAGTGCGCTGTCTGAATTTATTAAGTTTGCT

[0679] GAAGGTAGGAGGGGAGCTGCTGAAGTGCTAGTTGTGCTGAGTGAACTCCCTGATTTCCTGGCTAAAAAAGGTGGAGAGGC AATGGATACCATCAGTGTGTTTCTCCACTCTGAGGAAGGCTCTAGGGCTTACCGCAATGCACTATCAATGATGCCTGAGGCAA TG ACAATAGTC ATG CTGTTTATACTG G CTG G ACTACTG ACATCG G G AATGGTC ATCTTTTTCATGTCTCCCAAAG G CATC AGTA GAATGTCTATGGCGATGGGCACAATGGCCGGCTGTGGATATCTCATGTTCCTTGGAGGCGTCAAACCCACTCACATCTCCTAT ATCATGCTCATATTCTTTGTCCTGATGGTGGTTGTGATCCCCGAGCCAGGGCAACAAAGGTCCATCCAAGACAACCAAGTGG CATACCTCATTATTGGCATCCTGACGCTGGTTTCAGCGGTGGCAGCCAACGAGCTAGGCATGCTGGAGAAAACCAAAGAGG ACCTCTTTGGGAAGAAGAACTTAATTCCATCTAGTGCTTCACCCTGGAGTTGGCCGGATCTTGACCTGAAGCCAGGAGCTGC CTGGACAGTGTACGTTGGCATTGTTACAATGCTCTCTCCAATGTTGCACCACTGGATCAAAGTCGAATATGGCAACCTGTCTC TGTCTGGAATAGCCCAGTCAGCCTCAGTCCTTTCTTTCATGGACAAGGGGATACCATTCATGAAGATGAATATCTCGGTCATA

[0680] ATGCTGCTGGTCAGTGGCTGGAATTCAATAACAGTGATGCCTCTGCTCTGTGGCATAGGGTGCGCCATGCTCCACTGGTCTCT CATTTTACCTGGAATCAAAGCGCAGCAGTCAAAGCTTGCACAGAGAAGGGTGTTCCATGGCGTTGCCAAGAACCCTGTGGT TGATGGGAATCCAACAGTTGACATTGAGGAAGCTCCTGAAATGCCTGCCCTTTATGAGAAGAAACTGGCTCTATATCTCCTTC TTGCTCTCAGCCTAGCTTCTGTTGCCATGTGCAGAACGCCCTTTTCATTGGCTGAAGGCATTGTCCTAGCATCAGCTGCCTTA

[0681] GGGCCGCTCATAGAGGGAAACACCAGCCTTCTTTGGAATGGACCCATGGCTGTCTCCATGACAGGAGTCATGAGGGGGAAT CACTATGCTTTTGTGGGAGTCATGTACAATCTATGGAAGATGAAAACTGGACGCCGGGGGAGCGCGAATGGAAAAACTTTG GGTGAAGTCTGGAAGAGGGAACTGAATCTGTTGGACAAGCGACAGTTTGAGTTGTATAAAAGGACCGACATTGTGGAGGT

[0682] GGATCGTGATACGGCACGCAGGCATTTGGCCGAAGGGAAGGTGGACACCGGGGTGGCGGTCTCCAGGGGGACCGCAAA GTTAAG GTG GTTCCATG AG CGTG G CTATGTCAAG CTG G A AG GTAG G GTG ATTG AC CTG G G GTGTG G CCG CG G AG G CTG GT GTTACTACGCTGCTGCGCAAAAGGAAGTGAGTGGGGTCAAAGGATTTACTCTTGGAAGAGACGGCCATGAGAAACCCATG AATGTG CAA AGTCTG G G ATG G AACATCATCACCTTCAAG G AC A AAACTG ATATCCACCG CCTAG AACC AGTG AAATGTG AC A CCCTTTTGTGTGACATTGGAGAGTCATCATCGTCATCGGTCACAGAGGGGGAAAGGACCGTGAGAGTTCTTGATACTGTAGA AAAATGGCTGGCTTGTGGGGTTGACAACTTCTGTGTGAAGGTGTTAGCTCCATACATGCCAGATGTTCTCGAGAAACTGGAA TTGCTCCAAAGGAGGTTTGGCGGAACAGTGATCAGGAACCCTCTCTCCAGGAATTCCACTCATGAAATGTACTACGTGTCTG GAGCCCGCAGCAATGTCACATTTACTGTGAACCAAACATCCCGCCTCCTGATGAGGAGAATGAGGCGTCCAACTGGAAAAG TGACCCTGGAGGCTGACGTCATCCTCCCAATTGGGACACGCAGTGTTGAGACAGACAAGGGACCCCTGGACAAAGAGGCC ATAGAAGAAAGGGTTGAGAGGATAAAATCTGAGTACATGACCTCTTGGTTTTATGACAATGACAACCCCTACAGGACCTGGC ACTACTGTG G CTCCTATGTC AC AAAAACCTCAG G AAGTGCG G CG AG CATG GTAAATG GTGTTATTAAAATTCTG ACATATCCA TGGGACAGGATAGAGGAGGTCACAAGAATGGCAATGACTGACACAACCCCTTTTGGACAGCAAAGAGTGTTTAAAGAAAA AGTTGACACCAGAGCAAAGGATCCACCAGCGGGAACTAGGAAGATCATGAAAGTTGTCAACAGGTGGCTGTTCCGCCACC TGGCCAGAGAAAAGAACCCCAGACTGTGCACAAAGGAAGAATTTATTGCAAAAGTCCGAAGTCATGCAGCCATTGGAGCTT ACCTGGAAGAACAAGAACAGTGGAAGACTGCCAATGAGGCTGTCCAAGACCCAAAGTTCTGGGAACTGGTGGATGAAGA AAGGAAGCTGCACCAACAAGGCAGGTGTCGGACTTGTGTGTACAACATGATGGGGAAAAGAGAGAAGAAGCTGTCAGAG Valneva Austria GmbH PAT070-PCT

[0683] TTTGGGAAAGCAAAGGGAAGCCGTGCCATATGGTATATGTGGCTGGGAGCGCGGTATCTTGAGTTTGAGGCCCTGGGATTC CTGAATGAGGACCATTGGGCTTCCAGGGAAAACTCAGGAGGAGGAGTGGAAGGCATTGGCTTACAATACCTAGGATATGTG

[0684] ATCAGAGACCTGGCTGCAATGGATGGTGGTGGATTCTACGCGGATGACACCGCTGGATGGGACACGCGCATCACAGAGGC AGACCTTGATGATGAACAGGAGATCTTGAACTACATGAGCCCACATCACAAAAAACTGGCACAAGCAGTGATGGAAATGAC ATACAAGAACAAAGTGGTGAAAGTGTTGAGACCAGCCCCAGGAGGGAAAGCCTACATGGATGTCATAAGTCGACGAGACC AGAGAGGATCCGGGCAGGTAGTGACTTATGCTCTGAACACCATCACCAACTTGAAAGTCCAATTGATCAGAATGGCAGAAG CAGAGATGGTGATACATCACCAACATGTTCAAGATTGTGATGAATCAGTTCTGACCAGGCTGGAGGCATGGCTCACTGAGCA CGGATGTAACAGACTGAAGAGGATGGCGGTGAGTGGAGACGACTGTGTGGTCCGGCCCATCGATGACAGGTTCGGCCTGG CCCTGTCCCATCTCAACGCCATGTCCAAGGTTAGAAAGGACATATCTGAATGGCAGCCATCAAAAGGGTGGAATGATTGGGA GAATGTGCCCTTCTGTTCCCACCACTTCCATGAACTACAGCTGAAGGATGGCAGGAGGATTGTGGTGCCTTGCCGAGAACA GGACGAGCTCATTGGGAGAGGAAGGGTGTCTCCAGGAAACGGCTGGATGATCAAGGAAACAGCTTGCCTCAGCAAAGCC TATG CC AACATGTG GTCACTG ATGTATTTTCAC AAA AG G G ACATG AG G CTACTGTC ATTG G CTGTTTCCTCAG CTGTTCCC AC CTCATGGGTTCCACAAGGACGCACAACATGGTCGATTCATGGGAAAGGGGAGTGGATGACCACGGAAGACATGCTTGAGG TGTGGAACAGAGTATGGATAACCAACAACCCACACATGCAGGACAAGACAATGGTGAAAAAATGGAGAGATGTCCCTTATC TAACCAAGAGACAAGACAAGCTGTGCGGATCACTGATTGGAATGACCAATAGGGCCACCTGGGCCTCCCACATCCATTTGGT CATCCATCGTATCCGAACGCTGATTGGACAGGAGAAATACACTGACTACCTAACAGTCATGGACAGGTATTCTGTGGATGCTG

[0685] ACCTGCAACTGGGTGAGCTTATCTGAAACACCATCTAACAGGAATAACCGGGATACAAACCACGGGTGGAGAACCGGACTC CCCACAACCTGAAACCGGGATATAAACCACGGCTGGAGAACCGGACTCCGCACTTAAAATGAAACAGAAACCGGGATAAAA ACTACGGATGGAGAACCGGACTCCACACATTGAGACAGAAGAAGTTGTCAGCCCAGAACCCCACACGAGTTTTGCCACTGC TAAGCTGTGAGGCAGTGCAGGCTGGGACAGCCGACCTCCAGGTTGCGAAAAACCTGGTTTCTGGGACCTCCCACCCCAGA GTAAAAAGAACGGAGCCTCCGCTACCACCCTCCCACGTGGTGGTAGAAAGACGGGGTCTAGAGGTTAGAGGAGACCCTCC AGGGAACAAATAGTGGGACCATATTGACGCCAGGGAAAGACCGGAGTGGTTCTCTGCTTTTCCTCCAGAGGTCTGTGAGCA CAGTTTGCTCAAGAATAAGCAGACCTTTGGATGACAAACACAAAACCACT

[0686] SEQ ID NO: 2 CHIKV

[0687] Nucleotide sequence of CHIKV-A5nsP3

[0688] GATGGCTGCGTGAGACACACGTAGCCTACCAGTTTCTTACTGCTCTACTCTGCAAAGCAAGAGATTAATAACCCATCATGGAT

[0689] CCTGTGTACGTGGACATAGACGCTGACAGCGCCTTTTTGAAGGCCCTGCAACGTGCGTACCCCATGTTTGAGGTGGAACCA AGGCAGGTCACACCGAATGACCATGCTAATGCTAGAGCGTTCTCGCATCTAGCTATAAAACTAATAGAGCAGGAAATTGACC CCGACTCAACCATCCTGGATATCGGCAGTGCGCCAGCAAGGAGGATGATGTCGGACAGGAAGTACCACTGCGTCTGCCCGA TGCGCAGTGCGGAAGATCCCGAGAGACTCGCCAATTATGCGAGAAAGCTAGCATCTGCCGCAGGAAAAGTCCTGGACAGA AACATCTCTGGAAAGATCGGGGACTTACAAGCAGTAATGGCCGTGCCAGACACGGAGACGCCAACATTCTGCTTACACACA GACGTCTCATGTAGACAGAGAGCAGACGTCGCTATATACCAAGACGTCTATGCTGTACACGCACCCACGTCGCTATACCACCA GGCGATTAAAGGGGTCCGAGTGGCGTACTGGGTTGGGTTCGACACAACCCCGTTCATGTACAATGCCATGGCGGGTGCCTA CCCCTCATACTCGACAAACTGGGCAGATGAGCAGGTACTGAAGGCTAAGAACATAGGATTATGTTCAACAGACCTGACGGA AGGTAGACGAGGCAAGTTGTCTATTATGAGAGGGAAAAAGCTAAAACCGTGCGACCGTGTGCTGTTCTCAGTAGGGTCAAC GCTCTACCCGGAAAGCCGCAAGCTACTTAAGAGCTGGCACCTGCCATCGGTGTTCCATTTAAAGGGCAAACTCAGCTTCACA TGCCGCTGTGATACAGTGGTTTCGTGTGAGGGCTACGTCGTTAAGAGAATAACGATGAGCCCAGGCCTTTATGGAAAAACCA CAGGGTATGCGGTAACCCACCACGCAGACGGATTCCTGATGTGCAAGACTACCGACACGGTTGACGGCGAAAGAATGTCAT TCTCGGTGTGCACATACGTGCCGGCGACCATTTGTGATCAAATGACCGGCATCCTTGCTACAGAAGTCACGCCGGAGGATGC ACAGAAGCTGTTGGTGGGGCTGAACCAGAGAATAGTGGTTAACGGCAGAACGCAACGGAATACGAACACCATGAAAAATT ATCTGCTTCCCGTGGTCGCCCAAGCCTTCAGTAAGTGGGCAAAGGAGTGCCGGAAAGACATGGAAGATGAAAAACTCCTG G G G GTC AG AG AAAG AACACTG ACCTGCTG CTGTCTATG G GCATTCAAG AAG CAG AAAACACAC ACG GTCTACAAG AG G CC TGATACCCAGTCAATTCAGAAGGTTCAGGCCGAGTTTGACAGCTTTGTGGTACCGAGTCTGTGGTCGTCCGGGTTGTCAATC CCTTTGAGGACTAGAATCAAATGGTTGTTAAGCAAGGTGCCAAAAACCGACCTGATCCCATACAGCGGAGACGCCCGAGAA GCCCGGGACGCAGAAAAAGAAGCAGAGGAAGAACGAGAAGCAGAACTGACTCGCGAAGCCCTACCACCTCTACAGGCAG

[0690] CACAGGAAGATGTTCAGGTCGAAATCGACGTGGAACAGCTTGAGGACAGAGCGGGCGCAGGAATAATAGAGACTCCGAG AGGAGCTATCAAAGTTACTGCCCAACCAACAGACCACGTCGTGGGAGAGTACCTGGTACTCTCCCCGCAGACCGTACTACGT AGCCAGAAGCTCAGTCTGATTCACGCTTTGGCGGAGCAAGTGAAGACGTGCACGCACAACGGACGAGCAGGGAGGTATG CGGTCGAAGCGTACGACGGCCGAGTCCTAGTGCCCTCAGGCTATGCAATCTCGCCTGAAGACTTCCAGAGTCTAAGCGAAA GCGCAACGATGGTGTATAACGAAAGAGAGTTCGTAAACAGAAAGCTACACCATATTGCGATGCACGGACCAGCCCTGAACA CCGACGAAGAGTCGTATGAGCTGGTGAGGGCAGAGAGGACAGAACACGAGTACGTCTACGACGTGGATCAGAGAAGATG CTGTAAGAAGGAAGAAGCCGCAGGACTGGTACTGGTGGGCGACTTGACTAATCCGCCCTACCACGAATTCGCATATGAAGG GCTAAAAATCCGCCCTGCCTGCCCATACAAAATTGCAGTCATAGGAGTCTTCGGAGTACCGGGATCTGGCAAGTCAGCTATTA TCAAGAACCTAGTTACCAGGCAGGACCTGGTGACTAGCGGAAAGAAAGAAAACTGCCAAGAAATCACCACCGACGTGATG AGACAGAGAGGTCTAGAGATATCTGCACGTACGGTTGACTCGCTGCTCTTGAATGGATGCAACAGACCAGTCGACGTGTTGT Valneva Austria GmbH PAT070-PCT

[0691] ACGTAGACGAGGCGTTTGCGTGCCACTCTGGAACGCTACTTGCTTTGATCGCCTTGGTGAGACCAAGGCAGAAAGTTGTAC TTTGTGGTGACCCGAAGCAGTGCGGCTTCTTCAATATGATGCAGATGAAAGTCAACTATAATCACAACATCTGCACCCAAGTG

[0692] TACCACAAAAGTATCTCCAGGCGGTGTACACTGCCTGTGACCGCCATTGTGTCATCGTTGCATTACGAAGGCAAAATGCGCAC

[0693] TACGAATGAGTACAACAAGCCGATTGTAGTGGACACTACAGGCTCAACAAAACCTGACCCTGGAGACCTCGTGTTAACGTGC TTCAGAGGGTGGGTTAAACAACTGCAAATTGACTATCGTGGATACGAGGTCATGACAGCAGCCGCATCCCAAGGGTTAACC AG A AAAG G AGTTTACG CAGTTAG ACAAAA AGTTAATG AAAACCCG CTCTATG C ATCAACGTCAG AG CACGTCA ACGTACTCC TAACGCGTACGGAAGGTAAACTGGTATGGAAGACACTTTCCGGCGACCCGTGGATAAAGACGCTGCAGAACCCACCGAAA G G AAACTTCAAAG C AACTATTA AG GAGTGGGAGGTGGAG CATG CATCAATAATG G CG G G CATCTG CAGTCACCAAATG ACC

[0694] TTCGATACATTCCAAAATAAAGCCAACGTTTGTTGGGCTAAGAGCTTGGTCCCTATCCTCGAAACAGCGGGGATAAAACTAA

[0695] ATGATAGGCAGTGGTCTCAGATAATTCAAGCCTTCAAAGAAGACAAAGCATACTCACCTGAAGTAGCCCTGAATGAAATATGT

[0696] ACGCGCATGTATGGGGTGGATCTAGACAGCGGGCTATTTTCTAAACCGTTGGTGTCTGTGTATTACGCGGATAACCACTGGG

[0697] ATAATAGGCCTGGAGGGAAAATGTTCGGATTTAACCCCGAGGCAGCATCCATTCTAGAAAGAAAGTATCCATTCACAAAAGG GAAGTGGAACATCAACAAGCAGATCTGCGTGACTACCAGGAGGATAGAAGACTTTAACCCTACCACCAACATCATACCGGCC AACAGGAGACTACCACACTCATTAGTGGCCGAACACCGCCCAGTAAAAGGGGAAAGAATGGAATGGCTGGTTAACAAGAT AAACG G CCACC ACGTG CTCCTG GTC AGTG G CTATAACCTTG CACTG CCTACTA AG AG AGTC ACTTG G GTAG CG CCGTTAG GT

[0698] GTCCGCGGAGCGGACTACACATACAACCTAGAGTTGGGTCTGCCAGCAACGCTTGGTAGGTATGACCTAGTGGTCATAAACA

[0699] TCCACACACCTTTTCGCATACACCATTACCAACAGTGCGTCGACCACGCAATGAAACTGCAAATGCTCGGGGGTGACTCATT GAGACTGCTCAAACCGGGCGGCTCTCTATTGATCAGAGCATATGGTTACGCAGATAGAACCAGTGAACGAGTCATCTGCGTA

[0700] TTGGGACGCAAGTTTAGATCGTCTAGAGCGTTGAAACCACCATGTGTCACCAGCAACACTGAGATGTTTTTCCTATTCAGCA

[0701] ACTTTGACAATGGCAGAAGGAATTTCACAACTCATGTCATGAACAATCAACTGAATGCAGCCTTCGTAGGACAGGTCACCCG AGCAGGATGTGCACCGTCGTACCGGGTAAAACGCATGGACATCGCGAAGAACGATGAAGAGTGCGTAGTCAACGCCGCTA

[0702] ACCCTCGCGGGTTACCGGGTGGCGGTGTTTGCAAGGCAGTATACAAAAAATGGCCGGAGTCCTTTAAGAACAGTGCAACAC

[0703] CAGTGGGAACCGCAAAAACAGTTATGTGCGGTACGTATCCAGTAATCCACGCTGTTGGACCAAACTTCTCTAATTATTCGGAG TCTGAAGGGGACCGGGAATTGGCAGCTGCCTATCGAGAAGTCGCAAAGGAAGTAACTAGGCTGGGAGTAAATAGTGTAGC TATACCTCTCCTCTCCACAGGTGTATACTCAGGAGGGAAAGACAGGCTGACCCAGTCACTGAACCACCTCTTTACAGCCATG GACTCGACGGATGCAGACGTGGTCATCTACTGCCGCGACAAAGAATGGGAGAAGAAAATATCTGAGGCCATACAGATGCGG

[0704] ACCCAAGTAGAGCTGCTGGATGAGCACATCTCCATAGACTGCGATATTGTTCGCGTGCACCCTGACAGCAGCTTGGCAGGCA

[0705] GAAAAGGATACAGCACCACGGAAGGCGCACTGTACTCATATCTAGAAGGGACCCGTTTTCATCAGACGGCTGTGGATATGG CGGAGATACATACTATGTGGCCAAAGCAAACAGAGGCCAATGAGCAAGTCTGCCTATATGCCCTGGGGGAAAGTATTGAATC GATCAGGCAGAAATGCCCGGTGGATGATGCAGACGCATCATCTCCCCCCAAAACTGTCCCGTGCCTTTGCCGTTACGCTATG

[0706] ACTCCAGAACGCGTCACCCGGCTTCGCATGAACCACGTCACAAGCATAATTGTGTGTTCTTCGTTTCCCCTCCCAAAGTACAA

[0707] AATAG AAG G AGTG C AAAA AGTCAAATG CTCTA AG GTAATG CTATTTG ACC ACAACGTG CCATCG CG CGTAAGTCC AAG G G CT TATAGAGGTGCCGCTGCCGGTAACCTTGCGGCCGTGTCTGATTGGGTAATGAGCACCGTACCTGTCGCGCCGCCCAGAAGA AGGCGAGGGAGAAACCTGACTGTGACATGTGACGAGAGAGAAGGGAATATAACACCCATGGCTAGCGTCCGATTCTTTAG GGCAGAGCTGTGTCCGGTCGTACAAGAAACAGCGGAGACGCGTGACACAGCAATGTCTCTTCAGGCACCACCGAGTACCG CCACGGAACCGAATCATCCGCCGATCTCCTTCGGAGCATCAAGCGAGACGTTCCCCATTACATTTGGGGACTTCAACGAAGG

[0708] AGAAATCGAAAGCTTGTCTTCTGAGCTACTAACTTTCGGAGACTTCTTACCAGGAGAAGTGGATGACTTGACAGACAGCGA CTGGTCCACGTGCTCAGACACGGACGACGAGTTAAGACTAGACAGGGCAGGTGGGTATATATTCTCGTCGGACACCGGTCC

[0709] AGGTCATTTACAACAGAAGTCAGTACGCCAGTCAGTGCTGCCGGTGAACACCCTGGAGGAAGTCCACGAGGAGAAGTGTT ACCCACCTAAGCTGGATGAAGCAAAGGAGCAACTATTACTTAAGAAACTCCAGGAGAGTGCATCCATGGCCAACAGAAGCA GGTATCAGTCGCGCAAAGTAGAAAACATGAAAGCAGCAATCATCCAGAGACTAAAGAGAGGCTGTAGACTATACTTAATGTC

[0710] AGAGACCCCAAAAGTCCCTACTTACCGGACTACATATCCGGCGCCTGTGTACTCGCCTCCGATCAACGTCCGATTGTCCAATC CCGAGTCCGCAGTGGCAGCATGCAATGAGTTCTTAGCTAGAAACTATCCAACTGTCTCATCATACCAAATTACCGACGAGTAT

[0711] GATGCATATCTAGACATGGTGGACGGGTCGGAGAGTTGCCTGGACCGAGCGACATTCAATCCGTCAAAACTCAGGAGCTAC

[0712] CCGAAACAGCACGCTTACCACGCGCCCTCCATCAGAAGCGCTGTACCGTCCCCATTCCAGAACACACTACAGAATGTACTGG CAGCAGCCACGAAAAGAAACTGCAACGTCACACAGATGAGGGAATTACCCACTTTGGACTCAGCAGTATTCAACGTGGAGT

[0713] GTTTCAAAAAATTCGCATGCAACCAAGAATACTGGGAAGAATTTGCTGCCAGCCCTATTAGGATAACAACTGAGAATTTAGC

[0714] AACCTATGTTACTAAACTAAAAGGGCCAAAAGCAGCAGCGCTATTCGCAAAAACCCATAATCTACTGCCACTACAGGAAGTA CCAATGGATAGGTTCACAGTAGATATGAAAAGGGACGTAAAGGTGACTCCTGGTACAAAGCATACAGAGGAAAGACCTAAG

[0715] GTGCAGGTTATACAGGCGGCTGAACCCTTGGCGACAGCATACCTATGTGGGATTCACAGAGAGCTGGTTAGGAGGCTGAAC GCCGTCCTCCTACCCAATGTACATACACTATTTGACATGTCTGCCGAGGATTTCGATGCCATCATAGCCGCACACTTTAAGCCA

[0716] GGAGACACTGTTTTGGAAACGGACATAGCCTCCTTTGATAAGAGCCAAGATGATTCACTTGCGCTTACTGCTTTGATGCTGTT

[0717] AGAGGATTTAGGGGTGGATCACTCCCTGCTGGACTTGATAGAGGCTGCTTTCGGAGAGATTTCCAGCTGTCACCTACCGACA GGTACGCGCTTCAAGTTCGGCGCCATGATGAAATCAGGTATGTTCCTAACTCTGTTCGTCAACACATTGTTAAACATCACCATC GCCAGCCGAGTGCTGGAAGATCGTCTGACAAAATCCGCGTGCGCGGCCTTCATCGGCGACGACAACATAATACATGGAGTC

[0718] GTCTCCGATGAATTGATGGCAGCCAGATGTGCCACTTGGATGAACATGGAAGTGAAGATCATAGATGCAGTTGTATCCTTGA AAGCCCCTTACTTTTGTGGAGGGTTTATACTGCACGATACTGTGACAGGAACAGCTTGCAGAGTGGCAGACCCGCTAAAAA Valneva Austria GmbH PAT070-PCT

[0719] GGCTTTTTAAACTGGGCAAACCGCTAGCGGCAGGTGACGAACAAGATGAAGATAGAAGACGAGCGCTGGCTGACGAAGT GATCAGATGGCAACGAACAGGGCTAATTGATGAGCTGGAGAAAGCGGTATACTCTAGGTACGAAGTGCAGGGTATATCAGT

[0720] TGTGGTAATGTCCATGGCCACCTTTGCAAGCTCCAGATCCAACTTCGAGAAGCTCAGAGGACCCGTCATAACTTTGTACGGC GGTCCTAAATAGGTACGCACTACAGCTACCTATTTTGCAGAAGCCGACAGCAAGTATCTAAACACTAATCAGCTACAATGGAG TTCATCCCAACCCAAACTTTTTACAATAGGAGGTACCAGCCTCGACCCTGGACTCCGCGCCCTACTATCCAAGTCATCAGGCC CAGACCGCGCCCTCAGAGGCAAGCTGGGCAACTTGCCCAGCTGATCTCAGCAGTTAATAAACTGACAATGCGCGCGGTACC ACAACAGAAGCCACGCAGGAATCGGAAGAATAAGAAGCAAAAGCAAAAACAACAGGCGCCACAAAACAACACAAATCAA AAGAAGCAGCCACCTAAAAAGAAACCGGCTCAAAAGAAAAAGAAGCCGGGCCGCAGAGAGAGGATGTGCATGAAAATCG AAAATGATTGTATTTTCGAAGTCAAGCACGAAGGTAAGGTAACAGGTTACGCGTGCCTGGTGGGGGACAAAGTAATGAAAC CAGCACACGTAAAGGGGACCATCGATAACGCGGACCTGGCCAAACTGGCCTTTAAGCGGTCATCTAAGTATGACCTTGAATG CGCGCAGATACCCGTGCACATGAAGTCCGACGCTTCGAAGTTCACCCATGAGAAACCGGAGGGGTACTACAACTGGCACCA CGGAGCAGTACAGTACTCAGGAGGCCGGTTCACCATCCCTACAGGTGCTGGCAAACCAGGGGACAGCGGCAGACCGATCT TCGACAACAAGGGACGCGTGGTGGCCATAGTCTTAGGAGGAGCTAATGAAGGAGCCCGTACAGCCCTCTCGGTGGTGACC TGGAATAAAGACATTGTCACTAAAATCACCCCCGAGGGGGCCGAAGAGTGGAGTCTTGCCATCCCAGTTATGTGCCTGTTGG CAAACACCACGTTCCCCTGCTCCCAGCCCCCTTGCACGCCCTGCTGCTACGAAAAGGAACCGGAGGAAACCCTACGCATGC TTGAGGACAACGTCATGAGACCTGGGTACTATCAGCTGCTACAAGCATCCTTAACATGTTCTCCCCACCGCCAGCGACGCAG CACCAAGGACAACTTCAATGTCTATAAAGCCACAAGACCATACTTAGCTCACTGTCCCGACTGTGGAGAAGGGCACTCGTGC CATAGTCCCGTAGCACTAGAACGCATCAGAAATGAAGCGACAGACGGGACGCTGAAAATCCAGGTCTCCTTGCAAATCGGA ATAAAGACGGATGACAGCCACGATTGGACCAAGCTGCGTTATATGGACAACCACATGCCAGCAGACGCAGAGAGGGCGGG GCTATTTGTAAGAACATCAGCACCGTGTACGATTACTGGAACAATGGGACACTTCATCCTGGCCCGATGTCCAAAAGGGGAA ACTCTGACGGTGGGATTCACTGACAGTAGGAAGATTAGTCACTCATGTACGCACCCATTTCACCACGACCCTCCTGTGATAGG TCGGGAAAAATTCCATTCCCGACCGCAGCACGGTAAAGAGCTACCTTGCAGCACGTACGTGCAGAGCACCGCCGCAACTAC CGAGGAGATAGAGGTACACATGCCCCCAGACACCCCTGATCGCACATTAATGTCACAACAGTCCGGCAACGTAAAGATCACA GTCAATG GCCAGACG GTG CG GTACA AGTGTAATTG CG GTG G CTCAAATG AAG G ACTAACAACTAC AG AC AAAGTG ATTAAT AACTGCAAGGTTGATCAATGTCATGCCGCGGTCACCAATCACAAAAAGTGGCAGTATAACTCCCCTCTGGTCCCGCGTAATGC

[0721] TGAACTTGGGGACCGAAAAGGAAAAATTCACATCCCGTTTCCGCTGGCAAATGTAACATGCAGGGTGCCTAAAGCAAGGAA CCCCACCGTGACGTACGGGAAAAACCAAGTCATCATGCTACTGTATCCTGACCACCCAACACTCCTGTCCTACCGGAATATGG GAGAAGAACCAAACTATCAAGAAGAGTGGGTGATGCATAAGAAGGAAGTCGTGCTAACCGTGCCGACTGAAGGGCTCGAG GTCACGTGGGGCAACAACGAGCCGTATAAGTATTGGCCGCAGTTATCTACAAACGGTACAGCCCATGGCCACCCGCATGAGA TAATTCTGTATTATTATGAGCTGTACCCCACTATGACTGTAGTAGTTGTGTCAGTGGCCACGTTCATACTCCTGTCGATGGTGGG TATGGCAGCGGGGATGTGCATGTGTGCACGACGCAGATGCATCACACCGTATGAACTGACACCAGGAGCTACCGTCCCTTTC CTGCTTAGCCTAATATGCTGCATCAGAACAGCTAAAGCGGCCACATACCAAGAGGCTGCGATATACCTGTGGAACGAGCAGC AACCTTTGTTTTGGCTACAAGCCCTTATTCCGCTGGCAGCCCTGATTGTTCTATGCAACTGTCTGAGACTCTTACCATGCTGCT GTAAAACGTTGGCTTTTTTAGCCGTAATGAGCGTCGGTGCCCACACTGTGAGCGCGTACGAACACGTAACAGTGATCCCGAA CACGGTGGGAGTACCGTATAAGACTCTAGTCAATAGACCTGGCTACAGCCCCATGGTATTGGAGATGGAACTACTGTCAGTC ACTTTGGAGCCAACACTATCGCTTGATTACATCACGTGCGAGTACAAAACCGTCATCCCGTCTCCGTACGTGAAGTGCTGCGG TACAGCAGAGTGCAAGGACAAAAACCTACCTGACTACAGCTGTAAGGTCTTCACCGGCGTCTACCCATTTATGTGGGGCGGC GCCTACTGCTTCTGCGACGCTGAAAACACGCAGTTGAGCGAAGCACACGTGGAGAAGTCCGAATCATGCAAAACAGAATTT GCATCAGCATACAGGGCTCATACCGCATCTGCATCAGCTAAGCTCCGCGTCCTTTACCAAGGAAATAACATCACTGTAACTGC CTATGCAAACGGCGACCATGCCGTCACAGTTAAGGACGCCAAATTCATTGTGGGGCCAATGTCTTCAGCCTGGACACCTTTC GACAACAAAATTGTGGTGTACAAAGGTGACGTCTATAACATGGACTACCCGCCCTTTGGCGCAGGAAGACCAGGACAATTT GGCGATATCCAAAGTCGCACACCTGAGAGTAAAGACGTCTATGCTAATACACAACTGGTACTGCAGAGACCGGCTGTGGGTA CG GTACACGTG CCATACTCTC AGG CACCATCTG GCTTTAAGTATTG G CTA AAAG AACG CGGGGCGTCGCTGCAG CAC ACAG CACCATTTGGCTGCCAAATAGCAACAAACCCGGTAAGAGCGGTGAACTGCGCCGTAGGGAACATGCCCATCTCCATCGACAT ACCGGAAGCGGCCTTCACTAGGGTCGTCGACGCGCCCTCTTTAACGGACATGTCGTGCGAGGTACCAGCCTGCACCCATTCC TCAGACTTTGGGGGCGTCGCCATTATTAAATATGCAGCCAGCAAGAAAGGCAAGTGTGCGGTGCATTCGATGACTAACGCC GTCACTATTCGGGAAGCTGAGATAGAAGTTGAAGGGAATTCTCAGCTGCAAATCTCTTTCTCGACGGCCTTAGCCAGCGCCG AATTCCGCGTACAAGTCTGTTCTACACAAGTACACTGTGCAGCCGAGTGCCACCCCCCGAAGGACCACATAGTCAACTACCC GGCGTCACATACCACCCTCGGGGTCCAGGACATCTCCGCTACGGCGATGTCATGGGTGCAGAAGATCACGGGAGGTGTGG

[0722] GACTGGTTGTTGCTGTTGCCGCACTGATTCTAATCGTGGTGCTATGCGTGTCGTTCAGCAGGCACTAACTTGACAATTAAGTA

[0723] TGAAGGTATATGTGTCCCCTAAGAGACACACTGTACATAGCAAATAATCTATAGATCAAAGGGCTACGCAACCCCTGAATAGTA ACAAAATACAAAATCACTAAAAATTATAAAAACAGAAAAATACATAAATAGGTATACGTGTCCCCTAAGAGACACATTGTATGT AGGTGATAAGTATAGATCAAAGGGCCGAATAACCCCTGAATAGTAACAAAATATGAAAATCAATAAAAATCATAAAATAGAAA AACCATAAAC AG AAGTAGTTCA AAG G G CTATAAA ACCCCTG AATAGTA AC AAAACATAAAATTAATAAAAATCAAATG AATAC CATAATTG G CAA ACG G AAG AG ATGTAG GTACTTAAG CTTCCTAAAAG CAG CCG AACTC ACTTTG AG AAGTAG G CATAG CATA CCGAACTCTTCCACGATTCTCCGAACCCACAGGGACGTAGGAGATGTTATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAA AAAAAAAA Valneva Austria GmbH PAT070-PCT

[0724] SEQ ID NO: 3 - CHIKV E2

[0725] STKDNFNVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQIGIKTDDSHDWTKLRYMDNHMPADAERAGLF VRTSAPCTITGTMGHFILARCPKGETLTVGFTDSRKISHSCTHPFHHDPPVIGREKFHSRPQHGKELPCSTYVQSTAATTEEIEVH MPPDTPDHTLMSQQSGNVKITVNGQTVRYKCNCGGSNEGLTTTDKVINNCKVDQCHAAVTNHKKWQYNSPLVPRNAELGD RKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVIMLLYPDHPTLLSYRNMGEEPNYQEEWVMH KKEVVLTVPTEGLEVTWGNN EPYKYWPQLSTNGTAHGHPH EIILYYYELYPTMTVVVVSVATFILLSMVGMAAGMCMCARRRCITPYELTPGATVPFLLSLICCIR TAKA

[0726] SEQ ID NO: 4 - YFV - E (286.778 aa of YFV polyprotein, see SEQ ID NO: 5)

[0727] AHCIGITDRDFIEGVHGGTWVSATLEQDKCVTVMAPDKPSLDISLETVAIDRPAEVRKVCYNAVLTHVKINDKCPSTGEAHLAEE NEGDNACKRTYSDRGWGNGCGLFGKGSIVACAKFTCAKSMSLFEVDQTKIQYVIRAQLHVGAKQENWTTDIKTLKFDALSGSQ EVEFIGYGKATLECQVQTAVDFGNSYIAEMETESWIVDRQWAQDLTLPWQSGSGGVWREMHH LVEFEPPHAATIRVLALGNQ EGSLKTALTGAMRVTKDTNDNN LYKLHGGHVSCRVKLSALTLKGTSYKICTDKMFFVKNPTDTGHGTVVMQVKVSKGAPCRIPV IVADDLTAAINKGILVTVNPIASTNDDEVUEVNPPFGDSYIIVGRGDSRLTYQWHKEGSSIGKLFTQTMKGVERLAVMGDTAWDF SSAGGFFTSVGKGIHTVFGSAFQGLFGGLNWITKVIMGVVLIWVGINTRNMTMSMSMILVGVIMMFLSLGVGA

[0728] SEQ ID NO: 5- YFV amino acid (AGO04419.1 polyprotein)

[0729] MSGRKAQGKTLGVNMVRRGVRSLSNKIKQKTKQIGNRPGPSRGVQGFIFFFLFNILTGKKITAHLKRLWKMLDPRQGLAVLRKV KRVVASLMRGLSSRKRRSHDVLTVQFLILGMLLMTGGVTLVRKNRWLLLNVTSEDLGKTFSVGTGNCTTNILEAKYWCPDSMEY NCPNLSPREEPDDIDCWCYGVENVRVAYGKCDSAGRSRRSRRAIDLPTHEN HGLKTRQEKWMTGRMGERQLQKIERWFVRN PFFAVTALTIAYLVGSNMTQRVVIALLVLAVGPAYSAHCIGITDRDFIEGVHGGTWVSATLEQDKCVTVMAPDKPSLDISLETVAID RPAEVRKVCYNAVLTHVKINDKCPSTGEAHLAEENEGDNACKRTYSDRGWGNGCGLFGKGSIVACAKFTCAKSMSLFEVDQTKI QYVIRAQLHVGAKQENWTTDIKTLKFDALSGSQEVEFIGYGKATLECQVQTAVDFGNSYIAEMETESWIVDRQWAQDLTLPWQ SGSGGVWREMHHLVEFEPPHAATIRVLALGNQEGSLKTALTGAMRVTKDTNDNN LYKLHGGHVSCRVKLSALTLKGTSYKICTD KMFFVKN PTDTGHGTVVMQVKVSKGAPCRIPVIVADDLTAAINKGILVTVNPIASTNDDEVUEVNPPFGDSYIIVGRGDSRLTYQ WHKEGSSIGKLFTQTMKGVERLAVMGDTAWDFSSAGGFFTSVGKGIHTVFGSAFQGLFGGLNWITKVIMGVVUWVGINTRN MTMSMSMILVGVIMMFLSLGVGADQGCAINFGKRELKCGDGIFIFRDSDDWLNKYSYYPEDPVKLASIVKASFEEGKCGLNSV DSLEHEMWRSRADEINAIFEENEVDISVVVQDPKNVYQRGTHPFSRIRDGLQYGWKTWGKNLVFSPGRKNGSFIIDGKSRKECP FSNRVWNSFQIEEFGTGVFTTRVYMDAVFEYTIDCDGSILGAAVNGKKSAHGSPTFWMGSHEVNGTWMIHTLEALDYKECEW

[0730] PLTHTIGTSVEESEMFMPRSIGGPVSSHNHIPGYKVQTNGPWMQVPLEVKREACPGTSVIIDGNCDGRGKSTRSTTDSGKVIPE WCCRSCTMPPVSFHGSDGCWYPMEIRPRKTHESH LVRSWVTAGEIHAVPFGLVSMMIAMEVVLRKRQGPKQMLVGGVVLLG AMLVGQVTLLDLLKLTVAVGLH FHEMNNGGDAMYMALIAAFSIRPGLLIGFGLRTLWSPRERLVLTLGAAMVEIALGGVMGGL

[0731] WKYLNAVSLCILTINAVASRKASNTILPLMALLTPVTMAEVRLAAMFFCAVVIIGVLHQNFKDTSMQKTIPLVALTLTSYLGLTQPFL GLCAFLATRI FGRRSIPVN EALAAAGLVGVLAGLAFQEMENFLGPIAVGGLLMMLVSVAGRVDGLELKKLGEVSWEEEAEISGSS ARYDVALSEQGEFKLLSEEKVPWDQVVMTSLALVGAALHPFALLLVLAGWLFHVRGARRSGDVLWDIPTPKIIEECEHLEDGIYGI FQSTFLGASQRGVGVAQGGVFHTMWHVTRGAFLVRNGKKLIPSWASVKEDLVAYGGSWKLEGRWDGEEEVQLIAAVPGKNV VNVQ.TKPSLFKVRNGGEIGAVALDYPSGTSGSPIVNRNGEVIGLYGNGILVGDNSFVSAISQ.TEVKEEGKEELQEIPTMLKKGMTT VLDFH PGAGKTRRFLPQI LAECARRRLRTLVLAPTRVVLSEMKEAFHGLDVKFHTQAFSAHGSGREVIDAMCHATLTYRMLEPTR VVNWEVIIMDEAH FLDPASIAARGWAAHRARANESATILMTATPPGTSDEFPHSNGEIEDVQ.TDIPSEPWNTGHDWILADKRP TAWFLPSIRAANVMAASLRKAGKSVVVLNRKTFEREYPTIKQKKPDFILATDIAEMGANLCVERVLDCRTAFKPVLVDEGRKVAIK GPLRISASSAAQRRGRIGRNPNRDGDSYYYSEPTSEN NAH HVCWLEASMLLDNMEVRGGMVAPLYGVEGTKTPVSPGEMRLR DDQRKVFRELVRNCDLPVWLSWQVAKAGLKTNDRKWCFEGPEEHEILNDSGETVKCRAPGGAKKPLRPRWCDERVSSDQSAL SEFIKFAEGRRGAAEVLVVLSELPDFLAKKGGEAMDTISVFLHSEEGSRAYRNALSMMPEAMTIVMLFILAGLLTSGMVIFFMSPK GISRMSMAMGTMAGCGYLMFLGGVKPTHISYIMUFFVLMVVVIPEPGQQRSIQDNQVAYLIIGILTLVSAVAANELGMLEKTKE DLFGKKNUPSSASPWSWPDLDLKPGAAWTVYVGIVTMLSPMLHHWIKVEYGNLSLSGIAQSASVLSFMDKGIPFMKMNISVI

[0732] MLLVSGWNSITVMPLLCGIGCAMLHWSULPGIKAQQSKLAQRRVFHGVAKNPVVDGNPTVDIEEAPEMPALYEKKLALYLLLA LSLASVAMCRTPFSLAEGIVLASAALGPLIEGNTSLLWNGPMAVSMTGVMRGNHYAFVGVMYNLWKMKTGRRGSANGKTLG EVWKRELNLLDKRQFELYKRTDIVEVDRDTARRHLAEGKVDTGVAVSRGTAKLRWFHERGYVKLEGRVIDLGCGRGGWCYYAA AQKEVSGVKGFTLGRDGHEKPMNVQSLGWNIITFKDKTDIHRLEPVKCDTLLCDIGESSSSSVTEGERTVRVLDTVEKWLACGV DN FCVKVLAPYMPDVLEKLELLQRRFGGTVIRNPLSRNSTHEMYYVSGARSNVTFTVNQTSRLLMRRMRRPTGKVTLEADVILP IGTRSVETDKGPLDKEAIEERVERIKSEYMTSWFYDNDNPYRTWHYCGSYVTKTSGSAASMVNGVIKILTYPWDRIEEVTRMAM TDTTPFGQQRVFKEKVDTRAKDPPAGTRKIMKVVNRWLFRHLAREKNPRLCTKEEFIAKVRSHAAIGAYLEEQEQWKTANEAV QDPKFWELVDEERKLHQQGRCRTCVYNMMGKREKKLSEFGKAKGSRAIWYMWLGARYLEFEALGFLNEDHWASRENSGGG Valneva Austria GmbH PAT070-PCT

[0733] VEGIGLQYLGYVIRDLAAMDGGGFYADDTAGWDTRITEADLDDEQEILNYMSPHHKKLAQAVMEMTYKNKVVKVLRPAPGGK

[0734] AYMDVISRRDQRGSGQVVTYALNTITNLKVQLIRMAEAEMVIHHQHVQDCDESVLTRLEAWLTEHGCNRLKRMAVSGDDCVV

[0735] RPIDDRFGLALSHLNAMSKVRKDISEWQPSKGWNDWENVPFCSHHFHELQLKDGRRIVVPCREQDEUGRGRVSPGNGWMI

[0736] KETACLSKAYANMWSLMYFHKRDMRLLSLAVSSAVPTSWVPQGRTTWSIHGKGEWMTTEDMLEVWNRVWITNNPHMQD

[0737] KTMVKKWRDVPYLTKRQDKLCGSLIGMTNRATWASHIHLVIHRIRTLIGQEKYTDYLTVMDRYSVDADLQLGEU

[0738] SEQ ID NO: 6

[0739] KLK peptide

[0740] KLKLLLLLKLK

[0741] SEQ ID NO: 7

[0742] Oligo-(dldC)i3 dldC dldC dldC dldC dldC dldC dldC dldC dldC dldC dldC dldC dldC

Claims

Valneva Austria GmbH PAT070-PCTCLAIMS1. A high-yield method of preparing a composition comprising yellow fever virus (YFV), wherein the method comprises: a. Culturing host cells in a serum-free culture media in a fix-bed bioreactor; b. Infecting the host cells with YFV; and c. Collecting the culture medium 60-84 hours post-infection to produce a first YFV harvest (i).

2. The method according to claim 1 , wherein the fixed-bed bioreactor uses a falling film for medium oxygenation, preferably wherein the shear stress and / or medium foaming are minimized by maintaining the Falling Film Height (FFH) at the lowest possible level while still maintaining required dissolved oxygen (DO) levels.

3. The method according to claim 2, wherein the dissolved oxygen (DO) level for the first 3 hours post-infection is not less than 10%, preferably not less than 15%, and / or wherein the DO level from 3 hours post-infection to collection of culture medium is between 40-60%.

4. The method according to any preceding claim, wherein the host cells are Vero cells.

5. The method according to any preceding claim, wherein the culture medium is replaced 1 day post-infection with fresh culture medium.

6. The method according to any preceding claim, wherein the culture medium is collected by perfusion between 60-84 hours post-infection.

7. The method according to any preceding claim, wherein the method further comprises: d) adding culture medium to the bioreactor, and collecting culture medium 7-9 days post-infection to produce a second YFV harvest (ii).

8. A high-yield method of preparing a composition comprising yellow fever virus (YFV), wherein the method comprises: a. Culturing host cells in a serum-free culture medium in roller bottles; b. Infecting the host cells with YFV; andValneva Austria GmbH PAT070-PCT c. Collecting the culture medium 78-102 hours post-infection to produce a YFV harvest (i).

9. The method according to any preceding claim, wherein the serum free culture media in (a) for culturing the host cells is OptiPRO-SFM or VP-SFM, suitably OptiPRO-SFM, and / or wherein the culture medium is replaced in (b) with VP-SFM.

10. The method according to any preceding claim, wherein the method further comprises: dialyzing the virus harvest (i) and / or (ii) to form a liquid formulation (iii); wherein said liquid formulation (iii) comprises a) at least one strain of YFV; b) 2% to 8% sugar; c) 1 mmol / kg to 20 mmol / kg phosphate; d) 1 mmol / kg to 50 mmol / kg of at least one carboxylate buffering agent; e) 1 mmol / kg to 10 mmol / kg MgCI2; f) 0.1 % to 5% D- sorbitol; g) 1 to 20 mmol / kg L-methionine; and h) 0.01 to 1 mg / mL human albumin (HA), wherein the formulation is at a pH of 7.0 to 7.5.

11. The method according to claim 10, wherein the sugar is sucrose at 5%, the phosphate is 5 mmol / kg, the at least one carboxylate buffering agent is 25 mmol / kg, the MgCI2 is 5 mmol / kg, the D-sorbitol is 0.5%, the L-methionine is 10 mmol / kg, and the HA is 0.01 %, optionally wherein the formulation is at a pH of 7.3, and / or wherein the sugar is sucrose at 5%, the phosphate is potassium phosphate at 5 mM, the at least one carboxylate buffering agent is sodium citrate at 25 mmol / kg, the MgCh is 5 mmol / kg, the D-sorbitol is 0.5%, the L-methionine is 10 mmol / kg, and the HA is 0.01 % , optionally wherein the formulation is at a pH of 7.3, and / or wherein the HA is a recombinant human albumin (rHA); and / or the formulation further comprises at least one diluent selected from the group consisting of tissue culture medium, saline and water.

12. The method of claim 10 or 11 , wherein the method further comprises lyophilizing the liquid formulation (ii) to produce a lyophilized YFV formulation (iii).

13. The method according to any of claims 10 to 12, wherein (b) further comprises dividing the formulation (ii) after dialysis to produce 102TCID50 to 106TCID50 per dose of YFV.Valneva Austria GmbH PAT070-PCT14. The method according to any preceding claim, wherein the YFV comprises or consists of a polynucleotide sequence with at least 80% identity to SEQ ID NO: 1, preferably wherein the YFV polynucleotide sequence comprises one or more mutations selected from: A2608C, A5641G, A7225G, C8212T, A10243G, G10559GT, and / or A 10722G mutations in SEQ ID NO: 1 , and / or wherein the YFV is a live, attenuated virus.

15. A pharmaceutical composition of YFV obtained or obtainable by the method of any one of claims 10 to 14.

16. A liquid yellow fever virus (YFV) formulation comprising: a) YFV particles; b) 2% to 8% sucrose; c) 1 mmol / kg to 20 mmol / kg phosphate; d) 1 mmol / kg to 50 mmol / kg of at least one carboxylate buffering agent; e) 1 mmol / kg to 10 mmol / kg MgCh; f) 0.1% to 5% D-sorbitol; g) 1 to 20 mmol / kg L-methionine; and h) 0.001 % to 0.1 % human albumin (HA), wherein the formulation is at a pH of 7.0 to 7.5, wherein the formulation is for preparation of a liquid frozen or lyophilized formulation.

17. A reconstituted lyophilized yellow fever virus (YFV) formulation comprising: a) YFV particles; b) 2% to 8% sucrose; c) 1 mmol / kg to 20 mmol / kg phosphate; d) 1 mmol / kg to 50 mmol / kg of at least one carboxylate buffering agent; e) 1 mmol / kg to 10 mmol / kg MgCh; f) 0.1% to 5% D-sorbitol; g) 1 to 20 mmol / kg L-methionine; and h) 0.001% to 0.1% human albumin (HA), wherein the formulation is at a pH of 7.0 to 7.5.

18. The formulation according to claim 16 or 17, wherein the YFV comprises or consists of a polynucleotide sequence with at least 80% sequence identity to SEQ ID NO: 1 , particularly wherein the YFV polynucleotide sequence comprises one or more mutations selected from: A2608C, A5641G, A7225G, C8212T, A10243G, G10559GT, and / or A10722G mutations in SEQ ID NO: 1 , and / or wherein the sugar is sucrose at 5%, the phosphate is 5 mmol / kg, the at least one carboxylate buffering agent is 25 mmol / kg, the MgCI2 is 5 mmol / kg, the D-sorbitol is 0.5%, the L-methionine is 10 mmol / kg, and the HA is 0.01%, optionally wherein the formulation is at a pH of 7.3, and / or wherein the sugar is sucrose at 5%, the phosphate is potassium phosphate at 5 mmol / kg, the at least one carboxylate buffering agent is sodium citrate at 25 mmol / kg,Valneva Austria GmbH PAT070-PCT the MgCh is 5 mmol / kg, the D-sorbitol is 0.5%, the L-methionine is 10 mmol / kg, and the HA is 0.01%, optionally wherein the formulation is at a pH of 7.3, and / or wherein the HA is a recombinant human albumin (rHA); and / or the YFV formulation further comprises at least one diluent selected from the group consisting of tissue culture medium, saline and water, and / or wherein the liquid or lyophilized formulation comprises an effective amount of at least one strain of YFV, optionally wherein the effective amount is 102TCIDso / dose to 106TCIDso / dose of YFV and / or wherein the YFV is a live attenuated virus, and / or wherein the formulation further comprises one or more enveloped viruses, optionally wherein the enveloped viruses are selected from Flaviviridae, Alphaviridae, Togaviridae, Coronavirus, Orthomyxoviruses, Paramyxoviridae, and Rhabdoviruses.

19. A method of preparing a YFV or CHIKV composition, wherein the method comprises: a. Producing a virus harvest (i); b. Dialyzing the virus harvest (i) to form a liquid formulation (ii); wherein said liquid formulation (ii) comprises a) at least one strain of virus; b) 2% to 8% sucrose; c) 1 mmol / kg to 20 mmol / kg phosphate; d) 1 mmol / kg to 50 mmol / kg of at least one carboxylate buffering agent; e) 1 mmol / kg to 10 mmol / kg MgCh; f) 0.1% to 5% D- sorbitol; g) 1 to 20 mmol / kg L-methionine; and h) 0.001 % to 0.1% human albumin (HA), wherein the formulation is at a pH of 7.0 to 7.5.

20. The method according to claim 19, further comprising mixing the YFV liquid formulation (ii) and / or CHIKV liquid formulation (ii) to produce a combined liquid formulation (iii).

21. The method of claim 19 or 20, further comprising lyophilizing the liquid formulation (ii) or the combined liquid formulation (iii) to produce a lyophilized formulation (iv).

22. The method of any one of claims 19 to 21 , wherein the YFV comprises or consists of a polynucleotide sequence with at least 80% sequence identity to SEQ ID NO: 1 , preferably wherein the YFV polynucleotide sequence comprises one or moreValneva Austria GmbH PAT070-PCT mutations selected from: A2608C, A5641G, A7225G, C8212T, A10243G, G10559GT, and / or A10722G mutations in SEQ ID NO: 1 , and / or wherein the CHIKV comprises or consists of a polynucleotide sequence with at least 80%, 90%, 95%, 99% identity to SEQ ID NO: 2, and / or wherein the sucrose is 5%, the phosphate is 5 mmol / kg, the at least one carboxylate buffering agent is 25 mmol / kg, the MgCh is 5 mmol / kg, the D-sorbitol is 0.5%, the L- methionine is 10 mmol / kg, and the HA is 0.01 %, optionally wherein the formulation is at a pH of 7.3, and / or wherein the sucrose is 5%, the phosphate is potassium phosphate at 5 mmol / kg, the at least one carboxylate buffering agent is sodium citrate at 25 mmol / kg, the MgCh is 5 mmol / kg, the D-sorbitol is 0.5%, the L-methionine is 10 mmol / kg, and the HA is 0.01 %, optionally wherein the formulation is at a pH of 7.3, and / or wherein the HA is a recombinant human albumin (rHA); and / or the formulation further comprises at least one diluent selected from the group consisting of tissue culture medium, saline and water, and / or wherein step (b) further comprises dividing the composition (ii) after dialysis to produce 102TCI D50to 106TCI D50per dose of YFV and / or 102TCI D50to 106TCI D50per dose of CHIKV and / or wherein the YFV and / or CHIKV viruses are live, attenuated viruses.

23. A pharmaceutical composition of YFV and / or CHIKV obtained or obtainable by the method of any one of claims 19 to 22.

24. A liquid or reconstituted lyophilized combination formulation comprising: a) YFV particles; b) CHIKV particles; c) 2% to 8% sugar; d) 1 mmol / kg to 20 mmol / kg phosphate; e) 1 mmol / kg to 50 mmol / kg of at least one carboxylate buffering agent; f) 1 mmol / kg to 10 mmol / kg MgCh; g) 0.1 % to 5% D-sorbitol; h) 1 to 20 mmol / kg L- methionine; and i) 0.001 % to 0.1 % human albumin (HA), wherein the formulation is at a pH of 7.0 to 7.5.Valneva Austria GmbH PAT070-PCT25. The formulation according to claim 24, wherein the CHIKV comprises or consists of a polynucleotide sequence with at least 80%, 90%, 95%, 99% identity to SEQ ID NO: 2, and / or wherein the YFV comprises or consists of a polynucleotide sequence with at least 80% sequence identity to SEQ ID NO: 1 , preferably wherein the YFV polynucleotide sequence comprises one or more mutations selected from: A2608C, A5641G, A7225G, C8212T, A10243G, G10559GT, and / or A10722G mutations in SEQ ID NO: 1, and / or wherein the sugar is sucrose at 5%, the phosphate is 5 mmol / kg, the at least one carboxylate buffering agent is 25 mmol / kg, the MgCh is 5 mmol / kg, the D-sorbitol is 0.5%, the L-methionine is 10 mmol / kg, and the HA is 0.01%, optionally wherein the formulation is at a pH of 7.3, and / or wherein the sugar is sucrose at 5%, the phosphate is potassium phosphate at 5 mmol / kg, the at least one carboxylate buffering agent is sodium citrate at 25 mmol / kg, the MgCh is 5 mmol / kg, the D-sorbitol is 0.5%, the L-methionine is 10 mmol / kg, and the HA is 0.01%, optionally wherein the formulation is at a pH of 7.3, and / or wherein the HA is a recombinant human albumin (rHA); and / or the formulation further comprises at least one diluent selected from the group consisting of tissue culture medium, saline and water, and / or wherein the liquid or lyophilized formulation comprises an effective amount of at least one strain of YFV, optionally wherein the effective amount is 102TCIDso / dose to 106TCIDso / dose of YFV and 102TCID50 / dose to 106TCID50 / dose, of CHIKV, and / or wherein the viruses are live, attenuated viruses, and / or wherein the formulation further comprises one or more enveloped viruses, optionally wherein the enveloped viruses are selected from Flaviviridae, Alphaviridae, Togaviridae, Coronavirus, Orthomyxoviruses, Paramyxoviridae, and Rhabdoviruses.Valneva Austria GmbH PAT070-PCT26. The formulation according to any one of claims 15 to 18 and 23 to 25, for use in preventing a YFV infection.

27. The formulation according to any one of claims 23 to 25, for use in preventing a YFV and / or a CHIKV infection.

28. A method of preventing a YFV infection comprising administering the formulation according to any one of claims 15 to18 and 23 to 25 to a subject.

29. A method of preventing a YFV and / or a CHIKV infection comprising administering the formulation according to any one of claims 23 to 25 to a subject.

30. The method according to claim 20, wherein the YFV and / or CHIKV liquid formulation is provided in a single-dose pre-filled syringe.

31. The method according to claim 20, wherein the YFV and / or CHIKV liquid formulation is provided in a single-dose pre-filled syringe for simultaneous administration.

32. The pre-filled syringe assembly of claims 30 and 31, wherein the YFV and / or CHIKV liquid formulation is stable after being stored at temperatures ranging from -15°C to - 80°C for at least 12 months.

33. A pharmaceutical composition comprising YFV and optionally CHIKV, wherein the formulation is a liquid frozen formulation.

34. A pharmaceutical composition comprising YFV and optionally CHIKV, wherein the formulation is a lyophilized formulation.

35. The pharmaceutical composition of claim 33 or 34, wherein the formulation is a single dose or multi-dose formulation.

36. The composition of claim 34 wherein the lyophilized formulation is reconstitution with water for Injection (WFI).

37. The composition of claim 35, wherein the single dose formulation is reconstituted in 0.5mL of WFI.

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