EA046342B1 - STABLE COMPOSITIONS OF VACCINES, INCLUDING, IN PARTICULAR, LIVE ATTENUATED RECOMBINANT FLAVIVIRUS AND THEIR PREPARATION PROCESS - Google Patents

Description

Field of invention

This disclosure relates to the field of biotechnology, in particular to the composition of a live attenuated flavivirus vaccine and a method for its preparation. The present disclosure also relates to improved methodology in the field of production of live attenuated flavivirus vaccines.

Initial data

The flavivirus genome consists of a single-stranded, positively polarized 11-kb RNA molecule containing one open reading frame. RNA is transformed into a polyprotein, which is processed into at least 10 gene products: 3 structural proteins - nucleocapsid or core (C), premembrane protein (prM), cell envelope (E) and 7 non-structural (NS) proteins - NS 1, 2A, 2B , 3, 4A, 4B and 5 (Lindenbach BD, et al., In: Fields Virology. Edited by Knipe DM, Howley PM, Griffin PE, et al.: Wolters Kluwer, Lippencott Williams and Wilkins; 2007. pp. 1101-1152) . (Fernandez-Garcia MD, et al., Cell Host Microbe, 2009, 5:318-328). Some of these flaviviruses use arthropods (eg, tick and/or mosquito bites) as a means of transmitting the virus to recipients. Such arthropod-borne viruses (i.e., arboviruses) represent a major public health concern worldwide due to their highly pathogenic nature in humans. (Fernandez-Garcia MD, et al., Cell Host Microbe, 2009, 5:318-328). Specifically, human arboviruses include yellow fever (YF), Japanese encephalitis (JE), dengue (DEN), West Nile (WN), and tick-borne encephalitis (TBE) viruses, which exist naturally in life cycles involving vectors : mosquitoes or ticks, and birds and/or mammals that are competent reservoir hosts. (Gubler D, et al., In: Fields Virology. Edited by Knipe DM, Howley PM, Griffin PE, etc. 5th edition. Philadelphia: Wolters Kluwer, Lippencott Williams and Wilkins; 2007. p. 11531252).

However, dengue virus (DENV) has become the most serious human arbovirus worldwide, with an estimated 500 million dengue infections occurring annually, resulting in more than 2 million people suffering from a serious disease known as dengue hemorrhagic fever/shock. dengue syndrome, and 21,000 people die from the disease. There are four serotypes of dengue virus (DENV1 (DENV2, DENV3 and DENV4).

Numerous methods are known for producing live attenuated recombinant flavivirus preparations for vaccination and other purposes. Formulations and methods are also known that are useful in freezing, lyophilizing, or otherwise storing viable viral preparations for laboratory or vaccine use in order to maintain their activity.

Aqueous formulations of flaviviruses do not provide good viral stability over long periods of time and at temperatures above 5°C. For example, bulk aqueous formulations of YF-DEN (yellow fever) chimera lose more than 4 logs stabilized in liquid after storage for 1 day at 37°C. Thermal stability is currently a major problem in subtropical dengue-endemic countries where temperature-controlled transportation is difficult.

Lyophilization is a common method for stabilizing vaccines. However, lyophilization results in loss of viral efficacy. Vaccines lose their effectiveness over time, and the rate at which it loses depends on temperature. Live viruses are subject to osmotic, thermal and vacuum shock. Evolved viruses have a lipid bilayer, which, due to its high fragility, is considered the less stable component of the virus. Live viruses are subject to various stresses during lyophilization steps such as freezing, primary drying, secondary drying, which can affect the physicochemical stability of the viruses. Due to the nature of their structure, loss of potential activity upon freeze-drying can be caused by protein destabilization (e.g., unfolding, degradation, and aggregation), degradation of nucleic acids, changes in the lipid layer (e.g., phase transition, mechanical damage), and stresses associated with changes in the internal (ice formation) and external (change in pH and osmolarity) viral environment. In some cases, lyophilization has been reported to result in a loss of up to 40% of viral efficacy.

Although a wealth of information is available on stress mechanisms and strategies for stabilization of pharmaceutical peptides, proteins and DNA during lyophilization, due to the molecular complexity of viruses, various destabilization pathways, and the lack of analytical methods to measure physicochemical changes in antigen structure during and after lyophilization, viruses pose a particular problem for lyophilization. The mechanisms of destabilization as well as the mechanisms of protection of live, attenuated viral vaccines during lyophilization are not well known.

Hansen et al. 2015 (Freeze-drying of live virus vaccines: A review, Hansen et al., Vaccine 33 (2015) 55075519) discloses a compilation of several freeze-dried vaccine formulations (c), in which most formulations mention preferential use of sugar alcohol/protein additive (e.g. sucrose + trehalose, sorbitol, hydrolyzed gelatin, lactalbumin hydrolysates) to produce lyophilized viral vaccine.

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The following flavivirus vaccine formulations have previously been reported: 1) sorbitol, trehalose, urea, 2) lactose, sorbitol, HSA, 3) lactose, mannitol, HSA; 4) poloxamer, human albumin, trehalose, PBS; 5) trehalose, recombinant HSA, F127 (block copolymer of polyoxyethylene and polyoxypropylene).

In the case of HSA, the inclusion of these materials may raise potential safety concerns if these materials are derived from sources that pose a risk to humans or animals. Such added proteins are of concern for two main reasons. The first is that animal and human derived protein may contain one or more adventitious agents. The second reason is that the protein, whether derived from animals or humans, can cause an allergic reaction in susceptible individuals. In addition, previously reported lyophilized vaccine formulations use proteins that, even if produced using processes that support high yields, have cost implications for formulations. Keeping the cost of the vaccine and its components, such as stabilizers, low is critical to widespread vaccine adoption in low-income areas. From a regulatory and safety perspective, it is also critical that the excipients and stabilizers used do not contain any substances of animal origin or components of animal origin. Animal-derived compounds pose a potential concern due to possible contamination with scarpi prion protein (PrPSC) and new variant Creutzfeldt-Jakob disease (vCJD).

Nonionic surfactants used in pharmaceutical formulations include Triton™ X-100, Pluronic® F-68, F-88 and F-127 (poloxamers), Brij 35 (polyoxyethylene alkyl ether), polyoxyl stearate 40, Cremophor® EL and alpha-tocopherol TPGS. What each of these surfactants have in common is that they all contain polyoxyethylene substances and thus, to varying degrees, have a similar problem, since polyoxyethylene substances autoxidize to form reactive peroxides, which leads to increased undesirable immunogenicity of the protein. (See Edward T. Maggio et al; Polysorbates, peroxides, protein aggregation, immunogenicity - a growing concern; Journal of Excipients and Food Chemicals 3(2):46-53; 2012).

PVP has been reported to destabilize live attenuated viral compounds. (See: JA White et al; Development of a stable liquid formulation of live attenuated influenza vaccine; Vaccine volume 34, issue 32, July 12, 2016, pp. 3676-3683; 2016).

Trehalose is expensive; To achieve stability, it must be combined with other sugars and protein additives (gelatin). In addition, other stabilizers are better than trehalose at increasing the stability of the lyophilized vaccine during storage.

Sorbitol has a low glass transition temperature (Tg) (-1.6 degrees C) and therefore cannot be used as a main component of the formulation. The short generation time of sorbitol limits its use. To achieve stability, sorbitol must be combined with other sugars and protein supplements (gelatin).

Typically, recombinant viruses are stored as frozen pellets containing casein and/or collagen hydrolysates in phosphate-buffered saline (PBS). These granules are then rehydrated in a pharmaceutically acceptable solution, for example 0.4-0.9% NaCl. However, there are significant disadvantages of such compositions. These include incompletely defined components, complex preparation procedures, high cost, and the inability to maintain certain desired characteristics of the virus.

Previously developed flavivirus vaccine formulations are stable at 2-8°C for 6 months, at 25°C for 7 days and at 37°C for 1-2 days. There remains a need to develop formulations that contain minimal amounts of excipients and provide long-term heat stability to flavivirus vaccines, in particular live attenuated recombinant/chimeric dengue viruses.

Such compositions/formulas and preparation processes are described herein.

SUMMARY.

The present disclosure provides an immunogenic formulation consisting of a live attenuated flavivirus, carbohydrates, and amino acids, wherein the formulation is amenable to rapid freeze-drying and the reconstituted formulation retains the desired characteristics of the virus, including viability, immunogenicity, and stability.

The present disclosure relates more to a lyophilized immunogenic composition consisting of:

a) live attenuated recombinant/chimeric dengue virus, which uses live attenuated dengue virus strains rDEN1A30-1545; rDEN2/4A30(ME)-1495. 7163; rDEN3A30/31-7164 and ΛΕΝ4Δ30-7132, 7163, 8308 from the National Institutes of Health (NIH) of the United States.

b) Sucrose about 3% w/o - about 6% w/o.

c) Glycine about 3% w/o to 6% w/o.

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The present disclosure further describes a method for preparing such vaccine composition/formula.

Goals.

Some of the purposes of this disclosure are as follows:

The purpose of the present disclosure is to address one or more problems of the prior art, or at least to provide a useful alternative.

Another object of the present disclosure is to provide stabilizing lyophilized vaccine compositions/formulas consisting of at least one flavivirus, at least one carbohydrate, at least one amino acid and, optionally, a base. In which the composition will maintain the desired characteristics of the virus, including viability, immunogenicity and stability.

It is a further object of the present disclosure to provide stabilizing lyophilized vaccine compositions/formulas including, but not limited to, live attenuated recombinant/chimeric dengue virus serotypes (DEN 1, DEN 2, DEN 3, DEN 4) suitable for treating or preventing dengue infection, or to prevent, improve or delay the onset or progression of its clinical manifestations.

Another purpose of the present disclosure is to provide a method for producing such a vaccine composition/formula.

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

Detailed description

Although the present disclosure may be embodied in several embodiments, some of them are shown in the illustrations following the detailed description, it is understood that the present disclosure may be taken as exemplary of the principles of the disclosure and is not intended to limit the scope of the disclosure to those illustrated and disclosed herein. description.

According to the first embodiment of the present disclosure, an immunogenic composition comprising one or more live attenuated flaviviruses, one or more carbohydrates and one or more amino acids, in which the composition is amenable to rapid freeze-drying and the reconstituted composition retains the desired characteristics of the virus, including the viability of the virus, immunogenicity and stability.

The term live is used in its usual meaning, a live virus is a virus that has not been inactivated, i.e. a virus that can replicate on permissive cells. A live attenuated flavivirus is a virus that does not cause disease caused by the corresponding wild-type virus in animals or humans and is capable of eliciting a specific immune response.

According to a second embodiment of the present disclosure, the one or more live attenuated flaviviruses are recombinant flaviviruses and/or chimeric flaviviruses.

According to a third embodiment of the present disclosure, the one or more live attenuated flaviviruses are selected from the group consisting of dengue virus (DEN), yellow fever virus (YF), Japanese encephalitis virus (JE), Kunjin virus, West Nile virus (WN), tick-borne virus encephalitis (TBE), St. Louis encephalitis virus, Murray Valley encephalitis virus, Zika virus or any related flavivirus.

However, in a preferred aspect of the third embodiment, the one or more live attenuated flaviviruses is dengue virus (DEN), which is a plurality of live attenuated dengue viruses (DEN) of different serotypes selected from the group DEN-1, DEN-2, DEN-3 and DEN-4.

According to a fourth embodiment of the present disclosure, the one or more live attenuated flaviviruses are selected from the group consisting of live attenuated chimeric/recombinant yellow fever (YF) viruses and/or live attenuated chimeric/recombinant Japanese encephalitis (JE) viruses and/or live attenuated chimeric/recombinant dengue (DEN) viruses and/or live attenuated chimeric/recombinant West Nile virus (WN) viruses and/or live attenuated chimeric/recombinant tick-borne encephalitis (TBE) viruses and/or chimeric dengue virus (yellow fever-dengue) and/or chimeric YF-WN (yellow fever-West Nile) virus and/or chimeric YF-JE (yellow fever-Japanese encephalitis) virus or any related flavivirus virus.

However, in a preferred aspect of the fourth embodiment, the one or more live attenuated flaviviruses are live attenuated chimeric/recombinant dengue viruses (DEN).

According to a fifth embodiment of this disclosure, the live attenuated recombinant/chimeric dengue viruses used in the immunogenic formulation are described below:

A) Brief description of recombinant strains from the National Institutes of Health/their design:

All activities related to the generation of attenuated vaccine strains of all four dengue virus serotypes (DEN 1, DEN 2, DEN 3 and DEN 4), explained below, were carried out at the National

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Institute of Health, USA. The contents of WO2002095075 and WO2008022196 are incorporated herein in their entirety.

Origin of the gene.

1. Each of the attenuated dengue virus serotype 1-4 strains (rDEN1..A3(.), rDEN2... 4Δ30(ΜΕ), rDEN3y.A30... 31 and ΦΕΝ4Δ30) was developed by removing about 30 nucleotides (Δ30) (an additional 31 nucleotides (Δ31) in the case of DEN-3) from the non-translational 3′ ends of wild-type strains. The Δ31 mutation can also be generated separately to determine the contribution of either Δ30 or Δ31 to the combined Δ30/31 deletion mutation.

2. Structurally, all four strains envelop positive RNA viruses with a size of 35-50 nanometers.

3. The strain γΙ)ΕΝ1Δ30-1545 used here encodes a single Lys^Arg mutation at amino acid residue number 484 (mutation A1545G) in the viral polyprotein.

4. 4. The strain used, ΦΕΝ2/4Δ30(ΜΕ)-1495, 7163, encodes the Ser^Phe mutation at amino acid residue 186 (C1495T mutation) and the Leu^Phe mutation at amino acid residue 112 (A7163C mutation) in the viral polyprotein.

5. 5. ΦΕΝ3Δ30/31 includes the initial Δ30 deletion and a non-contiguous 31 nucleotide deletion that removes both the original TL-2 and TL-3 structures. The resulting strain γΟΕΝ3Δ30/31-7164, used in this case, encodes the Val^Ala mutation with a residual number of amino acids of 115 (mutation T7164C) in the viral polyprotein.

6. 6. The strain used ΦΕΝ4Δ30-7132, 7163, 8308 encodes the Thr^Ile mutation with a residual number of amino acids of 102 (mutation C7132T), the Leu^Phe mutation with a residual number of amino acids of 112 (mutation A7163C) and the Lys^Arg mutation with a residual number of amino acids 249 (mutation A8308G) in the viral polyprotein.

Figures depicting the RNA sequence and viral structure of the DEN vaccine strains:

Images 1, 2 and 3.

The wild-type strains used to generate vaccine strains are given in Table. below:

Table 1

Nomenclature of wild-type strains and vaccines

Serotype Wild type strain Vaccine strain DEN 1 Western Pacific strain rDENlA30-1545 DEN 2 New Guinea strain rDEN2/4A30(ME)-1495.7163 DEN 3 Sleman/78 rDEN3A30/31-7164 DEN 4 Dominica rDEN4A30-7132,7163,8308

A) Transformation procedure:

To create vaccine strains against dengue virus, mainly the following steps were taken:

1. A plasmid containing a full-length copy of the wild-type DEN virus cDNA was generated by generating short DNA segments using reverse transcriptase and PCR. The fragments thus obtained were suitably ligated to generate intact double-stranded DNA consisting of the full-length genomic strand of wild-type DEN cDNA cloned into a plasmid.

2. The Δ30 mutation was introduced by mutating a subfragment of the 3'UTR and replacing the wild-type DEN 3'UTR with a subfragment containing the Δ30 region. Specific mutations were introduced by site-specific PCR mutagenesis.

3. To generate the vaccine strain DEN 2, the structural genes M and E from DEN 2 were cloned into a plasmid and used to replace the structural genes in the DEN 4 cloned plasmid containing the Δ30 mutation. To generate the DEN 3 vaccine strain, two deletions of 30 and 31 nucleotides were introduced into the wild-type clone.

4. Copied RNA transcripts were synthesized from linearized plasmids using the AmpliCap SP6 Message Maker Kit (EpiCentre Technologies, Madison) and RNA purified using the RNeasy Mini kit (Qiagen, Valencia, CA). Veroelements (C6/36 for dengue 3) were transfected with purified RNA transcripts using DOTAP liposomal transfection reagents (Roche, Indianapolis, IN) to recover the desired virus. The rescued viruses were subjected to amplification, terminal dilution cloning, and final amplification to generate seed virus in Vero cells. Details of the number of amplification and terminal dilution cycles undertaken for each strain are given in Table. 2.

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table 2

Amplification and terminal dilution cycle to prepare seed virus

Virus strain DEN 1 DEN 2 DEN 3 DEN 4 Generation in Vero Vero C6/36* Vero Amplification Nil Nil 6X ZH Cloning by limiting dilution 2X 2X ZH ZH Amplification 2X 2X 2X 2X

* All further work on amplification and cloning by limiting dilution was carried out in Vero cells.

According to the first aspect of the fifth embodiment, the chimeric viruses have the property of exhibiting the characteristics of live attenuated viruses as defined above. Therefore, in the context of the disclosure, any chimeric virus expressing an envelope protein or one or more epitopes of one or more envelope proteins of one or more flaviviruses and inducing a specific immune response consisting of antibodies that neutralize the strain, or at least one of the strains, can be used. from which the coat protein or said epitope is derived.

According to a second aspect of the fifth embodiment, the live attenuated recombinant dengue virus nucleic acid further includes a mutation generating a mutation with a phenotype selected from the group consisting of temperature sensitivity in Vero cells or the human liver cell line HuH-7, host cell limitation in mosquito cells, or the human liver cell line HuH-7, host cell adaptation for enhanced replication in Vero cells, or attenuation in mice or monkeys, which includes a member selected from the group consisting of:

(1) ΙΌΕΝΙΔ30, rDEN2A30, rDEN3A30, rDEN4A30, (2) ΙΌΕΝΙΔ30, rDEN2A30, rDEN3A30, rDEN4/IA30, (3) rDENIA30, ΓΟΕΝ2Δ30, rDEN3A30, Γ ΟΕΝ4/2Δ30, (4) rDENIA30, rDEN2A30, rDEN3A30, rDEN4/3A30, (5) ΓΟΕΝΙΔ30, ΓΟΕΝ2Δ30, rDEN3/IA30, rDEN4A30, (6) ΓΟΕΝΙΔ30, ΓΟΕΝ2Δ30, rDEN3/IA30, rDEN4/IA30, (7) rDENIA30, ΓΟΕΝ2 Δ30, rDEN3/IA30, rDEN4/2A30, (8) ΓΟΕΝΙΔ30, ΓΟΕΝ2Δ30, rDEN3/IA30, rDEN4/3A30, (9) rDENIA30, ΓΟΕΝ2Δ30, rDEN3/2A30, rDEN4A30, (10) rDENIA30, rDEN2A30, rDEN3/2A30, rDEN4/IA30, (11) rDENIA30, rDEN2A30, rDEN3/ 2A30, ΓΟΕΝ4/2Δ30 , (12) rDENIA30, rDEN2A30, rDEN3/2A30, rDEN4/3A30, (13) rDENIA30, rDEN2A30, rDEN3/4A30, rDEN4A30, (14) rDENIA30, rDEN2A30, rDEN3/4A30, rDEN4/IA30,

- 5 046342 (15) YUEMDZO, rDEN2A30, rDEN3/4A30, rDEN4/2A30, (16) rDENIA30, rDEN2A30, rDEN3/4A30, rDEN4/3A30, (17) rDENIA30, rDEN2/IA30, rDEN3A30, rDEN4A30, ( 18) rDENIA30 Rden2/IA30, Rden3A30, Rden4/IA30, (19) Rdenia30, Rden2/IA30, Rden3A30, Rden4/2A30, (20) Rdenia30, Rden2/IA30, Rden3A30, Rden4/3A30, (21) Rdenia30, Rden2/IA30, rDEN3/IA30, rDEN4A30, (22) rDENIA30, rDEN2/IA30, rDEN3/IA30, rDEN4/IA30, (23) rDENIA30, rDEN2/IA30, rDEN3/IA30, rDEN4/2A30, (24) rDENIA30, rDEN2/IA30, rDEN3 /IA30, rDEN4/3A30, (25) rDENIA30, rDEN2/IA30, rDEN3/2A30, rDEN4A30, (26) rDENIA30, rDEN2/IA30, rDEN3/2A30, rDEN4/IA30, (27) YuEMDZO, rDEN2/IA30, rDEN3/ 2A30, rDEN4/2A30, (28) rDENIA30, rDEN2/IA30, rDEN3/2A30, rDEN4/3A30, (29) rDENIA30, rDEN2/IA30, rDEN3/4A30, rDEN4A30, (30) rDENIA30, rDEN2/IA30, rDEN3/4A30 , rDEN4/IA30, (31) rDENIA30, rDEN2/IA30, rDEN3/4A30, rDEN4/2A30, (32) rDENIA30, rDEN2/IA30, rDEN3/4A30, rDEN4/3A30, (33) rDENIA30, rDEN2/3A30, YuEYZDZO, Rden4a30, (34) Rdenia30, Rden2/3a30, Yuezdzo, Rden4/IA30, (35) Rdenia30, Rden2/3A30, Yueyzzzo, Rden4/2A30, (36) Rdenia30, Rden2/3a30, Yuezdzo, Rden4/3A30,

- 6 046342 (37) rDENIA30, rDEN2/3A30, rDEN3/IA30, rDEN4A30, (38) rDENIA30, rDEN2/3A30, rDEN3/IA30, rDEN4/IA30, (39) rDENIA30, rDEN2/3A30, rDEN3/IA30, rDEN4/ 2A30, (40) Rdenia30, Rden2/3a30, Rden3/IA30, Rden4/3a30, (41) Rdenia30, Rden2/3a30, Rden3/2A30, Rden4A30, (42) Rdenia30, Rden2/3a30, Rden3/2A30, Rden4/IA30 , (43) rDENIA30, rDEN2/3A30, rDEN3/2A30, rDEN4/2A30, (44) rDENIA30, rDEN2/3A30, rDEN3/2A30, rDEN4/3A30, (45) rDENIA30, rDEN2/3A30, rDEN3/4A30, rDEN4A30, (46) rDENIA30, rDEN2/3A30, rDEN3/4A30, rDEN4/IA30, (47) rDENIA30, rDEN2/3A30, rDEN3/4A30, rDEN4/2A30, (48) rDENIA30, rDEN2/3A30, rDEN3/4A30, rDEN4/3A30 , (49) rDENIA30, rDEN2/4A30, rDEN3A30, rDEN4A30, (50) rDENIA30, rDEN2/4A30, rDEN3A30, rDEN4/IA30, (51) rDENIA30, rDEN2/4A30, rDEN3A30, rDEN4/2A30, (52) rDENIA 30, rDEN2 /4A30, rDEN3A30, rDEN4/3A30, (53) rDENIA30, rDEN2/4A30, rDEN3/IA30, rDEN4A30, (54) rDENIA30, rDEN2/4A30, rDEN3/IA30, rDEN4/IA30, (55) rDENIA30, rDEN2/4A30, rDEN3/IA30, rDEN4/2A30, (56) rDENIA30, rDEN2/4A30, rDEN3/IA30, rDEN4/3A30, (57) rDENIA30, rDEN2/4A30, rDEN3/2A30, rDEN4A30, (58) rDENIA30, rDEN2/4A30, rDEN3 /2A30, rDEN4/IA30,

- 7 046342 (59) rDENIA30, rDEN2/4A30, rDEN3/2A30, rDEN4/2A30, (60) rDENIA30, rDEN2/4A30, rDEN3/2A30, rDEN4/3A30, (61) ΓΟΕΝΙΔ30, ΓΟΕΝ2/4 Δ30, rDEN3/4A30, ΓΟΕΝ4Δ30, (62) rDENIA30, rDEN2/4A30, rDEN3/4A30, rDEN4/IA30, (63) rDENIA30, rDEN2/4A30, rDEN3/4A30, rDEN4/2A30, (64) ΓΟΕΝΙΔ30, ΓΟΕ Ν2/4Δ30, rDEN3/4A30, ΓΟΕΝ4 /3Δ30, (65) rDENI/2A30, rDEN2A30, rDEN3A30, rDEN4A30, (66) rDENI/2A30, rDEN2A30, rDEN3A30, rDEN4/IA30, (67) rDENI/2A30, rDEN2A30, rDEN3A30, rDEN4/2A30, (68 ) rDENI /2A30, rDEN2A30, rDEN3A30, rDEN4/3A30, (69) rDENI/2A30, rDEN2A30, rDEN3/IA30, rDEN4A30, (70) ΓΟΕΝΙ/2Δ30, rDEN2A30, rDEN3/IA30, rDEN4/IA30, (71) rDE NI/2A30, rDEN2A30, rDEN3/IA30, rDEN4/2A30, (72) rDENI/2A30, rDEN2A30, rDEN3/IA30, rDEN4/3A30, (73) rDENI/2A30, rDEN2A30, rDEN3/2A30, rDEN4A30, (74) rDENI/2A30, rDEN2A 30 , rDEN3/2A30, rDEN4/IA30, (75) rDENI/2A30, rDEN2A30, rDEN3/2A30, rDEN4/2A30, (76) rDENI/2A30, rDEN2A30, rDEN3/2A30, rDEN4/3A30, (77) rDENI/2A30, rDEN2A30, rDEN3/4A30, rDEN4A30, (78) rDENI/2A30, rDEN2A30, rDEN3/4A30, rDEN4/IA30, (79) rDENI/2A30, rDEN2A30, rDEN3/4A30, rDEN4/2A30, i (80) rDENI/2A30, rDEN2A30, rDEN3/4A30, rDEN4/3A30,

- 8 046342 (81) rDENI/2A30, rDEN2/IA30, rDEN3A30, rDEN4A30, (82) rDENI/2A30, rDEN2/IA30, rDEN3A30, rDEN4/IA30, (83) rDENI/2A30, rDEN2/IA30, rDEN3A30, rDEN4/ 2A30, (84) ΓΟΕΝΙ/2Δ30, ΓΟΕΝ2/ΙΔ30, ΓΟΕΝ3Δ30, ΓΟΕΝ4/3Δ30, (85) rDENI/2A30, rDEN2/IA30, rDEN3/IA30, rDEN4A30, (86) rDENI /2A30, rDEN2/IA30, rDEN3/IA30 , rDEN4/IA30, (87) rDENI/2A30, rDEN2/IA30, rDEN3/IA30, rDEN4/2A30, (88) rDENI/2A30, rDEN2/IA30, rDEN3/IA30, rDEN4/3A30, (89) rDENI/2A30, rDEN2/IA30, rDEN3/2A30, rDEN4A30, i (90) rDENI/2A30, rDEN2/IA30, rDEN3/2A30, rDEN4/IA30, (91) rDENI/2A30, rDEN2/IA30, rDEN3/2A30, rDEN4/2A30, ( 92) rDENI/2A30, rDEN2/IA30, rDEN3/2A30 ₅ rDEN4/3A30, (93) rDENI/2A30, rDEN2/IA30, rDEN3/4A30, rDEN4A30, (94) rDENI/2A30, rDEN2/IA30, rDEN3/4A30, rDEN4/IA30, (95) ΓΟΕΝΙ/2Δ30, ΓΟΕΝ2/ΙΔ30, rDEN3/4A30, ΓΟΕΝ4/2Δ30, (96) rDENI/2A30, rDEN2/IA30, rDEN3/4A30, rDEN4/3A30, (97) rDE NI/2A30, rDEN2 /3A30, rDEN3A30, rDEN4A30, (98) rDENI/2A30, rDEN2/3A30, rDEN3A30, rDEN4/IA30, (99) rDENI/2A30, rDEN2/3A30, rDEN3A30, rDEN4/2A30, i (100) rDENI/2A30, rDEN2 /3A30, rDEN3A30, rDEN4/3A30, (101) rDENI/2A30, rDEN2/3A30, rDEN3/IA30, rDEN4A30, (102) rDENI/2A30, rDEN2/3A30, rDEN3/IA30, rDEN4/IA30,

- 9 046342 (103) rDENI/2A30, rDEN2/3A30, rDEN3/IA30, rDEN4/2A30, (104) rDENI/2A30, rDEN2/3A30, rDEN3/IA30, rDEN4/3A30, (105) rDENI/2A30, rDEN2/ 3A30, rDEN3/2A30, rDEN4A30, (106) ΓΟΕΝΙ/2Δ30, ΓΟΕΝ2/3Δ30, ΓΟΕΝ3/2Δ30, ΓΟΕΝ4/ΙΔ30, (107) rDENI/2A30, rDEN2/3A30, r DEN3/2A30, rDEN4/2A30, (108) rDENI /2A30, rDEN2/3A30, rDEN3/2A30, rDEN4/3A30, (109) rDENI/2A30, rDEN2/3A30, rDEN3/4A30, rDEN4A30, (110) rDENI/2A30, rDEN2/3A30, rDEN3/4A30, rDEN4/IA30 , (111) rDENI/2A30, rDEN2/3A30, rDEN3/4A30, rDEN4/2A30, (112) rDENI/2A30, rDEN2/3A30, rDEN3/4A30, rDEN4/3A30, (113) rDENI/2A30, rDEN2/4A30, rDEN3A30, rDEN4A30, (114) rDENI/2A30, rDEN2/4A30, rDEN3A30, rDEN4/IA30, (115) ΓΟΕΝΙ/2Δ30, rDEN2/4A30, rDEN3A30, rDEN4/2A30, (116) rDENI/2A30, rD EN2/4A30, rDEN3A30 , rDEN4/3A30, (117) ΓΟΕΝΙ/2Δ30, rDEN2/4A30, rDEN3/IA30, rDEN4A30, (118) rDENI/2A30, rDEN2/4A30, rDEN3/IA30, rDEN4/IA30, (119) rDENI/2A30, rDEN2/ 4A30, rDEN3/IA30, rDEN4/2A30, (120) rDENI/2A30, rDEN2/4A30, rDEN3/IA30, rDEN4/3A30, (121) rDENI/2A30, rDEN2/4A30, rDEN3/2A30 ₅ rDEN4A30, (122) rDENI /2A30, rDEN2/4A30, rDEN3/2A30, rDEN4/IA30, (123) rDENI/2A30, rDEN2/4A30, rDEN3/2A30, rDEN4/2A30, (124) rDENI/2A30, rDEN2/4A30, rDEN3/2A30, rDEN4 /3A30,

- 10 046342 (125) rDENI/2A30, rDEN2/4A30, rDEN3/4A30, rDEN4A30, (126) rDENI/2A30, rDEN2/4A30, rDEN3/4A30, rDEN4/IA30, (127) rDENI/2A30, rDEN2/4A30, rDEN3/4A30, rDEN4/2A30, (128) rDENI/2A30, rDEN2/4A30, rDEN3/4A30, rDEN4/3A30, (129) rDENI/3A30, rDEN2A30, rDEN3A30, rDEN4A30, (130) rDENI/3A30, rDEN2A30, rDEN3A30 , rDEN4/IA30, (131) rDENI/3A30, rDEN2A30, rDEN3A30, rDEN4/2A30, (132) rDENI/3A30, rDEN2A30, rDEN3A30, rDEN4/3A30, (133) rDENI/3A30, rDEN2A30, rDEN3/IA30, rD EN4A30, (134) rDENI/3A30, rDEN2A30, rDEN3/IA30, rDEN4/IA30, (135) rDENI/3A30, rDEN2A30, rDEN3/IA30, rDEN4/2A30, (136) rDENI/3A30, rDEN2A30, rDEN3/IA30, rDEN4/3A30 , (137) rDENI/3A30, rDEN2A30, rDEN3/2A30, rDEN4A30, (138) rDENI/3A30, rDEN2A30, rDEN3/2A30, rDEN4/IA30, (139) rDENI/3A30, rDEN2A30, rDEN3/2A30, rDEN4/2A30 , (140) Rdeni/3a30, Rden2a30, Rden3/2A30, Rden4/3a30, (141) Rdeni/3a30, Rden2a30, Rden3/4A30, Rden4A30, (142) Rdeni/3A30, Rden2A30, Rden3/4A30, Rden4/IA30, (( 143) rDENI/3A30, rDEN2A30, rDEN3/4A30, rDEN4/2A30, (144) rDENI/3A30, rDEN2A30, rDEN3/4A30, rDEN4/3A30, (145) ΓϋΕΝΙ/3Δ30, ΓΟΕΝ2/ΙΔ30 , ΓΟΕΝ3Δ30, ΓΟΕΝ4Δ30, (146 ) rDENI/3A30, rDEN2/IA30, rDEN3A30, rDEN4/IA30,

- 11 046342 (147) rDENI/ZDZO, rDEN2/IA30, rDEN3A30, rDEN4/2A30, (148) rDENI/ZDZO, rDEN2/IA30, rDEN3A30, rDEN4/3A30, (149) rDENI/ZDZO, rDEN2/IA30, rDEN3/ IA30, rDEN4A30, (150) rDENI/ZDZO, rDEN2/IA30, rDEN3/IA30, rDEN4/IA30, (151) rDENI/ZDZO, rDEN2/IA30, rDEN3/IA30, rDEN4/2A30, (152) rDENI/ZDZO, rDEN2 /IA30, rDEN3/IA30, rDEN4/3A30, (153) rDENI/ZDZO, rDEN2/IA30, rDEN3/2A30, rDEN4A30, (154) rDENI/ZDZO, rDEN2/IA30, rDEN3/2A30, rDEN4/IA30, (155) rDENI/ZDZO, rDEN2/IA30, rDEN3/2A30, rDEN4/2A30, (156) rDENI/ZDZO, rDEN2/IA30, rDEN3/2A30, rDEN4/3A30, (157) rDENI/ZDZO, rDEN2/IA30, rDEN3/4A30, rDEN4A30, (158) rDENI/ZDZO, rDEN2/IA30, rDEN3/4A30, rDEN4/IA30, (159) rDENI/ZDZO, rDEN2/IA30, rDEN3/4A30, rDEN4/2A30, (160) rDENI/ZDZO, rDEN2/IA30 , rDEN3/4A30, rDEN4/3A30, (161) rDENI/ZDZO, ΓϋΕΝ2/3Δ30, ΓϋΕΝ3Δ30, ΓϋΕΝ4Δ30, (162) rDENI/3A30, rDEN2/3A30, rDEN3A30, rDEN4/IA30, (163) rDENI/3A30, rDEN2/ 3A30, rDEN3A30, rDEN4/2A30, (164) rDENI/3A30, rDEN2/3A30, rDEN3A30, rDEN4/3A30, (165) rDENI/3A30, rDEN2/3A30, rDEN3/IA30, rDEN4A30, (166) rDENI/3A30, rDEN 2 /3A30, rDEN3/IA30, rDEN4/IA30, (167) rDENI/3A30, rDEN2/3A30, rDEN3/IA30, rDEN4/2A30, (168) rDENI/3A30, rDEN2/3A30, rDEN3/IA30, rDEN4/3A30,

- 12 046342 (169) rDENI/ZDZO, rDEN2/3A30, rDEN3/2A30, rDEN4A30, (170) rDENI/ZDZO, rDEN2/3A30, rDEN3/2A30, rDEN4/IA30, (171) rDENI/ZDZO, rDEN2/3A30, rDEN3/2A30, rDEN4/2A30, (172) rDENI/ZDZO, rDEN2/3A30, rDEN3/2A30, rDEN4/3A30, (173) rDENI/ZDZO, rDEN2/3A30, rDEN3/4A30, rDEN4A30, (174) rDENI/3A30 , rDEN2/3A30, rDEN3/4A30, rDEN4/IA30, (175) rDENI/3A30, rDEN2/3A30, rDEN3/4A30, rDEN4/2A30, (176) rDENI/3A30, rDEN2/3A30, rDEN3/4A30, rDEN4/3A30 , (177) rDENI/3A30, rDEN2/4A30, rDEN3A30, rDEN4A30, (178) rDENI/3A30, rDEN2/4A30, rDEN3A30, rDEN4/IA30, (179) rDENI/3A30, rDEN2/4A30, rDEN3A30, rDEN4/2A30 , (180) rDENI/3A30, rDEN2/4A30, rDEN3A30, rDEN4/3A30, (181) rDENI/3A30, rDEN2/4A30, rDEN3/IA30, rDEN4A30, (182) rDENI/3A30, rDEN2/4A30, rDEN3/IA30, rDEN4 /IA30, (183) rDENI/3A30, rDEN2/4A30, rDEN3/IA30, rDEN4/2A30, (184) rDENI/3A30, rDEN2/4A30, rDEN3/IA30, rDEN4/3A30, (185) rDENI/3A30, rDEN2/ 4A30, rDEN3/2A30, rDEN4A30, (186) rDENI/3A30, rDEN2/4A30, rDEN3/2A30, rDEN4/IA30, (187) rDENI/3A30, rDEN2/4A30, rDEN3/2A30, rDEN4/2A30, (188) rDENI /3A30, rDEN2/4A30, rDEN3/2A30, rDEN4/3A30, (189) rDENI/3A30, rDEN2/4A30, rDEN3/4A30, rDEN4A30, (190) rDENI/3A30, rDEN2/4A30, rDEN3/4A30, rDEN4/IA30 ,

- 13 046342 (191) rDENI/ZDZO, rDEN2/4A30, rDEN3/4A30, rDEN4/2A30, (192) rDENI/ZDZO, rDEN2/4A30, rDEN3/4A30, rDEN4/3A30, (193) rDENI/4A30, rDEN2A30, rDEN3A30, rDEN4A30, (194) rDENI/4A30, rDEN2A30, rDEN3A30, rDEN4/IA30, (195) rDENI/4A30, rDEN2A30, rDEN3A30, rDEN4/2A30, (196) rDENI/4A30, rDEN2A30, rDEN3A30 , rDEN4/3A30, ( 197) Rdeni/4a30, Rden2a30, Rden3/IA30, Rden4A30, (198) Rdeni/4A30, Rden2A30, Rden3/IA30, Rden4/IA30, (199) Rden/4A30, Rden2A30, Rden3/IA30, Rden4/2A30, (200 ) Rdeni/4a30, Rden2a30, Rden3/IA30, Rden4/3a30, (201) Rden/4A30, Rden2A30, Rden3/2A30, Rden4A30, (202) Rdeni/4A30, RDEN2A30, Rden3/2A30, Rden4/IA30, (203) rDENI/4A30, rDEN2A30, rDEN3/2A30, rDEN4/2A30, (204) rDENI/4A30, rDEN2A30, rDEN3/2A30, rDEN4/3A30, (205) rDENI/4A30, rDEN2A30, rDEN3/4A30, rDEN4A30, (206) rDENI /4A30, rDEN2A30, rDEN3/4A30, rDEN4/IA30, (207) rDENI/4A30, rDEN2A30, rDEN3/4A30, rDEN4/2A30, (208) rDENI/4A30, rDEN2A30, rDEN3/4A30, rDEN4/3A30, (209) rDENI/4A30, rDEN2/IA30, rDEN3A30, rDEN4A30, (210) rDENI/4A30, rDEN2/IA30, rDEN3A30, rDEN4/IA30, (211) rDENI/4A30, rDEN2/IA30, rDEN3A30, rDEN4/2A30, (212) rDENI /4A30, rDEN2/IA30, rDEN3A30, rDEN4/3A30,

- 14 046342 (213) rDENI/4A30, rDEN2/IA30, rDEN3/IA30, rDEN4A30, (214) rDENI/4A30, rDEN2/IA30, rDEN3/IA30, rDEN4/IA30, (215) rDENI/4A30, rDEN2/IA30, rDEN3/IA30, rDEN4/2A30, (216) rDENI/4A30, rDEN2/IA30, rDEN3/IA30, rDEN4/3A30, (217) rDENI/4A30, rDEN2/IA30, rDEN3/2A30, rDEN4A30, (218) rDENI/4A30 ₅ rDEN2/IA30, rDEN3/2A30, rDEN4/IA30, (219) rDENI/4A30, rDEN2/IA30, rDEN3/2A30, rDEN4/2A30, (220) rDENI/4A30, rDEN2/IA30, rDEN3/2A30, rDEN4/3A30 , (221) rDENI/4A30, rDEN2/IA30, rDEN3/4A30, rDEN4A30, (222) rDENI/4A30, rDEN2/IA30, rDEN3/4A30, rDEN4/IA30, (223) rDENI/4A30, rDEN2/IA30 ₅ rDEN3/ 4A30, rDEN4/2A30, (224) rDENI/4A30, rDEN2/IA30, rDEN3/4A30, rDEN4/3A30, (225) rDENI/4A30, rDEN2/3A30, rDEN3A30, rDEN4A30, (226) rDENI/4A30, rDEN2/3A30 , rDEN3A30, rDEN4/IA30, (227) rDENI/4A30, rDEN2/3A30, rDEN3A30, rDEN4/2A30, (228) rDENI/4A30, rDEN2/3A30, rDEN3A30, rDEN4/3A30, (229) rDENI/4A30, rDEN2/ 3A30, rDEN3/IA30, rDEN4A30, (230) rDENI/4A30, rDEN2/3A30, rDEN3/IA30, rDEN4/IA30, (231) rDENI/4A30, rDEN2/3A30, rDEN3/IA30, rDEN4/2A30, (232) rDENI /4A30, rDEN2/3A30, rDEN3/IA30, rDEN4/3A30, (233) rDENI/4A30, rDEN2/3A30, rDEN3/2A30, rDEN4A30, (234) rDENI/4A30, rDEN2/3A30, rDEN3/2A30, rDEN4/IA30 ,

- 15 046342 (235) rDENI/4A30, rDEN2/3A30, rDEN3/2A30, rDEN4/2A30, (236) rDENI/4A30, rDEN2/3A30, rDEN3/2A30, rDEN4/3A30, (237) rDENI/4A30, rDEN2/ 3A30, rDEN3/4A30, rDEN4A30, (238) rDENI/4A30, rDEN2/3A30, rDEN3/4A30, rDEN4/IA30, (239) rDENI/4A30, rDEN2/3A30, rDEN3/4A30, rDEN4/2A30, (240) rDENI /4A30, rDEN2/3A30, rDEN3/4A30, rDEN4/3A30, (241) rDENI/4A30, rDEN2/4A30, rDEN3A30, rDEN4A30, (242) rDENI/4A30, rDEN2/4A30, rDEN3A30, rDEN4/IA30, (243) Rdeni/4a30, Rden2/4A30, Rden3a30, Rden4/2A30, (244) Rdeni/4A30, Rden2/4A30, Rden3A30, Rden4/3A30, (245) Rden/4A30, Rden2/4A30, Rden3/IA30, RDEN4A30, (246, 246 ) rDENI/4A30, rDEN2/4A30, rDEN3/IA30, rDEN4/IA30, (247) rDENI/4A30, rDEN2/4A30, rDEN3/IA30, rDEN4/2A30, (248) rDENI/4A30, rDEN2/4A30, rDEN3/IA30 , rDEN4/3A30, (249) rDENI/4A30, rDEN2/4A30, rDEN3/2A30, rDEN4A30, (250) rDENI/4A30, rDEN2/4A30, rDEN3/2A30, rDEN4/IA30, (251) rDENI/4A30, rDEN2/ 4A30, rDEN3/2A30, rDEN4/2A30, (252) rDENI/4A30, rDEN2/4A30, rDEN3/2A30, rDEN4/3A30, (253) rDENI/4A30, rDEN2/4A30, rDEN3/4A30, rDEN4A30, (254) rDENI /4A30, rDEN2/4A30, rDEN3/4A30, rDEN4/IA30, (255) rDENI/4A30, rDEN2/4A30, rDEN3/4A30, rDEN4/2A30, and (256) ΓΟΕΝΙ/4Δ30, ΓϋΕΝ2/4Δ30, Γ ϋΕΝ3/4Δ30, ΓΟΕΝ4/3Δ30.

According to the sixth embodiment of the present disclosure, one or more carbohydrates include, but are not limited to, natural carbohydrates, synthetic carbohydrates, polyols, glass promoters, monosaccharides, disaccharides, trisaccharides, oligosaccharides and their corresponding sugar alcohols, polyhydroxy compounds, such such as carbohydrate derivatives and chemically modified carbohydrates, hydroxyethyl starch and sugar copolymers. Both natural and synthetic carbohydrates are suitable for use. Synthetic carbohydrates include, in particular, those in which the glycosidic bond is replaced by a thiol or carbon bond. Both D- and L-forms of carbohydrates can be used. A carbohydrate can be reducing or non-reducing. When using a reducing carbohydrate, it is preferable to add Maillard reaction inhibitors. Reducing carbohydrates suitable for use in the formulation are already known and include, but are not limited to, glucose, sucrose, maltose, lactose, fructose, galactose, mannose, maltulose and lactulose. Non-reducing carbohydrates include, in particular, non-reducing glycosides of polyhydroxyl compounds selected from sugar alcohols and other straight chain polyalcohols. Other beneficial carbohydrates include raffinose, stachyose, melezitose, dextran, cellibiose, mannobiose, and sugar alcohols. Sugar alcohol glycosides are preferably monoglycosides, in particular those obtained by reduction of disaccharides such as lactose, maltose, lactulose and maltulose. The glass former is selected from the group consisting of sucrose, mannitol, trehalose, mannose, raffinose, lactitol, lactobionic acid, glucose, maltulose, isomaltulose, maltose, lactose sorbitol, dextrose, fucose, or a combination thereof.

- 16 046342

However, according to a preferred aspect of the sixth embodiment, the immunogenic composition consists of sucrose as a suitable carbohydrate stabilizer in the range of 1% to 20% w/v, preferably in the range of 1-10%, more preferably in the range of 3-6%, most preferably in the range of less than or equal to 5% (w/v).

According to a seventh embodiment of the present disclosure, the one or more amino acids include, but are not limited to, leucine, isoleucine, histidine, glycine, glutamine, arginine, lysine, alanine, or a combination of amino acids, a peptide, a hydrolyzed protein, or a protein such as serum albumin.

However, in accordance with a preferred aspect of the seventh embodiment, the immunogenic composition consists of glycine as a suitable amino acid stabilizer in the range of 1% to 20% w/v, preferably in the range of 1-10%, more preferably in the range of 3-6% , most preferably less than or equal to 5% (w/v).

According to the eighth embodiment of the present disclosure, the immunogenic composition may further consist of a buffering agent selected from the group consisting of carbonate, phosphate, citrate, lactate, gluconate and tartrate buffering agents, as well as more complex organic buffering agents, including a phosphate buffering agent containing phosphate sodium and/or potassium phosphate in the ratio selected to achieve the desired pH. In another example, the buffering agent contains tris(hydroxymethyl)aminomethane, or Tris, formulated to achieve the desired pH. In another example, the buffering agent may be Hanks' salts minimum essential medium.

According to the ninth embodiment of the present disclosure, the immunogenic composition may further consist of a preservative selected from the group consisting of 2-phenoxyethanol, benzethionium chloride (famerol), phenol, m-cresol, thiomersal, formaldehyde, methyl and propylparabens, benzalkonium chloride, benzyl alcohol, chlorobutanol, p-chloro-cresol, or benzyl alcohol or a combination thereof.

According to the tenth embodiment of this disclosure, the immunogenic composition may further consist of pharmaceutically acceptable excipients selected from the group consisting of surfactants, polymers and salts. Examples of surfactants include nonionic surfactants such as polysorbate 20, polysorbate 80, etc. Examples of polymers may include dextran, carboxymethylcellulose, hyaluronic acid, cyclodextrin, etc. Examples of salts include NaCl, MgCl ₂ , KCl, CaCl ₂ , etc.

According to an eleventh embodiment of this disclosure, the immunogenic composition may further consist of an adjuvant selected from the group consisting of aluminum salt, aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate and potassium aluminum sulfate.

According to the twelfth embodiment of the present disclosure, the immunogenic composition may further consist of an immunostimulating component selected from the group to which it belongs: oil-in-water emulsion, MF-59, liposome, lipopolysaccharide, saponin, lipid A, lipid A derivatives, monophosphoryl lipid A, 3 -deacylated monophosphoryl lipid A, AS01, AS03, oligonucleotide, oligonucleotide consisting of at least one unmethylated CpG and/or liposome, Freund's adjuvant, complete Freund's adjuvant, incomplete Freund's adjuvant, polymers, copolymers, such as copolymers of polyoxyethylene and polyoxypropylene, including block copolymers, p 1005 polymer, CRL-8300 adjuvant, muramyl dipeptide, TLR-4 agonists, flagella, gram-negative bacteria-derived flagella, TLR-5 agonists, flagella fragments capable of binding to TLR-5 receptors, QS-21, ISCOMS, saponin combination with sterols and lipids.

According to the thirteenth embodiment of this disclosure, the specified immunogenic composition is lyophilized (frozen dried).

According to the fourteenth embodiment of the present disclosure, the lyophilized immunogenic composition is stable at 2-8°C for 12-36 months; at 25°C for 2-6 months; at 37°C for 1 week-4 weeks; at 42°C for 2-7 days; at 55°C for 2-7 days.

According to a fifteenth embodiment of the present disclosure, a method for reconstituting a lyophilized immunogenic formulation, comprising the step of reconstituting the lyophilized immunogenic formulation with an aqueous solution, optionally saline or water for injection (WFI).

According to the sixteenth embodiment of the present disclosure, the final pH of the immunogenic composition after reconstitution should be in the range of pH 6.0-pH 8.0; preferably in the pH range 7.0-pH 8.0; more preferably in the range pH 7.2-pH 7.9; and most preferably in the range of pH 7.5-pH 7.9.

According to a seventeenth embodiment of the present disclosure, the process of preparing a live attenuated chimeric/recombinant tetravalent dengue (DEN) vaccine includes any subset or all of the following steps:

a) Veroelements were revived and adapted for growth in Minimum Nutrient Medium (MNM) with Hank's saline solution and 10% Fetal Bovine Serum.

b) Verocells were initially amplified in tissue culture flasks (TCF with 175 cm ² of surface area available for cell growth), creating master and working verocell banks.

c) Cryopreserved cells from the working cell bank were restored, strengthened and further

- 17 046342 were moved in roller bottles (850 cm ² surface area available for cell growth) and incubated at 37 ± 1°C to obtain monolayers.

d) Verocellular monolayers in roller bottles were infected with working seed of dengue virus serotypes 1, 2, 3 and 4.

e) All roller bottles were incubated at 34 ± 1°C for 20 min and the volume replenished to 120 ml per bottle using minimal broth (MMM) with Hanks' saline and 2% fetal bovine serum. Next, all roller vials were incubated at 34±1°C for 2 days and a rolling speed of 0.7 rpm.

f) On day 2, monolayers in roller bottles were washed with fresh virus medium lacking fetal bovine serum and kept at 34 ± 1°C for 3 days at a rolling speed of 0.7 rpm.

g) On day 5 postinfection, cell supernatant was collected from all inoculated roller bottles and the bottles were re-added with fresh virus medium lacking fetal bovine serum.

h) Multiple harvests were taken and processed separately to obtain clarified monovalent virus pools (CMVPs).

i) Filtration of the viral harvest using forward flow filtration (DFF), through at least one clarifying filter,

j) I) Treatment of the virus harvest with a non-specific endonuclease to degrade cellular DNA.

k) The processed viral harvest was subjected to tangential flow filtration.

l) Stabilization of the virus yield with a stabilizing agent consisting of at least one amino acid and at least one hydrocarbon forms a stabilized virus yield.

m) Sterilization of the stabilized virus harvest by direct flow filtration through at least one sterilization filter.

n) Clarified monovalent viral pools (CMVP) of each dengue virus serotype were stored in polycarbonate bottles at -60°C or below.

o) Clarified monovalent viral pools (CMVP) of all four virus serotypes were mixed together to obtain the final mass, which is filled into vials and lyophilized to obtain the drug product, i.e. recombinant tetravalent dengue vaccine (live attenuated).

In the first aspect of the seventeenth embodiment, the Vero cell line was used in ATCC CCL-81 (cGMP Vero, kidney cells derived from the African green monkey (Cercopithecus aeothiops; available from ATCC, Manassas, VA, USA).

In accordance with the second aspect of the seventeenth option, multiple harvests were carried out at an appropriate time interval of approximately 4-5 times - preferably 4 times on the 5th, 7th, 9th and 11th days before discarding the starting material and processed separately for obtaining clarified monovalent viral pools (CMVP). With multiple harvests, the same amount of input material results in higher yields compared to the traditional single harvest method. This also saves time and overall processing costs, i.e. to amplify cells for infection.

According to the third aspect of the seventeenth option, the viral medium includes minimal nutritional medium (MNM) with Hanks' saline solution additionally containing dextrose, L-glutamine and sodium bicarbonate.

According to the fourth aspect of the seventeenth option, the medium containing the virus is clarified, as a rule, through filters of decreasing pore sizes (for example, 6 μ, 0.8 μ, 0.45 μ, 0.2 μ). Suitable commercial filters and filter devices are well known and can be selected by those skilled in the art. Exemplary filtration devices include, for example, Millipak (Millipore), Kleenpak (Pall) and Sartobran™ P filter devices.

According to the fifth aspect of the seventeenth embodiment, the filtered harvest was treated with a non-specific endonuclease, preferably benzonase, at a concentration in the range of 1-10 units/ml, at a temperature in the range of 4-37°C and at intervals of 2 hours to 12 hours.

In accordance with the sixth aspect of the seventeenth embodiment, the benzonase-treated crop was subjected to tangential flow filtration (TFF), typically through filters with a molecular weight cutoff (MWCO) of 500KD, more preferably 300KD, and most preferably 100KD.

According to the seventh aspect of the seventeenth option, the viral harvest was subjected to tangential flow filtration (TFF), which resulted in the concentration of the viral harvest at least 10 times and further resulted in the removal of residual impurities.

However, it is preferable to use residual impurities consisting of residual DNA, residual bovine serum albumin (BSA) and residual host cell protein.

According to the eighth aspect of the seventeenth embodiment, the process described above produces a purified and concentrated flavivirus preparation, which is more preferable for use

- 18 046342 dengue virus preparation, which contains concentrated live attenuated dengue virus particles, traces of residual cellular DNA (<10 g/dose), residual BSA (<50 g/dose) and residual cellular proteins. In addition, according to the process described above, the overall recovery of purified viruses is at least 50%.

According to the ninth aspect of the seventeenth embodiment, stabilizers consisting of a solution of one or more amino acids and one or more carbohydrates were mixed with concentrated virus (TFF concentrate) in a ratio of 60:40 or 50:50 or 40:60 virus to stabilizer to obtain the final formulation.

However, the preferred stabilizers, consisting of a solution of sucrose at a concentration of 7.5-15% (w/v) and glycine at a concentration of 7.5-15% (w/v), were mixed with concentrated virus (TFF concentrate) in a ratio of 60:40 or 50:50 or 40:60 of virus to stabilizer to obtain the final formulation consisting of sucrose at a concentration of 3 to 6% (w/v) and glycine at a concentration of 3 to 6% (w/v).

However, the preferred stabilizers, consisting of a solution of sucrose at a concentration of 12.5% (w/v) and glycine at a concentration of 12.5% (w/v), were mixed with the concentrated viral stock (TFF concentrate) in a ratio of 60:40 virus to stabilizer to obtain the final formulation consisting of sucrose at a concentration of 5% (w/v) and glycine at a concentration of 5% (w/v).

However, the preferred stabilizers, consisting of a solution of sucrose at a concentration of 11.25% (w/v) and glycine at a concentration of 12.5% (w/v), were mixed with the concentrated viral stock (TFF concentrate) in a ratio of 60:40 viral to stabilizer to obtain the final formulation consisting of sucrose at a concentration of 4.5% (w/v) and glycine at a concentration of 5% (w/v).

However, the preferred stabilizers, consisting of a solution of sucrose at a concentration of 15% (w/v) and glycine at a concentration of 15% (w/v), were mixed with concentrated virus (TFF concentrate) in a ratio of 60:40 virus to stabilizer to obtain the final formulation, consisting of sucrose at a concentration of 6% (w/v) and glycine at a concentration of 6% (w/v).

According to the tenth aspect of the seventeenth embodiment, the multiplicity of infection (MOI) of the flavivivirus, more preferably the dengue virus, for producing mother and worker seeds is in the range of 0.01 to 0.1 for each dengue serotype.

However, it is desirable that the multiplicity of infection (MOI) of dengue virus for obtaining basal seed and working seed is 0.01.

According to the eleventh aspect of the seventeenth embodiment, the immunogenic composition includes a dengue flavivirus, most preferably a dengue virus at a dose of at least 2.5 log ₁₀ PFU/0.5 ml of each of dengue virus serotypes 1, 2, 3 and 4.

According to the twelfth aspect of the seventeenth embodiment, the immunogenic composition includes dengue virus at a dose of 103 to 105 PFU/0.5 ml, preferably 103 to 104 PFU/0.5 ml, most preferably 103 PFU/0.5 ml of each dengue virus serotypes 1, 2, 3 and 4.

According to the eighteenth embodiment of this disclosure, the method of lyophilization (freeze drying) of an immunogenic composition includes the steps of freezing, primary drying and secondary drying.

However, it is preferred that the method of lyophilization (freeze drying) of a live attenuated chimeric/recombinant tetravalent dengue (DEN) vaccine composition includes any subset or all of the following steps:

a) Loading the product at a temperature of 20 to 5°C.

b) Step-by-step freezing with holding at each temperature, and the freezing stage should include freezing at a temperature of -30°C to -45°C with a freezing rate of 0.5 to 1°C/min for approximately 60 minutes to 930 minutes.

c) Annealing at -20°C for 5 hours followed by freezing at -45°C.

d) Primary drying with a temperature increase of approximately 0.5°C per minute to 1.0°C per minute to achieve a temperature of -25°C to -32°C, holding for about 600 minutes to 1980 minutes at a pressure of about 55 μbar;

e) Secondary drying involves heating the product at a rate of 0.5 to 1.0°C/min to achieve a temperature of 25°C to 30°C, holding for approximately 360-600 minutes at a pressure of 55 μbar.

The total duration of the lyophilization cycle ranges from 48 hours to 56 hours. Variations in temperature and cycle time are provided in accordance with the vial specification and the design of the lyophilizer. The product is freeze-dried based on a predetermined cycle to achieve a target moisture content of 2.0% to 3.5% by weight.

As used herein, the terms freeze-drying, freeze-drying or lyophilization imply lyophilization and refer to a process in which a suspension is frozen and then the water is removed by low-pressure sublimation. In this case, the term sublimation means a change in the physical properties of the composition, in which the composition changes directly from

- 19 046342 solid into gaseous state without turning into liquid.

According to a nineteenth embodiment of the present disclosure, the immunogenic composition is formulated for use in a method of reducing the onset or prevention of a health condition associated with administering to a human body an effective amount of the immunogenic composition intramuscularly, intravenously, subcutaneously, transcutaneously, or intradermally.

According to the twentieth embodiment of the present disclosure, the health condition is selected from the group consisting of dengue virus infection, Zika virus infection, West Nile virus infection, Japanese encephalitis infection, Kunjin virus infection, tick-borne encephalitis, St. Louis virus infection, Murray Valley encephalitis virus infection, yellow fever viral infection.

According to the twenty-first embodiment of this disclosure, the immunogenic composition can be administered subcutaneously, intradermally, or intramuscularly at a dose effective to produce neutralizing antibodies and protection. Vaccines are administered in a manner consistent with the dosage formulation and in quantities that will be prophylactically and/or therapeutically effective. The immunogenic composition of the currently disclosed vaccine may be administered as primary prophylactic agents to adults or children at risk of infection, or may be used as secondary agents to treat infected patients. For example, the tetravalent live attenuated dengue (DEN) vaccine formulation disclosed herein may be used by adults or children at risk of contracting dengue virus, or may be used as secondary agents to treat infected patients suffering from DEN.

According to a twenty-second embodiment of the present disclosure, the immunogenic composition may be presented in single-dose ampoules, multi-dose ampoules, or pre-filled syringes, wherein said immunogenic composition may be provided in a single-dose schedule, or preferably in a multiple-dose schedule, in which the primary course of vaccination may be with 1-2 separate doses, with subsequent doses given at subsequent intervals necessary to maintain and or enhance the immune response, for example, over 1-4 months for the second dose, and, if necessary, for the subsequent one(s) dose(s) over several months or years. The dosage schedule will also be determined, at least in part, by the need for additional dosage needed to provide protective immunity.

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

As used herein, the term include, or variations thereof such as include or include, will be understood to mean the inclusion of the claimed element, in whole or in part, or group of elements, in whole or in part, but not to the exclusion of any other element, in whole or in part, or group of elements, in whole or in part, and may mean includes, including, etc.; the term consisting of or consisting of also has the meaning under US Patent Law and the term is non-limiting and admits of more than what is cited to the extent that the essential or novel characteristics of what is cited are not altered by the presence of more than what is cited quoted.

The term immunogenic composition covers any composition that induces an immune response against an antigen or immunogen expressed by vectors of infection; for example, once introduced into the human body, induces an immune response against the target immunogen or antigen of interest. The terms vaccine composition and vaccine cover any composition that elicits a protective immune response against the antigen of interest or that effectively protects against the antigen; for example, when administered or injected into the body of a subject, it induces a protective immune response against the antigen or immunogen of interest or provides effective protection against an antigen or immunogen expressed by vectors of infection.

The use of the expression at least or at least one implies the use of one or more elements or ingredients or quantities. Although certain embodiments of the invention have been described, these embodiments have been presented by way of example only and are not intended to limit the scope of the invention. Changes or modifications in the wording of this invention, within the scope of the invention, may occur to one skilled in the art upon consideration of the disclosure herein. Such changes or modifications are fully consistent with the principle of the present invention.

The numerical values given for the various physical parameters, dimensions and quantities are approximate only, and values greater than the numerical value assigned to the physical parameters, dimensions and quantities are intended to fall within the scope of the invention unless otherwise stated in the specification.

Likewise, the components used in purification, eg filters, columns, are not exclusive and may be replaced by other components to achieve the same purpose at the discretion of the practitioner.

- 20 046342

Although this document places considerable emphasis on the specific features of the preferred embodiment, many additional features can be added to it and many changes can be made to the preferred embodiment without departing from the principles of the disclosure. These and other changes to the preferred embodiment of the disclosure will be apparent to one skilled in the art, and it is clearly understood that the foregoing is to be interpreted merely as an illustration of the disclosure and not as a limitation.

Advantages.

The above disclosure describes an invention that has a number of technical improvements and advantages, including, without limitation, the preparation of a stable lyophilized immunogenic composition consisting of live attenuated recombinant dengue viruses, at least one carbohydrate, at least one amino acid, and a method of manufacture such a composition. Compared to other lyophilized immunogenic formulations, this invention has the following advantages:

1. The minimum number of components in the vaccine.

2. The reconstituted vaccine retains the desired characteristics of the virus, including viability, immunogenicity and stability.

3. Improved stability at 2-8°C, 25°C, 37°C, 42°C and 55°C over long periods of time.

4. Does not contain preservatives, polymers or surfactants.

5. An improved method of producing the described stable composition/formulation, resulting in increased yield.

Examples

The examples below are provided to demonstrate preferred embodiments of the invention. Those skilled in the art will understand that the compositions and methods disclosed in the following examples represent methods discovered by the inventor that have been shown to be effective under the specific conditions described herein, and therefore those conditions are considered preferred when practicing the invention. However, those skilled in the art will appreciate that many changes can be made to specific embodiments of the invention that will still provide identical or similar results within the general spirit and scope of the invention.

Example 1.

Multiple harvests versus single harvests.

Certain experiments were conducted to initially determine a method for preparing an immunogenic formulation suitable for preclinical and clinical testing, as well as the use of flavivirus immunogenic formulations or vaccines. In some of the reported methods, live, attenuated recombinant/chimeric dengue viruses were used as flaviviruses in various formulations for preclinical and clinical trials. The dengue virus strains for creating the experimental vaccine were obtained from the US National Institutes of Health (NIH).

The manufacturing process for live attenuated chimeric/recombinant tetravalent dengue virus (DEN) vaccine involves any combination or all of the following steps:

1. Vero cells were revived and adapted for culture in minimal essential medium (MEM) with Hanks' saline solution and 10% fetal bovine serum (FBS);

2. Vero cells were initially amplified in tissue culture flasks (TCF with a culture area of 175 cm ² ), resulting in master banks and working banks of Vero cells.

3. Cryopreserved cells from the working cell bank were revived, amplified and then transferred to roller bottles (RB) (with a culture area of 850 cm ² ) and incubated at a temperature of 37 ± 1°C with a rotation speed of 0.7 rpm to obtain monolayers.

4. Monolayers of Vero cells in roller bottles were infected with working seed of dengue virus serotypes 1, 2, 3 and 4 at a multiplicity of infection (MOI) of 0.01.

5. All roller bottles were incubated at 34±1°C for 20 minutes; with volume replenishment to 120 ml per RB using minimal essential medium (MEM) with Hanks' saline solution and 2% fetal bovine serum. Next, all roller bottles were incubated at a temperature of 34 ± 1°C for 2 days with a rotation speed of 0.7 rpm.

6. On day 2, monolayers in roller bottles were washed with fresh virus medium, purified from fetal bovine serum, to remove traces of fetal bovine serum, and incubated at a temperature of 34 ± 1°C with a rotation speed of 0.7 rpm for 3 days.

7. On the 5th day after infection, the cell supernatant was collected from all infected roller bottles and fresh viral medium was placed in the bottles, including minimal nutrition

- 21 046342 body medium with Hanke's saline solution, and additionally containing dextrose, L-glutamine and sodium bicarbonate, without fetal bovine serum.

Table 3

Composition of minimal culture medium with Hanks' saline solution

Component mg/l Calcium Chloride (anhydrous) 140 Magnesium Sulfate (Anhydrous) 98 Potassium chloride 400 Monopotassium phosphate (anhydrous) 60 Sodium chloride 8000 Sodium phosphate disubstituted (anhydrous) 48 L-arginine hydrochloride 126 L-cystine hydrochloride 31 L-glutamine 292 L-histidine hydrochloride monohydrate 42 L-isoleucine 52 L-leucine 52 L-lysine hydrochloride 73 L-methionine 15 L-phenylalanine 32 L-threonine 48 L-tryptophan 10 L-tyrosine disodium dihydrate 52 L-valine 46 Choline chloride 1 Folic acid 1 1-inositol 2 Niacinamide 1 D-pantothenic acid (hemicalcium) 1 Pyridoxal hydrochloride 1 Riboflavin 0.1 Thiamine hydrochloride 1 Glucose 1000 Phenolsulfophthalein 10

Various approaches have been used to collect dengue virus cells, including single and multiple collections of cells at different times (from day 4 to day 12 postinfection) - daily, odd days, even days, etc. A comparison was made of virus titers in terms of yield during a single collection in comparison with multiple collections.

Table 4

Cell collection time

Set no. Qty RB Cell/Sample Collection Details 1 3 Daily cell collection on days 4, 5, 6, 7, 8, 9 2 3 Cell collection on even days on days 4, 6, 8 3 3 Cell collection on odd days on days 5, 7, 9, 11 4 3 Daily sample collection on days 4, 5, 6, 7, 8, 9, 10, 11 5 3 Single cell collection on day 6 6 3 Single cell collection on day 7

Starting from day 4, cells from infected RBs were collected in sets (sets 1 to 4) on the corresponding days. After collecting the cells, fresh viral medium (BC) was placed in the RB, and incubation was carried out at a temperature of 34°C until the next collection of material. A single collection of cell supernatant was also performed on day 6 (set 5) and day 7 (set 6), respectively.

Collected samples were tested to determine virus titers (CCID50) using the Spearman-Kerber method.

- 22 046342

Table 5

DEN 1 virus titers (CCID50)___________

Material collection days DEN 1 virus titers (SSJ ₅₀ /ml) Odd day Honest day Daily One-time fee Growth curve Day 4 5.5 5.5 6 Day 5 6.125 6.375 6.3 Day 6 6.625 6.375 6.975 7 Day 7 7.375 7 7.1 7.25 Day 8 7.125 6.625 7.625 Day 9 7.25 6.5 7 Day 10 6.5 6.125 6.875 Day 11 6.25 6.5 7 Day 12 6.25

The growth curve obtained for dengue virus serotype 1 (DEN 1) showed that multiple collections on days 5, 7, 9 and 11 yielded good virus titers and would therefore be used for further batches (see Fig. 4).

Another test of the effectiveness of multiple collections versus single collections was conducted using 18 RBs for Den 2, 3 and 4.

Table 6

DEN virus titers 2, 3, 4 (CCID ₅₀ )

Material collection days Virus titers (SSI ₅₀ /ml) DEN 2 DEN 3 DEN 4 Multiple collection Day 5 6.621 5.825 7.432 Day 7 6.73 6.021 7.588 Day 9 6.485 5.76 7.67 Day 11 6.202 5.64 7.321 One-time fee Day 6 6.691 5.882 7.575

In fig. Figure 5 shows the final titers of the DEN 2, 3, 4 virus using 90 roller bottles (for multiple collections and single collections of material).

The total yield from multiple harvests of cells from the same batch was significantly higher (about 0.4-0.6 log) than the yield obtained from a single harvest. Since 0.3 log is equivalent to twice the absolute value; this difference is more significant. Thus, the approach of multiple cell collection is more advantageous and preferable to single collection.

Example 2:

Dengue virus is grown on Vero cells. In this regard, it is necessary to remove impurities from the crop. Contaminants such as host cell DNA are treated with benzonase.

The influence of benzonase concentration and temperature on the content of cellular DNA and virus titer.

1. Multiple harvests were collected and processed separately to obtain purified monovalent viral pools (CMVPs).

2. Multiple harvests are purified using a 6 μ+0.45 μ filter and treated with benzonase to degrade cellular DNA.

3. Various experiments were conducted to determine the ideal benzonase concentration and temperature for crop treatment.

In this experiment, benzonase was added at four different concentrations of 500 U/L, 1250 U/L, 2500 U/L and 5000 U/L at 34 ± 1°C for 2 hours. It was found that optimal DNA degradation was achieved at benzonase concentrations of 1250, 2500 and 5000 U/L. Based on the results obtained, a concentration of 1250 U/L was selected as the working concentration.

Table 7

Effect of benzonase concentration on DNA content (ng/ml)

Control 500 units/l 1250 units/l 2500 units/l 5000 units/l Day 5 127 54 5 6 4 Day 7 310 36 7 9 3 Day 9 169 19 2 1 5

See fig. 6: Effect of benzonase concentration on DNA content.

See fig. 7: Effect of temperature on cellular DNA content and virus titer.

- 23 046342

The dengue virus is sensitive to prolonged exposure to temperature, so the experiment used a short exposure period, i.e. 2 hours at different temperatures. It was observed that at 37°C there was a loss of titer, although low DNA content was achieved. At 34°C, no loss of virus titer was observed, but the DNA content was low. At 25°C and 2-8°C there was no loss of virus titer, but DNA decay was also low. Therefore, a temperature of 34°C was selected for the process.

Example 3: Filtration and concentration.

1. Dengue virus is grown on Vero cells. In this regard, it is necessary to remove impurities from the crop.

2. Impurities are considered to be host cell DNA, host cell protein, residual BSA, and residual benzonase.

3. Tangential flow filtration (TFF) was used to remove impurities.

4. The benzonase treated crop was subjected to tangential flow filtration to remove impurities.

In initial experiments, tangential flow filtration was performed using a Millipore V Series 300 KD and Pall T Series 300 KD sieve cassette. Virus loss in the filtrate was observed to be >3.5 log. Thus, the plan was to change the cassette size to 100 KD. During the experiments, it was found that when using the Millipore cassette, there was no virus in the filtrate.

Through various tangential flow filtration options, the desired product was obtained using Merck Millipore 100KD V 10X sieve cassettes. Virus concentration is carried out in order to reduce the required storage area for bulk vaccine and remove impurities. The table below shows that after treatment with benzonase and tangential flow filtration, there was no significant loss of virus titer with significant removal of host cell DNA.

Table 8

DNA concentration in the host cell in ng/ml

Harvest day Intermediate stage manufacturing 5 7 9 DEN 1 C.V.P. 1754.5 2255.9 1631.2 BCVP 186.42 98.813 88.201 CMVP 2.238 3.583 4.134 DEN 2 C.V.P. 2430.3 2160.1 2419.2 BCVP 215.809 176.026 95.958 CMVP 15.285 8.236 6.409 DEN 3 C.V.P. 1578.6 1340.3 522.7 BCVP 93.877 22.9 10.37 CMVP 4.564 4.07 3.43 DEN 4 C.V.P. 1283.1 350.8 110.1 BCVP 30.19 53.14 26.81 CMVP 7.22 5.93 8.13

CVP:

Purified viral pool (after harvest filtration) BCVP: Purified viral pool after benzonase treatment.

CMVP: Purified monovalent viral pool (after tangential flow filtration, addition of stabilizer and after 0.2 m filtration).

Table 9

Host cell DNA in the final product

Serial number Batch number DNA (ng/dose) 1 Batch 1 0.345 2 Batch 2 0.043 3 Batch 3 0.580

Table 10

Average virus recovery from 3 consecutive batches (%)

Serotype Batch 1 Batch 2 Batch 3 Average allocation DEN 1 88.6 92.4 87.1 89.4 DEN 2 99.1 96.8 94.7 96.9 DEN 3 88.4 94.6 89.2 90.7 DEN 4 98.9 101.4 90.2 96.8

- 24 046342

Example 4:

Research of various stabilizers and optimization of the stabilizer formulation.

The stability of live, attenuated flavivirus immunogenic formulations was tested as a function of loss of activity using various stabilizing formulations (eg, loss of titer or Log ₁₀ PFU/dose).

The monovalent dengue vaccine was formulated using various combinations of stabilizers, which are given in Table. 11 and 12. The main components of these stabilizers were gelatin, sorbitol, sucrose, glycine, phosphates (KH ₂ PO ₄ , K ₂ HPO ₄ ), glutamate, lactalbumin hydrolysate (LAH) and amino acids in the form of L-histidine, L-arginine hydrochloride, L-alanine, tricine, etc.

Table 11

Different combinations of stabilizers

Number Stabilizer composition A Gelatin 2% + sucrose 20% + amino acids (2X) IN Gelatin 2% + sucrose 10% + amino acids (2X) WITH Gelatin 2% + LAH 0.70% + sucrose 20% + amino acids (2X) D Sucrose-phosphate-glutamate (2X) + LAH 4% + glycine 10% + amino acids (2X) E Gelatin 12.5% - sorbitol 25% + stabilizer-11 - Mixing ratio 80:20:10 F Gelatin 12.5% - sorbitol 25% + stabilizer-11 - Mixing ratio 1:1 G Sucrose 12.5% w/o + glycine 12.5% w/o (60:40)

Stabilizer-II contains L-histidine (2.1%), L-alanine (1%), tricine (3%), L-arginine hydrochloride (16%), lactalbumin hydrolysate (3.5%). The resulting volume of virus concentrate after tangential flow filtration (FTP conc.) was stabilized using various combinations of stabilizers described below:

Table 12

Final recipe

Number Recipe A 50 ml conc. FTP + 50ml stabilizer A (2X) = 100 ml IN 50 ml conc. FTP + 50ml stabilizer B (2X) = 100 ml WITH 50 ml conc. FTP + 50ml stabilizer C (2X) = 100 ml D 50 ml conc. FTP + 50ml stabilizer D (2X) = 100 ml E 320 ml conc. FTP + 120 ml stabilizer E = 440 ml F 50 ml conc. FTP + 50ml stabilizer F (2X) = 100 ml Lyo 1 320 ml conc. FTP + 120 ml stabilizer E = 440 ml Lyo 2 50 ml conc. FTP + 33ml stabilizer G = 83 ml

All of these formulations were tested for thermal stability at 37°C for 7 days. Samples were tested for CCID ₅₀ infectivity titers at 0, 1, 3, 5 and 7 days intervals.

The results of a study of infectivity titers of various liquid formulations showed a significant decrease, followed by complete loss (after 1-5 days), of the corresponding dengue virus titers.

The identified inability of the liquid formulation to maintain stability led to testing of lyophilized formulations. A monovalent dengue vaccine containing gelatin + sorbitol + stabilizer II (stabilizer E) was lyophilized and tested for thermal stability at 37°C for 7 days. Samples were tested for CCID ₅₀ infectivity titers, 0, 1, 3, 5 and 7 days apart. The results of the study of infectivity titers showed a higher stability profile compared to liquid formulations with significant retention of viral titers. Thus, the approach of lyophilizing a monovalent dengue mixture successfully addressed the problem of low stability and significantly reduced the loss of viral titers. Another stabilizer G was also tested, containing sucrose + glycine, which in lyophilized form showed excellent results for all four types of dengue virus. Samples were tested for CCID ₅₀ infectivity titers, 0, 1, 3, 5 and 7 days apart. The results of the study of infectivity titers showed a higher stability profile compared to other stabilizers, for example, the composition of gelatin + sorbitol + stabilizer II. In addition, it is easy to prepare and use, as it is of non-animal origin.

- 25 046342

Table 13

Titres of DEN 1 virus SSGO ₅₀ /ml (Liquid form compared to lyophilized)

Time stored AND I A IN WITH D E F LYO 1 LYO2 0 7.21 7.35 7.07 7.07 7.21 7.21 5.78 5.62 1 5.21 4.07 4.07 4.78 2.78 2.78 5.78 5.59 3 2.92 2.78 0 2.64 2.64 2.64 5.35 5.46 5 0 0 0 0 0 0 4.78 5.31 7 - - - - - - 4.64 5.18

See fig. 8: Titers of DEN 1 virus SSGO ₅₀ / ml (Liquid form compared to lyophilized).

Table 14

Virus titers DEN 2 SSGO ₅₀ / ml (Liquid form compared to lyophilized)

Time stored A IN WITH D LYO 1 LYO2 0 5.625 5.75 4.88 5 4.64 4.7 1 0 0 0 0 4.21 4.52 3 - - - - 4.07 4.43 5 - - - - 3.75 4.26 7 - - - - 3.5 4.15

See fig. 9: Titers of DEN 2 virus SSGO ₅₀ / ml (Liquid form compared to lyophilized).

Table 15

Virus titers DEN 3 CCHD _5o mt (Liquid versus lyophilized)

Storage time A IN WITH D F LYO 1 LY0 2 0 6.92 7.07 6.35 6.92 6.78 6.83 6.76 1 4.21 4.92 3.6 4.07 0 6.69 6.65 3 0 0 0 0 NT 6.58 6.56 5 NT NT NT NT NT 6.43 6.44 7 NT NT NT NT NT 6.35 6.33

NT - Not tested.

See fig. 10: Titers of DEN 3 virus SSGO ₅₀ / ml (Liquid composition compared to lyophilized).

Table 16

Virus titres DEN 4 CCJ ₅₀ /ml (Liquid form compared to lyophilized form)During storage LYO 1 LYO2 0 4.64 4.78 1 4.64 4.75 3 4.64 4.74 5 4.25 4.57 7 3.87 4.32

See fig. 11: Virus titers DEN 4 SSGO ₅₀ / ml (Lyophilized form).

Considering all the above stability study results, the best stability profile was obtained with LYO 2 (sucrose and glycine). Thus, the composition stabilizer sucrose and glycine was optimized to obtain a more stable formulation.

Example 5:

Lyophilization conditions.

To develop a dengue vaccine, we first tested different stabilizer formulations for the liquid vaccine, but it was found that the virus became unstable in the liquid formulation and there was a significant loss of titer in the liquid formulation within 5 days at 37°C. Liquid formulation is not suitable for dengue vaccine. Therefore, we decided to further test the dengue vaccine using lyophilization.

Various lyophilization trials were planned and carried out on monovalent dengue vaccine as well as tetravalent vaccine to study the suitability of the stabilizer for vaccine preparation and the stability of all four serotypes of the virus in the tetravalent vaccine stored at low temperature.

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Our studies have shown that the virus remains more stable in lyophilized form compared to liquid, and there was no loss of virus titer after lyophilization, as illustrated in Example 4.

We have chosen a lyophilized form of dengue vaccine to ensure better product stability. Lyophilization tests were conducted using gelatin-sorbitol or sucrose-glycine formulations as stabilizers for dengue vaccine CMVP, as shown in Example 4. Since gelatin is of porcine origin, there are currently some ethical issues regarding its use in vaccine formulations. The complexity of its preparation is also high, since it requires hydrolysis of gelatin at high temperatures. When using a gelatin sorbitol stabilizer, there is no consistency in the infectivity titers of all four serotypes and there is a significant loss of virus from the thawing stage to the lyophilization stage.

After a number of dengue virus vaccine trials using lyophilization and using the gelatin + sorbitol + stabilizer II formulation, we switched to the sucrose + glycine formulation as shown in example 4, and trials using lyophilization showed higher stability of the virus using the LYO 2 formulation according to compared to the LYO 1 formulation.

Table 17

Optimization of the lyophilization cycle

Test No. Lyophilization cycle in hours Loss of titer (PFU/ml) Moisture content (w/o%) 1 43 hours 0.056 2.761 2 42 hours 0.089 3.280 3 27 hours +0.017 3.343 4 35 hours 0.292 3.098 5 39 hours 0.154 2.575 6 45 hours 0.282 2.814 7 56 hours 0 2.495

In tests No. 1-7, the duration of the lyophilization cycle was changed from the standard cycle duration of 56 hours. Reducing the lyophilization cycle hours affected titer loss and moisture content. There was no significant loss of viral titer, however, moisture content increased from 2.50% to 3.34% by weight as cycle time decreased. Thus, the duration of the lyophilization cycle was set to about 56 hours.

Table 18

Lyophilization Process Parameters

Action Options Freezing -45°С int. 13.5±2 hours Cooling in capacitor < 1 hour Removing from the camera < 1 hour Primary drying -45°C to 25°C during 33± 3 hours Secondary drying -25°C during 8± 0.5 h.

See fig. 12: Lyophilization cycle.

Example 6: Stability data after lyophilization of a monovalent mass of dengue virus at 37±1°C for 14 days.

Monovalent masses of dengue virus (DEN 1,2,3,4) were formed with different concentrations of sucrose and glycine to obtain the final formulation including the concentration of sucrose and glycine as indicated in Table. 19 (SG1, SG2, SG3, SG4, SG5, SG6). The stabilized monovalent drugs were lyophilized using a standard lyophilization protocol. All lyophilized monovalent and tetravalent dengue virus preparations given in Table. below, were subjected to thermal stability testing at 37±1°C for 14 days. Samples were collected at appropriate time points and tested for infectivity titers.

Table 19

Formulations including sucrose + glycine

Composition code Sucrose Glycine SG1 5% m/o 3% m/o SG2 3% m/o 5% m/o SG3 5% m/o 5% m/o SG4 10% m/o 7% m/o SG5 4.5% m/o 5% m/o SG6 6% m/o 6% m/o

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Table 20A

Monovalent dengue vaccine composition SG1

Component Quantity per 0.5 ml dose Dengue virus serotype 1 (rDEN 1Δ30) NLT 1od ₁₀ 2.5 PFU Sucrose 5% m/o Glycine 3% m/o

Table 20B

Monovalent dengue vaccine composition SG2

Component Quantity per 0.5 ml dose Dengue virus serotype 1 (rDEN 1Δ30) NLT 1od ₁₀ 2.5PFU Sucrose 3% m/o Glycine 5% m/o

Table 20C

Monovalent dengue vaccine composition SG3

Component Quantity per 0.5 ml dose Dengue virus serotype 1 (rDEN 1Δ30) NLT 1od ₁₀ 2.5PFU Sucrose 5% m/o Glycine 5% m/o

Table 20D

Monovalent dengue vaccine composition SG4

Component Quantity per 0.5 ml dose Dengue virus serotype 1 (rDEN 1Δ30) NLT 1od ₁₀ 2.5 PFU Sucrose 10% m/o Glycine 7% m/o Table 20E Monovalent dengue vaccine composition SG5 Component Quantity per 0.5 ml dose Dengue virus serotype 1 (rDEN 1Δ30) NLT 1od ₁₀ 2.5 PFU Sucrose 4.5% m/o Glycine 5% m/o — Table 20F Monovalent dengue vaccine composition SG6 Component Quantity per 0.5 ml dose Dengue virus serotype 1 (rDEN 1Δ30) NLT 1od ₁₀ 2.5PFU Sucrose 6% m/o Glycine 6% m/o Table 21A Monovalent dengue vaccine composition SG1 Component Quantity per 0.5 ml dose Dengue virus serotype 2 (rDEN 2/4Δ30) NLT 1od ₁₀ 3.0 PFU Sucrose 5% m/o Glycine 3% m/o

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Table 21B

Monovalent dengue vaccine composition SG2

Component Quantity per 0.5 ml dose Dengue virus serotype 2 (rDEN 2/4Δ30) NLT 1od ₁₀ 3.0 PFU Sucrose 3% m/o Glycine 5% m/o

Table 21C

Monovalent dengue vaccine composition SG3

Component Quantity per 0.5 ml dose Dengue virus serotype 2 (rDEN 2/4Δ30) NLT 1od ₁₀ 3.0 PFU Sucrose 5% m/o Glycine 5% m/o

Table 21D

Monovalent dengue vaccine composition SG4

Component Quantity per 0.5 ml dose Dengue virus serotype 2 (rDEN 2/4Δ30) NLT 1od ₁₀ 3.0 PFU Sucrose 10% m/o Glycine 7% m/o

Table 21E

Monovalent dengue vaccine composition SG5

Component Quantity per 0.5 ml dose Dengue virus serotype 2 (rDEN 2/4Δ30) NLT 1od ₁₀ 3.0 PFU Sucrose 4.5% m/o Glycine 5% m/o

Table 21F

Monovalent dengue vaccine composition SG6

Component Quantity per 0.5 ml dose Dengue virus serotype 2 (rDEN 2/4Δ30) NLT 1od ₁₀ 3.0 PFU Sucrose 6% m/o Glycine 6% m/o

Table 22A

Monovalent dengue vaccine composition SG1

Component Quantity per 0.5 ml dose Dengue virus serotype 3 (rDEN 3Δ30/31) NLT 1od ₁₀ 2.5PFU Sucrose 5% m/o Glycine 3% m/o Monovalent dengue vaccine Table 22B :tav SG2 Component Quantity per 0.5 ml dose Dengue virus serotype 3 (rDEN 3Δ30/31) NLT 1od ₁₀ 2.5 PFU Sucrose 3% m/o Glycine 5% m/o

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Table 22C

Monovalent dengue vaccine composition SG3

Component Quantity per 0.5 ml dose Dengue virus serotype 3 (rDEN 3Δ30/31) NLT 1od ₁₀ 2.5 PFU Sucrose 5% m/o Glycine 5% m/o

Table 22D

Monovalent dengue vaccine composition SG4

Component Quantity per 0.5 ml dose Dengue virus serotype 3 (rDEN 3Δ30/31) NLT 1od ₁₀ 2.5 PFU Sucrose 10% m/o Glycine 7% m/o

Table 22E

Monovalent dengue vaccine composition SG5

Component Quantity per 0.5 ml dose Dengue virus serotype 3 (rDEN 3Δ30/31) NLT 1od ₁₀ 2.5 PFU Sucrose 4.5% m/o Glycine 5% m/o Table 22F Monovalent dengue vaccine composition SG6 Component Quantity per 0.5 ml dose Dengue virus serotype 3 (rDEN 3Δ30/31) NLT 1od ₁₀ 2.5PFU Sucrose 6% m/o Glycine 6% m/o Table 23A Monovalent dengue vaccine composition SG1 Component Quantity per 0.5 ml dose Dengue virus serotype 4 (rDEN 4Δ30) NLT log ₁₀ 2.5 PFU Sucrose 5% w/o Glycine 3% w/o Table 23B Monovalent dengue vaccine composition SG2 Component Qty per 0.5 ml dose Dengue virus serotype 4 (rDEN 4Δ30) NLT log ₁₀ 2.5 PFU Sucrose 3% w/o Glycine 5% w/o Table 23C Monovalent dengue vaccine composition SG3 Component Quantity per 0.5 ml dose Dengue virus serotype 4 (rDEN 4Δ30) NLT 1od ₁₀ 2.5PFU Sucrose 5% m/o Glycine 5% m/o

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Table 23D

Monovalent dengue vaccine composition SG4

Component Quantity per 0.5 ml dose Dengue virus serotype 4 (rDEN 4Δ30) NLT 1od ₁₀ 2.5PFU Sucrose 10% m/o Glycine 7% m/o

Table 23E

Monovalent dengue vaccine composition SG5

Component Quantity per 0.5 ml dose Dengue virus serotype 4 (rDEN 4Δ30) NLT 1od ₁₀ 2.5 PFU Sucrose 4.5% m/o Glycine 5% m/o

Table 23F

Monovalent dengue vaccine composition SG6

Component Quantity per 0.5 ml dose Dengue virus serotype 4 (rDEN 4Δ30) NLT 1od ₁₀ 2.5PFU Sucrose 6% m/o Glycine 6% m/o

Table 24

Tetravalent dengue vaccine composition SG1

Component Quantity per 0.5 ml dose Dengue virus serotype 1 (rDEN 1Δ30) NLT 1od ₁₀ 2.5PFU Dengue virus serotype 2 (rDEN 2/4Δ30) NLT 1od ₁₀ Z.OBOE Dengue virus serotype 3 (rDEN 3Δ30/31) NLT 1od ₁₀ 2.5PFU Dengue virus serotype 4 (rDEN 4Δ30) NLT 1od ₁₀ 2.5PFU Sucrose 5% m/o Glycine 3% m/o

Table 25

Tetravalent dengue vaccine composition SG2

Component Quantity per 0.5 ml dose Dengue virus serotype 1 (rDEN 1Δ30) NLT 1od ₁₀ 2.5PFU Dengue virus serotype 2 (rDEN 2/4Δ30) NLT 1od ₁₀ 3.0 PFU Dengue virus serotype 3 (rDEN 3Δ30/31) NLT 1od ₁₀ 2.5PFU Dengue virus serotype 4 (rDEN 4Δ30) NLT 1od ₁₀ 2.5PFU Sucrose 3% m/o Glycine 5% m/o

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Tetravalent dengue vaccine Component Dengue virus serotype 1 (rDEN 1Δ30) Dengue virus serotype 2 (rDEN 2/4Δ30) Dengue virus serotype 3 (rDEN 3Δ30/31) Table 26 SG3 Quantity per 0.5 ml dose NLT 1 unit ₁₀ 2.5 PFU NLT 1 unit ₁₀ 3.0 PFU NLT 1 unit ₁₀ 2.5 PFU Dengue virus serotype 4 (rDEN 4Δ30) NLT 1od ₁₀ 2.5 PFU Sucrose 5% m/o Glycine 5% m/o

Table 27

Tetravalent dengue vaccine composition SG4___________

Component Quantity per 0.5 ml dose Dengue virus serotype 1 (rDEN 1Δ30) NLT 1od ₁₀ 2.5PFU Dengue virus serotype 2 (rDEN 2/4Δ30) NLT 1od ₁₀ 3.0 PFU Dengue virus serotype 3 (rDEN 3Δ30/31) NLT 1od ₁₀ 2.5PFU Dengue virus serotype 4 (rDEN 4Δ30) NLT 1od ₁₀ 2.5PFU Sucrose 10% m/o Glycine 7% m/o

Table 28

Tetravalent dengue vaccine composition SG5

Component Quantity per 0.5 ml dose Dengue virus serotype 1 (rDEN 1Δ30) NLT 1od ₁₀ 2.5PFU Dengue virus serotype 2 (rDEN 2/4Δ30) NLT 1od ₁₀ 3.0 PFU Dengue virus serotype 3 (rDEN 3Δ30/31) NLT 1od ₁₀ 2.5PFU Dengue virus serotype 4 (rDEN 4Δ30) NLT 1od ₁₀ 2.5PFU Sucrose 4.5% m/o Glycine 5% m/o Tetravalent dengue vaccine co<Component Table 29 called SG6 Quantity per 0.5 ml dose Dengue virus serotype 1 (rDEN 1Δ30) NLT 1od ₁₀ 2.5 PFU Dengue virus serotype 2 (rDEN 2/4Δ30) NLT 1od ₁₀ 3.0 PFU Dengue virus serotype 3 (rDEN 3Δ30/31) NLT 1od ₁₀ 2.5 PFU Dengue virus serotype 4 (rDEN 4Δ30) NLT 1od ₁₀ 2.5 PFU Sucrose 6% m/o Glycine 6% m/o

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Table 30

DEN 1 Titer log ₁₀ PFU/ml after lyophilization_________

Storage time SG1 SG2 SG3 SG4 SG5 SG6 0 7.105 7.024 7.217 7.185 7.060 6.994 1 7.020 7.051 7.300 7.242 7.021 6.950 3 6.898 6.917 7.152 7.082 6.935 6.886 5 6.925 6.720 6.954 6.981 6.724 6.817 7 6.700 6.510 6.872 6.732 6.600 6.746 14 6.346 6.180 6.585 6.500 6.312 6.395

See fig. 13: DEN-1 titer log ₁₀ PFU/ml after lyophilization.

Table 31

DEN 2 Titer log ₁₀ PFU/ml after lyophilization

Storage time SG1 SG2 SG3 SG4 SG5 SG6 0 6.775 6.821 6.824 6.780 6.900 6.772 1 6.650 6.760 6.797 6.802 6.855 6.780 3 6.684 6.617 6.712 6.689 6.723 6.665 5 6.521 6.588 6.610 6.584 6.692 6.610 7 6.246 6.405 6.482 6.410 6.536 6.538 14 6.060 6.144 6.227 6.197 6.272 6.294

See fig. 14: DEN-2 titer log ₁₀ PFU/ml after lyophilization.

Table 32

DEN 3 Titer log ₁₀ PFU/ml after lyophilization_________

Storage time SG1 SG2 SG3 SG4 SG5 SG6 0 5.929 5.864 5.882 5.877 5.765 5.800 1 5.788 5.712 5.800 5.762 5.692 5.728 3 5.621 5.644 5.725 5.688 5.566 5.610 5 5.546 5.571 5.622 5.621 5.487 5.543 7 5.440 5.473 5.496 5.492 5.351 5.414 14 5.132 5.082 5.184 5.131 5.139 5.220

See fig. 15: DEN-3 titer log ₁₀ PFU/ml after lyophilization.

Table 33

DEN 4 Titer log ₁₀ PFU/ml after lyophilization_________

Storage time SG1 SG2 SG3 SG4 SG5 SG6 0 7.429 7.441 7.512 7.464 7.500 7.471 1 7.366 7.400 7.504 7.471 7.432 7.388 3 7.302 7.343 7.438 7.402 7.386 7.295 5 7.210 7.219 7.393 7.330 7.277 7.197 7 7.157 7.134 7.268 7.225 7.150 7.065 14 6.809 6.792 7.021 6.96 6.880 6.782

See fig. 16: DEN-4 titer log ₁₀ PFU/ml after lyophilization.

The above results of thermal stability of lyophilized monovalent preparations containing various concentrations of stabilizers sucrose and glycine indicate that the concentration of both sucrose and glycine plays an important role in maintaining the titer of viral infectivity.

Relatively large differences in virus titers were observed in formulations SG1 and SG2. There was no significant difference with formulations SG3, SG4, SG 5, SG 6 in all four serotypes. However, SG3, i.e. A 5% sucrose and 5% glycine formulation was chosen as the stabilizer for subsequent batches.

Example 7:

Stability data for tetravalent dengue vaccine, live attenuated vaccine (recombinant, lyophilized).

Dengue tetravalent vaccine (DTV) (live, attenuated, recombinant), having a combination of serotypes (DEN-1, DEN 2, DEN-3, DEN-4), stabilized using a sugar composition

- 33 046342 zy-glycine (SG) as shown in example 6, and further lyophilized according to example 5 in 3 ml USP type 1 glass vials. The container closure system consists of bromobutyl rubber stoppers and hinged aluminum and plastic caps. The stability and quality of the SG-stabilized vaccine were assessed in containers with the above-mentioned closure system for subsequent studies in accordance with the ICG requirement to maintain the shelf life of the drug.

Stability parameters indicating long-term/real-time stability were as follows:

1. Virus titers in each serotype.

2. pH.

3. Moisture content.

1. Stability data for tetravalent dengue vaccine at 2-8°C up to 12 months after lyophilization:

Table 34A

DEN-1 titer Log ₁₀ PFU/0.5 ml data after lyophilization at 2-8°C. up to 12 months

No. 0 1 2 3 6 9 12 Batch 4 4.62 4.43 4.29 4.44 4.61 4.4 4.65 Batch 5 5.17 4.8 4.69 4.85 4.83 4.67 4.59 Batch 6 4.88 4.57 4.57 4.7 4.55 4.62 4.59

See fig. 17A: DEN-1 titer Log ₁₀ PFU/0.5 ml data after lyophilization at 2-80°C for up to 12 months.

Table 34B

DEN-2 titer Log ₁₀ PFU/0.5 ml data after lyophilization at 2-8°C, up to 12 months

No. 0 1 2 3 6 9 12 Batch 4 5.22 5.06 4.93 4.92 5.14 4.85 4.88 Batch 5 5.03 4.79 4.82 4.81 4.80 4.78 4.38 Batch 6 5.01 4.93 4.85 4.86 4.88 4.63 4.24

See fig. 17B: DEN-2 titer Log ₁₀ PFU/0.5 ml data after lyophilization at 2-80°C for up to 12 months.

Table 34C

DEN-3 titer Log ₁₀ PFU/0.5 ml data after lyophilization at 2-80°C up to 12 months

No. 0 1 2 3 6 9 12 Batch 4 4.62 4.31 4.25 4.38 4.56 4.33 4.52 Batch 5 5.30 4.85 4.51 5.05 4.96 4.69 4.45 Batch 6 5.00 4.57 4.52 4.69 4.59 4.54 4.05

See fig. 17C: DEN-3 titer Log ₁₀ PFU/0.5 ml data after lyophilization at 2-80°C for up to 12 months.

Table 34D

DEN-4 titer Log ₁₀ PFU/0.5 ml data after lyophilization at 2-80°C for up to 12 months

No. 0 1 2 3 6 9 12 Batch 4 5.28 5.09 4.89 4.92 5.06 4.91 4.90 Batch 5 5.27 5.02 4.93 4.9 5.04 4.84 4.38 Batch 6 4.78 4.34 4.53 4.49 4.66 4.36 3.97

See fig. 17D: DEN-4 titer Log ₁₀ PFU/0.5 ml data after lyophilization at 2-80°C for up to 12 months.

pH and moisture content were estimated to be between 2-80°C for up to 12 months. The pH remained between 7.6 and 7.8 (Fig. 18). Residual moisture remained below 3.0% m/d up to 12 months of storage (Fig. 19).

2. Stability data for tetravalent dengue vaccine at 25°C±2°C and 60%±5% relative humidity up to 6 months after lyophilization:

Table 35A

DEN-1 titer Log ₁₀ PFU/0.5 ml data after lyophilization at 25°C for up to 6 months

No. 0 1 2 3 6 Batch 4 4.32 4.19 4.44 4.47 4.39 Batch 5 4.87 4.82 4.87 4.81 4.74 Batch 6 4.57 4.70 4.77 4.62 4.61

See fig. 20A: DEN-1 titer Log ₁₀ PFU/0.5 ml data after lyophilization at 25°C±2°C for up to 6 months.

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Table 35B

DEN-2 titer Log ₁₀ PFU/0.5 ml data after lyophilization at 25°C for up to 6 months

No. 0 1 2 3 6 Batch 4 4.92 4.95 5.11 5.10 4.87 Batch 5 4.73 4.69 4.89 4.91 4.74 Batch 6 4.71 4.72 4.85 4.81 4.67

See fig. 20B: DEN-2 titer Log ₁₀ PFU/0.5 ml data after lyophilization at 25°C±2°C for up to 6 months.

Table 35C

DEN-3 titer Log ₁₀ PFU/0.5 ml data after lyophilization at 25°C up to 6 months

No. 0 1 2 3 6 Batch 4 4.32 4.36 4.35 4.37 4.40 Batch 5 4.99 4.86 4.91 5.03 4.85 Batch 6 4.69 4.66 4.76 4.84 4.65

See fig. 20C: DEN-3 titer Log ₁₀ PFU/0.5 ml data after lyophilization at 25°C±2°C for up to 6 months.

Table 35D

DEN-4 titer Log ₁₀ PFU/0.5 ml data after lyophilization at 25°C for up to 6 months

No. 0 1 2 3 6 Batch 4 4.98 4.95 5.11 5.08 4.91 Batch 5 4.97 4.86 5.08 5.03 4.89 Batch 6 4.48 4.43 4.57 4.39 4.36

See fig. 20D: DEN-4 titer Log ₁₀ PFU/0.5 ml data after lyophilization at 25°C±2°C for up to 6 months.

pH and humidity were assessed at the starting and ending time points at 25°C±2°C for up to 6 months (6 months post-exposure). When stored under accelerated conditions for 6 months, there was no change in pH compared to the initial values (p<0.001); the mean ±3SD changed from 7.60-7.79 to 7.54-7.71 (Fig. 21). The residual moisture content remained within the upper limit of 3% ppm, average ±3SD - 1.845-2.774% ppm after 6 months after storage (Fig. 22).

3. Stability data for tetravalent dengue vaccine at 37°C±1°C up to 7 days after lyophilization:

The batches were exposed to 37°C±1°C for 7 days. Viral serotypes (DEN1-4) were titrated and loss in titers was calculated. The loss in credits was constant in all batches. The mean Log10 loss in viral titers and standard deviation of dengue 1 to dengue 4 serotypes in lyophilized DTVs were 0.604±0.117, 0.607±0.066, 0.548±0.130, 0.684±0.109, respectively (Fig. 23).

Table 36

Stability data for tetravalent dengue vaccine at 37°C±1°C up to 7 days after lyophilization

Π.Νο. DEN1 DEN2 DEN3 DEN4 Batch 1 0.60 0.68 0.60 0.68 Batch 2 0.68 0.61 0.69 0.62 Batch 3 0.50 0.54 0.41 0.61 Batch 4 0.67 0.67 0.52 0.76 Batch 5 0.69 0.50 0.65 0.69 Batch 6 0.71 0.65 0.63 0.87

See fig. 23: Stability data for tetravalent dengue vaccine at 37°C±1°C up to 7 days after lyophilization.

4. Stability data for tetravalent dengue vaccine at 42°C±1°C up to 7 days after lyophilization.

The stability of dengue tetravalent vaccine (DTV) (live, attenuated) was assessed on a representative batch. Lyophilized ampoules of finished products were subjected to thermal stress at 42°C for 7 days. Each virus serotype (DEN1-4) was titrated at the end of the exposure period against the baseline titer and the loss in titer was calculated.

(See Fig. 24).

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Data about

Table 37 dengue vaccine at 42°C±1°C up to 7 days after lyophilization

Days after exposure 1_od ₁₀ DTV virus titers (PFU/O.5 ml) DEN1 DEN2 DEN3 DEN4 0 days 4.41 4.43 4.26 4.06 7 days 3.91 3.81 3.83 3.42 Loss in credits 0.50 0.62 0.43 0.64

5. Stability data for tetravalent dengue vaccine at 55°C±1°C up to 2 days after lyophilization.

The stability of dengue tetravalent vaccine (DTV) (live, attenuated) was assessed on a representative batch. Lyophilized ampoules of finished products were subjected to thermal stress at 55°C for 2 days. Each virus serotype (DEN1-4) was titrated at the end of the exposure period against the baseline titer and the loss in titer was calculated.

Although the present invention has been described with respect to certain preferred embodiments, those skilled in the art will appreciate that various changes and modifications may be made without departing from the scope of the invention as defined in the following claims.

Claims (19)

1. Immunogenic composition, including: a) one or more live attenuated dengue viruses (DEN), including a plurality of live attenuated dengue viruses (DEN) of different serotypes selected from the group DEN-1, DEN-2, DEN-3 and DEN-4, in which the dengue virus present at a dose of 2.5-3.0 log ₁₀ PFU/0.5 ml; b) sucrose in an amount of 3-6% (w/v); And c) glycine in an amount of 3-6% (w/v); wherein the formulation is lyophilized and the reconstituted formulation retains the desired characteristics of the virus, including its viability, immunogenicity and stability. 2. The immunogenic composition as claimed in claim 1, wherein the live attenuated dengue viruses (DEN) are tetravalent, including dengue virus serotypes DEN-1, DEN-2, DEN-3 and DEN-4. 3. The immunogenic composition as claimed in claim 2, wherein live attenuated dengue viruses (DEN) are recombinant dengue viruses and/or chimeric dengue viruses containing a first nucleotide sequence encoding at least one structural protein from the first dengue virus, and a second nucleotide sequence encoding non-structural proteins from the second dengue virus. 4. An immunogenic composition as claimed in any one of claims 1 to 3, in which said live attenuated dengue virus serotypes DEN-1, DEN-2, DEN-3 and DEN-4 carry 30 nucleotide deletions, designated by the Δ30 mutation, and/or 31 nucleotide deletion, designated mutation Δ31, in the 3' untranslated region of the dengue virus genome. 5. An immunogenic composition as claimed in any one of claims 1 to 4, wherein said live attenuated dengue virus serotypes DEN-1, DEN-2, DEN-3 and DEN-4 have a phenotype that is temperature sensitivity in Vero cells or lines human liver cells HuH-7. 6. The immunogenic formulation as claimed in claim 1, wherein the lyophilized formulation is reconstituted with an aqueous solution selected from saline and WFI (water for injection), and wherein the final pH of the immunogenic formulation is 7-8. 7. Immunogenic composition according to claim 1, including: a) dengue virus serotype 1 (rDEN 1Δ30), b) dengue virus serotype 2 (rDEN 2/4Δ30), c) dengue virus serotype 3 (rDEN 3Δ30/31), d) dengue virus serotype 4 (rDEN 4Δ30), e) sucrose in an amount of 3-6% (w/v), f) glycine in an amount of 3-6% (w/v). 8. Immunogenic composition according to claim 7, including: a) dengue virus serotype 1 (rDEN 1Δ30), b) dengue virus serotype 2 (rDEN 2/4Δ30), c) dengue virus serotype 3 (rDEN 3Δ30/31), d) dengue virus serotype 4 (rDEN 4Δ30), e) sucrose in an amount of 4-5% (w/v) and f) glycine in an amount of 4-5% (w/v). - 36 046342 9. Immunogenic composition according to claim 7, including: a) dengue virus serotype 1 (rDEN 1Δ30), not less than 2.5 log ₁₀ PFU/0.5 ml, b) dengue virus serotype 2 (rDEN 2/4Δ30), not less than 2.5 log ₁₀ PFU/0.5 ml, c) dengue virus serotype 3 (rDEN 3Δ30/31), not less than 2.5 log ₁₀ PFU/0.5 ml, d) dengue virus serotype 4 (rDEN 4Δ30), not less than 2.5 log ₁₀ PFU/0.5 ml, e) sucrose 5% (w/v) and f) glycine 5% (w/v). 10. Immunogenic composition according to claim 7, including: a) dengue virus serotype 1 (rDEN 1Δ30), not less than 2.5 log ₁₀ PFU/0.5 ml, b) dengue virus serotype 2 (rDEN 2/4Δ30), not less than 2.5 log ₁₀ PFU/0.5 ml, c) dengue virus serotype 3 (rDEN 3Δ30/31), not less than 2.5 log ₁₀ PFU/0.5 ml, d) dengue virus serotype 4 (rDEN 4Δ30), not less than 2.5 log ₁₀ PFU/0.5 ml, e) sucrose in the amount of 4.5% (w/v) and f) glycine in an amount of 5% (w/v). 11. Immunogenic composition according to claim 7, including: a) dengue virus serotype 1 (rDEN 1Δ30), not less than 2.5 log ₁₀ PFU/0.5 ml, b) dengue virus serotype 2 (rDEN 2/4Δ30), not less than 2.5 log ₁₀ PFU/0.5 ml, c) dengue virus serotype 3 (rDEN 3Δ30/31), not less than 2.5 log ₁₀ PFU/0.5 ml, d) dengue virus serotype 4 (rDEN 4Δ30), not less than 2.5 log ₁₀ PFU/0.5 ml, e) sucrose in the amount of 6% (w/v) and f) glycine in an amount of 6% (w/v). 12. A method for producing an immunogenic composition according to any one of claims 1 to 11, including the following steps, such as: a) Multiple collections of a supernatant containing at least one serotype of dengue virus in minimal culture medium with Hanks' saline solution additionally containing dextrose, L-glutamine and sodium bicarbonate, with multiple collections being carried out in one batch; b) Filter the resulting viral harvest using direct flow filtration (DFF) through at least one clarifying filter with a pore size of 6 to 0.45 µm. c) Treatment of the resulting viral harvest with a non-specific endonuclease at 34±1°C for at least 2 hours, the non-specific endonuclease being benzonase having a concentration in the range of 0.5 to 5 units/ml; d) Concentration of the resulting viral harvest by tangential flow filtration (TFF) using a 100 kDa molecular weight cutoff (MWCO) membrane; e) Stabilization of the resulting viral harvest using a stabilizing agent consisting of sucrose at a concentration of 3-6% w/v and glycine at a concentration of 3-6% w/v to form a stabilized viral harvest, and f) Sterilization of the stabilized virus harvest by direct filtration of the stream through at least one clarification filter with a pore size of 0.8 to 0.2 μm to form a sterilized purified virus harvest where the overall recovery of purified viruses is at least 50%, and g) freeze-drying of the sterilized virus harvest, including the stages of freezing, primary drying and secondary drying, where i. the freezing step includes freezing at -45°C for 690 to 930 minutes; ii. the primary drying stage includes heating at a temperature from +0.5 to 1.0°C/min to achieve a temperature of -25°C with a holding time of 1800 to 1980 minutes; and iii. the secondary drying stage involves heating at a temperature of +0.5 to 1.0°C/min to reach a temperature of +25°C with a holding time of 420 to 540 minutes. 13. The method stated in paragraph 12, in which the viral harvest is treated with benzonase with a concentration of 1.25 units/ml. 14. The method stated in claim 12, in which the viral harvest is subjected to tangential flow filtration (TFF), as a result of which the concentration of the viral harvest increases at least 10 times. 15. The method stated in claim 12, in which the stabilizing agent consists of sucrose at a concentration of 4-5% (w/v) and glycine at a concentration of 4-5% (w/v). 16. The method stated in claim 12, in which the stabilizing agent consists of sucrose at a concentration of 5% (w/v) and glycine at a concentration of 5% (w/v). 17. The method stated in claim 12, in which the stabilizing agent consists of sucrose at a concentration of 4.5% (w/v) and glycine at a concentration of 5% (w/v). 18. The method stated in claim 12, in which the stabilizing agent consists of sucrose at a concentration of 6% (w/v) and glycine at a concentration of 6% (w/v). 19. Kit for reconstitution of lyophilized (sublimated) immunogenic composition, about - 37 046342 made according to claims 12 to 18, including a first container containing an immunogenic composition according to any of the previous claims, a second container containing an aqueous solution selected from saline or water for injection (WFI).