BE1024160A9 - IMMUNOGENIC FORMULATION - Google Patents

Abstract

The present invention provides formulations of immunogenic compositions comprising one or more strains of inactivated and purified dengue virus.

Description

IMMUNOGENIC FORMULATION

The present disclosure relates to formulations of immunogenic compositions. In particular, the present disclosure relates to formulations of immunogenic compositions containing one or more purified and inactivated dengue virus strains.

Dengue viruses (DEN or DENV) are members of the flaviviridae family. In the prototype family, the yellow fever virus (YF), the dengue viruses are enveloped single-stranded RNA viruses having a size of about 50 nm (detailed description in Henchal and Putnak, Clin Microbiol. Rev. 3: 376-96 (1990)). In the dengue group, there are four serotypes, dengue type one (dengue-1 or DEN1), dengue type 2 (dengue-2 or DEN2), dengue type 3 (dengue-3 or DEN3) and dengue fever type 4 (dengue-4 or DEN4). Although there is considerable genetic and antigenic similarity between serotypes, there is no significant cross-neutralization (Calisher et al., J. Gen. Virol 70: 37-43 (1989)).

The dengue virus can be transmitted to humans by mosquitoes, causing an acute viral illness. The viral dengue disease is endemic to the tropical and subtropical regions of the world. Infection with any of the four serotypes of DENV may result in asymptomatic infection, mild non-specific viral disease, classical dengue fever, or severe dengue fever with bleeding tendency. Although relatively rare, dengue haemorrhagic fever (DHF) and dementia shock syndrome (DSS) are important causes of death in children.

After an infection with a dengue virus serotype, humans who are once again infected with a different serotype of dengue virus have an increased risk of developing a serious disease. The mechanisms responsible for the subsequent enhancement of the disease are not well understood (see, for example, Halstead, Rev. Infect Dis 11: Suppl 4: S830-S839 (1989), Rothman, Nat Rev. Immunol 11: 532-543 (2011), Guzman and Isturiz, Int J Antimicrob Agents 36: S40-S42 (2010)).

The prevention and the fight against a human disease caused by the dengue virus are mainly carried out, at the present time, by the control of the mosquito vector. Therefore, there remains a need for safe and effective vaccines against all serotypes of dengue virus, and methods for producing them.

The development of an effective dengue vaccine has been hampered by problems with the stability of viral preparations after virus purification. In particular, serotype DEN4 exhibits reduced stability at room temperature (relative to other serotypes), which increases the difficulty of preparing DEN4 PIV under the form of a bulk vaccine component. There is a need for immunogenic formulations comprising DEN4 that are stable during storage.

One aspect of the present invention is an immunogenic composition comprising an inactivated and purified serotype 4 dengue virus, a buffering agent, a surfactant and a stability enhancing excipient.

In a further aspect of the present invention, the stability enhancing excipient is selected from an inorganic salt and a sugar, such as CaCl 2, MgSO 4, and sucrose.

In a further aspect of the present invention, the stability enhancing excipient is selected from MgSO 4 at a concentration of at least about 1.8 mM, at least about 3.75 mM, at least or about 7.5 mM, at least or about 15 mM, at least or about 30 mM, and at least about 45 mM.

In a further aspect of the invention, the stability enhancing excipient is selected from CaCl 2 at a concentration of at least about 1.8 mM, at least about 3.7 mM. at least about 7.5 mM, at least about 15 mM, at least about 30 mM, and at least about 45 mM.

In a further aspect of the invention, the stability enhancing excipient is sucrose at a concentration of at least about 8% w / v (w / v), or at least about 10% in w / v.

In a further aspect, the immunogenic composition of the invention comprises sucrose and at least one further excipient selected from CaCl 2 and

MgSCb.

In a further aspect of the invention, the composition further comprises an adjuvant. 1 / adjuvant may be an adjuvant without aluminum, or an aluminum salt, such as aluminum hydroxide or aluminum phosphate.

In a further aspect of the invention, the composition further comprises at least one additional serotype of inactivated and purified dengue virus.

In a further aspect of the invention, the composition comprises an inactivated and purified serotype 1 dengue virus (PIV DEN1), an inactivated and purified serotype 2 dengue virus (PIV DEN2) and a dengue virus of inactivated and purified serotype 3 (PIV DEN3).

A further aspect of the invention is a method of formulating a bulk preparation of inactivated and purified dengue virus or a finished vaccine formulation thereof, comprising providing a solution comprising water sterile, buffering agent, surfactant and stability enhancing excipient; and adding to the solution of an inactivated and purified dengue virus.

Fig. 1 is a graph showing the DLS results for a DEN4 PIV formulation supplemented with the tested excipients listed in Table 1 at time 0. The Y axis is the average size Z of the nanometer (nrn) viral particles. The control (CTRL PB) is the basic formulation containing the drug PIV DEN4 (without any excipient tested).

Figure 2 is a graph showing the DLS results for a DEN4 PIV formulation supplemented with the excipients tested listed in Table 1, after storage for 48 hours at 25 ° C (T48h25 ° C).

The units of the Y axis and the control (CTRL PB) are as in FIG. 1.

Figure 3 is a graph depicting HP-SEC-UV recovery for DEN4 PIV fonnulation supplemented with the excipients tested listed in Table 1, after 48 hours of storage at 25 ° C. The Y axis is the% recovery from the standard curve. The controls are the basic formulation containing the PIV DEN4 mediocentric substance (without any tested excipients).

Figure 4 is a graph showing the DLS results for a DEN4 PIV formulation supplemented with the tested excipients listed in Table 2, at time 0. The Y axis is the average size Z of the nanometer (nm) viral particles. The control (CTRL PB) is the basic formulation containing the drug PIV DEN4 (without any excipient tested).

Figure 5 is a graph showing the DLS results for a DEN4 PIV formulation supplemented with the excipients tested listed in Table 2, after storage for 48 hours at 25 ° C (T48h25 ° C). The units of the Y axis and the control (CTRL PB) are as in Figure 4.

Fig. 6 is a graph showing HP-SEC-UV recovery for a DEN4 PIV formulation supplemented with the tested excipients listed in Table 2 after 48 hours storage at 25 ° C. The Y axis is the% recovery from the standard curve. The controls are PIV DEN4 formulated without any excipient tested (basic formulation).

Fig. 7 is a graph showing the DLS results for a DEN4 PIV formulation supplemented with the excipients tested listed in Table 3, after 48 hours of storage at 25 ° C. The Y axis is the average size Z of viral particles in nanometers (nrn). The control (CTRL PB) is the basic formulation containing the drug substance PIV DEN4 (without any tested excipient).

Fig. 8 is a graph showing the DLS results for a DEN4 PIV formulation supplemented with the excipients tested listed in Table 3, after storage for 7 days at 25 ° C. The units of the Y-axis and the control are as in FIG. 7,

Figure 9 is a graph depicting the results of nephelometry on a DEN4 PIV formulation supplemented with the tested excipients listed in Table 3, after 48 hours storage at 25 ° C. The units of the Y axis are ONE (nephelometry unit); UTN stands for nephrometric turbidity unit. The control (PB) is the formulation of PIV DEN4 without any excipient tested (basic formulation).

Figure 10 is a graph showing nephelometry results on a DEN4 PIV formulation supplemented with the tested excipients listed in Table 3, after 7 days of storage at 25 ° C. The units and the witness are as in Figure 9.

Figure 11 is a graph showing the results of HP-SEC-UV on a DEN4 PIV formulation supplemented with the excipients tested listed in Table 3, after 48 hours of storage at 25 ° C. The Y axis is the% recovery from the standard curve. The control (PB) is the formulation of PIV DEN4 without any tested excipient (basic formulation).

Figure 12 is a graph showing the results of HP-SEC-UV on a DEN4 PIV formulation supplemented with the excipients tested listed in Table 3, after 7 days of storage at 25 ° C. The Y axis is the% recovery from the standard curve. The control (PB) is the formulation of PIV DEN4 without any excipient tested (basic formulation).

Fig. 13 is a graph showing the results of an ELISA test on a DEN4 PIV formulation supplemented with the excipients tested listed in Table 3, after 48 hours of storage at 25 ° C. The Y axis is the% recovery from the theoretical antigenicity that should be found on the basis of the applied dilution factor. The control (PB) is the formulation of PIV DEN4 without any excipient tested (basic formulation).

Fig. 14 is a graph showing the results of an ELISA test on a DEN4 PIV formulation supplemented with the excipients tested listed in Table 3, after 7 days of storage at 25 ° C. The Y axis is the% recovery from the theoretical antigenicity that should be found on the basis of the applied dilution factor. The control (PB) is the formulation of PIV DEN4 without any tested excipient (basic form).

Fig. 15 is a graph showing the results of IF on a DEN4 PIV formulation supplemented with the tested excipients listed in Table 3 after 48 hours of storage at 25 ° C. The Y axis is the% recovery from the standard curve. The control (PB) is the formulation of PIV DEN4 without any excipient tested (basic formulation).

Figure 16 is a graph showing the results of IF on a DEN4 PIV formulation supplemented with the excipients tested listed in Table 3, after 7 days of storage at 25 ° C. The Y axis is the% recovery from the standard curve. The control (PB) is the formulation of PIV DEN4 without any excipient tested (basic formulation).

Fig. 17 is a graph showing DLS results for a tetravalent base formulation containing DEN PIVs supplemented with CaC1.2 at various concentrations, MgSCg at various concentrations, or 8% sucrose, after 6 days of storage. at 25 ° C. The control is the tetravalent formulation without added excipient (basic formulation). 1 / Y axis is the average size Z of viral particles in nanometer (nm).

Fig. 18 is a graph showing the results of an ELI SA test on DEN1 obtained with the tetravalent formulations, as described for Fig. 17. The Y axis is the% recovery from the theoretical antigenicity which should be found on the basis of the applied dilution factor.

Fig. 19 is a graph showing the results of an ELI SA test on DEN2 obtained with the tetravalent formulations, as described for Fig. 17. The Y axis is the% recovery from the theoretical antigenicity which should be found on the basis of the applied dilution factor.

Fig. 20 is a graph showing the results of an ELTSA test on: DEN 3 obtained with the tetravalent formulations, with reference to Fig. 17. The Y axis is the% recovery from the theoretical antigenicity which should be to be found on the basis of the applied dilution factor.

Fig. 21 is a graph showing the results of an ELISA assay on DEN4 obtained with the tetravalent formulations, as described for Fig. 17. The Y axis is the% recovery from the theoretical antigenicity that should be found based on the applied dilution factor.

Fig. 22 is a graph showing the results of an ELISA assay on DEN1 obtained with non-adjuvant tetravalent formulations with CaCl2 (15 mM), MgSO4 (15 mM), and added sucrose (8% total), to provide the % recovery compared to bulk purified substance (2% sucrose total). "NC" means non-centrifuged sample, and "C" means centrifuged.

Fig. 23 is a graph showing the results of an ELISA test on DEN2 obtained with the tetravalent formulations without adjuvant, as described for Fig. 22.

Fig. 24 is a graph showing the results of an ELTSA test on DEN 3 obtained with tetravalent formulations without adjuvant, as described for Fig. 22.

Fig. 25 is a graph showing the results of an ELISA test on DEN3 obtained with tetravalent formulations without adjuvant, as described for Example 22.

Fig. 26 is a graph showing the results of DLS analysis of tetravalent formulations without adjuvant, as described for Fig. 22, measured after seven days of storage at different temperatures (4 ° C, 25 ° C, and 37 ° C) . "Fresh" indicates the basic formulation evaluated at TO.

Figure 27 is a graph of the protein content of tetravalent formulations without adjuvant, as described for Figure 22, evaluated by SEC HPLC with UV detection (results provided as% recovery vs. protein content of the substance purified in bulk).

Figure 28 is a graph of the results of nephelometry on non-adjuvanted tetravalent formulations as described for Figure 22. The units of the Y axis are UN (nephelometric unit).

Figure 29 shows the results of static light scattering (SLS) for measuring particles <10 μm, in tetravalent formulations with Al (OH) 3 as an adjuvant.

Figure 30 shows the results of a static light scattering (SLS) for measuring particles <100 gm, in tetravalent formulations with Al (OH) 3 as an adjuvant.

Fig. 31 is a graph showing the results of a DLS (Z-axis Y-axis) analysis of a DS DEN4 with MgSOi at various time points (TO, 1 week) and under various conditions (22 ° C, 30 ° C, and after 1, 2 or 3 cycles of freezing / thawing). The control (CTRL) is a composition of DEN4 without MgSCy.

Overview of Dengue Vaccination

The present disclosure relates to formulations of immunogenic compositions. In particular, the present disclosure relates to formulations of compositions, such as bulk vaccine preparations and finished vaccine formulations, containing one or more strains of inactivated and purified dengue virus (DEN PIV). The formulations disclosed herein enhance the recovery and stability of PIV DEN in the immunogenic compositions, which facilitates the production, storage and distribution of these compositions. The enhanced recovery and stability of the formulations disclosed herein apply to all stages of the vaccine manufacturing process, including bulk vaccine preparations and finished vaccine formulations.

Effective vaccination against the four dengue virus serotypes (DENV) is desirable, as infection with any of the serotypes can cause human disease, and multiple DENV serotypes are often present in a single geographical area. In addition, the predominant serotype may vary with time and geographical location.

Several live attenuated DENV candidate vaccines have been described (Walface et al., Opinion in Virology 3: 352-56 (2013), Halstead, Vaccine 31: 4501-07 (2013), Thomas et al., Am. J. Trop, Meel Hyg 88: 73-88 (2013), Bauer et al., Am J. Trop Med Hyg 14-0625 (July 2015 epub), Watanaveeradej et al., Amo Trop, Med Hyg 91 (1): 119-28 (2014)). A non-replicating recombinant DENV subunit vaccine has also been described. Coller et al., Vaccine 29: 7267-7275 (2011).

An inactivated tetravalent dengue purified vaccine (TDEN PIV) has been shown to induce protective antibody responses against DENV challenge in rhesus macaques. Fernandez et al., Am. J. Trop. Med. Hyg. 92 (4): 698-708 (2015).

Stabilization of DEN4

In one aspect, the present disclosure relates to methods of increasing the compositional stability comprising inactivated and purified dengue virus serotypes, such as PIV DEN4, by formulation of the inactivated dengue virus (s) with excipients. improving stability as described herein.

In another aspect, the present disclosure relates to a method for improving the recovery of an inactivated dengue virus (or a plurality thereof) of preserved antigenicity, in a composition containing it, by the formulation of the or inactivated dengue viruses with stability enhancing excipients as described herein.

As used herein, the stability of a dengue VIP formulation is measured by changing the average size of virus particles over time (aggregation or flocculation of the virus). Therefore, the stability of a PIV formulation of the nib can be evaluated by measuring any increase in the average size of the virus particles over time (aggregation or flocculation), another measure of the stability of a formulation of PIV of dengue is the change in antigenicity over time, with less stable formulations showing greater decrease in antigenicity. Thus, a "less stable" formulation is a formulation in which the average size of the virus particles increases and / or whose antigenicity decreases, compared to a comparison formulation under identical or similar conditions and for identical periods of time. or the like. An excipient which stabilizes a formulation is an excipient which decreases aggregation over time and / or which maintains antigenicity over time, compared to an identical formulation but which does not contain the excipient or which contains 1 ' excipient in a lesser amount. In one embodiment of the present invention, the stabilities of two formulations are compared after storage for 48 hours at 25 ° C; in another embodiment, the formulations are compared after storage for six days at 25 ° C, in another embodiment, the formulations are. compared after storage for seven days at 25 ° C.

Compositions of the present invention

A first aspect of the present disclosure relates to immunogenic compositions which comprise one or more inactivated and purified dengue viruses, which are at least one of the above. Such compositions can be bulk preparations of commercially produced inactivated and purified dengue virus (DEN), and suitable for formulation into pharmaceutical compositions (e.g., vaccines for preventing infection with and / or or a disease caused by the dengue virus); or finished vaccine formulations (drug product). The addition of a selected excipient, as disclosed, improves the stability of the composition, as compared to formulations which do not contain the excipients mentioned or where the selected excipient is selected. at significantly lower rates. These formulations containing a selected excipient possess the favorable character of reducing the aggregation of the inactivated virus. for example during storage, and / or to maintain antigenicity over time (both being compared to a formulation that does not contain the selected excipient or that contains the selected excipient at significantly lower levels. used here, a "significantly lower" rate is less than half, less than 40%, less than 25% or less than 20% of a comparison rate.

Accordingly, an embodiment of the present invention is an immunogenic composition comprising one or more inactivated and purified dengue virus serotypes, the immunogenic composition further comprising a stability enhancing excipient selected from a sugar, an amino acid, or a salt that provides a divalent ion in solution. Suitable excipients include sucrose, CaCl 2 and MgSCg. The aggregation of viral particles decreases in the immunogenic compositions of the present invention (relative to formulations not containing the stability enhancing excipient), for example after storage at 25 ° C for 48 hours, and / or after storage at 25 ° C for six days, and / or after storage at 25 ° C for seven days. The aggregation of viral particles can be measured by any suitable method known in the art.

Alternatively or additionally, the antigenicity is preserved in the immunogenic compositions of the present invention (as compared to formulations not containing the stability enhancing excipient), for example after storage at 25 ° C for 48 hours, and / or after storage at 25 ° C for six days, and / or after storage at 25 ° C for seven days. Antigenicity can be measured by any suitable method known in the art, such as an ELISA assay.

Alternatively or additionally, the recovery of the inactivated and purified dengue virus (or viral antigens) is improved in the immunogenic compositions of the present invention, for example more than 50%, 60%, 70%, 80%, 90%. %, (or more than 95%) of the viral material is recovered in the final preparation after storage at 25 ° C for 48 hours, and / or after storage at 25 ° C for six days, and / or after storage at 25 ° C for seven days.

In one embodiment of the present invention, the excipient selected is sucrose, and the final sucrose concentration is present in the immunogenic composition (e.g., bulk preparation or finished vaccine formulation) at a high level, e.g. at least about 10% w / v (w / v), or at least about 9% w / v, or at least about 8% w / v. v, or at least or about 6% w / v. In some embodiments, the final concentration of sucrose is from about 5% to about 20% w / v; between about 6% and about 10% w / v, or between about 7% and about 9% w / v. In a further aspect, the immunogenic composition of the invention comprises sucrose and at least one additional excipient selected from CaCl 2 and MgSO 4.

In one embodiment of the present invention, the selected excipient is CaCl 2, and is present in the immunogenic composition (e.g., bulk preparation or finished vaccine formulation) at a final concentration of at least or about 5 mM, at least or about 10 mM, at least or about 15 mM, at least or about 20 mM, morris or about 30 mM, or at least or about 45 mM. CaC'1.2 can be provided in the form of dehydrated calcium chloride (CaCl2.2H2O). In some embodiments, the final CaCl 2 concentration is between about 1 mM and about 50 mM; between about 5 mM and about 25 mM; or between about 10 mM and about 20 mM. In a further embodiment, the immunogenic composition of the invention comprises CaCl 2 and at least one additional excipient selected from sucrose and MgS O 4.

In one embodiment of the present invention, the selected excipient is MgSO 4, and is present in the immunogenic composition (e.g., bulk preparation or finished vaccine formulation) at a final concentration of at least or about 5 mM, at least or about 10 mM, at least or about 15 mM, at least or about 20 mM, at least or about 30 mM, or at least or about 45 mM. The amount of MgSO 4 can be adjusted depending on the amount of the product. i n d i n d i n d i n d i o n i o n i o n o n o n o stability by using an appropriate amount of MgSCg. MgSCt can be provided in the form of magnesium sulfate heptahydrate (MgSO4.7H2O). In some embodiments, the final MgSO4 concentration is between about 1 mM and about 50 mM; between about 5 mM and about 25 mM; or between about 10 mM and about 20 mM. In a further embodiment, the immunogenic composition of the invention comprises MgSO 4 and at least one additional excipient selected from sucrose and CaCl 2.

One embodiment of the invention is an immunogenic composition comprising an inactivated and purified virus of the four dengue virus serotypes (a tetravalent PIV DEK composition), further comprising an excipient selected from CaCl2, MgSCy, and the like. In a real-time mode, the e x c i p i ent t s. MgSO4 has a final concentration of at least about 7.5 mM, at least about 15 mM, at least about 30 mM, or at least about 45 mM. . The immunogenic composition may further be adjuvanted, for example with an aluminum-based adjuvant.

One embodiment of the invention is an immunogenic compost comprising inactivated and purified DEN-4, further comprising at least one excipient selected from CaCl2, MgSCt, and sucrose. In one embodiment, the excipient is MgSCg at a final concentration of at least about 7.5 mM, at least about 15 mM, at least about 30 mM, or df at least or about 45 mM. The immunogenic composition may further be adjuvanted, for example with an aluminum-based adjuvant.

The compositions disclosed herein may include one or more dengue virus serotypes. In one embodiment, the compositions comprise at least DEN-4. Generally, the compositions comprise a plurality of dengue viruses derived from more than one serotype, i.e., dengue serotype 1, dengue serotype 2, dengue serotype 3 and / or dengue serotype 4 (DEN-1, DEN-2, DEN-3 and / or DEN-4, respectively). For example, the composition may comprise two, three or four different serotypes of the dengue virus. In one embodiment, the composition comprises inactivated and purified dengue viruses of the four serotypes, and initiates an immune response directed against each of DEN-1, DEN-2, DEN-3 and DEN-4, the virus or viruses. can be selected from wild-type viruses (that is, propagated from or corresponding to a virulent virus from a naturally occurring isolate), or the virus (s) can be selected from attenuated viruses, recombinant viruses and / or chimeric viruses. A single composition may comprise one or more wild-type viruses, one or more attenuated viruses, one or more recombinant viruses and / or one or more chimeric viruses, in any combination.

The purified dengue virus can be inactivated using a chemical agent, a physical agent and / or inactivating radiation, alone or in any combination. The purified dengue virus can be inactivated by exposure to formaldehyde, formaldehyde, betapropiolactone (GLP), hydrogen peroxide, ultraviolet radiation and gamma radiation, or a combination of any of these techniques. Descriptions of these methods can be found, for example, in published PCT Application No. WO 2010/094,663 (US publication No. 2011 318,407), and US publication No. 2007,031,451, which are incorporated herein by reference. This is a reference for illustrating examples of dengue virus inactivation methods.

Traditionally, a human dose of the immunogenic composition contains an amount of PIV DEN that induces an immunoprotective response without significant adverse side effects in a typical subject; Obtaining an immunoprotective effect may require administration of more than one dose to an individual, depending on the selected immunization schedule. As used herein, immunoprotective does not require complete protection against infection; it means a decrease in the severity or incidence of an infection, a disease or the symptoms of a disease. The antigen content may be measured by the total protein content of a purified or partially purified viral antigen, or by immunological methods, for example by ELISA, or by a quantitative immunoprecipitation method, such as immunodiffusion. Generally, it is expected that each human dose will comprise at least about 0.1 μg, at least about 0.2 μg, at least about 0.25 μg, at least about less or about 0.33 μg, at least or about 0.4 μg, at least or about 0.5 μg, at least or about 1.0 μg, at least or about 2.0 μg, at least or about 3, 0 μg, at least or about 4.0 μg, at least or about 5.0 μg, at least or about 8.0 μg, at least or about 10.0 μg, or at least about 20 μg (or n any amount between 0.1 and 20.0 μg) of each virus serotype. Generally, a single human dose of the immunogenic composition contains at most 100 μg of each virus serotype, for example, at most 90 μg, or at most 80 μg, or at most 75 μg, or at most 70 μg, or plus 60 yg, or not more than 50 yg, or not more than 40 yg, or not more than 30 yg, or not more than 20 yg, or not more than 10 yg, or not more than 8 yg, or not more than 4 ug, or not more than 2 yg (or any amount between 2 and 100 μg) of each virus serotype. In some embodiments, the immunogenic composition for use in humans is a liquid formulation, for example a solution or a suspension. In other embodiments, the composition is prepared, lyopnized and resuspended prior to administration to the subject.

The amount of PIV DEN 'used in an immunogenic composition is chosen according to the population of subjects for which it is intended (for example, adults, infants). An optimal amount for a particular composition can be determined by conventional studies involving observation of antibody titers and other responses in subjects.

In some embodiments of the present invention, the immunogenic composition comprises one or more PTV DENV serotypes and an adjuvant. The adjuvant may be an aluminum salt. Suitable aluminum salts include hydrated aluminum hydroxide (Al (OH) 3), aluminum phosphate (AlPO 4), aluminum oxide hydroxide (AlO (OH)) and hydroxyphosphate of aluminum. 'aluminum. In some embodiments, the inactivated and purified dengue virus is adsorbed on aluminum salts. When a plurality of dengue viruses are included in the composition, each can be adsorbed on the same aluminum salt, or different viruses can be adsorbed on different aluminum salts. When aluminum is present, the amount is generally between about 100 μg and 1 mg, from about 100 μg, to about 200 μg, about 500 μg, about 750 μg, or about 1000 μg. as about 500 μg per dose. In formulations in which an aluminum salt is used, the inactivated and purified dengue virus (s) may be previously adsorbed onto the aluminum salt prior to formulation in the compositions disclosed herein. Alternatively, the aluminum salt may be added to a liquid composition, or included in the liquid in which a freeze-dried immunogenic composition is delivered to it. The adjuvant may alternatively be a liposomal formulation of immunostimulants. An appropriate liposomal formulation of this type is the AS01 adjuvant system (GlaxoSmithKline), which contains monophosphoryl lipid A (MPL, a detoxified lipopolysaccharide derivative (LPS)) and QS21 (a saponin extracted from the Quillaja saponaria tree). See, for example, WO 94/00153 (PCT / EP 93/01 524), WO 94/21,292 (PCT / EP 94/00818), WO 07/068907 (PC1 / GB 06/004 634). ), WO 96/33739 (PCT / EP 96/01464)). The adjuvant may be an oil-in-water emulsion, such as the adjuvant system AS03 (GlaxoSmithKline) containing alpha-tocopherol and squalene (see, for example, WO 95/17210 (PCT / EP 94 / 04246), WO 08/043 774 (PCT / EP 07/060 743)). The composition may be aluminum-free, or may include both an aluminum salt adjuvant and a non-alum adjuvant (such as lipopolysaccharide, saponin, or oligo-nucleotide). In one embodiment, the immunogenic composition comprises a DENV PIV of the four serotypes, and an adjuvant (e.g., a tetravalent adjuvanted vaccine based on PIV DEN). In one embodiment, the tetravalent adjuvanted vaccine is based on PIV DEN. comprises an aluminum-based adjuvant, such as Al (OH) 3.

The immunogenic compositions disclosed herein may further comprise one or more surfactants; many surfactants are known in the art for use in pharmaceutical formulations. Each surfactant of this type is selected to be suitable for administration to a subject, particularly a human subject. In some embodiments, the surfactant is selected to be suitable for parenteral administration, for example for intramuscular, subcutaneous, transcutaneous or intradermal administration. Suitable surfactants for the dengue compositions disclosed herein include poloxamer surfactants, polysorbate surfactants, octoxinol surfactants, polidocanol surfactants, polyoxylstearate surfactants, polyoxylated castor oil surfactants, N-octyl glucoside surfactants, macrogol 15-hydroxystearate, and combinations thereof. In some embodiments, the poloxamer surfactants are particularly suitable for formulations in which the inactivated and purified dengue virus (s) are not adsorbed on an aluminum salt.

The poloxamer surfactants are linear copolymers of polyethylene-polypropylene glycol. In commerce, they are often called pluronic surfactants. In some embodiments, the poloxamer surfactant is selected from a polyethylene-polypropylene glycol copolymer having an average molecular weight of at least about 1000 kD, and an average molecular weight of at most about 15,000 kD. In a specific embodiment, the immunogenic composition is formulated with a polyethylene-poly-propylene glycol copolymer, poloxamer 188, which is commercially marketed under the trade names Pluronic ™ F-68, Lutrol ™ F-68, and Kolliphor ™ P188, which has a molecular weight of 860 kD, with a polyoxypropylene molecular weight of 1800 g / mole and a polyoxyethylene content of 80%.

The immunogenic compositions disclosed herein may further comprise one or more buffering agents. The buffering agent (s) are generally chosen so as to maintain the pH of the composition at a pH greater than or equal to the neutral pH, for example at a pH equal to or close to the physiological pH of 7.4, and in some cases at a pH of pH equal to or greater than 7.5, equal to or greater than 8.0, equal to or greater than 8.3, or equal to or greater than 8.5.

Suitable buffering agents include carbonate, phosphate, citrate, lactate, gluconate and tartrate, as well as more complex organic buffering agents. In some examples, the buffering agent comprises a phosphate buffering agent which contains sodium phosphate and / or potassium phosphate. Generally, such a buffering agent, or buffer system, comprises in the form of sodium phosphate and potassium phosphate in a ratio of one to one in order to obtain the desired pH. In another example, the buffering agent contains tris (hydroxymethyl) aminomethane, or "Tris", formulated to obtain the desired pH. Methods of buffer formulation to achieve the desired pH are well known to those skilled in the art, and a suitable composition can be determined without extensive experimentation on the basis of the desired pH.

Immunogenic compositions (e.g., bulk preparations and finished vaccine formulations) disclosed herein may include additional components, such as one or more mineral salts, e.g. to modify or maintain tonicity within a desired range, such as Isotonic or near isotonic. The appropriate amount differs depending on the other components in the formulation, and can be determined without undue experimentation by those skilled in the art. One of these suitable mineral salts is NaCl. Other mineral salts and ions may also be used, for example potassium, calcium, magnesium, manganese, zinc salts, as well as other pharmaceutically acceptable salts and ions. The pharmaceutically acceptable salts and their selection are discussed in detail, for example, in Pharmaceutical Salts: Properties, Selection, and Use, 2nd Revised Edition, P. Heinrich Stahl (Editor), Camille G. Wermuth (Publisher), Wiley, 2011.

Immunogenic compositions (e.g., bulk preparations and finished vaccine formulations) disclosed herein may further comprise one or more sugar or polyol excipients, for example chorsis in the group consisting of: sucrose, trehalose mannose, mannitol, raffinose, lactitol, sorbitol and lactobionic acid, glucose, maltulose, iso-maltulose, lactulose, maltose, lactose, isoformose, maltitol , palatinite, stachyose, melezitose, dextran or a combination thereof. In a specific embodiment, the excipient comprises sucrose. Optionally, the sugar or polyol may be used in combination with an amino acid, such as glycine, alanine, arginine, lysine and / or glutamine.

In one embodiment, the immunogenic composition comprises 5 mM Tris, 150 mM NaCl, optionally with a surfactant, for example, poloxamer 188, and a sugar, for example, 2% w / v sucrose.

Formulation processes

Another aspect of the present disclosure relates to methods for formulating immunogenic compositions (e.g., bulk preparations or finished vaccine formulations) comprising one or more inactivated and purified dengue viruses. Such methods include: providing a solution and mixing with the solution of one or more inactivated and purified dengue viruses. The solution may contain one or more buffering agents and one or more surfactants. In some embodiments, the inactivated and purified dengue virus (s) is adsorbed on an aluminum salt (e.g., to produce a pre-adsorbed bulk preparation of an inactivated dengue virus) prior to mixing with the solution. Generally, only one serotype or single strain of inactivated and purified dengue virus is adsorbed on an aluminum salt (for example, aluminum hydroxide, aluminum phosphate or aluminum hydroxyphosphate) for produce a bulk mono-substance previously adsorbed. To produce a multivalent immunogenic eposition, bulk mono-substances of different serotypes of dengue are then combined to the desired ratio (eg, 1/1/1 / l on a porcine basis, or adjusted on the basis of 1 relative immunogenicity) with the solution containing the buffering agent and the surfactant. Generally, the inactivated and purified dengue virus (s) are added to a suitable solution (in a final formulation) for parenteral administration. In some embodiments, the solution is an isotonic solution and comprises sterile water free of endotoxin. As disclosed herein, the solution also includes one or more excipients to enhance stability during storage, such as a sugar, an amine acid or a divalent ion. Suitable excipients for increasing stability include sucrose, CaCl 2, MgSCy.

In some embodiments, after adding the inactivated and purified dengue virus to the solution containing buffer and surfactant (and optionally additional components) as described herein, the formulated immunogenic composition is stored as a bulk liquid, for example at room temperature, at 0-4 ° C, or at 0 ° C (as at or about -20 ° C, or at about -70 ° C to -80 ° C), before final filling in the form of drug product has been changed,

An example of a method of formulating a bulk immunogenic composition based on DEN PIV, tetravalent and adjuvanted, according to the present invention is as follows: with gentle stirring, add sterile water for injection: saffron to obtain a concentration final 2% w / v; Tris to obtain a final concentration of 5 mM (in an alternative formulation, Tris is provided at a final concentration of 3.5 mM); NaCl to obtain a final concentration of 150 mM; the pluronic mixture (TM) / lutrol (TM) F68 to obtain a final concentration of 0.10% in p o 1 d s / v o 1 urn; MgSCg for a final concentration of 15 mM (high dose) or 7.5 mM (low dose); Al (OH) 3 to obtain a final concentration of 1000 μg / ml (high dose) or 500 μg / ml (low dose); after stirring for 15 to 30 minutes, add PIV DEN1 to provide 8 μg / ml (high dose) or 4 μg / ml (low dose) PIV DEN2 to provide 8 μg / ml (high dose) or 4 μg / ml ( low dose) PIV DEN3 to provide 8 μg / ml (high dose) or 4 μg / ml (low dose) PIV DEN4 to provide 8 μg / ml (high dose) or 4 μg / ml (low dose) stop 1 ' stirring 10 to 15 minutes after the addition of the last PTV DEN; check the pH and add if necessary

## EQU1 ## where to store in bulk until filled.

In the above process, the aluminum-based adjuvant is added to the composition prior to addition of DEN PIV; in an alternative method, the PIV DEN viruses are previously adsorbed on the aluminum-based adjuvant. Other examples of suitable formulations for DEN PIVs can be found in WO 2012/160 199.

In some embodiments, the formulation process then involves lyophilization of the solution (e.g., the bulk preparation containing the inactivated and purified dengue virus (s)) to produce a lyophilized composition. In embodiments involving lyophilization of the immunogenic composition, for example for storage and / or dispensing purposes, the lyophilized composition is generally resuspended in a suitable amount, for example 0.05 to 2 ml. generally between 0.5 and 1.5 ml, for example 0.5 or 1.0 or 1.5 ml, of a pharmaceutically acceptable solution, such as sterile water for injection free of endotoxin, before 1 '. administration. Optionally, the pharmaceutically acceptable solution used for reconstitution may comprise an adjuvant, as described in US Pat.

The present invention provides formulations and vaccines for use in medicine, more specifically as a method of treating a mammal, particularly a human, suffering from dengue infection or at risk of being infected with dengue fever. The use of the formulations and vaccines of the present invention is also provided in the preparation of an immunoprophylactic and / or immunotherapeutic product for the treatment or prophylaxis of dengue infection (e.g., a prophylactic or an immuno-therapeutic vaccine).

The finished vaccine compositions of the invention may be lyophilized or in aqueous form,

Solutions or suspensions. The liquid formulations allow the compositions to be administered directly from their packaged form, without the need to reconstitute them in an aqueous medium. In some embodiments, an adjuvant may be admixed with the active ingredient of the vaccine at the time of clinical administration, therefore the adjuvant and antigen may be kept separate in a packaged or dispensed vaccine, ready for formulation final at the time of administration. In other embodiments, an adjuvant is mixed with the antigen during preparation, therefore the composition is packaged in an adjuvanted form.

The finished vaccine compositions may be packaged in any suitable manner, for example in pre-filled vials or syringes. Syringes can be supplied with or without a needle. A syringe usually contains a single dose of the vaccine formulation, while a vial may comprise a single dose or multiple doses. In one embodiment, the dose is for a human. In an additional embodiment, the dose is for an adult, a teenager, a toddler, an infant or a human who is less than one year old. Effective dosages can be established using methods known in the art. In one embodiment, the dosage is from 0.1 μg to 10.0 μg of antigen for each dengue virus serotype, from 0.5 μg to 8.0 μg d. antigen for each dengue virus serotype, or 1 μg to 4.0 μg of antigen for each dengue virus serotype.

Although the finished vaccine composition can be administered by different routes, the most common administration is intramuscular, subcutaneous or intradermal administration. Generally, the finished vaccine composition is administered at an effective dose to produce neutralizing antibodies directed against each of the DENV conterrus serotypes in the composition. The amount of VIP to be administered depends on the subject to be treated, the ability of the subject's immune system to synthesize antibodies, and the desired degree of protection. The precise amounts of vaccine to administer may depend on the judgment of the attending physician and may be specific to each subject. The vaccine may be given in a single dose schedule, or preferably a multi-dose schedule in which a primary phase of vaccination is followed by additional doses at subsequent time intervals to maintain and / or enhance the desired immune response. .

The present disclosure relates to the formulation of immunogenic compositions, such as bulk vaccine preparations and finished vaccine formulations, containing serotype 4 inactivated and purified dengue virus. The formulations disclosed herein enhance the stability of the immunogenic compositions ( for example, a bulk or finished vaccine comprising PIV DEN4), which facilitates the manufacture, production, storage and distribution of vaccines comprising PIV DEN4.

A first aspect of the present disclosure relates to immunogenic compositions (e.g., bulk vaccine compositions and finished vaccine formulations) which comprise inactivated and purified DEN4, in combination with a buffering agent, a surfactant and a stability enhancing excipient. As described herein, the selected excipients have been found to improve the stability of the inactivated antigenicity-inactivated DEN4 virus in a formulation, relative to formulations not containing the selected excipient. DEN4 PIV virus formulations containing one or more selected excipients as described herein (a "stability enhancing" agent or excipient) have the favorable characteristics of limiting the increase in average size of viruses over time. and / or to limit the reduction of antigenicity over time (e.g., during storage), compared with formulations not containing the at least one excipient, the excipient enhancing the stability selected may be added at any time or at several times during the preparation of the finished vaccine product, for example added to an individual serotype of DS prior to the addition of any adjuvant; added to individual adjuvant serotypes, especially those adsorbed on an alum adjuvant; added to a composition comprising multiple serotypes, with or without other excipients or adjuvants; or added to a compound of the drug product that is being used.

The compositions disclosed herein may comprise only a single dengue virus serotype (a monovalent composition containing, for example, DEN-4), or the compositions may comprise more than one serotype of dengue (multivalent). In one embodiment, the composition comprises a plurality of dengue viruses from more than one serotype. For example, the composition may comprise one, two or three serotypes of dengue in addition to DEN-4. In a specific example, the composition comprises four serotypes of inactivated and purified dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4), and the composition is capable of eliciting a specific immune response for each of the four serotypes in a subject hu ma in. In another embodiment, the composition comprises at least DEN-4.

The dengue viruses used can be selected from any suitable strain (or strains) of dengue virus. For example, one strain of virus may be chosen for each serotype, which is chosen on the basis of its compliance with a defined sequence (eg, consensus) for the serotype. Such a virus can be of natural or synthetic origin. For example, a strain of virus may be chosen to correlate with a prevalent strain (eg, a naturally occurring or "wild-type" strain) in the area where the population in the vaccine is intended to be. administered. Another option is to select appropriate strains for each serotype based on availability or previous experience. Dengue strains are described in US Pat. No. 6,254,873, which is incorporated by reference herein. Additional strains are disclosed, for example, in US Pat. No. 7,226,602, which is also incorporated herein by reference. Other strains can be found, for example, in the VBRC viral genome database (available at http: //athena.b ioc.uv ic.ca/organisms/

Flaviviridae / Dengue / Curated genes), and the dengue virus database (available at http: // www. Broad. Mit. Edu / annotation / viral / Dengue / Projectlnfo.html).

In the context of an inactivated and purified dengue virus vaccine, virulent or attenuated strains may be used. Generally, virulent strains propagate at a higher titre in host cells, which facilitates commercial scale production. However, virulent strains should be handled with care. The attenuated strains, for example developed by adaptation of production in cultured cells and selection for reduced virulence and / or reduced replication in mosquito dengue vectors, require less precautions in handling but may to be more difficult to produce. Examples of attenuated strains suitable for use in the context of an immunogenic composition containing inactivated dengue virus are described in WO 2000/057907 and US Patent No. 6,638,514, and in WO 2000 / 058,444 and US Patent No. 6,613,556, and WO 2002/066621 (US publication No. 2004 052 818), WO 2000/057904 (US Patent No. 6,528,065, WO 2000/05790). 8, WO 2000/057909 (IJS Patent No. 6,511,667), WO 2000/057,910 (US Patent No. 6,537,557), WO 2002/095,075 (for example, US Patent No. 7,226,602). ) and WO 2UJ2 / 102288 (US Patent No. 7,569,383), which are hereby incorporated by reference.

Therefore, the inactivated and purified virus (s) may be selected from wild type (ie, propagated from or corresponding to a virulent virus from a naturally occurring isolate) virus, or or the viruses may be selected from attenuated viruses. A selected virus may be a recombinant virus. For example, a recombinant virus may be a chimeric virus, for example a virus having a nucleic acid derived from a dengue virus and a nucleic acid from another flavivirus, such as a different dengue virus, a yellow fever, or an encephalitis virus. Genera ry, a v i r u s t h e m a n dengue M protein and / or Dengue protein E protein. Examples of chimeric dengue viruses can be found, for example, in WO 98/37911 (US Patent Nos. 6,696,281; 6,962,708), WO 96/40933 and; WO 2001 06084 / (US Patent No. 7,094,411;

No. 7,641,909; No. 8,025,887) and EP 1,159,968. Methods for producing such chimeric dengue viruses can also be found in WO 03/101397. A single composition can comprise one or more wild-type viruses, a or more attenuated viruses, one or more recombinant viruses and / or one or more chimeric viruses, in any combination.

Production process of DENV

Methods of producing dengue viruses are known in the art, and are described in sufficient detail to guide those skilled in the art, for example in published POT application No. WO 2010/094 663, USNo Publication. 2011 318 407. The virus can be propagated under non-animal conditions, that is, when it is used in the case of an animal that has its original source. animal (eg bovine serum f eet al). Methods of producing viruses under serum-free conditions can also be found, for example, in US Publication No. 2006 0 183 224. The disclosures of these published patent applications are hereby incorporated by reference to provide useful information. Additional details regarding the propagation and purification of dengue viruses for inclusion in immunogenic compositions (e.g., bulk and finished vaccine preparations) disclosed herein. In one embodiment, the virus-containing medium is clarified by filtration, concentrated, and the medium is exchanged (for example, by ultrafiltration and diafiltration) for a suitable buffer (for example, phosphate buffered saline (PBS), 125 mM citrate, pH 7.6). of the

Suitable buffer solutions may be chosen by those skilled in the art. The initial concentration and buffer exchange may be followed by additional filtration and size exclusion chromatography.

Prior to inactivation of the virus, a surfactant, such as a poloxamer surfactant as disclosed herein, and selected for inclusion in the immunogenic composition (e.g., bulk preparation and / or formulation finished vaccine), can be added to the buffered viral composition. Alternatively, the surfactant may be added after inactivation. The inactivated and purified dengue virus is then sterilized by filtration to produce a bulk preparation of inactivated dengue virus.

Terms and definitions

All references disclosed in this document are incorporated by reference in their entirety.

To facilitate review of the various embodiments of the present disclosure, the following explanations of terms are provided. Additional terms and explanations may be provided in the context of this disclosure.

Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as are understood by those skilled in the art to which this disclosure belongs. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (eds.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). Definitions of common terms used in vaccinology can be found in Vaccines 6th Edition, Plot, Kin, Grenst and Of Fit (Eds.), Saunders, 2012.

The singular terms "a", "an", "the" and "the" include plural referents, unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "plurality" refers to two or more than two. In addition, numerical limitations with respect to concentrates or levels of a substance, such as an antigen, are intended to be approximate. Thus, (for example) when it is indicated that a concentration is at least 200 μg, it is expected that it will be understood that the concentration is at least approximately (or "about" or "~ 2 0 0 pg.

Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present disclosure, suitable methods and materials are described herein. The term "includes" means "includes". Therefore, unless the context otherwise requires, it will be understood that the word "includes" and variations such as "include" and "include" imply the inclusion of a named compound or a specified composition (eg for example, a nucleic acid, polypeptide, antigen) or indicated step, or a group of compounds or steps, not exclude any other compound, composition, step or group thereof. 1 / abbreviation "e.g." is derived from the Latin exernpli grat ia, and is used herein to indicate a non-limiting example. Therefore, the abbreviation "e.g." is synonymous with the term "for example".

As used herein, a drug substance (DS) refers to an active pharmaceutical ingredient (especially an active vaccine component, such as an antigen or an inactivated virus), i.e., a material that exerts a physiological action when administered to a human subject (including the induction of an immune response). When manufactured and / or stored in bulk preparations, a DS may be called bulk preparation, bulk purified substance (PB), bulk intermediate or (in the case of vaccines) bulk vaccine, vaccine component in bulk form. bulk or bulk antiqenes.

To provide a finished drug product (DP) (especially a finished vaccine), one or more drug substances are generally formulated with inactive ingredients (e.g., vehicles, buffers, excipients, binders). Inactive ingredients are included in the formulation for a variety of reasons, for example to facilitate manufacture, improve stability or improve other characteristics of a product. As used herein, a finished or final vaccine formulation is considered to be a medicinal product.

A viral drug substance of inactivated and purified dengue (or an active ingredient) refers to a dengue virus in the final antigenic form, relative to purification and inactivation, and intended for administration to a subject . A bulk preparation or a bulk formulation of a drug substance based on PIV DEN virus can be further processed, for example by dilution, concentration, as by lyophilization and resuspension, and / or packaged, for example in flasks. or multi - dose and single dose syringes with rins of administration. i o n c o mm e co mp o s i t. i o n v a nc a nc e nn i

As used herein, an "adjuvant" is an agent that increases the production of an antigen-specific immune response to the administration of the antigen in the absence of the agent. T h e w a n d s c o u r a n t. Scoops are aluminum-containing adjuvants which include suspensions of minerals (or inorganic salts, such as aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate) on which The antigen is adsorbed. Other adjuvants include one or more immunostimulatory components that contribute to the production of an enhanced immune response specific for an antigen. Immunostimulatory components include oil and water emulsions, such as an oil-in-water and oil-in-water emulsion (and variants thereof, including doubled emulsions and reversible emulsions), liposaccharides, lipopolysaccharides, immunostimulatory nucleic acids (such as CpG oligonucleotides), liposomes, Toll receptor agonists (in particular, TLR2, TLR4, TLR7 / 8 and TLR9 agonists), and various combinations of these components. Adjuvants may include combinations of immunostimulant components.

As used herein, an "antigen" refers to a substance, including a peptide and a protein (such as a glycoprotein), which induces an immune response in a mammal, including a human.

As used herein, a "buffering agent" is a compound or composition that alone or in combination increases the ability of a solution to maintain or resist a change in pH when an acid or base is added. outed. The term buffering agent encompasses a wide variety of compounds and compositions, usually weak acids or weak bases which, when present in solution with their base or conjugated acicie, can be used to maintain the pH at a desired value or within a desired range.

As used herein, a "bulk" amount refers to a volume greater than the volume of a product, medicated and marketed. Therefore, a "bulk vaccine component" refers to a vaccine DS or active ingredient that is maintained, contained or stored in a volume greater than the volume of the vaccine product ini. The concentration of a vaccine active ingredient (such as a DEN PIV virus) in a bulk preparation is generally greater than the concentration of the active ingredient in the finished vaccine product. A "bulk substance" is an intermediate product in the commercial manufacture of vaccines. A bulk vaccine component may be univalent (monovalent), that is, it contains a vaccine active ingredient (such as a single dengue virus serotype), bivalent or multivalent. A bulk substance may contain a purified antigen or PIV at a concentration different from that present in a final (finished) vaccine formulation (the formulation to be dispensed, sold commercially or used clinically). The final vaccine formulations may be polyvalent, including multiple dengue VIP serotypes (e.g., it may be a mixture of multiple serotype PIV bulk substances), and may include antigens from additional pathogens. Bulk substances can be stored until they are used for the preparation of the end-use product.

An "immune response" is a response of an immune system cell, such as a B lymphocyte, a T cell or a monocyte, to a stimulus. An immune response may be a B-cell response that results in the production of specific antibodies, such as antigen-specific antigen-specific antibodies. An immune response may also be a lymphocyte-like response, such as a CD4 + response or a CD8 + response. In some cases, the response is specific for a particular antigen (i.e., an "antigen-specific response"). If the antigen is from a pathogen, the specific response of an antigen is a "pathogen specific response". A "protective immune response" is an immune response that inhibits a pathogenic function or activity of a pathogen, reduces infection with a pathogen, or decreases the symptoms (including death) that result from infection with the pathogen. A protective immune response can be measured, for example, by inhibiting viral replication or plaque formation in a plaque reduction assay or ELISA neutralization assay, or by measuring the resistance to challenge with the virus. pathogen in vivo.

As used herein, an "immunogenic composition" is a composition of matter suitable for administration to a human or animal subject (e.g., in a clinical or experimental setting), and which is capable of eliciting an immune response. in a subject to which it is administered, for example directed against a pathogen, such as the dengue virus. As such, an immunogenic composition comprises one or more antigens (e.g., an integer whole virus or antigenic subunits, e.g., polypeptides thereof) of the antigenic epitopes. An immunogenic composition may also include one or more additional components, such as an excipient, carrier and / or adjuvant. In some instances, the immunogenic compositions are administered to elicit an immune response that protects the subject against pathogen-induced symptoms or conditions. In the context of the present disclosure, it will be understood that the term immunogenic composition encompasses compositions that are intended to be administered to a subject or population of subjects in order to elicit a protective or palliative immune response against dengue (ie, that is, vaccine compositions or vaccines). The immunogenic response may be an immunoprotective response triggered in a human subject.

The term "inactivated" in the context of a dengue virus vaccine means that the antigenic component (e.g., a virus) is unable to replicate in vivo or in vitro. For example, the term inactivated includes a virus that has been replicated, for example in vitro, and then killed using a chemical or physical medium such that it is not able to replicate at all. The term may also include antigens produced by other treatments (e.g., by fractionation, fragmentation, and the like), and components produced by recombinant means, e.g., in a cell culture.

As used in this document. a "mixture" comprises at least two different elements, for example a mixture comprising the molecules of a polypeptide species and the molecules of an adjuvant species, or comprising the molecules of two different polypeptides.

As used herein, the terms "peptide", "polypeptide" and "protein" are interchangeable and refer to a polymer of amino acids, regardless of size. Although "protein" is often used in reference to relatively large polypeptides, and "peptide" is often used in reference to small polypeptides, the use of these terms in the art overlaps and varies. The term "polypeptide" as used herein refers to peptides, polypeptides and proteins unless otherwise indicated. As used herein, the terms "protein", "polypeptide" and "peptide" refer to both expressed gene products and chemically synthesized entities, and include inactivated protein glycoproteins and toxins (toxoid). .es).

As used herein, the term "purification" (e.g., with respect to a pathogen or a composition containing a pathogen, such as a dengue virus) refers to the method of removing undesirable components. of a composition. The term purification is a relative term, and does not require that all traces of the undesirable component be removed from the composition (does not require absolute purity). In the context of vaccine production, purification may include such methods as centrifugation, dialysis, ion exchange chromatography and size exclusion chromatography, affinity purification or precipitation. Therefore, for example, a purified virus preparation is a preparation in which the virus is more enriched than it is in its generative environment, for example within a cell or cell population within the cell. which replicates itself naturally or in an artificial environment, such as a culture. A substantially pure virus preparation may be purified so that the desired virus or viral component is at least 50% of the total protein content of the preparation. In some embodiments, a substantially pure virus will comprise at least 60% or at least 70%, such as at least 80%, at least 85%, at least 90% or at least 95% or more of the total protein content of the protein. the preparation. Alternatively, purification of a virus preparation can be evaluated by reducing contaminants, such as host cell proteins, in the preparation. Therefore, a substantially pure virus preparation (e.g., an inactivated and purified dengue virus) generally comprises less than 30%, or at least 25%, of residual proteins of the host cell. For example, an immunogenic composition (e.g., a bulk preparation or a finished vaccine formulation) comprising an inactivated and purified dengue virus may comprise less than 20% residual protein from the host cell, less than 15%, or % or less (for example, measured on a weight / weight basis).

As used herein, a "subject" is a living multicellular vertebrate organism. In the context of the present disclosure, the subject may be an experimental subject, such as a non-human animal, for example a mammal such as a mouse, a cotton rat or a non-human primate. Alternatively, the subject may be a human subject, for example in a clinical setting.

As used herein, a "surfactant" or surface active agent is an amphiphilic molecule characterized by a hydrophilic head and a hydrophobic tail. When adsorbed on the surface of a liquid, a surfactant acts to decrease the surface tension of the liquid, the interfacial tension between two liquids, or the tension of the liquid and a solid. A surfactant may act as a detergent, a wetting agent, an emulsifier, a foaming agent and / or a dispersant.

As used in this document, the term "includes" means "includes". Therefore, unless the context otherwise requires, it will be understood that the word "includes" and variations such as "include" and "include" imply the inclusion of a named compound or a specified composition ( for example, an antigen or excipient) or indicated step, or a group of compounds or steps, but not the exclusion of any other compound, composition, step or group thereof. When it is described that resilient modes include certain components, these embodiments are intended to include embodiments of the indicated components.

The following examples are provided to illustrate features and / or embodiments. These examples should not be construed as limiting the invention to the particular features or embodiments described. It will be understood by those skilled in the art that quantities, e.g., volumes, are only provided as an example, and that the scale may be varied (increased or decreased) at the discretion of the attending physician. Similarly, the components used in the assays, for example the filters, the columns, are not intended to limit or exclude in any way whatsoever, and may be replaced by other components to achieve the same objective, as will be understood by those skilled in the art.

Examples

Example 1 Preparation of a VIP of Dengue (DEN)

The inactivated and purified dengue viruses of serotypes DEN1, DEN2, DEN3 and DEN4 were prepared as previously described; see Simmens et al., Virology 396: 280-288 (2010) and Fernandez et al., Clin. Vaccine Immunol. 18 (4): 523 ~ 32 (2011). Briefly, the viruses were propagated in a Vero cell line, and the virus in the culture supernatant was concentrated, purified and inactivated with formalin.

Example 2: assay methods ELI SA: monoclonal antibodies {mAb) specif ic of DENV serotypes were identified and confirmed for DEN1 serotypes, DEN2, DEN3 and DEN4 (respectively, the mAb El0 3, DV4 4, DV3 / 1I and E88). An enzyme immunoassay protocol (ELISA) on DENV was established using the appropriate DENV specific mAb and detection with the same biotin-conjugated mAb. This ELISA test was used to determine the antigenicity of the serotype.

An inhibition-based antigenicity ELISA was developed to evaluate the antigen content of the formulations in which the antigens were adsorbed on aluminum. In brei, alum adsorbed DPIV antigens are initially incubated with the biotin conjugated mAbs and specific for the DENV serotypes described above. The antigen / mAb mixtures are then centrifuged before transferring the supernatant to ELISA plates previously coated with a purified bulk DENV (PB) material. MAb binding is revealed by the addition of the streptavidin substrate Amdex-HRP (Amersham) and TMB (tetramethylbenzidine peroxidase).

Dynamic Light Diffusion (DLS): A Wyatt DynaPro plate reader (Wyatt Technology Corp) was used to determine the size distribution profile of small suspended particles. The protocol used multiple 96-well plates, UV-transparent background (Corning, NY), 22 ° C temperature, 14% laser power, 77% attenuation, 5 second acquisition time. Five measurements were obtained

The results of DLS analysis are expressed as the Z average, which is obtained by analysis of DLS data using the cumulant technique (see, for example, Koppel, J. Chem Phys 57: 4814-4820 (1972). Thomas, J. Colloid Interface Sci 117: 187-192 (1987)). The average size Z increases as the particle size increases, and thus provides a reliable measure of the average size of a size distribution of

High Performance Sterile Exclusion Chromatography (UV Defect): Liquid phase size exclusion chromatography conditions for determining protein content by UV detection have been developed. The liquid chromatography system was HPLC Waters Alliance e2695, UV 2998; TSKGel G600 PWXL column, Dl 7.8 mm X L 30 cm; temperature of the heating device 40 ° C; injection volume 100 μl; sample IV temperature 15 ° C / analysis time 25 minutes; mobile phase: 5 mM TRIS, 150 mM NaCl, 0.1% PX188, pH 7.4; UV wavelength 210 nm; flow rate 1.0 ml / minute. Nephelometer: Nephelometry estimates the turbidity or turbidity of a solution due to the presence of suspended particles. The examples provided herein have used a Nephelostar microplate nephelometer (BMG Labtech) and multiple 9-well plates (UV-transparent background, Corning, NY). The sample volume was 100 μL. Nephelostar instrument included a dedicated optical system to measure light scattering caused by turbidity. Optical settings: gain 55; laser intensity (%) 90; beam focusing (mm) 1.5. General settings: positioning delay (seconds) 0.1; horizontal direction reading. The nephelometric unit (UN) is a dimensionless unit. UTN means nephelometric turbidity unit, UTN (e.g., as shown in FIGS. 9 and 10) corresponding to the limit of detection by the eye and being determined for each experiment with an internal control comprising the diluted formazine molecule up to at a specific concentration (10 NTU = 500 UN with the laboratory instrument described in this document). An increase in nephelometry indicates an increase in the turbidity of a solution which can be attributed, in the present examples, to an aggregation phenomenon.

Static Light Diffusion: The static light scattering test for particle size estimation used the Malvern Mastersizer 2000 instrument (Malvern Instruments, Ltd.). Dispersant was 150 mM NaCl plus 5 mM Tris; the temperature was 20-25 ° C, the darkness was about 5 to 7%, the recirculation rate was 1250 rpm, the size standard was 5 microns latex polymer microspheres. . Sample diluents were used for bottom measurement. Five measurements were used to calculate mean size distributions and reported values.

Intrinsic Fluorescence: Evaluating the intrinsic fluorescence (IF) protein content was performed with a Varioskan Flash system (Thermo Scientific) using multiple 96-well UV-transparent (Corning) plates containing 100 μl of sample. The intrinsic fluorescence of the proteins is caused by protein excitation with ultraviolet light at 280 nm and the observation is at about 320 nm. For concentration determination and to reduce interference and variability, a calibration curve was established with each serotype of dengue, and each sample was introduced into three wells.

Example 3 Drug Substance PIV DEN4

A composition comprising a PIV DEN4 was formulated and combined with a buffer (50 mM NaCl, 5 mM Tris (tris (hydroxymethyl) aminomethane), 3% w / v sucrose and 0.1% w / v. poloxamer 188. The final pH was 8.0 and the final DEN4 content was 34 μg / ml, and in this example, this basic formulation was used as a control, the stability of the PIV DEN4 in this formulation. This was found to be limited, with an increase in the size of the virus particles and a fall in antigenicity occurring after 48 hours at room temperature (25 ° C).

Example 4 Screening of Excipients with Drug Substance PIV DEN4

Various excipients to be tested (Tables 1 and 2) were added to the PIV DEN4 base formulation (as described above) to determine if the DEN4 serotype could be stabilized. The excipients tested were sugars, amino acids, divalent ions, surfactants and co-solvents. The base formulation containing PIV DEN4 (before addition of excipients tested) contained 3% sucrose; when sucrose was used as excipients to be tested, it was present in addition to the 3% sucrose of the base formulation. The base formulation containing PIV DEN4 also contained 0.1% poloxamer 188; when poloxamer 188 was used as the exciprent to be tested, it was present in addition to the 0.1% 0.1% poloxamer of the base formulation.

Stability was evaluated after storage for 48 hours at 25 ° C, using dynamic light scattering (DLS) and HP-SEC-UV (high performance steric exclusion chromatography using ultraviolet detection). The control ("CTRL PB") for Figures 3 and 6 was a calibration curve prepared with a bulk purified DEN4 substance: freshly thawed and diluted. Results: Figures 1 to 3 present the results for the drug formulation based on PTV DEN 4 supplemented with the test excipients listed in Table 1. Figure 1 provides the DLS results at time 0, Figure 2 provides the results. DLS results after 48 hours of storage at 25 ° C, and Figure 3 provides the results of HP-SEC-UV recovery after 48 hours of storage at 25 ° C. The controls were the base formulation containing PIV DEN4 without excipient to be tested (indicated by bulk purified material ("PB") in the figures).

Figures 4 to 6 show the results for the test excipients listed in Table 2. Figure 4 provides the DLS results at time 0, Figure 5 provides the DLS results after 48 hours of storage at 25 ° C, and the Figure 6 provides the results of HP-SEC-UV recovery after 48 hours of storage at 25 ° C. The controls were the base formulation containing PIV DEN4 (without excipient to be tested). ιο

Όΐ 4-> co

CD

-P

CO 4-1

C G) Ή O-i

* H 0 X Q)

v-H 3 rö 0) 1 -i

S

EH α>

Γ "I Ή Μ -α> + j ω

(D &; -1-4 :: 5

Ci) ϋ rd

Sdd dd

Cu cd o Ή 4-1

Cd! --- 1

O

CO

(Dcd dd -1-4cc

Cl) cd o

'H -1-4 iÖ Ή

O

Cu Ή

O

O Ή 2 ® (d ^ 8 s 03

^ 0Q s

E-i O • --- · r-i O fti c o of the $ Όs' * E-H ° \ 1

Ci) 4-> hd c cd * Cd Cl) 4-1 cd o ü ιο

Όΐ 4-> co

CD

-P

CO 4-1

C G) Ή O-i

* H 0 X Q)

CM 03 03 Q) 1 -i

S

EH

Of the initial tested excipients tested (Tables 1 and 2), most were discarded based on DLS and HP-SEC-UV results after 48 hours at 25 ° C; Ten excipients to be tested were selected to continue the study.

Example 5: Additional tests of the test excipients selected with the drug substance PIV DEN 4

Based on the results of the above examples, ten test excipients were selected for further tests, some being evaluated at more than one concentration (Table 3).

Table 3: excipients to be tested

The excipients to be tested were added to PIV DEN4 (basic formulation described in Example 4 above). Stability was evidenced at time zero, after 48 hours at 25 ° C, and after 7 days at 25 ° C. Four samples were tested for each excipient at each ternporous period (n = 4). As a guideline, the PIV DEN4 base formulation was also evaluated at these time periods.

Samples were evaluated by DLS, HP-SEC-UV, nephelometry, ELISA and intrinsic fluorescence (IF).

The results are shown in Figures 7 to 16, where the bars are the average of four samples tested (n = 4), with the standard error indicated. ELISA: The Y axis is the% recovery from the theoretical antigenicity that would have been found based on the applied dilution factor. IF: The Y axis is the% recovery from a calibration curve prepared with the drug substance DEN4 which has been defrosted and diluted.

The results demonstrate that the presence of some tested excipients (including 8% sucrose, 15 mM CaCl 2 and 15 mM MgSCy) reduced virus aggregation after 7 days of storage at 25 ° C (evaluated by DLS and nephelometry). ), compared to the witness. In addition, antigenicity was conserved over time (as evaluated by HP-SEC-UV, ELISA and IE).

Example 6: non-adjuvanted tetravalent formulation

To further examine the effect of CiaCLg, MgSO4, and a high sucrose concentration on the stability of PIV DEN4, CaCl2 dose ranges (1.8 mM, 3.7 mM, 7.5 mM, 15mM, 30mM, 45mM) and MgSO4 (1.8mM, 3.7mM, 7.5mM, 15mM, 30mM, 45mM), as well as 8% sucrose. weight / volume, were added to the non-adjuvanted tetravalent base composition of dengue virus. The tetravalent base formulation contained 8 μg / ml of each of DEN1, DEN2, DEN3 and DEN4, with 0 mM NaCl, 5 mM Tris, 2% w / v sucrose, 0.1% w / v. of pxl88, pH 8.5 (basic tetravalent formulation). The control ("Ctrl tetra") was the basic tetravalent formulation (without CaCl2, Mg504, or additional sucrose).

Stability after 6 days at 25 ° C was evaluated by DLS and ELI SA. The results are shown in Fig. 17 (DLS-evaluated diameter (nm)), and Figs. 18-21 (antigenicity of DEN1, DEN2, DEN3 and DEN4, respectively, evaluated by ELISA). The bars shown in FIGS. 17 to 21 are the average of four samples (n = 4), with the standard error indicated.

Example 7

Based on the foregoing experiments, three excipient conditions (8% total sucrose, 15 mM MgSO 4 and 15 mM CaCl 2) were selected for side-by-side comparison in the tetravalent formulation of non-adjuvanted PIV DEN (as described above). above). In this example, the control group is a tetravalent formulation with 2% total sucrose, which is the residual amount of sucrose from the drug substance.

The control (2% sucrose) and tested formulations were incubated for 7 days at 4 ° C, 25 ° C and 37 ° C. The readings were then applied to non-centrifuged samples (NC) and centrifuged samples (C) to assess the loss of grade if aggregation was present.

Figures 22 to 25 show graphs of the antigenicity of serotypes DEN1, DEN2, DEN3 and DEM4, respectively, from the tetravalent ηon adjuvanted formulation evaluated by ELISA. "NC" indicates the non-centrifuged sample and "C" indicates the centrifuged sample. Figure 26 is a graph of results from the DLS analysis, showing an increase in the average particle size in the formulation maintained at different temperatures. "Fresh" indicates the basic formulation evaluated at TO.

Figure 27 is a graph of the protein content determined by SEC HPLC with UV detection, and provides the results as% recovery versus protein content of the bulk purified product. As before centrifugation, centrifugation would have been performed, while non-centrifuged samples were evaluated. Figure 28 is a graph of the results of the nephelometry evaluation.

Example 8 Adjuvanted Formulation

The selected test excipients were evaluated using Al (OH) 3 adjuvanted tetravalent forinulation containing 4 μg of each of the four DENV serotypes, and 500 μg Al (OH) 3 adjuvant. The formulation comprised 150 mM NaCl. mM Tris, 2% w / v saffrose, 0.1% w / v pxl88, pH 8.5 (tetravalent base formulation). This example used samples of about 20 ml (rather than well microplates). The excipients tested were 15 mM CaCl 2, 15 mM MgSO 4 and sucrose had a total content of 8% w / v. The control contained 2% w / v of sucrose from the drug substance. The samples were evaluated after 7 days at 4 ° C, after 7 days at 25 ° C, and after 7 days at 37 ° C.

Fig. 29 shows the results of static light scattering (SLS) for mesuring particles <10 μm, and Fig. 30 shows SLS for measuring particles <100 μm.

Example 9: reduced aggregation after 7 days at 30 ° C., with MgSCg as an excipient

The stability of DS PIV DEN4 supplemented with 15 mM MgSO4 was evaluated by DLS at time zero (T0), after one week (1W, 7 days) of storage at 22 ° C., after 1W of storage at 30 ° C. after a freeze / thaw cycle (1F / T), after two freeze / thaw cycles (2F / T) and after three freeze / thaw cycles (3F / T). See Figure 31.

The comparison was made with respect to the control (CTRL, DS DEN4 DS without MgSO4).

The MgSCn at 15 mM stabilized the size of the DEN4 virus after storage for one week at 30 ° C relative to a composition of MgS04 sausage (compare the fifth and sixth bar in Figure 31), the Y axis is the average size Z (diameter) of viral particles in nanometers.

Claims (29)

An immunogenic composition, comprising: (a) an inactivated and purified serotype 4 dengue virus; (b) a buffering agent; (c) a surfactant; and (d) a stability enhancing excipient. The immunogenic composition of claim 1, wherein said stability enhancing excipient is selected from an inorganic salt and a sugar. An immunogenic composition according to any one of the preceding claims, wherein said stability enhancing excipient is selected from CaCl 2, MgSO 4 and sucrose. An immunogenic composition according to any one of the preceding claims wherein said stability enhancing excipient is selected from MgSO 4 at a concentration of at least about 1.8 mM, at least or about 3, 75mM, at least or about 7.5mM, at least or about 15mM, at least or about 30mM, and at least or about 45mM. An immunogenic composition according to any one of claims 1 to 3, wherein said stability enhancing excipient is selected from CaCl 2 at a concentration of at least or about 1.8 mM, at least or about 3.75 mM, at least or about 7.5 mM, at least or about 15 mM, at least or about 30 mM, and at least or about 45 mM mM. An immunogenic composition according to any one of claims 1 to 3, wherein said stability enhancing excipient is sucrose at a concentration of at least about 8% w / v (w / v), or at least at least or about 10% w / v. An immunogenic composition according to any one of the preceding claims, wherein said composition is liquid. Immunogenic composition according to any one of the preceding claims, further comprising an adjuvant. The immunogenic composition of claim 8, wherein said adjuvant is an aluminum salt. 10. An immunogenic composition according to any one of claims 8 to 9, wherein said adjuvant comprises at least one of aluminum hydroxide and aluminum phosphate. Immunogenic composition according to any one of claims 7 to 10, further comprising at least one additional immunostimulant component. The immunogenic composition of claim 11, wherein the at least one additional immunostimulatory component comprises one or more oil and water emulsions, liposome, lipopolysaccharide, saponin and oligonucleotide. The immunogenic composition of claim 8, wherein said adjuvant is a non-aluminum adjuvant. The immunogenic composition of claim 13, wherein the aluminum-free adjuvant is selected from an oil-in-water emulsion, a liposome, a lipopolysaccharide, a saponin, and an oligonucleotide. 15. An immunogenic composition according to any one of the preceding claims, further comprising at least one serotype of the additional inactivated and purified dengue virus. An immunogenic composition according to any one of the preceding claims, further comprising an inactivated and purified serotype 1 dengue virus (PIV DEN1), an inactivated and purified serotype 2 dengue virus (PIV DEN2) and a virus inactivated and purified serotype 3 dengue (PIV DEN3). The immunogenic composition of claim 16, wherein the composition elicits an immune response against each of DEN1, DEN2, DEN3 and DEN4 in an adult human. 18. An immunogenic composition according to any one of the preceding claims, wherein the surfactant is a poloxamer. 19. An immunogenic composition according to any one of the preceding claims, wherein the surfactant is present in an amount of at least 0.001 M (w / v). 20. An immunogenic composition according to any one of the preceding claims, wherein the surfactant is present in an amount of at most 1.0% (w / v). 21. An immunogenic composition according to any one of the preceding claims comprising a phosphate buffering agent. 22. Immunogenic composition according to claim 21, comprising a phosphate buffer agent selected from sodium phosphate and potassium phosphate. 23. An immunogenic composition according to any one of claims 1 to 20, wherein the buffering agent comprises tris (hydroxymethyl) aminomethane. 24. An immunogenic composition according to any one of the preceding claims, which is a bulk preparation. 25. A method for formulating a bulk preparation of inactivated and purified dengue virus or a finished vaccine formulation thereof, comprising: (a) providing a solution comprising sterile water, a buffering agent a surfactant and a stability enhancing excipient; (b) adding to the solution of (a) an inactivated and purified serotype 4 dengue virus. The method of claim 25, wherein said stability enhancing excipient is MgSCy. The method of any one of claims 25 to 26, wherein step (b) further comprises adding additional serotypes of inactivated and purified dengue virus. The method of any one of claims 25 to 27, wherein step (b) further comprises adding inactivated and purified dengue viruses of serotype 1, serotype 2 and serotype 3. The method of any of claims 25 to 27, wherein said solution of step (a) further comprises an aluminum-based adjuvant, such as Al (OH) 3.