WO2017109698A1

WO2017109698A1 - Immunogenic formulation

Info

Publication number: WO2017109698A1
Application number: PCT/IB2016/057827
Authority: WO
Inventors: Brigitte CLAES; Thierry DEBLOCK; Frédéric Mathot; Maria Dolores MORALES AIRA
Original assignee: Glaxosmithkline Biologicals Sa
Priority date: 2015-12-22
Filing date: 2016-12-20
Publication date: 2017-06-29
Also published as: BE1024160A9; BE1024160B9; BE1024160A1; BE1024160B1

Abstract

Formulations of immunogenic compositions comprising one or more strains of purified inactivated Dengue virus.

Description

IMMUNOGENIC FORMULATION

TECHNICAL FIELD This disclosure relates to formulations of immunogenic compositions. In particular, this disclosure relates to formulations of immunogenic compositions containing one or more strains of purified inactivated Dengue virus.

BACKGROUND

Dengue viruses (DEN or DENV) are members of the family flaviviradae. Like the family prototype, yellow fever (YF) virus, Dengue viruses are enveloped, single-stranded RNA viruses approximately 50 nm in size (Reviewed in Henchal and Putnak, Clin. Microbiol. Rev. 3:376-96 (1990)). Within the dengue group 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-type-4 (dengue-4 or DEN4). While there is considerable genetic and antigenic similarity among the serotypes, there is no significant cross-neutralization (Calisher et al. J. Gen. Virol. 70: 37-43 (1989)).

Dengue virus can be transmitted to humans via mosquitoes, causing an acute viral disease. Dengue viral disease is endemic in the tropics and subtropics of the world. Infection with any one of the four DENV serotypes can cause an asymptomatic infection, a mild nonspecific viral illness, classic dengue fever, or a severe dengue illness with hemorrhagic tendencies. Although relatively rare, Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS) are significant causes of death in children.

After infection with one Dengue virus serotype, humans who experience a subsequent infection with a different dengue virus serotype are at increased risk for severe disease. The mechanisms responsible for subsequent enhanced disease are not well understood (see, e.g., Halstead, Rev. Infect. Dis. l l :Suppl 4:S830-S839 (1989); Rothman, Nat. Rev. Immunol. 11 :532-543 (2011), Guzman and Isturiz, Int. J. Antimicrob. Agents 36:S40-S42 (2010)).

Prevention and control of human disease caused by Dengue virus is primarily achieved, at present, via controlling the mosquito vector. Thus, there remains a need for safe and effective vaccines against all serotypes of Dengue virus, and methods of producing the same.

The development of an effective Dengue vaccine has been hampered by issues with stability of viral preparations after viral purification. In particular, the DEN4 serotype has shown reduced stability at room temperature (compared to other serotypes), which increases the difficulty of manufacturing DEN4 PIV as a bulk vaccine component. There is a need for immunogenic formulations comprising DEN4 which exhibit stability during storage. SUMMARY OF THE INVENTION

One aspect of the present invention is an immunogenic composition, comprising purified inactivated Dengue Virus serotype 4, 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 CaCh, MgSOi, and sucrose.

In a further aspect of the present invention, the stability-enhancing excipient is selected from MgS04 at a concentration of at least or about 1.8mM, 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.

In a further aspect of the invention, the stability -enhancing excipient is selected from

CaCh at a concentration of at least or about 1.8mM, 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.

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

In a further aspect, the immunogenic composition of the invention comprises sucrose and at least one additional excipient selected from CaCh and MgSOi.

In a further aspect of the invention, the composition further comprises an adjuvant. The adjuvant may be an aluminum-free adjuvant, 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 purified inactivated Dengue virus serotype.

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

A further aspect of the invention is a method of formulating a bulk preparation of purified inactivated Dengue virus or a finished vaccine formulation thereof, by providing a solution comprising sterile water, a buffering agent, a surfactant, and a stability -enhancing excipient; and adding to the solution purified inactivated Dengue Virus. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 graphs DLS results for DEN4 PIV formulation supplemented with the Test Excipients as listed in Table 1, at Time 0. The Y-axis is the Z-average size of viral particles in nanometers (nm). Control (CTRL PB) is the DEN4 PIV Drug Substance base formulation (without any Test Excipient).

Figure 2 graphs DLS results for DEN4 PIV formulation supplemented with the Test Excipients as listed in Table 1, after storage for 48 hours at 25°C (T48h25°C). Y-axis units and Control (CTRL PB) are as in Figure 1.

Figure 3 graphs HP-SEC-UV recovery for DEN4 PIV formulation supplemented with the Test Excipients as listed in Table 1, after 48 hours storage at 25°C The Y-axis is % recovery compared to the calibration curve. Controls were the DEN4 PIV Drug Substance base formulation (without any Test Excipient).

Figure 4 graphs DLS results for DEN4 PIV formulation supplemented with the Test

Excipients as listed in Table 2, at Time 0. The Y-axis is Z-average size of viral particles in nanometers (nm). Control (CTRL PB) is the DEN4 PIV Drug Substance base formulation (without any Test Excipient).

Figure 5 graphs DLS results for DEN4 PIV formulation supplemented with the Test Excipients as listed in Table 2, after storage for 48 hours at 25°C (T48h25°C). Y-axis units and Control (CTRL PB) is as in Figure 4.

Figure 6 graphs HP-SEC-UV recovery for DEN4 PIV formulation supplemented with the Test Excipients as listed in Table 2, after 48 hours storage at 25°C. The Y-axis is % recovery compared to the calibration curve. Controls were the DEN4 PIV formulated without any Test Excipient (base formulation).

Figure 7 graphs DLS results for DEN4 PIV formulation supplemented with the Test Excipients as listed in Table 3, after 48 hours storage at 25°C The Y-axis is the Z-average size of viral particles in nanometers (nm). Control (CTRL PB) is the DEN4 PIV Drug Substance base formulation (without any Test Excipient).

Figure 8 graphs DLS results for DEN4 PIV formulation supplemented with the Test

Excipients as listed in Table 3, after 7 days storage at 25 °C. Y-axis units and Control is as in Figure 7. Figure 9 graphs results of nephelometry on DEN4 PIV formulation supplemented with the Test Excipients as listed in Table 3, after 48 hours storage at 25°C. The Y-axis units are NU (nephelometric units); NTU stands for Nephelometric Turbidity Unit. Control (PB) is DEN4 PIV formulation without any Test Excipients (base formulation).

Figure 10 graphs results of nephelometry on DEN4 PIV formulation supplemented with the Test Excipients as listed in Table 3, after 7 days storage at 25°C. Units and Control are as in Figure 9.

Figure 11 graphs results of HP-SEC-UV on DEN4 PIV formulation supplemented with the Test Excipients as listed in Table 3, after 48 hours storage at 25°C. The Y-axis is % recovery compared to the calibration curve. Control (PB) is DEN4 PIV formulation without any Test Excipients (base formulation).

Figure 12 graphs results of HP-SEC-UV on DEN4 PIV formulation supplemented with the Test Excipients as listed in Table 3, after 7 days storage at 25°C. The Y-axis is % recovery compared to the calibration curve . Control (PB) is DEN4 PIV formulation without any Test Excipients (base formulation).

Figure 13 graphs results of ELISA on DEN4 PIV formulation supplemented with the Test Excipients as listed in Table 3, after 48 hours storage at 25°C. The Y-axis is % recovery compared to the theoretical antigenicity that should be found based on the dilution factor applied. Control (PB) is DEN4 PIV formulation without any Test Excipients (base formulation).

Figure 14 graphs results of ELISA on DEN4 PIV formulation supplemented with the Test Excipients as listed in Table 3, after 7 days storage at 25 °C. The Y-axis is % recovery compared to the theoretical antigenicity that should be found based on the dilution factor applied. Control (PB) is DEN4 PIV formulation without any Test Excipients (base formulation).

Figure 15 graphs results of IF on DEN4 PIV formulation supplemented with the Test Excipients as listed in Table 3, after 48 hours storage at 25°C. The Y-axis is % recovery compared to the calibration curve. Control (PB) is DEN4 PIV formulation without any Test Excipients (base formulation).

Figure 16 graphs results of IF on DEN4 PIV formulation supplemented with the Test

Excipients as listed in Table 3, after 7 days storage at 25 °C. The Y-axis is % recovery compared to the calibration curve. Control (PB) is DEN4 PIV formulation without any Test Excipients (base formulation). Figure 17 graphs DLS results for Tetravalent DEN PIV base formulation supplemented with CaC12 at varied concentrations, MgS04 at varied concentrations, or sucrose 8%, after 6 days storage at 25°C. Control is the tetravalent formulation without added excipient (base formulation). The Y-axis is the Z-average size of viral particles in nanometers (nm).

Figure 18 graphs ELISA results of DEN 1 from the tetravalent formulations as described for Figure 17. The Y-axis is % recovery compared to the theoretical antigenicity that should be found based on the dilution factor applied.

Figure 19 graphs ELISA results of DEN2 from the tetravalent formulations as described for Figure 17. The Y-axis is % recovery compared to the theoretical antigenicity that should be found based on the dilution factor applied.

Figure 20 graphs ELISA results of DEN 3 from the tetravalent formulations as described for Figure 17. The Y-axis is % recovery compared to the theoretical antigenicity that should be found based on the dilution factor applied.

Figure 21 graphs ELISA results of DEN4 from the tetravalent formulations as described for Figure 17. The Y-axis is % recovery compared to the theoretical antigenicity that should be found based on the dilution factor applied.

Figures 22 graphs ELISA results of DEN 1 from unadjuvanted tetravalent formulations with CaC12 (15mM), MgS04 (15mM), and added sucrose (8% total), to provide the % recovery compared to Purified Bulk (2% sucrose total) . "NC" indicates non-centrifuged sample, and "C" indicates centrifuged.

Figure 23 graphs ELISA results of DEN2 from unadjuvanted tetravalent formulations as described for Figure 22.

Figure 24 graphs ELISA results of DEN3 from unadjuvanted tetravalent formulations as described for Figure 22.

Figure 25 graphs ELISA results of DEN3 from unadjuvanted tetravalent formulations as described for Figure 22.

Figure 26 graphs results from DLS analysis of unadjuvanted tetravalent formulations as described for Figure 22, measured after seven days of storage at different temperatures (4°C,

25°C, and 37°C). "Fresh" indicates base formulation assessed at TO.

Figure 27 graphs the protein content of unadjuvanted tetravalent formulations as described for Figure 22, assessed by SEC HPLC with UV detection (results provided as the % recovery compared to the protein content of the Purified Bulk). Figure 28 graphs results of nephelometry on unadjuvanted tetravalent formulations as described for Figure 22. Y-axis units are NU (nephelometric units).

Figure 29 shows results of Static Light Scattering (SLS) to measure particles <10μιη, in Al(OH)3 adjuvanted tetravalent formulations.

Figure 30 shows results of Static Light Scattering (SLS) to measure particles <100μιη, in

Al(OH)3 adjuvanted tetravalent formulations.

Figure 31 graphs results of DLS (Z -average on Y-axis) analysis of DEN4 DS with MgS04 at various time points (TO, 1 Week) and conditions (22°C, 30°C, and after 1, 2, or 3 Freeze/Thaw cycles). Control (CTRL) was DEN4 composition without MgS04.

DETAILED DESCRIPTION

Overview of vaccination against Dengue:

This disclosure relates to formulations of immunogenic compositions. In particular, this disclosure relates to formulations of compositions, such as bulk vaccine preparations and finished vaccine formulations, containing one or more strains of purified inactivated Dengue virus (PIV DEN). The formulations disclosed herein increase recovery and stability of PIV DEN in immunogenic compositions, facilitating the production, storage and distribution of such compositions. The increased 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 all four serotypes of dengue virus (DENV) is desirable, as infection with any serotype may cause human disease, and multiple DENV serotypes are often present within a single geographic area. Further, the predominant serotype may vary over both time and geographic location.

Several live-attenuated DENV vaccine candidates have been described (Wallace et al,

Curr. Opinion in Virology 3:352-56 (2013); Halstead, Vaccine 31 :4501-07 (2013), Thomas et al., Am. J. Trop. Med Hyg 88:73-88 (2013); Bauer et al, Am J. Trop Med. Hyg.14-0625 (July 2015 epub), Watanaveeradej et al, Am J. Trop. Med. Hyg. 91(1): 119-28 (2014)). A recombinant, non-replicating DENV subunit vaccine has also been described. Coller et al., Vaccine 29:7267-7275 (2011). A tetravalent dengue purified-inactivated vaccine (TDEN PIV) has been shown to induce protective antibody responses against DENV challenge in rhesus macaques. Fernandez et al., Am. J. Trap. Med. Hyg. 92(4):698-708 (2015).

Stabilization of DEN4

In one aspect, the present disclosure concerns methods for increasing stability of compositions comprising purified inactivated Dengue Virus serotypes, such as PIV DEN4, by formulating the inactivated Dengue virus(es) with stability-enhancing excipients as described herein.

In yet another aspect, this disclosure relates to a method for enhancing recovery of an antigenically preserved inactivated Dengue virus (or plurality thereof), in a composition containing the same, by formulating the inactivated Dengue virus(es) with stability-enhancing excipients as described herein.

As used herein, one measure of the stability of a formulation of PIV dengue is the change in average size of viral particles over time (aggregation or flocculation of virus). Thus the stability of a formulation of PIV Dengue virus can be assessed by measuring any increase in average viral particle size over time (aggregation or flocculation). Another measure of the stability of a formulation of PIV dengue is changes in antigenicity over time, with less stable formulations showing greater decreases in antigenicity. Thus, a 'less stable' formulation is one demonstrating increased average viral particle size, and/or decreased antigenicity, as compared to a comparator formulation under the same or similar conditions and over the same or similar time periods. An excipient that stabilizes a formulation is one that decreases aggregation over time, and/or maintains antigenicity over time, compared to a formulation that is identical except that it does not contain the excipient, or contains the excipient in a lesser amount. In one embodiment of the present invention, the stability 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 this disclosure relates to immunogenic compositions that include one or more purified inactivated Dengue viruses, in combination with at least one excipient. Such compositions may be bulk preparations of purified inactivated Dengue virus (PIV DEN) produced at commercial scale, and suitable for formulation into pharmaceutical compositions (e.g., vaccines to prevent infection by and/or disease due to Dengue virus); or finished vaccine formulations (Drug Product). Addition of a selected excipient as disclosed herein enhances the stability of the composition, as compared to formulations that do not include the selected excipient or that contain the selected excipient at significantly lower levels. Such formulations containing a selected excipient possess the favorable characteristic of reducing aggregation of the inactivated virus, e.g., during storage, and/or maintaining antigenicity over time (both as compared to a formulation that does not contain the selected excipient, or that contains the selected excipient at significantly lower levels. As used herein, a 'significantly lower' level is less than half, less than 40%, less than 25%, or less than 20% of a comparator level.

Thus one embodiment of the present invention is an immunogenic composition comprising one or more purified inactivated Dengue virus serotype(s), where the

immunogenic composition further comprises a stability -enhancing excipient selected from a sugar, an amino acid, or a salt that provides a divalent ion when in solution. Suitable excipients include sucrose, CaCh, and MgS04.

Aggregation of viral particles is decreased in the immunogenic compositions of the present invention (compared to formulations without the stability-enhancing excipient), e.g., 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. Aggregation of viral particles may be measured by any suitable method as is known in the art.

Alternatively or in addition, antigenicity is preserved in the immunogenic compositions of the present invention (compared to formulations without the stability-enhancing excipient), e.g., 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 may be measured by any suitable method as is known in the art, such as an ELISA assay.

Alternatively or in addition, recovery of purified inactivated Dengue virus (or viral antigens) is enhanced in the immunogenic compositions of the present invention, such that greater than 50%, 60%, 70%, 80%, 90%, (or greater 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 selected excipient 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 or about 10% weight/volume (w/v), or at least or about 9% w/v, or at least or about 8% w/v, or at least or about 6% w/v. In some embodiments, the final sucrose concentration is between about 5% to about 20% w/v; between about 6% to about 10% w/v, or between about 7% to about 9% w/v. In a further aspect, the immunogenic composition of the invention comprises sucrose and at least one additional excipient selected from CaCh and MgS04.

In one embodiment of the present invention, the selected excipient is CaC12, and is present in the immunogenic composition (e.g., bulk preparation or finished vaccine formulation) at a final concentration of at least or about 5mM, at least or about 10 mM, at least or about 15mM, at least or about 20mM, at least or about 30mM, or at least or about 45mM. CaC12 may be provided as calcium chloride dehydrate (CaCh.2H20). In some embodiments, the final CaCh concentration is between about 1 mM to about 50 mM; between about 5 mM to about 25 mM; or between about 10 mM to about 20 mM. In a further embodiment, the immunogenic composition of the invention comprises CaC12 and at least one additional excipient selected from sucrose and MgS04.

In one embodiment of the present invention, the selected excipient is MgS04, and is present in the immunogenic composition (e.g., bulk preparation or finished vaccine formulation) at a final concentration of at least or about 5mM, at least or about 10 mM, at least or about 15mM, at least or about 20mM, at least or about 30mM, or at least or about 45mM. The amount of MgS04 may be adjusted depending on the amount of antigen present in the composition, in order to achieve stability using a suitable amount of MgS04. MgS04 may be provided as Magnesium sulfate heptahydrate (MgSC JIrhO). In some embodiments, the the final MgS04 concentration is between about 1 mM to about 50 mM; between about 5 mM to about 25 mM; or between about 10 mM to about 20 mM. In a further embodiment, the immunogenic composition of the invention comprises MgSC and at least one additional excipient selected from sucrose and CaCh.

One embodiment of the invention is an immunogenic composition comprising purified inactivated virus of all four Dengue viral serotypes (a tetravalent PIV DEN composition), further comprising an excipient selected from CaCh, MgS04, and sucrose. In one embodiment the excipient is MgS04 at a final concentration of at least or about 7.5 mM, at least or about 15mM, at least or about 30 mM, or at least or about 45mM. The immunogenic composition may further be adjuvanted, such as with an aluminum adjuvant. One embodiment of the invention is an immunogenic composition comprising purified inactivated DEN-4, further comprising at least one excipient selected from CaCh, MgS04, and sucrose. In one embodiment the excipient is MgS04 at a final concentration of at least or about 7.5 mM, at least or about 15mM, at least or about 30 mM, or at least or about 45mM. The immunogenic composition may further be adjuvanted, such as with an aluminum adjuvant.

The compositions disclosed herein can include one or more than one serotype of Dengue virus. In one embodiment, the compositions include at least DEN-4. Commonly, the compositions include a plurality of Dengue viruses from more than one serotype, that is 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 can include two, three or four different serotypes of Dengue virus. In one embodiment the composition includes purified inactivated Dengue virus of all four serotypes, and elicits an immune response to each of DEN-1, DEN-2, DEN-3 and DEN-4. The virus(es) can be selected from among wild-type viruses (i.e., propagated from or corresponding to virulent virus from a naturally occurring isolate), or the virus(es) can be selected from attenuated viruses, recombinant viruses, and/or chimeric viruses. A single composition can include one or more wild-type virus, one or more attenuated virus, one or more recombinant virus, and/or one or more chimeric virus, in any combination.

Purified Dengue virus can be inactivated using chemical, physical and/or irradiating inactivating agents, alone or in any combination. The purified Dengue virus can be inactivated by exposure to formaldehyde, formalin, betapropiolactone (BPL), hydrogen peroxide, ultraviolet irradiation and gamma irradiation, or combination of any of these techniques. Details of such methods can be found, e.g., in published PCT Application No. WO

2010/094663 (US Publication No. 2011318407), and in US Publication No. 20070031451, which are incorporated herein by reference for the purpose of illustrating exemplary methods of inactivating Dengue viruses.

Typically, a human dose of the immunogenic composition contains an amount of PIV DEN that induces an immunoprotective response without significant adverse side effects in the typical subject; achieving an immunoprotective effect may require administration of more than one dose to an individual, according to a selected immunization schedule. As used herein, immunoprotective does not require complete protection against infection; it means a lessening of the severity or incidence of infection, disease, or symptoms of disease. Antigen content can be measured in terms of μg total protein content of a purified or partially purified virus antigen, or by immunological methods, e.g., ELISA, or by a quantitative immunoprecipitation method such as radial immunodiffusion.

Generally, it is expected that each human dose will comprise at least or about 0.1 μg, at least or about 0.2 μg, at least or about 0.25 μg, at least or about 0.3 μg, at least 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 or about 20 μg (or any amount between 0.1 and 20.0 μg) of each serotype of virus. Typically, a single human dose of the immunogenic composition contains no more than 100 μg of each serotype of virus, for example, no more than 90 μg, or no more than 80 μg, or no more than 75 μg, or no more than 70 μg, or no more than 60 μg, or no more than 50 μg, or no more than 40 μg, or no more than 30 μg, or no more than 20 μg, or no more than 10 μg, or no more than 8 μg, or no more than 4 μg, or no more than 2 μg (or any amount between 2 and 100 μg) of each serotype of virus. In certain embodiments, the immunogenic composition for human use is a liquid formulation, e.g., a solution or suspension. In other embodiments, the composition is prepared, lyophilized, and resuspended prior to administration to the subject.

The amount of PIV DEN utilized in an immunogenic composition is selected based on the intended subject population (e.g., adults, infants). An optimal amount for a particular composition can be ascertained by standard studies involving observation of antibody titres and other responses in subjects.

In certain embodiments of the present invention, the immunogenic composition comprises one or more PIV DENV serotypes and an adjuvant. The adjuvant may be an aluminum salt. Suitable aluminum salts include hydrated aluminum hydroxide (Al(OH)3), aluminum phosphate (AIPO4), aluminum oxide hydroxide (AIO(OH)) and aluminum hydroxyphosphate. In some embodiments the purified inactivated Dengue virus is adsorbed onto the aluminium salt. Where a plurality of Dengue viruses is included in the composition, each can be adsorbed onto the same aluminum salt, or different viruses can be adsorbed onto different aluminum salts. Where aluminum is present, the amount is typically between about 100 μg and 1 mg, such as from about 100 μg, or about 200 μg, to about 500 μg, to about 750 μg, to about 1000 μg, such as about 500 μg per dose. In formulations in which an aluminum salt is employed, the purified inactivated Dengue virus(es) can be pre-adsorbed onto the aluminum salt prior to formulation in the compositions disclosed herein. Alternatively, the aluminum salt can be added to a liquid composition, or included in the liquid in which a lyophilized immunogenic composition is resuspended.

The adjuvant may alternatively be a liposomal formulation of immunostimulants. One such suitable liposomal formulation is the adjuvant system AS01 (GlaxoSmithKline), which contains monophosphoryl lipid A (MPL, a detoxified lipopolysaccharide (LPS) derivative) and QS21 ( a saponin extracted from the bark of the Quillaja saponaria tree). See e.g., WO 94/00153 (PCT/EP93/01524), WO 94/21292 (PCT/EP94/00818), WO 07/068907

(PCT/GB06/004634), WO 96/33739 (PCT/EP96/01464)). The adjuvant may be an oil-in- water emulsion, such as the AS03 adjuvant system (GlaxoSmithKline) containing alpha- tocopherol and squalene (see, e.g., WO 95/17210 (PCT/EP94/04246), WO 08/043774

(PCT/EP07/060743)). The composition may be aluminum-free, or may comprise both an aluminum salt adjuvant and a further non-alum adjuvant (such as a lipopolysaccharide, a saponin, or an oligonucleotide,). In one embodiment the immunogenic composition comprises PIV DENV of all four serotypes, and an adjuvant (e.g., a tetravalent adjuvanted DEN PIV vaccine). In one embodiment the tetravalent adjuvanted DEN PIV vaccine comprises an aluminum adjuvant, such as Al(OH)3.

The immunogenic compositions disclosed herein may further comprise one or more surfactants; numerous surfactants are known in the art, for use in pharmaceutical formulations. Each such surfactant is selected to be suitable for administration to a subject, particularly a human subject. In certain embodiments, the surfactant is selected to be suitable for parenteral administration, e.g., for intramuscular, subcutaneous, transcutaneous or intradermal administration. Surfactants suitable for the Dengue compositions disclosed herein include poloxamer surfactants, polysorbate surfactants, octoxinol surfactants, polidocanol surfactants, polyoxyl stearate surfactants, polyoxyl castor oil surfactants, N-octyl-glucoside surfactants, macrogol 15 hydroxy stearate, and combinations thereof. In certain embodiments, Poloxamer surfactants are particularly suitable for formulations in which the purified inactivated Dengue virus(es) are not adsorbed onto an aluminum salt.

Poloxamer surfactants are polyethylene-polypropylene glycol linear copolymers.

Commercially, these are often referred to as Pluronic surfactants. In certain embodiments, the poloxamer surfactant is selected from a polyethylene -polypropylene glycol copolymer with an average molecular weight of at least about 1000 kD, and an average molecular weight of no more than about 15,000 kD. In one specific embodiment, the immunogenic composition is formulated with a polyethylene-polypropylene glycol copolymer, poloxamer 188, which is sold commercially under the trademarks Pluronic™ F-68, Lutrol™ F-68, and Kolliphor™ P188, which has an average molecular weight of 8600 kD, with a polyoxypropylene molecular weight of 1800 g/mole and an 80% polyoxyethylene content.

The immunogenic compositions disclosed herein may further comprise one or more buffering agents. The buffering agent(s) is typically selected to maintain the pH of the composition at or above neutral, e.g., at or near physiological pH of 7.4, and in some instance at or above pH 7.5, such as at or above pH 8.0, at or above pH8.3, or at or above pH 8.5.

Suitable buffering agents include carbonate, phosphate, citrate, lactate, gluconate and tartrate buffering agents, as well as more complex organic buffering agents. In certain examples, the buffering agent includes a phosphate buffering agent that contains sodium phosphate and/or potassium phosphate. Typically, such a buffering agent, or system, includes both sodium phosphate and potassium phosphate in a 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. Methods of formulating buffers to the desired pH are well known to those of skill in the art, and a suitable composition can be determined without undue experimentation based on the pH desired.

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

The immunogenic compositions (e.g., bulk preparations and finished vaccine formulations) disclosed herein may further include one or more sugar or polyol excipients, e.g., selected from the group consisting of: sucrose, trehalose, mannose, mannitol, raffinose, lactitol, sorbitol and lactobionic acid, glucose, maltulose, iso-maltulose, lactulose, maltose, lactose, iso-maltose, maltitol, palatinit, stachyose, melezitose, dextran or a combination thereof. In one specific embodiment, the excipient comprises sucrose. Optionally, the sugar or polyol can 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, e.g., Poloxamer 188 and a sugar, e.g., sucrose 2% w/v.

Methods of Formulation

Another aspect of this disclosure relates to methods for formulating immunogenic compositions (e.g., bulk preparations and finished vaccine formulations) comprising one or more purified inactivated Dengue viruses. Such methods comprise: providing a solution and admixing with the solution one or more purified inactivated Dengue viruses. The solution may contain buffering agent(s) and surfactant(s). In some embodiments, the one or more purified inactivated Dengue viruses are adsorbed onto an aluminum salt (e.g., to produce a pre-adsorbed bulk preparation of inactivated Dengue virus) prior to admixing with the solution. Typically, a single serotype or strain of purified inactivated Dengue virus is adsorbed onto an aluminum salt (e.g., aluminum hydroxide, aluminum phosphate or aluminum hydroxyphosphate) to produce a pre-adsorbed monobulk. To produce a multivalent immunogenic composition, monobulks of different Dengue serotypes are then combined in the desired ratio (e.g., 1 : 1 : 1 : 1 based on weight, or adjusted based on relative immunogenicity) with the solution containing the buffering agent and the surfactant.

Typically, the purified inactivated Dengue virus(es) are added to a solution suitable (in final formulation) for parenteral administration. In some embodiments, the solution is an isotonic solution and comprises sterile, endotoxin-free water. As disclosed herein, the solution also includes one or more excipients to increase stability during storage, such as a sugar, amino acid, or bivalent ion. Suitable excipients for increasing stability include sucrose, CaCh, and MgS04.

In certain embodiments, following addition of the purified inactivated Dengue virus to the solution containing the buffer and the surfactant (and optionally, additional components) as described herein, the formulated immunogenic composition is stored as a bulk liquid, e.g., at room temperature, at 0-4°C, or below 0°C (such as at or about -20°C, or at or about - 70°C to -80°C), prior to final filling in the Drug Product form. An example of a method of formulating a bulk adj wanted tetravalent PIV DEN immunogenic composition according to the present invention is as follows:

Under gentle agitation, add to sterile water for injection:

Sucrose to provide a final concentration of 2% w/v;

Tris to provide a final concentration of 5mM (in an alternate formulation Tris is provided in a final concentration of 3.5mM);

NaCl to provide a final concentration of 150mM;

Pluronic(TM)/Lutrol(TM) F68 to provide final concentration of 0.10% w/v;

MgS04 to provide a final concentration of 15mM (high dose) or 7.5mM (low dose);

Al(OH)3 to provide a final concentration of 1000 μg/ml (high dose) or 500 μg/ml (low dose); After stirring for 15-30 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 stirring 10-15 minutes after addition of final PIV DEN.

Check pH and adjust if necessary to 7.9 +/- 0.3

Store as bulk until filling In the above method, the aluminum adjuvant is added to the composition prior to addition of PIV DEN; in an alternative method, the PIV DEN virus are preadsorbed onto the aluminum adjuvant. Further examples of formulations suitable for PIV DEN may be found in WO 2012/160199.

In some embodiments, the formulation method then involves lyophilizing the solution (e.g., the bulk preparation containing the purified inactivated Dengue virus(es)) to produce a lyophilized composition. In embodiments involving the lyophilization of the immunogenic composition, e.g., for storage and/or distribution, the lyophilized composition is typically resuspended in a suitable amount, e.g., 0.05-2 mis, typically between 0.5 and 1.5 mis, for example, 0.5 or 1.0 or 1.5 mis, of a pharmaceutically acceptable solution, such as sterile endotoxin-free water for injection, prior to administration. Optionally, the pharmaceutically acceptable solution used for reconstitution may include an adjuvant, as disclosed herein.

The present invention provides for formulations and vaccines for use in medicine, specifically as a method of treating a mammal, particularly a human, suffering from or at risk of dengue infection. Also provided for is the use of the formulations and vaccines of the present invention in the manufacture of an immunoprophylactic and/or immunotherapeutic for the treatment or prophylaxis of dengue infection (e.g., a prophylactic vaccine or an immunotherapeutic vaccine).

Finished vaccine compositions of the invention may be lyophilised or in aqueous form, solutions or suspensions. Liquid formulations allow the compositions to be administered direct from their packaged form, without the need for reconstitution in an aqueous medium. In some embodiments an adjuvant may be mixed with the vaccine active ingredient at the time of clinical administration, and thus adjuvant and antigen may be kept separate in a packaged or distributed vaccine, ready for final formulation at the time of administration. In other embodiments an adjuvant is mixed with antigen during manufacture, and thus the composition is packaged in an adjuvanted form.

Finished vaccine compositions may be packaged in any suitable manner, e.g., in vials or in ready-filled syringes. The syringes may be supplied with or without needles. A syringe typically contains a single dose of the vaccine formulation, whereas a vial may include a single dose or multiple doses. In one embodiment the dose is for a human. In a further embodiment the dose is for an adult, adolescent, toddler, infant or less than one-year-old human. Effective dosages can be established using methods as are known in the art. In one embodiment the dosage is from 0. ^g to lO.C^g of antigen per each Dengue virus serotype, from O^g to 8.C^g of antigen per each Dengue virus serotype, or from ^g to 4.C^g of antigen per each Dengue virus serotype.

Although the finished vaccine composition can be administered by a variety of different routes, most commonly delivery is by intramuscular, subcutaneous or intradermal administration. Generally, the finished vaccine composition is administered in a dose effective for the production of neutralizing antibodies to each of the DENV serotypes contained in the composition. The quantity of PIV to be administered, depends on the subject to be treated, capacity of the subject's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of the vaccine to be administered may depend on the judgment of the practitioner and may be specific to each subject. The vaccine may be given in a single dose schedule, or preferably a multiple dose schedule in which a primary course of vaccination is followed by additional doses given at subsequent time intervals to maintain and or reinforce the desired immune response. The present disclosure concerns the formulation of immunogenic compositions, such as bulk vaccine preparations and finished vaccine formulations, containing purified inactivated Dengue virus of serotype 4. The formulations disclosed herein increase stability of immunogenic compositions (e.g., bulk or finished vaccine comprising DEN4 PIV), thus facilitating the manufacture, production, storage and distribution of vaccines comprising the DEN4 PIV.

A first aspect of this disclosure relates to immunogenic compositions (e.g., bulk vaccine compositions and finished vaccine formulations) that comprise purified inactivated DEN4, in combination with a buffering agent, a surfactant and a stability-enhancing excipient. As described herein, selected excipients have been found to enhance the stability of antigenically preserved inactivated DEN4 virus in formulation, as compared to formulations that do not include the selected excipient. Formulations of PIV DEN4 virus containing a selected excipient(s) as described herein (a "stability-enhancing" agent or excipient) possess the favorable characteristics of limiting average viral size increase over time, and/or as limiting reductions in antigenicity over time (e.g., during storage), as compared to formulations that do not contain the selected excipient(s).

The selected stability-enhancing excipient may be added at any one or more of various points in the manufacture of a finished vaccine product, e.g., added to individual serotype DS prior to addition of any adjuvant; added to individual adjuvanted serotypes, including those adsorbed to alum adjuvant; added to a composition comprising multiple serotypes, with or without other excipients or adjuvants; or added to an otherwise complete vaccine drug product.

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

The Dengue viruses used can be selected from any suitable strain (or strains) of Dengue virus. For example, a virus strain can be selected for each serotype, which is chosen based on its conformity to a defined (e.g., consensus) sequence for the serotype. Such a virus can be naturally occurring or synthetic. For example, a virus strain can be selected to correlate with a strain prevalent (e.g., a naturally occurring or "wild type" strain) in the area or population in which the vaccine is intended to be administered. Another option is to select suitable strains for each serotype based on availability or prior experience. Dengue strains are described in U.S. Pat. No. 6,254,873, which is incorporated by reference herein. Additional strains are disclosed, e.g., in U.S. Pat. No. 7,226,602, which is also incorporated herein by reference. Additional strains can be found, for example, in the VBRC viral genome database (available at http:// athena.bioc.uvic.ca/organisms/ Flaviviridae/ Dengue/ Curated_genes), and the Dengue Virus Database (available at http:// www. broad, mit. edu/ annotation/ viral/ Dengue/

Proj ectlnfo .html) .

In the context of a purified inactivated Dengue virus vaccine, either virulent or attenuated strains can be used. Typically virulent strains propagate to higher titer in host cells, facilitating production at commercial scale. However, virulent strains require handling precautions. Attenuated strains, e.g., developed by adaptation to production in cultured cells and selection for reduced virulence and/or reduced replication in the mosquito vectors of Dengue, require fewer handling precautions but can be more difficult to produce. Exemplary attenuated strains suitable for use in the context of an immunogenic composition containing an inactivated Dengue virus are described in WO 2000/057907 and U.S. Pat. No. 6,638,514, and WO 2000/058444 and U.S. Pat. No. 6,613,556, WO 2002/066621 (US Publication No.

2004052818), WO 2000/057904 (U.S. Pat. No. 6,528,065, WO 2000/057908, WO

2000/057909 (U.S. Pat. No. 6,511,667); WO 2000/057910 (U.S. Pat. No. 6,537,557), WO 2002/095075 (e.g., U.S. Pat. No. 7,226,602) and WO 2002/102828 (U.S. Pat. No. 7,569,383), which are incorporated herein by reference.

Thus the purified, inactivated virus(es) can be selected from among wild-type viruses

(i.e., propagated from or corresponding to virulent virus from a naturally occurring isolate), or the virus(es) can be selected from attenuated viruses. A selected virus can be a recombinant virus. For example, a recombinant virus can be a chimeric virus, e.g., a virus having a nucleic acid from a Dengue virus and a nucleic acid from another flavivirus, such as a different Dengue virus, a Yellow Fever virus, or a Japanese Encephalitis virus. Typically, a chimeric virus includes one or both of a Dengue M and a Dengue E protein. Examples of chimeric Dengue viruses can be found in, e.g., WO 98/37911 (U.S. Pat. Nos. 6,696,281; 6,962,708), WO 96/40933 and WO 2001060847 (U.S. Pat. Nos. 7,094,411; 7,641,909; 8,025,887) and EP1159968 Methods for producing such chimeric Dengue virus can also be found in WO 03/101397. A single composition can include one or more wild-type virus, one or more attenuated virus, one or more recombinant virus, and/or one or more chimeric virus, in any combination.

Methods of Producing DENV

Methods for producing Dengue virus(es) are known in the art, and are described in detail sufficient to guide one of ordinary skill in the art in, e.g., published PCT Application No. WO 2010/094663, US publication No. 2011318407. The virus may be propagated in animal -free conditions, i.e., produced without the use of any material from animal origin (e.g., fetal bovine serum). Methods for producing virus in serum-free conditions can also be found, for example, in US Publication No. 20060183224. The disclosures of these published patent applications are incorporated herein by reference to provide additional details regarding the propagation and purification of Dengue viruses for inclusion in the immunogenic compositions (e.g. bulk and finished vaccine preparations) disclosed herein. In one embodiment, medium containing the virus is clarified by filtration, concentrated, and the medium exchanged (e.g., by ultrafiltration and diafiltration) for a suitable buffer (for example, phosphate buffered saline (PBS), 125 mM Citrate, pH 7.6). Suitable buffer solutions can be selected by those of skill in the art. Initial concentration and buffer exchange may be followed by further 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 finished vaccine formulation), can be added to the buffered viral composition. Alternatively, the surfactant can be added following inactivation. The purified inactivated Dengue virus is then sterile filtered to produce a bulk preparation of inactivated Dengue virus.

Terms and Definitions

All references disclosed herein are incorporated by reference in their entirety.

To facilitate review of the various embodiments of this disclosure, the following explanations of terms are provided. Additional terms and explanations can be provided in the context of this disclosure. Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill 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 (ed.), 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 6^th Edition, Plotkin, Orenstein and Offit (Eds.), Saunders, 2012.

The singular terms "a," "an," and "the" include plural referents unless 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. Additionally, numerical limitations given with respect to concentrations or levels of a substance, such as an antigen, are intended to be approximate. Thus, (for example) where a concentration is indicated to be at least 200 pg, it is intended that the concentration be understood to be at least approximately (or "about" or "~") 200 pg.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described herein. The term "comprises" means "includes." Thus, unless the context requires otherwise, the word "comprises," and variations such as "comprise" and

"comprising" will be understood to imply the inclusion of a stated compound or composition (e.g. , nucleic acid, polypeptide, antigen) or step, or group of compounds or steps, but not to the exclusion of any other compounds, composition, steps, or groups thereof. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g. " is synonymous with the term "for example."

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

To provide a finished Drug Product (DP) (including a finished vaccine), one or more Drug Substances are typically formulated with inactive ingredients (e.g., vehicles, buffers, excipients, binders). Inactive ingredients are included in the formulation for various reasons, e.g., to facilitate manufacture, enhance stability, or enhance other product characteristics. As used herein, a finished or final vaccine formulation is considered a Drug Product.

A purified, inactivated Dengue viral Drug Substance (or active ingredient) refers to a Dengue virus in the final antigenic form, with respect to purification and inactivation, and intended for administration to a subject. A bulk preparation or bulk formulation of PIV DEN virus drug substance can be further processed, e.g., by dilution, concentration, such as by lyophilization and resuspension, and/or packaged, e.g., into multidose or single dose vials or syringes for administration as an immunogenic composition or vaccine.

As used herein, "adjuvant" is an agent that enhances the production of an antigen- specific immune response as compared to administration of the antigen in the absence of the agent. Common adjuvants include aluminum-containing adjuvants that include suspensions of minerals (or mineral salts, such as aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate) onto which antigen is adsorbed. Other adjuvants include one or more immuno stimulatory component that contributes to the production of an enhanced antigen- specific immune response. Immunostimulatory components include oil and water emulsions, such as water-in-oil, and oil-in-water (and variants thereof, including double emulsions and reversible emulsions), liposaccharides, lipopolysaccharides,

immunostimulatory nucleic acids (such as CpG oligonucleotides), liposomes, Toll-like Receptor agonists (particularly, TLR2, TLR4, TLR7/8 and TLR9 agonists), and various combinations of such components. Adjuvants can include combinations of

immunostimulatory components.

As used herein an "antigen" refers to a substance, including peptides and proteins (including glycoproteins), that 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 change in pH when an acid or an alkali is added. The term buffering agent encompasses a wide variety of compounds and compositions, typically, either weak acids or weak bases, which when present in solution with their conjugate base or acid, respectively, 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 larger than the volume of a finished, commercialized Drug Product. Thus a "bulk vaccine component" refers to a vaccine DS or active ingredient which is kept, contained, or stored in, a volume larger than the volume of the finished vaccine product. The concentration of vaccine active ingredient (such as a PIV DEN virus) in a bulk preparation is typically higher than the concentration of the active ingredient in the finished vaccine product. A "bulk" is an intermediate product in the commercial manufacture of vaccines. A bulk vaccine component may be univalent (monovalent), i.e., containing one vaccine active ingredient (such as a single serotype of Dengue virus), bivalent, or multivalent. A bulk may contain a purified antigen or PIV in different concentrations than will be present in a final (finished) vaccine formulation (the formulation for distribution, commercial sale, or clinical use). Final vaccine formulations may be polyvalent, comprising multiple serotypes of dengue PIV (e.g., may be a mixture of multiple serotype PIV bulks), and may comprise antigens from additional pathogens. Bulks may be stored until needed for the preparation of the final vaccine formulation.

An "immune response" is a response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. An immune response can be a B cell response, which results in the production of specific antibodies, such as antigen specific neutralizing antibodies. An immune response can also be a T cell response, such as a CD4+ response or a CD8+ response. In some cases, the response is specific for a particular antigen (that is, an "antigen-specific response"). If the antigen is derived from a pathogen, the antigen-specific response is a "pathogen-specific response." A "protective immune response" is an immune response that inhibits a detrimental function or activity of a pathogen, reduces infection by a pathogen, or decreases symptoms (including death) that result from infection by the pathogen. A protective immune response can be measured, for example, by the inhibition of viral replication or plaque formation in a plaque reduction assay or ELISA-neutralization assay, or by measuring resistance to pathogen challenge 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 that is capable of eliciting a specific immune response in the subject to which it is administered, e.g., against a pathogen, such as Dengue virus. As such, an immunogenic composition includes one or more antigens (for example, whole purified virus or antigenic subunits, e.g., polypeptides, thereof) or antigenic epitopes. An immunogenic composition can also include one or more additional components, such as an excipient, carrier, and/or adjuvant. In certain instances, immunogenic compositions are administered to elicit an immune response that protects the subject against symptoms or conditions induced by a pathogen. In the context of this disclosure, the term immunogenic composition will be understood to encompass compositions that are intended for administration to a subject or population of subjects for the purpose of eliciting a protective or palliative immune response against Dengue (that is, vaccine compositions or vaccines). The immunogenic response may be an immunoprotective response elicited in a human subject.

The term "inactivated" in the context of a Dengue virus vaccine means that the antigenic component (e.g., virus) is incapable of replication in vivo or in vitro. For example, the term inactivated encompasses a virus that has been replicated, e.g., in vitro, and then killed using chemical or physical means such that it is no longer capable of replicating. The term can also include antigens produced by further processing (e.g., splitting, fractionation, and the like), and components produced by recombinant means, e.g., in cell culture.

As used herein, a "mixture" comprises at least two different elements, e.g., a mixture comprising molecules of a polypeptide species and molecules of an adjuvant species, or comprising molecules of two different polypeptides.

As used herein, the terms "peptide", "polypeptide", and "protein" are interchangeable and mean 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, usage of these terms in the art overlaps and varies. The term "polypeptide" as used herein refers to peptides, polypeptides and proteins, unless otherwise noted. As used herein, the terms "protein", "polypeptide" and "peptide" refer to both expressed gene products and chemically synthesized entities, and encompass glycoproteins and inactivated protein toxins (toxoids).

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 process of removing undesired components from a composition. 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 processes as centrifugation, dialization, ion-exchange chromatography, and size-exclusion chromatography, affinity-purification or precipitation. Thus, for example, a purified virus preparation is one in which the virus is more enriched than it is in its generative environment, for instance within a cell or population of cells in which it is replicated naturally or in an artificial environment such as a culture. A preparation of substantially pure viruses can be purified such that the desired virus or viral component represents at least 50% of the total protein content of the preparation. In certain embodiments, a substantially pure virus will represent 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 preparation. Alternatively, the purification of a virus preparation can be assessed as the reduction in contaminants, such as host cell proteins, in the preparation. Accordingly, a preparation of substantially pure virus (e.g., purified inactivated Dengue virus) typically includes less than 30%, or less than 25%, residual host cell proteins. For example, an immunogenic composition (e.g. bulk preparation or finished vaccine formulation) comprising a purified inactivated Dengue virus can include less than 20% residual host cell protein, less than 15%, or 10% or less (e.g., measured on a wt/wt basis).

As used herein, a "subject" is a living multi-cellular vertebrate organism. In the context of this disclosure, the subject can be an experimental subject, such as a non-human animal, e.g., a mammal such as a mouse, a cotton rat, or a non-human primate.

Alternatively, the subject can be a human subject, e.g., 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 at the surface of a liquid, a surfactant acts to lower the surface tension of the liquid, the interfacial tension between two liquids, or the tension between the liquid and a solid. A surfactant may act as detergent, wetting agent, emulsifier, foaming agent, and/or dispersant.

As used herein, the term "comprises" means "includes." Thus, unless the context requires otherwise, the word "comprises," and variations such as "comprise" and

"comprising" will be understood to imply the inclusion of a stated compound or composition (e.g., antigen or excipient) or step, or group of compounds or steps, but not to the exclusion of any other compounds, composition, steps, or groups thereof. Embodiments described as comprising certain components are intended to include embodiments consisting of the indicated components.

The following examples are provided to illustrate features and/or embodiments. These examples should not be construed to limit the invention to the particular features or embodiments described. It will be appreciated by those of skill in the art that the amounts, e.g., volumes, are provided as examples only, and that the scale can be modified (either increased or decreased) at the option of the practitioner. Similarly, the components used in the assays, e.g., filters, columns, are not intended to be in any way limiting or exclusionary, and can be substituted for other components to achieve the same purpose as will be apparent to one of ordinary skill in the art.

EXAMPLES

Example 1: Preparation of Dengue (DEN) PIV

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

Example 2: Assay Methods

ELISA: DENV serotype-specific monoclonal antibodies (mAbs) were identified and confirmed for serotypes DEN1, DEN2, DEN3 and DEN4 (respectively, the mAbs El 03, DV44, DV3/1I, and E88). A DENV Enzyme Linked Immunosorbent Assay (ELISA) protocol was established, using the appropriate DENV-specific mAb and detection with the same mAb conjugated with biotin. This ELISA was used to determine serotype-specific in vitro antigenicity.

An inhibition-based antigenicity ELISA was developed to evaluate the antigen content of formulations in which the antigens were adsorbed onto Aluminum. Briefly, Alum-adsorbed DPIV antigens are initially incubated with biotin-conjugated DENV serotype-specific mAbs described above. Antigens/mAbs mixtures are then centrifuged prior to transferring the supernatant to ELISA plates previously coated with DENV Purified Bulk (PB). Binding of mAbs is revealed by adding Streptavidine Amdex-HRP Substrate (Amersham) and TMB (tetra-methyl-bensidine peroxidise). Dynamic Light Scattering (DLS): A Wyatt DynaPro Plate Reader (Wyatt Technology Corp) was used to determine the size distribution profile of small particles in suspension. The protocol used multi 96-well plates, UV-bottom transparent (Corning, NY), temperature 22°C, laser power 14 %, attenuation 77%, acquisition time 5 seconds. Five measurements were obtained per sample.

Results of DLS analysis are expressed in terms of the Z-average, which is obtained by analyzing DLS data using the technique of cumulants (see e.g., Koppel, J. Chem. Phys 57:4814-4820 (1972); Thomas, J. Colloid Interface Sci. 117: 187-192 (1987)). The Z-average size increases as the particle size increases, and therefore provides a reliable measure of the average size of a particle size distribution.

High Performance Size Exclusion Chromatography (UV detection): Size exclusion liquid chromatographic conditions to determine protein content by UV detection were developed. The Liquid chromatography system was an HPLC Waters Alliance e2695, UV 2998; column a TSKGel G600 PWXL 7.8mm ID X 30 cm L; heater temperature 40°C; injection volume 100 μΐ; sample temperature 15°C; run time 25 minutes; mobile phase: TRIS 5mM, NaCl 150mM, PX188 0.1%, pH 7.4; UV wavelength 210 nm; flow rate 1.0 ml/minute. Nephelometry: Nephelometry assesses the cloudiness, or turbidity, of a solution caused by the presence of suspended particles. The examples provided herein used a Nephelostar (BMG Labtech) microplate nephelometer and multi-96 well plates (UV bottom transparent, Corning, NY). Volume of the sample was ΙΟΟμυ. The Nephelostar instrument comprised a dedicated optical system to measure light scattering caused by turbidity. Optic settings: Gain 55; laser intensity (%) 90; beam focus (mm) 1.5. General settings: Positioning delay (seconds) 0.1; reading direction horizontal.

Nephelometric Unit (NU) is a dimensionless unit. NTU stands for Nephelometric Turbidity Unit, where (e.g., as indicated in Figures 9-10) 10 NTU corresponds to the eye detection limit and is determined for each run with an internal control comprising the formazin molecule diluted to a specific concentration (10 NTU ~= 500 NU with the laboratory instrumentation described herein). An increase in nephelometry indicates an increase in the solution turbidity that can be ascribed, in the present examples, to an aggregation phenomenon.

Static Light Scattering: Static light scattering assay to assess particle size used the Malvern Mastersizer 2000 (Malvern Instruments, Ltd.). Dispersant was NaCl 150mM plus Tris 5mM; temperature was 20-25 °C, obscuration was about 5-7%, recirculation speed 1250 rpm, size standard was latex polymer microspheres 5μιη. The sample diluents was used for background measurement. Five measurements were used to calculate the average size distributions and reported values.

Intrinsic Fluorescence: assessment of protein content by Intrinsic Fluorescence (IF) was performed with a Varioskan Flash system (Thermo Scientific) using Multi-96 well plates UV -bottom transparent (Corning) containing ΙΟΟμί of sample. Intrinsic protein fluorescence is caused by exciting the protein with 280 nm ultraviolet light and observing at approximately 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 dispensed into three wells.

Example 3: DEN4 PIV drug substance

A composition comprising DEN4 PIV was formulated and combined with buffer (50 mM

NaCl, 5 mM Tris (tris(hydroxymethyl)aminomethane), 3% w/v sucrose, and 0.1% w/v poloxamer 188. Final pH was 8.0 and final DEN4 virus content was 34μg/ml. For purposes of this Example, this base formulation was used as a control. Stability of the DEN4 PIV in this base formulation was found to be limited, with an increase in virus particle size and a drop in antigenicity occurring after 48 hours at room temperature (25 °C).

Example 4: Screening of Excipients with DEN4 PIV Drug Substance

Various Test Excipients (Tables 1 and 2) were added to the DEN4 PIV base formulation (as described above) to determine whether the DEN4 serotype could be stabilized. Excipients tested included sugars, amino acids, bivalent ions, surfactants and co-solvents. The DEN4 PIV base formulation (prior to addition of Test Excipients) contained 3% sucrose; where sucrose was used as a Test Excipient it was in addition to the 3% sucrose of the base formulation. The DEN4 PIV base formulation also contained 0.1% poloxamer 188; where poloxamer 188 was used as a Test Excipient, it was in addition to the 0.1% poloxamer 188 of the base formulation.

Stability was assessed after storage for 48 hours at 25°C, using Dynamic Light Scattering (DLS), and HP-SEC-UV (High Performance Size Exclusion Chromatography using

Ultraviolet detection). The control ("CTRL PB") for Figures 3 and 6 was a calibration curve made with freshly thawed and diluted DEN4 purified bulk.

Results: Figures 1 - 3 show results for DEN4 PIV Drug Substance formulation supplemented with the Test Excipients as listed in Table 1. Figure 1 provides DLS results at Time 0, Figure 2 provides DLS results after 48 hours storage at 25°C, and Figure 3 provides results of HP-SEC-UV recovery after 48 hours storage at 25°C. Controls were the DEN4 PIV base formulation without Test Excipient (indicated as Purified Bulk ('ΡΒ') in the Figures).

Figures 4-6 show results for the Test Excipients as listed in Table 2. Figure 4 provides DLS results at Time 0, Figure 5 provides DLS results after 48 hours storage at 25°C, and Figure 6 provides results of HP-SEC-UV recovery after 48 hours storage at 25°C. Controls were the DEN4 PIV base formulation (without Test Excipient).

TABLE1 : Test Excipients

Excipient Valine 1 1 .4mM

Valine 1 14mM

Alanine 34mM Serine 1 1 .4mM

Alanine 341 mM Serine 1 14mM

Phenylalanine 3.4mM Proline 34mM

Phenylalanine 34.1 mM Proline 341 mM

Leucine 3mM Arginine 30mM

Leucine 30mM Arginine 300mM

Glutamic Acid 1 mM Glycine 30mM

Glutamic Acid 10mM Glycine 300mM

Aspartic Acid 1 .14mM Asparagine 3.41 mM

Aspartic Acid 1 1 .4mM Asparagine 34.1 mM

Lysine 1 mM Histidine 30mM

Lysine 10mM Histidine 30mM

Threonine 10mM Methionine 30mM

Threonine 30mM Methionine 30mM

Taurine 1 1 .4mM CaCI₂ & Arginine 15mM & 30 mM

Taurine 1 14mM CaC & Arginine 15mM & 300 mM CaCI₂& Histidine 15mM & 30 mM CaCI₂ 15mM

CaCI₂& Histidine 15mM & 30 mM CaCI₂ 15mM

MgS0₄ & Arginine 15mM & 30 mM MgS0₄ 15mM

MgS0₄ & Histidine 15mM & 30 mM TRAVASOL(TM) 1 .5%

MgS0₄ & Histidine 15mM & 300 mM TRAVASOL(TM) 3.0%

ZnC 15mM TRAVASOL(TM) 5.0%

15mM CTRL DEN4 PB -

ZnC

CTRL DEN4 PB

ZnCI₂& Arginine 15mM & 30 mM -

CTRL DEN4 PB

ZnCI₂& Arginine 15mM & 300 mM -

ZnCI₂& Histidine 15mM & 30 mM

*TRAVASOL(TM) (Baxter Corporation) is a sterile pharmaceutical solution containing 10% amino acids.

Table 2: Test Excipients

Excipient: Cone.

Maltose 5.7%

Maltose 1 1 .4%

Glucose 5.7%

Glucose 1 1 .4%

Dextran 5.7%

Dextran 1 1 .4%

Trehalose 3%

Trehalose 5%

Trehalose 10%

Mannitol 3%

Mannitol 5%

Mannitol 10%

Glycerol 3%

Glycerol 5%

Glycerol 10%

HP-b-CD 3%

HP-b-CD 5%

HP-b-CD 10%

PEG300 3%

PEG300 5%

PEG300 10% Of the initial Test Excipients evaluated (Tables 1 and 2), most were eliminated based on the results of the DLS and HP-SEC-UV analysis after 48 hours at 25°C; ten Test Excipients were selected for further investigation.

Example 5: Further Testing of Selected Test Excipients with DEN4 PIV Drug

Substance

Based on the results of above Examples, ten Test Excipients were selected for further testing, with some evaluated at more than one concentration (Table 3). Table 3: Test Excipients

The Test Excipients were added to the DEN4 PIV (base formulation as described in Example 4, above). Stability was assessed 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 time period (n=4). As control, the DEN4 PIV base formulation was also assessed at these time periods.

Samples were assessed with DLS, HP-SEC-UV, nephelometry, ELISA and Intrinsic Fluorescence (IF).

Results are shown in Figures 7-16, where the bars are the average of the four samples tested (n=4), with Standard Error indicated. ELISA: The Y-axis is % recovery compared to the theoretical antigenicity that should be found based on the dilution factor applied. IF: The Y-axis is % recovery compared to a calibration curve made with freshly thawed and diluted DEN4 drug substance.

The results demonstrate that the presence of certain Test Excipients (including Sucrose 8%, CaCh 15mM, and MgS04 15mM) reduced the virus aggregation after 7 days storage at 25 °C (assessed by DLS and nephelometry), compared to control. Additionally, the antigenicity was conserved over time (as assessed by HP-SEC-UV, ELISA and IF).

Example 6: Unadivanted Tetravalent Formulation

To further examine the effect of CaCh, MgS04, and high-concentration sucrose on the stability of DEN4 PIV, dose ranges of CaCh (1.8mM, 3.75mM, 7.5mM, 15mM, 30mM, 45mM) and MgSCU (1.8mM, 3.75mM, 7.5mM, 15mM, 30mM, 45mM), as well as sucrose 8% w/v, were added to an unadjuvanted tetravalent base composition of Dengue virus. The tetravalent base formulation contained 8 μg/ml each of DEN 1, DEN2, DEN3, and DEN4, with 150 mM NaCl, Tris 5mM, 2% sucrose w/v, 0.1% pxl88 w/v, pH 8.5 (tetravalent base formulation). Control ('ctrl tetra') was the tetravalent base formulation (without CaCh, MgS04, or additional sucrose).

Stability up to 6 days at 25°C was assessed by DLS and ELISA. Results are shown in Figure 17 (diameter (nm) assessed by DLS), and Figures 18-21 (antigenicity of DEN 1, DEN2, DEN3 and DEN4, respectively, assessed by ELISA). The bars shown in Figures 17- 21 the average of four samples (n=4), with Standard Error indicated.

Example 7

Based on the preceding experiments, three excipient conditions (total sucrose 8%, MgS04 at 15 mM, and CaCh at 15 mM) were selected for side-by-side comparison in unadjuvanted tetravalent DEN PIV formulation (as described 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 test formulations were incubated for 7 days at 4°C, 25°C and 37°C. Read-outs were then applied on both non-centrifuged samples (NC) and centrifuged samples (C) to evaluate the loss in content if aggregation was present. Figures 22-25 graph the antigenicity of the DEN1, DEN2, DEN3, and DEN4 serotypes, respectively, from the unadjuvanted tetravalent formulation, assessed using ELISA. "NC" indicates non-centrifuged sample, and "C" indicates centrifuged. Figure 26 graphs results from DLS analysis, showing any increase in average particle size in the formulations held at different temperatures. "Fresh" indicates base formulation assessed at TO.

Figure 27 graphs the protein content as assessed by SEC HPLC with UV detection, and provides results as the % recovery compared to the protein content of the Purified Bulk product. As centrifugation prior to analysis would remove aggregates, only non-centrifuged samples were assessed. Figure 28 graphs results of nephelometry assessment.

Example 8: Adjuvanted formulation

The selected Test Excipients were assessed using an Al(OH)3 adjuvanted tetravalent formulation containing 4μg of each of the four DENV serotypes, and 500μg of Al(OH)3 adjuvant . The formulation comprised 150 mM NaCl, Tris 5mM, 2% sucrose w/v, 0.1% pxl 88 w/v, pH 8.5 (tetravalent base formulation). This example used samples of approximately 20 ml (rather than microwell plates). Test Excipients were 15mM CaCh, 15mM MgS04 and total sucrose 8% w/v. The control contained 2% w/v sucrose from the Drug Substance. Samples were assessed after 7 days at 4°C, after 7 days at 25°C, and after 7 days at 37°C.

Figure 29 shows results of Static Light Scattering (SLS) to measure particles <10μπι, and Figure 30 shows SLS to measure particles <100 μπι.

Example 9: Reduced Aggregation at 7 days, 30°C, with MgSQ4 excipient

Stability of DEN4 PIV DS supplemented with 15mM MgS04 was assessed using DLS at Time Zero (TO), after one week (1W, seven days) storage at 22°C, after 1W storage at 30°C, after one freeze-thaw cycle (1F/T), after two freeze-thaw cycles (2F/T) and after three freeze- thaw cycles (3F/T). See Figure 31. Comparison was to Control (CTRL, DEN4 DS without

MgS04 at 15mM stabilized the DEN4 virus size after one week's storage at 30°C compared to MgS04-free composition (compare fifth and sixth bars on Fig. 31). Y-axis is the Z-average size (diameter) of viral particles in nanometers.

Claims

CLAIMS We claim:

1. An immunogenic composition, comprising:

(a) purified inactivated Dengue Virus serotype 4;

(b) a buffering agent;

(c) a surfactant; and

(d) a stability-enhancing excipient.

2. The immunogenic composition of claim 1, where said stability-enhancing excipient is selected from an inorganic salt and a sugar.

3. The immunogenic composition of any preceding claim, where said stability-enhancing excipient is selected from CaCh, MgS04, and sucrose.

4. The immunogenic composition of any preceding claim, where said stability -enhancing excipient is selected from MgS04 at a concentration of at least or about 1.8mM, 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.

5. The immunogenic composition of any of claims 1-3, where said stability-enhancing excipient is selected from CaCh at a concentration of at least or about 1.8mM, 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.

6. The immunogenic composition of any of claims 1-3, where said stability-enhancing excipient is sucrose at a concentration of at least or about 8% weight/volume (w/v), or at least or about 10% w/v.

7. The immunogenic composition of any preceding claim where said composition is a liquid.

8. The immunogenic composition of any preceding claim, further comprising an adjuvant.

9. The immunogenic composition of claim 8, where said adjuvant is an aluminum salt.

10. The immunogenic composition of any one of claims 8-9, where said adjuvant comprises at least one of aluminum hydroxide and aluminum phosphate.

11. The immunogenic composition of any one of claims 7-10, further comprising at least one additional immunostimulatory component.

12. The immunogenic composition of claim 11, wherein the at least one additional immuno stimulatory component comprises one or more of an oil and water emulsion, a liposome, a lipopolysaccharide, a saponin, and an oligonucleotide.

13. The immunogenic composition of claim 8, where said adjuvant is an aluminum-free adjuvant.

14. The immunogenic composition of claim 13 where the aluminum-free adjuvant is selected from an oil in water emulsion, a liposome, a lipopolysaccharide, a saponin, and an oligonucleotide.

15. The immunogenic composition of any preceding claim further comprising at least one additional purified inactivated Dengue virus serotype.

16. The immunogenic composition of any preceding claim further comprising purified inactivated Dengue virus serotype 1 (PIV DEN1), purified inactivated Dengue virus serotype 2 (PIV DEN2), and purified inactivated Dengue virus serotype 3 (PIV DEN3).

17. The immunogenic composition of claim 16, where the composition elicits an immune response to each of DEN 1, DEN2, DEN3 and DEN4 in an adult human.

18. The immunogenic composition of any preceding claim where the surfactant is a poloxamer.

19. The immunogenic composition of any preceding claim where the surfactant is present is an amount of at least 0.001% (wt/v).

20. The immunogenic composition of any preceding claim where the surfactant is present is an amount of no more than 1.0% (wt/v).

21. The immunogenic composition of any preceding claim comprising a phosphate buffering agent.

22. The immunogenic composition of claim 21, comprising a phosphate buffering agent selected from sodium phosphate and potassium phosphate.

23. The immunogenic composition of any of claims 1-20 where the buffering agent comprises Tris(hydroxymethyl)aminomethane,

24. The immunogenic composition of any preceding claim which is a bulk preparation.

25. A method of formulating a bulk preparation of purified inactivated 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), purified inactivated Dengue Virus serotype 4.

26. The method of claim 25, where said stability enhancing agent is MgS04.

27. The method of any one of claims 25-26, where step (b) further comprises adding additional purified inactivated Dengue Virus serotypes.

28. The method of any one of claims 25-27, where step (b) further comprises adding purified inactivated Dengue Virus of serotype 1, serotype 2, and serotype 3.

29. The method of anyone of claim 25-27, where said solution of step (a) further comprises an aluminum adjuvant, such as Al(OH)3.