Compositions and methods for vaccinating against dengue virus in children and young people

Abstract

Embodiments herein relate to compositions, methods and uses for inducing an immune response against all four dengue virus serotypes in children or young people at an age of about 1 to about 20 years. Some embodiments relate to compositions, including but not limited to dengue virus chimeras, which can be used, alone or in combination with other constructs, for vaccine compositions against all four dengue virus serotypes. In certain embodiments, compositions may comprise constructs exceeding one dengue virus serotype in various concentrations or ratios, such as dengue--1 (DEN-1) virus, dengue-2 (DEN-2) virus, dengue-3 (DEN-3) virus, and / or dengue-4 (DEN-4) virus, to improve protection from infection in children and young people. In certain embodiments, the viruses of the formulations are limited to dengue virus serotypes. Other embodiments relate to methods of administering an immunogenic composition against a dengue virus, which may comprise a chimeric dengue construct and a live attenuated dengue virus, using a single, dual, or other regimen.

Description

[0001] This application is a divisional application of a patent application with an application date of April 13, 2017, a priority date of April 13, 2016, an application number of 201780028507.X, and an invention title of "Compositions and Methods for Vaccinating Against Dengue Virus in Children and Young Adults".

[0002] Related Applications

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 322,167, filed on April 13, 2016, which is hereby incorporated by reference in its entirety for all purposes. Field of the Invention

[0004] The embodiments disclosed herein relate to compositions, methods, and uses for inducing an immune response against all four dengue virus serotypes in children or young adults (young adults) from about 1 year of age to about 20 years of age. Some embodiments relate to compositions that can include, but are not limited to, chimeric and non-chimeric flavivirus constructs, which alone or in combination can be used in vaccine compositions to induce an immune response against all four dengue virus serotypes. In certain embodiments, the composition can contain various concentrations or ratios of constructs of more than one dengue virus serotype, such as dengue-1 (DEN-1) virus, dengue-2 (DEN-2) virus, dengue-3 (DEN-3) virus, and / or dengue-4 (DEN-4) virus, to improve protection against dengue infection in children and young adults. Other embodiments relate to methods of administering vaccine compositions that can include chimeric dengue constructs and live attenuated dengue virus using single, dual, or other dosing regimens. Background of the Invention

[0006] Dengue fever is a mosquito-borne disease caused by infection with the dengue virus. Infection with the dengue virus can lead to symptoms of weakness and pain, including sudden high fever, headache, joint and muscle pain, nausea, vomiting, and rash. To date, four dengue virus serotypes have been identified: dengue-1 (DEN-1), dengue-2 (DEN-2), dengue-3 (DEN-3), or dengue-4 (DEN-4). Other subtypes may be discovered in the future (e.g., DEN-5). Dengue virus serotypes 1-4 can also cause dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). In the most severe cases, DHF and DSS can be life-threatening. The dengue virus causes 50 million to 100 million cases of debilitating dengue fever, 500,000 cases of DHF / DSS, and more than 20,000 deaths each year, with a large proportion of these being children. To date, there is no effective vaccine to prevent dengue fever and no drug treatment for the disease. Mosquito control efforts have been ineffective in preventing dengue outbreaks in endemic areas or in preventing further geographical spread of the disease. An estimated 3.5 billion people are at risk of infection with the dengue virus. In addition, the dengue virus is a major cause of fever in travelers to endemic areas such as Asia, Central and South America, and the Caribbean. In addition, DHF / DSS is a major cause of severe illness and death in children in some Asian and Latin American countries.

[0007] All four dengue virus serotypes are endemic in the tropical regions of the world and constitute the most important mosquito-borne viral threat to humans in the tropical regions of the world. The dengue virus is mainly transmitted to humans by Aedes aegypti. Infection with one dengue virus serotype confers lifelong protection against reinfection with that serotype, but does not prevent secondary infection with one of the other three dengue virus serotypes. In fact, previous infection with one dengue virus serotype increases the risk of severe disease (DHF / DSS) following secondary infection with a different serotype. The development of an effective vaccine (especially in children and young adults) is an important approach to preventing and controlling this global disease. Summary of the Invention

[0009] The embodiments disclosed herein relate to compositions, methods, and uses of chimeric dengue virus constructs. In some embodiments, the composition can comprise a chimeric dengue virus construct, either alone or in combination with a live attenuated dengue virus, having an attenuated dengue virus backbone with structural genes from at least one other dengue virus serotype. Other embodiments relate to a combination of at least one live attenuated virus with one or more chimeric dengue viruses, the chimeric dengue viruses containing structural elements against at least one additional dengue virus serotype. Other embodiments can include chimeric dengue compositions having a modified DEN-2 backbone (e.g., PDK-53 as the starting backbone in P1 (passage-1) and PDK-53 modified using passage variability and selection, as indicated for P2, P3...P8..P10, etc.) and one or more structural components of DEN-1, DEN-2, DEN-3, or DEN-4. In some embodiments, an immunogenic composition can be produced, which, when introduced into a subject, generates an immune response in the subject against one or more dengue viruses. Thus, the constructs encompassed herein can be generated and passaged, and each passage provides an attenuated dengue virus for use in a pharmaceutically acceptable vaccine composition.

[0010] The embodiments disclosed herein relate to compositions, methods, and uses for inducing an immune response against all four dengue virus serotypes in children or young adults between the ages of about 1 year and about 45 years. Current formulations are ineffective in young children, and there is an unmet need for a dengue vaccine for use against dengue infection in all age groups. According to these embodiments, the compositions and methods disclosed herein are for use in children between the ages of about 1 to 20 years, about 1 to 15 years, about 1 to 11 years, or about 2 to about 9 years, or about 1 to about 5 years.

[0011] In certain embodiments, a chimeric dengue virus construct for a dengue virus serotype for use in a vaccine can comprise a passage 8 (P8) live, attenuated virus or chimeric virus having a nucleic acid sequence identified by SEQ ID NOs: 1, 3, 5, and 7 or a polypeptide sequence indicated by SEQ ID NOs: 2, 4, 6, and 8. Encompassed herein is that any passage of any of the live attenuated viruses described herein can be used in an immunogenic composition to induce an immune response against the represented dengue virus serotypes (e.g., serotypes 1-4). According to these embodiments, an immunogenic composition comprising a P-8 isolated live attenuated virus can be administered to a subject to induce an immunogenic response against one or more dengue virus serotypes according to the selected construct. In addition, the live attenuated virus can be combined with one or more of these chimeric viruses. This is considered for each live attenuated virus isolated / generated in each subsequent cell passage (e.g., African green monkey Vero cell generated, hereinafter: Vero cells or other suitable cell line). Encompassed herein is that any cell line capable of producing dengue virus (e.g., GMP approved) is used for passage of any viral construct at manufacturing scale or is considered herein for subsequent use in a vaccine or immunogenic composition against dengue virus.

[0012] In other embodiments, the compositions encompassed herein can be combined with other immunogenic compositions against other Flaviviruses, such as Zika virus, West Nile virus, Japanese encephalitis, St. Louis encephalitis virus, yellow fever virus, or any other flavivirus chimeric construct and / or live attenuated virus. In certain embodiments, a single composition can be used against multiple flaviviruses. In some embodiments, even if the vaccine composition is limited to dengue virus constructs and live attenuated dengue virus, the compositions disclosed herein are capable of inducing an immune response against Zika virus.

[0013] In certain embodiments, the immunogenic composition of the invention can comprise a chimeric dengue virus against one or more of DEN-1, DEN-2, DEN-3, and / or DEN-4, alone or in combination with a live attenuated dengue virus composition.

[0014] In other embodiments, the construct can include constructs having adaptive mutations in the structural or non-structural regions of the virus, which increase growth or production upon introduction into a subject without affecting the attenuation or safety of the virus. In certain embodiments, any of the covered chimeric dengue virus constructs can include a live-attenuated DEN-2 virus with specific mutations used as a backbone, wherein the live-attenuated DEN-2 PDK virus can further contain the structural proteins of one or more of the prM (premembrane) and E (envelope) structural proteins of other dengue virus serotypes. Additionally, the DEN-2 backbone can include additional mutations or reversion of mutations in order to enhance the immune response to a predetermined composition in a subject upon administration (e.g., chimeric dengue virus 2 / 1, 2 / 3, or 2 / 4).

[0015] In some embodiments, the structural protein genes can include the prM and E genes of DEN-1, DEN-2, DEN-3, or DEN-4 having one or two reversions on the DEN-2 backbone to improve immunogenicity. For example, in certain embodiments, the dengue constructs can include those constructs designated TDV-1-A, TDV-2-F, TDV-3-F, and TDV-4-F (see the Examples section), wherein the DEN-2 backbone has one or more reversions to wild-type DEN-2 from the live-attenuated DEN-2 virus (e.g., in the non-coding region (NCR) or non-structural region (NS1, etc.) or other mutations not found in P1 or other previously passaged viruses), and the live-attenuated DEN-2 virus has previously been shown to be safe and effective for inducing an immune response. The live-attenuated DEN-2 virus of the present application is an improved form of the originally used live-attenuated DEN-2 virus. The chimeric constructs of some embodiments disclosed herein can include a modified attenuated DEN-2 PDK-53 backbone having one or more structural proteins of a second dengue virus serotype, wherein the structural proteins can contain additional mutations to increase the immunogenic response to the chimeric construct. In some embodiments, certain mutations obtained through attenuated DEN-2 PDK-53 can be reverted to a control or another amino acid to produce a chimeric construct different from the P1 construct, which can result in increased immunogenicity, increased growth, increased plaque size without affecting vaccine safety and attenuation and can affect the growth and / or replication of the live-attenuated virus.

[0016] In other embodiments, the live attenuated DEN-2 genome can be used to generate chimeric constructs of dengue virus serotype 1 (DEN-1), dengue virus serotype 3 (DEN-3), and dengue virus serotype 4 (DEN-4), wherein one or more structural protein genes of the DEN-2 viral genome can be replaced, respectively, by one or more structural protein genes of DEN-1, DEN-3, or DEN-4. In some embodiments, the structural protein can comprise the C, prM, or E protein of a third dengue virus. In certain embodiments, the structural protein gene can comprise the prM and E genes of DEN-1, DEN-3, or DEN-4. According to these embodiments, these chimeric viruses can express the surface antigens of DEN-1, DEN-3, or DEN-4 while retaining the attenuated phenotype of the parental attenuated DEN-2. In addition, the chimeric constructs for vaccines for children and young adults disclosed herein can include chimeric constructs of DEN-4, DEN-2, DEN-1, and DEN-3 that use the attenuated DEN-2 virus as a backbone to express the DEN-1, DEN-3, and DEN-4 surface antigens.

[0017] In certain embodiments, the compositions of the invention can comprise a composition that can contain a single chimeric dengue virus construct disclosed herein and a pharmaceutically acceptable carrier or excipient. In other embodiments, the compositions disclosed herein can contain two or more, or three or more, chimeric dengue virus constructs and a pharmaceutically acceptable carrier or excipient. According to these embodiments, one or more of the dengue virus chimeric constructs encompassed herein can be combined with one or more live attenuated dengue viruses. In certain embodiments, the live attenuated virus can be a live attenuated DEN-2 virus, wherein a reversion to wild-type amino acids in the NCR, NS1 region, or other regions contributes to an increased immune response, increased virus growth, or other improvements in the improved live attenuated dengue virus constructs used in the formulations disclosed herein.

[0018] In certain embodiments, the live attenuated dengue virus can include mutations or substitutions at nucleotides 5’NCR-57-T, NS1-53-Asp, and NS3-250-Val of DENV-2, which can be shared by the common PDK-53 virus-specific genetic background of four TDV construct viruses. The genetic sequences of the three attenuated loci, as well as the previously established in vitro and in vivo attenuated phenotypes of these vaccine candidates, have been carefully monitored for cGMP-produced TDV seeds. Strategies for generating master virus seeds (MVS) for the preparation of dengue virus vaccine compositions, as well as other related passages of dengue virus, are disclosed herein. These MVS can be used to prepare clinical materials and ultimately for the preparation of commercial vaccine supplies.

[0019] In certain embodiments, the immunogenic composition can comprise a trivalent or tetravalent formulation capable of inducing an immune response against at least three or all four dengue virus serotypes in children and young adults. For example, the immunogenic composition can include pre-master virus seed, master virus seed (MVS), working virus seed (WVS), and bulk virus seed (BVS) constructs for each of the four dengue virus serotypes. In other embodiments, the immunogenic composition can comprise one or more polynucleotides having a nucleic acid sequence encoding a modified live attenuated dengue-2 virus serotype, the nucleic acid sequence represented by SEQ ID NO: 9 (pre-master), 11 (MVS), 13 (WVS), or 15 (BVS); a dengue-1 / dengue-2 chimeric polynucleotide having a nucleic acid sequence encoding non-structural proteins from a modified live attenuated dengue-2 virus serotype and structural proteins from a dengue-1 virus serotype, the nucleic acid sequence represented by SEQ ID NO: 1 (pre-master), 3 (MVS), 5 (WVS), or 7 (BVS); a dengue-3 / dengue-2 chimeric polynucleotide having a nucleic acid sequence encoding non-structural proteins from a modified live attenuated dengue-2 virus serotype and structural proteins from a dengue-3 virus serotype, the nucleic acid sequence represented by SEQ ID NO: 17 (pre-master), 19 (MVS), 21 (WVS), or 23 (BVS); a dengue-4 / dengue-2 chimeric polynucleotide having a nucleic acid sequence encoding non-structural proteins from a modified live attenuated dengue-2 virus serotype and structural proteins from a dengue-4 virus serotype, the nucleic acid sequence represented by SEQ ID NO: 25 (pre-master), 27 (MVS), 29 (WVS), or 31 (BVS). Some embodiments disclose immunogenic compositions capable of eliciting an immune response against one, two, three, or all four dengue virus serotypes in children or young adults.

[0020] In certain embodiments, the immunogenic composition can include a formulation in which the concentration ratio of the polynucleotide encoding the modified live attenuated dengue-2 virus serotype in the composition is at least 0.5 - 1 log PFU lower than the log (phage forming unit) PFU of one or more dengue virus polynucleotides. In other embodiments, the immunogenic composition can include a formulation in which the concentration ratio of the polynucleotide encoding the modified live attenuated dengue-2 virus serotype in the composition is at least 1.5 or greater than 2 or 3 log PFU lower than the log phage forming unit (PFU) of one or more polynucleotides.

[0021] In certain embodiments, an immunogenic composition against dengue, alone or in combination with other viral vaccines, can be a mixture of polypeptides and polynucleotides. According to these embodiments, the immunogenic composition can comprise the polynucleotides described herein or other flavivirus polynucleotides with or without various polypeptides encoded by the polynucleotides. For example, the polypeptides can include polypeptides corresponding to a modified live attenuated dengue-2 virus serotype represented by SEQ ID NO:10 (premajor), 12 (MVS), 14 (WVS), or 16 (BVS); polypeptides corresponding to a dengue-1 / dengue-2 chimeric polynucleotide represented by SEQ ID NO:2 (premajor), 4 (MVS), 6 (WVS), or 8 (BVS); polypeptides corresponding to a dengue-3 / dengue-2 chimeric polynucleotide represented by SEQ ID NO:18 (premajor), 20 (MVS), 22 (WVS), or 24 (BVS); and polypeptides corresponding to a dengue-4 / dengue-2 chimeric polynucleotide represented by SEQ ID NO:26 (premajor), 28 (MVS), 30 (WVS), or 32 (BVS).

[0022] In certain embodiments, the immunogenic compositions disclosed herein can include various concentration ratios of polypeptides and / or polynucleotides, including a ratio of dengue-1 / dengue-2 chimera to modified live attenuated dengue-2 virus serotype to dengue-3 / dengue-2 chimera to dengue-4 / dengue-2 chimera of 4:4:5:5 plaque forming units (PFU). In other embodiments, the ratio of dengue-1 / dengue-2 chimera to modified live attenuated dengue-2 virus serotype to the dengue-3 / dengue-2 chimera to the dengue-4 / dengue-2 chimera can be about 5:4:5:5 log PFU. Other ratios encompassed herein can include compositions in which the live attenuated dengue-2 and / or dengue-1 chimeric virus is reduced compared to at least one of the dengue-3 or dengue-4 chimeras. In other embodiments, the tetravalent formulation ratio can be about 5:3:5:5, 3:3:5:5, or any ratio that in certain instances has a lower amount of live attenuated dengue-2 or reduced presentation or both live attenuated dengue-2 and dengue-1 / dengue-2 chimera in the formulation.

[0023] In other embodiments, the compositions disclosed herein can be pharmaceutical compositions that comprise one or more of the immunogenic compositions disclosed herein and a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition can comprise an immunogenic composition that consists of a live attenuated dengue virus and / or a dengue chimera having polynucleotides and polypeptides against one or more dengue serotypes.

[0024] Other embodiments may include a kit having at least one immunogenic composition of the present disclosure and at least one container for use against dengue virus infection in children and young adults.

[0025] Certain embodiments include methods for immunizing children and young adults against at least one to all four dengue virus serotypes. The methods may include administering to a subject one or more doses of a pharmaceutical composition disclosed herein. According to these embodiments, the ratios having these characteristics may include live attenuated viruses and / or chimeras that induce an immune response against all four dengue virus serotypes in the subject.

[0026] In certain embodiments, the methods may include administering to a child or young adult an immunogenic composition that, in addition to a polynucleotide, includes one or more polypeptides encoded by the polynucleotides disclosed herein. The methods may include polypeptides that include one or more of the following: polypeptides represented by SEQ ID NO: 10 (premajor), 12 (MVS), 14 (WVS), or 16 (BVS); polypeptides represented by SEQ ID NO: 2 (premajor), 4 (MVS), 6 (WVS), or 8 (BVS); polypeptides represented by SEQ ID NO: 18 (premajor), 20 (MVS), 22 (WVS), or 24 (BVS); and polypeptides represented by SEQ ID NO: 26 (premajor), 28 (MVS), 30 (WVS), or 32 (BVS). In certain embodiments, the methods may include administering to a subject in the age range of about 1 year to about 20 years an immunogenic composition having all four dengue virus serotypes represented in the immunogenic compositions disclosed herein and eliciting an immune response against all four dengue virus serotypes in the subject (a quadrivalent formulation). In other embodiments, the methods may include using an immunogenic composition disclosed herein in a subject in the age range of about 1 year to about 45 years.

[0027] In certain embodiments, the methods may include administering to a subject one or more doses of a quadrivalent formulation of the present disclosure by any acceptable means, including, for example, subcutaneously, intravenously, intradermally, transdermally, orally, by inhalation, vaginally, topically, intranasally, or rectally. According to these embodiments, the formulations disclosed herein may be administered to a subject as a single dose or in two or more doses. In some embodiments, the immunogenic formulations disclosed herein may be administered within about 90 days of each other, within about 60 days of each other, within about 30 days of each other, and within less than about 30 days of each other. In some embodiments, the immunogenic formulations disclosed herein may be administered to a child or young adult on day 0 and again about 3 months from the first administration.

[0028] In certain embodiments, the tetravalent formulations disclosed herein can elicit an immune response against all four dengue virus serotypes in at least 60% of children and young adults who have received one or more doses of the tetravalent composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The following drawings form a part of this specification and are included to further illustrate certain embodiments. Some embodiments may be better understood by referring to one or more of these drawings, either alone or in combination with the detailed description of the specific embodiments presented.

[0031] Figure 1 Represents an exemplary figure that reflects the exemplary chimeric construct DEN-2 / DEN-4 of the invention compared to previously generated constructs and wild-type dengue virus.

[0032] Figure 2 Represents an exemplary histogram that compares various responses using a live attenuated DEN-2 backbone (with additional mutations) and a second dengue virus serotype as a structural component replacing the dengue-2 structural component (e.g., TDV-1MVS). This figure shows the plaque size of TDV MVS. Wild-type dengue virus and previously published investigational vaccine candidate viruses are included for control and comparison. The figure shows the generation of an improved dengue virus construct compared to the control dengue virus chimeric construct.

[0033] Figure 3 Represents an exemplary histogram that represents the temperature sensitivity of TDV MVS (master virus seed, denoted herein as DENVax). Wild-type dengue virus and previously published investigational vaccine candidate viruses are included for comparison with the MVS level.

[0034] Figure 4 Represents an exemplary histogram that represents the viral growth of TDV (also known as DENVax) MVS in C6 / 36 cells compared to the control. Wild-type dengue virus and investigational vaccine candidate viruses are included for comparison with TDV MVS (denoted herein as DENVax).

[0035] Figures 5A - 5C Represents an exemplary figure of neurovirulence in neonatal mice. Summary results of several experiments, summarized for the neurovirulence of the wt DENV-2 16681 virus in CDC-ICR (n = 72) and Taconic-ICR (n = 32) neonatal mice challenged with 10 4 pfu of viral ic. The neurovirulence of the wt DENV-2 16681 virus in neonatal mice challenged with 10 4 pfu (B) or 10 3Neurovirulence of TDV MVS (Master Virus Seed, herein designated DENVax) tested at pfu(C) doses in Taconic-ICR mice. Indicates the number of animals tested per group in one experiment (n = 16) or two combined experiments (n = 31 or 32).

[0036] Figure 6 Represents an exemplary histogram showing plaque sizes of TDV MVS, WVS, and BVS. Mean plaque diameter ± SD (error bars) of viral plaques in Vero or LLC-MK2 cells under agarose overlay measured at 9 days pi. Includes wild-type DENV and previously published investigational vaccine candidate viruses for control and comparison.

[0037] Figure 7 Represents an exemplary histogram showing growth of TDV (herein designated DENVax) MSV, WVS, and BVS in C6 / 36 cells at two incubation temperatures to verify retention of this in vitro attenuation marker after large-scale preparation. Includes certain wild-type dengue viruses and previously published investigational vaccine candidate viruses for comparison.

[0038] Figure 8 Represents an exemplary histogram depicting restricted growth of TDV MVS (Master Virus Seed, herein designated DENVax), WVS, and BVS in C6 / 36 cells. Mean titer ± SD (error bars) of virus replicated in C6 / 36 cells at 7 days pi. Includes certain wild-type dengue viruses and previously published investigational vaccine candidate viruses for comparison.

[0039] Figures 9A - 9B Represents an exemplary graph of neurovirulence data for TDV MVS (Master Virus Seed, herein designated DENVax) in neonatal ICR mice. (A) IC inoculation with virus at a dose of 10 4 PFU. (B) IC inoculation with virus at a dose of 10 3 PFU.

[0040] Figure 10 Represents an exemplary graph comparing the new live-attenuated dengue-2 virus with a previously generated live-attenuated dengue-2 virus.

[0041] Figure 11A Is a representative diagram of the live-attenuated dengue virus and dengue-dengue chimeric construct (designated TDV) of the present disclosure, including representative protein domains and mutation sites.

[0042] Figure 11B Is a representative formulation of a tetravalent formulation according to one embodiment of the present disclosure.

[0043] Figure 12A Is a representative graph of a dosing regimen in a subject as part of a clinical trial according to an embodiment of the present disclosure.

[0044] Figure 12B Is a representative graph of a dosing regimen in a subject as part of a clinical trial according to an embodiment of the present disclosure.

[0045] Figure 13A Is a representative line graph depicting the results of a microneutralization test (MNT) according to an embodiment of the present disclosure, wherein the neutralizing antibody response after administration of a quadrivalent formulation to seropositive subjects.

[0046] Figure 13B Is a representative line graph depicting the results of a microneutralization test (MNT) according to an embodiment of the present disclosure, wherein the neutralizing antibody response after administration of a quadrivalent formulation to dengue-naïve subjects.

[0047] Figure 14 Is a representative bar graph depicting the percentage of clinical trial subjects who are seropositive for all four dengue serotypes at various time points after administration of a quadrivalent formulation according to an embodiment of the present disclosure.

[0048] Figure 15 Is a representative bar graph depicting the percentage of clinical trial subjects who are seropositive for all four dengue serotypes at various time points after administration of a quadrivalent formulation according to an embodiment of the present disclosure.

[0049] Figure 16 Is a representative bar graph depicting the percentage of seropositive clinical trial subjects who were naïve before receiving a pharmaceutical composition for all four dengue serotypes at various time points after administration of a quadrivalent formulation according to an embodiment of the present disclosure.

[0050] Figure 17 Is a representative bar graph depicting the percentage of seropositive clinical trial subjects (children and adults) for multiple serotypes after receiving a pharmaceutical composition for all four dengue serotypes according to an embodiment of the present disclosure.

[0051] Definitions

[0052] As used herein, "a" or "an" can mean one or more than one item.

[0053] As used herein in the specification, "subject" can include, but is not limited to, mammals such as humans (e.g., children to adults) or mammalian species (domesticated or wild), such as dogs, cats, other household pets (e.g., hamsters, guinea pigs, mice, rats), ferrets, rabbits, pigs, horses, cows, prairie dogs, wild rodents, or zoo animals.

[0054] As used herein, the terms "viral chimera", "chimeric virus", "flavivirus chimera", and "chimeric flavivirus" can refer to constructs that contain a portion of the nucleotide sequence of dengue-2 virus and additional nucleotide sequences that do not originate from dengue-2 virus or that originate from a different flavivirus. A "dengue chimera" contains at least two different dengue virus serotypes, but does not include different flaviviruses. Thus, examples of other dengue viruses or flaviviruses can include, but are not limited to, sequences from dengue-1 virus, dengue-3 virus, dengue-4 virus, Zika virus, West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, tick-borne encephalitis virus, yellow fever virus, and any combination thereof.

[0055] As used herein, "nucleic acid chimera" can refer to constructs of the present disclosure that contain nucleic acids having a portion of the nucleotide sequence of a dengue virus (e.g., dengue-2 virus) and additional nucleotide sequences that are not of the same origin as this dengue virus. According to these embodiments, any chimeric dengue or flavivirus chimera disclosed herein can be considered an example of a nucleic acid chimera.

[0056] As used herein, "live attenuated virus" can refer to a wild-type virus that has been mutated or selected for traits useful in a vaccine or other immunogenic composition, where some of these traits can include reduced virulence, safety, efficacy, or improved growth, among others.

[0057] As used herein, "dengue virus formulation" or "immunogenic composition" or "vaccine formulation" or a pharmaceutical formulation thereof can include various combinations of polynucleotides and / or polypeptides disclosed herein that can be administered to a subject and that can elicit an immune response in the subject against one or more dengue virus serotypes.

[0058] Description

[0059] In the following sections, various exemplary compositions and methods are described to elaborate on various embodiments. It will be apparent to those skilled in the art that not all or even some of the specific details outlined herein are required to practice the various embodiments, and that concentrations, times, and other specific details can be modified by routine experimentation. In some cases, well-known methods or components have not been included in the specification.

[0060] In accordance with embodiments of the present disclosure, conventional molecular biology, protein chemistry, microbiology, and recombinant DNA techniques within the skill of the art can be used. These techniques are explained fully in the literature. See, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Edition 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Animal Cell Culture, R.I. Freshney, ed., 1986).

[0061] Embodiments disclosed herein relate to compositions, methods, and uses for inducing an immune response to one or up to all four dengue virus serotypes in a subject, such as a child or young person from about 1 year of age to about 20 years of age. Other embodiments relate to compositions, methods, and uses for inducing an immune response to one or more dengue virus serotypes in a subject, either alone or simultaneously. In accordance with these embodiments, attenuated dengue viruses and nucleic acid chimeras can be generated and used in the vaccine compositions disclosed herein. Some embodiments relate to modified or mutated dengue constructs or chimeras. Other embodiments relate to introducing mutations to modify the amino acid sequence of the structural proteins of the dengue virus, wherein the mutations increase the immunogenicity of the virus.

[0062] Live attenuated dengue viruses of all four serotypes have been developed by passage of wild-type virus in cell culture. These are some of the most promising live attenuated vaccine candidates for immunization against flaviviruses, particularly dengue virus infection and / or disease. These vaccine candidates have been named by a combination of their dengue serotype, the cell line in which they were passaged, and the number of times they were passaged. Thus, the dengue serotype 1 wild-type virus passaged 13 times in PDK cells is named the DEN-1PDK-13 virus. Other candidate vaccines are DEN-2PDK-53, DEN-3PGMK-30 / FRhL-3 (e.g., passaged 30 times in primary green monkey kidney cells and then 3 times in fetal rhesus lung cells) and DEN-4PDK-48. These four candidate vaccine viruses were obtained by tissue culture passage of the wild-type parents DEN-1 16007, DEN-2 16681, DEN-3 16562, and DEN-4 1036 viruses, respectively.

[0063] The DEN-2PDK-53 virus vaccine candidate (hereinafter PDK-53) has several measurable biological markers associated with attenuation, including temperature sensitivity, small plaque size, reduced replication in mosquito C6136 cell cultures, reduced replication in intact mosquitoes, loss of neurovirulence in suckling mice, and reduced incidence of viremia in monkeys. Clinical trials of the candidate PDK-53 vaccine have demonstrated its safety and immunogenicity in humans. In addition, the PDK-53 vaccine induces dengue virus-specific T cell memory responses in human vaccinees. Some embodiments herein describe improved DEN-2PDK-53 for use in the chimeric constructs disclosed herein for inducing an immune response against dengue virus in children and young adults.

[0064] Immunogenic flavivirus chimeras having a dengue-2 virus backbone and at least one structural protein of another dengue virus serotype can be used to prepare dengue virus chimeras, and methods of producing the dengue virus chimeras are described. The immunogenic dengue virus chimeras are provided alone or in combination in an immunogenic composition in a pharmaceutically acceptable carrier to minimize, inhibit infection by, or immunize an individual against one or more serotypes alone or in combination, such as dengue virus serotypes DEN-1, DEN-2, DEN-3, and DEN-4. When combined, the immunogenic dengue virus chimeras can be used as multivalent vaccines (e.g., bivalent, trivalent, and tetravalent) to confer protection against infection by more than one flavivirus species or strain simultaneously. In certain embodiments, the dengue virus chimeras can be combined in an immunogenic composition that serves as a bivalent, trivalent, or tetravalent vaccine against known dengue virus serotypes and that can confer immunity against other pathogenic flaviviruses by including nucleic acids encoding one or more proteins from different flaviviruses.

[0065] In some embodiments, the avirulent, immunogenic dengue virus chimeras provided herein contain the nonstructural protein genes of an attenuated dengue-2 virus (e.g., PDK-53) or an equivalent thereof and one or more of the structural protein genes or immunogenic portions of a flavivirus against which immunogenicity is to be induced in a subject. For example, some embodiments relate to chimeras having the genome of the attenuated dengue-2 virus PDK-53 as a viral backbone and replacing one or more of the structural protein genes or combinations thereof encoding the capsid, pre-membrane / membrane, or envelope of the PDK-53 genome with the corresponding structural protein genes from DEN-1, DEN-3, or DEN-4 or other flaviviruses against which protection is to be provided, such as different flaviviruses or different dengue virus serotypes. According to these embodiments, the nucleic acid chimeras disclosed herein can have the functional properties of an attenuated dengue-2 virus and be avirulent, but express antigenic epitopes of the structural gene products of DEN-1, DEN-3, or DEN-4 and be immunogenic in the context of other flaviviruses (e.g., induce an immune response to the gene products in a subject). These constructs are then passaged 1 or more times beyond passage 53 to produce live attenuated virus compositions that are used to generate immunogenic compositions against one or more dengue virus serotypes (e.g., P1-P10).

[0066] In another embodiment, the nucleic acid chimera can be a nucleic acid chimera having, but not limited to, a first nucleotide sequence encoding a nonstructural protein from an attenuated dengue-2 virus and a second nucleotide sequence encoding a structural protein from a dengue-4 virus, alone or in combination with another flavivirus. In other embodiments, the attenuated dengue-2 virus can be the vaccine strain PDK-53, which has one or more revertant amino acids to wild-type amino acids (see Examples) that are selected for improved physical properties or increased immunogenicity. These specific revertants confer the desired properties for use as a live attenuated dengue-2 or as a chimeric construct as described herein in a regimen against dengue virus infection in children and young adults. Some embodiments include one or more of the structural proteins of the C, prM, or E protein of a second dengue virus.

[0067] In other embodiments disclosed herein, the nucleotide and amino acid sequences can include, for example, substitutions, deletions, or insertions in the PDK-53 dengue-2 genome to reduce interference with other targeted dengue virus serotypes. These modifications can be made in the structural and nonstructural proteins alone or in combination with the exemplary modifications disclosed herein and can be generated by passaging the attenuated virus and obtaining improved compositions for inducing an immune response against one or more dengue virus serotypes.

[0068] Certain embodiments disclosed herein provide methods for preparing chimeric flaviviruses using recombinant techniques, e.g., by inserting replacement sequences of one serotype into the backbone of another serotype. Other embodiments herein relate to passaging a confirmed (e.g., safe and effective) live attenuated chimeric virus to effect additional improvements and selecting for desired traits. In certain embodiments, the live attenuated dengue-2 used herein can include one or more of the mutations shown in Table 3. In other embodiments, the dengue-dengue chimeras of the present application can include one or more of the mutations shown in Table 3. In other embodiments, the dengue-dengue chimeras can include all of the mutations for each chimera, as shown in Table 3 for Den-2 / Den-1, Den-2 / Den-3, or Den-2 / Den-4. Pharmaceutical compositions comprising the live attenuated viruses represented by the constructs of Table 3 are encompassed. For example, monovalent, bivalent, trivalent, or tetravalent compositions are encompassed that use the dengue-dengue chimeras and the live attenuated dengue-2 virus as shown in Table 3 herein.

[0069] In certain embodiments, the live attenuated DEN-2 variants encompassed herein can be formulated into a pharmaceutical composition, wherein the pharmaceutical composition can be administered alone or in combination with a dengue-dengue chimera or a dengue-flavivirus chimera. In certain embodiments, the bivalent, trivalent, or tetravalent compositions can be administered to a subject in a single application or in multiple applications.

[0070] Flavivirus chimera

[0071] Dengue viruses types 1-4 (DEN-1 to DEN-4) are mosquito-borne flavivirus pathogens. The flavivirus genome contains a 5'-noncoding region (5'-NC), a capsid protein (C) coding region, a pre-membrane / membrane protein (prM) coding region, an envelope protein (E) coding region, a region encoding nonstructural proteins (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5), and a 3' noncoding region (3'NC). Flavivirus structural proteins include C, prM, and E. Nonstructural proteins include NS1-NS5. The structural and nonstructural proteins are translated as a single polyprotein and processed by cellular and viral proteases.

[0072] A flavivirus chimera can be a construct formed by fusing a non-structural protein gene of one type or serotype of a virus species from dengue virus or a virus of the Flaviviridae family with a protein gene (e.g., a structural protein gene) of a different type or serotype from dengue virus or another flavivirus. In other embodiments, a flavivirus chimera construct can be formed by fusing a non-structural protein gene of one type or serotype of a virus species from dengue virus or the Flaviviridae family with other nucleotide sequences that direct the synthesis of a polypeptide or protein selected from other dengue virus serotypes or other flaviviruses. In other embodiments, sequence replacements from one flavivirus to another are encompassed.

[0073] In other embodiments, a dengue chimera can contain a non-structural protein gene of a live attenuated dengue virus or its equivalent and one or more structural protein genes or antigenic portions thereof of a dengue virus or other flavivirus against which immunogenicity is conferred.

[0074] Other suitable dengue viruses for constructing dengue chimeras can be wild-type, virulent DEN-1 16007, DEN-2 16681, DEN-3 16562, and DEN-4 1036, as well as the attenuated vaccine strains DEN-1 PDK-13, DEN-2 PDK-53, DEN-3 PMK-30 / FRhL-3, and DEN-4 PDK-48. Genetic differences between wild-type / attenuated virus pairs of DEN-1, DEN-2, DEN-3, and DEN-4 are encompassed, as well as changes in the amino acid sequences encoded by the viral genomes.

[0075] In some embodiments, the dengue-2 virus used herein can include the DEN-2 PDK-53-V variant, in which genomic nucleotide position 5270 is mutated from A to T, and amino acid position 1725 of the polyprotein or amino acid position 250 of the NS3 protein contains a valine residue of the dengue-2 full-length sequence. A DEN-2 PDK-53 variant without this nucleotide mutation, DEN-2 PDK-53-E, differs from PDK-53-V at this position. DEN-2 PDK-53-E has an A at nucleotide position 5270 and a glutamate at polypeptide position 1725, amino acid position 250 of the NS3 protein. In some embodiments, the modified PDK 53 dengue-2 can contain one or more reversions to these positions of the native sequence to obtain more desirable traits for use in the vaccine compositions encompassed herein.

[0076] In certain embodiments, a dengue-dengue chimeric virus can comprise a DEN-2 virus-specific infectious clone having a modified backbone and structural genes (prM-E or C-prM-E) inserted from other dengue viruses or other flaviviruses. In some embodiments, dengue-2 backbone variants can be generated from the dengue-2 16681 strain (P), PDK-53-E (E), or PDK-53-V (V); the last letter indicates the C-prM-E structural gene from the parental (P) strain or its vaccine derivative (V) or the prM-E structural gene from the parental (P) or its vaccine derivative (V1). For example; DEN-2 / 1-VP denotes a chimeric virus that comprises an attenuated DEN-2 PDK-53V backbone containing valine at NS3-250 and the C-prM-E gene from wild-type DEN-1 16007; DEN-2 / 1-VV denotes the DEN-2 PDK-53V backbone with dengue-1, the DEN-1 PDK-13 vaccine strain; DEN-2 / 1-VP1 denotes the DEN-2 PDK-53V backbone and the prM-E gene from wild-type DEN-1 16007; DEN-2 / 3-VP1 denotes the DEN-2 PDK-53V backbone and the prM-E gene from wild-type DEN-3 16562; DEN-2 / 4-VP1 denotes the DEN-2 PDK-53V backbone and the prM-E gene from wild-type DEN-4 1036. Other chimeric viruses disclosed herein are designated using a similar nomenclature.

[0077] In some embodiments, the dengue-dengue chimeric viruses disclosed herein can contain the attenuated dengue-2 virus PDK-53 genome as a backbone, wherein the structural protein genes encoding the C, prM, and E proteins of the PDK-53 genome or combinations thereof can be replaced with the corresponding structural protein genes from dengue-1, dengue-3, or dengue-4 viruses and optionally another flavivirus against which protection is desired, such as a different flavivirus (e.g., Zika virus or yellow fever or combinations thereof) or a different dengue virus strain. The live attenuated dengue-2 PDK-53 virus strain has a mixed genotype at nucleotide position 5270. A substantial portion (about 29%) of the viral population encodes the unmutated NS3-250-Glu present in wild-type DEN-2 16681 rather than the NS3-250-Val mutation. Since both of these genetic variants are avirulent, this mutation may not be required in the avirulent chimeric viruses.

[0078] In certain embodiments, single mutations at NS1-53, double mutations at NS1-53 and 5’NC-57, double mutations at NS1-53 and NS3-250, and triple mutations at NS1-53, 5’NC-57, and NS3-250 result in attenuation of the DEN-2 virus. Thus, the genome of any dengue-2 virus containing such non-conservative amino acid substitutions or nucleotide substitutions at these loci can be used as a base sequence for deriving the modified PDK-53 virus disclosed herein. Optionally, another mutation in the stem of the stem / loop structure in the 5’ non-coding region can provide additional stability of the avirulent phenotype. Mutations in this region disrupt potential secondary structures important for viral replication.

[0079] The mutations disclosed herein can be achieved by any method known in the art, including but not limited to selected clones with additional characteristics after passage in a cell line of interest (e.g., Vero cells). Those skilled in the art will understand that virulence screening assays, as described herein and well known in the art, can be used to distinguish virulent and avirulent backbone constructs.

[0080] Construction of flavivirus chimeras

[0081] The flavivirus chimeras disclosed herein can be produced by using recombinant engineering to remove the corresponding PDK-53 gene and replace it with a dengue-1, dengue-3, or dengue-4 virus gene or other genes known in the art, by splicing one or more of the structural protein genes of a flavivirus against which the desired immunity is directed into the PDK-53 dengue-2 virus genome backbone, or by other methods known in the art.

[0082] Alternatively, using the sequences provided in the sequence listing, nucleic acid molecules encoding flavivirus proteins can be synthesized using known nucleic acid synthesis techniques and inserted into a suitable vector. The live attenuated viruses disclosed herein can be produced using recombinant engineering techniques known to those skilled in the art.

[0083] Suitable chimeric viruses or nucleic acid chimeras containing nucleotide sequences encoding structural proteins of other flaviviruses or dengue virus serotypes can be evaluated for their utility as vaccines by screening them for the aforementioned attenuated phenotype markers indicative of avirulence and by screening them for immunogenicity. Conventional screening methods known to those skilled in the art can be used to evaluate antigenicity and immunogenicity using in vitro or in vivo reactivity with flavivirus antibodies or immune reactive sera.

[0084] Dengue virus vaccines

[0085] In certain embodiments, chimeric viruses and nucleic acid chimeras can provide live attenuated viruses that can be used as immunogens or vaccines. Some embodiments include chimeras that exhibit high immunogenicity against dengue-4 virus without producing dangerous pathogenic or lethal effects.

[0086] To reduce the occurrence of DHF / DSS in children and young adults vaccinated against only one dengue virus serotype, a tetravalent vaccine is needed to provide simultaneous immunity against all four virus serotypes. A tetravalent vaccine can be produced by combining the live attenuated dengue-2 virus of the present application with the dengue-2 / 1, dengue-2 / 3, and dengue-2 / 4 chimeras or other dengue virus constructs described herein in a suitable pharmaceutical carrier for administration as a multivalent vaccine.

[0087] In certain embodiments, the chimeric viruses or nucleic acid chimeras of the present disclosure can include the structural genes of wild-type or live attenuated viruses on an attenuated DEN-2 virus backbone. For example, a dengue-2 / dengue-4 chimera can express the structural protein genes of wild-type DEN-41036 virus.

[0088] In certain methods, the viruses used in the chimeras described herein can be cultured using techniques known in the art. Then, viral plaque titrations can be performed and the plaques counted to assess the viability and phenotypic characteristics of the growth culture. Wild-type viruses can be passaged through cultured cell lines to obtain attenuated candidate starting materials.

[0089] In certain embodiments, chimeric clones can be constructed from various dengue serotype clones available to those skilled in the art. Cloning of virus-specific cDNA fragments can also be achieved. cDNA fragments containing structural or nonstructural protein genes can be amplified from dengue virus RNA using various primers by reverse transcriptase-polymerase chain reaction (RT-PCR). The amplified fragments can be cloned into the cleavage sites of other intermediate clones. The intermediate chimeric dengue virus clones can then be sequenced to verify the accuracy of the inserted dengue virus-specific cDNA.

[0090] Full-genome length chimeric plasmids constructed by inserting the structural protein and / or nonstructural protein gene regions of dengue serotype viruses into a vector can be obtained using recombinant techniques well known to those skilled in the art.

[0091] Nucleotide and Amino Acid Analysis

[0092] The NS1-53 mutation in the DEN-2PDK-53 vaccine virus is significant for the attenuated phenotype of this virus because the NS1-53-Gly of the DEN-2 16681 virus is conserved in almost all flaviviruses sequenced to date, including tick-borne viruses. The DEN-4 vaccine virus can also contain an amino acid mutation in the NS1 protein at position 253. This locus (which is a Gln to His mutation in the DEN-4PDK-48 vaccine virus) is Gln in all four dengue virus wild serotypes. This Gln residue is unique to dengue viruses within the genus Flavivirus. The NS1 protein is a glycoprotein secreted from flavivirus-infected cells. It is present on the surface of infected cells, and NS1-specific antibodies are present in the sera of virus-infected individuals. Protection has been reported in animals immunized with the NS1 protein or passively immunized with NS1-specific antibodies. The NS1 protein appears to be involved in early viral RNA replication.

[0093] In certain embodiments, the mutations occurring in the NS2A, NS2B, NS4A, and NS4B proteins of the DEN-1, -2, -3, and -4 attenuated strains are essentially conserved. The NS4A-75 and NS4A-95 mutations of the DEN-2 and DEN-4 vaccine viruses respectively occur at amino acid conserved sites among dengue viruses but generally not among flaviviruses.

[0094] A nucleic acid sequence encoding a DEN-4, DEN-3, or DEN-1 virus (e.g., a structural element) can be inserted into a vector (e.g., a plasmid) and recombinantly expressed in a living organism (e.g., into a dengue-2 backbone) to produce recombinant dengue virus peptides and / or polypeptides and / or viruses.

[0095] Nucleic acid detection methods

[0096] The present disclosure provides rapid genetic tests diagnostic for each of the vaccine viruses described herein. This embodiment of the present disclosure enhances the analysis of viruses isolated from the sera of vaccinated humans who develop viremia, as well as the characterization of viremia in non-human primates immunized with candidate vaccine viruses.

[0097] These sequences can include diagnostic TaqMan probes that are used to report the detection of cDNA amplicons amplified from a viral genomic RNA template by using reverse transcriptase / polymerase chain reaction (RT / PCR) and forward and reverse amplification primers designed to amplify the cDNA amplicons, as described below. In some cases, one of the amplification products has been designed to contain a vaccine virus-specific mutation at the 3' end terminus of the amplicon, which effectively makes the test more specific for the vaccine strain because primer extension and thus amplification at the target site only occur if the viral RNA template contains the specific mutation.

[0098] A PCR-based automated nucleic acid sequence detection system, or other known techniques for nucleic acid detection, can be used. The TaqMan assay is a highly specific and sensitive assay that allows for the automated real-time visualization and quantification of amplicons generated by PCR from a sample nucleic acid template. TaqMan can assay for the presence or absence of a specific sequence. In this assay, forward and reverse primers are designed to anneal upstream and downstream, respectively, of the target mutation site. A specific detector probe constitutes the third primer component of the assay, and the specific detector probe is designed to have a melting temperature approximately 10 °C higher than either of the amplification primers and to contain a vaccine virus-specific nucleotide mutation or its complement (depending on the strand of the RT / PCR amplicon being detected).

[0099] Probes designed to specifically detect a mutant locus in one of the vaccine virus genomes will contain a vaccine-specific nucleotide in the middle of the probe. If the viral RNA template is vaccine virus-specific, this probe will generate detectable fluorescence in the TaqMan assay. However, genomic RNA templates from wild-type DEN virus have reduced probe hybridization efficiency due to a single nucleotide mismatch (in the case of the parental virus DEN virus) or potentially more than one mismatch (as can occur in other wild-type DEN viruses) and will not result in appreciable fluorescence. DNA polymerase is more likely to displace a mismatched probe from the RT / PCR amplicon template than to cleave the mismatched probe to release the reporter dye (TaqMan allelic discrimination assay, Applied Biosystems).

[0100] One strategy for diagnostic genetic testing utilizes molecular beacons. The molecular beacon strategy also utilizes primers for RT / PCR amplification of the amplicon and detects a specific sequence within the amplicon by a probe that contains a reporter and a quencher dye at the ends of the probe. In this assay, the probe forms a stem-loop structure. The molecular beacon assay employs a quencher and a reporter dye that are different from those used in the TaqMan assay.

[0101] Pharmaceutical composition

[0102] Embodiments herein provide for the administration of a composition to a subject in a biocompatible form suitable for in vivo drug administration. "Biocompatible form suitable for in vivo administration" refers to the form of the active agent to be administered (e.g., the agent of the embodiments), wherein the therapeutic effect of the active agent exceeds any toxic effects. Administration of a therapeutically effective amount of a therapeutic composition is defined as an amount effective over the dosage and period of time necessary to achieve the desired result. For example, the therapeutically active amount of a compound can vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the antibody to elicit the desired response in the individual. The dosage regimen can be adjusted to provide the optimal therapeutic response.

[0103] In one embodiment, a compound (e.g., the live attenuated virus of the embodiment) can be administered in a convenient manner, e.g., subcutaneously, intravenously, by oral administration, inhalation, intradermally, transdermal application, intravaginal application, topical application, intranasally or rectally. Depending on the route of administration, the active compound can be contained in a protective buffer (e.g., albumin and trehalose, poloxamer 407 / trehalose / albumin (FTA)). In one embodiment, the composition can be administered orally. In another embodiment, the composition can be administered intravenously. In one embodiment, the composition can be administered intranasally, e.g., by inhalation. In another embodiment, the composition can be administered intradermally using a needleless system (e.g., ) or other intradermal administration system.

[0104] In certain embodiments, the composition can be administered to children or young adults in a suitable carrier or diluent. In certain embodiments, the live attenuated dengue virus vaccine can be administered to children or young adults in a stable formulation (e.g., albumin and trehalose; FTA, or other formulations for stabilizing live attenuated viruses). As used herein, "pharmaceutically acceptable carrier" can also include diluents, such as saline and aqueous buffer solutions. It may be necessary to combine the live attenuated virus formulation with materials that prevent its inactivation or co-administer the compound with materials that prevent its inactivation. In certain embodiments, one or more formulations of the live attenuated dengue virus can be administered subcutaneously or intradermally to children or young adults as an initial dose, followed by a booster. Dispersants can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils. Under ordinary storage and use conditions, these formulations can contain preservatives to prevent the growth of microorganisms or other stabilizing formulations (e.g., FTA or other stabilizing formulations for stabilizing live attenuated flaviviruses or their chimeras).

[0105] Pharmaceutical compositions suitable for injectable use can be administered by means known in the art. For example, sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions can be used. In some embodiments, the composition can be sterile and can be fluid to facilitate injectability. The pharmaceutically acceptable carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycols, etc.) and suitable mixtures thereof. For example, the required particle size can be maintained in the case of dispersions by using coating materials such as lecithin and the appropriate fluidity can be maintained by using surfactants. The prevention of microorganisms can be achieved by adding various antibacterial or antifungal agents or other agents.

[0106] In some embodiments, upon formulation, the solution can be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. According to these embodiments, the formulation can be readily administered in a variety of dosage forms, such as the types of injectable solutions described above. In certain instances, the live attenuated dengue virus and / or chimeras can be delivered in a formulation having a specific ratio, such as 5:4:5:5, 4:4:5:5, or other ratios (e.g., PFU of a given dengue virus serotype), wherein the dengue-3 and / or dengue-4 constructs in the trivalent or quadrivalent formulation are at least 0.5 log greater in terms of pfu than the dengue-1 and / or dengue-2 represented in the formulation. In certain embodiments, the pfu concentration of the dengue-3 and dengue-4 live attenuants or chimeras is at least 1 log greater than the concentration of dengue-1 and / or dengue-2 in the quadrivalent formulation. In some embodiments, the DEN2 / 4 chimera can be present at a higher concentration than other dengue virus serotypes, such as live attenuated dengue-1 and / or live attenuated dengue-2.

[0107] In certain embodiments, a single dose or multiple doses of the dengue formulation can also be administered to children or young adults from about 1 year to about 20 years of age. In some embodiments, children or young adults can be treated with a single dose formulation. In other embodiments, children or young adults can be treated with at least two doses of the live attenuated dengue virus formulation. In certain embodiments, children or young adults can be administered a composition of the dengue-dengue formulation on day 0 and a booster dose within about 3 months of the first dose. In certain embodiments, the children and young adults are naive subjects (seronegative) who have never been exposed to the dengue virus. In other embodiments, the children and young adults can have been previously exposed to the dengue virus and / or dengue virus infection (seropositive). According to these embodiments, seronegative children and / or seronegative young adults can be treated on day 0 and then receive a booster dose within 6 months, 5 months, 4 months, 3 months, or less of the first dose in order to generate an enhanced immune response to the dengue virus. In certain embodiments, the children can be children from about 2 years to about 17 years of age. In other embodiments, the children are 2 to 17 years of age.

[0108] In another embodiment, a nasal solution or spray, aerosol, or inhalant can be used to deliver the live attenuated dengue virus formulation to a subject. Certain formulations can contain excipients such as, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.

[0109] A pharmaceutical composition can be prepared with a carrier that protects the active ingredient from rapid elimination from the body, such as a timed-release formulation or a coating material. Such carriers can include controlled-release formulations, such as, but not limited to, microencapsulated delivery systems and biodegradable biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and other known substances.

[0110] In certain embodiments, the dose range of the live attenuated dengue-dengue chimeras, live attenuated dengue viruses, or flavivirus chimeras can be from about 10 2 to about 10 6 PFU initially administered, and optionally, then at least a second administration as needed within 30 days or up to 12 months thereafter. In certain embodiments, a subject can receive a dual administration of a monovalent, bivalent, trivalent, or tetravalent composition as disclosed herein, where the compositions are a single composition mixture or a pre-determined composition with different dengue virus serotypes.

[0111] It is evident that for any particular child or young person or any particular aged child, a specific dosage regimen can be adjusted according to individual needs. In some embodiments, the dosage regimen is adjusted to accommodate adolescent subjects, such as children up to about 20 years of age, or up to about 18 years of age, or up to about 15 years of age, or up to about 12 years of age, or up to about 9 years of age, or up to about 6 years of age, or up to about 3 years of age, or up to about 1.5 years of age. In certain embodiments, the child can be between 2 and 17 years of age. In other embodiments, the child can be between 4 and 16 years of age.

[0112] In certain embodiments, the immunogenic compositions disclosed herein can be administered to children or young adults in one or more doses. In some embodiments, the immunogenic compositions disclosed herein can be administered as a single dose or two or more doses within a predetermined time period, including but not limited to within about 6 months, within about 120 days, within about 90 days, within about 80 days, within about 70 days, within about 60 days, within about 50 days, within about 40 days, within about 30 days, within about 20 days, within about 10 days, within about 5 days or less, or on the same day within a few hours or minutes to a subject or simultaneously at the same or different anatomical locations. In some embodiments, the immunogenic compositions disclosed herein can be administered within about 90 days of each other, within about 60 days of each other, within about 30 days of each other, and within less than about 30 days of each other. In some embodiments, the compositions disclosed herein can be administered subcutaneously or intradermally to a subject. Administration at two or more anatomical sites can include any combination of administrations, including by the same mode at two or more anatomical sites or by two different modes including two separate anatomical sites. According to these embodiments, two or more anatomical sites can include different limbs or different regions of the body. In certain embodiments, two doses of the vaccine composition can be administered sequentially to a subject at the same or multiple anatomical locations on day 0, for example to provide protection against all dengue serotypes (e.g., cross-protection). In certain embodiments, the immunogenic compositions disclosed herein can include but are not limited to a single dose that elicits an immunogenic response against all four serotypes (e.g., a tetravalent formulation) in a subject, which is capable of inducing an immune response against all dengue virus serotypes. In certain embodiments, a subject can receive a single dose of the composition, wherein the single dose is capable of inducing a sufficient immune response against all four dengue virus serotypes. In other embodiments, the immunogenic composition can include a combination of a live attenuated dengue virus serotype and other immunogenic reagents against other flaviviruses (e.g., Zika virus, Japanese encephalitis, West Nile virus, St. Louis encephalitis virus, yellow fever or other viruses). In certain embodiments, the vaccines against dengue virus disclosed herein can be used to reduce other related viral infections, such as Zika virus infection.

[0113] In some embodiments, the pharmaceutical compositions disclosed herein can be used to increase the immune response to a target formulation in children or young adults by combining an immunogenic composition against dengue virus with a reagent that enhances CD8 + T cell responses or other immune responses against one or more dengue virus serotypes (e.g., one or more structural or non-structural components of the dengue virus) in children or young adults who receive such pharmaceutical compositions against dengue virus.

[0114] Local administration is accomplished by topically applying creams, gels, rinses, etc. containing a therapeutically effective amount of a serine protease inhibitor. Transdermal administration is accomplished by applying creams, rinses, gels, etc. that enable the serine protease inhibitor to penetrate the skin and enter the bloodstream. Additionally, an osmotic pump can be used for administration. The necessary dosage will vary with the specific condition being treated, the method of administration, and the rate of clearance of the molecule from the body.

[0115] Other embodiments relate to methods of reducing inactivation of live attenuated viruses, including but not limited to combining one or more live attenuated viruses with a composition capable of reducing inactivation of the live attenuated virus (such as a flavivirus). These compositions can include but are not limited to one or more protein reagents; one or more sugar or polyol reagents; and optionally one or more EO-PO block copolymers, wherein the composition can reduce inactivation or stabilize the live attenuated virus.

[0116] In certain embodiments, the compositions covered herein can be partially or completely dehydrated or hydrated. In other embodiments, the stabilizing protein reagents for the pharmaceutical or non-pharmaceutical compositions used herein can include but are not limited to whey protein, human serum albumin, recombinant human serum albumin (rHSA), bovine serum albumin (BSA), other serum albumins or albumin gene family members. The sugar or polyol reagents can include but are not limited to monosaccharides, disaccharides, sugar alcohols, trehalose, sucrose, maltose, isomaltose, cellobiose, gentiobiose, laminaribose, xylobiose, mannotriose, lactose, fructose, sorbitol, mannitol, lactitol, xylitol, erythritol, raffinose, amylase, cyclodextrin, chitosan or cellulose. In certain embodiments, the surfactants can include but are not limited to nonionic surfactants, such as alkyl poly(ethylene oxide), copolymers of poly(ethylene oxide) and poly(propylene oxide) (EO-PO block copolymers), poly(vinylpyrrolidone), alkyl polyglucosides (such as sucrose monostearate, lauryl diglucoside or sorbitan monolaurate, octyl glucoside and decyl maltoside), fatty alcohols (cetyl alcohol or oleyl alcohol) or cocamides (cocamide MEA, cocamide DEA and cocamide TEA).

[0117] In other embodiments, the surfactants can include but are not limited to poloxamer 407 (such as Pluronic )), a replacement for poloxamer 407 or poloxamer 335, 338 or 238 other than poloxamer 407, or other EO-PO block copolymers with similar properties.

[0118] In some embodiments, the vaccine composition can include, but is not limited to, one or more protein reagents, which are serum albumin; one or more trehalose saccharides; and one or more surfactant polymer reagents, such as EO-PO block copolymers, poloxamer 407 or more specifically, Pluronic

[0119] In other embodiments, the formulation for stabilizing live virus can contain one or more live flaviviruses, one or more carbohydrate reagents, and one or more amino acids or their salt, ester, or amide derivatives. In other embodiments, the formulation used herein stabilizes live attenuated flaviviruses for commercial use. In some embodiments, the composition further contains a buffer. According to these embodiments, the buffer can include, but is not limited to, phosphate buffered saline (PBS). According to these embodiments, the buffer can include at least one of sodium chloride (NaCl), monosodium phosphate, and / or disodium phosphate (Na2HPO4), potassium chloride (KCl), and potassium phosphate (KH2PO4). In some embodiments, the buffer of the composition can contain sodium chloride at a concentration of 25 mM to 200 mM. In other embodiments, the compositions disclosed herein can contain urea and / or other suitable reagents such as MSG.

[0120] In some embodiments, live attenuated flaviviruses such as dengue virus can be stabilized in a formulation comprising, but not limited to, recombinant HSA at a concentration of 0.1% to 0.2% (w / v); and / or sucrose at a concentration of about 4.0% to about 6.0% (w / v); and / or mannitol at a concentration of about 2% to 4% (w / v); and / or alanine at a concentration of about 8.0 mM to about 22.0 mM; and / or methionine at a concentration of about 1.0 mM to about 5.0 mM; and / or MSG at a concentration of about 8.0 mM to 12.0 mM; and / or urea at a concentration of about 0.1% to about 0.3% (w / v). In certain embodiments, the composition can comprise recombinant HSA, trehalose, mannitol, alanine, methionine, MSG, and urea. In other embodiments, the stabilized composition can comprise HSA at a concentration of about 0.1% to about 0.2% (w / v); trehalose at a concentration of about 4% to about 6% (w / v); mannitol at a concentration of about 2% to about 4% (w / v); wherein the alanine concentration is 8 mM to 22 mM; wherein the methionine concentration is 1 mM to 5 mM; wherein the MSG concentration is 8 mM to 12 mM; wherein the urea concentration is 0.1% to 0.3% (w / v). Certain formulations for stabilizing live attenuated viruses can comprise, but not limited to, recombinant HSA, sucrose, alanine, and urea. According to these embodiments, the HSA concentration can be about 0.1% to about 0.2% (w / v); the sucrose concentration can be about 4% to about 6% (w / v); the alanine concentration can be about 8.0 mM to about 22 mM; and the urea concentration can be about 0.1% to about 0.3% (w / v). Other stabilized formulations can include recombinant HSA, sucrose, methionine, and urea. The recombinant HSA concentration can be about 0.1% to 0.2% (w / v); the sucrose concentration can be about 4.0% to about 6.0% (w / v); the methionine concentration can be about 1.0 mM to about 5.0 mM; the urea concentration can be about 0.1% to about 0.3% (w / v). In other embodiments, the stabilized formulation can comprise recombinant HSA, sucrose, arginine, and urea, wherein the recombinant HSA concentration can be 0.1% to 0.2% (w / v); the sucrose concentration can be 4% to 6% (w / v); the arginine concentration can be 10 mM to 50 mM; the urea concentration can be 0.1% to 0.3% (w / v). Other stabilized formulations can include recombinant HSA, trehalose, arginine, and urea, wherein the recombinant HSA concentration is about 0.1% to 0.2% (w / v); the trehalose concentration is about 4% to 6% (w / v); the arginine concentration is about 10 mM to 50 mM; the urea concentration is about 0.1% to 0.3% (w / v). In other embodiments, the stabilized composition can include recombinant HSA, trehalose, MSG, and urea.According to these embodiments, the recombinant HSA concentration can be from about 0.1% to about 0.2% (w / v); the trehalose concentration can be from about 4.0% to about 6.0% (w / v); wherein the MSG concentration is 8 mM to 12 mM; and wherein the urea concentration is 0.1% to 0.3% (w / v).

[0121] Some embodiments herein relate to live attenuated virus compositions that are partially or fully dehydrated for transport or other reasons. According to these embodiments, the composition can be 20% or more; 30% or more; 40% or more; 50% or more; 60% or more; 70% or more; 80% or more; or 90% or more dehydrated. According to these embodiments, the viral vaccine composition can be dehydrated and rehydrated in any known stabilizing composition prior to administration of the pharmaceutically acceptable composition to a child or young person.

[0122] In certain embodiments, the subject can be a mammal, such as a human or a veterinary and / or domestic or farm or wild animal. In certain embodiments, the immunogenic composition of the present disclosure can effectively immunize adolescent subjects, such as human children 20 years of age or younger. Although the prior art related to currently available dengue virus vaccines / immunogenic compositions for use in children (e.g., dengue / yellow fever chimeras) reports low efficacy and / or immunogenicity in children 9 years of age or younger, the immunogenic compositions disclosed herein can generate an effective immune response in children from about 1 year to about 17 years or from about 1 year to about 9 years of age or older. Compared to the prior art, the immunogenic compositions disclosed herein exhibit excellent efficacy and immunogenicity.

[0123] In an exemplary method, an immunogenic composition disclosed herein having all four dengue virus serotypes presented (e.g., a tetravalent formulation) can be administered to a subject, wherein the immunogenic composition can elicit an immune response in the subject against all four dengue virus serotypes. In certain embodiments, the immunogenic compositions of the present disclosure can include combinations of polynucleotides and / or polypeptides of various dengue virus serotype constructs or live attenuated dengue viruses disclosed herein. In other embodiments, the immunogenic composition can include a polynucleotide having a nucleic acid sequence encoding a modified live attenuated dengue-2 virus serotype represented by SEQ ID NO:9 (premajor), 11 (MVS), 13 (WVS), or 15 (BVS); a dengue-1 / dengue-2 chimeric polynucleotide having a nucleic acid sequence encoding a non-structural protein from a modified live attenuated dengue-2 virus serotype and a structural protein from a dengue-1 virus serotype, represented by SEQ ID NO:1 (premajor), 3 (MVS), 5 (WVS), or 7 (BVS); a dengue-3 / dengue-2 chimeric polynucleotide having a nucleic acid sequence encoding a non-structural protein from a modified live attenuated dengue-2 virus serotype and a structural protein from a dengue-3 virus serotype, represented by SEQ ID NO:17 (premajor), 19 (MVS), 21 (WVS), or 23 (BVS); and a dengue-4 / dengue-2 chimeric polynucleotide having a nucleic acid sequence encoding a non-structural protein from a modified live attenuated dengue-2 virus serotype and a structural protein from a dengue-4 virus serotype, represented by SEQ ID NO:25 (premajor), 27 (MVS), 29 (WVS), or 31 (BVS). These polynucleotides can be included with or without the various polypeptides they encode. In certain embodiments, the immunogenic compositions disclosed herein can include one or more polypeptides corresponding to a modified live attenuated dengue-2 virus serotype represented by SEQ ID NO:10 (premajor), 12 (MVS), 14 (WVS), or 16 (BVS); one or more polypeptides corresponding to a dengue-1 / dengue-2 chimeric polynucleotide represented by SEQ ID NO:2 (premajor), 4 (MVS), 6 (WVS), or 8 (BVS); one or more polypeptides corresponding to a dengue-3 / dengue-2 chimeric polynucleotide represented by SEQ ID NO:18 (premajor), 20 (MVS), 22 (WVS), or 24 (BVS); and / or one or more polypeptides corresponding to a dengue-4 / dengue-2 chimeric polynucleotide represented by SEQ ID NO:26 (premajor), 28 (MVS), 30 (WVS), or 32 (BVS).

[0124] The concentrations of the various polynucleotides and polypeptides (e.g., TDV-1, TDV-2, TDV-3, and TDV-4) disclosed herein can be approximately the same, or certain dengue virus serotypes can be presented in the immunogenic composition in excess of other serotypes, depending on the need or outbreak of one or more serotypes. According to these embodiments, the various concentrations or ratios can be expressed as log PFU concentrations relative to each other, as Figures 11A - 11B shown. For example, based on the ratio of TDV-1 to TDV-2 to TDV-3 to TDV-4, the concentrations can be applied to the various tetravalent formulations of the immunogenic compositions disclosed herein. The concentrations can include, but are not limited to, log PFU concentrations of 3:3:3:3, 4:4:4:4, 5:5:5:5, 5:1:5:5, 3:2:3:3, 4:2:4:4, 5:3:5:5, 4:3:4:4, 5:4:5:5, 4:4:5:5, or any concentration ratio of any dengue virus serotype, depending on, for example, the number of serotypes represented in the formulation, the predetermined response, and the desired effect, as will be readily appreciated by those skilled in the art based on this disclosure.

[0125] In certain embodiments, the concentration of live attenuated dengue-2 virus can be 0.5, 1, 1.5, 2, 2.5, 3, or 4 log PFU lower than one or more other dengue virus serotypes in the immunogenic composition to induce a more balanced immune response to all four dengue virus serotypes and reduce viral interference. Although prior art related to existing dengue virus vaccines / immunogenic compositions reports lower potency and / or immunogenicity of dengue-2 virus, the immunogenic compositions disclosed herein provide formulations that can generate an effective immune response to all four dengue virus serotypes, including dengue-2, in greater than 60% of subjects and in some cases approximately 85% (see, for example, Figures 15 - 16 ), even when the live attenuated dengue-2 virus can be at least 1 log PFU lower than other dengue virus serotypes. For subjects 20 years of age or younger, the immunogenic compositions of the present disclosure exhibit excellent efficacy and immunogenicity compared to the prior art and provide a more balanced immune response to all four dengue virus serotypes at any age. In certain embodiments, the seroconversion rate can be evaluated to analyze the efficacy of a particular formulation over time after administration of one or more doses of the live attenuated vaccine.

[0126] In certain embodiments, the immunogenic composition against dengue virus can include one or more of the following: a dengue-1 / dengue-2 chimera at a concentration of about 1.0x1 3 to about 5x1 5 PFU; a concentration of about 1.0x1 3 to about 5x1 5Live attenuated dengue-2 of PFU; concentration about 5.0x 10 3 to about 5x10 5 Dengue-3 / dengue-2 chimera of PFU; and / or concentration about 1.0x 10 4 to about 5x 10 6 Dengue-4 / dengue-2 chimera of PFU. In some embodiments, the tetravalent formulation may comprise in the trivalent or tetravalent formulation a concentration of dengue-3 / dengue-2 chimera that is at least 0.5 to 1 log greater than the dengue-1 / dengue-2 chimera and live attenuated dengue-2, while dengue-4 / dengue-2 may comprise in the trivalent or tetravalent formulation a concentration that is at least 0.5 to 1 log greater than the dengue-1 / dengue-2 chimera and live attenuated dengue-2. In certain embodiments, the concentrations of dengue-3 / dengue-2 and dengue-4 / dengue-2 chimeras may be the same or the concentration of the dengue-4 / dengue-2 chimera is greater than dengue-3 / dengue-4 in the bivalent, trivalent or tetravalent formulations encompassed herein. In some embodiments, a subject treated with the compositions and methods disclosed herein may be treated 2 times a year, annually, every 18 months or a similar schedule, depending on, for example, the location of the subject and travel plans. In certain embodiments, the immunogenic composition against dengue virus may comprise one or more of the following: concentration about 2.0x 10 4 PFU of dengue-1 / dengue-2 chimera; concentration about 5.0x 10 3 PFU of live attenuated dengue-2; concentration about 1x 10 5 PFU of dengue-3 / dengue-2 chimera; and / or concentration about 3.0x 10 5 PFU of dengue-4 / dengue-2 chimera.

[0127] In certain embodiments, any symptoms or signs associated with dengue infection or other flavivirus infections may be evaluated in children or young adults receiving the compositions disclosed herein over a period of time. For example, the onset of dengue or other symptoms or signs associated with dengue infection in children or young adults may be evaluated.

[0128] Treatment methods

[0129] In one embodiment of the present disclosure, the method provides for inducing an immune response against dengue virus serotypes using mono-, bi-, tri- or tetravalent formulations of the live attenuated and / or chimeric virus constructs encompassed herein.

[0130] The embodiments of the present invention are further illustrated by the following non-limiting examples, which should not be construed in any way as limiting its scope. On the contrary, it should be clearly understood that various other embodiments, their modifications and equivalents can be adopted, and after reading the description herein, those skilled in the art can conceive them without departing from the spirit of the present invention or the scope of the appended claims. Example

[0131] The following examples are included to illustrate certain embodiments provided herein. Those skilled in the art should understand that the techniques disclosed in the following examples represent techniques found to work well in the practice disclosed herein and can thus be considered to constitute a preferred mode of its practice. However, based on the present disclosure, those skilled in the art should understand that many changes can be made in the specific embodiments disclosed without departing from the spirit and scope herein and still obtain the same or similar results.

[0132] Example 1

[0133] In some exemplary methods, compositions referred to herein as "master virus seeds (MVS)" are disclosed. These compositions can be derived from one or more live attenuated dengue viruses, such as DEN-1, DEN-2, DEN-3, and DEN-4. In certain methods, the compositions can be derived from one or more live attenuated dengue viruses, including but not limited to the specific constructs referred to herein as TDV-1, TDV-2, TDV-3, and TDV-4 (e.g., previously referred to as DENVax). In other exemplary methods, strategies for generating and characterizing these compositions are provided. In other embodiments, tetravalent dengue virus formulations and the genetic and phenotypic characterization of these formulations are provided. These constructs can be used to generate immunogenic compositions for treating children or young adults between 1 and 20 years of age.

[0134] Major TDV Viruses Before Production and Analysis

[0135] Certain procedures are carried out to generate pre-master dengue virus seeds, such as serial amplification and purification of dengue virus (e.g., TDV). First, the TDV virus is re-derived by transfecting viral RNA transcribed from full-length recombinant TDV cDNA into production-verified cells (e.g., Vero cells) to generate the P1 (passage 1) virus seed. Then, four P1 viruses from each of dengue-1 to dengue-4 are amplified and plaque purified to obtain candidate pre-master vaccine P7 seeds (see Table 1). Certain tests are carried out to analyze the passage of the dengue virus. For example, full-length genome sequencing demonstrates that all four P2 (passage 2) seed viruses are genetically identical to their homologous ancestors, research-derived, research-grade candidate vaccine viruses. The initial plaque phenotype is also retained in the P2 viruses. Six plaque-purified viruses (P3A-F) are isolated for each serotype of the dengue virus (e.g., TDV1-4) from the P2 seeds, and each isolated plaque is directly plaque purified two more times. The third plaque purification (P5) of each virus is amplified twice (P6 A-F and P7 A-F) in Vero cells to generate potential pre-master P7 TDV seeds (Table 1).

[0136] Table 1 Examples of cGMP re-derivation of TDV virus in WCB-Vero cells

[0137]

[0138] Further tests were conducted to characterize the P7 TDV seeds, such as analyzing the genomic sequences and plaque phenotypes of the P7 seeds, and comparing them with the P2 seeds (Table 2). The plaque phenotype of the P7 virus was generally similar to that of the P2 seeds. In some exemplary experiments, the virus titers were monitored. The virus titers of most P7 seeds reached over 6.0 log pfu / ml, except for 5 viruses. Genomic sequencing of over 60 candidate vaccine virus seeds after 10 or more serial passages in Vero cells identified no reversion events at NS1-53 and NS3-250, the three major attenuation determinants of the DENV-2 PDK-53 genetic vector, indicating that these two loci were highly stable in the candidate vaccine virus seeds. A sensitive mismatch amplification assay (TaqMAMA) was developed to accurately measure the reversion rate at the 5'NCR-57 locus by real-time RT-PCR. In some studies, the 5'NCR-57 reversion rates of all 24 P7 seeds were measured by TaqMAMA. Depending on the input viral RNA concentration of each virus in the assay, the sensitivity limit of TaqMAMA was between 0.01% and 0.07% reversion, which was much more sensitive than the 10 - 30% reversion sensitivity limit detectable by consensus genomic sequence analysis. The data obtained showed that 15 of the 24 P7 viruses had minimal or undetectable reversion (<0.07%), 1 virus (TDV-3-D) had nearly 100% reversion, and 8 viruses (e.g., TDV-1, 1TDV-2, 2TDV-3, and 4 TDV-4) had partial reversion of 0.08% to 12.85% (Table 2). Full-length genomic sequencing was performed on 16 of the 24 P7 viruses with low levels of 5'NCR57 reversion as measured by TaqMAMA. All sequenced viruses maintained the other two TDV attenuation determinants (NS1-53, NS3-250), and all viruses acquired additional mutations that were not present in the initial engineered recombinant cDNA clones (Table 2). In one exemplary target vaccine composition, TDV-1-A, TDV-2-F, TDV-3-F, and TDV-4-F were selected as the pre-master seeds for each serotype because their genotypes and plaque phenotypes were most closely similar to those of the initially designed vaccine recombinants. TDV-1-A, TDV-2-F, and TDV-4-F had two non-synonymous mutations, and TDV-3-F had 1. Evidence suggested that these additional mutations observed in these 4 pre-master seeds did not cause safety concerns or immunogenic changes in the virus. These pre-master seeds were further amplified to produce MVS (master seeds, designated P8, Table 1).

[0139] The exemplary methods provided herein use purified in vitro transcribed viral RNA from cloned cDNA plasmids as a pure source to transfect vaccine-validated Vero cells, thereby generating vaccine virus. Serial plaque purification and whole genome sequence analysis are incorporated into the manufacturing process to ensure the manufactured vaccine seeds with optimal purity and genetic stability. Six cloned viruses are prepared as potential pre-master seeds for each TDV serotype. Through genomic analysis, including TaqMAMA and whole genome sequencing, and characterization of the viral plaque phenotype, pre-master seeds are selected to facilitate the production of master virus seeds for each serotype (serotypes 1 - 4). The selected pre-master seeds have undetectable revertants (<0.01% or <0.07%) at the 5'NCR-57 locus, have 1 or 2 amino acid substitutions in their genomes, and retain the previously observed small plaque phenotype.

[0140] Table 2. Characterization of Pre-Master (P7) Seeds

[0141]

[0142]

[0143] Example 2

[0144] In another exemplary method, the compositions of master virus seeds, working virus seeds, and bulk virus seeds, as well as their genetic and phenotypic characteristics, are described. These compositions are provided for the manufacture of clinical materials and ultimately for commercial vaccine supply. Serial plaque purification and whole genome sequence analysis are incorporated into the manufacturing process to ensure the composition of vaccine seeds with optimal safety and genetic stability for the manufacture of clinical trial materials.

[0145] Production and Manufacturing Quality Control of MVS, WVS, and BVS

[0146] In some studies, MVS of 4 TDVs are generated by amplifying pre-master P7 seeds in validated Vero cells. In other studies, large amounts of WVS are prepared in cell factories using MVS. In addition, bulk stocks of BVS of TDV are amplified from WVS and used in human clinical trials. Quality control of product release is performed in some exemplary methods, including all tests for identity, infectious titer, sterility, mycoplasma, and adventitious agents in vitro and in vivo for MVS, WVS, and BVS. All seeds pass the viral identity test using serotype-specific RT-PCR assays, which show positive amplification corresponding to their serotype and negative for heterologous serotypes (data not shown). No detectable mycoplasma or adventitious agents are detected in the MVS, WVS, or bulk stocks of BVS.

[0147] In an exemplary method, the immunogenic compositions disclosed herein having all four dengue virus serotypes presented (e.g., tetravalent) can be administered to children or young adults, wherein the immunogenic composition can elicit an immune response against all four dengue virus serotypes in the children or young adults.

[0148] Genetic analysis of MVS, WVS, and BVS

[0149] In certain exemplary methods, after generating MVS from the selected pre-MVS (P7), the strains selected above are generated and the corresponding viral RNA is sequenced again. Full-length genome sequencing shows that the MVS of TDV-1 is the same as its pre-major seed, while WVS and subsequent BVS acquire 2 additional substitutions at E-483 and NS4B-108 (see Tables 2 and 3). The Ala substitution at E-483 represents a part of the genotype in MVS but becomes the major genotype in BVS. TDV-2 and TDV-3 are the same as their respective pre-major seeds (Tables 2 and 3). The TDV-2 MVS is the same as its pre-major seed, and WVS and BVS have 2 additional mutations at NS4A-36 and NS4B-111. Both mutations are partial in WVS and are the major genotypes in BVS. The MVS of TDV-3 is again the same as the pre-major seed, but WVS and BVS contain an additional amino acid substitution at NS4A-23. During the generation of MVS, the TDV-4 MVS acquires an additional amino acid mutation at locus NS2A-99 (from Lys to Lys / Arg mixed genotype) (Table 3). Its WVS and BVS retain the NS2A-99 Lys / Arg mixed genotype, and BVS has an additional NS4B-238 Ser / Phe mixed genotype. The consensus sequence results also confirm that MVS, WVS, and BV retain three attenuated genetic determinants at the 5'NCR-57, NS1-53, and NS3-250 loci. Analysis of the attenuated loci with the least stability by TaqMAMA shows that the 5'NCR-57 reversion rate in MVS is between <0.7% and 0.13%, ≤0.07% in WVS, and between <0.07 and 0.21% in BVS. A 3% reversion at the 5'NCR-57 locus is considered the maximum allowable rate for vaccine batch acceptance (Table 3).

[0150] Table 3 Nucleotide and amino acid substitutions in TDV seeds

[0151]

[0152]

[0153] Whole genome sequence analysis revealed additional amino acid mutations that formed in TDV-4MVS, while the other three TDVMVS batches retained the consensus genomic sequence of their pre-master seeds. From the P1 seed to the pre-master (P7) seed, only 1 or 2 non-synonymous mutations occurred in a given seed. From P1 to the MVS (P8) seed, 2 to 7 nucleotide substitutions were identified in any given TDV seed, and only 2 to 3 of these substitutions resulted in amino acid changes. None of the silent mutations in MVS were within the 5' or 3' NCR that could affect virus replication. Only the prM-52Lys-Glu change in TDV-2 and the NS2A-66Asp-Gly substitution in TDV-4 were non-conservative changes. The NS2A-66 mutation in TDV-4 was located in the non-structural backbone portion of DENV-2 PDK-53. Although the NS2A-66 locus is usually Asp in various strains of DENV-2, it is usually Gly for DENV-4. The Asp to Gly change in TDV-4 may be related to the fitness of TDV-4 in Vero cells. The TDV-2 prM-52 mutation was located in the C-terminal portion of prM, which is excised from the mature virus particle. In some exemplary methods, phenotypic characterization was performed to confirm that none of the mutations in the MVS seeds significantly altered the attenuated phenotype of the vaccine.

[0154] The TDV virus exhibited high genetic stability during the manufacturing process. Three defined DENV-2 PDK-53 attenuating loci located in the 5' NCR, NS1-53, and NS3-250 remained stable in the consensus genomic sequence after continuous passage of TDV from the pre-master strain to the bulk vaccine formulation. The highly sensitive TaqMAMA at the 5' NCR-57 locus showed minimal or undetectable revertants in the MVS, WVS (P9 / working), and BVS (bulk virus seed of the vaccine) of dengue virus serotypes. The 5' NCR-57 revertant rate of the TDV BVS formulation (P10 equivalent) was significantly lower than the 5' NCR-57 revertant rate (4 - 74% revertants) evolved in the research-grade vaccine candidates after 10 consecutive passages in Vero cells. The strategy for large-scale manufacturing of the TDV seeds provided herein yielded genetically stable vaccine seeds that retained the markers of attenuation in the candidate vaccine virus.

[0155] All TDV virus constructs can be stored in stabilizing buffers such as FTA, poloxamer 407 (0.01% to about 3.0% w / v) and about 5% to about 50% (w / v) trehalose, 2 and about 0.01% to about 3.0% albumin (such as rHSA)

[0156] Safety and in vivo immunogenicity

[0157] In this example, it was demonstrated that the exemplary compositions are safe and substantially immunologically inert after subcutaneous injection. Four different exemplary compositions were selected for testing in mice as follows (data not shown).

[0158] Formulation 1: 15% trehalose, 2% F-127, 1% rHSA

[0159] Formulation 2: 15% trehalose, 2% F-127, 1% rHSA, 1 mM CaCl2 / 0.5 mM MgSO4 Formulation 3: 15% trehalose, 2% F-127, 1% rHSA, 0.5% chitosan

[0160] Formulation 4: 22.5% trehalose, 3% F-127, 1.5% rHSA

[0161] Formulation 5: Plaque phenotype of PBSTDV MVS

[0162] In one exemplary method, the plaque phenotype of TDV MVS was compared with wild-type dengue virus and its homologous research-grade chimeric virus in Vero cells ( Figure 2 ). All MVSs of TDV-1, -2, and -3 produced plaques in Vero cells that were significantly smaller than their wild-type homologs and were very similar to their homologous research-grade viruses (within 0.4 mm difference). TDV-4 MVS was also significantly smaller than wild-type DENV-4 but slightly larger than the initial laboratory-derived D2 / 4-V chimera (0.9 mm difference).

[0163] Figure 2 Represents an exemplary histogram showing the plaque sizes of TDV MVS in contrast to control wild-type virus and research-grade vaccine candidate virus. Mean plaque diameter (mm) ± SD (error bars) of viral plaques in Vero cells under agarose overlay measured at 9 days pi. Included are wild-type DEN virus represented by black bars and previously published research-grade vaccine candidate virus represented by white bars for comparison with the control of the TDV master vaccine seed represented by gray bars.

[0164] Temperature sensitivity of TDV MVS

[0165] In another exemplary method, the temperature sensitivity of TDV MVS was tested in Vero cells and compared with its homologous wild-type and initial research-grade chimeric vaccine viruses. Wild-type (WT) DENV-3 16562 is not temperature-sensitive. Wt dengue virus serotype 1 and dengue virus serotype-4 are moderately temperature-sensitive at 39 °C (titers are approximately 1.0 log lower at 39 °C than at 37 °C 10 pfu / ml, Figure 3) Dengue virus serotype-2 16681 is the most temperature-sensitive among the WT dengue viruses tested and causes a 100-fold decrease in titer at 39 °C. TDV-1, -2, and -3 are as temperature-sensitive as their original homologous research-grade chimeric vaccine viruses ( Figure 2 ) For these TDV strains, the titer decreases by 2.0 to 3.0 logs at 39 °C 10 pfu / ml. TDV-4 is also temperature-sensitive, showing a 5-fold decrease in titer. However, the initial research-grade D2 / 4-V showed a ~10-fold decrease in titer. The final stabilized TDV-4MVS contains poloxamer 407, (as well as other reagents (FTA or bivalent formulation, albumin, and trehalose) that stabilize these formulations), which have previously been shown to enhance the thermal stability of dengue virus. The presence of in TDV-4MVS may contribute to the less obvious temperature sensitivity of the virus in the Vero cell culture assay. In different experiments, the temperature sensitivity of MVS-derived TDV-4 strains was further evaluated in the absence of . To remove poloxamer 407 from the strain, viral RNA was isolated from the TDV-4 bulk virus preparation and transfected into Vero cells. In the absence of poloxamer 407, this TDV-4 virus appeared to be as temperature-sensitive as the D2 / 4V research strain (a 1.5-log decrease in titer 10 pfu / ml) on day 3 post-infection ( Figure 3 ).

[0166] Figure 3 The exemplary histogram shown shows the temperature sensitivity of TDV MVS. Wild-type dengue virus and previously published research-grade vaccine candidate viruses are included for comparison. TDV-4MVS contains additional poloxamer 407, which can mask the temperature sensitivity results of the virus in this assay. Also included are different experiments analyzing the alternative TDV-4 in the absence of . Mean titers ± SD (error bars) of viruses replicating in Vero cells at 37 °C or 39 °C.

[0167] Replication of TDV MVS in mosquito C6 / 36 cells

[0168] In some exemplary methods, based on the knowledge of the attenuated phenotype of the backbone DENV-2 PDK53 virus retained by the investigational chimeric vaccine viruses in these mosquito cells, the TDV MVS were cultured in C6 / 36 cells to verify their retention of the attenuated phenotype in vitro. Compared to wt dengue virus, TDV-1, TDV-2, and TDV-4 MVS showed a significant reduction in C6 / 36 cell growth (at least 3 log 10 pfu / ml reduction) on day 6 post-infection (pi) ( Figure 4 ). Compared to wt DENV-3 16562, TDV-3 MSV also showed reduced growth, but the reduction was less pronounced (1-2 log 10 pfu / ml reduction). However, the C6 / 36 titers of the TDV-3 seed lot were similar to those of the initial investigational chimeric D2 / 3-V vaccine virus (within 1 log 10 pfu / ml difference).

[0169] Figure 4 Exemplary histograms are shown that plot the restricted growth of TDV MVS (gray bars) in C6 / 36 cells compared to wt dengue virus (black bars) and investigational vaccine virus (white bars). Mean titers ± SD (error bars) of virus replicating in C6 / 36 cells on day 6 pi.

[0170] Virus infection, dissemination, and transmission rates in whole mosquitoes

[0171] In some exemplary methods, the infection and dissemination rates of TDV were compared to their parental wt dengue viruses. In certain exemplary experiments, oral infection experiments were conducted in Aedes aegypti. Infective blood meals were retitrated to measure virus titers, and only experiments with similar virus titers (less than 1 log 10 pfu / ml difference) in the blood meal between the parental dengue virus and the TDV of each serotype were included for comparison in Table 4. TDV-1, TDV-2, and the investigational D2 PDK-53-VV45R did not infect mosquitoes by oral feeding, which was significantly different from their parental viruses DENV-1 16007 (44% infection) and DENV-2 16681 (43.3% infection) (p < 0.0001). Since no mosquitoes were infected with TDV-1 and -2, there was little concern about transmission for these two vaccine viruses. Although TDV-4 did infect some mosquitoes (2 out of 55) by oral feeding, the infection rate was significantly lower than that of its parental wild-type virus DENV-4 1036 (8 out of 50) (p < 0.05). TDV-3 had a blood meal virus titer of 5.2 ± 0.02 log 10In two experiments at 6.0 log pfu / ml, no mosquitoes were infected (Table 4), and in different experiments at 6.0 log pfu / ml, only 1 out of 30 mosquitoes was infected (data not shown). However, wild-type dengue virus-3 6562 also had a very low infection rate (8%) at 5.2 log pfu / ml, and this ratio did not increase in different experiments with a higher bloodmeal virus titer of 6.2 log pfu / ml (3%, 1 positive out of 30 mosquitoes, data not shown). Although the infection rates of wild-type (WT) dengue virus-3 and dengue virus-4 were significantly lower than those of wild-type dengue virus-1 and dengue virus-2, the average virus titers in infected mosquitoes were similar (3.1 to 3.9 log pfu / mosquito). In contrast, the titers of TDV-4 from two infected mosquitoes were both minimal (0.7 log pfu / mosquito), which was 1000-fold lower than the titer from mosquitoes infected with wild-type dengue virus serotype-4 1036 (3.9 ± 1.5 pfu / mosquito). 10 For those infected mosquitoes, the transmission outside the midgut can be evaluated by determining the presence of virus in the legs. The four parental DENV led to transmission rates ranging between 36.3% and 62.5%, and their average virus titers from the legs (in log pfu) were between 0.9 ± 0.3 and 2.2 ± 0.7 (excluding negative samples). Neither of the two TDV-4-infected mosquitoes resulted in virus transmission to the legs (Table 4). Although transmissible virus was detectable in the legs, none of the four wt dengue viruses was detected in the saliva of orally infected mosquitoes, indicating that the oral feeding conditions might not be sensitive enough for measuring the transmission rates of these DENVs. Therefore, in other exemplary methods, highly stringent artificial mosquito infections by direct IT inoculation were subsequently performed (Table 4). Except for TDV-4, all viruses (wt and TDV) achieved 100% infection of Aedes aegypti by IT inoculation. The virus titer of the TDV-4 inoculum was slightly lower than those of the other three virus inocula, but it still successfully infected 70% of the inoculated mosquitoes. Despite the high body infection rate achieved by IT inoculation, all four TDV viruses showed significantly lower (p < 0.005) or undetectable transmission rates (0 - 10%) compared to wt dengue viruses (43 - 87%, Table 4). The TDV viruses indicated little to no infection and transmission after oral feeding, and the highly stringent IT results confirmed the minimal transmission ability of these TDV viruses in Aedes aegypti. 10 10 10 10

[0172] 10

[0173] Table 4: Virus infection, dissemination, and transmission rates in whole mosquitoes

[0174]

[0175] Vector competence is an important safety component of live attenuated flavivirus vaccine viruses. Previously, the research-grade DENV-2 PDK-53-VV45R virus and wt revertant derivatives were tested in Aedes aegypti mosquitoes, and the NS1-53-Asp attenuating mutation was found to be the major determinant of impaired mosquito replication. Two other major attenuating loci of the DENV-2 PDK-53 vaccine, nucleotide 5'NCR-57-T and NS3-250-Val, also exhibited some inhibitory effects on mosquito replication, thus providing additional redundant restrictions on mosquito vector competence. Some of the exemplary methods described herein were used to test oral and IT infection and replication of all four TDV strains in mosquitoes. TDV-1, -2, and -3 did not infect any Aedes aegypti mosquitoes by oral infection (Table 4). TDV-4 infected 3.6% of orally exposed mosquitoes with an average in vivo replication titer that was only lower than the average replication titer of mosquitoes infected with wt DENV-4, a level significantly lower than that of wt DENV-4. Surprisingly, TDV-4 was detected in the legs of infected mosquitoes, indicating that TDV-4 could not disseminate from the mosquito midgut after oral infection. The infection rates of TDV-1, -2, and -4 were all significantly lower than those of their wild-type counterparts, but the difference between TDV-3 and WT DENV-3 16562 was not significant because the infection rates of both viruses were very low. Compared with other wild-type DENV strains evaluated in Aedes aegypti mosquitoes collected from the same Mae Sot province in Thailand, the parental wild-type dengue virus strains used to engineer the TDV strains appeared to have lower infection and dissemination rates by oral infection. The wt DENV-1 PUO359, DENV-2 PUO218, DENV-3 PaH881 / 88, and DENV-4 1288 used to engineer the ChimeriVax-DEN vaccines based on the yellow fever (YF) 17D vaccine had infection rates ranging from 47-77%. In contrast, the YF 17D vaccine could not infect Aedes aegypti mosquitoes. Although the ChimeriVax strains contain prM-E from these highly infectious wt DENVs, the ChimeriVax strains retain the mosquito-attenuated phenotype of their YF 17D replication backbone. The results provided herein also indicate that mosquito attenuation of the DENV-2 PDK-53 backbone is maintained in the TDV strains. In addition, using wt dengue virus strains with lower mosquito infectivity in the constructs included in the compositions described herein provides additional safety features.

[0176] In an exemplary method, oral infection results indicated that TDV had minimal mosquito infectivity and dissemination ability. Additionally, more sensitive and rigorous IT infection experiments were conducted to further analyze the potential of TDV transmission by Aedes aegypti. The IT results demonstrated that all four TDV viruses had undetectable or minimal mosquito transmission potential compared to their wt counterparts. TDV transmission could only theoretically occur under the following conditions: (1) the vector feeds on a vaccinated individual with a sufficient viremia titer to infect the mosquito midgut, (2) the virus is able to replicate in the midgut epithelium and is then able to disseminate from the midgut, and (3) the disseminated virus can replicate in the salivary glands and expel sufficient virus in the saliva for transmission. The threshold of human viremia required for mosquito infection has not been fully established, but human viremia can be 10 6 -10 8 mosquito infectious dose 50 (MID 50 ) / ml. This MID 50 was based on direct IT inoculation of mosquitoes with diluted human plasma. Analysis of TDV in non-human primates indicated that the viremia titers after TDV immunization were very low (less than 2.4 log 10 pfu / ml) and persisted for 2 - 7 days. Given the low viremia levels and low mosquito infectivity, dissemination, and transmission ability of TDV, these vaccine viruses are unlikely to be naturally transmitted by mosquitoes or cause viremia.

[0177] Therefore, it is proposed that any passage (P1 - P10) of any serotype can be used in the composition to produce a safe and effective vaccine against one, two, three, or all four dengue virus serotypes.

[0178] Neurovirulence in suckling mice

[0179] The initial research-grade vaccine viruses were highly attenuated in terms of neurovirulence in neonatal ICR mice maintained within DVBD / CDC. All of these mice survived an ic (intracerebral) challenge with 10 4 pfu of each vaccine virus. On the other hand, the wt dengue virus serotype-2 16681 virus caused mortality rates of 62.5% - 100% in these CDC-ICR mice in various experiments. In some experiments, commercially available ICR mice obtained from Taconic Labs (Taconic-ICR) were used to study neurovirulence in neonatal mice. The neonatal Taconic-ICR mice were observed to be significantly more susceptible to dengue virus serotype-2 infection than the previous CDC-ICR mice. Figure 5A Summarized with 10 4Neurovirulence of wt dengue virus serotype-2 16681 in Taconic-ICR neonatal mice and CDC-ICR colonies attacked by pfu virus ic. Taconic-ICR mice (100% mortality in 32 mice, mean survival time 8.3 ± 0.5 days) were more susceptible to ic dengue virus serotype-2 16681 than previous CDC-ICR mice (91% mortality in 72 mice, mean survival time 14.6 ± 2.3 days).

[0180] In other exemplary methods, to evaluate the neurovirulence of TDVMVS, initially in one (n = 16) or two (n = 31 - 32) experiments, Taconic-ICR mice were attacked ic (intracerebrally) with a dose of approximately 10 4 pfu of wt dengue virus serotype-2 16681, D2PDK-53VV45R, D2 / 3-V, or TDV 1-4 viruses. At this dose, the D2 / 3-V research-grade virus and TDV-1 and TDV-3 MVS exhibited a fully attenuated neurovirulence phenotype (no disease or death). As expected, the wt dengue virus serotype-2 was found to be "lethal", with a mean mouse survival time (AST) of 8.3 ± 0.8 days. In these dengue virus serotype-2 susceptible Taconic-ICR mice, the D2 PDK-53-VV45R research-grade virus caused 81.3% mortality. TDV-2 MVS and TDV-4 MVS were both lethal in Taconic-ICR, showing AST values of 9.8 ± 1.7, 10.2 ± 1.4, and 11.3 ± 0.4 days, respectively.

[0181] In some exemplary methods, at a 10-fold lower dose (10 3 pfu, Figure 5C) The neurovirulence of the wt dengue virus serotype-2 16681 virus was compared with that of D2 PDK-53VV45R, TDV-2MVS, and TDV-4MVS, as well as the D2 / 4-V investigational virus. At this lower challenge dose, the wt dengue virus serotype-2 retained a uniformly lethal neurotoxic phenotype with an AST of 9.0 ± 1.4 days. The other four viruses exhibited an intermediate neurovirulence phenotype, and the degree of neurovirulence was serotype-specific. The D2 PDK-53-VV45R virus and its TDV-2MVS correlate showed significant attenuation (32.3% survival at an AST of 13.1 ± 3.8 days and 31.2% survival at an AST of 10.5 ± 3.4 days, respectively). Both the TDV-4MVS and investigational D2 / 4-V viruses were highly attenuated in terms of neurovirulence (81.3% survival at an AST of 18.8 ± 5.8 days and 100% survival, respectively). The results indicate that the MVS of TDV-1 and -3 exhibit complete attenuation of neurovirulence, while the TDV-2 and -4MVS batches retain an attenuated phenotype very similar to their homologous investigational virus candidate vaccines.

[0182] Figures 5A - 5C Exemplary graphs showing neurovirulence in neonatal mice tested with various compositions comprising wt dengue virus serotype-2 and different attenuated dengue viruses. The pooled results of many experiments summarize the neurovirulence of the wt dengue virus serotype-2 16681 virus in CDC-ICR (n = 72) and Taconic-ICR (n = 32) neonatal mice challenged with 10 4 pfu of virus (A) ic. The neurovirulence of TDV MVS tested in Taconic-ICR mice at a dose of 10 4 pfu (B) or 10 3 pfu (C). The number of animals tested per group in one experiment (n = 16) or two combined experiments (n = 31 or 32) is indicated.

[0183] WVS and BVS plaque phenotypes

[0184] Certain studies were conducted to compare the plaque phenotypes of WVS and BVS with MVS, wt dengue virus, and its homologous laboratory-derived investigational chimeras in Vero cells ( Figure 6)。The average plaque size was calculated from 10 plaques for each vaccine virus, but for the reduced numbers of wt DENV-1, -3, and -4, the average plaque size was calculated. All MVS viruses of TDV-1, -2, and -3 produced plaques that were significantly smaller in Vero cells than their wt counterparts and were very similar (within 0.4 mm difference) to their homologous research-grade viruses. TDV-4 MVS was also significantly smaller than wt DENV-4 but was slightly (0.9 mm) larger than the initial laboratory-derived D2 / 4-V chimeric virus. Except for TDV-2, all WVS and BVS of TDV-1, -3, -4 retained plaque sizes that were significantly smaller than the plaque sizes produced from their wt counterparts. The plaques produced by TDV-2 WVS and BVS were similar to the plaques of the wt DENV-2 virus in Vero cells, but when tested in LLC-MK2 cells, the plaques produced by all TDV-2 produced seeds were slightly smaller than the plaques of wt DENV-2 (1.4 ± 0.4) and were similar to the laboratory-derived D2 PDK-53-VV45R (1.0 ± 0.3)( Figure 6 )。

[0185] Assessment of phenotypic markers of virus attenuation was evaluated for the composition of the MVS bulk, including the small plaque phenotype, temperature sensitivity, reduced replication in mosquito cells, reduced mosquito infection / dissemination / transmission, and reduced neurovirulence in neonatal ICR mice. The results indicate that all TDVs retain the expected attenuated phenotype similar to the initial research-grade vaccine viruses. Given that the mutations responsible for attenuation are conserved in all MVS, WVS, and BV, it can be expected that the attenuated phenotype will be retained in the material manufactured for human clinical testing.

[0186] Figure 6 Exemplary histograms showing the plaque sizes of TDV MVS, WVS, and BVS. The mean plaque diameter ± SD (error bars) of virus plaques in Vero or LLC-MK2 cells under agarose overlay measured at 9 days pi. Wild-type DENV and previously published research-grade vaccine candidate viruses were included for control and comparison.

[0187] Virus replication in mosquito C6 / 36 cells

[0188] Previous studies have demonstrated that PDK-53-based research-grade chimeric vaccine viruses retain the attenuated phenotype of the backbone DENV-2 PDK53 virus in C6 / 36 cells. In some exemplary methods, TDV MSV, WVS, and BVS are cultured in C6 / 36 cells to verify the retention of this in vitro attenuation marker after large-scale manufacture. Compared to wild-type dengue virus, except for TDV-3, the produced seeds showed a significant growth reduction (at least 3 log) in C6 / 36 cells at 6 days pi 10(PFU / ml reduction) Figure 7 )。Compared to wt DENV-3 16562, TDV-3 seeds also showed reduced growth, but the reduction was not significant (1-2 log 10 PFU / ml reduction). However, the titers of TDV-3 seed batches were similar to those of the initial research-grade chimeric D2 / 3-V vaccine virus (within 1 log 10 PFU / ml difference).

[0189] Figure 8 Exemplary histograms showing restricted growth of TDV MVS, WVS, and BVS in C6 / 36 cells. Mean titers ± SD (error bars) of virus replicated in C6 / 36 cells at 7 days pi. wt dengue virus and previously published research-grade vaccine candidate viruses were included for comparison.

[0190] Neurovirulence in suckling mice

[0191] Additional experiments were conducted to analyze neurovirulence in neonatal ICR mice. At an intracranial dose of 10 4 PFU, the survival of wt DENV-2 16681 and D2 PDK-53-VV45R in ICR mice was 0% and 18.8% respectively( Figure 9A ), but in CDC ICR mice, it was approximately 20% for wt DENV-2 16681 and 100% for D2 PDK-53-VV45R. In this study, TDV-1 and TDV-3 MVS were attenuated (100% survival) for mice at a dose of 10 4 PFU, but TDV-2 and TDV-4 MVS at doses above 10 4 PFU resulted in 100% mortality( Figure 5A ). However, when tested at a viral dose of 10 3 PFU, TDV-2 (31.3% survival) and TDV-4 (81.3% survival) showed reduced neurovirulence compared to wild-type dengue virus serotype-2 16681 (0% survival), and their survival rates were similar to those of the research-grade vaccine candidates D2 PKD-53-VV45R (32.3%) and D2 / 4-V (100%) respectively( Figure 9B)。Although wild-type DENV-1, -3, or -4 were not included in this study for comparison, previous work demonstrated that wild-type DENV-1 16007 was attenuated in CDC-ICR mice via the ic route, while wild-type DENV-3 16562 and DENV-4 1036 were highly virulent (0% survival) for CDC-ICR mice. These three wild-type DENVs may exhibit similar or higher virulence in more susceptible Taconic ICR mice. Therefore, including these wild-type dengue viruses for comparison with their homologous TDV MVSs was considered uninformative. This study indicated that all four TDV MVSs and the initial laboratory-derived candidate vaccine viruses exhibited comparable mouse-attenuated phenotypes relative to wild-type DENV-2 16681.

[0192] Figures 9A - 9B Exemplary graphs representing the neurovirulence data of TDV MVSs in neonatal ICR mice. (A) Intracerebral (IC) inoculation with a virus dose of 10 4 PFU. (B) Intracerebral (IC) inoculation with a virus dose of 10 3 PFU.

[0193] Exemption of all seed lots of TDV for identity, sterility, and adventitious agents testing. Whole genome sequence analysis revealed that an additional amino acid mutation evolved in TDV-4MVS, while the other 3 TDV MVSs retained the consensus genomic sequence of their parental master seeds. In the WVS lots, TDV-3 acquired an additional amino acid mutation, while the other 3 serotypes accumulated 2 additional amino acid substitutions relative to their parental master seeds. The genomic sequences of all 4 BVS lots were identical to their WVS lots. Overall, only 1 or 2 non-silent mutations occurred in a given seed from P2 seed to parental master (P7) seed. Between parental master and BCS (P10) seeds, only 1 to 2 nucleotide substitutions were observed, and all of these substitutions occurred in NS2A, 4A, or 4B except for a single nucleotide change that resulted in conserved glycine and alanine at residue E-483. From P2 to BVS (P10) seeds, a total of 3 to 8 nucleotide substitutions were identified in any given TDV seed, and only 2 to 4 of these substitutions resulted in amino acid changes. None of the silent mutations in BVS were within the 5'- or 3'-NCR regions that may affect virus replication. These results indicate that the TDV virus is highly genetically stable during the manufacturing process. Three defined DENV-2 PDK-53 attenuation loci located in 5'NCR, NS1-53, and NS3-250 remained unchanged in the consensus genomic sequence during successive passages of TDV to produce BVS bulk. The highly sensitive TaqMAMA of the 5'-NCR-57 locus showed minimal or undetectable revertants in MVS, WVS, and BVS of TDV. The highest revertant rate of 0.21% was identified in TDV-2BVS. The revertant rates of P10 equivalent BVS (<0.07% to 0.21%) were significantly lower than the revertant rates evolved in other vaccine candidates after successive passages in Vero cells (4 - 74% revertants by P10). This indicates that this strategy for large-scale manufacturing of TDV seeds is successful in terms of maintaining genetic stability and retention of attenuation markers in the candidate vaccine virus.

[0194] Since the MVS bulk disclosed herein will be used for future manufacture of WVS and BVS lots, a complete set of virus attenuation phenotypic evaluations was performed on all MVSs or their equivalent alternative bulks, including small plaque phenotype, temperature sensitivity, reduced replication in mosquito cells, reduced infection / dissemination / transmission in whole mosquitoes, and reduced neurovirulence in neonatal ICR mice. For WVS and BVS bulks, plaque size and infectivity in mosquito cells were also performed to confirm their attenuation. The results indicated that all MVS bulks of the 4 TDV serotypes retained the expected attenuated phenotypes, such as small plaques, reduced replication in C6 / 36 cells, and reduced mouse neurovirulence, similar to the initial laboratory-derived vaccine virus ( Figure 6 、 8and 9). Except for TDV-4, all three other MVS master batches of TDV are TS at 39°C, as Figure 3 and Figure 7 shown.

[0195] For the WVS and BVS master batches, two attenuated phenotypes / minute plaques and restricted replication in C6 / 36 cells were analyzed and confirmed. Due to the very small genetic changes between MVS and BVS, they are expected to retain the attenuated phenotype as MVS. In addition to the experiments described in this report, the manufactured TDV was also tested for safety and immunogenicity in Ag129 mice and non-human primates.

[0196] This document provides exemplary methods to demonstrate the manufacture of TDV MVS, WVS, and BVS master batches under cGMP. The BVS master batch is used to formulate the tetravalent TDV currently under evaluation in human clinical trials. Unique manufacturing strategies for optimizing the genetic stability and safety of the manufactured MVS are provided in some of the exemplary methods. Since the major attenuated loci of TDV have been fully characterized previously and a highly sensitive and quantifiable SNP assay, TaqMAMA was developed to integrate genomic sequences and TaqMAMA to identify the best pre-major seed for preparing MVS. The genetic and phenotypic characteristics of MVS were comprehensively analyzed to confirm that these viruses maintain the desired attenuation for vaccine safety. This can be the only live attenuated virus vaccine that can effectively analyze all major attenuated genetic loci from the pre-major master batch to the BVS master batch during the manufacturing process. The results provided in this document illustrate the advantages of a strategically designed live attenuated vaccine in terms of vaccine safety.

[0197] Example 3

[0198] In another exemplary method, in addition to one or more constructs encoding the non-structural protein domain of dengue-2 serotype and the structural protein from any one of dengue-1 (TDV-1), dengue-3 (TDV-3), or dengue-4 (TDV-4) serotypes, the construct may further include a modified live attenuated dengue virus based on the live attenuated dengue-2 serotype (TDV-2). Additionally, the individual constructs in the immunogenic compositions disclosed herein can be formulated at a ratio or concentration of PFU based on the log PFU compared to each other. For example, the immunogenic composition can be formulated such that TDV-1 is present at a concentration of approximately 2x10 4 PFU, TDV-2 is present at a concentration of approximately 5x10 4 PFU, TDV-3 is present at a concentration of approximately 1x10 5 PFU, and TDV-4 is present at a concentration of 3x10 5 PFU, as Figure 11BAs shown. The relative concentrations of the different dengue serotype constructs in the immunogenic formulation can also be expressed as a ratio of the logarithms of the PFU-based numbers, e.g., about 4:4:5:5, corresponding to the ratio of TDV-1:TDV-2:TDV-3:TDV-4, as Figure 11B shown. In some embodiments, the ratio can be about 5:4:5:5 and other predetermined ratios, where one or more of the live attenuated dengue virus serotypes are higher or lower compared to the other dengue virus serotypes. In one example, the relative concentration of TDV-2 can be decreased compared to TDV-1, TDV-3, and TDV-4, and when all four dengue serotypes are administered to a subject, this can result in an immunogenic composition with a better balanced response. For example, TDV-1, TDV-2, TDV-3, and TDV-4 can induce neutralizing antibodies against all four dengue virus serotypes (chimeras of live attenuated dengue-2 and dengue virus-1, -3, and -4), and using dengue-2 as a backbone can induce a multifunctional and cross-reactive CB8+ T cell response to dengue non-structural proteins.

[0199] In another exemplary method, to test the efficacy and tolerability of the dengue immunogenic compositions disclosed herein in children (a group that has historically been difficult to effectively immunize against dengue virus and in which previous vaccines have failed to work, especially children 9 years of age and younger), a two-part randomized, double-blind, placebo-controlled clinical trial was conducted. Part 1 was conducted in a descending age manner, in which a single dose of a dengue vaccine composition containing all four constructs, TDV-1, TDV-2, TDV-3, and TDV-4, was administered to subjects 21 - 45 years of age (n = 38), subjects 12 - 20 years of age (n = 36), subjects 6 - 11 years of age (n = 38), and subjects 1.5 - 5 years of age (n = 36) ( Figure 12A ). Part II was conducted in healthy child subjects 1.5 - 11 years of age (n = 211) as an extension of Part I ( Figure 12B ). A vaccine dose was administered on Day 0, and tissue samples (e.g., blood samples) were taken from the subjects on Day 0 and at various time points throughout the trial to test for viremia, immunogenicity, and overall safety ( Figures 12A - 12B ). For Part I, the randomized doses of vaccine and placebo were administered at a vaccine-to-placebo ratio of approximately 2:1. For Part II, the randomized doses of vaccine and placebo were administered at a vaccine-to-placebo ratio of approximately 3:1. In the immunogenic composition, TDV-1 was present at a concentration of about 2x10 4 PFU, TDV-2 was present at a concentration of about 5x10 4 PFU, TDV-3 was present at a concentration of about 1x10 5The concentration of PFU exists, and TDV-4 exists at a concentration of about 3x10 5 PFU.

[0200] In another method, to study the tolerability of the immunogenic compositions against dengue disclosed herein in children, the incidence of adverse events (AE) was studied (data not shown). The incidence of pain, itching, erythema, and edema at the injection site was determined (self-reported) between TDV and placebo administrations. Various AEs were investigated within 14 days after the first administration of TDV (n = 249) or placebo (n = 111), and within 14 days after the second administration of TDV (n = 249) or placebo (n = 111). Overall, no serious vaccine-related AEs were reported in the study subjects, and there were no significant changes in blood chemistry or hematology, indicating that the disclosed dengue vaccine compositions are well tolerated in children. (Data not shown)

[0201] In another exemplary method, to test the efficacy of the immunogenic compositions disclosed herein in children, the presence of neutralizing antibodies produced by the subjects after injection of the TDV vaccine composition was measured at various time points up to 720 days after injection using a microneutralization assay. As Figures 13A - 13B shown, TDV vaccine administration elicited a neutralizing antibody response that persisted up to day 720, regardless of the initial immune status of the subjects ( Figure 13A seropositive at baseline as shown in Figure 13B ; dengue-naive at baseline as shown in

[0202] In another embodiment, to test whether the dengue immunogenic compositions can elicit a tetravalent response in children regardless of the initial immune status, the seropositivity of the study subjects was measured at various time points up to 720 days after injection of the TDV vaccine composition. As Figure 14As shown, at all post - injection time points tested for all dengue serotypes, greater than about 60% of the study subjects were seropositive. At all post - injection time points tested for three or more dengue serotypes, greater than about 80% of the subjects were seropositive. And at all post - injection time points tested for two or more dengue serotypes, nearly 100% of the subjects were seropositive. Data at days 0, 28, 90, and 120 were obtained from parts I and II of the clinical trial (n = 249), while data at days 180, 360, and 720 were obtained from part I of the clinical trial (n = 90). Seropositivity was determined by MNT 50 Titer > 10

[0203] In another exemplary method, tests were conducted to determine whether the immunogenic compositions for dengue disclosed herein were capable of eliciting an immune response against all four dengue virus serotypes in children who were seronegative (dengue - naive) at baseline. As Figure 15 shown, at all post - injection time points tested for all dengue serotypes, greater than about 40% of the study subjects were seropositive. At all post - injection time points tested for three or more dengue serotypes, greater than about 60% of the subjects were seropositive. And at all post - injection time points tested for two or more dengue serotypes, nearly 100% of the subjects were seropositive. Data at days 0, 28, 90, and 120 were obtained from parts I and II of the clinical trial (n = 133), while data at days 180, 360, and 720 were obtained from part I of the clinical trial (n = 40). Seropositivity was determined by MNT 50 Titer > 10. It should be noted that the tetravalent compositions disclosed herein are capable of inducing an immune response against all four dengue virus serotypes, which can persist up to 720 days after administration.

[0204] Example 4

[0205] In one method, participants were enrolled; 1,794 received two doses of TDV subcutaneously at months 0 and 3 (n = 200; group 1); one dose at month 0 (n = 398; group 2); one dose at month 0 and a booster at month 12 (n = 998; group 3); or placebo (n = 198; group 4). TDV elicited neutralizing antibodies against all DENVs, which peaked at month 1 and remained above baseline at month 6. The month 6 GMT (95% CI) in groups 1 - 4 were: for DENV-1, 89 (321, 746), 434 (306, 615), 532 (384, 738), 62 (32, 120); for DENV-2, 1,565 (1,145, 2,140), 1,638 (1,286, 2,088), 1,288 (1,031, 1,610), 86 (44, 169); for DENV-3, 160 (104, 248), 151 (106, 214), 173 (124, 240), 40 (23, 71); and for DENV-4, 117 (79, 175), 110 (80, 149), 93 (69, 125), 24 (15, 38). In initially seronegative participants, two doses of TDV elicited higher mean GMTs and seroconversion rates to DENV-3 and -4 than one dose, and the four-valent seroconversion rate at month 6 was 85.0% compared with 67.6% after one TDV dose.

[0206] Study design and participants

[0207] These studies were multicenter, randomized, double-blind, placebo-controlled studies conducted at three hospitals / clinics in Panama, the Philippines, and the Dominican Republic. Healthy participants aged 2 - 17 years were enrolled and their eligibility was assessed, then randomized at a 1:2:5:1 ratio to receive two doses of TDV at months 0 and 3 (group 1); one dose at month 0 (group 2); one dose at month 0 and a booster at month 12 (group 3); or placebo (group 4). The 1:2:5:1 randomization ratio was chosen to provide data on single-dose regimens with or without a booster to support the potential use of this dosing regimen in phase 3 development. Previous studies of TDV had established the safety and immunogenicity of a 2-dose schedule (months 0 - 3), and this group (group 1) was used for comparison with the larger one-dose groups (groups 2 and 3).

[0208] Procedures

[0209] The serotype composition of TDV in the lyophilized formulation was 2.5x10 4 、6·3x10 3 、3·2x104 and 4·0x10 5 The TDV-1, TDV-2, TDV-3, and TDV-4 of phage-forming units (PFU). The placebo was phosphate-buffered saline; the TDV was reconstituted in water for injection at the time of administration and 0.5 mL of TDV or placebo (e.g., deltoid region) was injected subcutaneously. The samples were previously lyophilized in a stabilization formulation.

[0210] Blood samples for measuring neutralizing antibodies were collected from participants in the immunogenic subset at months 0 and 3, and months 1 and 6 prior to the administration of the study treatment and analyzed centrally.

[0211] One endpoint was immunogenicity, which was shown by the geometric mean titer (GMT) of neutralizing antibodies against each of the four DENV serotypes at months 1, 3, and 6 using the microneutralization test [MNT 50 . The secondary immunogenic endpoint was the seropositivity rate for each of the four DENV serotypes at months 1, 3, and 6 (where seropositivity was defined by the MNT 50 as a reciprocal neutralizing titer ≥ 10). The immunogenic endpoints were summarized for the per protocol set (PPS), which included all participants from the immunogenic subset who had no major protocol violations and for whom valid pre-dose and post-dose blood samples were available.

[0212] Statistical analysis

[0213] This trial was designed to be primarily descriptive and not based on testing formal null hypotheses. Therefore, the sample size was not determined based on a formal statistical power calculation. The planned sample size of 1800 participants (600 in the immunogenic subset) was considered to provide a reasonable number of participants for evaluating the persistence of the immune response after a single dose and the effect of the booster dose, and to provide an adequate safety database to support a phase 3 efficacy trial in approximately 20000 participants.

[0214] The GMT and seropositivity rate of dengue neutralizing antibodies were calculated at baseline and at months 1, 3, and 6 separately for each of the four TDV serotypes with a 95% confidence interval (CI). The percentages of participants with at least bivalent, trivalent, and tetravalent seropositivity were summarized by group at each study visit. These data were also presented by baseline dengue serostatus; seropositivity was defined as the reciprocal MNT for ≥ 1 DENV serotype 50≥10. Describe the safety data in summary, presenting the requested AEs by age (<6 and ≥6 years).

[0215] At month 6, 1743 participants received the planned regimen. The mean age of the study participants was 7.3 years (data not shown). The demographic data of the PPS (immunogenic subset) generally reflected the demographic data of the safety group, except that a smaller proportion in the PPS was aged 2 - 5 years than in the safety group. The proportion of participants seropositive for any DENV at baseline in the PPS was similar between study groups (overall 54.7%; range 51.2–57.4%). Among vaccinated participants, TDV elicited neutralizing antibodies against all 4 dengue serotypes. The highest levels of dengue neutralizing antibodies were observed against DENV-2 ( Figure 2 ), followed by DENV-1, and lower levels were observed against DENV-3 and DENV-4. The GMT for each serotype peaked at month 1 and remained elevated from baseline at month 6 (day 180). The month 6 GMTs (95% CI) in groups 1 - 4 were: DENV-1: 489 (321, 746), 434 (306, 615), 532 (384, 738), 62 (32, 120); DENV-2: 1565 (1145, 2140), 1638 (1286, 2088), 1288 (1031, 1610), 86 (44, 169); DENV-3: 160 (104, 248), 151 (106, 214), 173 (124, 240), 40 (23, 71); and DENV-4: 117 (79, 175), 110 (80, 149), 93 (69, 125), 24 (15, 38). Among participants seronegative at baseline, the two-dose schedule elicited higher DEN-3 and DEN-4 GMTs at month 6 (group 1) than the single dose given to participants in groups 2 and 3, although the CIs overlapped (data not shown). Among participants seropositive at baseline, the GMT responses were similar in all TDV groups (data not shown).

[0216] By month 1, the seropositivity rates for individual DENV among TDV-vaccinated participants in all study groups increased to 87.3–100%, and remained high for each DENV at month 6 (85.0–100%; Figure 3, (left panel). Among participants who were seronegative at baseline, the two-dose schedule led to higher seropositivity rates for DENV-3 (97·5%) and DENV-4 (87·5%) at month 6 than the one-dose schedule (85·7% and 85·3% for DENV-3 and 81·4% and 69·3% for DENV-4 – data not shown). However, the 95% CIs overlapped.

[0217] At baseline, 44·7% of participants were overall quadrivalent seropositive for DENV. By month 1, more than 80% of participants who received TDV vaccine in each study group were quadrivalent seropositive, and 96% or more were at least trivalent seropositive (data not shown). Multivalent seropositivity rates were maintained at month 6, with slightly higher rates 6 months after two TDV doses (data not shown). Among baseline seronegative participants, the 6-month quadrivalent seropositivity rate was higher in participants who received two doses (85% in group 1) than in those who received one dose (70% and 65·3% in groups 2 and 3), although the 95% CIs overlapped.

[0218] Overall, 1402 participants received one TDV dose and 194 received two doses. None of the 40 SAEs reported by 32 participants (i.e., 1·8% of the safety cohort) were related to the study vaccine or procedure. Three SAEs led to study withdrawal (an allergic reaction to food coloring, immune thrombocytopenic purpura, and acute glomerulonephritis). One death unrelated to the study vaccine or procedure occurred after the 6-month analysis cut-off (due to pneumonia, pulmonary tuberculosis, and septic shock). Two pregnancies led to study interruption, but the participants subsequently delivered normally and their infants were healthy.

[0219] No major differences in unsolicited AE rates were seen between TDV and placebo, either after the first dose versus the second dose or in relation to seropositivity at baseline, and most were not related to study vaccination (Table 3). Overall, 15 of 562 participants (2·7%) in the immunogenicity subset reported vaccine-related unsolicited AEs. Unsolicited AEs were mild in 161 of 186 participants (86·6%). Among baseline seropositive participants, relative to 1 of 47 (2 · 1%) who received placebo and 2 of 252 (0 ·8%),5 out of 250 (2.0%) and none out of 45 (0%) reported unsolicited AEs related to vaccination after the first and second TDV injections, respectively. Among the initially seronegative participants, 4 out of 202 (2.0%) and 1 out of 42 (2.4%) reported unsolicited AEs related to the vaccine after the first and second TDV injections, respectively, as compared to none out of 43 (0%) and 2 out of 203 (1.0%) who received placebo.

[0220] Seropositivity after vaccination can be an important measure of vaccine performance as it provides evidence of a measurable response to vaccination. In the absence of correlates of protection, it is of course impossible to say what magnitude of response is required for protection. However, a vaccine that elicits humoral and cellular immunity and shows measurable seroconversion to all dengue serotypes in the majority of individuals, even those without prior exposure to dengue, suggests that it is suitable for evaluation in large-scale vaccine efficacy trials and can be an effective vaccine against dengue virus in children and adolescents. The dosing schedule selected for the trial should generate a multivalent response in the largest proportion of initially seronegative subjects.

[0221] In this exemplary large study, the Phase 2 cohort was drawn from two dengue-endemic regions (Asia and Latin America) and approximated the real-world population that would be vaccinated with TDV. In this first study evaluating a 1-for-2 TDV dose given at 3-month intervals, the induced humoral immunogenicity was shown to remain robust at 6 months after the initial dose, only slightly reduced in initially seronegative vaccinees, but the administration of the second dose did help to mitigate these reductions (e.g., DENV-4) and increase the proportion of subjects with an immunological response to vaccination (e.g., DENV-3). Among 1596 participants vaccinated with TDV vaccine, no new safety concerns emerged within 6 months. Regardless of the dengue serostatus at vaccination, TDV was confirmed to be safe and well-tolerated in children and young people starting at 2 years of age.

[0222] Although other vaccines of flavivirus chimeras have been licensed in several regions, these are not approved for children under 9 years of age. These flavivirus chimeras induce 35 - 50% serotype-specific protection against DENV-1 and -2 and lower efficacy in dengue-naïve recipients. Thus, there remains a need for a safe and effective vaccine against all four DENV serotypes in recipients of all ages, particularly those less than 9 years old (regardless of prior dengue exposure and infecting serotype). The promising Phase 2 results of TDV support the initiation of a Phase 3 evaluation of a two-dose schedule in studies designed to support the use of TDV across a wide age range, regardless of dengue seropositivity at vaccination (particularly in young children).

[0223] Materials and Methods

[0224] Viruses and Cells

[0225] DENV-1 16007, DENV-2 16681, DENV-3 16562, and DENV-4 1034 served as wild-type (wt) DENV controls and they were the parental genotype viruses of four recombinant TDV vaccine candidates. The TDV ancestral research-level viruses designated D2 / 1-V, D2 PDK-53-VV45R, D2 / 3-V, and D2 / 4-V were previously prepared and characterized. Vero (African green monkey kidney) cells used for the preparation of the master and working cell banks for vaccine production were derived from the American Type Culture Collection (ATCC) CCL81 cell line, which has been characterized by the World Health Organization (WHO) for vaccine production (WCB-Vero cells).

[0226] Derivation of Live Recombinant TDV Viruses from cDNA Clones

[0227] To re-derive the candidate vaccine viruses under cGMP manufacturing conditions, the previously engineered DENV infectious cDNA clones pD2-PDK-53-VV45R, pD2 / 1-V, pD2 / 4-V, and in vitro ligated pD2 / 3-V containing the full-genome length viral cDNA were used to prepare fresh viral RNA transcripts by in vitro transcription as previously described. Briefly, the XbaI-linearized DENV genomic cDNA was treated with proteinase K, extracted with phenol / chloroform and precipitated in ethanol to remove any residual protein, and then suspended in RNase-free Tris-EDTA buffer prior to transcription. In vitro transcription was performed using the AmpliScribe T7 High Yield Transcription Kit (Epicenter Technologies) according to the manufacturer's recommended protocol. The RNA cap analog m7G(5’)ppp(5’)A (New England BioLabs) was incorporated during the 2-hour transcription reaction to add a 5'-terminal A-cap to the RNA transcripts. The samples were then treated with DNase I to digest the template cDNA, followed by low-pH phenol / chloroform extraction and ethanol precipitation to remove residual DNA and protein. The purified RNA transcripts suspended in RNase-free water were aliquoted into 20-μl portions and stored at -80 °C until ready to transfect cells. The integrity and concentration of the RNA transcripts were analyzed by agarose gel electrophoresis. Each 20-μl aliquot was estimated to contain sufficient genome-length viral RNA to allow transfection of 0.4 - 1 x 10 7 production-certified Vero cells.

[0228] Each RNA transcript was transfected into WCB-Vero cells in Shantha Biotechnics' cGMP facility. The TDV RNA transcript was thawed, mixed with 400 μl of Vero cell suspension (1 x 10 7 cells / ml), and transferred to a pre-chilled sterile electroporation dish (4-mm gap) and electroporated using the Gene Pulser Xcell total system (BioRad Laboratories). Each sample was pulsed once at 250 V / ∞ Ohms / 500 μF, incubated at room temperature for 10 - 15 minutes, transferred to a 75-cm 2 flask containing 30 ml of cell growth medium (MEM containing 10% FBS), and incubated at 36°C ± 1°C, 5% CO2 for 6 to 11 days. The medium was harvested, clarified by centrifugation, stabilized, and stored in small aliquots at below -60°C. The virus titer of the candidate vaccine bulk (designated P1, representing passage level 1) generated by transfection was determined by plaque titration assay in Vero cells and used for further propagation of the TDV seed.

[0229] Manufacture of TDV virus seeds

[0230] P1 virus seeds were used to propagate TDV pre-master, master, working, and bulk virus seed lots by a strategy designed to ensure optimal genetic stability and safety of the manufactured batches. The strategy included three consecutive plaque purifications, as well as genetic analysis of viruses at various passage levels to select the best clonal virus population for successive seed production (Table 1). Briefly, the P1 seeds harvested from transfected cells were amplified by infecting Vero cells at an MOI of 0.001 once to generate P2 seeds. Aliquots of the P2 seed bulk were evaluated by plaque morphology and complete viral genome sequencing. Genetically confirmed P2 bulk was inoculated onto a monolayer of Vero cells with overlay medium as described in the plaque titration section below to generate well-separated plaques. After visualization with neutral red, six individual plaques (plaque clones A to F) from each of the 4 vaccine virus serotypes were isolated and pooled into 0.5 ml of medium (passage P3). Each of the six plaque suspensions was subjected to two additional rounds of plaque purification to generate virus seeds with two and three plaque purifications at passages P4 and P5, respectively. The P5 virus was amplified by two consecutive Vero passages to generate the P7 seed bulk.

[0231] Perform genetic analysis of three major TDV attenuation loci using point sequencing and / or Taqman-based mismatch amplification mutation assay (TaqMAMA) as previously disclosed, and perform plaque phenotype analysis to screen all 24 P7 seeds. Then, further characterize the seeds with appropriate initial characteristics by complete genome sequencing. As a result of these analyses, one of the six (clone AF) P7 seeds of each TDV serotype was selected as the pre-master seed based on the presence of the DENV-2 PDK-53 attenuation mutation, minimal genomic sequence alterations, and the expected plaque phenotype. Amplify each selected pre-master seed into a master virus seed (MVS or P8) by passaging the virus once at an MOI of 0.001 in multiple 175 cm 2 Vero cell culture flasks. Except for TDV-4 MVS, harvest the master virus seeds at 8 - 10 days post-infection (pi). Harvest the MVS bulk at 6 - 10 days post-infection (pi), clarify by centrifugation, and stabilize by adding a sucrose / phosphate / glutamate solution (final concentrations of 7.5% sucrose, 3.4 mM potassium dihydrogen phosphate, 7.2 mM dipotassium hydrogen phosphate, 5.4 mM sodium glutamate) and 0.95 to 1.90% FBS (final concentration). Prepare TDV-4 MVS in a different manner to optimize its yield. Briefly, in 0.1% F-127 TM , poloxamer 407 (other EO-PO block copolymers have been evaluated and can be substituted herein, see the issued patent), infect multiple cell flasks with the TDV-4 pre-master seed at an MOI of 0.001. Harvest the infectious medium at 6 - 10 days post-infection, and stabilize with 17% FBS (final concentration), pool and freeze. Store all four TDV MVS bulks as 1 ml aliquots at below -60 °C.

[0232] Prepare TDV working virus seeds (WVS) by passaging once at an MOI of 0.001 in the Vero cell culture of MVS. The procedures are similar to the production of MVS, except that they are cultured in multi-layer cell factories (6360 cm 2 ). Filter the WVS bulk through 10 μM and 0.45 μM filters, stabilize with the same stabilizer used for MVS, aliquot into 30 ml PETG bottles or 2.0 ml cryovials, and store at below -60 °C.

[0233] In some methods, produce bulk virus seeds (BVS) by infecting multiple cell factories (each 6360 cm 2 ) of confluent Vero cells with 90 mL of diluted WVS to obtain an MOI of 0.001. The medium used to dilute the WVS inoculum contains 0.1% F-127 TM, serum-free. After 1.5 hours of adsorption, the cells were washed 3 times with PBS, and 800 ml of serum-free DMEM medium was added to each cell factory, and the factory was incubated at 36 (±1) °C in 5 (±0.5) % CO2. After 4 days of incubation, a small aliquot of the medium was collected for sterility testing. The virus was harvested between 5th and 10th day post-infection, immediately clarified by passing through a 0.45 μm pore size filter, and stabilized by adding 500 ml of 3x FTA buffer (PBS, 15% trehalose, 1.0% F-127 TM poloxamer 407, 0.1% human serum USP) per liter of each clarified virus pool. The stabilized virus was aliquoted into 1-L PETG bottles and stored frozen at below -60 °C for subsequent pooling and quality control testing. All stabilized virus harvests with virus titers higher than 10 5 PFU / ml and acceptable residual DNA levels were rapidly thawed in a water bath at 32 °C, then aseptically pooled and mixed. Each pooled monovalent BVS was aliquoted into labeled PETG containers and stored at below -60 °C until further use.

[0234] Manufactured Product Quality Control

[0235] Test the identity, sterility, and detectable adventitious agents of MVS, WVS, and BVS seeds. Confirm the identity of each vaccine bulk by RT-PCR using TDV serotype-specific primers. The amplified cDNA fragment contains the E / NS1 chimeric junction site to identify each of the four TDV serotypes. Each seed was tested in all 4 serotype-specific RT-PCR reactions to confirm virus identity and freedom from cross-contamination with heterologous TDV serotypes. Sterility testing was performed according to USP 71 (United States Pharmacopeia, Section 71). Mycoplasma testing was performed.

[0236] The following in vitro and in vivo tests for virus contamination were performed using the unclarified and unstabilized TDV harvests collected during seed production. The harvested infectious medium was first neutralized with DENV rabbit polyclonal antiserum (Inviragen) for 1 hour at 36 ± 1 °C to inactivate DENV. For in vitro testing, the neutralized seed was inoculated into 25 cm 2In three indicator cell lines MRC5, VERO, and MA104 in flasks. Echovirus (CPE control) or mumps virus (hemadsorption control) was used as the positive CPE or hemadsorption control, respectively. The CPE of all cells was monitored daily for a total of 14 days. At the end of 14 days, the culture supernatant was removed and replaced with 10 mL of guinea pig red blood cell (RBC) solution (3 mL of 0.5% guinea pig RBC in phosphate-buffered saline, made up to 10 mL with cell growth medium). The flasks were then incubated at 5 ± 3 °C for 30 minutes and then at room temperature for 30 minutes. The monolayer was washed with PBS, and the presence of any star-shaped RBC clumps for hemadsorption was observed at 10X magnification.

[0237] In vivo tests of the foreign agent were conducted in suckling mice, weaned mice, and guinea pigs. Suckling mice were inoculated intraperitoneally (ip) with 0.1 mL or 0.01 mL of the seed sample neutralized with DENV-antiserum (10 mice per dose group). Similarly, 10 weaned mice were each inoculated intraperitoneally with 0.5 mL or 0.03 mL of the sample. Each guinea pig (5 per group) was inoculated intraperitoneally with 5.0 mL. The morbidity and mortality of suckling mice were observed daily for a total of 14 days after inoculation. After inoculation, weaned mice were observed for a total of 28 days, and guinea pigs were observed for a total of 42 days. If ≥80% of the inoculated animals remained healthy throughout the observation period, the test article met the acceptance criteria.

[0238] In vivo tests for contaminants were also conducted in embryonated eggs. For each sample, 10 embryonated eggs (9 days old) were each inoculated with 0.5 mL of the sample neutralized with DENV antiserum into the allantoic fluid and incubated at 35 °C for 3 days. The allantoic fluid from these 10 eggs was collected, pooled, and passaged into the allantoic fluid of 10 fresh embryonated eggs (10 - 11 days old; 0.5 mL / egg) and incubated at 35 °C for another 3 days. Similarly, for each sample, 10 embryonated eggs (6 - 7 days old) were each inoculated with 0.5 mL / egg (TDV-2 monovalent BVS) or 0.25 mL / egg (TDV-1, TDV-3, and TDV-4 BVS) by injection into the yolk sac and incubated at 35 °C for 9 days. The yolk sacs from these 10 eggs were collected and pooled, and a 10% suspension was passaged into the yolk sacs of 10 fresh embryonated eggs (6 - 7 days old; 0.5 mL / egg) and incubated at 35 °C for another 9 days. The survival rate of the eggs inoculated into the allantoic fluid (both initial and passaged inoculations) was observed after 3 days of incubation. The hemagglutination activity of the two pooled allantoic fluids was tested using chicken, guinea pig, and human type O red blood cells at 4 °C and 25 °C. The survival rate of the eggs inoculated into the yolk sac (both initial and passaged inoculations) was observed after 9 days of incubation.

[0239] Viral plaque assay and immunofocus assay

[0240] The viral titers were measured using Vero cells by plaque assay or immunofocus assay. As previously described, plaque assays were performed in a double-agarose overlay in six-well plates of confluent Vero cells, and they were also used to evaluate the plaque phenotype of the TDV seed. For accurate comparison, the plaque sizes of all viruses were measured and compared in the same experiment. After visualization with neutral red on day 9 post-infection, the average plaque size of up to 10 well-separated plaques of each virus was measured and calculated. The smaller plaques of wt DENV-1, -3, and -4 were measured, as their larger plaque sizes generally did not allow measurement of 10 well-separated plaques.

[0241] Since the quadrivalent TDV contains all four DENV serotypes, DENV serotype-specific immunofocus assays were developed to quantify each TDV component in the quadrivalent formulation. The immunofocus assay of each individual TDV MVS was compared with the plaque assay to ensure comparable virus titration results between the two assays. The immunofocus assay was performed in six-well plates of confluent Vero cells infected with serial dilutions of the virus. The cells were overlaid with balanced salt medium (BSS / YE-LAH medium) containing 0.7% high-viscosity carboxymethylcellulose (Sigma) and incubated at 37 °C, 5% CO2 for 7 days. After removal of the overlay, the cell sheets were washed three times with PBS, fixed with cold 80% acetone at -20 °C for 30 minutes, washed once with PBS, and blocked with blocking buffer containing 2.5% (w / v) non-fat dry milk, 0.5% Triton X-100, 0.05% Tween-20 in PBS at 37 °C for 30 minutes. The blocked cells were incubated with diluted DENV serotype-specific MAbs, 1F1 (DENV-1), 3H5 (DENV-2), 8A-1 (DENV-3), or 1H10 (DENV-4) in blocking buffer at 37 °C for 1 hour or overnight at 4 °C, washed three times with wash buffer (0.05% Tween-20 in PBS), and incubated with affinity-purified goat anti-mouse IgG conjugated to alkaline phosphatase or horseradish peroxidase (HRP) (Jackson Immuno Research Laboratories) at 37 °C for 45 - 60 minutes. The plates were washed three times and then the appropriate substrate was added, 1-step NBT / BCIP plus inhibitor (Pierce) for alkaline phosphatase or Vector-VIP kit (Vector Labs) for HRP for color development. When the foci were fully developed, the color development was stopped by rinsing with water. The stained immunoglobulins were visualized directly and counted on a light box.

[0242] Gene sequence

[0243] Sequencing of the full-length genomes of MVS and WVS was performed (see below). Briefly, viral RNA was extracted from TDV seeds using the QIAamp Viral RNA kit (Qiagen), and overlapping cDNA fragments covering the entire genome were amplified using the Titan One Tube RT-PCR kit (Roche Applied Science, Inc.). The amplified cDNA fragments were gel purified and then sequenced using both forward and reverse primers with the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems). The sequence reactions were cleaned using the BigDye XTerminator purification kit (Applied Biosystems) and run on the 3130xl Genetic Analyzer (Applied Biosystems) at DVBD / CDC. Lasergene SeqMan software (DNAStar, Inc) was used for genome analysis and comparison.

[0244] Taqman-based Mismatch Amplification Mutation Assay (TaqMAMA)

[0245] TaqMAMA is a sensitive quantitative single nucleotide polymorphism assay that was developed to allow for a more refined assessment of the revertant levels at the 5'NC-57 attenuation site and was further optimized for this study. Viral RNA extracted from MVS and WVS was analyzed by TaqMAMA using two sets of primers / Taqman probes specific for the wt or vaccine 5'NC-57 region. The forward primers for detecting DENV-2 wt and vaccine sequences were D2-41-GC and D2-40-TT, respectively. The 3'-terminal nucleotide of each forward primer matched the specific 5'NCR-57 nucleotide of each virus, while the nucleotide adjacent to the 3'-terminal nucleotide in each primer differed from the DENV-2 viral genomic sequence to enhance the mismatch effect. The reverse primer CD-207 and Taqman probe CD-169F were the same for both the wt and vaccine groups. The sequences of the primers and probes and the cycling conditions have been previously described. Real-time RT-PCR was performed in a 25-μl reaction containing 5 μl of viral RNA template, 0.4 μM of each primer, and 0.2 μM of probe using an iR5 or CFX-95 system (BioRad) with the BioRadiScript RT-PCR (for probes) kit. Triplicate reactions for each wt and vaccine-specific assay were performed for each sample. Genomic copy numbers were determined relative to a standard curve prepared for each viral genotype, where the RNA standards were transcripts from plasmids containing nt 1-2670 of each genotype-specific cDNA. In addition, the specificity of the assay was confirmed by testing each RNA standard with a heterologous genotype primer / probe set to ensure minimal cross-reactivity in each experiment. Results were reported as the percentage of viral genomes showing revertants. Previously, due to a higher cross-reactive background that limited the input RNA levels for this assay, the initial detection sensitivity was approximately 0.1% revertants (discrimination ability). Since then, the assay has been further optimized using improved real-time PCR equipment and reaction kits, and the cross-reactive background has been considerably reduced at much higher levels of RNA template input (7-8 log 10 copies). This optimization has led to a significant improvement in detection sensitivity, down to 0.01-0.07% revertants.

[0246] Viral Replication in Mosquito C6 / 36 Cells and Temperature Sensitivity in Mammalian Vero Cells

[0247] The replication phenotypes of four TDVMVS stocks and wt DENV-1, -2, -3, and -4 viruses were evaluated in C6 / 36 mosquito cells (Aedes albopictus). C6 / 36 cells cultured in 6-well plates were infected in duplicate with each virus at an MOI of 0.001 and incubated at 28 °C in a 5% CO2 incubator with DMEM medium containing 2% FBS at 4 ml / well. At 6 days post-infection, small aliquots of the culture supernatants were collected for each virus, mixed with an equal volume of medium containing 40% FBS, and stored at -80 °C until ready for virus plaque titration.

[0248] Temperature sensitivity was determined by comparing virus growth at 39 °C with that at 37 °C at 5 days post-infection in Vero cells in 6-well plates. Cells were infected in quadruplicate with each virus at an MOI of 0.001 at 37 °C. After virus adsorption, the infected cultures were incubated with DMEM medium containing 2% FBS at 4 ml / well in 2 different 5% CO2 incubators, one set (duplicate plates) at 37 °C and the other at 39 °C. At 5 days post-infection, aliquots (50-μl) of the culture supernatants were collected, mixed with an equal volume of DMEM containing 40% FBS, and stored at -80 °C until ready for virus plaque titration. The incubator temperatures were calibrated with an NIST-traceable factory-calibrated thermometer (-1 to 51 °C; ERTCO).

[0249] Mosquito infection, dissemination, and transmission

[0250] Aedes aegypti mosquitoes used in the study were from a colony established in 2002 from a village near Mae Sot, Thailand (16’N, 33’E). After emergence from larvae, adult mosquitoes were maintained at 28 °C on a 16:8 (light:dark) photoperiod with 10% sucrose solution provided ad libitum. Five- to seven-day-old female mosquitoes were used for infectious blood meal feeding or intrathoracic (IT) inoculation. Freshly cultured aliquots of TDV and wt DENV were used immediately after harvest (without any freeze-thaw cycles) to prepare virus blood meals for oral infection as described below. The remaining virus supernatants were supplemented with FBS to a final concentration of 20%, and aliquots were stored at -80 °C for future virus plaque titration and IT inoculation experiments. Freshly prepared TDV seeds used in these experiments were amplified from the pre-master seed in Vero cells and were considered TDV MVS equivalents.

[0251] Infectious blood meal was prepared by mixing fresh virus with defibrinated chicken blood (Colorado Serum Company) at a 1:1 ratio on the day of oral infection. Mosquitoes were sugar-starved overnight and then provided with the virus:blood mixture for 1 hour using a Hemotek membrane feeding system (Discovery Workshops). Fifty microliter aliquots of the blood meal were retained at -80 °C for back titration of the virus dose. Fully engorged females were sorted under cold anesthesia and placed in cardboard boxes with 10% sucrose solution provided ad libitum. The cardboard boxes were placed at 28 °C with a photoperiod of 16:8 h (light:dark). Fourteen days later, 25 - 30 mosquitoes from each virus group were anesthetized by exposure to triethylamine ( Carolina Biological Supply Company) and one hind leg was removed and placed in 0.5 ml DMEM containing 10% FBS and 5% penicillin / streptomycin (100 U / ml and 100 μg / ml, respectively). Saliva was collected by inserting the proboscis of the anesthetized mosquito into a capillary tube containing 2.5% FBS and 25% sucrose solution. The mosquitoes were allowed to salivate for at least 15 minutes and then the capillary tube and the body were placed in separate tubes containing DMEM. The mosquito bodies, legs, and saliva were stored at -80 °C until they were homogenized and assayed for infectious virus. For IT inoculation, the mosquitoes were cold anesthetized and inoculated with approximately 50 pfu of virus in 0.34 μl of inoculum. The inoculated mosquitoes were kept for 7 days under the same conditions as described above. Then the mosquitoes were anesthetized and their saliva and bodies were collected as described above. The samples were stored at -80 °C until further processing.

[0252] To process samples for virus titration, the body and leg samples were homogenized at 24 cycles per second for 4 minutes using a Mixer Mill with copper-coated BBs (Crossman Corporation, NY) and then clarified by centrifugation at 3,000 x g for 3 minutes. The saliva samples were centrifuged at 3,000 × g for 3 minutes to expel the fluid from the capillary tubes. Ten-fold dilutions of the body and leg homogenates and saliva samples were tested for the presence of infectious virus by plaque assay. Results from the body, leg, and saliva were used to determine the infection, dissemination, and transmission rates, respectively.

[0253] Mouse neurovirulence

[0254] Timed pregnant female ICR mice were obtained from Taconic Labs and monitored several times a day to determine the approximate time of birth of a litter of pups. In a given experiment, approximately 12 - 24 hours after birth, intracranial (ic) inoculation using a 30-gauge needle was performed with 10 in 20 μl of diluent 3 to 10 4The virus of pfu attacks 8 offspring in each of the two litters of each virus (n = 16). The animals were monitored at least 3 times a day after the attack for at least 32 days. At the first signs of the disease (rough fur, hunchback, weight loss, abnormal movement, paralysis or lethargy), the animals were euthanized by lethal anesthesia with isoflurane gas followed by cervical dislocation. The number of days post-infection at euthanasia represents the "time to disease / onset" or "survival time" of the animals. Animal experiments were conducted according to the animal protocol approved by DVBD / CDCIACUC.

[0255] Derivation of the master seed virus

[0256] TDV - 1 Master Virus Seed (MVS)

[0257] This article provides the nucleotide sequences of the chimeric virus genomes and the deduced amino acid sequences of the translated proteins. For example, the TDV-1 polynucleotide sequences include those represented by SEQ ID NO:1, 3, 5, and 7, and the TDV-1 polypeptide sequences include those represented by SEQ ID NO:2, 4, 6, and 8. Most of the prM-E gene (nt 457 to -2379, underlined) is specific to the wild-type (wt) DEN-1 16007 virus; the remaining genome is specific to the DEN-2 PDK-53 virus. All engineered substitutions are different from the wt viruses (D1 16007 or D2 16681), and additional mutations detected in the MVS (changes from the engineered cDNA clones) are marked.

[0258] Substitutions included in the genome and protein:

[0259] Junction sites between D1 (prM-E) and D2 backbone:

[0260] a. MluI (nt 451-456): Engineered silent mutation, nt -453A to G

[0261] b. NgoMIV (nt 2380-2385): Engineered mutation, nt -2381 / 2382TG to CC (resulting in E-482Val to Ala change)

[0262] D2 PDK-53 virus backbone (changed from wt D2 16681): All in bold

[0263] a. 5'-untranslated region (NCR)-57 (nt -57C to T): Major attenuation locus (in red)

[0264] b. NS1-53 Gly to Asp (nt -2579G to A): Major attenuation locus (in red)

[0265] c. NS2A-181 Leu to Phe (nt-4018 C to T)

[0266] d. NS3-250 Glu to Val (nt-5270 A to T): major attenuation locus (in red)

[0267] e. nt-5547 (NS3 gene) T to C silent mutation

[0268] f. NS4A-75 Gly to Ala (nt-6599 G to C)

[0269] * The nt-8571 C to T silent mutation of PDK-53 was not engineered in the vaccine virus

[0270] DEN-1 prM-E (altered from wt D1 16007)

[0271] a. Engineered nt-1575 T to C silent mutation to remove the native XbaI site

[0272] Additional substitutions found in the vaccine seed (0.03% nt different from the initial clone)

[0273] TDV-2 master virus seed (MVS)

[0274] a. NS2A-116 Ile to Leu (nt-3823 A to C, in bold)

[0275] b. NS2B-92 Glu to Asp (nt-4407 A to T, in bold)

[0276] c. nt-7311 A to G silent mutation (in bold)

[0277] The nucleotide sequences of the chimeric virus genomes and the deduced amino acid sequences of the translated proteins are provided herein. For example, the TDV-2 polynucleotide sequences include those represented by SEQ ID NO: 9, 11, 13, and 15, and the TDV-2 polypeptide sequences include those represented by SEQ ID NO: 10, 12, 14, and 16. The engineered virus is based on the D2 PDK-53 virus. All engineered substitutions that differ from the wild-type DEN-2 16681 virus (which is also the parental virus of PDK-53), as well as the additional mutations detected in the MVS (changes from the engineered cDNA clone), are marked.

[0278] Substitutions contained in the genome and protein:

[0279] D2 PDK-53 virus backbone (altered from wt D2 16681): all in bold

[0280] a. 5'-untranslated region (NCR) - 57 (nt - 57C to T): major attenuation locus (in red)

[0281] b. prM - 29Asp to Val (nt - 524A to T)

[0282] c. nt - 2055C to T (E gene) silent mutation

[0283] d. NS1 - 53 Gly to Asp (nt - 2579G to A): major attenuation locus (in red)

[0284] e. NS2A - 181Leu to Phe (nt - 4018C to T)

[0285] f. NS3 - 250 Glu to Val (nt - 5270A to T): major attenuation locus (in red)

[0286] g. nt - 5547 (NS3 gene) T to C silent mutation

[0287] h. NS4A - 75Gly to Ala (nt - 6599G to C)

[0288] *The nt - 8571C to T silent mutation of PDK - 53 was not engineered in the vaccine virus

[0289] Engineered cloning markers (silent mutations):

[0290] a. nt - 900T to C silent mutation: infectious clone marker

[0291] Additional substitutions found in the vaccine seed (0.02% nt different from the initial clone)

[0292] a. prM - 52Lys to Glu (nt - 592A to G), in bold

[0293] b. NS5 - 412 Ile to Val (nt - 8803A to G), in bold

[0294] TDV - 3 master virus seed (MVS)

[0295] This document provides the nucleotide sequences of chimeric viral genomes and the deduced amino acid sequences of the translated proteins. For example, the TDV-3 polynucleotide sequences include those represented by SEQ ID NO: 17, 19, 21, and 23, and the TDV-3 polypeptide sequences include those represented by SEQ ID NO: 18, 20, 22, and 24. Most of the prM-E gene (nt -457 to -2373, underlined) is specific to the wild-type (wt) DEN-3 16562 virus; the remaining nucleotide sequence is specific to the DEN-2 PDK-53 virus. The E protein of the DEN-3 virus has two fewer amino acids than the E protein of DEN-2. Therefore, the nt position starting from NgoMIV is 6 nt smaller than the initial DEN-2 PDK-53 nt position. All engineered substitutions are different from the wt virus (DEN-3 16562 or DEN-2 16681), and additional mutations (changes from the engineered cDNA clones) are marked.

[0296] Substitutions contained in the genome and protein:

[0297] Junction points:

[0298] a. MluI (nt 451-456): Engineered silent mutation, nt -453 A to G

[0299] b. NgoMIV (nt 2374-2379): Engineered mutation, nt -2375 / 2376 TG to CC (resulting in E-480 Val to Ala change)

[0300] D2 PDK-53 virus backbone (altered from wt D2 16681): In bold

[0301] a. 5'-untranslated region (NCR)-57 (nt -57 C to T): Major attenuation locus (in red)

[0302] b. NS1-53 Gly to Asp (nt -2573 G to A): Major attenuation locus (in red)

[0303] c. NS2A-181 Leu to Phe (nt -4012 C to T)

[0304] d. NS3-250 Glu to Val (nt -5264 A to T): Major attenuation locus (in red)

[0305] e. nt -5541 (NS3 gene) T to C silent mutation

[0306] f. NS4A-75 Gly to Ala (nt -6593 G to C)

[0307] *The nt-8565 C to T silent mutation of PDK-53 is not engineered in the vaccine virus

[0308] Engineered mutations in DEN-3 prM-E (changes from wt D3 16562)

[0309] a. Engineered nt-552 C to T silent mutation: Cloning marker

[0310] b. Engineered E-345 His to Leu (nt-1970 A to T) for efficient replication in culture of additional substitutions found in the vaccine seed (0.02% nt different from the initial clone)

[0311] a. E-223 Thr to Ser mutation (nt-1603 A to T, in bold)

[0312] b. nt-7620 A to G silent mutation (in bold)

[0313] TDV-4 master virus seed (MVS)

[0314] The nucleotide sequences of the chimeric virus genomes and the deduced amino acid sequences of the translated proteins are provided herein. For example, the TDV-4 polynucleotide sequences include those represented by SEQ ID NO: 25, 27, 29, and 31, and the TDV-4 polypeptide sequences include those represented by SEQ ID NO: 26, 28, 30, and 32. Most of the prM-E gene (nt-457 to -2379, underlined) is specific to the wild-type (wt) DEN-4 1036 virus; the remaining nucleotide sequences are specific to the DEN-2 PDK-53 virus. All engineered substitutions are different from the wt virus (DEN-3 16562 or DEN-2 16681), and additional mutations (changes from the engineered cDNA clone) are marked.

[0315] Substitutions included in the genome and protein:

[0316] Junction points:

[0317] a. MluI (nt 451-456): Engineered silent mutation, nt-453 A to G

[0318] b. NgoMIV (nt 2380-2385): Engineered mutation, nt-2381 / 2382 TG to CC (resulting in E-482 Val to Ala change)

[0319] D2 PDK-53 virus backbone (changes from wt D2 16681)

[0320] a. 5'-untranslated region (NCR) - 57 (nt - 57C to T): major attenuation locus (in red)

[0321] b. NS1 - 53 Gly to Asp (nt - 2579G to A): major attenuation locus (in red)

[0322] c. NS2A - 181 Leu to Phe (nt - 4018C to T, in bold)

[0323] d. NS3 - 250 Glu to Val (nt - 5270A to T): major attenuation locus (in red)

[0324] e. nt - 5547 (NS3 gene) T to C silent mutation (in bold)

[0325] f. NS4A - 75 Gly to Ala (nt - 6599G to C, in bold)

[0326] * The nt - 8571C to T silent mutation of PDK - 53 was not engineered in the vaccine virus

[0327] Engineered substitutions in the cDNA clone

[0328] a. Engineered C - 100 Arg to Ser (nt - 396A to C): can improve virus replication in culture

[0329] b. Engineered nt - 1401A to G silent mutation

[0330] c. Engineered E - 364 Ala to Val (nt - 2027C to T): can improve virus replication in culture

[0331] d. Engineered E - 447 Met to Leu (nt - 2275A to C): can improve virus replication in culture

[0332] Additional substitutions found in the vaccine seed (0.06% nt different from the initial clone)

[0333] a. nt - 225 (C gene) A to T silent mutation (in bold)

[0334] b. NS2A - 66 Asp to Gly (nt - 3674A to G) mutation (in bold)

[0335] c. NS2A - 99 Lys to Lys / Arg mixture (nt - 3773A to A / G mixture, in bold)

[0336] d. nt - 5391C to T (NS3 gene) silent mutation (in bold)

[0337] e. NS4A-21 Ala to Val (nt-6437 C to T, bolded)

[0338] f. nt-7026 T to C / T mixture silent mutation (bolded)

[0339] g. nt-9750 A to C silent mutation (bolded)

[0340] *******************

[0341] According to the present disclosure, all of the compositions and methods disclosed and claimed herein can be made and implemented without undue experimentation. Although the compositions and methods have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the compositions and methods and to the steps or the sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically related reagents can be substituted for the reagents described herein while the same or similar results would be obtained. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

Claims

1. A method for treating children or young adults against dengue virus infection, comprising: Administering a pharmaceutical composition to the child or young person, the pharmaceutical composition comprising at least one of a live attenuated dengue virus and a dengue-dengue chimeric virus, wherein administering the pharmaceutical composition comprises administering a first dose of the pharmaceutical composition on day 0 and administering at least a second dose of the same or a different pharmaceutical composition against dengue virus within 180 days of the first administration, wherein the composition induces an immune response against dengue virus in the child or young person.

2. The method according to claim 1, wherein the pharmaceutical composition comprises all four dengue virus serotypes, i.e., a tetravalent formulation, and the pharmaceutical composition induces an immune response against the four dengue virus serotypes in the child or young person.

3. The method according to claim 1 or 2, wherein the pharmaceutical composition comprises at least one of the following: A polynucleotide comprising a nucleic acid sequence encoding a live attenuated dengue-2 virus serotype, the nucleic acid sequence being represented by SEQ ID NO: 9 (premajor), 11 (MVS), 13 (WVS), or 15 (BVS); A dengue-1 / dengue-2 chimeric polynucleotide comprising a nucleic acid sequence encoding a non-structural protein from a modified live attenuated dengue-2 virus serotype and a structural protein from a dengue-1 virus serotype, the nucleic acid sequence being represented by SEQ ID NO: 1 (premajor), 3 (MVS), 5 (WVS), or 7 (BVS); A dengue-3 / dengue-2 chimeric polynucleotide comprising a nucleic acid sequence encoding a non-structural protein from a modified live attenuated dengue-2 virus serotype and a structural protein from a dengue-3 virus serotype, the nucleic acid sequence being represented by SEQ ID NO: 17 (premajor), 19 (MVS), 21 (WVS), or 23 (BVS); and A dengue-4 / dengue-2 chimeric polynucleotide comprising a nucleic acid sequence encoding a non-structural protein from a modified live attenuated dengue-2 virus serotype and a structural protein from a dengue-4 virus serotype, the nucleic acid sequence being represented by SEQ ID NO: 25 (premajor), 27 (MVS), 29 (WVS), or 31 (BVS).

4. The method according to any one of claims 1-3, wherein the pharmaceutical composition comprises all four dengue virus serotypes, and wherein the concentration ratio of the polynucleotide encoding the live attenuated dengue-2 virus serotype in the composition is at least 0.5 log PFU lower than the log (log) plaque forming units (PFU) of one or more other dengue virus serotypes, and wherein the immunogenic composition elicits a balanced immune response against all four dengue virus serotypes in the subject.

5. The method according to any one of claims 1-4, further comprising one or more polypeptides encoded by the polynucleotide of claim 3.

6. The method according to claim 5, wherein the polypeptide corresponding to the modified live attenuated dengue-2 virus serotype is represented by SEQ ID NO.10 (premajor), SEQ ID NO.12 (MVS), SEQ ID NO.14 (WVS), or SEQ ID NO.16 (BVS); wherein the polypeptide corresponding to the dengue-1 / dengue-2 chimeric polynucleotide is represented by SEQ ID NO.2 (premajor), SEQ ID NO.4 (MVS), SEQ ID NO.6 (WVS), or SEQ ID NO.8 (BVS); wherein the polypeptide corresponding to the dengue-3 / dengue-2 chimeric polynucleotide is represented by SEQ ID NO.18 (premajor), SEQ ID NO.20 (MVS), SEQ ID NO.22 (WVS), or SEQ ID NO.24 (BVS); and wherein the polypeptide corresponding to the dengue-4 / dengue-2 chimeric polynucleotide is represented by SEQ ID NO.26 (premajor), SEQ ID NO.28 (MVS), SEQ ID NO.30 (WVS), or SEQ ID NO.32 (BVS).

7. The method according to any one of claims 1 to 6, wherein the ratio of the dengue-1 / dengue-2 chimera to the live attenuated dengue-2 virus serotype to the dengue-3 / dengue-2 chimera to the dengue-4 / dengue-2 chimera is 4:4:5:5 plaque forming units (PFU).

8. The method according to any one of claims 1 to 5, wherein the ratio of the dengue-1 / dengue-2 chimera to the live attenuated dengue-2 virus serotype to the dengue-3 / dengue-2 chimera to the dengue-4 / dengue-2 chimera is 5:4:5:5 PFU.

9. The method according to any one of claims 7 or 8, wherein a dose is administered to the child or young person on day 0, and a booster is administered 120 days or less after the dose on day 0, and an immune response to all four dengue virus serotypes is induced in the subject.

10. The method according to claim 9, wherein two doses of the immunogenic composition are administered to the subject within 30 days or within 30, 60, 90, or 120 days of each other.

11. The method according to any one of claims 1-10, which comprises administering the pharmaceutical composition to a child or young person aged 1 to 20 years.

12. The method according to any one of claims 1-11, which comprises administering the pharmaceutical composition to a child or young person aged 2 to 17 years.

13. The method according to any one of claims 1-12, which comprises administering the pharmaceutical composition to a child or young person aged 1 to 11 years.

14. The method according to any one of claims 1-13, which comprises administering the pharmaceutical composition to a child or young person aged 1.5 to 9 years.

15. A method according to any one of claims 1-6 and 9-14, wherein the pharmaceutical composition comprises a formulation comprising at least one of a dengue-1 / dengue-2 chimera having a concentration of 1.0 x 10 3 - 5 x 10 5 pfu; a live attenuated dengue-2 having a concentration of 1.0 x 10 3 - 5 x 10 5 pfu; a dengue-3 / dengue-2 chimera having a concentration of 5.0 x 10 3 - 5 x 10 5 pfu; and a dengue-4 / dengue-2 chimera having a concentration of 1.0 x 10 4 - 5 x 10 6 pfu.

16. A method according to any one of claims 1-6 and 9-15, wherein the pharmaceutical composition comprises a tetravalent formulation, the tetravalent formulation comprising 2.5 x 10 4 pfu of dengue-1 / dengue-2 chimera, 6.3 x 10 3 pfu of live attenuated dengue-2, 3.2 x 10 4 pfu of dengue-3 / dengue-2 chimera and 4.0 x 10 5 pfu of dengue-4 / dengue-2 chimera.

17. The method according to any one of claims 1-16, which comprises administering the pharmaceutical composition to the child or young person subcutaneously, intravenously, intradermally, intradermally, transdermally, orally, by inhalation, vaginally, topically, intranasally or rectally.

18. The method according to any one of claims 1-17, wherein the pharmaceutical composition comprises a tetravalent composition and elicits an immune response against all four dengue virus serotypes in at least 60% of the children or young persons who receive one or more doses of the immunogenic composition.

19. The method according to any one of claims 1-18, wherein at least one of the live attenuated dengue virus and the dengue-dengue chimeric virus further comprises a stabilizing buffer to reduce degradation of the dengue virus.

20. The method according to claim 20, wherein the stabilizing buffer comprises trehalose and albumin and optionally poloxamer 407.

21. The method according to claim 20, wherein poloxamer 407 has a concentration of 0.1 to 3.0% (w / v), trehalose has a concentration of 5.0 to 50% (w / v), and albumin has a concentration of 0.01 to 3.0% (w / v).

22. The method according to any one of claims 1-8 and 11-21, wherein the child or young person is seronegative or unimmunized against the dengue virus prior to administration of the pharmaceutical composition.

23. The method according to any one of claims 1-8 and 11-22, wherein the child or young person is seropositive for the dengue virus prior to administration of the pharmaceutical composition.

24. The method according to claim 23, wherein the child or young person is seropositive, and the pharmaceutical composition is administered to the seropositive child or young person on day 0 without subsequent booster administration.

25. The method according to claim 22, wherein the child or young person is traveling to a dengue endemic area for the first time.

26. The method according to any one of claims 1-25, wherein at least 60% of the children or young persons are seropositive for all four dengue virus serotypes after administration of the pharmaceutical composition.

27. The method according to any one of claims 1-26, wherein at least 80% of the children or young persons are seropositive for all four dengue virus serotypes after administration of the pharmaceutical composition.

28. A method for treating children aged 1.5 to 11 years for dengue virus infection, comprising: A pharmaceutical composition is administered to the child, the pharmaceutical composition comprising at least one of a live attenuated dengue virus and a dengue-dengue chimeric virus, wherein administering the pharmaceutical composition comprises administering a first dose of the pharmaceutical composition on day 0 and administering at least a second dose of the pharmaceutical composition within 180 days after the first dose, wherein the composition induces an immune response against the dengue virus in human children.

29. The method according to claim 28, wherein the pharmaceutical composition comprises a formulation, the formulation comprising at least one of a dengue-1 / dengue-2 chimera having a concentration of 1.0 x 10 3 - 5 x 10 5 pfu; a live attenuated dengue-2 having a concentration of 1.0 x 10 3 - 5 x 10 5 pfu; a dengue-3 / dengue-2 chimera having a concentration of 5.0 x 10 3 - 5 x 10 5 pfu; and a dengue-4 / dengue-2 chimera having a concentration of 1.0 x 10 4 - 5 x 10 6 pfu.

30. The method according to claim 28 or 29, wherein the pharmaceutical composition comprises at least one of the following: A polynucleotide comprising a nucleic acid sequence encoding a live attenuated dengue-2 virus serotype, said nucleic acid sequence being represented by SEQ ID NO:9 (premajor), 11 (MVS), 13 (WVS) or 15 (BVS); A dengue-1 / dengue-2 chimeric polynucleotide comprising a nucleic acid sequence encoding non-structural proteins from a modified live attenuated dengue-2 virus serotype and structural proteins from a dengue-1 virus serotype, said nucleic acid sequence being represented by SEQ ID NO:1 (premajor), 3 (MVS), 5 (WVS) or 7 (BVS); A dengue-3 / dengue-2 chimeric polynucleotide comprising a nucleic acid sequence encoding non-structural proteins from a modified live attenuated dengue-2 virus serotype and structural proteins from a dengue-3 virus serotype, said nucleic acid sequence being represented by SEQ ID NO:17 (premajor), 19 (MVS), 21 (WVS) or 23 (BVS); and A dengue-4 / dengue-2 chimeric polynucleotide comprising a nucleic acid sequence encoding non-structural proteins from a modified live attenuated dengue-2 virus serotype and structural proteins from a dengue-4 virus serotype, said nucleic acid sequence being represented by SEQ ID NO:25 (premajor), 27 (MVS), 29 (WVS) or 31 (BVS).

31. The method according to any one of claims 28-30, wherein the child is between 2 and 9 years of age.

32. The method according to any one of claims 28 - 31, wherein the pharmaceutical composition comprises a tetravalent formulation, the tetravalent formulation comprising 2.0 x 10 4 pfu of dengue-1 / dengue-2 chimera, 5.0 x 10 3 pfu of live attenuated dengue-2, 1.0 x 10 5 pfu of dengue-3 / dengue-2 chimera and 3.0 x 10 5 pfu of dengue-4 / dengue-2 chimera and a pharmaceutically acceptable excipient.

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