Methods for preventing dengue and hepatitis a

Abstract

To provide a method for preventing dengue disease and hepatitis A in a subject or subject population by simultaneously administering a unit dose of a dengue vaccine composition and a hepatitis A vaccine on the same day.SOLUTION: The unit dose of a dengue vaccine composition includes constructs of each dengue serotype, such as TDV-1, TDV-2, TDV-3 and TDV-4, at various concentrations in order to improve protection from dengue infection.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for the simultaneous administration of a unit dose of a dengue vaccine composition together with a Hepatitis A vaccine to a subject or a population of subjects on the same day. The unit dose according to the present invention provides an immune response against all serotypes of dengue virus, i.e. DENV-1, DENV-2, DENV-3 and DENV-4, as well as against Hepatitis A virus. [Background technology]

[0002] Vaccines for protection against viral infections have been used effectively to reduce the incidence of human disease. One of the most successful techniques for viral vaccines is to immunize animals or humans with weakened or attenuated virus strains ("live attenuated viruses"). Due to limited replication after immunization, attenuated virus strains do not cause disease. However, even with limited viral replication, it is still sufficient to express the full repertoire of viral antigens and generate a strong and long-lasting immune response against the virus. Thus, immunized individuals are protected from disease upon subsequent exposure to a pathogenic virus strain. These live attenuated virus vaccines are among the most successful vaccines in public health.

[0003] Dengue disease is a mosquito-borne disease caused by infection with the dengue virus. Dengue virus infection can lead to debilitating and painful symptoms including sudden high fever, headache, joint and muscle pain, nausea, vomiting, and skin rash. So far, four serotypes of dengue virus have been identified: dengue-1 (DENV-1), dengue-2 (DENV-2), dengue-3 (DENV-3), and dengue-4 (DENV-4). 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 50-100 million cases of debilitating dengue fever, 500,000 cases of DHF / DSS, and over 20,000 deaths each year, the majority of which are in children. All four dengue virus serotypes are endemic throughout the tropical regions of the world and represent the most significant mosquito-borne threat to humans in the tropics. Dengue virus is transmitted to humans primarily by the Aedes aegypti mosquito, but also by the Aedes albopictus mosquito. Infection with one dengue virus serotype confers lifelong protection from reinfection with that serotype, but does not prevent secondary infection with one of the other three dengue virus serotypes. Indeed, previous infection with one dengue virus serotype may lead to an increased risk of severe disease (DHF / DSS) during secondary infection with a different serotype.

[0004] To date, only one vaccine, the yellow fever backbone-based tetravalent dengue vaccine CYD-TDV (Dengvaxia®, Sanofi Pasteur, Lyon, France), has been licensed in several countries based on clinical demonstration of overall vaccine efficacy (VE) against virologically confirmed dengue (VCD) in 56-61% of children in Asia and Latin America (Capeding MR et al. Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in Asia: a phase 3, randomised, observer-masked, placebo-controlled trial. Lancet 2014,384:1358-65; Villar LA et al. Safety and immunogenicity of a recombinant tetravalent dengue vaccine in 9-16 year olds: a randomized, controlled, phase II trial in Latin America. Pediatr Infect Dis J 2013,32:1102-9). However, clinical trials have shown that Dengvaxia® may enhance rather than reduce the risk of severe disease from dengue infection in individuals who have not been previously infected with dengue virus (seronegative population). Therefore, Dengvaxia® is only recommended for use in individuals who have been previously infected with at least one dengue virus serotype (seropositive population). More specifically, according to the European Medicines Agency's European Public Assessment Report (EPAR) for the product, Dengvaxia® is only for use in people aged 9-45 years who have previously been infected with dengue virus and who live in areas where this infection is endemic. Endemic areas are areas where the disease occurs regularly throughout the year.See also Sridhar S et al.Effect of Dengue Serostatus on Dengue Vaccine Safety and Efficacy. N Engl J Med 2018,379:327-40; and World Health Organization. Dengue vaccine:WHO position paper-September 2018. Wkly. Epidemiol. Rec.2018,93:457-476. S.R. Adinegoro et al., in the New England Journal of Medicine,Vol.373,page 1195, "Efficacy and Long-Term Safety of a Dengue Vaccine in Regions of Endemic Disease", report a pooled risk of 1.58 for hospitalization for virologically confirmed dengue disease in children under 9 years of age, indicating an increased risk of severe dengue in vaccinated populations. Thus, there remains a significant unmet need for an effective vaccine with a good safety profile in both dengue-naive and seropositive individuals (including dengue-naive populations living in endemic areas, younger individuals who may never have developed any seropositive response to or been exposed to dengue, and travelers and individuals from non-endemic areas). There is also a need for outbreak control or travel vaccination that reduces the risk of dengue after only one dose.

[0005] One further drawback of Dengvaxia®, the only currently approved dengue vaccine, is that it must only be offered to people who have had a positive test result indicating previous infection with the dengue virus, i.e., individuals with a known serostatus for dengue (EPAR). Thus, individuals with unknown dengue serostatus cannot be vaccinated with Dengvaxia®.

[0006] There is therefore a need for a dengue vaccine and corresponding vaccination regimen that stimulates an immune response against all dengue serotypes, preferably a balanced immune response against all serotypes, in both seronegative and seropositive populations, that is safe for a wider age group, in particular for subjects under 9 years of age, and protects against dengue disease of any severity (including DSS, DHF).The development of a safe and effective vaccine capable of protecting all populations, including both seronegative and seropositive populations, in particular children and young adults and elderly subjects, in epidemic situations and for travel purposes represents an important approach to the prevention and control of this global disease.

[0007] Thus, there is a medical need for a dengue vaccine and corresponding methods of administration that are safe and effective across a wide range of age groups, regardless of serostatus. There is a need for a dengue vaccine and corresponding methods of administration that avoid costly and time-consuming serostatus testing or seroprevalence studies. There is a need for a dengue vaccine and corresponding methods of administration that can be used in pandemic settings. Furthermore, there is a medical need for a dengue vaccine that is not only safe and effective, but can also be administered to individuals with unknown dengue serostatus, children under the age of 9, and seronegative individuals.

[0008] There is also a need for vaccines that are administered in fewer doses than the current Dengvaxia® schedule of three doses administered six months apart, for example, a vaccine that could be administered in as few as two doses or even just one dose to be effective.

[0009] The above objectives are in line with the WHO research priorities on dengue vaccines: WHO position paper - September 2018 (Wkly. Epidemiol. Rec. 2018, 93:457-476).

[0010] Hepatitis A is a liver disease caused by the hepatitis A virus (HAV). The virus spreads primarily when an uninfected (and unvaccinated) person ingests food or water contaminated with the feces of an infected person. The disease is closely associated with unsafe water or food, improper sanitation, and poor personal hygiene. The virus can also be transmitted through close physical contact with an infected person. Unlike hepatitis B and C, hepatitis A infection does not cause chronic liver disease and is rarely fatal, but it can cause debilitating symptoms and fulminant hepatitis (acute liver failure), which is often fatal. Hepatitis A tends to occur sporadically around the world and recur periodically.

[0011] Hepatitis A virus is one of the most frequent causes of foodborne infections. Epidemics associated with contaminated food or water can occur explosively, such as the 1988 epidemic in Shanghai that affected approximately 300,000 people. Hepatitis A virus can persist in the environment and survive food preparation processes routinely used to inactivate and / or control bacterial pathogens. The disease can lead to significant economic and social consequences in communities. It may take weeks or months for people who have recovered from the disease to return to work, school, or daily activities. The impact on food establishments where the virus is identified, and on overall local productivity, can be significant. In developing countries with poor sanitation and poor hygiene standards, most children (90%) have been infected with hepatitis A virus before the age of 10.

[0012] The number of people travelling internationally has increased significantly in recent decades. According to the United Nations World Tourism Organisation (UNWTO), over 1.1 billion tourists travelled abroad in 2014. The risk of contracting a disease during international travel depends on a variety of factors, such as the area of ​​the world visited, the length of the trip, and the variety of activities planned. Vaccination recommendations form an important part of health preparation before international travel. Vaccination against the Hepatitis A virus is generally recommended for travellers to risk areas around the world, including Asia, Africa, and Central and South America.

[0013] For conventional hepatitis A vaccination, a two-dose schedule is recommended, especially for travelers and immunocompromised individuals who are at substantial risk of contracting hepatitis A. However, in healthy individuals, comparable efficacy has been achieved with a single dose. The vaccination schedule for children / young adults (12 months to 18 years) and adults (19 years and older) consists of a primary dose administered intramuscularly and a further booster dose administered intramuscularly 6 to 18 months later.

[0014] Available Hepatitis A vaccines include HAVRIX® and VAQTA®.

[0015] Thus, there is a need for a safe and effective method of simultaneously preventing dengue disease and hepatitis A. In particular, there is a need for hepatitis A and dengue vaccines that provide non-inferiority when administered simultaneously to a subject or population of subjects, as well as suitable dosing schedules to achieve a synergistic effect.

[0016] Furthermore, there is a need for effective and safe prevention of dengue and hepatitis A in subjects who do not know their hepatitis A and / or dengue serostatus, particularly in subjects from non-dengue endemic countries traveling to dengue and hepatitis A endemic countries. Summary of the Invention

[0017] It is an object of the present invention to provide safe and effective protection against dengue disease and hepatitis A.

[0018] It is an object of the present invention to provide a method of administration for preventing Hepatitis A and Dengue disease that is useful in typical vaccination situations where subjects do not know their dengue and / or hepatitis A serostatus and corresponding serologic tests are unavailable, impractical, or unreliable.

[0019] It is an object of the present invention to provide safe and effective protection against dengue disease and hepatitis A for travelers from non-endemic countries, particularly those vaccinated in travel clinics. In these situations, it would be beneficial to be able to vaccinate against multiple diseases simultaneously, avoiding duplication during the same visit.

[0020] It is an object of the present invention to provide a safe and effective vaccine for Hepatitis A and Dengue disease in a subject or subject population and corresponding methods of preventing Hepatitis A and Dengue disease in subjects or subject populations originating from dengue endemic and non-dengue endemic areas for subjects across a broad age range, specifically, subjects aged 2-60 years, preferably 18-60 years, regardless of previous exposure to any dengue virus serotype and / or Hepatitis A virus, and regardless of the corresponding seropositivity or seronegativity for dengue and / or hepatitis A prior to vaccination.

[0021] It is an object of the present invention to provide a vaccine and a corresponding method for preventing Hepatitis A and Dengue diseases, which avoids testing the individual dengue and / or hepatitis A serostatus before administering the Hepatitis A and Dengue vaccine to a subject or a population of subjects individually, or avoids analysis of the seroprevalence of dengue and / or hepatitis A in the subject or population of subjects to be vaccinated.

[0022] It is an object of the present invention to provide dengue and hepatitis A vaccines that can be safely co-administered with TDV as a travel vaccine prior to a subject traveling internationally to a country where HAV and dengue are endemic, and methods for safely administering these vaccines.

[0023] Thus, the present invention is directed to methods of preventing dengue disease and hepatitis A.

[0024] The present invention is further directed to a method for preventing Hepatitis A and Dengue disease in a subject or population of subjects comprising concurrently administering on the same day unit doses of a Hepatitis A vaccine and a Dengue vaccine composition, the unit dose comprising a tetravalent dengue virus composition comprising four live attenuated dengue virus strains.

[0025] The present invention provides, for example, the following: 1. A unit dose of a hepatitis A vaccine and a dengue vaccine composition for use in a method for preventing hepatitis A and dengue disease in a subject or population of subjects comprising administering said unit doses of said hepatitis A vaccine and said dengue vaccine composition simultaneously on the same day, said unit dose comprising a tetravalent dengue virus composition comprising four live attenuated dengue virus strains. 2. The Hepatitis A vaccine and unit dose for use according to paragraph 1, wherein said Hepatitis A vaccine is an inactivated virus vaccine. 3. A Hepatitis A vaccine and unit dose for use according to paragraph 1 or 2, wherein the dengue vaccine composition upon reconstitution with 0.5 mL of a pharma- ceutical acceptable diluent comprises: (i) a concentration of at least 3.3 log10 pfu / 0.5 mL for the chimeric dengue serotype 2 / 1 strain; (ii) for dengue serotype 2, a concentration of at least 2.7 log pfu / 0.5 mL; (iii) for chimeric dengue serotype 2 / 3 strains, a concentration of at least 4.0 log pfu / 0.5 mL; and (iv) for chimeric dengue serotype 2 / 4 strains, a concentration of at least 4.5 log10 pfu / 0.5 mL; 4. The Hepatitis A vaccine and unit dose for use according to any one of the preceding clauses, wherein said subject population or said subject is seronegative for all dengue serotypes. 5. The Hepatitis A vaccine and unit dose for use according to any one of the preceding clauses, wherein said unit dose of said Dengue vaccine composition is administered by subcutaneous injection and said Hepatitis A vaccine is administered by intramuscular injection, said injection being preferably administered in the arm, more preferably in the deltoid area of ​​the arm. 6. The Hepatitis A vaccine and unit dose for use according to clause 5, wherein the unit dose of the dengue vaccine composition and the Hepatitis A vaccine are administered in different anatomical sites (e.g., opposite arms). 7. The Hepatitis A vaccine and unit dose for use according to any one of the preceding claims, wherein two of said unit doses of said dengue vaccine composition are administered within 12 months or more, or within 6 months, or within 3 months. 8. Hepatitis A vaccine and unit dose for use according to clause 7, comprising administering two of said unit doses of said dengue vaccine composition and one dose of said hepatitis A vaccine, specifically according to the following schedule: on day -1, a first unit dose of the dengue vaccine composition and a first simultaneous administration of the hepatitis A vaccine; and - A second administration of a second unit dose of said dengue vaccine composition following said first co-administration (eg, about 3 months later). 9. The Hepatitis A vaccine and unit dose for use according to any one of the preceding claims, wherein the subject population or the subject is aged between 2 and 60 years. 10. The Hepatitis A vaccine and unit dose for use according to any one of the preceding clauses, wherein said subject population or said subject is from a dengue endemic area. 11. The Hepatitis A vaccine and unit dose for use according to any one of clauses 1 to 9, wherein the subject population or the subject is from a non-dengue endemic area, preferably a non-dengue and non-Hepatitis A endemic area. 12. A Hepatitis A vaccine and unit dose for use according to any one of the preceding claims, wherein said Hepatitis A vaccine comprises a Hepatitis A virus derived from the Hepatitis A virus strain HM-175. 13. The Hepatitis A vaccine and unit dose for use according to any one of the preceding claims, wherein said Hepatitis A vaccine comprises an inactivated Hepatitis A virus, said inactivated Hepatitis A virus being derived from the Hepatitis A virus strain HM-175. 14. A Hepatitis A vaccine and unit dose for use according to any one of the preceding claims, wherein said Hepatitis A vaccine comprises an inactivated Hepatitis A virus, said inactivated Hepatitis A virus being adsorbed to aluminium. 15. The Hepatitis A vaccine and unit dose for use according to paragraph 14, wherein the aluminum is aluminum hydroxide or aluminum hydroxyphosphate sulfate. 16. A Hepatitis A vaccine and unit dose for use according to any one of the preceding claims, wherein said Hepatitis A vaccine comprises an inactivated Hepatitis A virus, and said Hepatitis A vaccine comprises phosphate buffered saline and excipients dissolved therein in amino acid form and polysorbate form. 17. A Hepatitis A vaccine and unit dose for use according to any one of the preceding claims, wherein said Hepatitis A vaccine comprises Hepatitis A virus expressing a viral antigen in a concentration ranging from 500 ELISA units (EL.U.) to 2000 ELISA units (EL.U.). 18. The method does not include a step of determining whether the subject population or the subject has previously been infected with dengue and / or previously been infected with hepatitis A prior to said administration of the hepatitis A vaccine and prior to said administration of the unit dose of the dengue vaccine composition; or The Hepatitis A vaccine and unit dose for use according to any one of the preceding claims, wherein the Hepatitis A serostatus and / or Dengue serostatus of the subject population or subject is unknown prior to said administration of the Hepatitis A vaccine and prior to said administration of the unit dose of the Dengue vaccine composition. 19. The method does not include determining whether the subject population or the subject has previously been infected with dengue and / or previously been infected with hepatitis A at any time before, during, or after administering the unit doses of the hepatitis A vaccine and the dengue vaccine composition; or 19. The Hepatitis A vaccine and unit dose for use according to clause 18, wherein the Hepatitis A serostatus and / or the Dengue serostatus of the subject population or subject is unknown at any time before, during or after the step of administering the unit dose of the Hepatitis A vaccine and Dengue vaccine composition. 20. The hepatitis A vaccine and unit dose for use according to any of the preceding paragraphs, wherein the method comprises a primary vaccination comprising the steps of: (A) selecting a subject for administration of the tetravalent dengue virus composition unit dose and the hepatitis A vaccine in need of protection against dengue infection and hepatitis A infection, without determining whether the subject has previously been infected with dengue and / or has previously been infected with hepatitis A; and (B) administering to the subject a first unit dose of the tetravalent dengue virus composition and a Hepatitis A vaccine, and optionally (C) administering to the subject at least one additional unit dose of the tetravalent dengue virus composition within 3 to 12 months of administration of the first unit dose, and optionally (D) administering to the subject at least one additional dose of the Hepatitis A vaccine within 6 to 18 months of administration of the first unit dose. 21. The Hepatitis A vaccine and unit dose for use according to any one of the preceding claims, wherein the method comprises a primary vaccination consisting of the steps of: (A) selecting a subject to receive said unit dose of said tetravalent dengue virus composition and said hepatitis A vaccine based on a need for protection against dengue infection and hepatitis A infection; and (B) administering to the subject a first unit dose of the tetravalent dengue virus composition and a Hepatitis A vaccine; and (C) administering to the subject two additional unit doses of the tetravalent dengue virus composition at about 6 months and about 12 months after administration of the first unit dose, and administering the Hepatitis A vaccine either at about 6 months or about 12 months after administration of the first unit dose. 22. Hepatitis A vaccine and unit dose for use according to paragraph 21, wherein step (A) is carried out without determining whether the patient has been previously infected with Hepatitis A. 23. The Hepatitis A vaccine and unit dose for use according to any one of clauses 3 to 22, wherein the dengue vaccine composition, upon reconstitution with a pharma- ceutically acceptable diluent, provides a total concentration of (i), (ii), (iii), and (iv), and, based on said total concentration of pfu / 0.5 ml, the concentration of (ii) at pfu / 0.5 ml is less than 10%, the concentration of (iv) at pfu / 0.5 ml is at least 50%, the concentration of (i) at pfu / 0.5 ml is at least 1%, and the concentration of (iii) at pfu / 0.5 ml is at least 6%, at least 8%, or at least 10%, or at least 12%, or at least 14%, or at least 16%, or at least 18%. 24. A Hepatitis A vaccine and unit dose for use according to any one of the preceding claims, wherein said method results in compatibility between said dengue vaccine composition and said Hepatitis A vaccine. 25. The Hepatitis A vaccine and unit dose for use according to any one of the preceding claims, wherein said method results in a synergistic effect between said dengue vaccine composition and said Hepatitis A vaccine. 26. The Hepatitis A vaccine and unit dose for use according to any one of the preceding clauses, wherein the method results in non-inferiority in a non-inferiority clinical trial involving at least 60 or at least 120 healthy subjects divided into one subject population and one control subject population, wherein the subject populations receive the unit doses of the Hepatitis A vaccine and the Dengue vaccine composition simultaneously on the same day, and wherein the control subject population receives Hepatitis A vaccine and placebo doses simultaneously on the same day. 27. The Hepatitis A vaccine and unit dose for use according to any one of the preceding clauses, wherein the Hepatitis A vaccine provides a Hepatitis A seroprotection rate of at least 95%, or at least 98%, 30 days after administration (day 0 / 1) in a subject population of at least 30, or at least 50 healthy subjects who are seronegative for Hepatitis A at baseline and seronegative for all dengue virus serotypes at baseline, receiving the unit doses of the Hepatitis A vaccine and the dengue vaccine composition simultaneously on the same day. 28. The method results in a difference in seroprotection rate for hepatitis A versus administration of a single agent for hepatitis A, the difference being determined in a non-inferiority clinical trial including at least 60 or at least 120 healthy subjects who are seronegative for hepatitis A at baseline and who are seronegative for all dengue virus serotypes at baseline; The healthy subject is e) a subject population of at least 30 or at least 50 healthy subjects receiving the unit doses of the Hepatitis A vaccine and the Dengue vaccine composition on the same day and at the same time (on day 0 / 1); f) a control population of at least 30 or 50 subjects receiving the Hepatitis A vaccine and a placebo on the same day and at the same time (day 0 / 1); The difference between the Hepatitis A seroprotection rate of the control subject population 30 days after the administration (day 0 / 1) and the Hepatitis A seroprotection rate of the subject population 30 days after the administration (day 0 / 1) is determined; 4. The Hepatitis A vaccine and unit dose for use according to any one of the preceding claims, wherein the upper limit of said difference within a two-sided 95% confidence interval is less than 10%. 29. The Hepatitis A vaccine and unit dose for use according to any one of clauses 1 to 26, wherein the Hepatitis A vaccine provides a Hepatitis A seroprotection rate of at least 95%, or at least 98%, or at least 99%, 30 days after administration (day 0 / 1) in a subject population of at least 30 or at least 50 healthy subjects who are seronegative for Hepatitis A at baseline receiving the unit doses of the Hepatitis A vaccine and the dengue vaccine composition simultaneously on the same day, wherein the healthy subjects include healthy subject(s) who are seropositive for at least one dengue virus serotype at baseline, and healthy subject(s) who are seronegative for all dengue virus serotypes at baseline. 30. The method results in a difference in seroprotection rates for hepatitis A versus single agent administration of hepatitis A, the difference being determined in a non-inferiority clinical trial including at least 60 or at least 120 healthy subjects who are seronegative for hepatitis A at baseline, the healthy subjects including healthy subject(s) who are seropositive for at least one dengue virus serotype at baseline and healthy subject(s) who are seronegative for all dengue virus serotypes at baseline; The healthy subject is e) a subject population of at least 30 or at least 50 healthy subjects receiving the unit doses of the Hepatitis A vaccine and the Dengue vaccine composition simultaneously on the same day (day 0 / 1), the subject population comprising healthy subject(s) who are seropositive for at least one Dengue virus serotype at baseline and healthy subject(s) who are seronegative for all Dengue virus serotypes at baseline; f) a control subject population of at least 30 or at least 50 healthy subjects receiving the Hepatitis A vaccine and a placebo simultaneously on the same day (day 0 / 1), the control subject population comprising healthy subject(s) who are seropositive for at least one dengue virus serotype at baseline and healthy subject(s) who are seronegative for all dengue virus serotypes at baseline; The difference between the Hepatitis A seroprotection rate of the control subject population 30 days after the administration (day 0 / 1) and the Hepatitis A seroprotection rate of the subject population 30 days after the administration (day 0 / 1) is determined; Item 27. The Hepatitis A vaccine and unit dose for use according to any one of items 1 to 26, wherein the upper limit of the difference within a two-sided 95% confidence interval is less than 10%. 31. The Hepatitis A vaccine and unit dose for use according to any one of the preceding clauses, wherein said subject or said subject population is exposed to a Hepatitis A virus pandemic and / or a Dengue virus pandemic. 32. The Hepatitis A vaccine and unit dose for use according to any one of the preceding clauses, wherein the method results in an anti-Hepatitis A virus antibody geometric mean concentration (GMC) of at least 70mIU / ml, or at least 80mIU / ml, or at least 90mIU / ml 30 days after administration (day 0 / 1) for a subject population of at least 30 or at least 50 healthy subjects who receive the unit doses of the Hepatitis A vaccine and Dengue vaccine composition simultaneously on the same day and who are seronegative for Hepatitis A at baseline and seronegative for all Dengue virus serotypes at baseline. 33. A Hepatitis A vaccine and unit dose for use according to any one of the preceding clauses, wherein said administration of said unit doses of said Hepatitis A vaccine and said Dengue vaccine composition on the same day and simultaneously to said subject or said population of subjects is safe. 34. A Hepatitis A vaccine and unit dose for use according to any one of the preceding claims, wherein there are no serious adverse events associated with said same-day administration. 35. The method comprises the steps of: a) determining the geometric mean titer (GMT) of neutralizing antibodies as measured by the following MNT50: - for dengue serotype 1, at least 110, or at least 140, or at least 150, for dengue serotype 2, at least 3000, or at least 3500, or at least 3900, - for dengue serotype 3, at least 100, or at least 120, or at least 140, and / or - for dengue serotype 4, at least 80, or at least 110, or at least 140, The Hepatitis A vaccine and unit dose for use according to any one of the preceding claims, provided 30 days after the unit doses of the Hepatitis A vaccine and the Dengue vaccine composition are administered simultaneously on the same day (day 0 / 1) to a population of at least 30 or at least 50 healthy subjects who are seronegative for Hepatitis A at baseline and seronegative for all Dengue virus serotypes at baseline. 36. The Hepatitis A vaccine and unit dose for use according to any one of the preceding claims, wherein the subject or subject population is aged between 18 and 60 years. 37. A kit for treating Hepatitis A and Dengue fever comprising: the kit comprising a box containing at least: (a) a first container holding a Hepatitis A vaccine; and (b) a second container holding a unit dose of a dengue vaccine composition, said unit dose comprising a tetravalent dengue virus composition comprising four live attenuated dengue virus strains.

[0026] definition In describing the present invention, the following terms should be used as indicated below: As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0027] As used herein, the terms "unit dose of a dengue vaccine composition," "unit dose," and "unit dose of the invention described herein" refer to an amount of a dengue vaccine administered to a subject in a single dose. In one embodiment, one unit dose is present in a vial, and this unit dose is administered to a subject (e.g., optionally after reconstitution). In one embodiment, multiple unit doses of a dengue vaccine composition may be present in a vial such that the contents of one vial can vaccinate multiple subjects.

[0028] A "lyophilized unit dose" or a "unit dose in lyophilized form" refers to a unit dose obtained by subjecting a given volume of a liquid dengue vaccine composition, such as 0.5 mL, to lyophilization. Thus, an aqueous formulation of a dengue vaccine composition produced by combining a pharma- ceutically acceptable excipient and a dengue virus composition comprising four dengue virus strains, preferably TDV-1 to TDV-4, is subjected to lyophilization to obtain a lyophilized unit dose.

[0029] A "reconstituted unit dose" or a "unit dose in reconstituted form" is obtained from a lyophilized dose by reconstitution with a pharma- ceutically acceptable diluent. The diluent does not contain dengue virus. The reconstituted unit dose is a liquid that can be administered to a subject by injection, e.g., subcutaneous injection.

[0030] As used herein, the term "when reconstituted in 0.5 mL" refers to the concentration of dengue virus present in a reconstituted unit dose when 0.5 mL of diluent is used for reconstitution, although it is not intended to limit reconstitution to being performed using 0.5 mL of diluent. Using a different volume for reconstitution (e.g., 0.8 mL) will result in a different concentration of dengue virus in the reconstituted unit dose, whereas administration of a total unit dose volume (e.g., 0.8 mL) will result in the same total amount of dengue virus being administered.

[0031] As used herein, "a concentration of at least Xlog10 pfu / 0.5 mL" refers to the concentration of a dengue serotype in 0.5 mL, but does not limit the unit dose to 0.5 mL. If the unit dose has a volume different from 0.5 mL, or is lyophilized from a volume different from 0.5 mL, or is reconstituted in a volume different from 0.5 mL, the concentration will be different from "a concentration of at least Xlog10 pfu / 0.5 mL". However, if the volume of the unit dose is 0.5 mL, or is lyophilized from a volume of 0.5 mL, or is reconstituted in a volume of 0.5 mL, the concentration will be "a concentration of at least Xlog10 pfu / 0.5 mL". Thus, while the concentration may vary, the total amount of virus in the unit dose remains the same.

[0032] As used herein, the term "dengue serotype" refers to a species of dengue virus that is defined by its cell surface antigens and thus can be distinguished by serological methods known in the art. Currently, four serotypes of dengue virus are known: dengue serotype 1 (DENV-1), dengue serotype 2 (DENV-2), dengue serotype 3 (DENV-3) and dengue serotype 4 (DENV-4).

[0033] As used herein, the term "tetravalent dengue virus composition" refers to a dengue virus composition that comprises four different immunogenic components derived from four different dengue serotypes DENV-1, DENV-2, DENV-3, and DENV-4, preferably four different live attenuated dengue viruses (each representing one dengue serotype), and that aims to stimulate an immune response against all four dengue serotypes.

[0034] As used herein, the term "live attenuated dengue virus" refers to a viable dengue virus that has been mutated to reduce pathogenicity. A live attenuated dengue virus may be a dengue virus in which all components are derived from the same dengue serotype, or it may be a chimeric dengue virus having portions from more than one dengue serotype, or a mixed chimeric dengue virus having portions from other flaviviruses.

[0035] A "virus strain" and specifically a "dengue virus strain" is a genetic subtype of a virus, specifically a dengue virus, characterized by a particular nucleic acid sequence. Dengue serotypes can include different strains with different nucleic acid sequences that have the same cell surface antigens. A dengue virus strain can be a dengue virus in which all components are derived from the same dengue serotype, or it can be a chimeric dengue virus that has parts from more than one dengue serotype.

[0036] As used herein, "TDV-2" refers to a molecularly characterized and cloned dengue serotype 2 strain derived from the live attenuated DEN-2 PDK-53 virus strain. The PDK-53 strain is described, for example, in Bhamarapravati et al. (1987) Bulletin of the World Health Organization 65(2):189-195. In one embodiment, the TDV-2 strain served as a backbone for chimeric TDV-1, TDV-3, and TDV-4 strains into which portions from the TDV-1, TDV-3, and TDV-4 strains were introduced.

[0037] A "non-chimeric dengue virus" or "non-chimeric dengue serotype strain" or "non-chimeric dengue strain" contains only parts from one dengue serotype. Specifically, a non-chimeric dengue virus does not contain parts from different flaviviruses (e.g., yellow fever virus, Zika virus, West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, tick-borne encephalitis virus). TDV-2 is an example of a non-chimeric dengue virus.

[0038] A "chimeric dengue virus" or "chimeric dengue serotype strain" or "chimeric dengue strain" comprises portions derived from at least two different dengue serotypes. As used herein, a chimeric dengue virus does not comprise portions derived from different flaviviruses (e.g., Yellow Fever Virus, Zika Virus, West Nile Virus, Japanese Encephalitis Virus, St. Louis Encephalitis Virus, Tick-Borne Encephalitis Virus). Specifically, the chimeric dengue viruses described herein do not comprise portions derived from Yellow Fever Virus. As used herein, a "chimeric dengue serotype 2 / 1 strain" or "DENV-2 / 1 chimera" or "TDV-1" refers to a dengue virus chimeric construct that comprises portions derived from both DENV-2 and DENV-1. Specifically, in a chimeric dengue serotype 2 / 1 strain, the prM and E proteins derived from DENV-1 replace the prM and E proteins derived from DENV-2, as detailed below. As used herein, "chimeric dengue serotype 2 / 3 strain" or "DENV-2 / 3 chimera" or "TDV-3" refers to a dengue virus chimeric construct that includes portions from both DENV-2 and DENV-3. Specifically, in a chimeric dengue serotype 2 / 3 strain, the prM and E proteins from DENV-3 replace the prM and E proteins from DENV-2, as detailed below. As used herein, "chimeric dengue serotype 2 / 4 strain" or "DENV-2 / 4 chimera" or "TDV-4" refers to a dengue virus chimeric construct that includes portions from both DENV-2 and DENV-4. Specifically, in a chimeric dengue serotype 2 / 4 strain, the prM and E proteins from DENV-4 replace the prM and E proteins from DENV-2, as detailed below. A mixed chimeric dengue virus has portions from other flaviviruses.

[0039] As used herein, "TDV" refers to a tetravalent live attenuated dengue vaccine comprising a mixture of four live attenuated dengue virus strains TDV-1, TDV-2, TDV-3, and TDV-4 expressing surface antigens from the four dengue serotypes DENV-1, DENV-2, DENV-3, and DENV-4, respectively. In one embodiment (e.g., also in the Examples), TDV-1 has a nucleotide sequence set forth in SEQ ID NO:1 and / or an amino acid sequence set forth in SEQ ID NO:2. In one embodiment, TDV-2 has a nucleotide sequence set forth in SEQ ID NO:3 and / or an amino acid sequence set forth in SEQ ID NO:4. In one embodiment, TDV-3 has a nucleotide sequence set forth in SEQ ID NO:5 and / or an amino acid sequence set forth in SEQ ID NO:6. In one embodiment, TDV-4 has a nucleotide sequence set forth in SEQ ID NO:7 and / or an amino acid sequence set forth in SEQ ID NO:8.

[0040] As used herein, the term "dengue disease" refers to a disease caused by infection with the dengue virus. Symptoms of dengue disease include sudden high fever, headache, joint and muscle pain, nausea, vomiting, and skin rash. The term dengue disease also includes more severe forms of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Symptoms of DHF include increased vascular permeability, hypovolemia, and abnormal blood clotting mechanisms. Subjects with DHF may show severe signs of plasma leakage and bleeding. If a subject with DHF goes into shock, the person is classified as having DSS. Symptoms of DSS include bleeding, which may appear as small dots of blood on the skin and large patches of blood under the skin. Sustained shock is the main factor associated with complications including massive gastrointestinal bleeding that may lead to death. As used herein, DHF cases are defined as VCD cases that meet the WHO 1997 DHF criteria. In the context of preventing dengue disease in elderly subjects, the term "preventing dengue disease" preferably includes preventing DHF and / or DSS. In the context of preventing dengue disease in elderly subjects, the term "preventing dengue disease" preferably includes preventing severe end-organ manifestations of dengue disease (e.g., hepatomegaly and acute renal failure).

[0041] As used herein, "preventing dengue disease" refers to preventing a subject from developing one or more symptoms of dengue disease due to infection with a dengue virus. Specifically, preventing dengue disease is accomplished by vaccinating or inoculating a subject with a dengue vaccine composition, such as a reconstituted unit dose, described herein. As used herein, the term "prophylactically treating dengue disease" is equivalent to "preventing dengue disease." In certain embodiments, preventing dengue disease includes preventing DHS and / or DSS.

[0042] As used herein, the term "virologically confirmed dengue disease", "VCD case", or "VCD fever" refers to a febrile illness or illness clinically suspected to be dengue fever disease with a positive serotype-specific reverse transcriptase polymerase chain reaction (RT-PCR). The term "virologically confirmable dengue" illness refers to a subject with a febrile illness or illness clinically suspected to be dengue fever disease, where testing of the subject (e.g., using RT-PCR) would confirm the presence of at least one dengue serotype. Severe forms of VCD fever are identified as follows: Dengue hemorrhagic fever (DHF) was defined according to WHO 1997 criteria. Severe dengue fever was defined through the evaluation of an independent Dengue Case Adjudication Committee that evaluates all hospitalized VCD cases (severe / non-severe) based on the criteria redefined in the Charter. All non-hospitalized cases are considered non-severe.

[0043] As used herein, the term "febrile illness" is defined as a body temperature of 38° C. or higher on any two out of three consecutive days.

[0044] As used herein, the term "virologically confirmed dengue disease with hospitalization" is considered as a substitute term for severe dengue, and "incidence of virologically confirmed dengue disease with hospitalization" is used as a safety parameter. As used herein, "relative risk for virologically confirmed dengue disease with hospitalization" means the number of events of virologically confirmed dengue disease with hospitalization divided by the number of subjects treated with a unit dose disclosed herein divided by the number of events of virologically confirmed dengue disease with hospitalization divided by the number of subjects treated with placebo. If the "relative risk for virologically confirmed dengue disease with hospitalization" is less than or equal to 1, the vaccine reduces the risk of virologically confirmed dengue disease with hospitalization to less than or equal to placebo and is considered "safe." In this context, the risk of virologically confirmed dengue disease involving hospitalization may be 0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, or 0.1 or less, particularly when determined 30 days after the second dose to 12 months after the second dose, particularly in an age group selected from the age group of subjects 4 to 16 years, the age group of subjects 4 to less than 9 years, the age group of subjects 2 to less than 9 years, the age group of subjects 4 to 5 years, the age group of subjects 6 to 11 years, and the age group of subjects 12 to 16 years.

[0045] Alternatively, as used herein, a vaccine is considered "safe" if it has a vaccine efficacy (VE) with respect to virologically confirmed dengue disease with hospitalization equal to or greater than 0%. This means that the vaccine reduces the likelihood of virologically confirmed dengue disease with hospitalization to or below placebo. A specifically considered "safety" is a combined vaccine efficacy with respect to virologically confirmed dengue disease with hospitalization for all four serotypes with a two-sided 95% confidence interval, specifically from about 30 days after the first dose or the second or last dose of the dosing schedule to at least 12 months, 12 to 18 months, 12 months, or 18 months after the second or last dose of the dosing schedule, when the unit dose or the placebo is administered at least twice within less than six months (e.g., within three months). , a combined vaccine efficacy with a lower limit of greater than 25% when measured against placebo in a subject population of at least 1,500 or at least 2,000 healthy subjects who are seronegative to all serotypes at baseline or seropositive to at least one serotype at baseline (specifically when measured in age groups selected from the age groups of subjects 4-16 years old, subjects 4 to less than 9 years old, subjects 2 to less than 9 years old, subjects 4 to 5 years old, subjects 6 to 11 years old, and subjects 12 to 16 years old). Specifically, the lower limit may be greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 65%, greater than 66%, greater than 67%, greater than 68%, greater than 70%, or greater than 75%. Specifically, the two-sided 95% confidence intervals of combination vaccine efficacy against virologically confirmed dengue fever with hospitalization for all four serotypes place the lower limit of the two-sided confidence interval within 10 percentage points, 15 percentage points, or 20 percentage points when comparing seropositive and seronegative subjects.In certain embodiments, "safe" means providing combined vaccine efficacy against virologically confirmed dengue fever with hospitalization for all four serotypes with a two-sided 95% confidence interval, with a lower limit of greater than 65%, when measured against placebo in a population of at least 5,000 healthy subjects aged 4-16 years, regardless of baseline serostatus, from the first dose of the dosing schedule to 12-18 months after the last dose of the dosing schedule.

[0046] A vaccine is considered safe within the meaning of the present invention if one of the criteria defined above for the term "safe" is met. In this context, safety in particular refers to a vaccine that is safe for all subjects, regardless of baseline serostatus. This means that the vaccine can be administered without the need for a determination of the occurrence of past dengue infection in the subject prior to administration. Preferably, the vaccine is safe as defined above for all age groups starting from 4 years of age, in particular for 4-60 years or 4-16 years, preferably regardless of serostatus. Relevant subgroups in this context are <9 years, 2-9 years, 4-9 years, 4-5 years, 6-11 years and any age group within the range of 12-16 years or 4-16 years. Further definitions of VE for virologically confirmed dengue disease with hospitalization are mentioned below in the disclosure regarding certain treatment methods.

[0047] As used herein, "vaccine efficacy" or "VE" measures the proportional reduction in cases in vaccinated people. Vaccine efficacy (VE) is measured by calculating the risk of disease in vaccinated and unvaccinated people and determining the percentage reduction in disease risk in vaccinated people versus unvaccinated people. The higher the percentage reduction in disease in the vaccinated group, the higher the vaccine efficacy. For example, a VE of 90% indicates a 90% reduction in disease incidence in the vaccinated group or a 90% reduction in the number of cases expected in the unvaccinated population. Vaccine efficacy is calculated by the formula: 100*(1-HR), where HR is the hazard ratio defined as the vaccine hazard rate (λv) divided by the placebo hazard rate (λc), i.e., HR=λv / λc. λv indicates the hazard rate in subjects vaccinated with the tetravalent dengue vaccine composition disclosed herein, and λc indicates the hazard rate in unvaccinated subjects, i.e., subjects given a placebo. Hazard rate ratios (HR) are estimated from Cox proportional hazards models with the test vaccine as a factor, adjusted for age, and stratified by region. As used herein, the term "composite vaccine efficacy against all four serotypes" is defined as vaccine efficacy with respect to risk of dengue disease regardless of the serotype causing virologically confirmed dengue disease and the subject's baseline serostatus. A vaccine is considered "effective" if the composite efficacy is greater than 30%. In this context, the composite vaccine efficacy may be 40% or more, 50% or more, 60% or more, 70% or more, 72% or more, or 80% or more, particularly when determined in an age group selected from the age group of subjects 4-16 years, the age group of subjects 4 to <9 years, the age group of subjects 2 to <9 years, the age group of subjects 4-5 years, the age group of subjects 6-11 years, and the age group of subjects 12-16 years, particularly when determined 30 days after the second dose to 12 months after the second dose or 18 months after the second vaccination. In this context, effective specifically refers to a vaccine that is effective for all subjects regardless of baseline serostatus.Preferably, the vaccine is effective for all age groups, preferably starting from 4 years regardless of serostatus, and in particular for 4-60 years or 4-16 years regardless of serostatus. Relevant subgroups in this context are <9 years, 2 to <9 years, 4 to <9 years, 4 to 5 years, 6 to 11 years, and 12 to 16 years or any age group within the range of 4 to 16 years. In certain embodiments, "effective" means providing a combined vaccine efficacy in preventing virologically confirmable dengue disease with a two-sided 95% confidence interval, with a lower limit of combined vaccine efficacy against all four serotypes greater than 60% when measured against placebo in a subject population of at least 5,000 healthy subjects aged 4 to 16 years, regardless of baseline serostatus, from the first dose of the dosing schedule to 18 months after the last dose of the dosing schedule. Further specific efficacies may be defined. As used herein, "combined vaccine efficacy against all four serotypes in seronegative subjects" refers to efficacy measured in subjects who are seronegative at baseline. As used herein, "vaccine efficacy against a particular serotype, e.g., serotype 1" refers to efficacy with respect to a particular serotype that is responsible for virologically confirmed dengue disease. As used herein, "combined vaccine efficacy against all four serotypes against virologically confirmed dengue with hospitalization" refers to efficacy where only virologically confirmed dengue cases with hospitalization are considered. Such vaccine efficacy can be determined for subjects who are seronegative or seropositive at baseline, and for different age groups.

[0048] As used herein, "relative risk" means the number of virologically confirmed dengue disease events divided by the number of subjects treated with a unit dose disclosed herein divided by the number of virologically confirmed dengue disease events divided by the number of subjects treated with a placebo. As used herein, the term "combined relative risk for all four serotypes" is defined as the relative risk for the serotype causing virologically confirmed dengue disease and the risk of dengue disease regardless of the subject's baseline serostatus.

[0049] As used herein, "vaccination" or "inoculation" refers to the administration of a vaccine to a subject, with the goal of preventing the subject from developing one or more symptoms of the disease. As used herein, "vaccination against dengue disease" or "inoculation against dengue disease" refers to the administration of a dengue vaccine composition to a subject, with the goal of preventing the subject from developing one or more symptoms of dengue disease. In principle, the method includes a primary vaccination and, optionally, one or more booster vaccinations. A primary vaccination is defined as a primary administration schedule for administering a composition or unit dose disclosed herein to establish a protective immune response, and consists of, for example, two administrations, for example, within three months. Whenever administration is mentioned within the scope of this disclosure, such administration refers to a primary vaccination unless specified as a booster vaccination. A booster vaccination refers to an administration or administration schedule that is administered after a primary vaccination, for example, at least one year after the last administration, for example, the second administration, of the primary vaccination schedule, or even 4 to 4.5 years, or 5 or 10 years after the last administration, for example, the second administration, of the primary vaccination schedule. Booster administration attempts to enhance or re-establish the immune response of the primary vaccination.

[0050] As used herein, the term "subject(s)" is limited to human subjects (e.g., infants, children, or adults). The term "elderly subject(s)" refers to subjects over 60 years of age, e.g., 61-100 years, 61-90 years, 61-80 years, 61-75 years, or 61-70 years.

[0051] As used herein, "subject population" refers to a group of subjects. A subject population can refer to at least 40 subjects, at least 50 subjects, at least 60 subjects, at least 100 subjects, or at least 1000 subjects, and is defined by certain parameters. Parameters that can be used to define a subject population include, but are not limited to, the age of the subject and the serostatus of the subject, whether the subject is from a dengue endemic or non-dengue endemic area.

[0052] As used herein, an "endemic area" refers to an area in which a disease or infectious agent is constantly present and / or normally prevalent in a population within the area. As used herein, a "non-endemic area" refers to an area in which a disease is not present or normally prevalent. Thus, a "dengue endemic area" refers to a geographic area in which infection with the dengue virus is constantly maintained at a baseline level. A "non-dengue endemic area" is a geographic area in which infection with the dengue virus is not constantly maintained at a baseline level. Thus, a subject population or subject "from a dengue endemic area" or "from a non-dengue endemic area" refers to a subject population or subject residing in a geographic area as defined above. Whether a geographic area or a subject population is dengue endemic or not can be determined by different calculation methods, such as those described in Bhatt et al. (2013) Nature 496(7446):504-507 and supplementary materials and Stanaway et al. (2016) Lancet Infect Dis. 16(6):712-723 and supplementary materials. Overviews of dengue endemic areas and dengue epidemiology are published regularly, for example by WHO or CDC. Typical dengue endemic areas are in Central and South America, Southeast Asia and the Pacific Islands, and dengue endemic countries include, but are not limited to, Australia, Brazil, Bangladesh, Colombia, China, Dominican Republic, Indonesia, India, Mexico, Malaysia, Nicaragua, Nigeria, Pakistan, Panama, Philippines, Puerto Rico, Singapore, Sri Lanka, Thailand and Vietnam. The infectivity of an area is measured by seroprevalence surveys, which are provided as seroprevalence rates. Areas with very high infectivity are considered to have a seroprevalence rate of more than 80%. As used herein, the term "area", when referring to seroprevalence, refers to a geographical area where seroprevalence can be determined or is known, such as a village, town, city, region, county, state, district or parts of the above or the entire country.

[0053] As used herein, "serostatus" refers to the amount of antibodies a subject has for a particular infectious agent, specifically dengue virus. As used herein, "seronegative" or "seronaive" means that a subject does not have neutralizing antibodies in serum to any one of the dengue serotypes DENV-1, DENV-2, DENV-3, and DENV-4. A seronegative or seronaive subject or subject population is defined by a neutralizing antibody titer of less than 10 for each one of the four dengue serotypes. A subject or subject population with a neutralizing antibody titer of 10 or greater for at least one dengue serotype is defined as "seropositive" for that dengue serotype. Serostatus at baseline refers to the serostatus prior to administration of the dengue vaccine composition described herein.

[0054] As used herein, "neutralizing antibody titer" refers to the amount of antibodies in a subject's serum that neutralize each dengue serotype. Neutralizing antibody titers against DENV-1, DENV-2, DENV-3, and DENV-4 are determined in a subject's serum sample using known methods, such as the plaque reduction neutralization test (PRNT) described in the WHO guidelines (World Health Organization Department of Immunization Vaccines Biologicals (2007) Guidelines for plaque reduction neutralization testing of human antibodies to dengue viruses, WHO / IVB / 07.07) or the microneutralization (MNT50) assay described herein. As used herein, "a ratio of dengue serotype 2 neutralizing antibody titer to dengue serotype 4 neutralizing antibody titer of 20 or less" means that the ratio obtained by dividing the dengue serotype 2 neutralizing antibody titer by the dengue serotype 4 neutralizing antibody titer is 20 or less. That is, the dengue serotype 2 neutralizing antibody titer is no more than 20-fold higher than the dengue serotype 4 neutralizing antibody titer in the subject.

[0055] As used herein, the terms "geometric mean neutralizing antibody titer" and "GMT" refer to the geometric mean of the titers of neutralizing antibodies to the corresponding dengue serotype in the sera of subjects in a subject population. The geometric mean is calculated by well-known formulas. As used herein, "a ratio of GMT of dengue serotype 2 to GMT of dengue serotype 4 of 20 or less" means that the ratio obtained by dividing the geometric mean neutralizing antibody titer of dengue serotype 2 (GMT DENV-2) by the geometric mean neutralizing antibody titer of dengue serotype 4 (GMT DENV-4) is 20 or less. That is, the geometric mean neutralizing antibody titer of dengue serotype 2 is 20 times or less higher than the geometric mean neutralizing antibody titer of dengue serotype 4 in the subject population.

[0056] As used herein, "immune response" refers to a subject's response to administration of a dengue vaccine. Specifically, the immune response includes the formation of neutralizing antibodies against one or more dengue serotypes. The immune response may also include the stimulation of a cell-mediated response or the formation of antibodies against nonstructural proteins (e.g., NS1). An immune response is stimulated by administration of a unit dose of the invention described herein if, after said administration of said unit dose, there is an increase in the titer of neutralizing antibodies against at least one dengue virus serotype, preferably all four dengue virus serotypes. An immune response is stimulated by administration of a unit dose of the invention described herein if, after said administration of said unit dose, there is an increase in the secretion of interferon gamma by peripheral blood mononuclear cells stimulated with a peptide derived from a dengue virus protein. An immune response is stimulated by administration of a unit dose of the invention described herein if, after said administration of said unit dose, there is an increase in the titer of antibodies against nonstructural proteins (e.g., NS1). In certain embodiments, administration of a reconstituted unit dose of the invention described herein stimulates the formation of neutralizing antibodies against one or more dengue serotypes, cell-mediated responses, and the formation of antibodies against nonstructural proteins (e.g., NS1).

[0057] As used herein, a "balanced immune response" means that the immune response against the four dengue serotypes is sufficient to provide protection against infection by all four dengue serotypes, and preferably the immune response against the four dengue serotypes is of similar strength. Specifically, the neutralizing antibody titers against the four dengue serotypes at 180 days or 365 days after administration of the first reconstituted unit dose of the invention described herein are similar, i.e., they differ by less than 30-fold, less than 25-fold, or less than 20-fold.

[0058] The "total concentration in pfu / 0.5ml", which serves as the reference value for calculation of the concentration percentage for each individual component of the tetravalent dengue vaccine, is shown for one exemplary tetravalent vaccine composition comprising dengue serotype 1 at a concentration of 3.60 log10 pfu / 0.5ml, dengue serotype 2 at a concentration of 4.00 log10 pfu / 0.5ml, dengue serotype 3 at a concentration of 4.60 log10 pfu / 0.5ml, and dengue serotype 4 at a concentration of 5.11 log10 pfu / 0.5ml. Typically, the logarithm of the concentration is converted to a numerical value. This conversion results in a 4 × 10 3 pfu / 0.5 ml, 1 × 10 for serotype 2 4 pfu / 0.5 ml, 4 × 10 for serotype 3 4 pfu / 0.5 ml and 1.3 × 10 for serotype 4 5 pfu / 0.5 ml. The total concentration in pfu / 0.5 ml is 1.84 x 10 5 The sum of the preceding figures gives pfu / 0.5ml.

[0059] The "concentration percentage" for each of serotypes 1, 2, 3, and 4 is obtained by dividing the concentration value of the individual serotype (expressed as pfu / 0.5 ml) by the total concentration (expressed as pfu / 0.5 ml) and multiplying the result by 100, i.e. Serotype 1 concentration percentage = (4 x 10 3 pfu / 0.5 ml ÷ 1.84 × 10 5 pfu / 0.5ml) x 100 = 2% Serotype 2 concentration percentage = (1 x 10 4 pfu / 0.5 ml ÷ 1.84 × 10 5 pfu / 0.5ml) x 100 = 5% Serotype 3 concentration percentage = (4 x 10 4 pfu / 0.5 ml ÷ 1.84 × 10 5 pfu / 0.5ml) x 100 = 22% Serotype 4 concentration percentage = (1.3 x 10 5 pfu / 0.5 ml ÷ 1.84 × 10 5 pfu / 0.5ml) x 100 = 71%. Concentration percentages are rounded to the nearest whole number.

[0060] As used herein, "concurrent" administration means administration of at least two different vaccines, such as a dengue vaccine and a hepatitis A vaccine, on the same day. "Same day" has its ordinary meaning of within 24 hours (e.g., within one calendar day). Concurrent administration can be administered by the same physician, for example, during the same medical visit.

[0061] As used herein, "sequential" administration means administration of at least two different vaccines (e.g., a dengue vaccine and a yellow fever vaccine, or a dengue vaccine and a hepatitis A vaccine) on different or subsequent days, e.g., within 90 days, but on a combined dosing schedule.

[0062] As used herein, the term "chronic disease or condition" includes diseases and conditions that persist for more than three months in an elderly subject. Specifically, it includes diabetes, hypertension, allergies, previous stroke, ischemic heart disease, chronic kidney disease, and chronic obstructive pulmonary disease.

[0063] As used herein, the term "immune dysfunction" means that at least one function of at least one component of the immune system is weaker than in younger subjects, i.e., subjects having an age less than 60 years old. These functions include a reduced antioxidant response of monocytes to oxidative stress induced by dengue virus and a reduced T cell response and cytokine production in response to dengue virus infection.

[0064] As used herein, "unsolicited systemic adverse events" in children under 6 years of age are defined as fever, irritability / tantrums, drowsiness, and loss of appetite occurring within 14 days after each vaccine dose, and in children 6 years of age and older, as fever, headache, asthenia, malaise, and muscle pain occurring within 14 days after each vaccine dose.

[0065] As used herein, a "non-spontaneous local adverse event" is injection site pain, injection site erythema, or injection site swelling occurring within 7 days after each vaccination.

[0066] As used herein, a "spontaneous adverse event" is any adverse event (AE) that is not an involuntary local or systemic AE as defined above.

[0067] As used herein, a "serious adverse event" or "SAE" is any unexpected medical occurrence or effect at any dose that results in death, is life-threatening, requires hospitalization or an extension of an existing hospitalization, results in a persistent or significant disability / incapacity, is a congenital anomaly / birth defect, or is medically significant for reasons other than the criteria above.

[0068] The relationship of each AE, including involuntary systemic AEs (involuntary local AEs believed to be related), to the study vaccine(s) will be assessed using the following categories: As used herein, "IP-related AE" or "vaccine-related AE" means that there is a relationship between the vaccine and the AE (without a determination of the degree of likelihood); there is a reasonable possibility that the vaccine contributed to the AE. As used herein, "non-IP-related" or "non-vaccine-related" means that there is no suspicion that there is a relationship between the vaccine and the AE; there are other more likely causes, and administration of the vaccine is not suspected to have contributed to the AE.

[0069] As used herein, a subject or subject population that is "2-60 years old" or "18-60 years old" refers to a subject or subject population that is 2-60 years old or 18-60 years old on day 1 of administration of a dengue vaccine composition described herein.

[0070] As used herein, "percentage points" refers to the percentage difference between two percentage values. For example, if two values ​​in % are within 5 percentage points, e.g., one value is 1% and the second value is 6%.

[0071] As used herein, the term "determination of previous dengue infection in a subject prior to dosing" means that previous dengue infection must be assessed prior to vaccination by a suitably validated serological test, e.g., a laboratory-confirmed history of dengue by the methods disclosed herein, such as the MNT50 test described in Example 2, or any serological test with adequate performance in terms of specificity and cross-reactivity based on local disease epidemiology.

[0072] As used herein, % w / v refers to % mg / ml, for example, 150 mg / ml is 15% w / v.

[0073] As used herein, the term "Hepatitis A virus" may be abbreviated as "HAV."

[0074] As used herein, the term "placebo" may be abbreviated as "Pbo."

[0075] As used herein, "hepatitis A seronegative at baseline" or "hepatitis A naive (at baseline)" respectively means that a subject does not have a predefined amount of anti-hepatitis A antibodies in serum. Quantitatively, a subject's hepatitis A seronegativity is defined as an anti-hepatitis A antibody level <10mIU / ml. When determining anti-hepatitis A antibodies by ELISA, the lower level of quantification is 12.5mIU / ml, which is the practical lower level of anti-HAV antibodies for determining seronegativity. A subject with an anti-hepatitis A antibody level ≥ 12.5mIU / ml is defined as hepatitis A seropositive. An ELISA for determining anti-hepatitis A antibodies is disclosed, for example, in Beck et al. J Travel Med 2004;11:201-207.

[0076] As used herein, "at baseline" refers to the time when a subject's serostatus was last measured prior to the first vaccination.

[0077] As used herein, the unit "mIU / ml" means milli-international units per millimeter. This concentration unit refers to the amount of anti-Hepatitis A antibodies in the serum of a subject (e.g., as measured before or after vaccination). As used herein, the "viral antigen activity of the Hepatitis A vaccine" of the present invention is expressed in terms of the WHO's standard recommendation using enzyme-linked immunosorbent assay (ELISA). This WHO recommendation (WHO Information Sheet "Observed Rate of Hepatitis A Vaccine Antibody Activity" published in June 2012) is based on the WHO's standard recommendation using enzyme-linked immunosorbent assay (ELISA). According to the Hepatitis A Vaccine Reactions - Hepatitis A Vaccine (see "E.V. Vaccine Reactions - Hepatitis A Vaccine"), the viral antigen activity of the Hepatitis A vaccine is expressed in terms of ELISA units (EL.U.). The viral antigen activity of the Hepatitis A vaccine can be determined by ELISA, for example, according to Andre FE., Hepburn A., D'Hondt E., "Inactivated candidate vaccines for hepatitis", A. Prog Med Virol 1990;37:72-95.

[0078] As used herein, the term "CCID" refers to the amount of virus (e.g., vaccine virus) that infects 50% of cell cultures. The CCID50 assay is a limiting dilution assay with statistical titer calculation (Morrison D et al, J Infect Dis. 2010; 201(3): 370-7)).

[0079] As used herein, "non-inferiority" with respect to simultaneous administration of hepatitis A vaccine and quadrivalent dengue vaccine on the same day is specifically concluded if the difference in seroprotection rate (SPR) between the SPR of subjects receiving HAV and placebo (same-day simultaneous subjects, i.e., control population) and the SPR of subjects receiving HAV and TDV (same-day simultaneous) has the upper limit of a two-sided 95% confidence interval lower than the non-inferiority margin set at 10%, where the seroprotection rate is based on measurements 30 days after simultaneous administration on day 1 and is calculated using the Newcomb score method. A non-inferiority clinical trial is a study designed to compare at least two treatment methods, in this case simultaneous administration of dengue vaccine and hepatitis A vaccine to administration of either dengue vaccine or hepatitis A vaccine as a single agent.

[0080] As used herein, the term "seroprotection rate" (abbreviated as "SPR") is defined by the proportion / percentage of subjects who are HAV- or DEN-naive at baseline who are seroprotected against HAV or DENV, respectively, 30 days (1 month) after their first vaccination.

[0081] As used herein, the term "control subject population" refers to a group of subjects in a clinical trial setting (e.g., a non-inferiority clinical trial) who receive one verum (e.g., a unit dose of a hepatitis A vaccine or a dengue vaccine composition) and a placebo on the same day, rather than receiving simultaneous unit doses of a hepatitis A vaccine and a dengue vaccine composition.

[0082] As used herein, the term "synergism" or "synergy" is defined as the effect of administering to a subject or population of subjects unit doses of a hepatitis A vaccine and a dengue vaccine composition on the same day simultaneously, which results in higher anti-hepatitis A antibody concentrations and / or higher mean titers of neutralizing antibodies for each serotype of dengue virus than the same day simultaneously administered corresponding hepatitis A vaccine and a placebo, and / or the same day simultaneously administered unit doses of a dengue vaccine composition and a placebo (monoadministration). Thus, higher antibody concentrations following simultaneous administration compared to monoadministration are an indication in favor of simultaneous administration. [Brief description of the drawings]

[0083] [Figure 1] Genetic structure of the four dengue strains contained in TDV. The open red triangles indicate the three attenuating mutations present in the 5'NCR, NS1 and NS3 proteins. The TDV-1, TDV-3 and TDV-4 strains are chimeric viruses in which the prM and E genes from dengue serotypes 1, 3 and 4, respectively, have been inserted into the TDV-2 backbone. [Diagram 2] Schematic diagram showing the microneutralization test (MNT) used to determine titers of neutralizing antibodies. [Diagram 3] FIG. 1 is a flow diagram of the clinical trial of Example 3. [Figure 4]Cumulative incidence of A) virologically confirmed dengue cases and B) hospitalized virologically confirmed dengue cases over time during the Part 1 study period by baseline serostatus (safety set data; data shown truncated at 18 months). The table shows the number of participants under follow-up at various time points until the end of the Part 1 study period. [Diagram 5] Study design for Phase III study described in Example 3. [Figure 6] Schematic of study design for the HAV and TDV co-administration study described in Example 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0084] Dengue virus strains Dengue virus is a single-stranded positive-sense RNA virus of the Flaviviridae family. The classification is summarized in Table 1. The Flaviviridae family includes three genera: Flavivirus, Hepacivirus, and Pestivirus. The Flavivirus genus contains highly pathogenic and potentially hemorrhagic fever viruses, such as yellow fever virus and dengue virus, encephalitis viruses, such as Japanese encephalitis virus, Murray Valley encephalitis virus, and West Nile virus, as well as a number of less pathogenic viruses.

[0085] [Table 1]

[0086] The flavivirus genome consists of the following sequences, from 5' to 3' (see Figure 1): -5'-non-coding region (5'-NCR), - the capsid protein (C) coding region, - the premembrane protein (prM) coding region, - the envelope protein (E) coding region, - the regions coding for the nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) and - 3' non-coding region (3'-NCR).

[0087] The viral structural proteins are C, prM and E, and the nonstructural proteins are NS1 to NS5. The structural and nonstructural proteins are translated as a single polyprotein and processed by cellular and viral proteases.

[0088] The unit dose of the invention described herein comprises a dengue virus composition comprising four live attenuated dengue virus strains (tetravalent dengue virus composition) representing dengue serotype 1, dengue serotype 2, dengue serotype 3, and dengue serotype 4. Preferably, the composition comprises a chimeric dengue virus and optionally at least one non-chimeric dengue virus, in particular a molecularly characterized and cloned dengue serotype 2 strain derived from the live attenuated DEN-2 PDK-53 virus strain (TDV-2), as well as the following chimeric dengue virus strains in which the structural proteins prM and E from the TDV-2 strain are replaced with the corresponding structural proteins from other dengue serotypes: -DENV-2 / 1 chimera (TDV-1), -DENV-2 / 3 chimera (TDV-3) and -DENV-2 / 4 chimera (TDV-4), which include three chimeric dengue strains derived from a TDV-2 strain by

[0089] The genetically modified tetravalent dengue vaccine TDV is based on the molecularly characterized and cloned dengue-2 virus strain (TDV-2). This attenuated TDV-2 strain was generated by cDNA cloning of the laboratory-derived attenuated DEN-2 PDK-53 virus strain originally isolated at Mahidol University, Bangkok, Thailand (Kinney et al. (1997) Virology 230(2):300-308). DEN-2 PDK-53 was generated by 53 serial passages at 32°C in primary canine kidney (PDK) cells (Bhamarapravati et al. (1987) Bull. World Health Organ. 65(2):189-195).

[0090] The attenuated DEN-2 PDK-53 strain (the precursor of TDV-2) is derived from the wild-type virus strain DEN-2 16681 (SEQ ID NO: 11) and differs from the wild-type in nine nucleotides as follows (Kinney et al. (1997) Virology 230(2):300-308): (i) 5'-noncoding region (NCR)-57 (nt-57 C to T): the major attenuation locus (ii) prM-29 Asp to Val (nt-524 A to T) (iii) nt-2055 C to T (E gene) silent mutation (iv) NS1-53 Gly to Asp (nt-2579 G to A): major attenuation locus (v) NS2A-181 Leu to Phe (nt-4018 C to T) (vi) NS3-250 Glu to Val (nt-5270 A to T): major attenuation locus (vii) nt-5547 (NS3 gene) T to C silent mutation (viii) NS4A-75 Gly to Ala (nt-6599 G to C) * nt-8571 C to T (NS5 gene) silent mutation

[0091] Three nucleotide changes located in the 5' non-coding region (NCR) (nucleotide 57) (mutation (i)), the NS-1 (amino acid 828 of SEQ ID NO:4) (mutation (iv)) and the NS-3 gene (amino acid 1725 of SEQ ID NO:4) (mutation (vi)) form the basis for the attenuated phenotype of the DEN-2 PDK-53 strain (Butrapet et al. (2000) J. Virol. 74(7):3111-3119) (Table 2). These three mutations are referred to herein as "attenuating mutations" and are contained in TDV-1, TDV-2, TDV-3 and TDV-4.

[0092] [Table 2]

[0093] In one embodiment, the TDV-2 contains, in addition to the three attenuating mutations: a) an adenine to thymidine mutation at nucleotide 524 in the prM gene, resulting in an amino acid change from asparagine to valine at position 143; and / or b) a silent mutation from cytosine to thymidine at nucleotide 2055 in the E gene, and / or c) a cytosine to thymidine mutation at nucleotide 4018 in the NS2A gene, resulting in an amino acid change from leucine to phenylalanine at position 1308; and / or d) a silent thymidine to cytosine mutation at nucleotide 5547 in the NS3 gene; and / or e) a guanine to cytosine mutation at nucleotide 6599 in the NS4A gene, resulting in an amino acid change from glycine to alanine at position 2168, and / or f) a silent thymidine to cytosine mutation at nucleotide 900 in the prM gene. The silent cytosine to thymidine mutation at nucleotide 8571 in the NS5 gene of DEN-2 PDK-53 is not present in TDV-2 strains.

[0094] In another embodiment, the TDV-2 contains, in addition to the three attenuating mutations: g) an adenine to guanine mutation at nucleotide 592 in the prM gene resulting in an amino acid change from lysine to glutamine at position 166; and / or h) an adenine to guanine mutation at nucleotide 8803 in the NS5 gene, resulting in an amino acid change from isoleucine to valine at position 2903.

[0095] In another embodiment, the TDV-2 comprises, in addition to the three attenuating mutations, mutations a) and g), preferably mutations a), g), c), e), and h), more preferably mutations a), g), c), e), h), and b), even more preferably mutations a), g), c), e), h), b), and d), most preferably mutations a) through h). The nucleotide and amino acid positions of TDV-2 refer to the nucleotide sequence shown in SEQ ID NO:3 and the amino acid sequence shown in SEQ ID NO:4.

[0096] The structural envelope (E) and premembrane (prM) proteins of dengue virus have been identified as the primary antigens that elicit neutralizing protective antibody responses (Plotkin 2001). To generate a tetravalent dengue vaccine (TDV), standard molecular genetic engineering methods (Huang et al. (2003) J. Virol. 77(21):11436-11447) were used to modify DENV-2 by replacing the nucleic acid sequences encoding the prM and E glycoproteins of DENV-2 with the corresponding wild-type prM and E glycoproteins encoding nucleic acid sequences from the wild-type strains of DENV-1, DENV-3, and DENV-4, DENV-1 16007, DENV-3 16562, or DENV-4 1036 viruses, respectively (see Table 3).

[0097] [Table 3]

[0098] A diagram of the four TDV strains included in the dengue vaccine composition is shown in FIG.

[0099] Chimeric dengue strains TDV-1, TDV-3, and TDV-4 express the surface antigens prM and E of DENV-1, DENV-3, or DENV-4 viruses, respectively, and retain the genetic changes responsible for the attenuation of TDV-2, as shown in Table 3. Thus, each of the TDV-1, TDV-3, and TDV-4 strains contains the attenuating mutations listed in Table 2.

[0100] In one embodiment, the TDV-1 comprises, in addition to the three attenuating mutations: c) a cytosine to thymidine mutation at nucleotide 4018 in the NS2A gene, resulting in an amino acid change from leucine to phenylalanine at position 1308; and / or d) a silent thymidine to cytosine mutation at nucleotide 5547 in the NS3 gene; and / or e) a guanine to cytosine mutation at nucleotide 6599 in the NS4A gene resulting in an amino acid change from glycine to alanine at position 2168; and / or i) a silent thymidine to cytosine mutation at nucleotide 1575 in the E gene, and / or j) a silent adenine to guanine mutation at nucleotide 453 at the junction between the prM-E gene and the DEN-2 PDK-53 backbone, and / or k) a thymidine-guanine to cytosine-cytosine mutation at nucleotides 2381 / 2382 at the junction site between the prM-E gene and the DEN-2 PDK-53 backbone, resulting in an amino acid change from valine to alanine at position 762.

[0101] In another embodiment, the TDV-1 comprises, in addition to the three attenuating mutations: l) an adenine to cytosine mutation at nucleotide 3823 in the NS2A gene, resulting in an amino acid change from isoleucine to leucine at position 1243; and / or m) an adenine to thymidine mutation at nucleotide 4407 in the NS2B gene, resulting in an amino acid change from glutamine to asparagine at position 1437; and / or n) a silent adenine to guanine mutation at nucleotide 7311 in the NS4B gene.

[0102] In another embodiment, the TDV-1 strain comprises, in addition to the three attenuating mutations, mutations l) and m), preferably mutations l), m), c), and e), even more preferably mutations l), m), c), e), d), and n), most preferably mutations l), m), c), e), d), n), i), j), and k). The nucleotide and amino acid positions of TDV-1 refer to the nucleotide sequence shown in SEQ ID NO:1 and the amino acid sequence shown in SEQ ID NO:2.

[0103] In one embodiment, the TDV-3 contains, in addition to the three attenuating mutations: c) a cytosine to thymidine mutation at nucleotide 4012 in the NS2A gene, resulting in an amino acid change from leucine to phenylalanine at position 1306; and / or d) a silent thymidine to cytosine mutation at nucleotide 5541 in the NS3 gene; and / or e) a guanine to cytosine mutation at nucleotide 6593 in the NS4A gene, resulting in an amino acid change from glycine to alanine at position 2166; and / or j) a silent adenine to guanine mutation at nucleotide 453 at the junction between the prM-E gene and the DEN-2 PDK-53 backbone, and / or k) a thymidine-guanine to cytosine-cytosine mutation at nucleotides 2375 / 2376 at the junction between the prM-E gene and the DEN-2 PDK-53 backbone, resulting in an amino acid change from valine to alanine at position 760; and / or o) a silent mutation from cytosine to thymidine at nucleotide 552 in the prM gene, and / or p) an adenine to thymidine mutation at nucleotide 1970 in the E gene, which results in an amino acid change from histidine to leucine at position 625.

[0104] In another embodiment, the TDV-3 contains, in addition to the three attenuating mutations: q) an adenine to thymidine mutation at nucleotide 1603 in the E gene, resulting in an amino acid change from threonine to serine at position 503, and / or r) a silent adenine to guanine mutation at nucleotide 7620 in the NS5 gene.

[0105] In another embodiment, TDV-3 comprises, in addition to the three attenuating mutations, mutations p) and q), preferably mutations p), q), c), and e), even more preferably mutations p), q), c), e), d), and r), and most preferably mutations p), q), c), e), d), r), j), k), and o). The nucleotide and amino acid positions of TDV-3 refer to the nucleotide sequence shown in SEQ ID NO:5 and the amino acid sequence shown in SEQ ID NO:6.

[0106] In one embodiment, the TDV-4 contains, in addition to the three attenuating mutations: c) a cytosine to thymidine mutation at nucleotide 4018 in the NS2A gene, resulting in an amino acid change from leucine to phenylalanine at position 1308; and / or d) a silent thymidine to cytosine mutation at nucleotide 5547 in the NS3 gene; and / or e) a guanine to cytosine mutation at nucleotide 6599 in the NS4A gene resulting in an amino acid change from glycine to alanine at position 2168; and / or j) a silent adenine to guanine mutation at nucleotide 453 at the junction between the prM-E gene and the DEN-2 PDK-53 backbone, and / or k) a thymidine-guanine to cytosine-cytosine mutation at nucleotides 2381 / 2382 at the junction between the prM-E gene and the DEN-2 PDK-53 backbone, resulting in an amino acid change from valine to alanine at position 762; and / or s) an adenine to cytosine mutation at nucleotide 396 in the C gene resulting in an amino acid change from arginine to serine at position 100; and / or t) a silent adenine to guanine mutation at nucleotide 1401 in the E gene, and / or u) a cytosine to thymidine mutation at nucleotide 2027 in the E gene, resulting in an amino acid change from alanine to valine at position 644; and / or v) an adenine to cytosine mutation at nucleotide 2275 in the E gene, resulting in an amino acid change from methionine to leucine at position 727.

[0107] In another embodiment, the TDV-4 contains, in addition to the three attenuating mutations: w) a silent adenine to thymidine mutation at nucleotide 225 in the C gene, and / or x) an adenine to guanine mutation at nucleotide 3674 in the NS2A gene, resulting in an amino acid change from asparagine to glycine at position 1193; and / or y) a mutation in the NS2A gene at nucleotide 3773 from adenine to adenine / guanine mix, resulting in an amino acid change at position 1226 from lysine to lysine / asparagine mix, and / or z) a silent mutation from cytosine to thymidine at nucleotide 5391 in the NS3 gene, and / or aa) a cytosine to thymidine mutation at nucleotide 6437 in the NS4A gene, resulting in an amino acid change from alanine to valine at position 2114; and / or bb) a silent mutation from thymidine to a thymidine / cytosine mix at nucleotide 7026 in the NS4B gene, and / or cc) a silent adenine to cytosine mutation at nucleotide 9750 in the NS5 gene.

[0108] In another embodiment, the TDV-4 comprises, in addition to the three attenuating mutations, mutations s), u), and v), preferably mutations s), u), v), c), e), x), y), and aa), even more preferably mutations s), u), v), c), e), x), y), aa), and w), even more preferably mutations s), u), v), c), e), x), y), aa), w), d), z), bb), and cc), and most preferably mutations s), u), v), c), e), x), y), aa), w), d), z), bb), cc), j), k), and t). The nucleotide and amino acid positions of TDV-4 refer to the nucleotide sequence shown in SEQ ID NO:7 and the amino acid sequence shown in SEQ ID NO:8.

[0109] In a preferred embodiment, TDV-1 has the nucleotide sequence of SEQ ID NO: 1, TDV-2 has the nucleotide sequence of SEQ ID NO: 3, TDV-3 has the nucleotide sequence of SEQ ID NO: 5, and / or TDV-4 has the nucleotide sequence of SEQ ID NO: 7. In a further preferred embodiment, TDV-1 has the amino acid sequence of SEQ ID NO: 2, TDV-2 has the amino acid sequence of SEQ ID NO: 4, TDV-3 has the amino acid sequence of SEQ ID NO: 6, and TDV-4 has the amino acid sequence of SEQ ID NO: 8. In a further preferred embodiment, TDV-1 has a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2, TDV-2 has a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4, TDV-3 has a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 6, and TDV-4 has a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 8.

[0110] [Table 4]

[0111] Thus, in certain preferred embodiments, a unit dose of the invention described herein comprises live attenuated dengue virus strains TDV-1, TDV-2, TDV-3, and TDV-4, which are based on TDV-2 and contain the prM and E regions of DENV-1, -3, and -4, respectively. In another particular preferred embodiment, TDV-1 is characterized by the nucleotide sequence set forth in SEQ ID NO:1 and the amino acid sequence set forth in SEQ ID NO:2, TDV-2 is characterized by the nucleotide sequence set forth in SEQ ID NO:3 and the amino acid sequence set forth in SEQ ID NO:4, TDV-3 is characterized by the nucleotide sequence set forth in SEQ ID NO:5 and the amino acid sequence set forth in SEQ ID NO:6, and TDV-4 is characterized by the nucleotide sequence set forth in SEQ ID NO:7 and the amino acid sequence set forth in SEQ ID NO:8.

[0112] The DENV-3 E protein has two fewer amino acids than the DENV-2 E protein, so the TDV-2 nucleotide and encoded amino acid backbone starting after the DENV-3 E region at nucleotide 2374 of SEQ ID NO:5 and amino acid 760 of SEQ ID NO:6 have six fewer nucleotides and two fewer amino acids than the original TDV-2 nucleotide and amino acid positions, respectively.

[0113] Dengue vaccine composition The present invention is directed, in part, to a unit dose of a dengue vaccine composition as described above. The dengue vaccine composition includes a tetravalent dengue virus composition, also referred to as a dengue virus composition, and a pharma- ceutically acceptable excipient.

[0114] Dengue virus composition, virus concentration and % concentration The invention relates, in part, to a unit dose of a dengue vaccine composition, the dengue vaccine composition comprising four live attenuated dengue virus strains: (i) dengue serotype 1, preferably at a concentration of at least 3.3 log10 pfu / 0.5 mL; (ii) dengue serotype 2, preferably at a concentration of at least 2.7 log pfu / 0.5 mL; (iii) dengue serotype 3, preferably at a concentration of at least 4.0 log pfu / 0.5 mL; and (iv) to a unit dose of a dengue vaccine composition comprising a tetravalent dengue virus composition, preferably comprising dengue serotype 4 at a concentration of at least 4.5 log10 pfu / 0.5 mL;

[0115] In one embodiment, the dengue vaccine composition comprises four live attenuated dengue virus strains: (i) dengue serotype 1, preferably at a concentration of at least 3.3 log10 pfu / 0.5 mL to 3.8 log10 pfu / 0.5 mL; (ii) dengue serotype 2, preferably at a concentration of at least 2.7 log pfu / 0.5 mL; (iii) dengue serotype 3, preferably at a concentration of at least 4.0 log pfu / 0.5 mL; and (iv) A tetravalent dengue virus composition comprising dengue serotype 4, preferably at a concentration of at least 4.5 log10 pfu / 0.5 ml or 4.6 log10 pfu / 0.5 ml, optionally up to 6.2 log10 pfu / 0.5 ml.

[0116] The invention further relates, in part, to a unit dose of a dengue vaccine composition, the dengue vaccine composition comprising four live attenuated dengue virus strains: (i) a chimeric dengue serotype 2 / 1 strain at a concentration of at least 3.3 log10 pfu / 0.5 mL; (ii) a dengue serotype 2 strain at a concentration of at least 2.7 log10 pfu / 0.5 mL; (iii) a chimeric dengue serotype 2 / 3 strain at a concentration of at least 4.0 log10 pfu / 0.5 mL; and (iv) to a unit dose of a dengue vaccine composition comprising a tetravalent dengue virus composition comprising a chimeric dengue serotype 2 / 4 strain at a concentration of at least 4.5 log10 pfu / 0.5 mL;

[0117] In one embodiment, the dengue vaccine composition comprises four live attenuated dengue virus strains: (i) a chimeric dengue serotype 2 / 1 strain with a concentration of at least 3.3 log10 pfu / 0.5 mL to 3.8 log10 pfu / 0.5 mL, (ii) a dengue serotype 2 strain with a concentration of at least 2.7 log10 pfu / 0.5 mL, (iii) a chimeric dengue serotype 2 / 3 strain with a concentration of at least 4.0 log10 pfu / 0.5 mL, and (iv) a tetravalent dengue virus composition comprising a chimeric dengue serotype 2 / 4 strain at a concentration of at least 4.5 log10 pfu / 0.5 mL or at least 4.6 log10 pfu / 0.5 mL, optionally up to 6.2 log10 pfu / 0.5 ml.

[0118] Preferably, the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3, and the chimeric dengue serotype 2 / 4 strain is TDV-4.

[0119] In one embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 log10 pfu / 0.5 mL to 5.3 log10 pfu / 0.5 mL; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 2.7 log10 pfu / 0.5 mL to 5.0 log10 pfu / 0.5 mL; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log10 pfu / 0.5 mL to 6.0 log10 pfu / 0.5 mL; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 4.5 log10 pfu / 0.5 mL to 6.5 log10 pfu / 0.5 mL.

[0120] In one such embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 log10 pfu / 0.5 mL to 5.0 log10 pfu / 0.5 mL; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 2.7 log10 pfu / 0.5 mL to 4.9 log10 pfu / 0.5 mL; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log10 pfu / 0.5 mL to 5.7 log10 pfu / 0.5 mL; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 4.5 log10 pfu / 0.5 mL to 6.2 log10 pfu / 0.5 mL.

[0121] In further such embodiments, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strains) have a concentration of 3.3 log10 pfu / dose to 5.0 log10 pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) have a concentration of 2.7 log10 pfu / dose to 4.9 log10 pfu / dose; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log10 pfu / dose to 5.7 log10 pfu / dose; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 4.5 log10 pfu / dose to 5.5 log10 pfu / dose.

[0122] In further such embodiments, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strains) have a concentration of 3.3 log10 pfu / dose to 4.1 log10 pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) have a concentration of 2.7 log10 pfu / dose to 3.6 log10 pfu / dose, (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log10 pfu / dose to 4.7 log10 pfu / dose, (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 4.5 log10 pfu / dose to 5.3 log10 pfu / dose.

[0123] In further such embodiments, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 log10 pfu / 0.5 mL to 3.6 log10 pfu / 0.5 mL; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 2.7 log10 pfu / 0.5 mL to 4.0 log10 pfu / 0.5 mL; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log10 pfu / 0.5 mL to 4.6 log10 pfu / 0.5 mL; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 4.5 log10 pfu / 0.5 ml or 4.6 log10 pfu / 0.5 mL to 5.1 log10 pfu / 0.5 mL.

[0124] In another embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) has a concentration of 4.3 log10 pfu / 0.5 mL to 4.4 log10 pfu / 0.5 mL; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 3.7 log10 pfu / 0.5 mL to 3.8 log10 pfu / 0.5 mL; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.5 log10 pfu / 0.5 mL to 5.0 log10 pfu / 0.5 mL; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 5.5 log10 pfu / 0.5 mL to 5.6 log10 pfu / 0.5 mL.

[0125] In a particularly preferred embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) has a concentration of 4.4 log pfu / 0.5 mL; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 3.8 log pfu / 0.5 mL; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.5 log10 pfu / 0.5 mL; (iv) Dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) has a concentration of 5.6 log10 pfu / 0.5 mL.

[0126] In another particularly preferred embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) has a concentration of 3.6 log pfu / 0.5 mL; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 4.0 log pfu / 0.5 mL; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.6 log10 pfu / 0.5 mL; (iv) Dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) has a concentration of 5.1 log10 pfu / 0.5 mL.

[0127] In another preferred embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, the arithmetic sum of all four serotypes is less than 6.7 log10 pfu / 0.5 mL, preferably less than 5.5 log10 pfu / 0.5 mL. In certain such embodiments, the arithmetic sum of all four serotypes is at least 4.6 log10 pfu / 0.5 mL. In a preferred embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, the arithmetic sum of all four serotypes is in the range of 4.6 log10 pfu / 0.5 mL to 6.7 log10 pfu / 0.5 mL, preferably in the range of 4.6 log10 pfu / 0.5 mL to 5.5 log10 pfu / 0.5 mL.

[0128] Preferably, in the aforementioned embodiment, the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3 and the chimeric dengue serotype 2 / 4 strain is TDV-4. More preferably, TDV-1 is characterized by a nucleotide sequence set forth in SEQ ID NO:1 and an amino acid sequence set forth in SEQ ID NO:2, TDV-2 is characterized by a nucleotide sequence set forth in SEQ ID NO:3 and an amino acid sequence set forth in SEQ ID NO:4, TDV-3 is characterized by a nucleotide sequence set forth in SEQ ID NO:5 and an amino acid sequence set forth in SEQ ID NO:6 and TDV-4 is characterized by a nucleotide sequence set forth in SEQ ID NO:7 and an amino acid sequence set forth in SEQ ID NO:8.

[0129] The invention relates, in part, to a unit dose of a dengue vaccine composition, the dengue vaccine composition comprising four live attenuated dengue virus strains: (i) dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain) at a concentration of at least 3.3 log10 pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) at a concentration of at least 2.7 log pfu / dose; (iii) dengue serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain) at a concentration of at least 4.0 log pfu / dose, and (iv) to a unit dose of a dengue vaccine composition comprising a tetravalent dengue virus composition comprising dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) at a concentration of at least 4.5 log10 pfu / dose;

[0130] In one embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strains) have a concentration of 3.3 log10 pfu / dose to 5.3 log10 pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) have a concentration of 2.7 log pfu / dose to 5.0 log pfu / dose; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log10 pfu / dose to 6.0 log10 pfu / dose; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 4.5 log10 pfu / dose to 6.5 log10 pfu / dose.

[0131] In one such embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strains) have a concentration of 3.3 log10 pfu / dose to 5.0 log10 pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) have a concentration of 2.7 log10 pfu / dose to 4.9 log10 pfu / dose; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log10 pfu / dose to 5.7 log10 pfu / dose, and (iv) dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 4.5 log10 pfu / dose to 6.2 log10 pfu / dose.

[0132] In further such embodiments, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strains) have a concentration of 3.3 log10 pfu / dose to 5.0 log10 pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) have a concentration of 2.7 log10 pfu / dose to 4.9 log10 pfu / dose; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log10 pfu / dose to 5.7 log10 pfu / dose; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 4.5 log10 pfu / dose to 5.5 log10 pfu / dose.

[0133] In further such embodiments, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strains) have a concentration of 3.3 log10 pfu / dose to 4.1 log10 pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) have a concentration of 2.7 log10 pfu / dose to 3.6 log10 pfu / dose; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log10 pfu / dose to 4.7 log10 pfu / dose; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 4.5 log10 pfu / dose to 5.3 log10 pfu / dose.

[0134] In further such embodiments, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strains) have a concentration of 3.3 log10 pfu / dose to 3.6 log10 pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) have a concentration of 2.7 log pfu / dose to 4.0 log pfu / dose; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log10 pfu / dose to 4.6 log10 pfu / dose; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 4.5 log10 pfu / dose 4.6 log10 pfu / dose to 5.1 log10 pfu / dose.

[0135] In another embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strains) have a concentration of 4.3 log10 pfu / dose to 4.4 log10 pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) have a concentration of 3.7 log10 pfu / dose to 3.8 log10 pfu / dose; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.5 log10 pfu / dose to 5.0 log10 pfu / dose; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 5.5 log10 pfu / dose to 5.6 log10 pfu / dose.

[0136] In a particularly preferred embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, where (i) dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain) has a concentration of 4.4 log10 pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 3.8 log pfu / dose; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.5 log10 pfu / dose; (iv) Dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) has a concentration of 5.6 log10 pfu / dose.

[0137] In another particularly preferred embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) has a concentration of 3.6 log10 pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 4.0 log pfu / dose; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.6 log10 pfu / dose; (iv) Dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) has a concentration of 5.1 log10 pfu / dose.

[0138] In another preferred embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, the arithmetic sum of all four serotypes is less than 6.7 log10 pfu / dose, preferably less than 5.5 log10 pfu / dose. In certain such embodiments, the arithmetic sum of all four serotypes is at least 4.6 log10 pfu / dose. In a preferred embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, the arithmetic sum of all four serotypes is in the range of 4.6 log10 pfu / dose to 6.7 log10 pfu / dose, preferably in the range of 4.6 log10 pfu / dose to 5.5 log10 pfu / dose.

[0139] In one embodiment, in the composition, (i), (ii), (iii), and (iv) have a total concentration of pfu / 0.5 mL, and based on that concentration, the concentration of (iii) is at least 10% of the total concentration in pfu / 0.5 mL.

[0140] In one embodiment, in the composition, (i), (ii), (iii), and (iv) have a total concentration of pfu / 0.5 mL, and based on the total concentration, the concentration of (ii) at pfu / 0.5 mL is less than 10%, the concentration of (iv) at pfu / 0.5 mL is at least 50%, the concentration of (i) at pfu / 0.5 mL is at least 1%, and the concentration of (iii) at pfu / 0.5 mL is at least 6%, at least 8%, or at least 10%, or at least 12%, or at least 14%, or at least 16%, or at least 18%.

[0141] The concentration in the reconstituted unit dose of (iii) in pfu / 0.5 mL is preferably at least 10%.

[0142] In one embodiment, in the composition, (i), (ii), (iii), and (iv) have a total concentration of pfu / 0.5 mL, and based on said total concentration, the concentration of (ii) at pfu / 0.5 mL is less than 2%, the concentration of (iv) at pfu / 0.5 mL is at least 50%, the concentration of (i) at pfu / 0.5 mL is at least 1%, and the concentration of (iii) at pfu / 0.5 mL is at least 6%.

[0143] Preferably, in the aforementioned embodiment, the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3 and the chimeric dengue serotype 2 / 4 strain is TDV-4. More preferably, TDV-1 is characterized by a nucleotide sequence set forth in SEQ ID NO:1 and an amino acid sequence set forth in SEQ ID NO:2, TDV-2 is characterized by a nucleotide sequence set forth in SEQ ID NO:3 and an amino acid sequence set forth in SEQ ID NO:4, TDV-3 is characterized by a nucleotide sequence set forth in SEQ ID NO:5 and an amino acid sequence set forth in SEQ ID NO:6 and TDV-4 is characterized by a nucleotide sequence set forth in SEQ ID NO:7 and an amino acid sequence set forth in SEQ ID NO:8.

[0144] The concentrations of the different dengue viruses are preferably determined by immunofocus assays known in the art. For example, the concentrations can be determined by immunofocus assays in which serial dilutions of dengue viruses are applied to a monolayer of adherent cells, such as Vero cells. After a period to allow the infectious viruses to bind and be taken up by the cells, an overlay containing a thickening agent, such as agarose or carboxymethylcellulose, is added to prevent the spread of the virus so that progeny viruses can only infect cells adjacent to the original infected cells. After an incubation period to allow for viral replication, the cells are fixed and stained using a serotype-specific anti-dengue monoclonal antibody and a secondary antibody, such as an antibody labeled with alkaline phosphatase. The bright spots are stained by adding a suitable substrate for the enzyme conjugated to the secondary antibody, such as 5-bromo-4-chloro-3-indolyl-phosphate / nitro blue tetrazolium phosphatase substrate. The number of plaques on the plate corresponds to the plaque-forming units of the virus in the solution applied to the cells. For example, a concentration of 1,000 pfu / μl indicates that 1 μl of solution applied to cells contains enough virus to produce 1,000 plaques in the cell monolayer.

[0145] The dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, a chimeric dengue serotype 2 / 1 strain, a dengue serotype 2 strain, a chimeric dengue serotype 2 / 3 strain, and a chimeric dengue serotype 2 / 4 strain, yielding a total concentration of pfu / 0.5 mL. The term "total concentration in pfu / 0.5 mL" or "total concentration in pfu / dose" is the sum of the concentrations of dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strains), dengue serotype 2 (e.g., dengue serotype 2 strains), dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) and dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains), preferably the sum of the concentrations of TDV-1, TDV-2, TDV-3, and TDV-4, and is defined as 100% dengue virus concentration determined by pfu (plaque forming units) in 0.5 mL or in a dose.

[0146] In one embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, wherein dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain), dengue serotype 2 (e.g., a dengue serotype 2 strain), dengue serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain), and dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) are obtained at a total concentration of pfu / 0.5 mL, whereby the concentration of dengue serotype 2 (e.g., a dengue serotype 2 strain) measured in pfu / 0.5 mL is less than 10% of the total concentration, or less than 8% of the total concentration, or less than 6% of the total concentration, and the concentration of dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) measured in pfu / 0.5 mL is at least 50% of the total concentration, or at least 60% of the total concentration, or at least 65% of the total concentration. In one embodiment, the concentration of dengue serotype 2 (e.g., dengue serotype 2 strain) measured in pfu / 0.5 mL is 0.3-10% or 0.5-8% of the total concentration, and the concentration of dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strain) measured in pfu / 0.5 mL is 50%-90% or 60%-88% of the total concentration, based on the total concentration. This means that the concentration of dengue serotype 2 (e.g., dengue serotype 2 strain) is lower than the concentration of dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strain).

[0147] In one such embodiment, the concentration of dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain) measured in pfu / 0.5 mL is at least 1% of the total concentration, and / or the concentration of dengue serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain) measured in pfu / 0.5 mL is at least 6% of the total concentration, or at least 7%, or 8%, 10%, 12%, 14%, 16%, or 18% of the total concentration. In one such embodiment, the concentration of dengue serotype 2 (e.g., chimeric dengue serotype 2 / 1 strains) measured in pfu / 0.5 mL is 1%-7% or 2%-6% or 2.0%-5.0% of the total concentration, and / or the concentration of dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) measured in pfu / 0.5 mL is 6%-25% or 7%-25% or 10%-25% or 18%-25% of the total concentration. This means that the concentration of dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strains) is lower than the concentration of dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains).

[0148] In a preferred embodiment, the concentration of dengue serotype 2 strains such as TDV-2 measured in pfu / 0.5 mL is less than 10%, preferably less than 6% or less than 2% of the total concentration, the concentration of dengue serotype 4 such as TDV-4 (e.g. chimeric dengue serotype 2 / 4 strains) measured in pfu / 0.5 mL is at least 50%, preferably at least 65% of the total concentration, the concentration of dengue serotype 1 such as TDV-1 (e.g. chimeric dengue serotype 2 / 1 strains) measured in pfu / 0.5 mL is at least 1%, preferably between 1%-7% or 2.0%-5.0% of the total concentration, and the concentration of dengue serotype 3 such as TDV-3 (e.g. chimeric dengue serotype 2 / 3 strains) measured in pfu / 0.5 mL is at least 6%, preferably between 6%-25% or 10%-25% or 18%-25% of the total concentration.

[0149] In a further preferred embodiment, a dengue virus composition is provided that includes dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strains), dengue serotype 2 (e.g., dengue serotype 2 strains), dengue serotype 1 (e.g., chimeric dengue serotype 2 / 3 strains), and dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains), such as TDV-1, TDV-2, TDV-3, and TDV-4, and is capable of detecting dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strains) measured in pfu / 0.5 mL. The concentration of dengue serotype 1 (e.g., dengue serotype 2 strains) measured in pfu / 0.5 mL is at least 1% of the total concentration, preferably between 1% and 7% or 2.0% and 5.0%, the concentration of dengue serotype 2 (e.g., dengue serotype 2 strains) measured in pfu / 0.5 mL is less than 10% of the total concentration, preferably less than 6% or less than 2%, and the concentration of dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) measured in pfu / 0.5 mL is at least 6% of the total concentration, preferably between 6% and 25% or 10% and 25% or 18% and 25%. It is particularly preferred that dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have the highest concentration of all four dengue serotypes.

[0150] In a further preferred embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, wherein the concentration of dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain) measured in pfu / 0.5 mL is 1%-7% of the total concentration, the concentration of dengue serotype 2 (e.g., a dengue serotype 2 strain) measured in pfu / 0.5 mL is less than 8% of the total concentration, e.g., in the range of 1%-8% of the total concentration, the concentration of dengue serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain) measured in pfu / 0.5 mL is at least 10% of the total concentration, and the concentration of dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) measured in pfu / 0.5 mL is at least 65% of the total concentration, e.g., in the range of 65%-80%. In certain such embodiments, the arithmetic sum of all four serotypes is within the range of 4.6 log10 pfu / 0.5 mL to 6.7 log10 pfu / 0.5 mL, preferably within the range of 4.6 log10 pfu / 0.5 mL to 5.5 log10 pfu / 0.5 mL.

[0151] In further preferred embodiments, dengue serotype 1, such as TDV-1 (e.g., a chimeric dengue serotype 2 / 1 strain) and dengue serotype 2, such as TDV-2 (e.g., a dengue serotype 2 strain), are each present at concentrations within 5 percentage points of each other, based on total concentration in pfu / 0.5 mL, and / or together are less than about 10% of the total concentration in pfu / 0.5 mL. In certain such embodiments, dengue serotype 3, such as TDV-3 (e.g., a chimeric dengue serotype 2 / 3 strain), is preferably at least about 10% of the total concentration in pfu / 0.5 mL, and more preferably dengue serotype 4, such as TDV-4 (e.g., a chimeric dengue serotype 2 / 4 strain), is at least about 70% of the total concentration in pfu / 0.5 mL. In certain such embodiments, dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) (e.g., TDV-4) represents the highest concentration in the composition of all four serotypes, preferably having at least about 70% of the total concentration in pfu / 0.5 mL, and dengue serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain) (e.g., TDV-3) represents the second highest concentration in the composition of all four serotypes, preferably having at least about 70% of the total concentration in pfu / 0.5 mL. or at least about 10% of the total concentration in pfu / 0.5 mL, with dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain) (e.g., TDV-1) and dengue serotype 2 (e.g., a dengue serotype 2 strain) (e.g., TDV-2) each representing a lower concentration than that of serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain) (e.g., TDV-3), and optionally together representing less than about 10% of the total concentration in pfu / 0.5 mL.

[0152] Preferably, in the aforementioned embodiment, the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3 and the chimeric dengue serotype 2 / 4 strain is TDV-4. More preferably, TDV-1 is characterized by a nucleotide sequence set forth in SEQ ID NO:1 and an amino acid sequence set forth in SEQ ID NO:2, TDV-2 is characterized by a nucleotide sequence set forth in SEQ ID NO:3 and an amino acid sequence set forth in SEQ ID NO:4, TDV-3 is characterized by a nucleotide sequence set forth in SEQ ID NO:5 and an amino acid sequence set forth in SEQ ID NO:6 and TDV-4 is characterized by a nucleotide sequence set forth in SEQ ID NO:7 and an amino acid sequence set forth in SEQ ID NO:8.

[0153] According to a further embodiment, the chimeric dengue serotype 2 / 4 strain, preferably TDV-4, has the highest concentration in the dengue vaccine composition, followed by the chimeric dengue serotype 2 / 3 strain, preferably TDV-3, then the chimeric dengue serotype 2 / 1 strain, preferably TDV-1, then the dengue serotype 2 strain, preferably TDV-2. It is particularly preferred that the dengue serotype 2 strain has the lowest concentration of the four strains present in the dengue vaccine composition.

[0154] Whenever a concentration / 0.5 ml is mentioned, this does not limit the volume of the unit dose described herein to 0.5 ml. 0.5 ml is the reference volume for the determination of the concentration of the virus strain in the composition in pfu / ml. The volumes and / or amounts per unit dose are described in the respective chapters.

[0155] Pharmaceutically acceptable excipients The present invention is directed, in part, to a unit dose of a dengue vaccine composition, the dengue vaccine composition including one or more pharma- ceutically acceptable excipients. In one embodiment, the dengue vaccine composition includes a non-reducing sugar, a surfactant, a protein, and an inorganic salt. Preferably, the non-reducing sugar is trehalose, the surfactant is poloxamer 407, the protein is human serum albumin, and the inorganic salt is sodium chloride.

[0156] In one embodiment, the unit dose of the dengue vaccine composition is formulated with one or more of the following pharma- ceutically acceptable excipients: from about 10% w / v to about 20% w / v of α,α-trehalose dihydrate or an equimolar amount of another form of α,α-trehalose, about 0.5% w / v to about 1.5% w / v of poloxamer 407, about 0.05% w / v to about 2% w / v human serum albumin, and - Approximately 70 mM to 140 mM sodium chloride.

[0157] In one embodiment, a unit dose of the dengue vaccine composition, when measured in 0.5 ml, comprises the following pharma- ceutically acceptable excipients: from about 10% w / v to about 20% w / v of α,α-trehalose or an equimolar amount of another form of α,α-trehalose, about 0.5% w / v to about 1.5% w / v of poloxamer 407, about 0.05% w / v to about 2% w / v human serum albumin, and - Approximately 70 mM to 140 mM sodium chloride, preferably It has a pH of -7 to 8.5.

[0158] In one embodiment, a unit dose of the dengue vaccine composition, when measured in 0.5 ml, comprises the following pharma- ceutically acceptable excipients: from about 143 mg / ml to about 185 mg / ml of α,α-trehalose dihydrate or an equimolar amount of another form of α,α-trehalose, from about 9.1 mg / ml to about 12.4 mg / ml of poloxamer 407, - from about 0.88% mg / ml to about 1.32 mg / ml of human serum albumin, and - Approximately 70 mM to 140 mM sodium chloride, preferably It has a pH of -7 to 8.5.

[0159] In a preferred embodiment, the lyophilized unit dose of the invention described herein is formulated with the following pharma- ceutically acceptable excipients: about 15% w / v of α,α-trehalose dihydrate, - approximately 1% w / v of poloxamer 407, about 0.1% w / v human serum albumin, and - about 100 mM sodium chloride.

[0160] In a preferred embodiment, the lyophilized unit dose of the invention described herein contains the following pharma- ceutically acceptable excipients when measured in 0.5 ml: about 15% w / v α,α-trehalose, - approximately 1% w / v of poloxamer 407, about 0.1% w / v human serum albumin, and - about 100 mM sodium chloride.

[0161] In a preferred embodiment, the lyophilized unit dose of the invention described herein is formulated with the following pharma- ceutically acceptable excipients: about 82.9 mg of α,α-trehalose dihydrate, - Approximately 5 mg of poloxamer 407, about 0.5 mg of human serum albumin, and Approximately 50 μmoles of sodium chloride.

[0162] In a preferred embodiment, the reconstituted unit dose of the invention described herein is formulated with the following pharma- ceutical acceptable excipients: about 15% w / v of α,α-trehalose dihydrate, - approximately 1% w / v of poloxamer 407, about 0.1% w / v human serum albumin, and - about 137 mM sodium chloride, preferably Has a pH of -7 to 8.5

[0163] In a preferred embodiment, the reconstituted unit dose of the invention described herein contains the following pharma- ceutically acceptable excipients when measured in 0.5 ml: about 15% w / v α,α-trehalose, - approximately 1% w / v of poloxamer 407, about 0.1% w / v human serum albumin, and preferably - about 137 mM sodium chloride, preferably It has a pH of -7 to 8.5.

[0164] In a preferred embodiment, the reconstituted unit dose of the invention described herein is formulated with the following pharma- ceutical acceptable excipients: about 82.9 mg of α,α-trehalose dihydrate, - Approximately 5 mg of poloxamer 407, about 0.5 mg of human serum albumin, and preferably about 68.5 μmol of sodium chloride, preferably It has a pH of -7 to 8.5.

[0165] The human serum albumin can be natural or recombinant human serum albumin (rHSA). The poloxamer 407 can be, for example, Pluronic F127.

[0166] In one embodiment, the unit dose further comprises a buffer. The buffer may be phosphate buffered saline (PBS). The buffer may comprise at least one of sodium chloride (NaCl), monosodium dihydrogen phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4), potassium chloride (KCl), and potassium dihydrogen phosphate (KH2PO4). In a preferred embodiment, the buffer may comprise disodium hydrogen phosphate (Na2HPO4), potassium chloride (KCl), and potassium dihydrogen phosphate (KH2PO4). The buffer may have a pH in the range of 7.0 to 8.5 at 25°C.

[0167] Unit Dose The present invention is directed, in part, to a unit dose of a dengue vaccine composition comprising a tetravalent dengue virus composition as described herein and a pharma- ceutically acceptable excipient as described herein.

[0168] The invention relates in part to, for example, (i) dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain) having a concentration of at least 3.3 log10 pfu / 0.5 mL; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) having a concentration of at least 2.7 log pfu / 0.5 mL; (iii) dengue serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain) having a concentration of at least 4.0 log pfu / 0.5 mL; and (iv) covering a unit dose of the above-mentioned dengue vaccine composition of dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) having a concentration of at least 4.5 log10 pfu / 0.5 mL.

[0169] Preferably, the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3 and the chimeric dengue serotype 2 / 4 strain is TDV-4. More preferably, TDV-1 is characterized by the nucleotide sequence set forth in SEQ ID NO: 1 and the amino acid sequence set forth in SEQ ID NO: 2, TDV-2 is characterized by the nucleotide sequence set forth in SEQ ID NO: 3 and the amino acid sequence set forth in SEQ ID NO: 4, TDV-3 is characterized by the nucleotide sequence set forth in SEQ ID NO: 5 and the amino acid sequence set forth in SEQ ID NO: 6 and TDV-4 is characterized by the nucleotide sequence set forth in SEQ ID NO: 7 and the amino acid sequence set forth in SEQ ID NO: 8.

[0170] In one embodiment, the unit dose is lyophilized. In one such embodiment, the lyophilized unit dose is obtained by subjecting to lyophilization a 0.5 mL volume of an aqueous dengue vaccine composition produced by combining a dengue vaccine composition comprising four dengue virus strains, specifically TDV-1 to TDV-4, as described herein with a pharma- ceutically acceptable excipient as described herein. In a preferred embodiment, the residual moisture content, as determined by Karl Fischer measurement, is 5.0% or less, preferably 3% or less.

[0171] In another embodiment, the unit dose is reconstituted. The reconstituted unit dose is obtained by subjecting the lyophilized unit dose to reconstitution with a pharma- ceutically acceptable diluent, preferably prior to administration of the dengue vaccine. In one such embodiment, reconstitution is achieved by adding a pharma- ceutically acceptable diluent, such as water for injection, phosphate buffered saline or aqueous sodium chloride solution, to the lyophilized unit dose. In one embodiment, an aqueous sodium chloride solution (e.g., an aqueous 37 mM sodium chloride solution) is added to the lyophilized unit dose for reconstitution. In one such embodiment, the lyophilized unit dose is reconstituted with 0.3-0.8 mL, or 0.4-0.7 mL, or 0.5 mL of diluent. In a preferred embodiment, the lyophilized unit dose is reconstituted with 0.3-0.8 mL, 0.4-0.7 mL, or 0.5 mL of an aqueous 37 mM sodium chloride solution. In a more preferred embodiment, the lyophilized unit dose is reconstituted with 0.5 mL of an aqueous 37 mM sodium chloride solution. The reconstituted unit dose can then be administered subcutaneously.

[0172] The lyophilized form of the unit dose is the final product and storage form of the unit dose after manufacture of the unit dose, while the reconstituted form of the unit dose is preferably prepared prior to administration of the unit dose to a subject.

[0173] The invention further comprises, in part, A unit dose of a dengue vaccine composition comprising a tetravalent virus composition comprising four live attenuated dengue virus strains, the unit dose being lyophilized and upon reconstitution with 0.5 mL of a pharma- ceutical acceptable diluent, (i) dengue serotype 1, such as a chimeric dengue serotype 2 / 1 strain, at a concentration of at least 3.3 log pfu / 0.5 ml; (ii) dengue serotype 2, such as a dengue serotype 2 strain at a concentration of at least 2.7 log pfu / 0.5 ml; (iii) dengue serotype 3, such as a chimeric dengue serotype 2 / 3 strain, at a concentration of at least 4.0 log pfu / 0.5 ml; and (iv) A unit dose of a dengue vaccine composition comprising dengue serotype 4, such as a chimeric dengue serotype 2 / 4 strain, at a concentration of at least 4.5 log10 pfu / 0.5 ml.

[0174] In one embodiment, the volume of a reconstituted unit dose is, for example, 0.5 mL, and upon reconstitution with a pharma- ceutically acceptable diluent, (i), (ii), (iii), and (iv) have a total concentration of pfu / 0.5 mL, based on which the concentration of (iii) is at least 10% of the total concentration in pfu / 0.5 mL.

[0175] In another embodiment, the volume of the reconstituted unit dose is, for example, 0.5 mL, and upon reconstitution with a pharma- ceutically acceptable diluent, (i), (ii), (iii), and (iv) have a total concentration of pfu / 0.5 mL, where based on that total concentration, the concentration of (ii) at pfu / 0.5 mL is less than 10%, the concentration of (iv) at pfu / 0.5 mL is at least 50%, the concentration of (i) at pfu / 0.5 mL is at least 1%, and the concentration of (iii) at pfu / 0.5 mL is at least 6%, at least 8%, or at least 10%, or at least 12%, or at least 14%, or at least 16%, or at least 18%.

[0176] The concentration in the reconstituted unit dose of (iii) in pfu / 0.5 mL is preferably at least 10%.

[0177] In one embodiment, the volume of a reconstituted unit dose is, for example, 0.5 mL, and upon reconstitution with a pharma- ceutically acceptable diluent, (i), (ii), (iii), and (iv) have a total concentration of pfu / 0.5 mL, where, based on the total concentration, (ii) has a concentration of less than 2% pfu / 0.5 mL, (iv) has a concentration of at least 50% pfu / 0.5 mL, (i) has a concentration of at least 1% pfu / 0.5 mL, and (iii) has a concentration of at least 6% pfu / 0.5 mL.

[0178] In one embodiment, the invention is directed to a lyophilized unit dose of a dengue vaccine composition, which upon reconstitution with 0.5 mL of a pharma- ceutical acceptable diluent comprises dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain) having a concentration of at least 3.3 log10 pfu / 0.5 mL, dengue serotype 2 (e.g., a dengue serotype 2 strain) having a concentration of at least 2.7 log10 pfu / 0.5 mL, dengue serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain) having a concentration of at least 4.0 log10 pfu / 0.5 mL, and dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) having a concentration of at least 4.5 log10 pfu / 0.5 mL, and a pharma-ceutical acceptable excipient as described herein, wherein the unit dose is preferably formulated in 0.5 mL prior to lyophilization. Preferably, the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3 and the chimeric dengue serotype 2 / 4 strain is TDV-4. More preferably, TDV-1 is characterized by the nucleotide sequence set forth in SEQ ID NO: 1 and the amino acid sequence set forth in SEQ ID NO: 2, TDV-2 is characterized by the nucleotide sequence set forth in SEQ ID NO: 3 and the amino acid sequence set forth in SEQ ID NO: 4, TDV-3 is characterized by the nucleotide sequence set forth in SEQ ID NO: 5 and the amino acid sequence set forth in SEQ ID NO: 6 and TDV-4 is characterized by the nucleotide sequence set forth in SEQ ID NO: 7 and the amino acid sequence set forth in SEQ ID NO: 8.

[0179] In one such embodiment, the lyophilized unit dose is obtained by lyophilizing 0.5 mL of a dengue vaccine composition comprising dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain) at a concentration of 3.3 log10 pfu / dose to 5.0 log10 pfu / 0.5 mL, dengue serotype 2 (e.g., a dengue serotype 2 strain) at a concentration of 2.7 log10 pfu / dose to 4.9 log10 pfu / 0.5 mL, dengue serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain) at a concentration of 4.0 log10 pfu / dose to 5.7 log10 pfu / 0.5 mL, and dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) at a concentration of 4.5 log10 pfu / dose to 5.5 log10 pfu / 0.5 mL, and a pharma- ceutically acceptable excipient as described herein. Preferably, the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3, and the chimeric dengue serotype 2 / 4 strain is TDV-4.

[0180] In one such embodiment, the lyophilized unit dose contains dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strains) at a concentration of 3.3 log10 pfu / 0.5 mL to 3.6 log10 pfu / 0.5 mL, dengue serotype 2 (e.g., dengue serotype 2 strains) at a concentration of 2.7 log10 pfu / 0.5 mL to 4.0 log10 pfu / 0.5 mL, dengue serotype 3 (e.g., dengue serotype 4 strains) at a concentration of 4.0 log10 pfu / 0.5 mL to 4.6 log10 pfu / 0.5 mL, The vaccine is obtained by freeze-drying 0.5 mL of a dengue vaccine composition comprising dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strain) at a concentration of 0.5 mL, and dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strain) at a concentration of 4.5 log10 pfu / 0.5 mL or 4.6 log10 pfu / 0.5 mL to 5.1 log10 pfu / 0.5 mL, and a pharma- ceutically acceptable excipient as described herein. Preferably, the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3, and the chimeric dengue serotype 2 / 4 strain is TDV-4.

[0181] In certain embodiments, a lyophilized unit dose refers to 0.5 mL prior to lyophilization in which TDV-2 and TDV-4 are present in certain relative amounts based on the total concentration of TDV-1, TDV-2, TDV-3, and TDV-4 in pfu / 0.5 mL, and the concentration of TDV-2 measured in pfu / 0.5 mL is less than 10%, or less than 8%, or less than 6%, and the concentration of TDV-4 measured in pfu / 0.5 mL is at least 50% or at least 65%. In some of these embodiments, the concentration of TDV-1 measured in pfu / 0.5 mL is at least 1%, and / or the concentration of TDV-3 measured in pfu / 0.5 mL is at least 6%, 7%, 8%, 10%, 12%, 14%, 16%, or at least 18%.

[0182] In certain embodiments, the volume of the reconstituted unit dose is 0.5 mL, TDV-2 and TDV-4 are present in certain relative amounts based on the total concentration of TDV-1, TDV-2, TDV-3, and TDV-4 in pfu / 0.5 mL, and the concentration of TDV-2 measured in pfu / 0.5 mL is less than 10%, or less than 8%, or less than 6%, and the concentration of TDV-4 measured in pfu / 0.5 mL is at least 50% or at least 65%. In some of these embodiments, the concentration of TDV-1 measured in pfu / 0.5 mL is at least 1%, and / or the concentration of TDV-3 measured in pfu / 0.5 mL is at least 6%, 7%, 8%, 10%, 12%, 14%, 16%, or at least 18%.

[0183] In a further preferred embodiment, the reconstituted unit dose has a volume of 0.5 mL and comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, wherein the concentration of dengue serotype 1 (e.g., a dengue serotype 2 / 1 strain) measured in pfu / 0.5 mL is between 1% and 7% of the total concentration, the concentration of dengue serotype 2 (e.g., a dengue serotype 2 strain) measured in pfu / 0.5 mL is less than 8% of the total concentration, e.g., in the range of 1% to 8% of the total concentration, the concentration of dengue serotype 3 (e.g., a dengue serotype 2 / 3 strain) measured in pfu / 0.5 mL is at least 10% of the total concentration, and the concentration of dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) measured in pfu / 0.5 mL is at least 65% of the total concentration, e.g., in the range of 65% to 80%. In certain such embodiments, the arithmetic sum of all four serotypes is within the range of 4.6 log10 pfu / 0.5 mL to 6.7 log10 pfu / 0.5 mL, preferably within the range of 4.6 log10 pfu / 0.5 mL to 5.5 log10 pfu / 0.5 mL.

[0184] In a further preferred embodiment, the reconstituted unit dose has a volume of 0.5 mL and comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, where dengue serotype 1, such as TDV-1 (e.g., a chimeric dengue serotype 2 / 1 strain) and dengue serotype 2, such as TDV-2 (e.g., a dengue serotype 2 strain), are each present at a concentration within 5 percentage points of each other, based on total concentration in pfu / 0.5 mL, and / or together are less than about 10% of the total concentration in pfu / 0.5 mL. In certain such embodiments, dengue serotype 3, such as TDV-3 (e.g., a chimeric dengue serotype 2 / 3 strain), is preferably at least about 10% of the total concentration in pfu / 0.5 mL, and more preferably dengue serotype 4, such as TDV-4 (e.g., a chimeric dengue serotype 2 / 4 strain), is at least about 70% of the total concentration in pfu / 0.5 mL. In certain such embodiments, dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) (e.g., TDV-4) represents the highest concentration in the composition of all four serotypes, preferably having at least about 70% of the total concentration in pfu / 0.5 mL, and dengue serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain) (e.g., TDV-3) represents the second highest concentration in the composition of all four serotypes, preferably having at least about 70% of the total concentration in pfu / 0.5 mL. or at least about 10% of the total concentration in pfu / 0.5 mL, with dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain) (e.g., TDV-1) and dengue serotype 2 (e.g., a dengue serotype 2 strain) (e.g., TDV-2) each representing a lower concentration than that of serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain) (e.g., TDV-3), and optionally together representing less than about 10% of the total concentration in pfu / 0.5 mL.

[0185] Lyophilized unit doses reconstituted in 0.5 mL will yield the above concentrations for the four dengue serotypes. While the unit doses of the dengue vaccine compositions described herein refer to the concentrations of dengue serotypes in 0.5 mL, the lyophilized unit doses can be reconstituted with other volumes of pharma- ceutically acceptable diluents, such as aqueous sodium chloride solution, without changing the absolute amount of virus administered or the ratio of viruses to each other.

[0186] In certain embodiments, the lyophilized unit dose of the present invention is prepared from a solution comprising a non-reducing sugar, a surfactant, a protein, and an inorganic salt.

[0187] In certain embodiments, the lyophilized unit dose of the present invention is prepared from a solution comprising trehalose, poloxamer 407, human serum albumin, and sodium chloride.

[0188] In certain embodiments, a lyophilized unit dose of the invention is prepared from a solution comprising about 10% w / v to about 20% w / v α,α-trehalose dihydrate or an equimolar amount of other forms of α,α-trehalose, about 0.5% w / v to about 1.5% w / v poloxamer 407, about 0.05% w / v to about 2% w / v human serum albumin, and about 70 mM to about 120 mM sodium chloride.

[0189] In a preferred embodiment, a lyophilized unit dose of the invention described herein is prepared from a solution comprising about 15% w / v α,α-trehalose dihydrate, about 1% w / v poloxamer 407, about 0.1% w / v human serum albumin, and about 100 mM sodium chloride.

[0190] In one embodiment, the solution from which the lyophilized unit dose is prepared further comprises a buffer. The buffer may be phosphate buffered saline (PBS). The buffer may comprise at least one of sodium chloride (NaCl), monosodium dihydrogen phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4), potassium chloride (KCl), and potassium dihydrogen phosphate (KH2PO4). In a preferred embodiment, the buffer may comprise disodium hydrogen phosphate (Na2HPO4), potassium chloride (KCl), and potassium dihydrogen phosphate (KH2PO4). The buffer may have a pH in the range of about 7.0 to about 8.5 at 25°C or a pH of about 6.8 to about 7.6 at 25°C, preferably about 7.2 at 25°C.

[0191] In a preferred embodiment, a reconstituted unit dose of the invention described herein comprises about 15% w / v α,α-trehalose dihydrate, about 1% w / v poloxamer 407, about 0.1% w / v human serum albumin, and about 137 mM sodium chloride. The reconstituted unit dose may have a pH of about 7.0 to about 8.5 at 25° C., preferably a pH of about 7.2 at 25° C.

[0192] The unit dose of the invention described herein activates multiple levers of the immune system (neutralizing antibodies, cell-mediated immunity, and anti-NS1 antibodies) in both seronegative and seropositive subject populations or subjects, and thus protects both dengue seronegative and dengue seropositive subject populations or subjects against dengue disease.

[0193] In one embodiment, a single unit dose is present in a container, preferably a vial, which is administered to a subject after reconstitution. In one embodiment, multiple unit doses of a dengue vaccine composition may be present in a container, preferably a vial, such that multiple subjects can be vaccinated with the contents of one container, preferably a vial. In one embodiment, a container containing multiple unit doses of the invention described herein is used to provide reconstituted unit doses for use in the methods of the invention described herein.

[0194] In certain embodiments, the container containing the unit dose of the invention is part of a kit. Thus, the invention is directed, in part, to a kit for preparing a reconstituted unit dose comprising a lyophilized unit dose of the invention described herein and a pharma- ceutical acceptable diluent for reconstitution.

[0195] In certain embodiments, the reconstitution diluent is provided in a container, preferably a vial, or a prefilled syringe.In some embodiments, the reconstitution diluent is selected from water for injection, phosphate buffered saline, or aqueous sodium chloride solution.In a preferred embodiment, the reconstitution diluent is 30-40 mM sodium chloride, for example, 37 mM sodium chloride.

[0196] In certain embodiments, the kit may further comprise a Hepatitis A vaccine, e.g., HAVRIX® or VAQTA®. In some embodiments, the Hepatitis A vaccine may be in a separate container, e.g., a vial. In another embodiment, the Hepatitis A vaccine and the unit dose of the invention may be in the same container. Thus, the present invention is directed, in part, to a combined Dengue / Hepatitis A vaccine, in which a unit dose of the invention described herein is combined with a Hepatitis A vaccine. Such a combined Dengue / Hepatitis A vaccine comprises a unit dose of the invention described herein and a Hepatitis A vaccine (e.g., HAVRIX® or VAQTA®) in the same formulation. In certain embodiments, the present invention is directed to a kit comprising such a combined Dengue / Hepatitis A vaccine and a unit dose of the invention described herein.

[0197] Hepatitis A vaccine In certain embodiments, the Hepatitis A vaccine is an inactivated Hepatitis A vaccine.

[0198] In certain embodiments, the Hepatitis A vaccine comprises a Hepatitis A virus derived from the Hepatitis A virus strain HM-175.

[0199] In certain embodiments, the Hepatitis A vaccine comprises an inactivated Hepatitis A virus, wherein the inactivated Hepatitis A virus is derived from the wild-type Hepatitis A virus strain HM-175.

[0200] In certain embodiments, the inactivated Hepatitis A virus is adsorbed to the carrier aluminum. In some of these embodiments, the aluminum is aluminum hydroxide or aluminum hydroxyphosphate sulfate.

[0201] In certain embodiments, the Hepatitis A vaccine comprises phosphate buffered saline and excipients dissolved therein in the form of amino acids and polysorbates, in such embodiments, the amino acids are present in a concentration of 0.2-0.8% w / v and / or the polysorbates are present in a concentration of 0.01-0.09 mg / ml.

[0202] In certain embodiments, the Hepatitis A vaccine comprises a Hepatitis A virus expressing a viral antigen at a concentration ranging from 500 ELISA units (EL.U.) to 2000 ELISA units (EL.U.), preferably from 700 EL.U. to 1600 EL.U., and most preferably from 1300 to 1550 EL.U. Alternatively, the concentration ranges from 500 EL.U. to 900 EL.U. In further embodiments, the concentration ranges from 200 to 400 EL.U.

[0203] In certain embodiments, the Hepatitis A vaccine is contained in a 1 ml or 0.5 ml dose of liquid.

[0204] An example of such a hepatitis A vaccine is GlaxoSmithKline's HAVRIX®, which is a sterile suspension of inactivated virus for intramuscular administration. HAVRIX® uses the hepatitis A virus strain HM-175, derived from wild-type hepatitis A virus (HAV) HM-175, the complete nucleotide sequence of which is disclosed in Cohen et al., Journal of Virology, Vol. 61, No. 1 (published Jan. 1987), pp. 50-59 (specifically, the complete nucleotide sequence of wild-type hepatitis A virus HM-175 is shown in Figure 1 of that publication).

[0205] The virus (HM175 strain) grows in MRC-5 human diploid cells. After removal of the cell culture medium, the cells are lysed to form a suspension. This suspension is purified by ultrafiltration and gel permeation chromatography procedures. Treatment of the lysate with formalin ensures viral inactivation. Viral antigen activity is referenced against standards using an enzyme-linked immunosorbent assay (ELISA) and is therefore expressed in terms of ELISA units (EL.U.). Each 1 mL dose of vaccine for adults (>= 18 years) contains 1440 EL.U. of viral antigen adsorbed to 0.5 mg of aluminum as aluminum hydroxide. Each 0.5 mL dose of vaccine for children and young adults (12 months to 18 years) contains 720 EL.U. of viral antigen adsorbed to 0.25 mg of aluminum as aluminum hydroxide. HAVRIX® contains the following excipients: amino acid supplement (0.3% w / v) and polysorbate 20 (0.05 mg / mL) in phosphate buffered saline. From the manufacturing process, HAVRIX® also contains residual MRC-5 cellular protein (<5 μg / mL), formalin (<0.1 mg / mL), and the aminoglycoside antibiotic neomycin sulfate (<40 ng / mL). HAVRIX® is formulated without preservatives.

[0206] VAQTA®, a hepatitis A vaccine from Merck Sharp & Dohme Corp., is an inactivated whole virus vaccine derived from hepatitis A virus grown in cell culture of human MRC-5 diploid fibroblasts. The vaccine contains inactivated virus from a strain originally derived by further serial passage of a proven attenuated strain. The virus is grown, harvested, purified by a combination of physical and high performance liquid chromatography techniques developed at Merck Research Laboratories, formalin inactivated, and then adsorbed to amorphous aluminum hydroxyphosphate sulfate. VAQTA® is a sterile suspension for intramuscular injection. One milliliter of vaccine is approximately 50 U of hepatitis A virus antigen, purified and formulated without preservatives. Within the limits of current assay availability, a 50 U dose of VAQTA® contains less than 0.1 μg of non-viral protein, 4×10 -6 <10 μg DNA -4 It contains less than 10 μg of bovine albumin and less than 0.8 μg of formaldehyde. Other process chemical residues, including neomycin, are less than 10 parts per billion (ppb). Each 0.5 mL pediatric dose contains 25 U of Hepatitis A virus antigen adsorbed to approximately 0.225 mg of aluminum provided as amorphous aluminum hydroxyphosphate sulfate in 0.9% sodium chloride, and 35 μg of sodium borate as a pH stabilizer. Each 1 mL adult dose contains 50 U of Hepatitis A virus antigen adsorbed to approximately 0.45 mg of aluminum provided as amorphous aluminum hydroxyphosphate sulfate in 0.9% sodium chloride, and 70 μg of sodium borate as a pH stabilizer.

[0207] Yellow fever vaccine Sanofi's Yellow Fever vaccine for subcutaneous use, YF-VAX®, is prepared by culturing the YF-17D strain of Yellow Fever virus in live Avian Leukosis Virus-free (ALV-free) chicken embryos. The vaccine contains sorbitol and gelatin (as stabilizers) and is lyophilized. No preservatives are added. YF-VAX maintains a 4.74 log per 0.5 mL dose throughout the life of the product. 10 It is formulated to contain at least pfu.

[0208] Combination vaccine compositions The present invention is further directed, in part, to combination vaccine compositions comprising a Hepatitis A antigen, such as HAVRIX® or VAQTA®, and a dengue antigen (e.g., the tetravalent dengue vaccine (TDV) disclosed herein or any other suitable tetravalent live-attenuated dengue virus vaccine).

[0209] In certain embodiments, the present invention provides a combination vaccine composition, the dengue vaccine composition comprising a tetravalent live attenuated dengue virus strain: (i) dengue serotype 1, preferably at a concentration of at least 3.3 log10 pfu / 0.5 mL; (ii) dengue serotype 2, preferably at a concentration of at least 2.7 log pfu / 0.5 mL; (iii) dengue serotype 3, preferably at a concentration of at least 4.0 log pfu / 0.5 mL; and (iv) to a unit dose of a dengue vaccine composition comprising a tetravalent dengue virus composition, preferably comprising dengue serotype 4 at a concentration of at least 4.5 log10 pfu / 0.5 mL;

[0210] In certain embodiments, the present invention provides a combination vaccine composition, the dengue vaccine composition comprising a tetravalent live attenuated dengue virus strain: (i) a chimeric dengue serotype 2 / 1 strain at a concentration of at least 3.3 log10 pfu / 0.5 mL to 3.8 log10 pfu / 0.5 mL; (ii) a dengue serotype 2 strain at a concentration of at least 2.7 log10 pfu / 0.5 mL; (iii) a chimeric dengue serotype 2 / 3 strain at a concentration of at least 4.0 log10 pfu / 0.5 mL; and (iv) a combination vaccine composition comprising a tetravalent dengue virus composition comprising a chimeric dengue serotype 2 / 4 strain at a concentration of at least 4.5 log10 pfu / 0.5 mL or at least 4.6 log10 pfu / 0.5 mL, optionally up to 6.2 log10 pfu / 0.5 ml.

[0211] Preferably, in the aforementioned embodiment, the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3 and the chimeric dengue serotype 2 / 4 strain is TDV-4. More preferably, TDV-1 is characterized by a nucleotide sequence set forth in SEQ ID NO:1 and an amino acid sequence set forth in SEQ ID NO:2, TDV-2 is characterized by a nucleotide sequence set forth in SEQ ID NO:3 and an amino acid sequence set forth in SEQ ID NO:4, TDV-3 is characterized by a nucleotide sequence set forth in SEQ ID NO:5 and an amino acid sequence set forth in SEQ ID NO:6 and TDV-4 is characterized by a nucleotide sequence set forth in SEQ ID NO:7 and an amino acid sequence set forth in SEQ ID NO:8.

[0212] In certain embodiments, the present invention is directed to a combination vaccine composition, wherein upon reconstitution of the dengue vaccine composition with a pharma- ceutically acceptable diluent, (i), (ii), (iii), and (iv) have a total concentration of pfu / 0.5 mL, and based on the total concentration of pfu / 0.5 mL, the concentration of (ii) at pfu / 0.5 mL is less than 10%, the concentration of (iv) at pfu / 0.5 mL is at least 50%, the concentration of (i) at pfu / 0.5 mL is at least 1%, and the concentration of (iii) at pfu / 0.5 mL is at least 6%, at least 8%, or at least 10%, or at least 12%, or at least 14%, or at least 16%, or at least 18%.

[0213] In certain embodiments, the present invention is directed to a combination vaccine composition, wherein the dengue vaccine composition comprises one or more pharma- ceutically acceptable excipients. In one embodiment, the dengue vaccine composition comprises a non-reducing sugar, a surfactant, a protein, and an inorganic salt. Preferably, the non-reducing sugar is trehalose, the surfactant is poloxamer 407, the protein is human serum albumin, and the inorganic salt is sodium chloride.

[0214] Furthermore, any vaccine excipient or combination thereof known to a person skilled in the art (e.g., those disclosed in WO2018 / 027075A1) can be used in the combination vaccine composition.

[0215] In one embodiment, the unit dose of the dengue vaccine composition is formulated with one or more of the following pharma- ceutically acceptable excipients: from about 10% w / v to about 20% w / v of α,α-trehalose dihydrate or an equimolar amount of another form of α,α-trehalose, about 0.5% w / v to about 1.5% w / v of poloxamer 407, about 0.05% w / v to about 2% w / v human serum albumin, and - Approximately 70 mM to 140 mM sodium chloride.

[0216] In certain embodiments, the present invention is directed to combination vaccine compositions, where the dengue vaccine composition includes another dengue vaccine (eg, Dengvaxia®). Dengvaxia®, a quadrivalent dengue vaccine containing a mixed chimeric dengue virus based on the yellow fever backbone, CYD-TDV (Dengvaxia®; Sanofi Pasteur, Lyon, France), has been licensed in several countries based on clinical demonstration of overall vaccine efficacy (VE) against virologically confirmed dengue fever (VCD) in 56-61% of children in Asia and Latin America (Capeding MR et al. Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in Asia: a phase 3, randomised, observer-masked, placebo-controlled trial. Lancet 2014,384:1358-65; Villar LA et al. Safety and immunogenicity of a recombinant tetravalent dengue vaccine in 9-16 year olds: a randomized, controlled, phase II trial in Latin America. Pediatr Infect Dis J 2013,32:1102-9). The preparation of these particular strains CYD1, CYD2, CYD3 and CYD4 is described in detail in International Patent Applications WO98 / 37911, WO03 / 101397, WO07 / 021672, WO08 / 007021, WO08 / 047023 and WO08 / 065315, to which reference may be made for a precise description of the process for their preparation. The corresponding nucleotide sequences of the prM-E regions of CYD1, CYD2, CYD3 and CYD4 are provided in WO2016034629, and the SEQ ID NOs are shown in Table 16 of this reference.

[0217] In a particular embodiment, the present invention provides a combination vaccine composition, wherein the amount of chimeric dengue virus in the CYD-TDV vaccine composition of the present invention is about 10 5 CCID50~about 10 6 The amount of live attenuated chimeric dengue virus of each of serotypes 1 to 4 contained in a CYD dosage form (eg, Dengvaxia®) is preferably equal.

[0218] In such an embodiment, CYD-TDV is dissolved / soluble in a solution containing 0.4% NaCl.

[0219] In certain embodiments, the present invention is directed to a combination vaccine composition, where the dengue vaccine composition includes another dengue vaccine (e.g., TV003 or TV005). TV003, developed by the National Institute of Allergy and Infectious Diseases, includes vaccine components rDEN1Δ30, rDEN2 / 4Δ30, rDEN3Δ30 / 31, and rDEN4Δ30, each of which is 3 log 10 PFU. TV005 is similar to TV003, except that the concentration of rDEN2 / 4Δ30 in TV005 is 4 log 10PFU. Vaccines TV003, TV005, and their vaccine components and their production are described in more detail in WO2008 / 022196A2 and SS Whitehead, Expert Rev Vaccines, 2016, 15(4):509 to 517. Using recombinant DNA technology, two attenuation strategies were utilized for the vaccine components TV003 or TV005: deletion of the 3' untranslated region and chimerization of structural genes. For example, component rDEN4Δ30 contains all structural and nonstructural proteins of wild-type DENV-4, but is attenuated by a deletion of 30 nucleotides in the 3' untranslated region (designated "Δ30"). The other vaccine component is also attenuated by a deletion of 30 nucleotides in the 3' untranslated region. In addition, rDEN3Δ30 / 31 contains a 31 nucleotide deletion in the 3' untranslated region (detailed in Figure 1c and Figure 13 of WO2008 / 022196A2). The rDEN2 / 4Δ30 component was generated by replacing the prM and E genes of DENV-2 into the rDEN4Δ30 genome. The complete genome sequences of the dengue strains that can be used to produce TV003 or TV005 are available under the Genbank accession numbers in Table A of WO2008 / 022196A1.

[0220] In certain embodiments, the present invention is directed to a combination vaccine composition, wherein the Hepatitis A vaccine is an inactivated Hepatitis A vaccine.

[0221] In certain embodiments, the Hepatitis A vaccine comprises a Hepatitis A virus derived from the Hepatitis A virus strain HM-175.

[0222] In certain embodiments, the present invention is directed to a combination vaccine composition, wherein the Hepatitis A vaccine comprises an inactivated Hepatitis A virus, and the inactivated Hepatitis A virus is derived from the wild-type Hepatitis A virus strain HM-175.

[0223] In certain embodiments, the present invention is directed to a combination vaccine composition in which the inactivated Hepatitis A virus is adsorbed to an aluminum carrier. In some of these embodiments, the aluminum is aluminum hydroxide or aluminum hydroxyphosphate sulfate.

[0224] In certain embodiments, the present invention is directed to a combination vaccine composition, in which the Hepatitis A vaccine comprises phosphate buffered saline and excipients dissolved therein in the form of an amino acid and a polysorbate, in such embodiments, the amino acid is present in a concentration of 0.2-0.8% w / v and / or the polysorbate is present in a concentration of 0.01-0.09 mg / ml.

[0225] In certain embodiments, the present invention is directed to a combination vaccine composition, wherein the Hepatitis A vaccine comprises a Hepatitis A virus expressing a viral antigen at a concentration ranging from 500 ELISA Units (EL.U.) to 2000 ELISA Units (EL.U.), preferably from 700 EL.U. to 1600 EL.U., and most preferably from 1300 to 1550 EL.U. Alternatively, the concentration is in the range of 500 EL.U. to 900 EL.U. In further embodiments, the concentration is in the range of 200 to 400 EL.U.

[0226] In certain embodiments, the present invention is directed to a combination vaccine composition, wherein the combination vaccine is contained in a dose that comprises a liquid, and the volume of the liquid is 0.5 ml, 1 ml, or 1.5 ml.

[0227] In certain embodiments, the combination vaccine composition is provided in a single vial in liquid or dehydrated form (eg, lyophilized form).

[0228] In certain embodiments, the combination vaccine composition results from mixing a unit dose of a dengue vaccine composition and a dose of a Hepatitis A vaccine in a syringe.

[0229] The invention is also directed, in part, to methods of administering any of the above combination vaccine compositions to a subject or population of subjects.

[0230] In certain embodiments, the present invention is also directed to such methods, wherein the combination vaccine composition is administered subcutaneously or intramuscularly.

[0231] Method for preventing dengue disease and hepatitis A, corresponding uses and corresponding kits - Patents.com The present invention is directed to methods for preventing Hepatitis A and Dengue diseases.

[0232] The present invention is directed, in part, to a method of preventing Hepatitis A and Dengue disease in a subject or population of subjects comprising concurrently administering on the same day unit doses of a Hepatitis A vaccine (e.g., HAVRIX® or VAQTA®) and a Dengue vaccine composition, the unit dose comprising a tetravalent dengue virus composition comprising four live attenuated dengue virus strains.

[0233] In certain embodiments, the present invention is directed to the aforementioned method, wherein the Hepatitis A vaccine (e.g., HAVRIX®) comprises an inactivated virus. Preferably, the Hepatitis A vaccine comprises an inactivated Hepatitis A virus, and the inactivated Hepatitis A virus is derived from Hepatitis A virus strain HM-175.

[0234] In certain embodiments, the Hepatitis A vaccine (eg, HAVRIX®) is derived from Hepatitis A virus strain HM-175.

[0235] In certain embodiments, the present invention is directed to the aforementioned method, wherein the Hepatitis A vaccine (e.g., HAVRIX®), preferably a virus derived from Hepatitis A virus strain HM-175, is adsorbed to aluminum. According to some of these embodiments, the aluminum is aluminum hydroxide or aluminum hydroxyphosphate sulfate.

[0236] In certain embodiments, the present invention is directed to the aforementioned method, wherein the Hepatitis A vaccine (e.g., HAVRIX®), preferably a Hepatitis A vaccine derived from Hepatitis A virus strain HM-175, comprises phosphate buffered saline and excipients dissolved therein in amino acid and polysorbate forms.

[0237] In certain embodiments, the present invention is directed to the aforementioned method, wherein the Hepatitis A vaccine (e.g., HAVRIX®) comprises a Hepatitis A virus expressing a viral antigen at a concentration ranging from 500 ELISA Units (EL.U.) to 2000 ELISA Units (EL.U.), preferably from 700 EL.U. to 1600 EL.U., and most preferably from 1300 to 1550 EL.U. Alternatively, the concentration is in the range of 500 EL.U. to 900 EL.U. In further embodiments, the concentration is in the range of 200 to 400 EL.U.

[0238] For example, the viral antigen activity of a hepatitis A vaccine can be measured according to the method disclosed in Andre FE., Hepburn A., D'Hondt E., "Inactivated candidate vaccines for hepatitis", A. Prog Med Virol 1990;37:72-95.

[0239] In certain embodiments, the present invention is directed to the aforementioned method, wherein the dengue vaccine composition upon reconstitution with 0.5 mL of a pharma- ceutical acceptable diluent comprises: (i) for chimeric dengue serotype 2 / 1 strains, a concentration of at least 3.3 log10 pfu / 0.5 mL; (ii) for dengue serotype 2, a concentration of at least 2.7 log pfu / 0.5 mL; (iii) for chimeric dengue serotype 2 / 3 strains, a concentration of at least 4.0 log pfu / 0.5 mL; and (iv) for chimeric dengue serotype 2 / 4 strains, a concentration of at least 4.5 log10 pfu / 0.5 mL;

[0240] According to some of these embodiments, the dengue vaccine composition upon reconstitution with a pharma- ceutically acceptable diluent provides a total concentration of (i), (ii), (iii), and (iv) at pfu / 0.5 mL, where, based on the total concentration of pfu / 0.5 mL, the concentration of (ii) at pfu / 0.5 mL is less than 10%, the concentration of (iv) at pfu / 0.5 mL is at least 50%, the concentration of (i) at pfu / 0.5 mL is at least 1%, and the concentration of (iii) at pfu / 0.5 mL is at least 6%, at least 8%, or at least 10%, or at least 12%, or at least 14%, or at least 16%, or at least 18%.

[0241] In certain embodiments, the invention is directed to the aforementioned methods, wherein the subject or subject population is seronegative for all dengue serotypes. According to some of these embodiments, the subject population or subject is seronegative for Hepatitis A at baseline.

[0242] In certain embodiments, the present invention is directed to the aforementioned methods, wherein a unit dose of the invention described herein and a Hepatitis A vaccine (e.g., HAVRIX® or VAQTA®) is administered on days 0 / 1.

[0243] In certain embodiments, the present invention is directed to the aforementioned methods, wherein a unit dose of the invention described herein is administered by subcutaneous injection and a Hepatitis A vaccine (e.g., HAVRIX® or VAQTA®) is administered by intramuscular injection. According to some embodiments, the injection is administered in the arm, preferably in the deltoid region of the arm. According to some of these embodiments, the subcutaneous injection of the unit dose of the invention described herein and the intramuscular injection of a Hepatitis A vaccine (e.g., HAVRIX® or VAQTA®) are administered at different anatomical sites (e.g., opposite arms).

[0244] In certain embodiments, the present invention is directed to the aforementioned method, wherein two unit doses of the dengue vaccine composition of the invention described herein are administered. In some embodiments, the two unit doses of the invention described herein are administered within 12 months or more, or within 6 months, or within 3 months (e.g., on days 0 / 1 and 90). According to some of these embodiments, an additional third unit dose of the invention described herein is administered after the second administration. Such a third administration may be administered 6-12 months after the first administration, e.g., 12 months after the first administration, or later than 12 months after the first administration, e.g., 12 months (1 year) after the second administration or even 5 years or more after the first or second administration, and may act as a booster.

[0245] In certain embodiments, the present invention provides that two unit doses of an invention described herein and one dose of a Hepatitis A vaccine (e.g., HAVRIX® or VAQTA®) are specifically administered according to the following schedule: - on day 0 / 1, a first unit dose and a first simultaneous administration of the Hepatitis A vaccine; and - A second administration of a second reconstituted unit dose following said first co-administration, for example 3 months later, preferably on day 90.

[0246] In certain embodiments, the invention is directed to the aforementioned methods, wherein a reconstituted unit dose of the invention described herein is administered subcutaneously to a subject or population of subjects, and a Hepatitis A vaccine (e.g., HAVRIX® or VAQTA®) is administered intramuscularly to a subject or population of subjects, and the subject or population of subjects is seronegative for all dengue serotypes. In other embodiments, the subject or population of subjects is seropositive for at least one dengue serotype.

[0247] In certain embodiments, the invention is directed to the aforementioned methods, wherein a unit dose of the invention described herein and a Hepatitis A vaccine (e.g., HAVRIX® or VAQTA®) are administered to a subject or population of subjects from a dengue endemic area. In certain embodiments, a reconstituted unit dose of the invention described herein is administered subcutaneously and a Hepatitis A vaccine (e.g., HAVRIX® or VAQTA®) is administered intramuscularly to a subject or population of subjects from a dengue endemic area.

[0248] In certain embodiments, the present invention is directed to the aforementioned method, wherein the subject or subject population is from a non-dengue endemic area, preferably a non-dengue and non-Hepatitis A endemic area.

[0249] According to some embodiments, a second dose of a hepatitis A vaccine (e.g., HAVRIX® or VAQTA®) is administered. The second dose of the hepatitis A vaccine may be administered after the first administration of the hepatitis A vaccine. Such a second administration may serve as a booster and may be administered 6-12 months or 6-18 months, such as 9 months (e.g., day 270) after the first administration of the hepatitis A vaccine.

[0250] In certain embodiments, the present invention is directed to the aforementioned method, wherein a reconstituted unit dose of the invention described herein is administered subcutaneously and a Hepatitis A vaccine (e.g., HAVRIX® or VAQTA®) is administered intramuscularly to a subject or subject population over 17 years of age, or over 18 years of age, or between 18 and 60 years of age. In further embodiments, the subject or subject population is an adult over 21 years of age, or between 21 and 60 years of age, or between 21 and 45 years of age. In some embodiments, the subject or subject population is from a dengue endemic area. In another embodiment, the subject or subject population is from a dengue non-endemic area, preferably a dengue non-endemic and hepatitis A non-endemic area. According to certain embodiments, the subject or subject population is seronegative for all four dengue serotypes.

[0251] In certain embodiments, the invention is directed to the aforementioned method, which does not include a step of determining whether the subject population or subject was previously infected with dengue and / or previously infected with hepatitis A prior to administration of the hepatitis A vaccine and the unit dose of the dengue vaccine composition, or the hepatitis A serostatus and / or dengue serostatus of the subject population or subject is unknown prior to administration of the hepatitis A vaccine and the unit dose of the dengue vaccine composition. According to certain embodiments, the method does not include a step of determining whether the subject population or subject was previously infected with dengue and / or previously infected with hepatitis A at any time before, during or after the step of administering the unit dose of the hepatitis A vaccine and the dengue vaccine composition, or the hepatitis A serostatus and / or dengue serostatus of the subject population or subject is unknown at any time before, during or after the step of administering the unit dose of the hepatitis A vaccine and the dengue vaccine composition.

[0252] In certain embodiments, the present invention is directed to a method as described above, comprising a primary vaccination comprising the steps of: (A) selecting a subject for administration of a unit dose of a tetravalent dengue virus composition and a hepatitis A vaccine in need of protection against dengue infection and hepatitis A infection, without determining whether the subject has had a previous dengue infection and / or a previous hepatitis A infection; and (B) administering to the subject a first unit dose of the tetravalent dengue virus composition and a hepatitis A vaccine on the same day and simultaneously, and optionally (C) administering to the subject a second unit dose of the tetravalent dengue virus composition within 3 to 12 months of administering the first unit dose, and optionally (D) administering to the subject at least one additional dose of a hepatitis A vaccine within 6 to 18 months of administration of the first unit dose.

[0253] In certain embodiments, the present invention is directed to a method as described above, comprising a primary vaccination comprising the steps of: (A) selecting a subject for administration of a unit dose of a tetravalent dengue virus composition and a hepatitis A vaccine based on the subject's need for protection against dengue infection and hepatitis A infection; and (B) administering to the subject a first unit dose of the quadrivalent dengue virus composition and a hepatitis A vaccine on the same day and simultaneously; and (C) administering to the subject two additional unit doses of the tetravalent dengue virus composition about 6 months and about 12 months after administration of the first unit dose, and administering an additional Hepatitis A vaccine either about 6 months or about 12 months after administration of the first unit dose. In some of these embodiments, the step (A) of selecting a subject is performed without a determination of past Hepatitis A infection.

[0254] In certain embodiments, the invention is directed to a combination vaccine composition as described above, wherein upon reconstitution of the unit dose with a pharma- ceutically acceptable diluent, (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL, and based on said total concentration of pfu / 0.5 mL, (ii) has a concentration of less than 10% at pfu / 0.5 mL, (iv) has a concentration of at least 50% at pfu / 0.5 mL, (i) has a concentration of at least 1% at pfu / 0.5 mL, and (iii) has a concentration of at least 6%, at least 8%, or at least 10%, or at least 12%, or at least 14%, or at least 16%, or at least 18% at pfu / 0.5 mL.

[0255] In certain embodiments, the method provides compatibility between the dengue vaccine composition and the Hepatitis A vaccine, specifically meaning that the immune response following co-administration is not inferior compared to a single administration of these vaccines.

[0256] In certain embodiments, the method results in synergy between the dengue vaccine composition and the Hepatitis A vaccine, specifically meaning that the immune response following co-administration is better than when one or both vaccines are administered alone.

[0257] In certain embodiments, the present invention is directed to the aforementioned method, which results in non-inferiority in a non-inferiority clinical trial involving at least 60 or at least 120 healthy subjects divided into one subject population and one control subject population, wherein the subject population receives unit doses of the Hepatitis A vaccine and the dengue vaccine composition simultaneously on the same day, and wherein the control subject population receives unit doses of the Hepatitis A vaccine and a placebo dose simultaneously on the same day.

[0258] In certain embodiments, the present invention is directed to the aforementioned method, wherein for a subject population of at least 30 or at least 50 healthy subjects who are seronegative for Hepatitis A at baseline and seronegative for all dengue virus serotypes at baseline receiving unit doses of the Hepatitis A vaccine and the Dengue vaccine composition simultaneously on the same day, the Hepatitis A vaccine provides a Hepatitis A seroprotection rate of at least 95%, or at least 98%, 30 days after administration (day 0 / 1).

[0259] In certain embodiments, the present invention relates to the aforementioned method that results in a difference in seroprotection rate for Hepatitis A versus single agent administration for Hepatitis A, the difference being determined in a non-inferiority clinical trial including at least 60 or at least 120 healthy subjects who are seronegative for Hepatitis A at baseline and seronegative for all Dengue virus serotypes at baseline; Healthy subjects: a) a subject population of at least 30 or at least 50 healthy subjects who receive unit doses of a Hepatitis A vaccine and a Dengue vaccine composition on the same day and at the same time (day 0 / 1); b) a control population of at least 30 or 50 subjects receiving the Hepatitis A vaccine and a placebo on the same day and at the same time (day 0 / 1); The difference between the hepatitis A seroprotection rate of the control subject population 30 days after dosing (day 0 / 1) and the hepatitis A seroprotection rate of the subject population 30 days after dosing (day 0 / 1) is determined; Methods are of interest where the upper limit of the difference within a two-sided 95% confidence interval is less than 10%.

[0260] In certain embodiments, the present invention is directed to the aforementioned method, wherein the hepatitis A vaccine provides a hepatitis A seroprotection rate of at least 95%, or at least 98%, or at least 99%, 30 days after administration (day 0 / 1) in a subject population of at least 30 or at least 50 healthy subjects who are seronegative for hepatitis A at baseline receiving unit doses of the hepatitis A vaccine and dengue vaccine composition simultaneously on the same day, wherein the healthy subjects include healthy subject(s) who are seropositive for at least one dengue virus serotype at baseline and healthy subject(s) who are seronegative for all dengue virus serotypes at baseline.

[0261] In certain embodiments, the invention relates to the aforementioned method, which results in a difference in seroprotection rate for Hepatitis A versus single agent administration for Hepatitis A, said difference being determined in a non-inferiority clinical trial including at least 60 or at least 120 healthy subjects who are seronegative for Hepatitis A at baseline, said healthy subjects including healthy subject(s) who are seropositive for at least one Dengue virus serotype at baseline and healthy subject(s) who are seronegative for all Dengue virus serotypes at baseline; Healthy subjects a) a subject population of at least 30 or at least 50 healthy subjects receiving unit doses of a Hepatitis A vaccine and a Dengue vaccine composition simultaneously on the same day (day 0 / 1), the subject population including healthy subject(s) who are seropositive for at least one Dengue virus serotype at baseline and healthy subject(s) who are seronegative for all Dengue virus serotypes at baseline; b) a control subject population of at least 30 or at least 50 healthy subjects receiving the Hepatitis A vaccine and a placebo simultaneously on the same day (day 0 / 1), the control subject population including healthy subject(s) who are seropositive for at least one dengue virus serotype at baseline and healthy subject(s) who are seronegative for all dengue virus serotypes at baseline; The difference between the hepatitis A seroprotection rate of the control subject population 30 days after dosing (day 0 / 1) and the hepatitis A seroprotection rate of the subject population 30 days after dosing (day 0 / 1) is determined; Methods are of interest where the upper limit of the difference within a two-sided 95% confidence interval is less than 10%.

[0262] In certain embodiments, the present invention is directed to the aforementioned method, wherein the subject or subject population has been exposed to a Hepatitis A vaccine pandemic and / or a Dengue virus pandemic. In certain embodiments, the present invention is directed to the aforementioned method, wherein for a subject population of at least 30 or at least 50 healthy subjects who are seronegative for Hepatitis A at baseline and seronegative for all dengue virus serotypes at baseline receiving unit doses of the Hepatitis A vaccine and the Dengue vaccine composition simultaneously on the same day, the method results in an anti-Hepatitis A virus antibody geometric mean concentration (GMC) 30 days after administration (day 0 / 1) of at least 70 mIU / ml, or at least 80 mIU / ml, or at least 90 mIU / ml.

[0263] An ELISA for determining anti-Hepatitis A antibodies is disclosed, for example, in Beck et al. J Travel Med 2004;11:201-207.

[0264] In certain embodiments, the present invention is directed to the aforementioned method, wherein the co-administration of unit doses of the Hepatitis A vaccine and the Dengue vaccine composition to a subject or population of subjects does not result in serious adverse events associated with the co-administration, and in addition, there have been no deaths associated with the co-administration.

[0265] In certain embodiments, the present invention relates to the aforementioned method, comprising administering to a subject a subject a geometric mean titer (GMT) of neutralizing antibodies as measured by the following MNT50: - for dengue serotype 1, at least 110, or at least 140, or at least 150, for dengue serotype 2, at least 3000, or at least 3500, or at least 3900, - for dengue serotype 3, at least 100, or at least 120, or at least 140, and / or - for dengue serotype 4, at least 80, or at least 110, or at least 140, The method involves administering unit doses of a hepatitis A vaccine and a dengue vaccine composition simultaneously on the same day (day 0 / 1) to a population of at least 30 or at least 50 healthy subjects who are seronegative for hepatitis A at baseline and seronegative for all dengue virus serotypes at baseline, 30 days after administration.

[0266] In some embodiments, the geometric mean neutralizing antibody titer (GMT) of a subject population or the neutralizing antibody titer of a subject is determined according to a microneutralization test (eg, according to the method described in Example 2).

[0267] The present invention is directed, in part, to a method of preventing Hepatitis A and Dengue disease in a subject or population of subjects comprising concurrently administering on the same day unit doses of a Hepatitis A vaccine and a Dengue vaccine composition, wherein the unit dose comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, wherein the four live attenuated dengue virus strains are different from those used in the unit doses defined above.

[0268] In one embodiment of the invention, the method is directed to the simultaneous administration of a Hepatitis A vaccine together with another Dengue vaccine (eg, Dengvaxia®) on the same day. Dengvaxia® is a tetravalent dengue vaccine, CYD-TDV (Dengvaxia®, Sanofi Pasteur, Lyon, France), based on the yellow fever backbone, which has been licensed in several countries based on clinical demonstration of overall vaccine efficacy (VE) against virologically confirmed dengue fever (VCD) in 56-61% of children in Asia and Latin America (Capeding MR et al. Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in Asia: a phase 3, randomised, observer-masked, placebo-controlled trial. Lancet 2014,384:1358-65; Villar LA et al. Safety and immunogenicity of a recombinant tetravalent dengue vaccine in 9-16 year olds: a randomized, controlled, phase II trial in Latin America. Pediatr Infect Dis J 2013,32:1102-9). The preparation of these particular strains CYD1, CYD2, CYD3 and CYD4 is described in detail in International Patent Applications WO98 / 37911, WO03 / 101397, WO07 / 021672, WO08 / 007021, WO08 / 047023 and WO08 / 065315, to which reference may be made for a precise description of the process for their preparation. The corresponding nucleotide sequences of the prM-E regions of CYD1, CYD2, CYD3 and CYD4 are provided in WO2016034629, and the SEQ ID NOs are shown in Table 16 of this reference.

[0269] In one such embodiment, the method comprises a vaccination comprising the steps of: (A) selecting a subject for administration of equal doses of a CYD-TDV composition (e.g., Dengvaxia®) and a hepatitis A vaccine (e.g., HAVRIX® or VAQTA®) based on a need for protection against dengue infection and hepatitis A infection; and (B) administering to the subject a first dose of a CYD-TDV composition (e.g., Dengvaxia®) and a Hepatitis A vaccine (e.g., HAVRIX® or VAQTA®) at month 0; (C) administering to the subject an additional dose of a CYD-TDV composition (e.g., Dengvaxia®), and optionally a Hepatitis A vaccine (e.g., HAVRIX® or VAQTA®), within 3 to 11 months, particularly at about 6 months, after administration of the first CYD-TDV dose; and (D) Administering to the subject a final dose of CYD-TDV (e.g., Dengvaxia®) and, optionally, a Hepatitis A vaccine (e.g., HAVRIX® or VAQTA®) at approximately 12 months.

[0270] In certain embodiments, the subject is between 2 and 60 years old.

[0271] In certain preferred embodiments, the subject is between 2 and 18 years old, between 4 and 16 years old, or between 18 and 60 years old.

[0272] Preferably, the exact amount of each component of the CYD-TDV administered may vary depending on the age and weight of the subject to be vaccinated, the frequency of administration, and the other components in the composition. The amount of chimeric dengue virus in the CYD-TDV contained in a dose of the vaccine composition is about 10 5 CCID50~about 10 6 The amount of live attenuated chimeric dengue virus of each of serotypes 1 to 4 contained in a CYD dosage form (e.g., Dengvaxia®) is preferably equal. Advantageously, the vaccine compositions described in this section contain an effective amount of a dengue antigen as defined herein.

[0273] In certain embodiments, the present invention is directed to the aforementioned method, wherein the dengue vaccine composition comprises another dengue vaccine (e.g., TV003 or TV005). TV003, developed by the National Institute of Allergy and Infectious Diseases, comprises vaccine components rDEN1Δ30, rDEN2 / 4Δ30, rDEN3Δ30 / 31, and rDEN4Δ30, each of which is 3 log 10 PFU. TV005 is similar to TV003, except that the concentration of rDEN2 / 4Δ30 in TV005 is 4 log 10 PFU. Vaccines TV003, TV005, and their vaccine components and their production are described in more detail in WO2008 / 022196A2 and SS Whitehead, Expert Rev Vaccines, 2016, 15(4):509 to 517. Using recombinant DNA technology, two attenuation strategies were utilized for the vaccine components TV003 or TV005: deletion of the 3' untranslated region and chimerization of structural genes. For example, component rDEN4Δ30 contains all structural and nonstructural proteins of wild-type DENV-4, but is attenuated by a deletion of 30 nucleotides in the 3' untranslated region (designated "Δ30"). The other vaccine component is also attenuated by a deletion of 30 nucleotides in the 3' untranslated region. In addition, rDEN3Δ30 / 31 contains a 31 nucleotide deletion in the 3' untranslated region (detailed in Figure 1c and Figure 13 of WO2008 / 022196A2). The rDEN2 / 4Δ30 component was generated by replacing the prM and E genes of DENV-2 into the rDEN4Δ30 genome. The complete genome sequences of the dengue strains that can be used to produce TV003 or TV005 are available under the Genbank accession numbers in Table A of WO2008 / 022196A1.

[0274] In certain embodiments, the present invention is directed to the aforementioned method, wherein a subject or population of subjects is administered, on the same day, simultaneously, a unit dose as disclosed herein, specifically a unit dose comprising a chimeric dengue serotype 2 / 1 strain, a live attenuated dengue serotype 2 strain, a chimeric dengue serotype 2 / 3 strain, and a chimeric dengue serotype 2 / 4 strain, Dengvaxia® as disclosed herein, and a Hepatitis A vaccine as disclosed herein.

[0275] In certain embodiments, the present invention is directed to the aforementioned method, wherein a subject or population of subjects is administered a unit dose as disclosed herein, specifically a unit dose comprising a chimeric dengue serotype 2 / 1 strain, a live attenuated dengue serotype 2 strain, a chimeric dengue serotype 2 / 3 strain, and a chimeric dengue serotype 2 / 4 strain, and a Hepatitis A vaccine as disclosed herein, on the same day and simultaneously, as a first administration, on day 0 / 1, and subsequently, the subject or population of subjects is administered Dengvaxia® as disclosed herein, as a second administration, within 3 months of the first administration, for example, on day 90 after the first administration. Alternatively, Dengvaxia® disclosed herein and the Hepatitis A vaccine disclosed herein are administered to a subject or population of subjects simultaneously on the same day on day 0 / 1 as a first administration, followed by administration of a unit dose disclosed herein (specifically comprising a chimeric dengue serotype 2 / 1 strain, a live attenuated dengue serotype 2 strain, a chimeric dengue serotype 2 / 3 strain, and a chimeric dengue serotype 2 / 4 strain) to the subject or population of subjects as a second administration within 3 months of the first administration, e.g., on day 90 after the first administration.

[0276] In certain embodiments, the present invention is directed to the aforementioned method, wherein a subject or a population of subjects is administered, on the same day and simultaneously, a unit dose as disclosed herein, specifically a unit dose comprising a chimeric dengue serotype 2 / 1 strain, a live attenuated dengue serotype 2 strain, a chimeric dengue serotype 2 / 3 strain, and a chimeric dengue serotype 2 / 4 strain, TV003 or TV005 as disclosed herein, and a Hepatitis A vaccine as disclosed herein.

[0277] In certain embodiments, the present invention is directed to the aforementioned method, wherein a subject or population of subjects is administered a unit dose as disclosed herein, specifically a unit dose comprising a chimeric dengue serotype 2 / 1 strain, a live attenuated dengue serotype 2 strain, a chimeric dengue serotype 2 / 3 strain, and a chimeric dengue serotype 2 / 4 strain, and a Hepatitis A vaccine as disclosed herein, on the same day and simultaneously on day 0 / 1 as a first administration, followed by administering to the subject or population of subjects TV003 or TV005 as disclosed herein as a second administration within 3 months of the first administration, such as on day 90 after the first administration. Alternatively, TV003 or TV005 disclosed herein and a Hepatitis A vaccine disclosed herein are administered to a subject or population of subjects simultaneously on the same day on day 0 / 1 as a first administration, followed by administration to the subject or population of subjects of a unit dose disclosed herein (specifically comprising a chimeric dengue serotype 2 / 1 strain, a live attenuated dengue serotype 2 strain, a chimeric dengue serotype 2 / 3 strain, and a chimeric dengue serotype 2 / 4 strain) as a second administration within 3 months of the first administration, for example on day 90 after the first administration.

[0278] The above methods are contemplated in the context of the use of a unit dose of the dengue vaccine disclosed herein for such methods of preventing dengue disease and hepatitis A, as well as in the context of the use of a unit dose of the dengue vaccine for the manufacture of a medicament for such methods of preventing dengue disease and hepatitis A.

[0279] The present invention further provides a kit for treating Hepatitis A and Dengue fever diseases, comprising: For kits, including boxes containing at least the following: (a) a first container holding a Hepatitis A vaccine (e.g., HAVRIX®) as defined above; and (b) a second container holding a unit dose of a dengue vaccine composition as defined above, said unit dose comprising a tetravalent dengue virus composition comprising four live attenuated dengue virus strains.

[0280] Methods and uses for preventing dengue and yellow fever disease The present invention is directed, in part, to methods of preventing dengue disease and yellow fever in a subject. Accordingly, in certain embodiments, the present invention is directed to a method of preventing dengue disease in a subject comprising administering to the subject a reconstituted unit dose of the invention described herein, the method further comprising preventing yellow fever in the subject by co-administration to the subject of a yellow fever vaccine, specifically YF-17D.

[0281] The present invention is directed, in part, to methods of preventing dengue disease and yellow fever in a population of subjects. Accordingly, in certain embodiments, the present invention is directed to a method of preventing dengue disease in a population of subjects comprising administering to the population a reconstituted unit dose of the invention described herein, the method further comprising preventing yellow fever in the population of subjects by concomitant administration to the population of a yellow fever vaccine, specifically YF-17D.

[0282] In certain embodiments, the present invention is directed to the aforementioned methods, wherein the unit dose of the invention described herein and the yellow fever vaccine (particularly, YF-17D) are administered simultaneously. In some of these embodiments, the simultaneous administration occurs on day 0 or day 90, preferably on day 0. In other embodiments, the administration of the unit dose of the invention described herein and the yellow fever vaccine (particularly, YF-17D) occurs sequentially, such that the yellow fever vaccine is administered before or after (e.g., within about 6 weeks, e.g., within about 4 weeks, e.g., within about 2 weeks, or e.g., within about 1 week) the unit dose of the dengue vaccine described herein.

[0283] In certain embodiments, the present invention is directed to the aforementioned methods, wherein a reconstituted unit dose of the invention described herein and a Yellow Fever vaccine (particularly, YF-17D) are administered by subcutaneous injection. According to some embodiments, the subcutaneous injection is administered in the arm, preferably in the deltoid region of the arm. According to some of these embodiments, the unit dose of the invention described herein and the subcutaneous injection of a Yellow Fever vaccine (particularly, YF-17D) are administered in different anatomical sites, e.g., in opposite arms, particularly when the vaccines are administered simultaneously.

[0284] In certain embodiments, the present invention is directed to the aforementioned methods, wherein two unit doses of the invention described herein are administered. In some embodiments, the two unit doses of the invention described herein are administered within 12 months or more, or within 6 months, or within 3 months, e.g., on days 0 / 1 and 90. According to some of these embodiments, an additional third unit dose of the invention described herein is administered after the second. Such a third dose may act as a booster and may be administered 6-12 months after the first dose, e.g., 12 months after the first dose, or later than 12 months after the first dose, e.g., 12 months (1 year) after the second dose, or even 5 years or more after the first or second dose.

[0285] In certain embodiments, the present invention is directed to the aforementioned method, wherein the two reconstituted unit doses of the invention described herein and one dose of Yellow Fever vaccine (specifically, YF-17D) are administered specifically according to the following schedule: Administration of the yellow fever vaccine on day -0; - a first administration of a first reconstituted unit dose, for example 3 months, preferably 90 days, after administration of said yellow fever vaccine; and - After said first administration of the reconstituted unit dose, for example 3 months later, preferably on day 180, a second administration of a second reconstituted unit dose.

[0286] In certain embodiments, the present invention is directed to the aforementioned method, wherein the two reconstituted unit doses of the invention described herein and one dose of Yellow Fever vaccine (specifically, YF-17D) are administered specifically according to the following schedule: - On day 0, a first administration of a first reconstituted unit dose; - a second administration of a second reconstituted unit dose after said first administration of the reconstituted unit dose, for example 3 months later, preferably on day 90; and - Administration of a Yellow Fever vaccine after said second administration of a reconstituted unit dose, for example 3 months later, preferably on day 180.

[0287] In certain embodiments, the present invention is directed to the aforementioned method, wherein the two reconstituted unit doses of the invention described herein and one dose of Yellow Fever vaccine (specifically, YF-17D) are administered specifically according to the following schedule: - on day 0, co-administration of a first reconstituted unit dose and the yellow fever vaccine; and - A second administration of a second reconstituted unit dose following said co-administration, for example 3 months later, preferably on day 90.

[0288] In a preferred embodiment, the unit doses of the yellow fever vaccine and the invention described herein are administered simultaneously on day 0 or simultaneously on day 90.

[0289] In certain embodiments, the invention is directed to the aforementioned methods, wherein the subject or population of subjects is seronegative for all dengue serotypes. In certain embodiments, the invention is directed to the aforementioned methods, wherein a reconstituted unit dose of the invention described herein is administered subcutaneously to the subject or population of subjects, and a yellow fever vaccine (specifically, the YF-17D vaccine) is administered subcutaneously to the subject or population of subjects, and the subject or population of subjects is seronegative for all dengue serotypes. In other embodiments, the subject or population of subjects is seropositive for at least one dengue serotype.

[0290] In certain embodiments, the present invention is directed to the aforementioned methods, wherein the unit dose and yellow fever vaccine of the invention described herein, particularly YF-17D, is administered to a subject or population of subjects originating from a dengue endemic area. In certain embodiments, the reconstituted unit dose and yellow fever vaccine of the invention described herein, particularly YF-17D, is administered subcutaneously to a subject or population of subjects originating from a dengue endemic area. In other embodiments, the subject or population of subjects originates from a non-dengue endemic area. Such populations of subjects or such subjects may be vaccinated according to the present invention in the context of travel to dengue endemic and yellow fever endemic areas.

[0291] In certain embodiments, the present invention is directed to the aforementioned method, wherein the reconstituted unit dose and yellow fever vaccine of the present invention (specifically YF-17D) described herein is administered subcutaneously to a subject or subject population over 17 years of age, or over 18 years of age, or between 18 and 60 years of age. In further embodiments, the subject or subject population is an adult over 21 years of age, or between 21 and 60 years of age, or between 21 and 45 years of age. In some embodiments, the subject or subject population is from a dengue endemic area. In another embodiment, the subject or subject population is from a dengue non-endemic area, preferably a dengue non-endemic and yellow fever non-endemic area. According to some of these embodiments, the subject or subject population is seronegative for all four dengue serotypes.

[0292] The above methods are contemplated both in the context of the use of a unit dose of a dengue vaccine disclosed herein for such methods and in the context of the use of a unit dose of a dengue vaccine for the manufacture of a medicament for such methods.

[0293] Methods and uses of prevention against dengue fever, methods and uses of vaccination The present invention is directed, in part, to methods of preventing dengue disease in a subject, particularly virologically confirmed dengue, VCD. Thus, in certain embodiments, the present invention is directed to methods of preventing dengue disease in a subject, comprising administering to the subject an inventive unit dose / tetravalent dengue virus composition described herein, particularly a reconstituted unit dose.

[0294] The present invention is directed, in part, to methods of preventing dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Accordingly, in certain embodiments, the present invention is directed to methods of preventing dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), comprising administering to a subject a reconstituted unit dose / tetravalent dengue virus composition of the invention described herein.

[0295] Thus, the present invention relates to a method of inoculating a subject against virologically confirmable dengue disease with a tetravalent dengue virus composition comprising four live attenuated dengue virus strains representing serotype 1, serotype 2, serotype 3, and serotype 4, specifically the tetravalent dengue virus composition comprises a chimeric dengue serotype 2 / 1 strain and a dengue serotype 2 strain and a chimeric dengue serotype 2 / 3 strain and a chimeric dengue serotype 2 / 4 strain, specifically the dengue serotype 2 strain is derived from the wild type virus strain DEN-2 16681 (SEQ ID NO: 11) and differs from the wild type in at least three nucleotides as follows: a) 5'-noncoding region (NCR)-57 (nt-57 C to T): the major attenuation locus b) NS1-53 Gly to Asp (nt-2579 G to A): major attenuation locus c) NS3-250 Glu to Val (nt-5270 A to T): major attenuation locus; The three chimeric dengue strains replaced the structural proteins prM and E from serotype 2 strains with the corresponding structural proteins from other dengue serotypes, resulting in the following chimeric dengue strains: - DENV-2 / 1 chimera; -DENV-2 / 3 chimeras and - the method is directed to a DENV-2 / 4 chimera, which is derived from a serotype 2 strain by resulting in a DENV-2 / 4 chimera.

[0296] Further information regarding the serotypes of the quadrivalent composition can be derived from the section "Dengue virus strains" above.

[0297] A tetravalent dengue virus composition for such a method comprises: (i) dengue serotype 1 at a concentration of at least 3.3 log10 pfu / 0.5 ml; (ii) dengue serotype 2 at a concentration of at least 2.7 log10 pfu / 0.5 ml; (iii) dengue serotype 3 at a concentration of at least 4.0 log pfu / 0.5 ml; and (iv) in the form of a unit dose containing dengue serotype 4 at a concentration of at least 4.5 log10 pfu / 0.5 ml.

[0298] Specifically, the unit dose is lyophilized and upon reconstitution with 0.5 mL of a pharma- ceutical acceptable diluent, (i) dengue serotype 1 at a concentration of at least 3.3 log10 pfu / 0.5 ml; (ii) dengue serotype 2 at a concentration of at least 2.7 log10 pfu / 0.5 ml; (iii) dengue serotype 3 at a concentration of at least 4.0 log pfu / 0.5 ml; and (iv) The method includes dengue serotype 4 at a concentration of at least 4.5 log10 pfu / 0.5 ml.

[0299] Further information regarding tetravalent compositions or unit doses can be derived from the above sections "Dengue vaccine compositions" and "Unit doses".

[0300] As such, the present invention provides a method and corresponding use, the method comprising the steps of: (A) administering to a subject a first unit dose of a tetravalent dengue virus composition; (B) administering to the subject a second unit dose of the tetravalent dengue virus composition within three months of administration of the first unit dose, comprising primary vaccination by administration of only two unit doses and corresponding uses. According to this embodiment, two doses administered within three months is sufficient to provide effective protection against subsequent dengue infection.

[0301] Such methods preferably provide a combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease with a two-sided 95% confidence interval greater than 60% when measured against placebo in a subject population of at least 5,000 healthy subjects aged 14-16 years, regardless of baseline serostatus, from the first dose of the dosing schedule through 18 months after the second dose of the dosing schedule.

[0302] Such methods also preferably provide a combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease with a two-sided 95% confidence interval of greater than 45% when measured against placebo in a subject population of at least 1,500 or at least 2,000 healthy subjects aged 14-16 years who are seronegative to all serotypes at baseline from 30 days after the second dose of the dosing schedule through 18 months after the second dose of the dosing schedule.

[0303] According to certain embodiments, the method of inoculation against virologically confirmed dengue disease is with dengue serotype 2 and / or dengue serotype 1. The method has very high efficacy against dengue serotype 2 and dengue serotype 1 and the highest efficacy against dengue serotype 2.

[0304] In certain embodiments, the invention is directed to the aforementioned method having vaccine efficacy against serotype 1 in preventing virologically confirmable dengue disease with a two-sided 95% confidence interval, where the lower limit is greater than 25% when measured against placebo in a subject population of at least 1,500, or at least 2,000, or at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4-16, regardless of baseline serostatus, from 30 days after the second dose through 12-18 months (e.g., at 12 or 18 months). In certain such embodiments, the lower limit is greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, or greater than 54%. In certain such embodiments, a subject population of at least 1,500 subjects is seronegative to all serotypes at baseline, and the lower limit is greater than 35%. In certain such embodiments, the seronegative and seropositive populations each receive vaccine efficacy against serotype 1 with a two-sided 95% confidence interval, the lower limit being within 10 percentage points.

[0305] In certain embodiments, the invention is directed to the aforementioned method, wherein the vaccine has vaccine efficacy against serotype 1 in preventing virologically confirmable dengue disease when measured against placebo in a subject population of at least 1,500, or at least 2,000, or at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4-16, regardless of baseline serostatus, from 30 days after the second dose through 12-18 months (e.g., at 12 or 18 months). In certain such embodiments, the vaccine efficacy is greater than 40%, greater than 50%, greater than 60%, or greater than 65%. In certain such embodiments, the subject population of at least 1,500 is seronegative to all serotypes at baseline. In certain such embodiments, the seronegative and seropositive populations each achieve vaccine efficacy against serotype 1 within 5 percentage points.

[0306] In certain embodiments, the invention is directed to the aforementioned method having vaccine efficacy against serotype 2 in preventing virologically confirmable dengue disease with a two-sided 95% confidence interval, where the lower limit is greater than 25% when measured against placebo in a subject population of at least 1,500, or at least 2,000, or at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4-16, regardless of baseline serostatus, from 30 days after the second dose through 12-18 months (e.g., at 12 or 18 months). In certain such embodiments, the lower limit is greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 85%. In certain such embodiments, the subject population of at least 1,500 is seronegative to all serotypes. In certain such embodiments, the seronegative and seropositive populations each receive vaccine efficacy against serotype 2 with a two-sided 95% confidence interval, the lower limit being within 5 percentage points.

[0307] In certain embodiments, the invention is directed to the aforementioned method, wherein the vaccine has vaccine efficacy against serotype 2 in preventing virologically confirmable dengue disease when measured against placebo in a subject population of at least 1,500, or at least 2,000, or at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4-16, regardless of baseline serostatus, from 30 days after the second dose through 12-18 months (e.g., at 12 or 18 months). In certain such embodiments, the vaccine efficacy is greater than 60%, greater than 70%, greater than 80%, or greater than 90%. In certain such embodiments, the subject population of at least 1,500 is seronegative to all serotypes at baseline. In certain such embodiments, the seronegative and seropositive populations each achieve vaccine efficacy against serotype 2 within 5 percentage points.

[0308] The effectiveness of this method is explained in further detail in this section below.

[0309] In certain embodiments, the unit dose is reconstituted and administered by subcutaneous injection. According to some of these embodiments, the subcutaneous injection is administered in the arm, preferably in the deltoid region of the arm.

[0310] According to one embodiment, such methods do not include a step of determining whether the subject has previously been infected with dengue prior to administration of the unit dose, or the serostatus of the subject is unknown prior to administration of the unit dose.

[0311] According to one embodiment, such methods preferably do not include a step of determining whether the subject has been previously infected with dengue fever, either before, during or after the administering step, or the serostatus of the subject is unknown, preferably at any time before, during or after the administering step.

[0312] The method according to the invention does not require testing of serostatus prior to vaccination, thus allowing immediate treatment and pandemic control. According to certain embodiments, the use is in a method where the subject is exposed to a dengue pandemic. In certain such embodiments, the pandemic is due to dengue serotype 2 and / or is due to serotype 1.

[0313] According to one embodiment of such a method, the subject is from an area where seroprevalence is unknown and / or where seroprevalence is less than 80%, or less than 70%, or less than 60%.

[0314] According to one embodiment of such a method, the subject is seronegative at baseline and originates from or travels to an area where seroprevalence is high for serotype 1 and / or serotype 2, i.e., 80%, or 90%, or greater.

[0315] According to this embodiment, the vaccine and corresponding method are safe for seronegative and seropositive subjects and thus do not require serostatus analysis or determination of past dengue infection or high seroprevalence in the area. Such methods preferably provide combined vaccine efficacy against virologically confirmed dengue with hospitalization for all four serotypes with a two-sided 95% confidence interval, with a lower limit of greater than 65% when measured against placebo in a population of at least 5,000 healthy subjects aged 4-16 years, regardless of baseline serostatus, from the first dose of the dosing schedule to 12-18 months after the second dose of the dosing schedule, and preferably in at least 1,500 healthy subjects aged 4-16 years who are seronegative at baseline. Preferably, the two-sided 95% confidence interval of combined vaccine efficacy against virologically confirmed dengue with hospitalization for all four serotypes shows a lower limit of the two-sided confidence interval within 10 percentage points, or within 15 percentage points, or within 20 percentage points when comparing seropositive and seronegative subjects. The method is preferably safe with respect to serotype 1 and serotype 2 and therefore can be used in pandemic situations due to serotype 1 and / or serotype 2 or even in seronegative subjects (e.g. travelers) or subjects with unknown serostatus in areas with very high seroprevalence (>80%) due to serotype 1 and / or 2.

[0316] The safety of the method is described in further detail in the section "Methods of Prevention, Methods of Vaccination."

[0317] According to one embodiment, such method does not include active monitoring of the subject for febrile illness after administration of the first and second unit doses. During active monitoring, any subject with febrile illness (defined as a fever of ≧38° C. on any two of three consecutive days) is asked by the investigator to return to the testing site for dengue evaluation. The subject / guardian is contacted at least once a week to ensure positive identification of febrile illness by reminding the subject / guardian of their obligation to return to the testing site in case of febrile illness. This contact is performed via appropriate methods (e.g., phone calls, text messages, home visits, school-based monitoring) that may vary in each study testing site.

[0318] According to one embodiment, such methods do not include a vaccine immunogenicity assay involving a GMT for dengue neutralizing antibodies.

[0319] According to one embodiment, such method does not include reactogenicity analysis, which concerns non-spontaneous local AEs (pain at injection site, erythema at injection site, and swelling at injection site) and non-spontaneous systemic AEs (children <6 years: fever, irritability / tantrums, drowsiness, and loss of appetite; children >6 years: asthenia, fever, headache, malaise, and myalgia), assessed, for example, by collection of diary cards, 7 days and 14 days after each vaccination (including the day of vaccination), respectively.

[0320] According to one embodiment, the method does not include active surveillance, immunogenicity analysis, and reactogenicity analysis.

[0321] According to such embodiments, the vaccine and the corresponding administration method are safe and therefore do not require further steps of monitoring or analysis.

[0322] In view of the above, a method according to one embodiment comprises the steps of: (A) selecting a subject for administration of a unit dose of a tetravalent dengue virus composition in need of protection against dengue infection, without determination of prior dengue infection; (B) administering to the subject a first unit dose of a tetravalent dengue virus composition; (C) administering to the subject a second unit dose of the tetravalent dengue virus composition within three months of administering the first unit dose. Thus, the method of inoculation is completed without determining whether the patient had previously been infected with dengue fever. The method further optionally includes a booster dose of the unit dose at least one year after administration of the second unit dose.

[0323] Selection of the subject may include all types of considerations, but preferably does not include a determination of past dengue infection. Selection may include consideration of age, health status, and risk of infection. Risk of infection includes consideration of seroprevalence, serotype-specific seroprevalence, and pandemic or serotype-specific pandemic status in the area where the subject normally resides or intends to travel. The subject may be selected due to its exposure to serotype 1 and / or serotype 2 or due to the fact that it requires protection against a particular dengue serotype, i.e., serotype 1 and / or serotype 2.

[0324] According to the present invention, the method is applicable to subjects of all ages.According to one embodiment, the subject is under 9 years old, or 4-5 years old, or 6-11 years old, or 12-16 years old, or 6-16 years old, or 4-16 years old, or 2-17 years old, or 9 years old, or over 9 years old, or 9-17 years old, or 18-45 years old, or 46-60 years old, or over 60 years old.

[0325] In particular, the present invention is directed to methods such that the methods are safe.

[0326] Specifically, the present invention is directed to a method of providing combination vaccine efficacy against virologically confirmed dengue fever with hospitalization for all four serotypes with a two-sided 95% confidence interval, the lower limit of which is greater than 65% when measured against placebo in a population of at least 5,000 healthy subjects aged 4-16 years, regardless of baseline serostatus, from the first dose of the dosing schedule to 12-18 months after the last dose of the dosing schedule.

[0327] In particular, the present invention is directed to such methods, in which the methods are effective.

[0328] Specifically, the present invention is directed to a method that provides combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease with a two-sided 95% confidence interval greater than 60% when measured against placebo in a subject population of at least 5,000 healthy subjects aged 14-16 years, regardless of baseline serostatus, from the first dose of the dosing schedule through 18 months after the last dose of the dosing schedule.

[0329] In certain embodiments, the present invention is directed to the aforementioned method, wherein the subject is seronegative for all dengue serotypes.

[0330] The present invention is directed, in part, to methods of preventing dengue disease (particularly virologically confirmed dengue, VCD) in a subject population. Thus, in certain embodiments, the present invention is directed to a method of preventing dengue disease in a subject population, comprising administering to the subject population a unit dose, particularly a reconstituted unit dose of the invention described herein.

[0331] The present invention is directed, in part, to the aforementioned method for preventing dengue disease (specifically, virologically confirmed dengue, VCD) in a subject population, comprising administering to the subject population at least a first reconstituted unit dose of the invention described herein, wherein a certain ratio of geometric mean neutralizing antibody titers (GMT) is achieved at 180 days or 365 days following administration of said first unit dose to the subject population. According to some embodiments, the geometric mean neutralizing antibody titers for dengue serotype 2 (GMT DENV-2) and the geometric mean neutralizing antibody titers for dengue serotype 4 (GMT DENV-4) provide a ratio of GMT DENV-2:GMT DENV-4 of 50 or less, or 40 or less, or 30 or less, or 20 or less, when tested in at least 40, or at least 50, or at least 60 subjects at 180 days or 365 days after at least a first administration of a reconstituted unit dose of the invention described herein and optionally after a second administration of a reconstituted unit dose of the invention described herein 90 days after the first administration. In some of these embodiments, the ratio of GMT DENV-2:GMT DENV-1 is 20 or less, or 18 or less, or 15 or less, 180 or 365 days after administration of the first reconstituted unit dose, and / or the ratio of GMT DENV-2:GMT DENV-3 is 20 or less, or 18 or less, or 15 or less, 180 or 365 days after administration of the first reconstituted unit dose.

[0332] The present invention is directed, in part, to the aforementioned method for preventing dengue disease (particularly virologically confirmed dengue, VCD) in a subject, comprising administering to the subject at least a first reconstituted unit dose of the invention described herein, wherein a certain ratio of neutralizing antibody titers is achieved at 180 days or 365 days after administration of said first unit dose to the subject. According to some embodiments, the neutralizing antibody titers against dengue serotype 2 and the neutralizing antibody titers against dengue serotype 4 at 180 days or 365 days after at least the first administration of a reconstituted unit dose of the invention described herein, and optionally after a second administration of a reconstituted unit dose of the invention described herein 90 days after the first administration, provide a ratio of neutralizing antibody titers against DENV-2:GMT DENV-4 of 50 or less, or 40 or less, or 30 or less, or 20 or less. In some of these embodiments, the ratio of DENV-2:DENV-1 neutralizing antibody titers is 20 or less, or 18 or less, or 15 or less, 180 or 365 days after administration of the first reconstituted unit dose, and / or the ratio of DENV-2:DENV-3 neutralizing antibody titers is 20 or less, or 18 or less, or 15 or less, 180 or 365 days after administration of the first reconstituted unit dose.

[0333] The geometric mean neutralizing antibody titer (GMT) of a subject population or the neutralizing antibody titer of a subject is determined according to the microneutralization test disclosed herein, for example, according to the method described in Example 2. Without wishing to be bound by any theory, it is currently understood that the method of inducing a more balanced immune response by administering the reconstituted unit dose of the invention described herein will be beneficial to the vaccinated subject or subject population in terms of less difference between the geometric mean neutralizing antibody titers (GMT) or neutralizing antibody titers of the four dengue serotypes. In particular, it is understood that a much larger response to any one of the four serotypes (e.g., DENV-2) is less beneficial compared to the other serotypes.

[0334] The present invention is directed, in part, to the aforementioned method for preventing dengue disease (specifically, virologically confirmed dengue, VCD) in a subject or population of subjects, comprising subcutaneous administration of two unit doses on days 1 and 90, resulting in a seropositivity rate in the subject population of at least 50 subjects, wherein the subjects in the subject population are seronegative to all dengue serotypes at baseline. In certain such embodiments, at least 80% of the subject population are seropositive for all four dengue serotypes at least one month after administration of the first unit dose, e.g., at day 30, and / or at least 80% of the subject population are seropositive for all four dengue serotypes prior to or at the time of administration of the second unit dose, e.g., at day 90, and / or at least 80%, or at least 85%, or at least 90%, or at least 95% of the subject population are seropositive for all four dengue serotypes after administration of the second unit dose, e.g., at day 120, and / or at least 80%, or at least 85%, or at least 90% of the subject population are seropositive for all four dengue serotypes after administration of the second unit dose, e.g., at day 270.

[0335] The invention is directed, in part, to the aforementioned method for preventing dengue disease (specifically virologically confirmable dengue, VCD) in a subject or subject population, comprising subcutaneous administration of two unit doses at days 1 and 90, resulting in a seropositivity rate in the subject population of at least 100 subjects, wherein the subjects in the subject population comprise 20%-40% subjects who are seronegative to all dengue serotypes and 60%-80% subjects who are seropositive to at least one dengue serotype at baseline, wherein at day 120 and / or day 270, the seropositivity rates for all four dengue serotypes in the seronegative part of the subject population do not deviate by more than 10 percentage points from the seropositivity rates for all four dengue serotypes in the seropositive part of the subject population, and / or at day 120, the seropositivity rates for all four dengue serotypes in the seronegative part of the subject population do not deviate by more than 5 percentage points from the seropositivity rates for all four dengue serotypes in the seropositive part of the subject population.

[0336] The present invention is directed, in part, to a method of preventing virologically confirmable dengue disease in a subject or population of subjects, comprising administering to the subject or population of subjects a reconstituted unit dose of a tetravalent dengue virus composition comprising four live, attenuated dengue serotypes (specifically the virus strains described herein).

[0337] The present invention is directed, in part, to a method of preventing virologically confirmable dengue disease accompanied by hospitalization in a subject or population of subjects, comprising administering to the subject or population of subjects a reconstituted unit dose of a tetravalent dengue virus composition comprising four live, attenuated dengue serotypes (specifically the virus strains described herein).

[0338] In certain embodiments, the method comprises a reconstituted unit dose of a dengue vaccine composition / tetravalent dengue virus composition administered to prevent dengue disease in a subject or population of subjects, the reconstituted unit dose comprises a tetravalent virus composition comprising four live attenuated dengue virus strains, the unit dose being lyophilized and upon reconstitution with 0.5 mL of a pharmacopoeia acceptable diluent, (i) dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain) at a concentration of at least 3.3 log10 pfu / 0.5 ml; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) at a concentration of at least 2.7 log pfu / 0.5 ml; (iii) dengue serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain) at a concentration of at least 4.0 log pfu / 0.5 ml; and (vi) A reconstituted unit dose is obtained that contains a concentration of dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) of at least 4.5 log10 pfu / 0.5 ml.

[0339] Preferably, the reconstituted unit dose / tetravalent dengue virus composition is used in the method of preventing dengue disease of the invention, wherein upon reconstitution of the unit dose with a pharma- ceutically acceptable diluent, (i), (ii), (iii), and (iv) have a total concentration of pfu / 0.5 mL, where based on said total concentration, the concentration of (ii) at pfu / 0.5 mL is less than 2%, the concentration of (iv) at pfu / 0.5 mL is at least 50%, the concentration of (i) at pfu / 0.5 mL is at least 1%, and the concentration of (iii) at pfu / 0.5 mL is at least 6%, and the subject or subject population is aged between 18 and 60 years.

[0340] In another preferred embodiment, the reconstituted unit dose / tetravalent dengue virus composition is used in the method of preventing dengue disease of the invention, wherein upon reconstitution with a pharma- ceutically acceptable diluent, (i), (ii), (iii), and (iv) have a total concentration of pfu / 0.5 mL, where based on the total concentration, the concentration of (ii) at pfu / 0.5 mL is less than 10%, the concentration of (iv) at pfu / 0.5 mL is at least 50%, the concentration of (i) at pfu / 0.5 mL is at least 1%, and the concentration of (iii) at pfu / 0.5 mL is at least 8%, and the subject or subject population is between 2 and 17 years of age.

[0341] In certain embodiments, the invention relates to the aforementioned method, wherein the unit dose comprises a tetravalent dengue virus composition comprising four live attenuated dengue serotypes (specifically virus strains as described herein), the serotypes having a certain concentration as described herein for the virus composition and unit dose, e.g., (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 log10 pfu / dose to 5.0 log10 pfu / dose, or 3.3 log10 pfu / 0.5 mL to 5.0 log10 pfu / 0.5 mL; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 2.7 log pfu / dose to 4.9 log pfu / 0.5 dose, or 2.7 log pfu / 0.5 ml to 4.9 log pfu / 0.5 ml; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log10 pfu / dose to 5.7 log10 pfu / 0.5 dose, or 4.0 log10 pfu / 0.5 mL to 5.7 log10 pfu / 0.5 mL; (iv) Dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) is directed to the method, having a concentration of 4.5 log10 pfu / dose to 5.5 log10 pfu / 0.5 dose, or 4.5 log10 pfu / 0.5 mL to 5.5 log10 pfu / 0.5 mL. In preferred such embodiments, the subject or subject population is aged between 2 and 17 years, such as between 4 and 16 years, preferably under the age of 9. In other preferred embodiments, the subject or subject population is aged between 4 and 5 years, between 6 and 11 years, or between 12 and 16 years.

[0342] In certain embodiments, the invention relates to the aforementioned method, wherein the unit dose, upon reconstitution with 0.5 mL of a pharma- ceutical acceptable diluent, has a concentration of 3.3 log10 pfu / 0.5 mL to 3.6 log10 pfu / 0.5 mL for dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strains) and 2.7 log10 pfu / 0.5 mL to 4.0 log10 pfu / 0.5 mL for dengue serotype 2 (e.g., dengue serotype 2 strains). u / 0.5mL, for dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) having a concentration of 4.0 log10 pfu / 0.5mL to 4.6 log10 pfu / 0.5mL, and for dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) having a concentration of 4.5 log10 pfu / 0.5mL or 4.6 log10 pfu / 0.5mL to 5.1 log10 pfu / 0.5mL. In preferred such embodiments, the subject or subject population is 2-17 years old, e.g., 4-16 years old, preferably less than 9 years old. In other preferred embodiments, the subject or subject population is 4-5 years old, 6-11 years old, or 12-16 years old.

[0343] In certain embodiments, the invention is directed to the aforementioned method, wherein the concentration of dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain) measured by pfu / 0.5 mL is 1%-7% of the total concentration, the concentration of dengue serotype 2 (e.g., a dengue serotype 2 strain) measured by pfu / 0.5 mL is less than 8% of the total concentration, e.g., in the range of 1%-8% of the total concentration, the concentration of dengue serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain) measured by pfu / 0.5 mL is at least 10% of the total concentration, and the concentration of dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) measured by pfu / 0.5 mL is at least 65% of the total concentration, e.g., in the range of 65%-80%. In certain such embodiments, the arithmetic sum of all four serotypes is in the range of 4.6 log10 pfu / 0.5 mL to 6.7 log10 pfu / 0.5 mL, preferably in the range of 4.6 log10 pfu / 0.5 mL to 5.5 log10 pfu / 0.5 mL. Preferably, in such embodiments, the subject or subject population is 2-17 years old, such as 4-16 years old, even more preferably under 9 years old. In other preferred embodiments, the subject or subject population is 4-5 years old, 6-11 years old, or 12-16 years old.

[0344] In a further preferred embodiment, the present invention is directed to the aforementioned method, wherein dengue serotype 1, such as TDV-1 (e.g., a chimeric dengue serotype 2 / 1 strain) and dengue serotype 2, such as TDV-2 (e.g., a dengue serotype 2 strain), are each present at a concentration within 5% points of each other based on total concentration in pfu / 0.5 mL, and / or together are less than about 10% of the total concentration in pfu / 0.5 mL. In certain such embodiments, dengue serotype 3, such as TDV-3 (e.g., a chimeric dengue serotype 2 / 3 strain), is preferably at least about 10% of the total concentration in pfu / 0.5 mL, and more preferably dengue serotype 4, such as TDV-4 (e.g., a chimeric dengue serotype 2 / 4 strain), is at least about 70% of the total concentration in pfu / 0.5 mL. In certain such embodiments, dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) (e.g., TDV-4) represents the highest concentration of all four serotypes in the composition, preferably at least about 70% of the total concentration in pfu / 0.5 mL, and dengue serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain) (e.g., TDV-3) represents the second highest concentration of all four serotypes in the composition, preferably at least about 70% of the total concentration in pfu / 0.5 mL. represents at least about 10% of the total concentration in pfu / 0.5 mL, with dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain) (e.g., TDV-1) and dengue serotype 2 (e.g., a dengue serotype 2 strain) (e.g., TDV-2) each representing a lower concentration than that of serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain) (e.g., TDV-3), and optionally together representing less than about 10% of the total concentration in pfu / 0.5 mL.

[0345] Preferably, the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3 and the chimeric dengue serotype 2 / 4 strain is TDV-4. More preferably, TDV-1 is characterized by the nucleotide sequence set forth in SEQ ID NO: 1 and the amino acid sequence set forth in SEQ ID NO: 2, TDV-2 is characterized by the nucleotide sequence set forth in SEQ ID NO: 3 and the amino acid sequence set forth in SEQ ID NO: 4, TDV-3 is characterized by the nucleotide sequence set forth in SEQ ID NO: 5 and the amino acid sequence set forth in SEQ ID NO: 6 and TDV-4 is characterized by the nucleotide sequence set forth in SEQ ID NO: 7 and the amino acid sequence set forth in SEQ ID NO: 8.

[0346] In certain embodiments, the present invention is directed to the aforementioned methods, wherein the reconstituted unit dose of the invention described herein is administered by subcutaneous injection. According to some of these embodiments, the subcutaneous injection is administered in the arm, preferably in the deltoid region of the arm.

[0347] In certain embodiments, the invention is directed to the aforementioned methods, where the reconstituted unit dose is administered to a subject with unknown serostatus and / or no testing has been performed to determine whether the subject is seropositive or seronegative (prior to) administering the unit dose described herein. In certain embodiments, the invention is directed to the aforementioned methods, where the method does not include a step of determining past dengue infection in one or more subjects. In certain embodiments, the invention is directed to the aforementioned methods, where the method does not include an analysis of seroprevalence in the area, or is performed in an area with less than 80%, less than 70%, or less than 60% seroprevalence. In certain embodiments, the invention is directed to the methods where the serostatus of the subject is unknown. In such embodiments, the serostatus is not determined at any time before and after administration for this method. In certain embodiments of the invention, the method is used in a pandemic setting. In certain embodiments, the invention is directed to the aforementioned methods, where the method is performed outside of a clinical trial.

[0348] In certain embodiments, the present invention is directed to the aforementioned method, wherein the subject or subject population is seronegative to all dengue serotypes.

[0349] In certain embodiments, the present invention is directed to the aforementioned method, wherein two unit doses of the invention described herein are administered. In some embodiments, the two unit doses are administered within 12 months or more, or within 6 months, or within 3 months, and optionally at least 4 weeks apart, e.g., on days 0 and 90, or on days 1 and 90. According to some of these embodiments, an additional third unit dose of the invention described herein is administered after the second administration. Such a third administration may act as a booster and may be administered 6 to 12 months after the first administration, e.g., 12 months after the first administration, or later than 12 months after the first administration, e.g., 12 months (1 year) after the second administration, or even 5 years or more after the first or second administration.

[0350] In certain embodiments, the methods of the invention comprise or consist of administering a single unit dose of the invention.

[0351] In certain embodiments, the present invention is directed to the aforementioned methods, wherein a reconstituted unit dose of the invention described herein is administered subcutaneously to a subject or population of subjects who are seronegative for all dengue serotypes, hi other embodiments, the subject or population of subjects is seropositive for at least one dengue serotype.

[0352] In certain embodiments, the present invention is directed to the aforementioned methods, wherein a unit dose of the invention described herein is administered to a subject or population of subjects originating from a dengue endemic area. In some of these embodiments, the subject or population of subjects originates from Singapore, Dominican Republic, Panama, Philippines, Colombia, Puerto Rico, or Thailand, particularly Singapore, Dominican Republic, Panama, or Philippines. In preferred embodiments, the subject or population of subjects originates from Asia Pacific or Central and South America. In other of these embodiments, the subject or population of subjects originates from Thailand, Sri Lanka, Philippines, Panama, Nicaragua, Dominican Republic, Colombia, or Brazil. In other embodiments, the subject or population of subjects originates from a non-dengue endemic area. Such a population of subjects or such subjects may be vaccinated according to the present invention in view of travel to a dengue endemic area. In certain embodiments, a reconstituted unit dose of the invention described herein is administered subcutaneously to a subject or population of subjects originating from a dengue endemic or non-dengue endemic area.

[0353] In certain embodiments, the present invention is directed to the aforementioned methods, wherein a reconstituted unit dose of the invention described herein is administered subcutaneously to a subject or subject population between the ages of 2 and 60. In some embodiments, the subject or subject population is an adult over 17 years of age, or over 18 years of age, or between 18 and 60 years of age. In further specific embodiments, the subject or subject population is an adult over 21 years of age, or between 21 and 60 years of age, or between 21 and 45 years of age.

[0354] In certain embodiments, the present invention is directed to the aforementioned methods, wherein a reconstituted unit dose of the invention described herein is administered subcutaneously to children and young adults between 2 and 17 years of age. In some embodiments, the subject or subject population is under 9 years of age, or under 4 years of age. In some embodiments, the subject or subject population is between 2 and 9 years of age, or between 2 and 5 years of age, or between 4 and 9 years of age, or between 6 and 9 years of age. In other embodiments, the subject or subject population is between 4 and 16 years of age. In some such embodiments, the subject or subject population is between 4 and 5 years of age, 6 and 11 years of age, or 12 and 16 years of age. Optionally, the subject or subject population is seronegative for all dengue serotypes.

[0355] In certain embodiments, the present invention is directed to the aforementioned methods, wherein a unit dose of the invention described herein is administered to a pediatric subject or population of pediatric subjects under 2 years of age, preferably between 2 months and 2 years of age, or between 2 months and 1.5 years of age, or between 2 months and 1 year of age. According to some of these embodiments, the pediatric subject or population of pediatric subjects is seronegative and is from a dengue endemic area.

[0356] In certain embodiments, the present invention is directed to the aforementioned methods, wherein a reconstituted unit dose of the invention described herein is administered, preferably by subcutaneous injection, to a pediatric subject or population of pediatric subjects under 2 years of age, preferably between 2 months and 2 years of age, or between 2 months and 1.5 years of age, or between 2 months and 1 year of age. According to some of these embodiments, the pediatric subject or population of pediatric subjects is seronegative and is from a dengue endemic area.

[0357] In certain embodiments, the invention is directed to the aforementioned methods, wherein the subject or subject population is 4-5 years old and of Asia-Pacific origin, 6-11 years old and of Asia-Pacific origin, or 12-16 years old and of Asia-Pacific origin. In other embodiments, the subject or subject population is 4-5 years old and of Latin American origin, 6-11 years old and of Latin American origin, or 12-16 years old and of Latin American origin.

[0358] In certain embodiments, the invention is directed to the aforementioned methods, wherein the subject or subject population is 4-5 years old and seropositive for at least one dengue serotype, 6-11 years old and seropositive for at least one dengue serotype, or 12-16 years old and seropositive for at least one dengue serotype. In other embodiments, the subject or subject population is 4-5 years old and seronegative for all dengue serotypes, 6-11 years old and seronegative for all dengue serotypes, or 12-16 years old and seronegative for all dengue serotypes.

[0359] In certain embodiments, the invention is directed to the aforementioned method, wherein the subject or subject population is of Asia-Pacific or Latin America origin and is seropositive for at least one dengue serotype at baseline, hi other embodiments, the subject or subject population is of Asia-Pacific or Latin America origin and is seronegative for all dengue serotypes at baseline.

[0360] In certain embodiments, the invention is directed to the aforementioned method, wherein the subject or subject population is of Asia-Pacific origin, is seropositive for at least one dengue serotype at baseline, and is 4-5 years old, 6-11 years old, or 12-16 years old. In other embodiments, the subject or subject population is of Asia-Pacific origin, is seronegative for all dengue serotypes at baseline, and is 4-5 years old, 6-11 years old, or 12-16 years old. In yet other embodiments, the subject or subject population is of Latin American origin, is seropositive for at least one dengue serotype at baseline, and is 4-5 years old, 6-11 years old, or 12-16 years old. In other embodiments, the subject or subject population is of America origin, is seronegative for all dengue serotypes at baseline, and is 4-5 years old, 6-11 years old, or 12-16 years old.

[0361] In certain embodiments, the present invention is directed to the aforementioned method, wherein the subject or subject population has been previously vaccinated against yellow fever. In other embodiments, the subject or subject population has been previously vaccinated against Japanese encephalitis. In yet other embodiments, the subject or subject population has not been previously vaccinated against yellow fever. In other embodiments, the subject or subject population has not been previously vaccinated against Japanese encephalitis. Prior vaccination refers to vaccination with a reconstituted unit dose described herein more than 30 days after the second administration (e.g., within 4 months after the first administration). For example, for vaccine efficacy (VE) determined in Example 6 from 30 days after the second vaccination, prior vaccination against yellow fever is defined as a yellow fever vaccination that occurs more than 30 days after the second vaccination. In certain embodiments, the subject or subject population has received Dengvaxia® within the dosing regimen described herein or within 4.5 years after the administration of the first dose.

[0362] In particular, unbalanced titers of neutralizing antibodies against the four dengue serotypes are observed in seronegative populations or subjects after administration of commercially available dengue vaccines. The present invention specifically shows that seronegative subjects exhibit a more balanced immune response against the four dengue serotypes after administration of a reconstituted unit dose of the invention described herein. It is therefore contemplated that the unit dose of the invention described herein and the method of the invention described herein may result in a stronger immune response in a subject population including both seropositive and seronegative subjects. This balanced response as well as balanced efficacy and safety are necessary to allow vaccination without prior serostatus analysis, which is a major advantage in vaccination programs, especially in pandemic situations.

[0363] The present invention is directed, in part, to a method of preventing virologically confirmable dengue disease in a subject, comprising administering to the subject a tetravalent dengue virus composition comprising four dengue virus strains representing serotype 1, serotype 2, serotype 3, and serotype 4, where the virus strains are optionally live-attenuated dengue virus strains.

[0364] The present invention is directed, in part, to a method of preventing virologically confirmable dengue disease in a subject, the method comprising administering to the subject a tetravalent dengue virus composition comprising four dengue virus strains representing serotype 1, serotype 2, serotype 3, and serotype 4, wherein the virus strains are optionally live-attenuated dengue virus strains.

[0365] In certain embodiments, the present invention is directed to the aforementioned methods, which do not include a step of determining the subject's serostatus at baseline, i.e., the aforementioned methods do not include determining the subject's past dengue infection history at baseline prior to administration of the tetravalent dengue virus composition. Specifically, such methods are safe and effective. Thus, in certain such embodiments, the subject has not been tested for the presence or absence of past dengue infection.

[0366] In certain embodiments, the present invention is directed to the aforementioned methods, wherein administration of the vaccine is safe, and particularly, such methods are also effective, regardless of whether or not the subject is determined to have had a previous dengue infection prior to administration of the tetravalent dengue virus composition.

[0367] In certain embodiments, the present invention is directed to the aforementioned methods, wherein the methods are safe and / or effective.

[0368] In certain embodiments, the present invention is directed to the aforementioned methods, wherein the composition comprises at least one chimeric dengue virus. In certain such embodiments, the present invention is directed to the aforementioned methods, wherein the composition comprises at least one non-chimeric dengue virus and at least one chimeric dengue virus, in particular chimeric dengue serotype 2 / 1 strains and dengue serotype 2 strains and chimeric dengue serotype 2 / 3 strains and chimeric dengue serotype 2 / 4 strains. Details of the compositions are described above.

[0369] Thus, in certain embodiments, the invention is directed to the aforementioned methods having vaccine efficacy, preferably combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease with a two-sided 95% confidence interval, greater than a lower limit of 25% when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 14-16, regardless of baseline serostatus, from 30 days after the first dose or the second / last dose until at least 12-18 months (e.g., at 12 or 18 months), wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or a placebo is administered, e.g., at least twice within less than six months (e.g., within three months). In embodiments, the invention is directed to the aforementioned methods having vaccine efficacy, preferably combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease with a two-sided 95% confidence interval, where the reconstituted unit dose or tetravalent dengue virus composition described herein or placebo is administered at least once, with a lower limit of greater than 25% when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects), regardless of baseline serostatus, until 15-21 months (e.g., 15 months or 21 months) after the first dose of the dosing schedule. In certain such embodiments, the lower limit is greater than 30%, greater than 40%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, or greater than 72%. Preferably, the reconstituted unit dose or placebo is administered subcutaneously within about 3 months, e.g., on days 0 and 90.

[0370] Thus, in certain embodiments, the invention is directed to the aforementioned methods having combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease with a two-sided 95% confidence interval, where the lower limit is greater than 60% when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4-16, regardless of baseline serostatus, from after the first dose through 18 months after the last dose, and where a reconstituted unit dose / tetravalent dengue virus composition described herein or placebo is administered at least twice, e.g., within less than six months (e.g., within three months). In these embodiments, the lower limit is, e.g., greater than 62%, greater than 64%, greater than 66%, greater than 68%, or greater than 69%.

[0371] In certain embodiments, the invention is directed to the aforementioned methods, which have a vaccine efficacy, preferably a combined vaccine efficacy against all four serotypes, of greater than 30% in preventing virologically confirmable dengue disease, as measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 14-16, regardless of baseline serostatus, from 30 days after the first dose or the second / last dose through at least 12 months or 12-18 months (e.g., at 12 or 18 months) after the second / last dose, wherein the reconstituted unit dose or tetravalent dengue virus composition described herein or placebo is administered at least twice within less than six months (e.g., within three months). In certain embodiments, the present invention is directed to the aforementioned methods, in which the vaccine efficacy, preferably the combined vaccine efficacy against all four serotypes, is greater than 30% when measured against a placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects), regardless of baseline serostatus, in preventing virologically confirmable dengue disease up to 15 months after the first dose of the dosing schedule, where a reconstituted unit dose or tetravalent dengue virus composition described herein or a placebo is administered at least once. In certain such embodiments, the vaccine efficacy is greater than 40%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 78%, greater than 79%, or about 80%. Preferably, the reconstituted unit dose or placebo is administered subcutaneously within about 3 months, e.g., on days 0 and 90.

[0372] Thus, in certain embodiments, the invention is directed to the aforementioned methods, in which the combined vaccine efficacy against all four serotypes is greater than 66% when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 14-16, regardless of baseline serostatus, from after the first dose through 18 months after the last dose, where a reconstituted unit dose or tetravalent dengue virus composition described herein or a placebo is administered at least twice, e.g., within less than six months (e.g., within three months). In these embodiments, the vaccine efficacy is, e.g., greater than 68%, greater than 70%, greater than 72%, or greater than 74%.

[0373] In certain embodiments, the invention is directed to the aforementioned methods having vaccine efficacy, preferably combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease with hospitalization with a two-sided 95% confidence interval, where the lower limit is greater than 0% when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects), regardless of baseline serostatus, from 30 days after the second administration to at least 18 months after the second administration, and where a reconstituted unit dose or tetravalent dengue virus composition as described herein or placebo is administered at least twice within less than 6 months (e.g., within 3 months). In certain such embodiments, the lower limit is greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, or greater than 80%, or greater than 90%.

[0374] In certain embodiments, the invention is directed to the aforementioned methods having a combined vaccine efficacy against all four dengue serotypes in seronegative subjects with a two-sided 95% confidence interval, when measured against placebo in a subject population of at least 1,500 or at least 2,000 healthy subjects who are seronegative for all serotypes at baseline from about 30 days after the second dose of the dosing schedule through at least 12 months or 12-18 months (e.g., at 12 or 18 months) after the second dose of the dosing schedule, the lower limit is greater than 25% and the unit dose / tetravalent dengue virus composition or the placebo is administered at least twice within less than six months (e.g., within three months). In certain such embodiments, the lower limit is greater than 30%, greater than 40%, greater than 50%, or greater than 55%.

[0375] In certain embodiments, the invention is directed to the aforementioned methods having combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease with a two-sided 95% confidence interval, where the lower limit is greater than 25% when measured against placebo in a subject population of at least 1,500, or at least 2,000, or at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4-16 who are seronegative to all serotypes at baseline from 30 days after the last dose through 12-18 months (e.g., at 12 or 18 months) after the last dose, and where a reconstituted unit dose / tetravalent dengue virus composition as described herein or a placebo is administered at least twice, e.g., within less than six months (e.g., within three months). In certain such embodiments, the lower limit is greater than 30%, greater than 35%, greater than 40%, or greater than 45%.

[0376] In certain embodiments, the invention is directed to the aforementioned methods having combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease with a two-sided 95% confidence interval, where the lower limit is greater than 25% when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4-16 who are seropositive at baseline from 30 days after the last dose through 12-18 months (e.g., at 12 or 18 months) after the last dose, and where a reconstituted unit dose / tetravalent dengue virus composition as described herein or a placebo is administered at least twice, e.g., within less than six months (e.g., within three months). In certain such embodiments, the lower limit is greater than 40%, greater than 45%, greater than 50%, greater than 60%, or greater than 65%.

[0377] In certain embodiments, the invention is directed to the aforementioned methods having combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease with a two-sided 95% confidence interval, where the lower limit exceeds 25% when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4-16 years who are seropositive at baseline or seronegative to all serotypes at baseline from 30 days after the last dose through 12-18 months (e.g., at 12 or 18 months) after the last dose, and where a reconstituted unit dose / tetravalent dengue virus composition as described herein or a placebo is administered at least twice, e.g., within less than six months (e.g., within three months). In certain such embodiments, the difference between the lower limit provided by subjects who are seropositive at baseline and subjects who are seronegative to all serotypes at baseline is 15 percentage points or less.

[0378] In certain embodiments, the invention is directed to the aforementioned methods, in which the combined vaccine efficacy against all four dengue serotypes in seronegative subjects is greater than 30% when measured against a placebo in a subject population of at least 1,500, or at least 2,000, healthy subjects who are seronegative for all serotypes at baseline, from 30 days after the second administration through at least 12 months or 12-18 months (e.g., at 12 or 18 months) after the second administration, where the unit dose / tetravalent dengue virus composition or the placebo is administered at least twice within less than six months (e.g., within three months). In certain such embodiments, the combined vaccine efficacy against all four dengue serotypes in seronegative subjects is greater than 40%, greater than 50%, greater than 60%, greater than 65%, or greater than 70%.

[0379] In certain embodiments, the invention is directed to the aforementioned methods having combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease when measured against placebo in a subject population of at least 1,500, or at least 2,000, or at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4-16 who are seronegative to all serotypes at baseline from 30 days after the last dose to 12-18 months (e.g., at 12 or 18 months) after the last dose, where the reconstituted unit dose / tetravalent dengue virus composition described herein or placebo is administered at least twice, e.g., within less than six months (e.g., within three months). In certain such embodiments, the vaccine efficacy is greater than 30%, greater than 40%, greater than 50%, greater than 55%, greater than 60%, or greater than 65%.

[0380] In certain embodiments, the invention is directed to the aforementioned methods having combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4-16 who are seropositive at baseline from 30 days after the last dose through 12-18 months (e.g., at 12 or 18 months) after the last dose, where a reconstituted unit dose / tetravalent dengue virus composition described herein or a placebo is administered at least twice, e.g., within less than six months (e.g., within three months). In certain such embodiments, the vaccine efficacy is greater than 40%, greater than 50%, greater than 60%, greater than 65%, greater than 70%, or greater than 75%.

[0381] In certain embodiments, the invention is directed to the aforementioned methods having combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease with a two-sided 95% confidence interval, where the lower limit exceeds 25% when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4-16 years who are seropositive at baseline or seronegative to all serotypes at baseline from 30 days after the last dose through 12-18 months (e.g., at 12 or 18 months) after the last dose, and where a reconstituted unit dose / tetravalent dengue virus composition as described herein or a placebo is administered at least twice, e.g., within less than 6 months (e.g., within 3 months). In certain such embodiments, the difference between the lower limits provided by subjects who are seropositive at baseline and subjects who are seronegative to all serotypes at baseline is 15 percentage points or less, or 10 percentage points or less.

[0382] In certain embodiments, the invention is directed to the aforementioned methods having a combined vaccine efficacy against all four dengue serotypes with a two-sided 95% confidence interval, when measured against placebo in a subject population of at least 1,000 healthy subjects aged 4-5 years at randomization, regardless of baseline serostatus, from about 30 days after the second dose of the dosing schedule to at least 12 months or 12-18 months after the second dose of the dosing schedule (e.g., at 12 or 18 months), the lower limit is greater than 25%, and the unit dose / tetravalent dengue virus composition or the placebo is administered at least twice within less than 6 months (e.g., within 3 months). In certain such embodiments, the lower limit is greater than 30%, greater than 40%, or greater than 45%.

[0383] In certain embodiments, the invention is directed to the aforementioned methods, in which the combined vaccine efficacy against all four dengue serotypes is greater than 30% when measured against placebo in a subject population of at least 1,000 healthy subjects aged 4-5 years at randomization, regardless of baseline serostatus, from 30 days after the second administration through at least 12 months or 12-18 months after the second administration (e.g., at 12 or 18 months), wherein the unit dose / tetravalent dengue virus composition or the placebo is administered at least twice within less than 6 months (e.g., within 3 months). In certain such embodiments, the combined vaccine efficacy against all four dengue serotypes is greater than 40%, greater than 50%, greater than 60%, greater than 65%, or greater than 70%.

[0384] In certain embodiments, the invention is directed to the aforementioned methods having a combined vaccine efficacy against all four dengue serotypes with a two-sided 95% confidence interval, when measured against placebo in a subject population of at least 1,000 healthy subjects aged 6-11 years at randomization, regardless of baseline serostatus, from about 30 days after the second dose of the dosing schedule through at least 12 months or 12-18 months after the second dose of the dosing schedule (e.g., at 12 or 18 months), the lower limit is greater than 25% and the unit dose / tetravalent dengue virus composition or the placebo is administered at least twice within less than 6 months (e.g., within 3 months). In certain such embodiments, the lower limit is greater than 30%, greater than 40%, greater than 50%, greater than 60%, or greater than 70%.

[0385] In certain embodiments, the invention is directed to the aforementioned methods, in which the combined vaccine efficacy against all four dengue serotypes is greater than 30% when measured against placebo in a subject population of at least 1,000 healthy subjects aged 6-11 years at the time of randomization, regardless of baseline serostatus, from 30 days after the second administration through at least 12 months or 12-18 months (e.g., at 12 or 18 months) after the second administration, wherein the unit dose / tetravalent dengue virus composition or the placebo is administered at least twice within less than six months (e.g., within three months). In certain such embodiments, the combined vaccine efficacy against all four dengue serotypes is greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 75%, or greater than 80%.

[0386] In certain embodiments, the invention is directed to the aforementioned methods having a combined vaccine efficacy against all four dengue serotypes with a two-sided 95% confidence interval, when measured against placebo in a subject population of at least 1,000 healthy subjects aged 12-16 years at randomization, regardless of baseline serostatus, from about 30 days after the second dose of the dosing schedule until at least 12 months or 12-18 months after the second dose of the dosing schedule (e.g., at 12 or 18 months), the lower limit is greater than 25% and the unit dose / tetravalent dengue virus composition or the placebo is administered at least twice within less than 6 months (e.g., within 3 months). In certain such embodiments, the lower limit is greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 65%, or greater than 68%.

[0387] In certain embodiments, the invention is directed to the aforementioned methods, in which from 30 days after the second administration to at least 12 months or 12-18 months after the second administration (e.g., at 12 or 18 months), the combined vaccine efficacy against all four dengue serotypes is greater than 30% when measured against placebo in a subject population of at least 1,000 healthy subjects aged 12-16 years at the time of randomization, regardless of baseline serostatus, wherein the unit dose / tetravalent dengue virus composition or the placebo is administered at least twice within less than six months (e.g., within three months). In certain such embodiments, the combined vaccine efficacy against all four dengue serotypes is greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 75%, or greater than 80%.

[0388] In certain embodiments, the invention is directed to the aforementioned methods having vaccine efficacy against serotype 1 with a two-sided 95% confidence interval, where the lower limit is greater than 25% when measured against a placebo in a subject population of at least 5,000 healthy subjects, or at least 10,000 healthy subjects, or at least 15,000 healthy subjects, regardless of baseline serostatus, from about 30 days after the second dose of the dosing schedule to at least 12 months or 12-18 months (e.g., at 12 or 18 months) after the second dose of the dosing schedule, and where the unit dose / tetravalent dengue virus composition or the placebo is administered at least twice within less than six months (e.g., within three months). In certain such embodiments, the lower limit is greater than 30%, greater than 40%, or greater than 50%.

[0389] In certain embodiments, the invention is directed to the aforementioned methods having vaccine efficacy against serotype 1 in preventing virologically confirmable dengue disease with a two-sided 95% confidence interval, where the lower limit is greater than 25% when measured against placebo in a subject population of at least 1,500, or at least 2,000, or at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4-16, regardless of baseline serostatus, from 30 days after the last dose through 12-18 months (e.g., at 12 or 18 months), and where a reconstituted unit dose / tetravalent dengue virus composition described herein or a placebo is administered at least twice, e.g., within less than six months (e.g., within three months). In certain such embodiments, the lower limit is greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, or greater than 54%. In certain such embodiments, a population of at least 1,500 subjects is seronegative to all serotypes at baseline, with a lower limit greater than 35%. In certain such embodiments, the seronegative and seropositive populations each achieve vaccine efficacy against serotype 1 with a two-sided 95% confidence interval, with a lower limit within 10 percentage points.

[0390] In certain embodiments, the invention is directed to the aforementioned methods, in which vaccine efficacy against dengue serotype 1 is greater than 30% when measured against a placebo in a subject population of at least 5,000 healthy subjects, or at least 10,000 healthy subjects, or at least 15,000 healthy subjects, regardless of baseline serostatus, from 30 days after the second administration through at least 12 months or 12-18 months (e.g., at 12 or 18 months) after the second administration, wherein the unit dose / tetravalent dengue virus composition or the placebo is administered at least twice within less than 6 months (e.g., within 3 months). In certain such embodiments, vaccine efficacy against dengue serotype 1 is greater than 40%, greater than 50%, greater than 60%, greater than 65%, or greater than 70%.

[0391] In certain embodiments, the invention is directed to the aforementioned methods having vaccine efficacy against serotype 1 in preventing virologically confirmable dengue disease when measured against a placebo in a subject population of at least 1,500, or at least 2,000, or at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4-16, regardless of baseline serostatus, from 30 days after the last dose through 12-18 months (e.g., at 12 or 18 months), wherein the reconstituted unit dose / tetravalent dengue virus composition described herein or a placebo is administered at least twice, e.g., within less than 6 months (e.g., within 3 months). In certain such embodiments, the vaccine efficacy is greater than 40%, greater than 50%, greater than 60%, or greater than 65%. In certain such embodiments, the subject population of at least 1,500 is seronegative to all serotypes at baseline. In certain such embodiments, the seronegative and seropositive populations each achieve vaccine efficacy against serotype 1 within 5 percentage points.

[0392] In certain embodiments, the invention is directed to the aforementioned methods having vaccine efficacy against serotype 2 with a two-sided 95% confidence interval, where the lower limit is greater than 25% when measured against placebo in a subject population of at least 5,000 healthy subjects, or at least 10,000 healthy subjects, or at least 15,000 healthy subjects, regardless of baseline serostatus, from about 30 days after the second dose of the dosing schedule to at least 12 months or 12-18 months after the second dose of the dosing schedule (e.g., at 12 or 18 months), and where the unit dose / tetravalent dengue virus composition or the placebo is administered at least twice within less than 6 months (e.g., within 3 months). In certain such embodiments, the lower limit is greater than 30%, greater than 40%, greater than 50, greater than 60, greater than 70, greater than 80, or greater than 90%.

[0393] In certain embodiments, the invention is directed to the aforementioned methods having vaccine efficacy against serotype 2 in preventing virologically confirmable dengue disease with a two-sided 95% confidence interval, where the lower limit is greater than 25% when measured against placebo in a subject population of at least 1,500, or at least 2,000, or at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4-16, regardless of baseline serostatus, from 30 days after the last dose through 12-18 months (e.g., at 12 or 18 months), and where a reconstituted unit dose / tetravalent dengue virus composition as described herein or a placebo is administered at least twice, e.g., within less than 6 months (e.g., within 3 months). In certain such embodiments, the lower limit is greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 85%. In certain such embodiments, a population of at least 1,500 subjects is seronegative to all serotypes. In certain such embodiments, the seronegative and seropositive populations each achieve vaccine efficacy against serotype 2 with a two-sided 95% confidence interval, the lower limit within 5 percentage points.

[0394] In certain embodiments, the invention is directed to the aforementioned methods, in which vaccine efficacy against dengue serotype 2 is greater than 30% when measured against a placebo in a subject population of at least 5,000 healthy subjects, or at least 10,000 healthy subjects, or at least 15,000 healthy subjects, regardless of baseline serostatus, from 30 days after the second administration through at least 12 months or 12-18 months (e.g., at 12 or 18 months) after the second administration, wherein the unit dose / tetravalent dengue virus composition or the placebo is administered at least twice within less than 6 months (e.g., within 3 months). In certain such embodiments, vaccine efficacy against dengue serotype 2 is greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0395] In certain embodiments, the invention is directed to the aforementioned methods having vaccine efficacy against serotype 2 in preventing virologically confirmable dengue disease when measured against a placebo in a subject population of at least 1,500, or at least 2,000, or at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4-16, regardless of baseline serostatus, from 30 days after the last dose through 12-18 months (e.g., at 12 or 18 months), wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or a placebo is administered at least twice, e.g., within less than 6 months (e.g., within 3 months). In certain such embodiments, the vaccine efficacy is greater than 60%, greater than 70%, greater than 80%, or greater than 90%. In certain such embodiments, the subject population of at least 1,500 is seronegative to all serotypes at baseline. In certain such embodiments, the seronegative and seropositive populations each achieve vaccine efficacy against serotype 2 within 5 percentage points.

[0396] In certain embodiments, the invention is directed to the aforementioned method having vaccine efficacy against serotype 3 with a two-sided 95% confidence interval, the lower limit being greater than 25% when measured against placebo in a subject population of at least 5,000 healthy subjects, or at least 10,000 healthy subjects, or at least 15,000 healthy subjects, regardless of baseline serostatus, from about 30 days after the second dose of the dosing schedule to at least 12 months after the second dose of the dosing schedule, and the unit dose / tetravalent dengue virus composition or the placebo is administered at least twice within less than six months (e.g., within three months). In certain such embodiments, the lower limit is greater than 30% and greater than 40%.

[0397] In certain embodiments, the invention is directed to the aforementioned method, in which vaccine efficacy against dengue serotype 3 is greater than 30% when measured against placebo in a subject population of at least 5,000 healthy subjects, or at least 10,000 healthy subjects, or at least 15,000 healthy subjects, regardless of baseline serostatus, from 30 days after the second administration to at least 12 months after the second administration, where the unit dose / tetravalent dengue virus composition or the placebo is administered at least twice within less than six months (e.g., within three months). In certain such embodiments, vaccine efficacy against dengue serotype 3 is greater than 40%, greater than 50%, greater than 55%, or greater than 60%.

[0398] In certain embodiments, the invention is directed to the aforementioned methods having combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease with hospitalization with a two-sided 95% confidence interval, the lower limit being greater than 25% when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4-16, regardless of baseline serostatus, from the first dose to 12-18 months (e.g., at 12 or 18 months) after the last dose, or from 30 days after the last dose to 12-18 months (e.g., at 12 or 18 months), wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or a placebo is administered at least twice, e.g., within less than six months (e.g., within three months). In certain such embodiments, the lower limit is greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 66%, greater than 67%, greater than 70%, greater than 75%, greater than 77%, or greater than 80%.

[0399] In certain embodiments, the invention is directed to the aforementioned methods having combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease with hospitalization when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4-16, regardless of baseline serostatus, from the first dose to 12-18 months (e.g., at 12 or 18 months) after the last dose, or from 30 days after the last dose to 12-18 months (e.g., at 12 or 18 months), where a reconstituted unit dose / tetravalent dengue virus composition described herein or a placebo is administered at least twice, e.g., within less than six months (e.g., within three months). In certain such embodiments, the vaccine efficacy is greater than 70%, greater than 75%, greater than 80%, or greater than 82%, or greater than 85%, greater than 88%.

[0400] In certain embodiments, the invention is directed to the aforementioned method having combined vaccine efficacy against all four serotypes against virologically confirmed dengue with hospitalization with a two-sided 95% confidence interval, where the lower limit is greater than 25% when measured against placebo in a subject population of at least 1,500 or at least 2,000 healthy subjects who are seronegative for all serotypes at baseline from about 30 days after the second dose of the dosing schedule through at least 12 months or 12-18 months after the second dose of the dosing schedule (e.g., at 12 or 18 months), and the unit dose / tetravalent dengue virus composition or placebo is administered at least twice within less than six months (e.g., within three months). In certain such embodiments, the lower limit is greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, or greater than 75%.

[0401] In certain embodiments, the invention is directed to the aforementioned methods having combined vaccine efficacy against all four serotypes in preventing virologically confirmed dengue disease with hospitalization at a two-sided 95% confidence interval, where the lower limit is greater than 25% when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4-16 who are seronegative to all serotypes at baseline from 30 days after the last dose through 12-18 months (e.g., at 12 or 18 months) after the last dose, and where a reconstituted unit dose / tetravalent dengue virus composition as described herein or a placebo is administered at least twice, e.g., within less than six months (e.g., within three months). In certain such embodiments, the lower limit is greater than 60%, greater than 65%, greater than 66%, greater than 67%, greater than 70%, greater than 75%, greater than 77%, or greater than 80%.

[0402] In certain embodiments, the invention is directed to the aforementioned methods, in which the combined vaccine efficacy against virologically confirmed dengue with hospitalization against all four serotypes is greater than 30% when measured against placebo in a subject population of at least 1,500 or at least 2,000 healthy subjects who are seronegative for all serotypes at baseline from 30 days after the second administration through at least 12 months or 12-18 months (e.g., at 12 or 18 months) after the second administration, wherein the unit dose / tetravalent dengue virus composition or the placebo is administered at least twice within less than six months (e.g., within three months). In certain such embodiments, the combined vaccine efficacy against virologically confirmed dengue with hospitalization against all four serotypes is greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0403] In certain embodiments, the invention is directed to the aforementioned methods having combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease with hospitalization when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4-16 who are seronegative for all serotypes at baseline from 30 days after the last dose to 12-18 months (e.g., at 12 or 18 months) after the last dose, where a reconstituted unit dose / tetravalent dengue virus composition described herein or a placebo is administered at least twice, e.g., within less than six months (e.g., within three months). In certain such embodiments, the vaccine efficacy is greater than 60%, greater than 65%, greater than 66%, greater than 67%, greater than 70%, greater than 75%, greater than 77%, greater than 80, or greater than 85%.

[0404] In certain embodiments, the invention is directed to the aforementioned methods having combined vaccine efficacy against virologically confirmed dengue disease with hospitalization for all four serotypes with a two-sided 95% confidence interval, where the lower limit is greater than 25% when measured against placebo in a subject population of at least 1,500 or at least 2,000 healthy subjects who are seropositive at baseline from about 30 days after the second dose of the dosing schedule through at least 12 months or 12-18 months after the second dose of the dosing schedule (e.g., at 12 or 18 months), and where the unit dose / tetravalent dengue virus composition or the placebo is administered at least twice within less than six months (e.g., within three months). In certain such embodiments, the lower limit is greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, or greater than 80%.

[0405] In certain embodiments, the invention is directed to the aforementioned methods having combined vaccine efficacy against all four serotypes in preventing virologically confirmed dengue disease with hospitalization with a two-sided 95% confidence interval, where the lower limit is greater than 25% when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4-16 who are seropositive at baseline from 30 days after the last dose through 12-18 months (e.g., at 12 or 18 months) after the last dose, and where a reconstituted unit dose / tetravalent dengue virus composition as described herein or a placebo is administered at least twice, e.g., within less than six months (e.g., within three months). In certain such embodiments, the lower limit is greater than 60%, greater than 65%, greater than 70%, greater than 75%, or greater than 80%.

[0406] In certain embodiments, the invention is directed to the aforementioned methods, in which the combined vaccine efficacy against virologically confirmed dengue with hospitalization against all four serotypes is greater than 30% when measured against placebo in a subject population of at least 1,500 or at least 2,000 healthy subjects who are seropositive at baseline from 30 days after the second administration through at least 12 months or 12-18 months after the second administration (e.g., at 12 or 18 months), where the unit dose / tetravalent dengue virus composition or the placebo is administered at least twice within less than six months (e.g., within three months). In certain such embodiments, the combined vaccine efficacy against virologically confirmed dengue with hospitalization against all four serotypes is greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0407] In certain embodiments, the invention is directed to the aforementioned methods having combined vaccine efficacy against all four serotypes from 30 days after the last dose through 12-18 months (e.g., at 12 months or 18 months) after the last dose in preventing virologically confirmable dengue disease with hospitalization when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4-16 who are seropositive at baseline, wherein a reconstituted unit dose / tetravalent dengue virus composition described herein or a placebo is administered at least twice, e.g., within less than six months (e.g., within three months). In certain such embodiments, the vaccine efficacy is greater than 75%, greater than 70%, greater than 80%, greater than 85%, or greater than 90%.

[0408] In certain embodiments, the invention is directed to the aforementioned methods having combined vaccine efficacy against all four serotypes in preventing virologically confirmed dengue disease with hospitalization at a two-sided 95% confidence interval, where the lower limit exceeds 25% when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4-16 who are seropositive at baseline or seronegative to all serotypes at baseline from 30 days after the last dose through 12-18 months (e.g., at 12 or 18 months) after the last dose, and where a reconstituted unit dose / tetravalent dengue virus composition as described herein or a placebo is administered at least twice, e.g., within less than six months (e.g., within three months). In certain such embodiments, the difference between the lower limit provided by subjects who are seropositive at baseline and subjects who are seronegative to all serotypes at baseline is 15 percentage points or less.

[0409] In certain embodiments, the invention is directed to the aforementioned methods having combined vaccine efficacy against all four serotypes in preventing hospitalized virologically confirmed dengue disease when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4-16 who are seropositive at baseline or seronegative to all serotypes at baseline, from 30 days after the last dose to 12-18 months (e.g., at 12 months or 18 months) after the last dose, where a reconstituted unit dose / tetravalent dengue virus composition described herein or a placebo is administered at least twice, e.g., within less than six months (e.g., within three months). In certain such embodiments, the difference between vaccine efficacy provided by subjects who are seropositive at baseline and subjects who are seronegative to all serotypes at baseline is 10 percentage points or less, or 5 percentage points or less.

[0410] In certain embodiments, the invention is directed to the aforementioned methods having a relative risk, preferably a combined relative risk for all four serotypes, with a two-sided 95% confidence interval, where the upper limit is less than 0.75 when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects), regardless of baseline serostatus, until 30 days after the second administration, at least 12 months after the second administration, and where a reconstituted unit dose / tetravalent dengue virus composition described herein or placebo is administered at least twice within less than six months (e.g., within three months). In certain such embodiments, the upper limit is less than 0.70, less than 0.65, less than 0.60, less than 0.55, less than 0.50, less than 0.45, less than 0.40, less than 0.35, less than 0.30, or less than 0.28. Preferably, said reconstituted unit dose or placebo is administered subcutaneously within about three months, for example, on days 0 and 90.

[0411] In certain embodiments, the invention is directed to the aforementioned methods, in which the relative risk, preferably the combined relative risk for all four serotypes, is less than 0.70 when measured against a placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects), regardless of baseline serostatus, from 30 days after the second administration to at least 12 months after the second administration, and in which a reconstituted unit dose / tetravalent dengue virus composition described herein or a placebo is administered at least twice within less than six months (e.g., within three months). In certain such embodiments, the relative risk is less than 0.65, less than 0.60, less than 0.55, less than 0.50, less than 0.45, less than 0.40, less than 0.35, less than 0.30, less than 0.25, or less than 0.23. Preferably, said reconstituted unit dose or placebo is administered subcutaneously within about three months, for example, on days 0 and 90.

[0412] In certain embodiments, the present invention is directed to the aforementioned method, wherein virologically confirmable dengue disease occurs in less than 2.5% of subjects when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects), regardless of baseline serostatus, from 30 days after the second administration to at least 12 months or at least 18 months after the second administration, and wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or a placebo is administered at least twice within less than 6 months (e.g., within 3 months). In certain such embodiments, virologically confirmable dengue disease occurs in less than 2.0% of subjects, less than 1.5% of subjects, less than 1.0% of subjects, less than 0.8% of subjects, or less than 0.6% of subjects. Preferably, said reconstituted unit dose or placebo is administered subcutaneously within about 3 months, e.g., on days 0 and 90.

[0413] In certain embodiments, the invention is directed to the aforementioned method having a combined vaccine efficacy against all four serotypes with a two-sided 95% confidence interval of greater than 61.0%, or greater than 65.0%, or greater than 70.0%, or greater than 72.0%, when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) from endemic areas selected from the group consisting of subjects 4-16 years of age at randomization, regardless of baseline serostatus, from about 30 days after the last dose of the dosing schedule to at least 12 or 13 months after the last dose of the dosing schedule, and wherein the unit dose / tetravalent dengue virus composition or the placebo is administered at least twice within 6 months or less.

[0414] In certain embodiments, the invention is directed to the aforementioned method, wherein the combined vaccine efficacy against all four serotypes is greater than 66%, or greater than 70%, or greater than 75%, or greater than 77%, or greater than 80.0%, when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) from endemic areas selected from the group consisting of subjects 4-16 years of age at the time of randomization, regardless of baseline serostatus, from about 30 days after the last dose of the dosing schedule to at least 12 or 13 months after the last dose of the dosing schedule, wherein the unit dose / tetravalent dengue virus composition or the placebo is administered at least twice within 6 months or less.

[0415] In certain embodiments, the present invention is directed to the aforementioned method, wherein the combined vaccine efficacy against all four serotypes is measured from about 30 days after the last administration of the dosing schedule to 12 or 13 months after the last administration of the dosing schedule.

[0416] In certain embodiments, the present invention is directed to the aforementioned method, wherein the unit dose or the placebo is administered at least twice within three months, specifically at about day 1 and about day 90, and the combined vaccine efficacy against all four serotypes is measured from 30 days after the second administration to 12 or 13 months after the second administration of the dosing schedule.

[0417] In certain embodiments, the present invention is directed to the aforementioned methods being effective and safe. In some of these embodiments, the subject or subject population is under 9 years old, under 4 years old, or under 2 years old, or between 2 and 9 years old, or between 2 and 5 years old, or between 4 and 9 years old, or between 6 and 9 years old. Optionally, the subject is seronegative for all dengue serotypes.

[0418] In certain embodiments, the invention is directed to the aforementioned methods, wherein the relative risk of virologically confirmed dengue fever with hospitalization is 1 or less, or 0.8 or less, or 0.6 or less, when measured against a placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects). In some of these embodiments, the subject or subject population is under 9 years of age, under 4 years of age, or under 2 years of age, or between 2 and 9 years of age, or between 2 and 5 years of age, or between 4 and 9 years of age, or between 6 and 9 years of age. Optionally, the subject is seronegative for all dengue serotypes.

[0419] In certain embodiments, the invention is directed to the aforementioned methods, wherein the healthy subjects of the subject population are between 4 and 16 years old. In some such embodiments, the healthy subjects of the subject population are between 4 and 5 years old, between 6 and 11 years old, or between 12 and 16 years old.

[0420] In certain embodiments, the present invention is directed to the aforementioned method, wherein healthy subjects of the subject population are defined as being healthy in terms of the exclusion criteria set forth in Example 6.

[0421] In certain embodiments, the present invention is directed to the aforementioned method, wherein the healthy subjects of the subject population are from Asia Pacific or Latin America.

[0422] In certain embodiments, the present invention is directed to the aforementioned method, wherein the healthy subjects of the subject population are seropositive for at least one serotype. In other embodiments, the healthy subjects of the subject population are seronegative for all serotypes.

[0423] In certain embodiments, the present invention is directed to the aforementioned method, wherein the healthy subjects of the subject population are 4-5 years old and of Asia-Pacific origin, 6-11 years old and of Asia-Pacific origin, or 12-16 years old and of Asia-Pacific origin. In other embodiments, the healthy subjects of the subject population are 4-5 years old and of Latin American origin, 6-11 years old and of Latin American origin, or 12-16 years old and of Latin American origin.

[0424] In certain embodiments, the invention is directed to the aforementioned method, wherein the healthy subjects of the subject population are 4-5 years old and seropositive for at least one dengue serotype, 6-11 years old and seropositive for at least one dengue serotype, or 12-16 years old and seropositive for at least one dengue serotype. In other embodiments, the healthy subjects of the subject population are 4-5 years old and seronegative for all dengue serotypes, 6-11 years old and seronegative for all dengue serotypes, or 12-16 years old and seronegative for all dengue serotypes.

[0425] In certain embodiments, the invention is directed to the aforementioned method, wherein the healthy subjects of the subject population are from Asia Pacific or Central and South America and are seropositive for at least one dengue serotype at baseline, hi other embodiments, the healthy subjects of the subject population are from Asia Pacific or Central and South America and are seronegative for all dengue serotypes at baseline.

[0426] In certain embodiments, the invention is directed to the aforementioned method, wherein the healthy subjects of the target population are from Asia Pacific, are seropositive for at least one dengue serotype at baseline, and are 4-5 years old, 6-11 years old, or 12-16 years old. In other embodiments, the healthy subjects of the target population are from Asia Pacific, are seronegative for all dengue serotypes at baseline, and are 4-5 years old, 6-11 years old, or 12-16 years old. In yet other embodiments, the healthy subjects of the target population are from Central or South America, are seropositive for at least one dengue serotype at baseline, and are 4-5 years old, 6-11 years old, or 12-16 years old. In other embodiments, the healthy subjects of the target population are from the Americas, are seronegative for all dengue serotypes at baseline, and are 4-5 years old, 6-11 years old, or 12-16 years old.

[0427] In certain embodiments, the present invention is directed to the aforementioned method, wherein the healthy subjects of the target population have received prior vaccination against yellow fever. In other embodiments, the healthy subjects of the target population have not received prior vaccination against yellow fever. Prior vaccination refers to vaccination with the reconstituted unit dose described herein prior to the first vaccination. For example, for vaccine efficacy (VE) determined in Example 6 from 30 days after the second vaccination, prior vaccination against yellow fever is defined as vaccination against yellow fever that is performed before 30 days after the second vaccination.

[0428] In certain embodiments, the present invention is directed to the aforementioned method, wherein the healthy subjects of the subject population have been previously vaccinated against Japanese encephalitis. In other embodiments, the healthy subjects of the subject population have not been previously vaccinated against Japanese encephalitis.

[0429] In certain embodiments, the invention is directed to the aforementioned method, wherein healthy subjects in the subject population received Dengvaxia® within the dosing regimen described herein or within 4.5 years of administration of the first dose. In certain embodiments, the invention is directed to the aforementioned method, wherein the incidence of vaccine-associated serious adverse events is less than 0.1%.

[0430] In certain embodiments, the present invention is directed to the aforementioned method, wherein the incidence of vaccine-associated spontaneous adverse events occurring within 4 weeks of administration is less than 2%.

[0431] In certain embodiments, the present invention is directed to the aforementioned method, wherein the incidence of vaccine-associated unsolicited adverse events occurring within two weeks of administration is less than 35%.

[0432] In certain embodiments, the present invention is directed to the aforementioned method, wherein the incidence of vaccine-associated non-spontaneous local reactions occurring within one week of administration is less than 40%.

[0433] In certain embodiments, the present invention is directed to the aforementioned methods, which do not increase the risk of virologically confirmed dengue fever accompanied by hospitalization in individuals (e.g., seronegative individuals).

[0434] The above methods are also contemplated in the context of the use of unit dosages for use in such methods, or for use in the manufacture of a medicament for such methods.

[0435] In certain embodiments, a tetravalent dengue vaccine (eg, Dengvaxia®) is used to vaccinate against dengue disease. Dengvaxia® is a tetravalent dengue vaccine, CYD-TDV (Dengvaxia®, Sanofi Pasteur, Lyon, France), based on the yellow fever backbone, which has been licensed in several countries based on clinical demonstration of overall vaccine efficacy (VE) against virologically confirmed dengue fever (VCD) in 56-61% of children in Asia and Latin America (Capeding MR et al. Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in Asia: a phase 3, randomised, observer-masked, placebo-controlled trial. Lancet 2014,384:1358-65; Villar LA et al. Safety and immunogenicity of a recombinant tetravalent dengue vaccine in 9-16 year olds: a randomized, controlled, phase II trial in Latin America. Pediatr Infect Dis J 2013,32:1102-9). The preparation of these particular strains CYD1, CYD2, CYD3 and CYD4 is described in detail in International Patent Applications WO98 / 37911, WO03 / 101397, WO07 / 021672, WO08 / 007021, WO08 / 047023...

Claims

[Claim 1] The invention described in the specification.

Images