Methods for preventing dengue fever and hepatitis A

Abstract

The present invention relates to a method for preventing dengue disease and hepatitis A in a subject or population of subjects by concurrently administering unit doses of a dengue vaccine composition and a hepatitis A vaccine on the same day. The unit doses of the dengue vaccine composition contain constructs of each dengue serotype (e.g., TDV-1, TDV-2, TDV-3, and TDV-4) at various concentrations to improve protection from dengue infection.

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 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, namely DENV-1, DENV-2, DENV-3 and DENV-4, as well as an immune response 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 technologies 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, they are still sufficient to express the full repertoire of viral antigens and can 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 fever 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. Four serotypes of dengue virus have been identified so far: dengue-1 (DENV-1), dengue-2 (DENV-2), dengue-3 (DENV-3), and dengue-4 (DENV-4). Dengue virus serotypes 1 to 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. Dengue virus causes 50 to 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 these regions. 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 protect against secondary infection with one of the other three dengue virus serotypes. Indeed, previous infection with one dengue virus serotype can 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 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, randomized, 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 increase, rather than reduce, the risk of severe disease from dengue infection in individuals without previous dengue virus infection (seronegative populations). Therefore, Dengvaxia® is only recommended for use in individuals previously infected with at least one dengue virus serotype (seropositive populations). 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 to 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. SR H Adinegoro et al., in "Efficacy and Long-Term Safety of a Dengue Vaccine in Regions of Endemic Disease," in the New England Journal of Medicine, Vol. 373, page 1195, reported a pooled risk of hospitalization for virologically confirmed dengue disease in children under 9 years of age of 1.58, indicating an increased risk of severe dengue in vaccinated individuals. 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, young 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 vaccinations that reduce the risk of dengue after only one dose.

[0005] One additional 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] Therefore, there is 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, particularly for subjects under 9 years of age, and that 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, particularly 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 administration method that is safe and effective across a wide range of age groups, regardless of serostatus. There is a need for a dengue vaccine and corresponding administration method that avoids costly and time-consuming serostatus testing or seroprevalence studies. There is a need for a dengue vaccine and corresponding administration method 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 a vaccine that can be administered in fewer doses than the current Dengvaxia® schedule of three doses given six months apart, for example, a vaccine that can be administered in as few as two doses or even just one dose to be effective.

[0009] The above objectives are consistent with the WHO research priorities for dengue vaccines: WHO policy 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 uninfected (and unvaccinated) individuals ingest food or water contaminated with the feces of an infected individual. The disease is closely associated with unsafe water or food, inadequate sanitation, and poor personal hygiene. The virus can also be transmitted through close physical contact with an infected individual. 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 Shanghai epidemic that affected approximately 300,000 people. Hepatitis A virus persists in the environment and can withstand food preparation processes routinely used to inactivate and / or control bacterial pathogens. The disease can lead to serious economic and social consequences in local communities. It can take weeks or months for people who recover from the illness to return to work, school, or daily activities. The impact on food establishments where the virus is confirmed 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 traveling internationally has increased significantly in recent decades. According to the United Nations World Tourism Organization (UNWTO), more than 1.1 billion tourists traveled to foreign countries in 2014. The risk of contracting a disease during international travel depends on various factors, such as the region of the world visited, the length of the trip, and the variety of planned activities. Vaccination recommendations are an important part of health preparation before international travel. Vaccination against hepatitis A virus is generally recommended for travelers to at-risk regions around the world, including Asia, Africa, and Latin America.

[0013] For conventional hepatitis A vaccination, a two-dose schedule is recommended, particularly for travelers and immunocompromised individuals who are substantially at risk of contracting hepatitis A. However, in healthy individuals, comparable efficacy is achieved with a single dose. The vaccination schedule for children / young adults (12 months to 18 years of age) 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] The object of the present invention is 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 when subjects do not know their dengue and / or hepatitis A serostatus and the corresponding serologic tests are unavailable, impractical, or unreliable.

[0019] The objective of the present invention is to provide safe and effective protection against dengue disease and hepatitis A to travelers from countries where hepatitis A and dengue are not endemic, particularly travelers receiving vaccinations at 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 a corresponding method for preventing hepatitis A and dengue disease in subjects or subject populations from dengue endemic and non-dengue endemic areas, for subjects across a wide age range, particularly those 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] The object of the present invention is to provide a vaccine and a corresponding method for preventing hepatitis A and dengue diseases, which avoids testing the serostatus of individual dengue and / or hepatitis A subjects or subject populations individually before administering hepatitis A and dengue vaccines to them, or avoids analyzing the seroprevalence of dengue and / or hepatitis A in the subjects or subject populations 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 travel vaccines before a subject travels internationally to countries where HAV and dengue are endemic, and methods for safely administering these vaccines.

[0023] Therefore, the present invention is directed to methods for 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 unit doses of a hepatitis A vaccine and a dengue vaccine composition on the same day, wherein the unit doses include a tetravalent dengue virus composition comprising four live-attenuated dengue virus strains.

[0025] 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.

[0026] 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 (e.g., optionally after reconstitution) is administered to a subject. 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.

[0027] A "lyophilized unit dose" or a "unit dose in lyophilized form" refers to a unit dose obtained by subjecting a given volume, such as 0.5 mL, of a liquid dengue vaccine composition to lyophilization. Thus, an aqueous formulation of a dengue vaccine composition produced by combining a pharmaceutically acceptable excipient and a dengue virus composition containing four dengue virus strains, preferably TDV-1 to TDV-4, is subjected to lyophilization to obtain a lyophilized unit dose.

[0028] A "reconstituted unit dose" or "unit dose in reconstituted form" is obtained from a lyophilized dose by reconstitution with a pharmaceutically 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.

[0029] As used herein, the term "upon reconstitution 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 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 administering a total unit dose volume (e.g., 0.8 mL) will result in the same total amount of dengue virus being administered.

[0030] As used herein, "a concentration of at least X log 10 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 other than 0.5 mL, or is lyophilized from a volume other than 0.5 mL, or is reconstituted in a volume other than 0.5 mL, the concentration will be different from "a concentration of at least X log 10 pfu / 0.5 mL." However, if the unit dose volume 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 X log 10 pfu / 0.5 mL." Thus, while the concentration may vary, the total amount of virus in the unit dose remains the same.

[0031] As used herein, the term "dengue serotype" refers to a species of dengue virus that is defined by its cell surface antigens and therefore 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).

[0032] 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.

[0033] 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 with portions from more than one dengue serotype, or a mixed chimeric dengue virus with portions from other flaviviruses.

[0034] A "virus strain," and specifically a "dengue virus strain," is a genetic subtype of 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 antigen. 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 with portions from more than one dengue serotype.

[0035] 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.

[0036] A "non-chimeric dengue virus" or "non-chimeric dengue serotype strain" or "non-chimeric dengue strain" contains portions derived from only one dengue serotype. Specifically, a non-chimeric dengue virus does not contain portions derived from a different flavivirus (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.

[0037] A "chimeric dengue virus" or "chimeric dengue serotype strain" or "chimeric dengue strain" contains portions derived from at least two different dengue serotypes. As used herein, a chimeric dengue virus does not contain 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 contain 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 containing 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 described in more detail below. As used herein, a "chimeric dengue serotype 2 / 3 strain" or "DENV-2 / 3 chimera" or "TDV-3" refers to a dengue virus chimeric construct containing portions derived from both DENV-2 and DENV-3. Specifically, in a chimeric dengue serotype 2 / 3 strain, the prM and E proteins derived from DENV-3 replace the prM and E proteins derived from DENV-2, as described in more detail below. As used herein, a "chimeric dengue serotype 2 / 4 strain" or "DENV-2 / 4 chimera" or "TDV-4" refers to a dengue virus chimeric construct containing portions derived from both DENV-2 and DENV-4. Specifically, in a chimeric dengue serotype 2 / 4 strain, the prM and E proteins derived from DENV-4 replace the prM and E proteins derived from DENV-2, as described in more detail below. A mixed chimeric dengue virus has portions from other flaviviruses.

[0038] 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, which express 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 the nucleotide sequence set forth in SEQ ID NO: 1 and / or the amino acid sequence set forth in SEQ ID NO: 2. In one embodiment, TDV-2 has the nucleotide sequence set forth in SEQ ID NO: 3 and / or the amino acid sequence set forth in SEQ ID NO: 4. In one embodiment, TDV-3 has the nucleotide sequence set forth in SEQ ID NO: 5 and / or the amino acid sequence set forth in SEQ ID NO: 6. In one embodiment, TDV-4 has the nucleotide sequence set forth in SEQ ID NO: 7 and / or the amino acid sequence set forth in SEQ ID NO: 8.

[0039] As used herein, the term "dengue fever disease" refers to a disease caused by infection with the dengue virus. Symptoms of dengue fever disease include sudden high fever, headache, joint and muscle pain, nausea, vomiting, and skin rash. The term 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, they are classified as having DSS. Symptoms of DSS include bleeding, which can appear as small dots of blood on the skin and large patches of blood under the skin. Sustained shock is a major factor associated with complications, including massive gastrointestinal bleeding, which can lead to death. As used herein, a DHF case is defined as a VCD case that meets 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 (e.g., hepatomegaly and acute renal failure).

[0040] 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 achieved 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.

[0041] As used herein, the terms "virologically confirmed dengue fever," "VCD case," or "VCD fever" refer 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 fever" 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 the WHO 1997 criteria. Severe dengue fever was defined through the evaluation of an independent Dengue Case Adjudication Committee, which evaluates all hospitalized VCD cases (severe / non-severe) based on the criteria redefined in the Charter. All non-hospitalized cases are considered non-severe.

[0042] As used herein, the term "fever illness" is defined as a body temperature of 38°C or higher on any two out of three consecutive days.

[0043] As used herein, the term "virologically confirmed dengue disease with hospitalization" is considered an alternative term for severe dengue, and the "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 1 or less, the vaccine confers a risk of virologically confirmed dengue disease with hospitalization equal to or less than that of placebo and is considered "safe." In this context, the risk of virologically confirmed dengue disease accompanied by 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 from 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 old, the age group of subjects 4 to less than 9 years old, the age group of subjects 2 to less than 9 years old, the age group of subjects 4 to 5 years old, the age group of subjects 6 to 11 years old, and the age group of subjects 12 to 16 years old.

[0044] 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. Particularly considered "safety" is the combined vaccine efficacy against virologically confirmed dengue disease with hospitalization for all four serotypes with a two-sided 95% confidence interval, particularly when the unit dose or the placebo is administered at least twice within less than six months (e.g., within three months), from approximately 30 days after the first dose or the second or last dose in the dosing schedule to at least 12 months, 12-18 months, 12 months, or 18 months after the second or last dose in the dosing schedule, particularly. , 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 (particularly when measured in age groups selected from the age groups of subjects 4 to 16 years old, 4 to less than 9 years old, 2 to less than 9 years old, 4 to 5 years old, 6 to 11 years old, and 12 to 16 years old). Particularly, the lower limit can 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 for combined 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 through 12-18 months after the last dose of the dosing schedule.

[0045] A vaccine is considered safe within the meaning of the present invention if it satisfies one of the criteria defined above for the term "safe." In this context, safety specifically 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 to determine the occurrence of previous 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, specifically for 4-60 years or 4-16 years, preferably regardless of serostatus. Relevant subgroups in this context are under 9 years of age, 2 to under 9 years of age, 4 to under 9 years of age, 4 to 5 years of age, 6 to 11 years of age, and any age group within the range of 12 to 16 years of age or 4 to 16 years of age. Further definitions of VE for virologically confirmed dengue disease with hospitalization are mentioned below in the disclosure regarding certain treatment methods.

[0046] As used herein, "vaccine efficacy" or "VE" measures the proportional reduction in cases among 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 among vaccinated people relative to unvaccinated people. The higher the percentage reduction in disease among the vaccinated group, the higher the vaccine efficacy. For example, a VE of 90% indicates a 90% reduction in disease incidence among 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 (λ) divided by the placebo hazard rate (λ), i.e., HR=λ / λ. λ represents the hazard rate among subjects vaccinated with the tetravalent dengue vaccine composition disclosed herein, and λ represents the hazard rate among unvaccinated subjects, i.e., subjects given a placebo. Hazard rate ratios (HRs) are estimated from Cox proportional hazards models using the test vaccine as a factor, adjusted for age, and stratified by region. As used herein, the term "combined vaccine efficacy against all four serotypes" is defined as vaccine efficacy with respect to the 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 its combined efficacy is greater than 30%. In this context, combined vaccine efficacy can be 40% or greater, 50% or greater, 60% or greater, 70% or greater, 72% or greater, or 80% or greater, particularly when determined in an age group selected from the age groups of subjects 4 to 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, and 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 of age, regardless of serostatus, and specifically for 4-60 years or 4-16 years of age, regardless of serostatus. Relevant subgroups in this context are <9 years of age, 2 to <9 years of age, 4 to <9 years of age, 4 to <9 years of age, 4 to 5 years of age, 6 to 11 years of age, and 12 to 16 years of age, or any age group within the range of 4 to 16 years of age. In certain embodiments, "effective" means providing a combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease within a two-sided 95% confidence interval, with a lower limit of combined vaccine efficacy 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 through 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 the particular serotype 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.

[0047] 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 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.

[0048] 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. Primary vaccination is defined as a primary administration schedule for administering a composition or unit dose disclosed herein to establish a protective immune response, consisting of, for example, two administrations, e.g., 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. Booster vaccination refers to an administration or administration schedule that occurs at least one year, or 4 to 4.5 years, or even 5 or 10 years after the last administration, e.g., the second administration, of the primary vaccination schedule after the primary vaccination. Booster administration attempts to enhance or re-establish the immune response of the primary vaccination.

[0049] As used herein, the term "subject(s)" is limited to a human subject (e.g., an infant, a child, or an adult). The term "elderly subject(s)" refers to a subject over 60 years of age, e.g., 61-100 years of age, 61-90 years of age, 61-80 years of age, 61-75 years of age, or 61-70 years of age.

[0050] As used herein, a "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 subjects and the serostatus of the subjects, whether the subjects are from a dengue-endemic or non-dengue-endemic area.

[0051] As used herein, an "endemic area" refers to an area where a disease or infectious agent is consistently present and / or typically prevalent in the population within that area. As used herein, a "non-endemic area" refers to an area where a disease is not present or typically prevalent. Thus, a "dengue-endemic area" refers to a geographic area where dengue virus infections are consistently maintained at a baseline level. A "non-dengue-endemic area" is a geographic area where dengue virus infections are not consistently maintained at a baseline level. Thus, a subject population or subject "from a dengue-endemic area" or "from a dengue-non-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 regularly published, for example, by the WHO or CDC. Typical dengue-endemic areas are found in Central and South America, Southeast Asia, and the Pacific Islands. Dengue-endemic countries include, but are not limited to, Australia, Brazil, Bangladesh, Colombia, China, the Dominican Republic, Indonesia, India, Mexico, Malaysia, Nicaragua, Nigeria, Pakistan, Panama, the Philippines, Puerto Rico, Singapore, Sri Lanka, Thailand, and Vietnam. The infectivity of an area is measured by seroprevalence surveys, which provide a seroprevalence rate. Areas with extremely high infectivity are considered to have a seroprevalence rate of over 80%. As used herein, the term "area," when referring to seroprevalence, refers to a geographic area where seroprevalence can be determined or is known, such as a village, town, city, region, county, state, district, or part of any of the above, or an entire country.

[0052] As used herein, "serostatus" refers to the amount of antibodies a subject has against a particular infectious agent, specifically dengue virus. As used herein, "seronegative" or "seronaive" means that a subject does not have neutralizing antibodies in their serum against 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 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. Baseline serostatus refers to the serostatus prior to administration of the dengue vaccine composition described herein.

[0053] 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 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, "the ratio of the neutralizing antibody titer of dengue serotype 2 to the neutralizing antibody titer of dengue serotype 4 is 20 or less" means that the ratio obtained by dividing the neutralizing antibody titer of dengue serotype 2 by the neutralizing antibody titer of dengue serotype 4 is 20 or less. That is, the dengue serotype 2 neutralizing antibody titer is no more than 20 times as high as the dengue serotype 4 neutralizing antibody titer in the subject.

[0054] 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 for dengue serotype 2 to GMT for dengue serotype 4 of 20 or less" means that the ratio obtained by dividing the geometric mean neutralizing antibody titer for dengue serotype 2 (GMT DENV-2) by the geometric mean neutralizing antibody titer for dengue serotype 4 (GMT DENV-4) is 20 or less. That is, the geometric mean neutralizing antibody titer for dengue serotype 2 is 20-fold or less higher than the geometric mean neutralizing antibody titer for dengue serotype 4 in the subject population.

[0055] 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 peptides derived from dengue virus proteins. 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).

[0056] As used herein, a "balanced immune response" means that the immune response to the four dengue serotypes is sufficient to confer protection against infection by all four dengue serotypes, and preferably the immune responses to the four dengue serotypes are of similar strength. Specifically, the neutralizing antibody titers to 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.

[0057] 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 log pfu / 0.5ml, dengue serotype 2 at a concentration of 4.00 log pfu / 0.5ml, dengue serotype 3 at a concentration of 4.60 log pfu / 0.5ml, and dengue serotype 4 at a concentration of 5.11 log pfu / 0.5ml. Typically, the logarithm of the concentration is converted to a numerical value. This conversion results in a 4 x 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 yields pfu / 0.5ml.

[0058] The "percentage concentration" 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 / 0.5ml ÷ 1.84 × 10 5 pfu / 0.5ml) x 100 = 2% Serotype 2 concentration percentage = (1 x 10 4 pfu / 0.5ml ÷ 1.84 × 10 5 pfu / 0.5ml) x 100 = 5% Serotype 3 concentration percentage = (4 x 10 4 pfu / 0.5ml ÷ 1.84 × 10 5 pfu / 0.5ml) x 100 = 22% Serotype 4 concentration percentage = (1.3 x 10 5 pfu / 0.5ml ÷ 1.84 × 10 5 pfu / 0.5ml) x 100 = 71%. Concentration percentages are rounded to the nearest whole number.

[0059] 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 conventional 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.

[0060] 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 administration schedule.

[0061] As used herein, the term "chronic disease or condition" includes diseases and conditions that persist for three months or longer in an elderly subject. Specifically, it includes diabetes, hypertension, allergies, previous stroke, ischemic heart disease, chronic kidney disease, and chronic obstructive pulmonary disease.

[0062] 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 aged less than 60 years. 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.

[0063] As used herein, an "unspontaneous systemic adverse event" in children under 6 years of age is defined as fever, irritability / tantrums, drowsiness, and loss of appetite occurring within 14 days after each vaccination, and in children 6 years of age and older is defined as fever, headache, asthenia, malaise, and muscle pain occurring within 14 days after each vaccination.

[0064] As used herein, an "unspontaneous local adverse event" is injection site pain, injection site erythema, or injection site swelling occurring within 7 days after each vaccination.

[0065] As used herein, a "spontaneous adverse event" is any adverse event (AE) that is not an involuntary local or systemic AE as defined above.

[0066] 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 extension of existing hospitalization, results in persistent or significant disability / incapacity, is a congenital anomaly / birth defect, or is medically significant for reasons other than those defined above.

[0067] The relationship of each AE, including involuntary systemic AEs (involuntary local AEs thought 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 determining 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.

[0068] 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.

[0069] 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, one value is 1% and the second value is 6%, for example.

[0070] As used herein, the term "determination of previous dengue infection in the subject prior to administration" means that previous dengue infection must be assessed prior to vaccination by an appropriately validated serological test, for example, 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.

[0071] As used herein, % w / v refers to % mg / ml, for example, 150 mg / ml is 15% w / v.

[0072] As used herein, the term "hepatitis A virus" may be abbreviated as "HAV."

[0073] As used herein, the term "placebo" may be abbreviated as "Pbo."

[0074] 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 antibody in serum. Quantitatively, a subject's hepatitis A seronegativity is defined as an anti-hepatitis A antibody level <10mIU / ml. When anti-hepatitis A antibody is determined by ELISA, the lower level of quantification is 12.5mIU / ml, which is the practical lower level of anti-HAV antibody 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 antibody is disclosed, for example, in Beck et al. J Travel Med 2004;11:201-207.

[0075] As used herein, "at baseline" refers to the last time a subject's serostatus was measured prior to the first vaccination.

[0076] As used herein, the unit "mIU / ml" refers to milli-international units per millimeter. This concentration unit refers to the amount of anti-hepatitis A antibodies in a subject's serum (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). According to this WHO recommendation (see WHO Information Sheet "Observed Rate of Vaccine Reactions - Hepatitis A Vaccine" published in June 2012), 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 F.E., Hepburn A., D'Hondt E., "Inactivated candidate vaccines for hepatitis", A. Prog Med Virol 1990;37:72-95.

[0077] 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 calculations (Morrison D et al., J Infect Dis. 2010;201(3):370-7).

[0078] 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 seroprotection rate is based on measurements 30 days after simultaneous administration on day 1 and 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 with administration of either dengue vaccine or hepatitis A vaccine alone.

[0079] 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.

[0080] 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.

[0081] As used herein, the term "synergism" or "synergy" is defined as the effect of simultaneous administration on the same day of unit doses of a hepatitis A vaccine and a dengue vaccine composition to a subject or population of subjects, which administration results in higher anti-hepatitis A antibody concentrations and / or higher mean titers of neutralizing antibodies for each serotype of dengue virus than simultaneous administration on the same day of the corresponding hepatitis A vaccine and placebo, and / or simultaneous administration on the same day of a unit dose of a dengue vaccine composition and placebo (monoadministration). Thus, higher antibody concentrations after simultaneous administration compared to monoadministration are an indication in favor of simultaneous administration. [Brief explanation of the drawings]

[0082] [Figure 1] Genetic structure of the four dengue strains contained in TDV. The open red triangles indicate three attenuating mutations present in the 5'NCR, NS1, and NS3 proteins. 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. [Figure 2] Schematic diagram showing the microneutralization test (MNT) used to determine the titer of neutralizing antibodies. [Figure 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. [Figure 5] Study design for the 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 INVENTION

[0083] Dengue virus strains Dengue virus is a single-stranded, positive-sense RNA virus in the Flaviviridae family. The taxonomy 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.

[0084] [Table 1]

[0085] The flavivirus genome, from 5' to 3' direction (see Figure 1): -5'-non-coding region (5'-NCR), - the capsid protein (C) coding region, - the premembrane protein (prM) coding region, - envelope protein (E) coding region, - the region encoding the nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) and -3' non-coding region (3'-NCR).

[0086] 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.

[0087] 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, specifically 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 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 includes three chimeric dengue strains derived from the TDV-2 strain by

[0088] 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).

[0089] The attenuated DEN-2 strain PDK-53 (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): 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

[0090] 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.

[0091] [Table 2]

[0092] In one embodiment, the TDV-2 contains, in addition to the three attenuating mutations: a) Adenine to thiazolinone at nucleotide 524 in the prM gene Min and a mutation to asparagine at position 143 acida mutation resulting in an amino acid change from α- to valine, and / or b) Cytosine to thiazolinone at nucleotide 2055 in the E gene Min and / or a silent mutation to c) Cytosine to thiazolinone at nucleotide 4018 in the NS2A gene Min resulting in an amino acid change from leucine to phenylalanine at position 1308; and / or d) The sequence at nucleotide 5547 in the NS3 gene Min a silent mutation from to cytosine, 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) The nucleotide 900 in the prM gene Min a silent mutation from α to cytosine. The cytosine at nucleotide 8571 in the NS5 gene of DEN-2 PDK-53 Min Silent mutations to are not present in TDV-2 strains.

[0093] 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 and a lysine to glutamine mutation at position 166 acid and / or h) The mutation in the NS5 gene from adenine to guanine at nucleotide 8803, resulting in an amino acid change from isoleucine to valine at position 2903.

[0094] 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), and most preferably mutations a) through h). The nucleotide and amino acid positions of TDV-2 refer to the nucleotide sequence set forth in SEQ ID NO:3 and the amino acid sequence set forth in SEQ ID NO:4.

[0095] The structural envelope (E) and premembrane (prM) proteins of dengue virus have been identified as primary antigens that elicit neutralizing and protective antibody responses (Plotkin 2001). To generate a tetravalent dengue vaccine (TDV), standard molecular genetic engineering techniques (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 those encoding the corresponding wild-type prM and E glycoproteins 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).

[0096] [Table 3]

[0097] A diagram of the four TDV strains included in the dengue vaccine composition is shown in Figure 1 .

[0098] 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.

[0099] In one embodiment, the TDV-1 contains, in addition to the three attenuating mutations: c) Cytosine to thiazolinone at nucleotide 4018 in the NS2A gene Min resulting in an amino acid change from leucine to phenylalanine at position 1308; and / or d) The nucleotide sequence at nucleotide 5547 in the NS3 gene Min a silent mutation from to cytosine, 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) TCR at nucleotide 1575 in the E gene Min a silent mutation from to cytosine, 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) The junction site between the prM-E gene and the DEN-2 PDK-53 backbone at nucleotides 2381 / 2382 Min - a guanine to cytosine-cytosine mutation resulting in an amino acid change at position 762 from valine to alanine.

[0100] In another embodiment, the TDV-1 contains, 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) Adenine to thiazolinone at nucleotide 4407 in the NS2B gene Min and a glutamine at position 1437 acid From asparagine acid and / or n) a silent adenine to guanine mutation at nucleotide 7311 in the NS4B gene.

[0101] 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 TDV-1 nucleotide and amino acid positions refer to the nucleotide sequence set forth in SEQ ID NO: 1 and the amino acid sequence set forth in SEQ ID NO: 2.

[0102] In one embodiment, the TDV-3 contains, in addition to the three attenuating mutations: c) Cytosine to thiazolinone at nucleotide 4012 in the NS2A gene Min resulting in an amino acid change from leucine to phenylalanine at position 1306; and / or d) The nucleotide sequence at nucleotide 5541 in the NS3 gene Min a silent mutation from to cytosine, 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) The junction site between the prM-E gene and the DEN-2 PDK-53 backbone at nucleotides 2375 / 2376 Min a guanine to cytosine-cytosine mutation resulting in an amino acid change from valine to alanine at position 760, and / or o) Cytosine to thiazolinone at nucleotide 552 in the prM gene Min and / or a silent mutation to p) Adenine to thiazolinone at nucleotide 1970 in the E gene Min which results in an amino acid change at position 625 from histidine to leucine.

[0103] In another embodiment, the TDV-3 contains, in addition to the three attenuating mutations: q) Adenine to thiazolinone at nucleotide 1603 in the E gene Min and / or r) a silent mutation from adenine to guanine at nucleotide 7620 in the NS5 gene.

[0104] In another embodiment, the 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 set forth in SEQ ID NO:5 and the amino acid sequence set forth in SEQ ID NO:6.

[0105] In one embodiment, the TDV-4 contains, in addition to the three attenuating mutations: c) Cytosine to thiazolinone at nucleotide 4018 in the NS2A gene Min resulting in an amino acid change from leucine to phenylalanine at position 1308; and / or d) The sequence at nucleotide 5547 in the NS3 gene Min a silent mutation from to cytosine, 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) The junction site between the prM-E gene and the DEN-2 PDK-53 backbone at nucleotides 2381 / 2382 Min a guanine to cytosine-cytosine mutation 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) Cytosine to thiazolinone at nucleotide 2027 in the E gene Min 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, which results in an amino acid change from methionine to leucine at position 727.

[0106] In another embodiment, the TDV-4 contains, in addition to the three attenuating mutations: w) Adenine to thiazolinone at nucleotide 225 in the C gene Min and / or a silent mutation to x) a mutation from adenine to guanine at nucleotide 3674 in the NS2A gene and an asparagine at position 1193 acid a mutation resulting in an amino acid change from α- to glycine, and / or y) A mutation from adenine to adenine / guanine mix at nucleotide 3773 in the NS2A gene and a lysine to lysine / guanine mutation at position 1226 Rugini mutations that result in amino acid changes to the nucleotide sequence, and / or z) Cytosine to thiazolinone at nucleotide 5391 in the NS3 gene Min and / or a silent mutation to aa) Cytosine to thiazolinone at nucleotide 6437 in the NS4A gene Minresulting in an amino acid change at position 2114 from alanine to valine; and / or bb) The nucleotide sequence at nucleotide 7026 in the NS4B gene Min Karachi Min / silent mutations to cytosine mixes, and / or cc) a silent adenine to cytosine mutation at nucleotide 9750 in the NS5 gene.

[0107] 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 set forth in SEQ ID NO:7 and the amino acid sequence set forth in SEQ ID NO:8.

[0108] 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.

[0109] [Table 4]

[0110] 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 particularly 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.

[0111] 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 are 6 nucleotides and 2 amino acids fewer than the original TDV-2 nucleotide and amino acid positions, respectively.

[0112] 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 pharmaceutically acceptable excipient.

[0113] 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 log 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 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.

[0114] 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 log pfu / 0.5 mL to 3.8 log 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.

[0115] 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 log pfu / 0.5 mL; (ii) dengue serotype 2 strains at a concentration of at least 2.7 log pfu / 0.5 mL; (iii) 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 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.

[0116] In one embodiment, the dengue vaccine composition comprises four live attenuated dengue virus strains: (i) a chimeric dengue serotype 2 / 1 strain at a concentration of at least 3.3 log pfu / 0.5 mL to 3.8 log pfu / 0.5 mL; (ii) dengue serotype 2 strains at a concentration of at least 2.7 log pfu / 0.5 mL; (iii) a chimeric dengue serotype 2 / 3 strain at a concentration of at least 4.0 log 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.

[0117] 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.

[0118] 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 log pfu / 0.5 mL to 5.3 log pfu / 0.5 mL; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 2.7 log pfu / 0.5 mL to 5.0 log pfu / 0.5 mL; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log pfu / 0.5 mL to 6.0 log pfu / 0.5 mL; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 4.5 log pfu / 0.5 mL to 6.5 log pfu / 0.5 mL.

[0119] 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 log pfu / 0.5 mL to 5.0 log pfu / 0.5 mL; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 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 log pfu / 0.5 mL to 5.7 log pfu / 0.5 mL; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 4.5 log pfu / 0.5 mL to 6.2 log pfu / 0.5 mL.

[0120] 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 log pfu / dose to 5.0 log pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) have a concentration of 2.7 log pfu / dose to 4.9 log pfu / dose; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log pfu / dose to 5.7 log 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.

[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 log pfu / dose to 4.1 log pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) have a concentration of 2.7 log pfu / dose to 3.6 log pfu / dose; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log pfu / dose to 4.7 log pfu / dose; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have concentrations of 4.5 log10 pfu / dose to 5.3 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 strain) has a concentration of 3.3 log pfu / 0.5 mL to 3.6 log pfu / 0.5 mL; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 2.7 log pfu / 0.5 mL to 4.0 log pfu / 0.5 mL; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log pfu / 0.5 mL to 4.6 log pfu / 0.5 mL; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 4.5 log pfu / 0.5 ml or 4.6 log pfu / 0.5 ml to 5.1 log pfu / 0.5 ml.

[0123] 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 log pfu / 0.5 mL to 4.4 log pfu / 0.5 mL; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 3.7 log pfu / 0.5 mL to 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 log pfu / 0.5 mL to 5.0 log pfu / 0.5 mL; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have a concentration of 5.5 log pfu / 0.5 mL to 5.6 log pfu / 0.5 mL.

[0124] 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 log pfu / 0.5 mL; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strain) has a concentration of 5.6 log10 pfu / 0.5 mL.

[0125] 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) has a concentration of 4.6 log pfu / 0.5 mL; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strain) has a concentration of 5.1 log10 pfu / 0.5 mL.

[0126] In another preferred embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, wherein the arithmetic sum of all four serotypes is less than 6.7 log pfu / 0.5 mL, preferably less than 5.5 log pfu / 0.5 mL. In certain such embodiments, the arithmetic sum of all four serotypes is at least 4.6 log 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, wherein the arithmetic sum of all four serotypes is within the range of 4.6 log pfu / 0.5 mL to 6.7 log pfu / 0.5 mL, preferably within the range of 4.6 log pfu / 0.5 mL to 5.5 log pfu / 0.5 mL.

[0127] Preferably, in the aforementioned embodiments, 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.

[0128] 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 log 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) 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.

[0129] 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 log pfu / dose to 5.3 log pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has 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 log pfu / dose to 6.0 log pfu / dose; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have concentrations of 4.5 log10 pfu / dose to 6.5 log10 pfu / dose.

[0130] 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 log pfu / dose to 5.0 log pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) have a concentration of 2.7 log pfu / dose to 4.9 log pfu / dose; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log pfu / dose to 5.7 log pfu / dose; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have concentrations of 4.5 log10 pfu / dose to 6.2 log10 pfu / dose.

[0131] 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 log pfu / dose to 5.0 log pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) have a concentration of 2.7 log pfu / dose to 4.9 log pfu / dose; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log pfu / dose to 5.7 log 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.

[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 log pfu / dose to 4.1 log pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) have a concentration of 2.7 log pfu / dose to 3.6 log pfu / dose; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.0 log pfu / dose to 4.7 log pfu / dose; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have concentrations of 4.5 log10 pfu / dose to 5.3 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 log pfu / dose to 3.6 log pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has 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 log pfu / dose to 4.6 log pfu / dose; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have concentrations of 4.5 log pfu / dose, 4.6 log pfu / dose, to 5.1 log pfu / dose.

[0134] 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 log pfu / dose to 4.4 log pfu / dose; (ii) dengue serotype 2 (e.g., dengue serotype 2 strains) has a concentration of 3.7 log pfu / dose to 3.8 log pfu / dose; (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains) have a concentration of 4.5 log pfu / dose to 5.0 log pfu / dose; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) have concentrations of 5.5 log10 pfu / dose to 5.6 log10 pfu / dose.

[0135] 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 / 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 log pfu / dose; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strain) has a concentration of 5.6 log10 pfu / dose.

[0136] 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 / 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) has a concentration of 4.6 log pfu / dose; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strain) has a concentration of 5.1 log10 pfu / dose.

[0137] In another preferred embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, wherein the arithmetic sum of all four serotypes is less than 6.7 log pfu / dose, preferably less than 5.5 log pfu / dose. In certain such embodiments, the arithmetic sum of all four serotypes is at least 4.6 log pfu / dose. In a preferred embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains, wherein the arithmetic sum of all four serotypes is in the range of 4.6 log pfu / dose to 6.7 log pfu / dose, preferably in the range of 4.6 log pfu / dose to 5.5 log pfu / dose.

[0138] 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.

[0139] 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) in pfu / 0.5 mL is less than 10%, the concentration of (iv) in pfu / 0.5 mL is at least 50%, the concentration of (i) in pfu / 0.5 mL is at least 1%, and the concentration of (iii) in 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%.

[0140] The concentration of (iii) in pfu / 0.5 mL in the reconstituted unit dose is preferably at least 10%.

[0141] 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%.

[0142] Preferably, in the aforementioned embodiments, 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.

[0143] The concentrations of different dengue viruses are preferably determined by immunofocus assays known in the art. For example, 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 allowing infectious virus to bind and be internalized by the cells, an overlay containing a viscosity-increasing agent, such as agarose or carboxymethylcellulose, is added to prevent viral spread so that progeny viruses can only infect cells adjacent to the original infected cell. After an incubation period to allow 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. Bright spots are stained by adding an appropriate substrate for the enzyme conjugated to the secondary antibody, such as 5-bromo-4-chloro-3-indolylphosphate / nitroblue tetrazolium phosphatase substrate. The number of plaques on the plate corresponds to the plaque-forming units of 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.

[0144] 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, to achieve 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.

[0145] 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, and based on that total concentration, 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%, or at least 60%, or at least 65% of the total concentration. In one embodiment, the concentration of dengue serotype 2 (e.g., dengue serotype 2 strains) measured in pfu / 0.5 mL is 0.3 to 10% or 0.5 to 8% of the total concentration, and the concentration of dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) measured in pfu / 0.5 mL is 50% to 90% or 60% to 88% of the total concentration. This means that the concentration of dengue serotype 2 (e.g., dengue serotype 2 strains) is lower than the concentration of dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains).

[0146] 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% to 7%, or 2% to 6%, or 2.0% to 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% to 25%, or 7% to 25%, or 10% to 25%, or 18% to 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).

[0147] 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% of the total concentration, preferably less than 6% or less than 2%; 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% of the total concentration, preferably at least 65%; 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% of the total concentration, preferably between 1% and 7% or 2.0% and 5.0%; 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% of the total concentration, preferably between 6% and 25%, or 10% and 25%, or 18% and 25%.

[0148] 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 the pfu / 0.5 mL of dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strains) is measured. 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%, 10% and 25%, or 18% and 25%. Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) is particularly preferred, having the highest concentration of all four dengue serotypes.

[0149] 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% to 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 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% to 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.

[0150] 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 account for 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 the concentration 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.

[0151] Preferably, in the aforementioned embodiments, 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.

[0152] 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.

[0153] 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 determining the concentration of the virus strain in the composition in pfu / ml. The volume and / or amount per unit dose is described in the respective chapter.

[0154] Pharmaceutically acceptable excipients The present invention is directed, in part, to a unit dose of a dengue vaccine composition, the dengue vaccine composition comprising one or more pharmaceutically 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.

[0155] In one embodiment, the unit dose of the dengue vaccine composition comprises one or more of the following pharmaceutically acceptable excipients: 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.

[0156] In one embodiment, a unit dose of the dengue vaccine composition, when measured in 0.5 ml, comprises the following pharmaceutically acceptable excipients: 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 - containing about 70 mM to 140 mM sodium chloride, preferably It has a pH of -7 to 8.5.

[0157] In one embodiment, a unit dose of the dengue vaccine composition, when measured in 0.5 ml, comprises the following pharmaceutically acceptable excipients: 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, about 0.88% mg / ml to about 1.32 mg / ml human serum albumin, and - containing about 70 mM to 140 mM sodium chloride, preferably It has a pH of -7 to 8.5.

[0158] In a preferred embodiment, the lyophilized unit dose of the invention described herein is prepared in the following pharmaceutically 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.

[0159] In a preferred embodiment, the lyophilized unit dose of the invention described herein contains, when measured in 0.5 ml, the following pharmaceutically acceptable excipients: 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.

[0160] In a preferred embodiment, the lyophilized unit dose of the invention described herein is prepared in the following pharmaceutically acceptable excipients: approximately 82.9 mg of α,α-trehalose dihydrate, -Approximately 5 mg of poloxamer 407, approximately 0.5 mg of human serum albumin, and - about 50 μmol of sodium chloride.

[0161] In a preferred embodiment, the reconstituted unit dose of the invention described herein is prepared using the following pharmaceutically 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 - approximately 137 mM sodium chloride, preferably Has a pH of -7 to 8.5

[0162] In a preferred embodiment, the reconstituted unit dose of the invention described herein contains, when measured in 0.5 ml, the following pharmaceutically acceptable excipients: about 15% w / v α,α-trehalose, approximately 1% w / v of poloxamer 407, about 0.1% w / v human serum albumin, and preferably - approximately 137 mM sodium chloride, preferably It has a pH of -7 to 8.5.

[0163] In a preferred embodiment, the reconstituted unit dose of the invention described herein is prepared using the following pharmaceutically acceptable excipients: approximately 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.

[0164] The human serum albumin can be natural or recombinant human serum albumin (rHSA). The poloxamer 407 can be, for example, Pluronic F127.

[0165] In one embodiment, the unit dose further comprises a buffer solution. The buffer solution may be phosphate buffered saline (PBS). The buffer solution 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 solution may comprise disodium hydrogen phosphate (Na2HPO4), potassium chloride (KCl), and potassium dihydrogen phosphate (KH2PO4). The buffer solution may have a pH in the range of 7.0 to 8.5 at 25°C.

[0166] 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 pharmaceutically acceptable excipient as described herein.

[0167] The present invention relates, in part, to, for example, (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) with a concentration of at least 3.3 log 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) A unit dose of the above-described 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.

[0168] 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.

[0169] In one embodiment, the unit dose is lyophilized. In one such embodiment, the lyophilized unit dose is obtained by lyophilizing a 0.5 mL volume of an aqueous dengue vaccine composition produced by combining a pharmaceutically acceptable excipient described herein with a dengue vaccine composition described herein that includes four dengue virus strains, specifically TDV-1 through TDV-4. In a preferred embodiment, the residual moisture content, as determined by Karl Fischer measurement, is 5.0% or less, preferably 3% or less.

[0170] In another embodiment, the unit dose is reconstituted. The reconstituted unit dose is obtained by subjecting the lyophilized unit dose to reconstitution with a pharmaceutically acceptable diluent, preferably prior to administration of the dengue vaccine. In one such embodiment, reconstitution is achieved by adding a pharmaceutically acceptable diluent, such as water for injection, phosphate-buffered saline, or aqueous sodium chloride solution, to the lyophilized unit dose. In one embodiment, aqueous sodium chloride solution (e.g., 37 mM aqueous 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 to 0.8 mL, 0.4 to 0.7 mL, or 0.5 mL of diluent. In a preferred embodiment, the lyophilized unit dose is reconstituted with 0.3 to 0.8 mL, 0.4 to 0.7 mL, or 0.5 mL of 37 mM aqueous sodium chloride solution. In a more preferred embodiment, the lyophilized unit dose is reconstituted with 0.5 mL of 37 mM aqueous sodium chloride solution. The reconstituted unit dose can then be administered subcutaneously.

[0171] The lyophilized form of the unit dose is the final product and storage form of the unit dose after manufacturing of the unit dose, and the reconstituted form of the unit dose is preferably prepared prior to administration of the unit dose to a subject.

[0172] The invention further comprises, in part, 1. A unit dose of a dengue vaccine composition comprising a tetravalent virus composition comprising four live attenuated dengue virus strains, wherein the unit dose is lyophilized and, upon reconstitution with 0.5 mL of a pharmaceutically 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 log pfu / 0.5 ml.

[0173] In one embodiment, the volume of the reconstituted unit dose is, for example, 0.5 mL, and upon reconstitution with a pharmaceutically acceptable diluent, (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.

[0174] In another embodiment, the volume of the reconstituted unit dose is, for example, 0.5 mL, and upon reconstitution with a pharmaceutically 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 6%, at least 8%, or at least 10%, or at least 12%, or at least 14%, or at least 16%, or at least 18%.

[0175] The concentration of (iii) in pfu / 0.5 mL in the reconstituted unit dose is preferably at least 10%.

[0176] In one embodiment, the volume of the reconstituted unit dose is, for example, 0.5 mL, and upon reconstitution with a pharmaceutically 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 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%.

[0177] In one embodiment, the invention is directed to a lyophilized unit dose of a dengue vaccine composition that, upon reconstitution with 0.5 mL of a pharmaceutically acceptable diluent, comprises dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain) having a concentration of at least 3.3 log pfu / 0.5 mL, dengue serotype 2 (e.g., a dengue serotype 2 strain) having a concentration of at least 2.7 log pfu / 0.5 mL, 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 dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) having a concentration of at least 4.5 log pfu / 0.5 mL, and a pharmaceutically 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.

[0178] 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 log pfu / dose to 5.0 log pfu / 0.5 mL, dengue serotype 2 (e.g., a dengue serotype 2 strain) at a concentration of 2.7 log pfu / dose to 4.9 log pfu / 0.5 mL, dengue serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain) at a concentration of 4.0 log pfu / dose to 5.7 log pfu / 0.5 mL, and dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) at a concentration of 4.5 log pfu / dose to 5.5 log pfu / 0.5 mL, and a pharmaceutically 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.

[0179] In one such embodiment, the lyophilized unit dose comprises a dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain) at a concentration of 3.3 log pfu / 0.5 mL to 3.6 log pfu / 0.5 mL, a dengue serotype 2 (e.g., a dengue serotype 2 strain) at a concentration of 2.7 log pfu / 0.5 mL to 4.0 log pfu / 0.5 mL, a dengue serotype 3 (e.g., a dengue serotype 3 strain) at a concentration of 4.0 log pfu / 0.5 mL to 4.6 log pfu / 0.5 mL, or a dengue serotype 4 (e.g., a dengue serotype 5 strain) at a concentration of 5.0 log pfu / 0.5 mL to 5.6 log pfu / 0.5 mL. The vaccine is obtained by lyophilizing 0.5 mL of a dengue vaccine composition containing dengue serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain) at a concentration of 0.5 mL and dengue serotype 4 (e.g., a 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 pharmaceutically 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 certain embodiments, a lyophilized unit dose refers to 0.5 mL before 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 TDV-2 concentration, measured in pfu / 0.5 mL, is less than 10%, or less than 8%, or less than 6%, and the TDV-4 concentration, measured in pfu / 0.5 mL, is at least 50% or at least 65%. In some of these embodiments, the TDV-1 concentration, measured in pfu / 0.5 mL, is at least 1%, and / or the TDV-3 concentration, measured in pfu / 0.5 mL, is at least 6%, 7%, 8%, 10%, 12%, 14%, 16%, or at least 18%.

[0181] 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%.

[0182] 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., dengue serotype 2 / 1 strain) measured in pfu / 0.5 mL is 1% to 7% of the total concentration, the concentration of dengue serotype 2 (e.g., 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., 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., 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 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.

[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 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 account for 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 present at 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 present at 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 the concentration 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.

[0184] A lyophilized unit dose 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 pharmaceutically acceptable diluents, such as aqueous sodium chloride solution, without changing the absolute amount of virus administered or the ratio of viruses to each other.

[0185] In certain embodiments, the lyophilized unit doses of the present invention are prepared from a solution comprising a non-reducing sugar, a surfactant, a protein, and an inorganic salt.

[0186] In certain embodiments, the lyophilized unit doses of the present invention are prepared from a solution comprising trehalose, poloxamer 407, human serum albumin, and sodium chloride.

[0187] In certain embodiments, lyophilized unit doses of the invention are prepared from a solution containing 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.

[0188] 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.

[0189] In one embodiment, the solution from which the lyophilized unit dose is prepared further comprises a buffer solution. The buffer solution may be phosphate buffered saline (PBS). The buffer solution 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 solution may comprise disodium hydrogen phosphate (Na2HPO4), potassium chloride (KCl), and potassium dihydrogen phosphate (KH2PO4). The buffer solution may have a pH ranging from about 7.0 to about 8.5 at 25°C, or a pH ranging from about 6.8 to about 7.6 at 25°C, preferably about 7.2 at 25°C.

[0190] 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.

[0191] 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 therefore protects both dengue-seronegative and dengue-seropositive subject populations or subjects against dengue disease.

[0192] 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 the contents of a single container, preferably a vial, can vaccinate multiple subjects. 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.

[0193] In certain embodiments, the container containing the unit dose of the invention is part of a kit. Accordingly, 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 pharmaceutically acceptable diluent for reconstitution.

[0194] In certain embodiments, the reconstitution diluent is provided in a container, preferably a vial, or a pre-filled 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.

[0195] 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 present in separate containers, e.g., vials. In other embodiments, the hepatitis A vaccine and the unit dose of the invention may be present 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.

[0196] Hepatitis A vaccine In certain embodiments, the hepatitis A vaccine is an inactivated hepatitis A vaccine.

[0197] In certain embodiments, the Hepatitis A vaccine comprises a Hepatitis A virus derived from Hepatitis A virus strain HM-175.

[0198] 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.

[0199] 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.

[0200] 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 at a concentration of 0.2-0.8% w / v and / or the polysorbates are present at a concentration of 0.01-0.09 mg / ml.

[0201] 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.

[0202] In certain embodiments, the Hepatitis A vaccine is contained in a 1 ml or 0.5 ml dose of liquid.

[0203] An example of such a hepatitis A vaccine is GlaxoSmithKline's HAVRIX®, a sterile suspension of inactivated virus for intramuscular administration. HAVRIX® uses the hepatitis A virus strain HM-175, which is derived from wild-type hepatitis A virus (HAV) HM-175, the complete nucleotide sequence of which is disclosed on pages 50-59 of Cohen et al., Journal of Virology, Vol. 61, No. 1 (published January 1987). (Specifically, the complete nucleotide sequence of wild-type hepatitis A virus HM-175 is shown in Figure 1 of that publication.)

[0204] The virus (HM175 strain) is propagated 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 to a standard using an enzyme-linked immunosorbent assay (ELISA) and is therefore expressed in terms of ELISA units (ELU). Each 1 mL dose of vaccine for adults (≥ 18 years) contains 1440 ELU 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 ELU 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), which are present in the cell growth medium. HAVRIX® is formulated without preservatives.

[0205] VAQTA®, a hepatitis A vaccine manufactured by Merck Sharp & Dohme Corp., is an inactivated whole virus vaccine derived from hepatitis A virus grown in cell culture in human MRC-5 diploid fibroblasts. The vaccine contains inactivated virus from a strain originally derived by 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 contains 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 nonviral protein, 4×10 -6 < μg DNA, 10 -4 It contains less than 1 μg of bovine albumin and less than 0.8 μg of formaldehyde. Chemical residues from other processes, 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.

[0206] 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 RI per 0.5 mL dose throughout the product's lifespan. 10 It is formulated to contain at least pfu.

[0207] 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).

[0208] In certain embodiments, the present invention provides a combination vaccine composition, wherein the dengue vaccine composition comprises a tetravalent live attenuated dengue virus strain: (i) dengue serotype 1, preferably at a concentration of at least 3.3 log 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 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.

[0209] In certain embodiments, the present invention provides a combination vaccine composition, wherein the dengue vaccine composition comprises a tetravalent live attenuated dengue virus strain: (i) a chimeric dengue serotype 2 / 1 strain at a concentration of at least 3.3 log pfu / 0.5 mL to 3.8 log pfu / 0.5 mL; (ii) dengue serotype 2 strains at a concentration of at least 2.7 log pfu / 0.5 mL; (iii) a chimeric dengue serotype 2 / 3 strain at a concentration of at least 4.0 log 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 log pfu / 0.5 mL or at least 4.6 log pfu / 0.5 mL, optionally up to 6.2 log pfu / 0.5 mL.

[0210] Preferably, in the aforementioned embodiments, 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.

[0211] In certain embodiments, the present invention is directed to a combination vaccine composition, wherein the dengue vaccine composition, upon reconstitution with a pharmaceutically 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%.

[0212] In certain embodiments, the present invention is directed to a combination vaccine composition, wherein the dengue vaccine composition comprises one or more pharmaceutically 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.

[0213] Additionally, any vaccine excipient or combination thereof known to those skilled in the art (e.g., those disclosed in WO2018 / 027075A1) can be used in the combination vaccine composition.

[0214] In one embodiment, the unit dose of the dengue vaccine composition comprises one or more of the following pharmaceutically acceptable excipients: 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.

[0215] 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 tetravalent dengue vaccine CYD-TDV (Dengvaxia®; Sanofi Pasteur, Lyon, France), containing a mixed chimeric dengue virus based on the yellow fever backbone, 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, randomized, 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 set out in Table 16 of this reference.

[0216] In certain embodiments, 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 each of the live attenuated chimeric dengue viruses of serotypes 1 to 4 contained in a CYD dosage form (eg, Dengvaxia®) is preferably equal.

[0217] In such an embodiment, CYD-TDV is dissolved / soluble in a solution containing 0.4% NaCl.

[0218] In certain embodiments, the present invention is directed to a combination vaccine composition, wherein 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 TV005 is similar to TV003, except that the concentration of rDEN2 / 4Δ30 in TV005 is 4 log 10The difference is that the vaccines TV003 and TV005 are PFU. Vaccines TV003 and TV005, 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 TV003 or TV005 vaccine components: deletion of the 3' untranslated region and chimerization of structural genes. For example, component rDEN4Δ30 contains all of the structural and nonstructural proteins of wild-type DENV-4, but is attenuated by a 30-nucleotide deletion in the 3' untranslated region (designated "Δ30"). The other vaccine component is also attenuated by a 30-nucleotide deletion in the 3' untranslated region. Additionally, rDEN3Δ30 / 31 contains a 31-nucleotide deletion in the 3' untranslated region (detailed in Figure lc 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 dengue strains that can be used to produce TV003 or TV005 are available under the Genbank accession numbers in Table A of WO2008 / 022196A1.

[0219] In certain embodiments, the present invention is directed to a combination vaccine composition, wherein the hepatitis A vaccine is an inactivated hepatitis A vaccine.

[0220] In certain embodiments, the Hepatitis A vaccine comprises a Hepatitis A virus derived from Hepatitis A virus strain HM-175.

[0221] 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.

[0222] In certain embodiments, the present invention is directed to a combination vaccine composition in which an inactivated hepatitis A virus is adsorbed to an aluminum carrier. In some of these embodiments, the aluminum is aluminum hydroxide or aluminum hydroxyphosphate sulfate.

[0223] In certain embodiments, the present invention is directed to a combination vaccine composition, wherein 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 at a concentration of 0.2-0.8% w / v and / or the polysorbate is present at a concentration of 0.01-0.09 mg / ml.

[0224] 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 ranges from 500 EL.U. to 900 EL.U. In further embodiments, the concentration ranges from 200 to 400 EL.U.

[0225] 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.

[0226] In certain embodiments, the combination vaccine composition is provided in a single vial in liquid or dehydrated form (eg, lyophilized form).

[0227] 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.

[0228] The present invention is also directed, in part, to methods of administering any of the above combination vaccine compositions to a subject or population of subjects.

[0229] In certain embodiments, the present invention is also directed to such methods wherein the combination vaccine composition is administered subcutaneously or intramuscularly.

[0230] Method for preventing dengue disease and hepatitis A, corresponding uses and corresponding kits The present invention is directed to methods for preventing Hepatitis A and Dengue fever diseases.

[0231] The present invention is directed, in part, to a method for preventing hepatitis A and dengue disease in a subject or population of subjects, comprising the simultaneous administration on the same day of unit doses of a hepatitis A vaccine (e.g., HAVRIX® or VAQTA®) and a dengue vaccine composition, wherein the unit dose comprises a tetravalent dengue virus composition comprising four live-attenuated dengue virus strains.

[0232] 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.

[0233] In certain embodiments, the Hepatitis A vaccine (eg, HAVRIX®) is derived from the Hepatitis A virus strain HM-175.

[0234] In certain embodiments, the present invention is directed to the aforementioned method, wherein a 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.

[0235] 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.

[0236] In certain embodiments, the present invention is directed to the aforementioned method, wherein the Hepatitis A vaccine (e.g., HAVRIX®) comprises 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.

[0237] 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.

[0238] 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 pharmaceutically acceptable diluent, comprises: (i) for chimeric dengue serotype 2 / 1 strains, a concentration of at least 3.3 log 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;

[0239] According to some of these embodiments, the dengue vaccine composition, upon reconstitution with a pharmaceutically 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%.

[0240] 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 subjects are seronegative for hepatitis A at baseline.

[0241] In certain embodiments, the present invention is directed to the aforementioned methods, wherein a unit dose of an invention described herein and a Hepatitis A vaccine (e.g., HAVRIX® or VAQTA®) is administered on day 0 / 1.

[0242] In certain embodiments, the present invention is directed to the aforementioned method, 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 injections are 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 in different anatomical locations (e.g., opposite arms).

[0243] In certain embodiments, the present invention is directed to the aforementioned method, wherein two unit doses of a 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 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, and may act as a booster.

[0244] 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 after said first co-administration, for example, 3 months later, preferably on day 90.

[0245] In certain embodiments, the present invention is directed to the aforementioned method, wherein a reconstituted unit dose of an 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 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.

[0246] In certain embodiments, the present invention is directed to the aforementioned method, 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.

[0247] 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.

[0248] 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 to 12 months or 6 to 18 months, such as 9 months (e.g., on day 270), after the first administration of the hepatitis A vaccine.

[0249] In certain embodiments, the present invention is directed to the aforementioned method, wherein a reconstituted unit dose of an 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 the age of 17, or over the age of 18, or between 18 and 60 years of age. In further embodiments, the subject or subject population is an adult over the age of 21, 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 other embodiments, 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.

[0250] In certain embodiments, the invention is directed to the aforementioned method, wherein the method does not include a step of determining whether the subject population or subjects have previously been infected with dengue and / or previously infected with hepatitis A prior to administering the hepatitis A vaccine and the unit dose of the dengue vaccine composition, or wherein the hepatitis A serostatus and / or dengue serostatus of the subject population or subjects is unknown prior to administering 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 subjects have previously been infected with dengue and / or previously infected with hepatitis A, or wherein the hepatitis A serostatus and / or dengue serostatus of the subject population or subjects is unknown prior to, during, or after the step of administering the unit dose of the hepatitis A vaccine and the dengue vaccine composition.

[0251] In certain embodiments, the present invention is directed to the aforementioned method, which comprises 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 hepatitis A vaccine within 6 to 18 months of administration of the first unit dose.

[0252] In certain embodiments, the present invention is directed to the aforementioned method, which comprises 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 tetravalent 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, step (A) of selecting a subject is performed without determining whether the subject has previously been infected with hepatitis A.

[0253] In certain embodiments, the invention is directed to the aforementioned method, wherein upon reconstitution of the unit dose with a pharmaceutically acceptable diluent, (i), (ii), (iii), and (iv) provide a total concentration of 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%.

[0254] In certain embodiments, the method results in compatibility between the dengue vaccine composition and the hepatitis A vaccine, specifically meaning that the immune response after co-administration is not inferior compared to a single administration of these vaccines.

[0255] In certain embodiments, the method results in synergy between the dengue vaccine composition and the hepatitis A vaccine, specifically meaning that the immune response after co-administration is better than when one or both vaccines are administered alone.

[0256] In certain embodiments, the 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 concurrently on the same day, and the control subject population receives hepatitis A vaccine and placebo doses concurrently on the same day.

[0257] 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 who receive 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).

[0258] In certain embodiments, the present invention relates to the aforementioned method, which results in a difference in hepatitis A seroprotection rate relative to single-agent administration of hepatitis A, wherein the difference is 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 simultaneously on the same day (day 0 / 1); b) a control population of at least 30 or 50 subjects receiving the Hepatitis A vaccine and placebo simultaneously on the same day (day 0 / 1); The difference between the hepatitis A seroprotection rate in the control subject population 30 days after administration (day 0 / 1) and the hepatitis A seroprotection rate in the subject population 30 days after administration (day 0 / 1) is determined; Methods are eligible for inclusion if the upper limit of the difference within a two-sided 95% confidence interval is less than 10%.

[0259] 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) for a subject population of at least 30 or at least 50 healthy subjects who are seronegative for hepatitis A at baseline and who receive 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.

[0260] In certain embodiments, the invention relates to the aforementioned method, which results in a difference in hepatitis A seroprotection rate relative to hepatitis A single agent administration, wherein the difference is 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, 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; 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 population of at least 30 or at least 50 healthy subjects receiving the hepatitis A vaccine and placebo simultaneously on the same day (day 0 / 1), the control 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 in the control subject population 30 days after administration (day 0 / 1) and the hepatitis A seroprotection rate in the subject population 30 days after administration (day 0 / 1) is determined; Methods are eligible for inclusion if the upper limit of the difference within a two-sided 95% confidence interval is less than 10%.

[0261] 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 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 who receive unit doses of the hepatitis A vaccine and the dengue vaccine composition simultaneously on the same day results in an anti-hepatitis A virus antibody geometric mean concentration (GMC) of at least 70 mIU / ml, or at least 80 mIU / ml, or at least 90 mIU / ml 30 days after administration (day 0 / 1).

[0262] An ELISA for determining anti-hepatitis A antibodies is disclosed, for example, in Beck et al. J Travel Med 2004;11:201-207.

[0263] In certain embodiments, the present invention is directed to the aforementioned method, wherein co-administration of unit doses of a hepatitis A vaccine and a dengue vaccine composition to a subject or population of subjects results in no serious adverse events associated with the co-administration, and no deaths associated with the co-administration.

[0264] In certain embodiments, the present invention relates to the aforementioned method, wherein the geometric mean titer (GMT) of neutralizing antibodies is measured by the following MNT: - 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, resulting in a 30-day survival benefit.

[0265] 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).

[0266] 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 unit doses of a hepatitis A vaccine and a dengue vaccine composition on the same day, 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 dose defined above.

[0267] In one embodiment of the invention, the method is directed to administering a hepatitis A vaccine together with another dengue vaccine (eg, Dengvaxia®) on the same day and at the same time. Dengvaxia®, a 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 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, randomized, 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 set out in Table 16 of this reference.

[0268] In one such embodiment, the method comprises vaccination comprising the steps of: (A) selecting a subject in need of protection against dengue fever infection and hepatitis A infection to receive equal doses of a CYD-TDV composition (e.g., Dengvaxia®) and a hepatitis A vaccine (e.g., HAVRIX® or VAQTA®); 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 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.

[0269] In certain embodiments, the subject is between 2 and 60 years old.

[0270] 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.

[0271] 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 1-4 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.

[0272] 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 TV005 is similar to TV003, except that the concentration of rDEN2 / 4Δ30 in TV005 is 4 log 10 The difference is that the vaccines TV003 and TV005 are PFU. Vaccines TV003 and TV005, 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 TV003 or TV005 vaccine components: deletion of the 3' untranslated region and chimerization of structural genes. For example, component rDEN4Δ30 contains all of the structural and nonstructural proteins of wild-type DENV-4, but is attenuated by a 30-nucleotide deletion in the 3' untranslated region (designated "Δ30"). The other vaccine component is also attenuated by a 30-nucleotide deletion in the 3' untranslated region. Additionally, rDEN3Δ30 / 31 contains a 31-nucleotide deletion in the 3' untranslated region (detailed in Figure lc 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 dengue strains that can be used to produce TV003 or TV005 are available under the Genbank accession numbers in Table A of WO2008 / 022196A1.

[0273] In certain embodiments, the present invention is directed to the aforementioned method, wherein a unit dose 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® disclosed herein, and a hepatitis A vaccine disclosed herein are administered to a subject or population of subjects simultaneously on the same day.

[0274] In certain embodiments, the present invention is directed to the aforementioned method, wherein a unit dose 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 disclosed herein are administered to a subject or population of subjects on the same day and simultaneously as a first dose on day 0 / 1, followed by administration of Dengvaxia® disclosed herein as a second dose within three months of the first dose, e.g., on day 90 after the first dose. 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 dose, 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 dose within 3 months of the first dose, e.g., on day 90 after the first dose.

[0275] In certain embodiments, the present invention is directed to the aforementioned method, wherein a unit dose 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 disclosed herein, and a hepatitis A vaccine disclosed herein are administered to a subject or population of subjects simultaneously on the same day.

[0276] In certain embodiments, the present invention is directed to the aforementioned method, wherein a subject or population of subjects is administered a unit dose disclosed herein, particularly 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 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 TV003 or TV005 disclosed herein as a second administration within 3 months of the first administration, for example, 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.

[0277] The above methods are contemplated in the context of the use of a unit dose of a 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 a dengue vaccine for the manufacture of a medicament for such methods of preventing dengue disease and hepatitis A.

[0278] The present invention further provides a kit for treating Hepatitis A and Dengue fever, comprising: For kits containing a box 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, wherein the unit dose comprises a tetravalent dengue virus composition comprising four live attenuated dengue virus strains.

[0279] Methods and uses for preventing dengue and yellow fever disease The present invention is directed, in part, to methods of preventing dengue fever disease and yellow fever disease in a subject. Accordingly, in certain embodiments, the present invention is directed to a method of preventing dengue fever 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 concomitant administration to the subject of a yellow fever vaccine (particularly, YF-17D).

[0280] The present invention is directed, in part, to methods of preventing dengue and yellow fever disease in a subject population. Accordingly, 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 reconstituted unit dose of the invention described herein, the method further comprising preventing yellow fever in the subject population by concomitant administration to the subject population of a yellow fever vaccine (specifically, YF-17D).

[0281] In certain embodiments, the present invention is directed to the aforementioned methods, wherein the unit dose of an invention described herein and a 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 an invention described herein and a yellow fever vaccine (particularly, YF-17D) is sequential, 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 a dengue vaccine described herein.

[0282] In certain embodiments, the present invention is directed to the aforementioned methods, wherein a reconstituted unit dose of an 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 an invention described herein and the subcutaneous injection of a yellow fever vaccine (particularly YF-17D) are administered in different anatomical locations, e.g., in opposite arms, particularly when the vaccines are administered simultaneously.

[0283] 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 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 to 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.

[0284] 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, e.g., three months, preferably 90 days, after administering the 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.

[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: - On day 0, the first administration of the first reconstituted unit dose; - a second administration of a second reconstituted unit dose after said first administration of the reconstituted unit dose, e.g., 3 months later, preferably at 90 days, 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.

[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, co-administration of a first reconstituted unit dose and the yellow fever vaccine; and - A second administration of a second reconstituted unit dose after said co-administration, for example, 3 months later, preferably on day 90.

[0287] In a preferred embodiment, the unit dose of the yellow fever vaccine and the invention described herein are administered simultaneously on day 0 or simultaneously on day 90.

[0288] 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 an invention described herein is administered subcutaneously to the subject or population of subjects, a yellow fever vaccine (particularly, 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.

[0289] In certain embodiments, the present invention is directed to the aforementioned methods, wherein the unit dose and yellow fever vaccine (particularly YF-17D) of the invention described herein is administered to a subject or population of subjects from a dengue-endemic area. In certain embodiments, the reconstituted unit dose and yellow fever vaccine (particularly YF-17D) of the invention described herein is administered subcutaneously to a subject or population of subjects from a dengue-endemic area. In other embodiments, the subject or population of subjects is from a non-dengue-endemic area. Such subject populations or such subjects may be vaccinated according to the present invention in the context of travel to dengue-endemic and yellow fever-endemic areas.

[0290] In certain embodiments, the present invention is directed to the aforementioned method, wherein the reconstituted unit dose and yellow fever vaccine of the invention described herein (particularly YF-17D) is administered subcutaneously to a subject or subject population over the age of 17, or over the age of 18, or between 18 and 60 years of age. In further embodiments, the subject or subject population is an adult over the age of 21, 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 other embodiments, 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.

[0291] 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.

[0292] Methods and uses for prevention against dengue fever, methods and uses for vaccination The present invention is directed, in part, to methods of preventing dengue disease (particularly virologically confirmed dengue, VCD) 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 an inventive unit dose / tetravalent dengue virus composition described herein, particularly a reconstituted unit dose.

[0293] The present invention is directed, in part, to methods for preventing dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Accordingly, in certain embodiments, the present invention is directed to methods for 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.

[0294] Thus, the present invention provides 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 comprising 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, specifically the dengue serotype 2 strains are derived from the wild-type virus strain DEN-2 16681 (SEQ ID NO: 11) and differ from its wild-type counterpart in at least three nucleotides as follows: a) 5'-noncoding region (NCR)-57 (nt-57 C to T): 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 - DENV-2 / 4 chimeras, derived from serotype 2 strains by resulting in

[0295] Further information regarding the serotypes of the tetravalent composition can be derived from the section "Dengue virus strains" above.

[0296] The tetravalent dengue virus composition for such a method comprises: (i) dengue serotype 1 at a concentration of at least 3.3 log pfu / 0.5 ml; (ii) dengue serotype 2 at a concentration of at least 2.7 log pfu / 0.5 ml; (iii) dengue serotype 3 at a concentration of at least 4.0 log pfu / 0.5 ml; and (iv) Dengue serotype 4 at a concentration of at least 4.5 log10 pfu / 0.5 ml.

[0297] The present invention specifically provides a unit dose that is lyophilized and upon reconstitution with 0.5 mL of a pharmaceutically acceptable diluent: (i) dengue serotype 1 at a concentration of at least 3.3 log pfu / 0.5 ml; (ii) dengue serotype 2 at a concentration of at least 2.7 log 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 log pfu / 0.5 ml.

[0298] Further information regarding tetravalent compositions or unit doses can be derived from the sections "Dengue vaccine compositions" and "Unit doses" above.

[0299] Therefore, the present invention provides a method and a corresponding use, said method comprising the following steps: (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 administering the first unit dose, and corresponding uses. According to this embodiment, two doses administered within three months are sufficient to provide effective protection against subsequent dengue infection.

[0300] Such methods preferably provide 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 study 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.

[0301] Such methods also preferably provide combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease with a two-sided 95% confidence interval greater than 45% when measured against placebo in a study 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.

[0302] 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.

[0303] 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, wherein 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) aged 4 to 16 years, regardless of baseline serostatus, from 30 days after the second dose through 12 to 18 months after the second dose (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 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.

[0304] 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) aged 4 to 16 years, regardless of baseline serostatus, from 30 days after the second dose through 12 to 18 months after the second dose (e.g., at 12 or 18 months). In certain such embodiments, 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.

[0305] 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) aged 4 to 16 years, regardless of baseline serostatus, from 30 days after the second dose through 12 to 18 months after the second dose (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.

[0306] 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) aged 4 to 16 years, regardless of baseline serostatus, from 30 days after the second dose through 12 to 18 months after the second dose (e.g., at 12 or 18 months). In certain such embodiments, 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.

[0307] The effectiveness of this method is explained in more detail further in this section below.

[0308] 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.

[0309] According to one embodiment, such a method does not include a step of determining whether the subject has previously been infected with dengue fever prior to administration of the unit dose, or the serostatus of the subject is unknown prior to administration of the unit dose.

[0310] According to one embodiment, such methods preferably do not include a step of determining whether the subject has previously been 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.

[0311] The method according to the invention does not require testing of serostatus before vaccination, thus allowing for immediate treatment and outbreak control. According to certain embodiments, the use is in a method where the subject is exposed to a dengue outbreak. In certain such embodiments, the outbreak is caused by dengue serotype 2 and / or by serotype 1.

[0312] 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%.

[0313] According to one embodiment of such a method, the subject is seronegative at baseline and originates from or travels to an area where the seroprevalence is high for serotype 1 and / or serotype 2, i.e., 80%, or 90%, or greater.

[0314] 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 previous 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 for 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, 15 percentage points, or 20 percentage points when comparing seropositive and seronegative subjects. The method is preferably safe with respect to serotype 1 and serotype 2 and can therefore be used in pandemic situations due to serotype 1 and / or serotype 2, or even in seronegative subjects (e.g., travelers) or subjects of unknown serostatus in areas with very high seroprevalence (>80%) due to serotype 1 and / or serotype 2.

[0315] The safety of the method is discussed in further detail in the section "Methods of prevention, methods of vaccination."

[0316] According to one embodiment, such a 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 weekly 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 carried out via appropriate methods (e.g., telephone, text message, home visit, school-based monitoring), which may vary at each study testing site.

[0317] According to one embodiment, such methods do not include vaccine immunogenicity assays involving GMTs for dengue neutralizing antibodies.

[0318] According to one embodiment, such methods do not include reactogenicity analysis, which relates to unsolicited local AEs (injection site pain, injection site erythema, and injection site swelling) and unsolicited systemic AEs (children <6 years: fever, irritability / tantrums, drowsiness, and loss of appetite; children ≥6 years: asthenia, fever, headache, malaise, and muscle pain), assessed, for example, by collecting diary cards for 7 days and 14 days (including the day of vaccination) after each vaccination, respectively.

[0319] According to one embodiment, the method does not include active surveillance, immunogenicity analysis, and reactogenicity analysis.

[0320] According to such embodiments, the vaccine and corresponding administration method are safe and therefore do not require further steps of monitoring or analysis.

[0321] In view of the above, a method according to one embodiment comprises the following steps: (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 vaccination is completed without determining whether the subject has 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.

[0322] Selection of subjects may include all types of considerations, but preferably does not include determining whether they have had a previous 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. Subjects may be selected due to their exposure to serotype 1 and / or serotype 2 or due to the fact that they require protection against specific dengue serotypes, i.e., serotype 1 and / or serotype 2.

[0323] 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.

[0324] In particular, the present invention is directed to methods such that the methods are safe.

[0325] 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 through 12-18 months after the last dose of the dosing schedule.

[0326] In particular, the present invention is directed to such methods, where the methods are effective.

[0327] 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 study 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.

[0328] In certain embodiments, the present invention is directed to the aforementioned method, wherein the subject is seronegative for all dengue serotypes.

[0329] The present invention is directed, in part, to methods of preventing dengue disease (particularly virologically confirmed dengue, VCD) in a subject population. Accordingly, 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.

[0330] The present invention is directed, in part, to the aforementioned method for preventing dengue disease (particularly 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 (GMTs) is achieved at 180 days or 365 days after 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 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 the reconstituted unit dose of the invention described herein and optionally after a second administration of the 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 days 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 days or 365 days after administration of the first reconstituted unit dose.

[0331] 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 administration of at least a first reconstituted unit dose of the invention described herein, and optionally after administration of a second reconstituted unit dose of the invention described herein 90 days after the first administration, result in 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.

[0332] The geometric mean neutralizing antibody titers (GMTs) of a subject population, or the neutralizing antibody titers of a subject, are 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 presently understood that the method of inducing a more balanced immune response by administering a reconstituted unit dose of the invention described herein will be beneficial to the vaccinated subject or subject population in terms of a smaller difference in the geometric mean neutralizing antibody titers (GMTs) against the four dengue serotypes or neutralizing antibody titers against the four dengue serotypes. Specifically, it is understood that a much larger response against any one of the four serotypes (e.g., DENV-2) will be less beneficial compared to the other serotypes.

[0333] The present invention is directed, in part, to the aforementioned method for preventing dengue disease (particularly virologically confirmable 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.

[0334] The invention is directed, in part, to the aforementioned method for preventing dengue disease (particularly virologically confirmable dengue, VCD) in a subject or subject population, which method results in a seropositivity rate in the subject population of at least 100 subjects comprising subcutaneous administration of two unit doses at days 1 and 90, 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, and 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.

[0335] 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 (particularly the virus strains described herein).

[0336] 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 (particularly the virus strains described herein).

[0337] In certain embodiments, the method comprises a reconstituted unit dose / tetravalent dengue virus composition of a dengue vaccine composition administered to prevent dengue disease in a subject or population of subjects, wherein the reconstituted unit dose comprises a tetravalent virus composition comprising four live attenuated dengue virus strains, and wherein the unit dose is lyophilized and, upon reconstitution with 0.5 mL of a pharmaceutically acceptable diluent, (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) at a concentration of at least 3.3 log 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., 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.

[0338] 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 pharmaceutically 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 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 18-60 years of age.

[0339] 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 pharmaceutically 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.

[0340] 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 (particularly the virus strains described herein), wherein the serotypes have a certain concentration as described herein relative to 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 log pfu / dose to 5.0 log pfu / dose, or 3.3 log pfu / 0.5 mL to 5.0 log 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 log pfu / dose to 5.7 log pfu / 0.5 dose, or 4.0 log pfu / 0.5 mL to 5.7 log pfu / 0.5 mL; (iv) Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strain) has a concentration of 4.5 log pfu / dose to 5.5 log pfu / 0.5 dose, or 4.5 log pfu / 0.5 mL to 5.5 log pfu / 0.5 mL. In preferred such embodiments, the subject or target population is aged 2-17 years, for example 4-16 years, preferably under 9 years. In other preferred embodiments, the subject or target population is aged 4-5 years, 6-11 years, or 12-16 years.

[0341] In certain embodiments, the invention relates to the aforementioned method, wherein the unit dose, upon reconstitution with 0.5 mL of a pharmaceutically acceptable diluent, has a concentration of 3.3 log pfu / 0.5 mL to 3.6 log pfu / 0.5 mL for dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain) and 2.7 log pfu / 0.5 mL to 4.0 log pfu / 0.5 mL for dengue serotype 2 (e.g., a dengue serotype 2 strain). The method is directed to a method for administering a dengue serotype 1 (e.g., chimeric dengue serotype 2 / 3 strain) containing a dengue serotype 1 (e.g., chimeric dengue serotype 2 / 3 strain) containing a dengue serotype 2 (e.g., chimeric dengue serotype 2 / 4 strain) containing a dengue serotype 1 ...17 strain) containing a dengue serotype 1 (e.g., chimeric dengue serotype 2 / 17 strain) containing a dengue serotype 1 (e.g., chimeric dengue serotype 2 / 17 strain) containing a dengue serotype 1 (e.g., chimeric dengue serotype 2 / 17 strain) containing a dengue serotype 1 (e.g., chimeric dengue serotype 2 / 17 strain) containing a dengue serotype 1 (e.g.,

[0342] In certain embodiments, the invention is directed to the aforementioned method, wherein the concentration of dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) measured by pfu / 0.5 mL is 1% to 7% of the total concentration, the concentration of dengue serotype 2 (e.g., 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% to 8% of the total concentration, the concentration of dengue serotype 3 (e.g., 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., 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% to 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 target population is 2 to 17 years old, e.g., 4 to 16 years old, even more preferably under 9 years old. In other preferred embodiments, the subject or target population is 4 to 5 years old, 6 to 11 years old, or 12 to 16 years old.

[0343] 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 concentrations within 5 percentage points of each other, based on total concentration in pfu / 0.5 mL, and / or together account for 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 / 0.5mL, 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 the concentration 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 / 0.5mL.

[0344] 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.

[0345] 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.

[0346] In certain embodiments, the present invention is directed to the aforementioned methods, wherein the reconstituted unit dose is administered to a subject whose serostatus is unknown and / or wherein the subject has not been tested to determine whether they are seropositive or seronegative (prior to administration of the unit dose described herein). In certain embodiments, the present invention is directed to the aforementioned methods, wherein the reconstituted unit dose is administered to a subject whose serostatus is unknown and / or the subject has not been tested to determine whether they are seropositive or seronegative (prior to administration of the unit dose described herein). In certain embodiments, the present invention is directed to the aforementioned methods, wherein the reconstituted unit dose is administered to a subject whose serostatus is unknown and / or wherein ...

[0347] In certain embodiments, the present invention is directed to the aforementioned method, wherein the subject or subject population is seronegative to all dengue serotypes.

[0348] 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.

[0349] In certain embodiments, the methods of the present invention comprise or consist of administering a single unit dose of the present invention.

[0350] 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 to a subject or population of subjects who are seronegative for all dengue serotypes. In other embodiments, the subject or population of subjects is seropositive for at least one dengue serotype.

[0351] 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 from a dengue-endemic area. In some of these embodiments, the subject or population of subjects is from Singapore, the Dominican Republic, Panama, the Philippines, Colombia, Puerto Rico, or Thailand, particularly Singapore, the Dominican Republic, Panama, or the Philippines. In preferred embodiments, the subject or population of subjects is from Asia-Pacific or Central and South America. In other embodiments, the subject or population of subjects is from Thailand, Sri Lanka, the Philippines, Panama, Nicaragua, the Dominican Republic, Colombia, or Brazil. In other embodiments, the subject or population of subjects is from a dengue-non-endemic area. Such a subject or population of subjects may be vaccinated according to the invention in light 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 from a dengue-endemic or non-dengue-endemic area.

[0352] 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 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 the age of 17, or over the age of 18, or between the ages of 18 and 60. In further specific embodiments, the subject or subject population is an adult over the age of 21, or between the ages of 21 and 60, or between the ages of 21 and 45.

[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 children and young adults between the ages of 2 and 17. 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, or between 6 and 11 years of age, or between 12 and 16 years of age. Optionally, the subject or subject population is seronegative for all dengue serotypes.

[0354] 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 the age of 2, 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 from a dengue-endemic area.

[0355] 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 the age of 2, 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 from a dengue-endemic area.

[0356] 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.

[0357] In certain embodiments, the invention is directed to the aforementioned method, 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.

[0358] 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. In other embodiments, the subject or subject population is of Asia-Pacific or Latin America origin and is seronegative for all dengue serotypes at baseline.

[0359] In certain embodiments, the invention is directed to the aforementioned method, wherein the subject or subject population is from Asia-Pacific, 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 from Asia-Pacific, 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 from Latin America, 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 from the Americas, is seronegative for all dengue serotypes at baseline, and is 4-5 years old, 6-11 years old, or 12-16 years old.

[0360] In certain embodiments, the present invention is directed to the aforementioned method, wherein the subject or subject population has received a prior vaccination against yellow fever. In other embodiments, the subject or subject population has received a prior vaccination against Japanese encephalitis. In yet other embodiments, the subject or subject population has not received a prior vaccination against yellow fever. In other embodiments, the subject or subject population has not received a prior vaccination 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 yellow fever vaccination is defined as yellow fever vaccination administered 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 of administering the first dose.

[0361] 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 demonstrates 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. Therefore, it is contemplated that the unit dose of the invention described herein and the methods of the invention described herein may result in a stronger immune response in a subject population that includes both seropositive and seronegative subjects. This balanced response, as well as balanced efficacy and safety, is necessary to allow vaccination without prior serostatus analysis, which is a major advantage in vaccination programs, especially in pandemic situations.

[0362] 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.

[0363] 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.

[0364] 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 methods do not include determining the subject's history of past dengue infection 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.

[0365] In certain embodiments, the present invention is directed to the aforementioned methods, wherein administration of the vaccine is safe, and particularly effective, regardless of whether the subject is determined to have had a previous dengue infection prior to administration of the tetravalent dengue virus composition.

[0366] In certain embodiments, the present invention is directed to the aforementioned methods, wherein the methods are safe and / or effective.

[0367] In certain embodiments, the invention is directed to the aforementioned methods, wherein the composition comprises at least one chimeric dengue virus. In certain such embodiments, the 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, particularly 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 provided above.

[0368] 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 second / final dose through at least 12-18 months (e.g., at 12 or 18 months), wherein 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 various embodiments, the present invention is directed to the aforementioned methods having vaccine efficacy, preferably combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease at a two-sided 95% confidence interval, wherein at least one reconstituted unit dose or tetravalent dengue virus composition described herein or placebo is administered, 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, for up to 15 to 21 months (e.g., 15 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.

[0369] 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%.

[0370] In certain embodiments, the invention is directed to the aforementioned methods, wherein the vaccine efficacy, preferably the combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease is greater than 30% 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 / final dose through at least 12 months or 12-18 months (e.g., at 12 or 18 months) after the second / final dose, wherein the reconstituted unit dose or tetravalent dengue virus composition described herein or the placebo is administered at least twice within less than six months (e.g., within three months). In certain embodiments, the invention is directed to the aforementioned methods, wherein a reconstituted unit dose or tetravalent dengue virus composition described herein or a placebo is administered at least once, resulting in vaccine efficacy, preferably combined vaccine efficacy against all four serotypes, of greater than 30% 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), regardless of baseline serostatus, for up to 15 months after the first dose of the dosing schedule. In certain such embodiments, 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 three months, e.g., on days 0 and 90.

[0371] Thus, in certain embodiments, the invention is directed to the aforementioned methods, in which 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 the reconstituted unit dose or 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, vaccine efficacy is, e.g., greater than 68%, greater than 70%, greater than 72%, or greater than 74%.

[0372] 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 accompanied by 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 dose through at least 18 months after the second dose, and where a 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 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%, greater than 80%, or greater than 90%.

[0373] In certain embodiments, the invention is directed to the aforementioned methods having combination 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 to 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, with a lower limit of greater than 25%, and 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 lower limit is greater than 30%, greater than 40%, greater than 50%, or greater than 55%.

[0374] 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 years 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 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%.

[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 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, 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 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%.

[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 at a two-sided 95% confidence interval, where the lower bound 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 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 bounds provided by subjects who are seropositive at baseline and subjects who are seronegative to all serotypes at baseline is 15 percentage points or less.

[0377] In certain embodiments, the invention is directed to the aforementioned method, wherein the combined vaccine efficacy against all four dengue serotypes in seronegative subjects 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 to all serotypes 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 all four dengue serotypes in seronegative subjects is greater than 40%, greater than 50%, greater than 60%, greater than 65%, or greater than 70%.

[0378] 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, as 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 years 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, wherein 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%.

[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 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, from 30 days after the last dose through 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 40%, greater than 50%, greater than 60%, greater than 65%, greater than 70%, or greater than 75%.

[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 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 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 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 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.

[0381] In certain embodiments, the invention is directed to the aforementioned methods having combination vaccine efficacy against all four dengue serotypes with a two-sided 95% confidence interval, when measured against placebo in a 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 through at least 12 months or 12-18 months after the second dose of the dosing schedule (e.g., at 12 or 18 months), with a lower bound of greater than 25%, and 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 lower bound is greater than 30%, greater than 40%, or greater than 45%.

[0382] In certain embodiments, the invention is directed to the aforementioned method, wherein the combined vaccine efficacy against all four dengue serotypes is greater than 30% when measured against placebo in a population of at least 1,000 healthy subjects aged 4-5 years at randomization, regardless of baseline serostatus, from 30 days after the second dose through at least 12 months or 12-18 months after the second dose (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%.

[0383] In certain embodiments, the invention is directed to the aforementioned methods having combination vaccine efficacy against all four dengue serotypes with a two-sided 95% confidence interval, where the lower limit is greater than 25% when measured against placebo in a study population of at least 1,000 healthy subjects 6-11 years of age 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), 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%, greater than 60%, or greater than 70%.

[0384] In certain embodiments, the invention is directed to the aforementioned method, wherein, from 30 days after the second dose through at least 12 months or 12-18 months after the second dose (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 population of at least 1,000 healthy subjects 6-11 years of age at 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%.

[0385] In certain embodiments, the invention is directed to the aforementioned methods having combination vaccine efficacy against all four dengue serotypes with a two-sided 95% confidence interval, where the lower limit is greater than 25% when measured against placebo in a study 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 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 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 65%, or greater than 68%.

[0386] In certain embodiments, the invention is directed to the aforementioned method, wherein, from 30 days after the second dose through at least 12 months or 12-18 months after the second dose (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 population of at least 1,000 healthy subjects aged 12-16 years at 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%.

[0387] 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 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 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 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%.

[0388] 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 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 bound of 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 bound within 10 percentage points.

[0389] In certain embodiments, the invention is directed to the aforementioned method, wherein vaccine efficacy against dengue serotype 1 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 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, vaccine efficacy against dengue serotype 1 is greater than 40%, greater than 50%, greater than 60%, greater than 65%, or greater than 70%.

[0390] 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 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 to 16, regardless of baseline serostatus, from 30 days after the last dose through 12 to 18 months after the last dose (e.g., at 12 or 18 months), wherein 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, 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.

[0391] 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 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 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%, greater than 80%, or greater than 90%.

[0392] 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) aged 4-16 years, 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 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 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.

[0393] In certain embodiments, the invention is directed to the aforementioned method, wherein vaccine efficacy against dengue serotype 2 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 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, 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%.

[0394] 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 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 to 16, regardless of baseline serostatus, from 30 days after the last dose through 12 to 18 months after the last dose (e.g., at 12 or 18 months), wherein 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, 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.

[0395] In certain embodiments, the invention is directed to the aforementioned methods 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 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 lower limit is greater than 30% or greater than 40%.

[0396] In certain embodiments, the invention is directed to the aforementioned method, wherein 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, 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, vaccine efficacy against dengue serotype 3 is greater than 40%, greater than 50%, greater than 55%, or greater than 60%.

[0397] 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 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 through 12-18 months after the last dose (e.g., at 12 or 18 months), or from 30 days after the last dose through 12-18 months after the last dose (e.g., at 12 or 18 months), wherein 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 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%.

[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 accompanied by hospitalization, 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 4-16, regardless of baseline serostatus, from the first dose through 12-18 months after the last dose (e.g., at 12 or 18 months), or from 30 days after the last dose through 12-18 months after the last dose (e.g., at 12 or 18 months), wherein 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, vaccine efficacy is greater than 70%, greater than 75%, greater than 80%, or greater than 82%, or greater than 85%, or greater than 88%.

[0399] In certain embodiments, the invention is directed to the aforementioned method having combined vaccine efficacy against all four serotypes against virologically confirmed dengue fever 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 to 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, and the unit dose / tetravalent dengue virus composition / 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%.

[0400] 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 years 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 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%.

[0401] In certain embodiments, the invention is directed to the aforementioned method, wherein the combined vaccine efficacy against virologically confirmed dengue with hospitalization for all four serotypes is greater than 30% when measured against placebo in a study 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 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 six months (e.g., within three months). In certain such embodiments, the combined vaccine efficacy against virologically confirmed dengue with hospitalization for all four serotypes is greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0402] In certain embodiments, the invention is directed to a method as described above having combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease accompanied by hospitalization, 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 4-16 years 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, wherein 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 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%.

[0403] In certain embodiments, the invention is directed to the aforementioned methods having combination vaccine efficacy against virologically confirmed dengue disease accompanied by 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 (e.g., at 12 or 18 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%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, or greater than 80%.

[0404] 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 years 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 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%.

[0405] In certain embodiments, the invention is directed to the aforementioned method, wherein the combined vaccine efficacy against virologically confirmed dengue with hospitalization for all four serotypes is greater than 30% when measured against placebo in a study 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 for all four serotypes is greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0406] 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 accompanied by hospitalization, 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, from 30 days after the last dose through 12-18 months (e.g., at 12 or 18 months) after the last dose, 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, vaccine efficacy is greater than 75%, greater than 70%, greater than 80%, greater than 85%, or greater than 90%.

[0407] 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 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 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 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.

[0408] In certain embodiments, the invention is directed to the aforementioned methods having combined vaccine efficacy against all four serotypes in preventing hospitalized virologically confirmable dengue disease, 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, wherein 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 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.

[0409] 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, of 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, through 30 days after the second administration, at least 12 months after the second administration, 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, the reconstituted unit dose or placebo is administered subcutaneously within about three months, eg, on days 0 and 90.

[0410] In certain embodiments, the invention is directed to the aforementioned methods, wherein the relative risk, preferably the combined relative risk for all four serotypes, is less than 0.70 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 through at least 12 months after the second administration, wherein 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 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, the reconstituted unit dose or placebo is administered subcutaneously within about three months, eg, on days 0 and 90.

[0411] In certain embodiments, the invention is directed to the aforementioned method, wherein virologically confirmable dengue disease occurs in less than 2.5% of subjects, regardless of baseline serostatus, 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), from 30 days after the second administration through at least 12 months or at least 18 months after the second administration, and wherein 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, 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, the reconstituted unit dose or placebo is administered subcutaneously within about three months, e.g., on days 0 and 90.

[0412] 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 six months or less.

[0413] 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 an endemic area 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, and wherein the unit dose / tetravalent dengue virus composition or the placebo is administered at least twice within six months or less.

[0414] 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 dose of the dosing schedule to 12 or 13 months after the last dose of the dosing schedule.

[0415] In certain embodiments, the 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 in the administration schedule to 12 or 13 months after the second administration.

[0416] 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 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.

[0417] In certain embodiments, the invention is directed to the aforementioned method, wherein the relative risk of virologically confirmed dengue fever resulting in hospitalization is 1 or less, or 0.8 or less, or 0.6 or less, 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). 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 subjects are seronegative for all dengue serotypes.

[0418] In certain embodiments, the invention is directed to the aforementioned methods, wherein the healthy subjects of the target population are between 4 and 16 years old. In some such embodiments, the healthy subjects of the target population are between 4 and 5 years old, between 6 and 11 years old, or between 12 and 16 years old.

[0419] 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.

[0420] 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.

[0421] 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.

[0422] In certain embodiments, the invention is directed to the aforementioned methods, wherein the healthy subjects of the target 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 target 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.

[0423] 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.

[0424] In certain embodiments, the invention is directed to the aforementioned method, wherein the healthy subjects of the subject population are from Asia-Pacific or Latin America and are seropositive for at least one dengue serotype at baseline. In other embodiments, the healthy subjects of the subject population are from Asia-Pacific or Latin America and are seronegative for all dengue serotypes at baseline.

[0425] In certain embodiments, the invention is directed to the aforementioned method, wherein the healthy subjects of the target population are from Asia-Pacific, seropositive for at least one dengue serotype at baseline, and 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, seronegative for all dengue serotypes at baseline, and 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, seropositive for at least one dengue serotype at baseline, and 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, seronegative for all dengue serotypes at baseline, and 4-5 years old, 6-11 years old, or 12-16 years old.

[0426] In certain embodiments, the present invention is directed to the aforementioned method, wherein healthy subjects in the target population have received a prior vaccination against yellow fever. In other embodiments, healthy subjects in the target population have not received a prior vaccination against yellow fever. Prior vaccination refers to a vaccination administered prior to the first vaccination with a reconstituted unit dose described herein. For example, for vaccine efficacy (VE) determined in Example 6 from 30 days after the second vaccination, prior yellow fever vaccination is defined as a yellow fever vaccination administered more than 30 days after the second vaccination.

[0427] In certain embodiments, the present invention is directed to the aforementioned method, wherein the healthy subjects of the subject population have received prior vaccination against Japanese encephalitis. In other embodiments, the healthy subjects of the subject population have not received prior vaccination against Japanese encephalitis.

[0428] 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%.

[0429] 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%.

[0430] 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%.

[0431] 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%.

[0432] 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).

[0433] The above methods are also contemplated in the context of the use of unit doses for use in such methods or for use in the manufacture of a medicament for such methods.

[0434] In certain embodiments, a tetravalent dengue vaccine (eg, Dengvaxia®) is used to vaccinate against dengue disease. Dengvaxia®, a 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 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, randomized, 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 set out in Table 16 of this reference.

[0435] In certain embodiments, the present invention is directed to the aforementioned methods, wherein a unit dose 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 Dengvaxia® disclosed herein are administered to a subject or population of subjects simultaneously on the same day.

[0436] In certain embodiments, the present invention is directed to the aforementioned methods, wherein a subject or population of subjects is administered a unit dose 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, as a first dose on day 0 / 1, and subsequently the subject or population of subjects is administered Dengvaxia®, as disclosed herein, as a second dose within 3 months of the first dose, e.g., on day 90 after the first dose. Alternatively, Dengvaxia® as disclosed herein is administered to a subject or population of subjects on day 0 / 1 as a first dose, and subsequently a unit dose 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, is administered to the subject or population of subjects as a second dose within 3 months of the first dose, for example, on day 90 after the first dose.

[0437] 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 TV005 is similar to TV003, except that the concentration of rDEN2 / 4Δ30 in TV005 is 4 log 10The difference is that the vaccines TV003 and TV005 are PFU. Vaccines TV003 and TV005, 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 TV003 or TV005 vaccine components: deletion of the 3' untranslated region and chimerization of structural genes. For example, component rDEN4Δ30 contains all of the structural and nonstructural proteins of wild-type DENV-4, but is attenuated by a 30-nucleotide deletion in the 3' untranslated region (designated "Δ30"). The other vaccine component is also attenuated by a 30-nucleotide deletion in the 3' untranslated region. Additionally, rDEN3Δ30 / 31 contains a 31-nucleotide deletion in the 3' untranslated region (detailed in Figure lc 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 dengue strains that can be used to produce TV003 or TV005 are available under the Genbank accession numbers in Table A of WO2008 / 022196A1.

[0438] In certain embodiments, the present invention is directed to the aforementioned method, wherein a unit dose 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 TV003 or TV005 disclosed herein are administered to a subject or population of subjects simultaneously on the same day.

[0439] In certain embodiments, the present invention is directed to the aforementioned method, wherein a subject or population of subjects is administered a unit dose disclosed herein, particularly 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, as a first dose on day 0 / 1, and subsequently the subject or population of subjects is administered TV003 or TV005 disclosed herein as a second dose within 3 months of the first dose, for example, on day 90 after the first dose. Alternatively, TV003 or TV005 disclosed herein is administered to a subject or population of subjects 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, for example, on day 90 after the first administration. [Example]

[0440] The following examples are included to demonstrate certain aspects and embodiments of the claimed invention. However, it should be understood by those of ordinary skill in the art that the following description is illustrative only and should not be construed as limiting the invention in any way.

[0441] Example 1: Preparation of dengue virus strains The methods used to generate the chimeric dengue strains TDV-1, -3, and -4 were standard molecular cloning and DNA engineering methods and are described in (2003) J. Virology 77(21):11436-11447. The following well-known methods were used to construct and introduce the prM-E genes of dengue serotypes 1, 3, and 4 into the TDV-2 backbone: reverse transcriptase PCR (RT-PCR), PCR, restriction enzyme digestion, DNA fragment ligation, bacterial transformation by electroporation, plasmid DNA preparation, in vitro transcription with T7 RNA polymerase, and transfection of Vero cells by electroporation.

[0442] As described in Huang et al. (2013) PLOS Neglected Dis, 7(5):e2243, different dengue serotypes are grown and purified separately and then mixed at certain concentrations as shown in Example 4. The mixture of dengue serotypes is present in the dengue vaccine composition and combined with a composition of pharmaceutically acceptable excipients to result in a dengue vaccine composition containing 15% w / v α,α trehalose dihydrate, 1% w / v poloxamer 407, 0.1% w / v human serum albumin, and 100 mM sodium chloride. The dengue vaccine composition is lyophilized and corresponds to a lyophilized unit dose of TDV. The lyophilized unit dose was reconstituted with 37 mM aqueous sodium chloride solution, and the reconstituted unit dose contained 15% w / v α,α trehalose dihydrate, 1% w / v poloxamer 407, 0.1% w / v human serum albumin, and 137 mM sodium chloride.

[0443] Example 2: Microneutralization test Immunogenicity was measured by microneutralization assay against each of the four dengue serotypes, with the titer defined as the dilution resulting in a 50% reduction in plaque count (MNT50). Briefly, on day 1, Vero cells were cultured in 96-well assay plates at 2.5 × 10 in DMEM and 10% FBS. 5Cells were seeded at a density of 1000 cells / ml and incubated at 37°C for 24 hours. On day 2, serial dilutions of heat-inactivated antibody-containing test and control serum samples (dilutions ranging from 1:10 to 1:20480) were prepared and mixed with a fixed concentration of dengue virus (specifically, DENV-1 strain 16007, DENV-2 strain 16681, DENV-3 strain 16562, and DENV-4 strain 1036) in 96-well microtiter plates (target 60-80 pfu / well) and incubated overnight at 2-8°C to allow neutralization of the virus by antibodies present in the serum. After incubation, the virus and antibody mixture was transferred to the 96-well plate containing Vero cells, and the plate was incubated at 37°C for 90-120 minutes to infect the Vero cells. A 1% methylcellulose overlay in DMEM was applied to the plates to limit the spread of progeny virus, and the plates were incubated at 34°C for 46–70 h depending on the dengue serotype: DENV1-66±2 hours DENV2-70±2 hours DENV3-66±2 hours DENV4-46±2 hours

[0444] After incubation, the cells were washed twice with PBS and fixed by adding cold methanol and incubating for 60 minutes at a temperature of ≤ -20°C. After fixation, the plates were dried and washed three times with washing buffer (1x PBS, pH 7.4 with 0.5% Tween), after which 50 μl of serotype-specific anti-dengue monoclonal antibody in blocking solution (2.5% nonfat dry milk in PBST) was added per well and incubated with the cells for 18 ± 4 hours at 2-8°C.

[0445] Monoclonal antibodies were generated as described in Gentry et al. (1982) Am. J. Trop. Med. Hyg. 31, 548-555; Henchal et al. (1985) Am. J. Trop. Med. Hyg. 34, 162-169; and Henchal et al. (1982) Am. J. Trop. Med. Hyg. 31(4):830-6. Briefly, anti-DENV-1 HBD was generated against the envelope of dengue 1 strain Hawaii, anti-DENV-2 was generated against isotype 1 of the envelope of dengue 2 strain New Guinea C, anti-DENV-3 HBD was generated against isotype 2A of the envelope of dengue 3 strain H87, and anti-DENV-4 HBD was generated against isotype 2A of the envelope of dengue 4 strain H241.

[0446] After incubation, the plate was washed three times with wash buffer, and 50 μl of secondary peroxidase-labeled goat anti-mouse IgG (H+L) (KPL Cat# 074-1806) in blocking solution was added and incubated at 37°C for 90-120 minutes. The plate was then washed three times with wash buffer, and 50 μl of precipitant substrate (2-amino-9-ethylcarbazole (AEC) tablets in 2.5 ml DMSO, 47.5 ml 50 mM acetate buffer, and 250 μl hydrogen peroxide) was added, and the mixture was incubated at room temperature for 20 minutes. Finally, the substrate was removed, and the plate was rinsed with dH2O and dried.

[0447] The titer of the sample is calculated using linear regression and reported as the MNT50 titer for each sample. Clinical data is reported as the geometric mean titer for all of the individual MNT50 titers in each treatment group. Briefly, the number of infectious puncta in each well was counted, and the neutralizing antibody titer was determined by comparing the reduction rate of infectious puncta centers in wells containing antibody (test sample) compared to wells containing virus only. MNT50 was calculated using the following linear regression formula: MNT50 = 10^[(50-c) / m], where c = the y-intercept of the regression line and m = the slope of the regression line.

[0448] Each test sample was tested in triplicate and titers were calculated from the mean of the triplicates. A schematic diagram of the steps performed in this study is shown in Figure 2.

[0449] Example 3: Phase III clinical trial in children A Phase III, double-blind, randomized, placebo-controlled trial was conducted to evaluate the efficacy, safety, and immunogenicity of a quadrivalent dengue vaccine (hereafter referred to as TDV (TDV-1, TDV-2, TDV-3, and TDV-4)) in 20,100 children aged 4 to 16 years residing in Thailand, Sri Lanka, the Philippines, Panama, Nicaragua, the Dominican Republic, Colombia, or Brazil. The trial included three parts. Part 1 evaluated vaccine efficacy (VE) and continued until both of the following two criteria were met: (i) 120 confirmed cases of dengue fever and (ii) a minimum duration of subject follow-up of 12 months after the second vaccination. Part 2 was an additional 6 months to evaluate VE and for secondary efficacy analyses. Part 3 evaluated long-term safety by following participants for side effects and continued for an additional 3 years.

[0450] Part 1: Active surveillance for the primary efficacy assessment in all subjects. During this period, subjects were contacted at least weekly to ensure identification of febrile illness potentially attributable to dengue fever. This part began on the day of vaccination and ended when both of the following two criteria were met: (i) 120 confirmed cases of dengue fever, and (ii) a minimum duration of subject follow-up of 12 months after the second vaccination. The end of Part 1 was defined for each subject so that the duration of follow-up after the second vaccination was approximately the same for all subjects. Virologically confirmed cases in Part 1 were counted toward the primary efficacy assessment if they occurred at least 30 days after the second vaccination. Part 1 ended 12 months after the second vaccination.

[0451] Part 2: Active surveillance for each subject after completion of Part 1 for an additional 6 months, i.e., 18 months after the second vaccination. During this period, subjects were contacted at least weekly to ensure identification of febrile illness potentially attributable to dengue fever. Virologically confirmed cases in Parts 1 and 2 will contribute towards secondary efficacy targets.

[0452] Part 3: Modified active surveillance for safety assessment in all subjects continuing for 3 years for each subject after completion of Part 2. Modified active surveillance during Part 3 will maintain at least weekly contact throughout Part 3 of the study but will modify the intensity of surveillance based on the need for hospitalization. Surveillance will identify febrile illness of any severity potentially attributable to dengue fever.

[0453] Selection criteria included: ●Subjects were between 4 and 16 years of age (inclusive) at the time of randomization. • Individuals who were in good health at the time of study entry as determined by medical history, physical examination (including vital signs), and the investigator's clinical judgment. • The subject and / or the subject's parent / guardian signed and dated the consent form / written informed consent form (if applicable), and any required privacy authorizations, after the nature of the study has been explained to them in accordance with local regulatory requirements and before the start of any study procedures. • Individuals who are able to comply with the study procedures and are available for the duration of the follow-up study.

[0454] Exclusion criteria included: 1. Febrile illness (temperature ≥ 38°C) or moderate or severe acute illness or infection at the time of randomization. 2. Any pre-existing medical history or any illness that, in the opinion of the investigator, may interfere with the outcome of the study or that may pose additional risks to the subject from participation in the study, including but not limited to: Known hypersensitivity or allergy to any of the vaccine components. b. Pregnant or lactating female subjects (after menarche). c. Individuals with any serious chronic or progressive disease (e.g., neoplasm, insulin-dependent diabetes mellitus, cardiac, renal, or liver disease, neurological or seizure disorder, or Guillain-Barre syndrome) as determined by the investigator. d. Known or suspected immune dysfunction / alteration, including: i. Chronic use of oral corticosteroids (equivalent to 20 mg / day prednisone for ≥ 12 weeks / ≥ 2 mg / kg body weight / day prednisone for ≥ 2 weeks) within 60 days prior to Day 1 (Month 0) (use of inhaled, intranasal, or topical corticosteroids is permitted). ii. Receiving parenteral steroids within 60 days prior to Day 1 (Month 0) (equivalent to 20 mg / day prednisone for ≥ 12 weeks / ≥ 2 mg / kg body weight / day prednisone for ≥ 2 weeks) iii. Have received immunoglobulin and / or any blood products within 3 months prior to Day 1 (Month 0) or are scheduled to receive such during the study. iv. Receipt of immunostimulants within 60 days prior to Day 1 (Month 0). v. Having received immunosuppressive therapy such as anticancer chemotherapy or radiation therapy within 6 months prior to Day 1 (Month 0). vi. Human immunodeficiency virus (HIV) infection or HIV-related illness. vii. Inherited immunodeficiency. 3. Having received any other vaccine within 14 days (for inactivated vaccines) or within 28 days (for live vaccines) prior to Day 1 (Month 0), or scheduled to receive any vaccine within 28 days of Day 1 (Month 0). 4. Participation in any clinical trial with another investigational agent 30 days prior to Day 1 (Month 0) or intention to participate in another clinical trial at any time during the conduct of this study. 5. Previous participation in any clinical trial of a dengue vaccine candidate or previous vaccination with a dengue vaccine. 6. First-degree relative of an individual involved in the study execution. 7. Females of childbearing potential who are sexually active and have not used any acceptable method of contraception for at least 2 months prior to Day 1 (Month 0). 8. Females of childbearing potential who are sexually active and refuse to use an acceptable method of contraception until 6 weeks after the second vaccination. 9. Is deprived of liberty by administrative or court order, or is in an emergency situation, or is in compulsory hospitalization. 10. Current alcohol abuse or drug dependence that may interfere with the subject's ability to comply with study procedures. 11. Identified as an employee of the investigator or testing center and directly involved in the proposed trial o...

Claims

1. a dengue vaccine, wherein the dengue vaccine is administered in combination with a hepatitis A vaccine, and wherein the dengue vaccine is administered without requiring a determination of the occurrence of a previous dengue infection in the subject; the dengue vaccine is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains representing serotype 1, serotype 2, serotype 3 and serotype 4, each of the four live attenuated dengue virus strains (i) non-chimeric dengue viruses that contain all components derived from the same dengue serotype; and (ii) Chimeric dengue viruses having portions from two dengue serotypes is selected from Dengue serotype 1 has the amino acid sequence of SEQ ID NO:2; Dengue serotype 2 has the amino acid sequence of SEQ ID NO: 4; Dengue serotype 3 has the amino acid sequence of SEQ ID NO:6, and Dengue serotype 4 has the amino acid sequence of SEQ ID NO:

8. Dengue vaccine.

2. a dengue vaccine administered in combination with a hepatitis A vaccine, wherein the dengue vaccine is administered without determining whether the subject has had a previous dengue infection; the dengue vaccine is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains representing serotype 1, serotype 2, serotype 3 and serotype 4, each of the four live attenuated dengue virus strains (i) non-chimeric dengue viruses containing all components derived from the same dengue serotype; and (ii) Chimeric dengue viruses having portions from two dengue serotypes is selected from Dengue serotype 2 strains are derived from the wild-type virus strain DENV-2 16681, differs from its wild-type counterpart in at least three nucleotides as follows: a) 5'-noncoding region (NCR)-57 (nt-57 C to T); b) NS1-53 Gly to Asp (nt-2579 G to A), and c) NS3-250 Glu to Val (nt-5270 A to T) dengue serotype 1 is a chimeric dengue serotype 2 / 1 strain; the dengue serotype 2 is a non-chimeric serotype 2 strain; dengue serotype 3 is a chimeric dengue serotype 2 / 3 strain; dengue serotype 4 is a chimeric dengue serotype 2 / 4 strain; wherein the three chimeric dengue strains are derived from serotype 2 strains by replacing the structural proteins prM and E from the serotype 2 strains with the corresponding structural proteins from other dengue serotypes; wherein in the DENV-2 / 1 chimeric strain, the structural proteins prM and E derived from the serotype 2 strain are replaced with the corresponding structural proteins derived from DENV-1 16007; In the DENV-2 / 3 chimeric strain, the structural proteins prM and E from the serotype 2 strain are replaced with the corresponding structural proteins from DENV-3 16562; and In the DENV-2 / 4 chimeric strain, the structural proteins prM and E from the serotype 2 strain were replaced with the corresponding structural proteins from DENV-4 1036. Dengue vaccine.

3. 1. A dengue vaccine, wherein the dengue vaccine is administered in combination with a hepatitis A vaccine, and the dengue vaccine is administered to a subject whose serostatus to dengue virus has not been determined, or who is seropositive or seronegative; the dengue vaccine is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains representing serotype 1, serotype 2, serotype 3 and serotype 4, each of the four live attenuated dengue virus strains (i) non-chimeric dengue viruses containing all components derived from the same dengue serotype; and (ii) Chimeric dengue viruses having portions from two dengue serotypes is selected from The dengue serotype 2 strain is derived from the wild-type virus strain DENV-2 16681; differs from its wild-type counterpart in at least three nucleotides as follows: a) 5'-noncoding region (NCR)-57 (nt-57 C to T); b) NS1-53 Gly to Asp (nt-2579 G to A), and c) NS3-250 Glu to Val (nt-5270 A to T) dengue serotype 1 is a chimeric dengue serotype 2 / 1 strain; the dengue serotype 2 is a non-chimeric serotype 2 strain; dengue serotype 3 is a chimeric dengue serotype 2 / 3 strain; dengue serotype 4 is a chimeric dengue serotype 2 / 4 strain; wherein the three chimeric dengue strains are derived from serotype 2 strains by replacing the structural proteins prM and E from the serotype 2 strains with the corresponding structural proteins from other dengue serotypes; wherein in the DENV-2 / 1 chimeric strain, the structural proteins prM and E derived from the serotype 2 strain are replaced with the corresponding structural proteins derived from DENV-1 16007; In the DENV-2 / 3 chimeric strain, the structural proteins prM and E from the serotype 2 strain are replaced with the corresponding structural proteins from DENV-3 16562; and In the DENV-2 / 4 chimeric strain, the structural proteins prM and E from the serotype 2 strain were replaced with the corresponding structural proteins from DENV-4 1036. Dengue vaccine.

4. a combination of a dengue vaccine and a hepatitis A vaccine, wherein the dengue vaccine is administered without the need to determine the occurrence of a previous dengue infection in the subject; the dengue vaccine is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains representing serotype 1, serotype 2, serotype 3 and serotype 4, each of the four live attenuated dengue virus strains (i) non-chimeric dengue viruses containing all components derived from the same dengue serotype; and (ii) Chimeric dengue viruses having portions from two dengue serotypes is selected from Dengue serotype 1 has the amino acid sequence of SEQ ID NO:2; Dengue serotype 2 has the amino acid sequence of SEQ ID NO: 4; Dengue serotype 3 has the amino acid sequence of SEQ ID NO:6, and Dengue serotype 4 has the amino acid sequence of SEQ ID NO:

8. Combination.

5. a combination of a dengue vaccine and a hepatitis A vaccine, wherein the dengue vaccine is administered without determining whether the subject has had a previous dengue infection; the dengue vaccine is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains representing serotype 1, serotype 2, serotype 3 and serotype 4, each of the four live attenuated dengue virus strains (i) non-chimeric dengue viruses containing all components derived from the same dengue serotype; and (ii) Chimeric dengue viruses having portions from two dengue serotypes is selected from Dengue serotype 2 strains are derived from the wild-type virus strain DENV-2 16681, differs from its wild-type counterpart in at least three nucleotides as follows: a) 5'-noncoding region (NCR)-57 (nt-57 C to T); b) NS1-53 Gly to Asp (nt-2579 G to A), and c) NS3-250 Glu to Val (nt-5270 A to T) dengue serotype 1 is a chimeric dengue serotype 2 / 1 strain; the dengue serotype 2 is a non-chimeric serotype 2 strain; dengue serotype 3 is a chimeric dengue serotype 2 / 3 strain; dengue serotype 4 is a chimeric dengue serotype 2 / 4 strain; wherein the three chimeric dengue strains are derived from serotype 2 strains by replacing the structural proteins prM and E from the serotype 2 strains with the corresponding structural proteins from other dengue serotypes; wherein in the DENV-2 / 1 chimeric strain, the structural proteins prM and E derived from the serotype 2 strain are replaced with the corresponding structural proteins derived from DENV-1 16007; In the DENV-2 / 3 chimeric strain, the structural proteins prM and E from the serotype 2 strain are replaced with the corresponding structural proteins from DENV-3 16562; and In the DENV-2 / 4 chimeric strain, the structural proteins prM and E from the serotype 2 strain were replaced with the corresponding structural proteins from DENV-4 1036. Combination.

6. A combination of a dengue vaccine and a hepatitis A vaccine, wherein the dengue vaccine is administered to a subject whose serostatus to dengue virus has not been determined, or who is seropositive or seronegative; the dengue vaccine is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains representing serotype 1, serotype 2, serotype 3 and serotype 4, each of the four live attenuated dengue virus strains (i) non-chimeric dengue viruses containing all components derived from the same dengue serotype; and (ii) Chimeric dengue viruses having portions from two dengue serotypes is selected from Dengue serotype 2 strains are derived from the wild-type virus strain DENV-2 16681, differs from its wild-type counterpart in at least three nucleotides as follows: a) 5'-noncoding region (NCR)-57 (nt-57 C to T); b) NS1-53 Gly to Asp (nt-2579 G to A), and c) NS3-250 Glu to Val (nt-5270 A to T) dengue serotype 1 is a chimeric dengue serotype 2 / 1 strain; the dengue serotype 2 is a non-chimeric serotype 2 strain; dengue serotype 3 is a chimeric dengue serotype 2 / 3 strain; dengue serotype 4 is a chimeric dengue serotype 2 / 4 strain; wherein the three chimeric dengue strains are derived from serotype 2 strains by replacing the structural proteins prM and E from the serotype 2 strains with the corresponding structural proteins from other dengue serotypes; wherein in the DENV-2 / 1 chimeric strain, the structural proteins prM and E derived from the serotype 2 strain are replaced with the corresponding structural proteins derived from DENV-1 16007; In the DENV-2 / 3 chimeric strain, the structural proteins prM and E from the serotype 2 strain are replaced with the corresponding structural proteins from DENV-3 16562; and In the DENV-2 / 4 chimeric strain, the structural proteins prM and E from the serotype 2 strain were replaced with the corresponding structural proteins from DENV-4 1036. Combination.

7. A kit for hepatitis A and dengue disease, said kit comprising: (a) a first container holding a hepatitis A vaccine; (b) a second container holding a dengue vaccine; and Equipped with the dengue vaccine is administered without the need to determine the occurrence of a previous dengue infection in the subject; the dengue vaccine is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains representing serotype 1, serotype 2, serotype 3 and serotype 4, each of the four live attenuated dengue virus strains (i) non-chimeric dengue viruses containing all components derived from the same dengue serotype; and (ii) Chimeric dengue viruses having portions from two dengue serotypes is selected from Dengue serotype 1 has the amino acid sequence of SEQ ID NO:2; Dengue serotype 2 has the amino acid sequence of SEQ ID NO: 4; Dengue serotype 3 has the amino acid sequence of SEQ ID NO:6, and Dengue serotype 4 has the amino acid sequence of SEQ ID NO:

8. kit.

8. A kit for hepatitis A and dengue disease, said kit comprising: (a) a first container holding a hepatitis A vaccine; (b) a second container holding a dengue vaccine; and Equipped with the dengue vaccine is administered without determining whether the subject has had a previous dengue infection; the dengue vaccine is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains representing serotype 1, serotype 2, serotype 3 and serotype 4, each of the four live attenuated dengue virus strains (i) non-chimeric dengue viruses containing all components derived from the same dengue serotype; and (ii) Chimeric dengue viruses having portions from two dengue serotypes is selected from Dengue serotype 2 strains are derived from the wild-type virus strain DENV-2 16681, differs from its wild-type counterpart in at least three nucleotides as follows: a) 5'-noncoding region (NCR)-57 (nt-57 C to T); b) NS1-53 Gly to Asp (nt-2579 G to A), and c) NS3-250 Glu to Val (nt-5270 A to T) dengue serotype 1 is a chimeric dengue serotype 2 / 1 strain; the dengue serotype 2 is a non-chimeric serotype 2 strain; dengue serotype 3 is a chimeric dengue serotype 2 / 3 strain; dengue serotype 4 is a chimeric dengue serotype 2 / 4 strain; wherein the three chimeric dengue strains are derived from serotype 2 strains by replacing the structural proteins prM and E from the serotype 2 strains with the corresponding structural proteins from other dengue serotypes; wherein in the DENV-2 / 1 chimeric strain, the structural proteins prM and E derived from the serotype 2 strain are replaced with the corresponding structural proteins derived from DENV-1 16007; In the DENV-2 / 3 chimeric strain, the structural proteins prM and E from the serotype 2 strain are replaced with the corresponding structural proteins from DENV-3 16562; and In the DENV-2 / 4 chimeric strain, the structural proteins prM and E from the serotype 2 strain were replaced with the corresponding structural proteins from DENV-4 1036. kit.

9. A kit for hepatitis A and dengue disease, said kit comprising: (a) a first container holding a hepatitis A vaccine; (b) a second container holding a dengue vaccine; and Equipped with the dengue vaccine is administered to a subject whose serostatus to dengue virus has not been determined, or who is seropositive or seronegative; the dengue vaccine is a tetravalent dengue virus composition comprising four live attenuated dengue virus strains representing serotype 1, serotype 2, serotype 3 and serotype 4, each of the four live attenuated dengue virus strains (i) non-chimeric dengue viruses containing all components derived from the same dengue serotype; and (ii) Chimeric dengue viruses having portions from two dengue serotypes is selected from Dengue serotype 2 strains are derived from the wild-type virus strain DENV-2 16681, differs from its wild-type counterpart in at least three nucleotides as follows: a) 5'-noncoding region (NCR)-57 (nt-57 C to T); b) NS1-53 Gly to Asp (nt-2579 G to A), and c) NS3-250 Glu to Val (nt-5270 A to T) dengue serotype 1 is a chimeric dengue serotype 2 / 1 strain; the dengue serotype 2 is a non-chimeric serotype 2 strain; dengue serotype 3 is a chimeric dengue serotype 2 / 3 strain; dengue serotype 4 is a chimeric dengue serotype 2 / 4 strain; wherein the three chimeric dengue strains are derived from serotype 2 strains by replacing the structural proteins prM and E from the serotype 2 strains with the corresponding structural proteins from other dengue serotypes; wherein in the DENV-2 / 1 chimeric strain, the structural proteins prM and E derived from the serotype 2 strain are replaced with the corresponding structural proteins derived from DENV-1 16007; In the DENV-2 / 3 chimeric strain, the structural proteins prM and E from the serotype 2 strain are replaced with the corresponding structural proteins from DENV-3 16562; and In the DENV-2 / 4 chimeric strain, the structural proteins prM and E from the serotype 2 strain were replaced with the corresponding structural proteins from DENV-4 1036. kit.

10. A method for preparing the combination of any one of claims 4 to 6 or the kit of any one of claims 7 to 9, said method comprising combining said dengue vaccine and said hepatitis A vaccine.

11. A combination of the dengue vaccine according to any one of claims 4 to 6 and a hepatitis A vaccine, said combination being a combined vaccine composition comprising the hepatitis A vaccine and the dengue vaccine.

12. A dengue vaccine as claimed in claim 1 or 2, a combination as claimed in claim 4 or 5, or a kit as claimed in claim 7 or 8, wherein the determination of past dengue infection in the subject is characterized by a laboratory-confirmed history of dengue or a validated serological test.

13. Each of the live attenuated dengue virus serotype 1, serotype 2 and serotype 4 strains further comprises: having a phenylalanine at amino acid position 1308 and an alanine at amino acid position 2168, and the live attenuated dengue virus serotype 3 strain further having a phenylalanine at amino acid position 1306 and an alanine at amino acid position 2166; 10. A dengue vaccine, combination or kit according to any one of claims 2, 3, 5, 6, 8 and 9.

14. the chimeric dengue serotype 2 / 1 strain further having a leucine at amino acid position 1243 and an aspartic acid at amino acid position 1437; the dengue serotype 2 strain further having a glutamic acid at amino acid position 166 and a valine at amino acid position 2903; the chimeric dengue serotype 2 / 3 strain has a serine at amino acid position 503; and the chimeric dengue serotype 2 / 4 strain has a glycine at amino acid position 1193, a lysine / arginine mix at amino acid position 1226, and a valine at amino acid position 2114.

14. A dengue vaccine, combination or kit according to any one of claims 2, 3, 5, 6, 8, 9 and 13.

15. 10. The dengue vaccine of any one of claims 1 to 3, the combination of any one of claims 4 to 6, or the kit of any one of claims 7 to 9, wherein the dengue vaccine is a dengue vaccine composition that provides a reduced risk of dengue disease resulting in hospitalization compared to unvaccinated subjects in a target population.

16. The hepatitis A vaccine is an inactivated virus vaccine. A dengue vaccine according to any one of claims 1 to 3, a combination according to any one of claims 4 to 6, or a kit according to any one of claims 7 to 9.

17. 10. The dengue vaccine of any one of claims 1 to 3, the combination of any one of claims 4 to 6, or the kit of any one of claims 7 to 9, wherein the dengue virus composition, upon reconstitution with 0.5 mL of a pharmaceutically acceptable diluent, comprises: (i) for chimeric dengue serotype 2 / 1 strains, a concentration of at least 3.3 log 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.

18. The subject is seronegative to all dengue serotypes. A dengue vaccine according to any one of claims 1 to 3, a combination according to any one of claims 4 to 6, or a kit according to any one of claims 7 to 9.

19. the dengue vaccine is administered by subcutaneous injection and the hepatitis A vaccine is administered by intramuscular injection; A dengue vaccine according to any one of claims 1 to 3, a combination according to any one of claims 4 to 6, or a kit according to any one of claims 7 to 9.

20. 20. The dengue vaccine, combination or kit of claim 19, wherein said dengue vaccine is administered by subcutaneous injection and said hepatitis A vaccine is administered by intramuscular injection, and said injections are administered in the arm.

21. 21. The dengue vaccine, combination or kit of claim 20, wherein said dengue vaccine is administered by subcutaneous injection and said hepatitis A vaccine is administered by intramuscular injection, said injection being administered in the deltoid region of the arm.

22. 22. The dengue vaccine, combination or kit of any one of claims 19 to 21, wherein the dengue vaccine and the hepatitis A vaccine are administered at different anatomical sites.

23. 23. The dengue vaccine, combination or kit of claim 22, wherein the dengue vaccine and the hepatitis A vaccine are administered in opposite arms.

24. two doses of the dengue vaccine are administered within 12 months or more, or within 6 months, or within 3 months; A dengue vaccine according to any one of claims 1 to 3, a combination according to any one of claims 4 to 6, or a kit according to any one of claims 7 to 9.

25. 10. The dengue vaccine of any one of claims 1 to 3, the combination of any one of claims 4 to 6, or the kit of any one of claims 7 to 9, wherein two doses of the dengue vaccine and one dose of the hepatitis A vaccine are administered.

26. 26. The dengue vaccine, combination or kit of claim 25, wherein the doses comprise administering according to the following schedule: on day -1, a first unit of the dengue vaccine and a first co-administration of the hepatitis A vaccine; and - a second administration of a second dose of said dengue vaccine after said first co-administration.

27. 27. The dengue vaccine, combination or kit of claim 26, wherein the second administration of the second dose of the dengue vaccine following the first co-administration occurs three months later.

28. The subject is 2 to 60 years old. A dengue vaccine according to any one of claims 1 to 3, a combination according to any one of claims 4 to 6, or a kit according to any one of claims 7 to 9.

29. the subject is over 60 years old; A dengue vaccine according to any one of claims 1 to 3, a combination according to any one of claims 4 to 6, or a kit according to any one of claims 7 to 9.

30. The dengue vaccine of any one of claims 1 to 3, the combination of any one of claims 4 to 6, or the kit of any one of claims 7 to 9, wherein the subject is from a dengue endemic area.

31. The dengue vaccine of any one of claims 1 to 3, the combination of any one of claims 4 to 6, or the kit of any one of claims 7 to 9, wherein the subject is from a dengue-free area.

32. 32. The dengue vaccine, combination, or kit of claim 31, wherein the subject is from a non-dengue and non-hepatitis A endemic area.

33. The dengue vaccine of any one of claims 1 to 3, the combination of any one of claims 4 to 6, or the kit of any one of claims 7 to 9, wherein the hepatitis A vaccine comprises a hepatitis A virus derived from the hepatitis A virus strain HM-175.

34. The dengue vaccine of any one of claims 1 to 3, the combination of any one of claims 4 to 6, or the kit of any one of claims 7 to 9, wherein 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.

35. The dengue vaccine according to any one of claims 1 to 3, the combination according to any one of claims 4 to 6, or the kit according to any one of claims 7 to 9, wherein the hepatitis A vaccine comprises an inactivated hepatitis A virus, and the inactivated hepatitis A virus is adsorbed to aluminum.

36. 36. The dengue vaccine, combination, or kit of claim 35, wherein the aluminum is aluminum hydroxide or aluminum hydroxyphosphate sulfate.

37. 10. The dengue vaccine of any one of claims 1 to 3, the combination of any one of claims 4 to 6, or the kit of any one of claims 7 to 9, wherein the hepatitis A vaccine comprises an inactivated hepatitis A virus and the hepatitis A vaccine comprises phosphate buffered saline with an amino acid and a polysorbate.

38. The dengue vaccine according to any one of claims 1 to 3, the combination according to any one of claims 4 to 6, or the kit according to any one of claims 7 to 9, 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.).

39. the hepatitis A vaccine does not include a step of determining whether the subject has previously been infected with hepatitis A and / or whether the subject has previously been infected with hepatitis A prior to administering the hepatitis A vaccine and prior to administering the dengue vaccine; or The dengue vaccine of any one of claims 1 to 3, the combination of any one of claims 4 to 6, or the kit of any one of claims 7 to 9, wherein the hepatitis A serostatus of the subject is unknown prior to said administration of the hepatitis A vaccine and prior to said administration of the dengue vaccine.

40. the hepatitis A vaccine does not include determining whether the subject has previously been infected with hepatitis A at any time before, during, or after administering the hepatitis A vaccine and the dengue vaccine; or 40. The dengue vaccine, combination or kit of claim 39, wherein the subject's hepatitis A serostatus is unknown at any time before, during or after the steps of administering the hepatitis A vaccine and the dengue vaccine.

41. A dengue vaccine according to any one of claims 1 to 3, a combination according to any one of claims 4 to 6, or a kit according to any one of claims 7 to 9, wherein primary vaccination comprises the following steps: (A) selecting a subject for administration of a dengue vaccine 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 at any time before, during, or after the step of administering the dengue vaccine, or without knowing the serostatus of the subject at any time before, during, or after the step of administering the dengue vaccine; and (B) administering to said subject a first dose of said dengue vaccine and a hepatitis A vaccine, and optionally (C) administering to the subject at least one additional dose of the dengue vaccine within 3 to 12 months of administration of the first 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 dose.

42. A dengue vaccine according to any one of claims 1 to 3, a combination according to any one of claims 4 to 6, or a kit according to any one of claims 7 to 9, wherein primary vaccination comprises the following steps: (A) selecting a subject to administer the dengue vaccine dose and the hepatitis A vaccine based on the need for protection against dengue infection and hepatitis A infection; and (B) the subject is administered a first dose of the dengue vaccine and a hepatitis A vaccine; and (C) administering to the subject two additional doses of the dengue vaccine at 6 months and 12 months after administration of the first dose, and administering the hepatitis A vaccine either 6 months or 12 months after administration of the first dose.

43. 43. The dengue vaccine, combination or kit of claim 41 or 42, wherein step (A) is performed without a step of determining whether the subject has previously been infected with hepatitis A, or the hepatitis A serostatus of the subject is unknown prior to the administration of the hepatitis A vaccine and prior to the administration of the dengue vaccine.

44. 10. The dengue vaccine of any one of claims 1 to 3, the combination of any one of claims 4 to 6, or the kit of any one of claims 7 to 9, wherein the dengue virus composition, upon reconstitution with a pharmaceutically acceptable diluent, gives a total concentration of (i), (ii), (iii), and (iv) in pfu / 0.5 mL, and the concentration of (ii) in pfu / 0.5 mL is less than 10%, the concentration of (iv) in pfu / 0.5 mL is at least 50%, the concentration of (i) in pfu / 0.5 mL is at least 1%, and the concentration of (iii) in 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%, based on the total concentration of pfu / 0.5 mL.

45. A dengue vaccine according to any one of claims 1 to 3, a combination according to any one of claims 4 to 6, or a kit according to any one of claims 7 to 9, wherein compatibility is provided between the dengue vaccine and the hepatitis A vaccine.

46. A dengue vaccine according to any one of claims 1 to 3, a combination according to any one of claims 4 to 6, or a kit according to any one of claims 7 to 9, wherein a synergistic effect is achieved between the dengue vaccine and the hepatitis A vaccine.

47. 10. The dengue vaccine of any one of claims 1 to 3, the combination of any one of claims 4 to 6, or the kit of any one of claims 7 to 9, wherein non-inferiority of the dengue vaccine and the hepatitis A vaccine is demonstrated in a non-inferiority clinical trial including at least 60 or at least 120 healthy subjects divided into one subject population and one control subject population, wherein the subject population receives the hepatitis A vaccine and the dengue vaccine simultaneously on the same day, and the control subject population receives the hepatitis A vaccine and a placebo dose simultaneously on the same day.

48. 10. The dengue vaccine of any one of claims 1 to 3, the combination of any one of claims 4 to 6, or the kit of any one of claims 7 to 9, wherein the hepatitis A vaccine confers 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 receive the hepatitis A vaccine and the dengue vaccine simultaneously on the same day and who are seronegative for hepatitis A at baseline and seronegative for all dengue virus serotypes at baseline.

49. a difference in seroprotection rate for hepatitis A relative to single-agent administration of hepatitis A is provided, 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 and seronegative for all dengue virus serotypes at baseline; wherein the healthy subject a) a subject population of at least 30 or at least 50 healthy subjects who receive the Hepatitis A vaccine and the Dengue vaccine simultaneously on the same day (day 0 / 1); b) a control population of at least 30 or 50 subjects receiving Hepatitis A vaccine and placebo simultaneously on the same day (day 0 / 1); The difference between the hepatitis A seroprotection rate in the control subject population 30 days after the administration (Day 0 / 1) and the hepatitis A seroprotection rate in the subject population 30 days after the administration (Day 0 / 1) is determined; The dengue vaccine of any one of claims 1 to 3, the combination of any one of claims 4 to 6, or the kit of any one of claims 7 to 9, wherein the upper limit of the difference within a two-sided 95% confidence interval is less than 10%.

50. 10. The dengue vaccine of any one of claims 1 to 3, the combination of any one of claims 4 to 6, or the kit of any one of claims 7 to 9, 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 and receive the hepatitis A vaccine and the dengue vaccine simultaneously on the same day, wherein the healthy subjects include one or more healthy subjects who are seropositive for at least one dengue virus serotype at baseline and one or more healthy subjects who are seronegative for all dengue virus serotypes at baseline.

51. A difference in seroprotection rate for hepatitis A relative to single-agent administration of hepatitis A is provided, said difference being determined in a non-inferiority clinical trial involving at least 60 or at least 120 healthy subjects who are seronegative for hepatitis A at baseline, said healthy subjects including one or more healthy subjects who are seropositive for at least one dengue virus serotype at baseline and one or more healthy subjects who are seronegative for all dengue virus serotypes at baseline, said healthy subjects comprising: a) a subject population of at least 30 or at least 50 healthy subjects receiving the hepatitis A vaccine and the dengue vaccine simultaneously on the same day (day 0 / 1), the subject population including one or more healthy subjects who are seropositive for at least one dengue virus serotype at baseline and one or more healthy subjects 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 placebo simultaneously on the same day (day 0 / 1), the control subject population comprising one or more healthy subjects who are seropositive for at least one dengue virus serotype at baseline and one or more healthy subjects who are seronegative for all dengue virus serotypes at baseline; The difference between the hepatitis A seroprotection rate in the control subject population 30 days after the administration (Day 0 / 1) and the hepatitis A seroprotection rate in the subject population 30 days after the administration (Day 0 / 1) is determined; The dengue vaccine of any one of claims 1 to 3, the combination of any one of claims 4 to 6, or the kit of any one of claims 7 to 9, wherein the upper limit of the difference within a two-sided 95% confidence interval is less than 10%.

52. The dengue vaccine of any one of claims 1 to 3, the combination of any one of claims 4 to 6, or the kit of any one of claims 7 to 9, wherein the subject has been exposed to a hepatitis A virus outbreak and / or a dengue virus outbreak.

53. 10. The dengue vaccine of any one of claims 1 to 3, the combination of any one of claims 4 to 6, or the kit of any one of claims 7 to 9, wherein, in a subject population of at least 30 or at least 50 healthy subjects who receive the hepatitis A vaccine and the dengue vaccine simultaneously on the same day and who are seronegative for hepatitis A at baseline and seronegative for all dengue virus serotypes at baseline, the anti-hepatitis A virus antibody geometric mean concentration (GMC) achieved 30 days after administration (day 0 / 1) is at least 70mIU / ml, or at least 80mIU / ml, or at least 90mIU / ml.

54. A dengue vaccine described in any one of claims 1 to 3, a combination described in any one of claims 4 to 6, or a kit described in any one of claims 7 to 9, wherein there are no serious adverse events associated with simultaneous administration on the same day.

55. Geometric mean titer (GMT) of neutralizing antibodies as measured by 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 dengue vaccine of any one of claims 1 to 3, the combination of any one of claims 4 to 6, or the kit of any one of claims 7 to 9, provided 30 days after the hepatitis A vaccine and the dengue vaccine 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.

56. The dengue vaccine of any one of claims 1 to 3, the combination of any one of claims 4 to 6, or the kit of any one of claims 7 to 9, wherein the subject is aged between 18 and 60 years.

57. A dengue vaccine according to any one of claims 1 to 3, a combination according to any one of claims 4 to 6, or a kit according to any one of claims 7 to 9, comprising one or more dengue booster vaccinations.

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