Dengue vaccine unit dose and administration thereof

Abstract

To provide a safe and effective vaccine against all serotypes of dengue virus for subjects of a wide range of ages, and a corresponding inoculation method.SOLUTION: The present invention relates to unit doses of dengue vaccine compositions and methods and uses for preventing dengue disease and methods for stimulating an immune response against all four dengue virus serotypes in a subject or subject population. A unit dose of a dengue vaccine composition comprises constructs of each dengue serotype, such as TDV-1, TDV-2, TDV-3 and TDV-4, at various concentrations to improve protection against dengue infection.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a safe and effective method of vaccination against dengue disease and a corresponding safe and effective dengue vaccine. In particular, the present invention relates to a safe and effective method of vaccination against dengue disease regardless of serostatus and a corresponding safe and effective dengue vaccine. The present invention also relates to a unit dose of a dengue vaccine composition and a method for administering the unit dose of the dengue vaccine composition to a subject or population of subjects across a wide age group. The present invention also relates to specific concomitant administration regimes in which the unit dose / vaccine composition is concomitantly administered with one or more of yellow fever (YF) vaccine, hepatitis A vaccine, human papillomavirus (HPV) vaccine, measles, mumps, and rubella (MMR) combined vaccine, tetanus, diphtheria, and pertussis (whooping cough) (Tdap) combined vaccine, and / or a combined vaccine for diphtheria, tetanus, pertussis, poliomyelitis, and Haemophilus influenzae type b (DTap / IPV / Hib), or any combination of the above concomitant administration regimes. 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. [Background technology]

[0002] Vaccines for protection against viral infections have been effectively used 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, the limited viral replication is sufficient to allow the full repertoire of viral antigens to be expressed, which can generate a strong and long-lasting immune response against the virus. Thus, if later exposed to a pathogenic virus strain, the immunized individual is protected from disease. 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. To date, four serotypes of dengue virus have been identified: dengue-1 (DENV-1), dengue-2 (DENV-2), dengue-3 (DENV-3), and dengue-4 (DENV-4). Dengue virus serotypes 1 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. The 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 constitute the most significant mosquito-borne viral threat to humans there. Dengue viruses are transmitted to humans primarily by the Aedes aegypti mosquito, although Aedes Dengue virus is also transmitted by the Dengue virus S. albopictus mosquito. Infection with one dengue virus serotype confers lifelong protection from reinfection with that serotype, but does not prevent secondary infection with one of the other three dengue virus serotypes. In fact, 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 quadrivalent dengue vaccine CYD-TDV (Dengvaxia®, Sanofi Pasteur, Lyon, France), has been shown to virologically inhibit 56–61% of children in Asia and Latin America. It has been licensed in several countries based on clinical demonstration of overall vaccine efficacy (VE) against experimentally confirmed dengue fever (VCD) (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 not previously infected with dengue virus (seronegative populations). Therefore, Dengvaxia® is only recommended for use in individuals previously infected with at least one dengue virus serotype (seropositive population). More specifically, according to the European Medicines Agency's European Public Assessment Report (EPAR) for the product, Dengvaxia® is only for use in people aged 9 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. SRHadinegoro et al., The New England Journal of Medicine, Vol. 373, page 1195, "Efficacy The study "Long-Term Safety of a Dengue Vaccine in Regions of Endemic Disease" reports a pooled risk of hospitalization for virologically confirmed dengue disease among people under 9 years of age of 1.58, indicating an increased risk for vaccinated people for severe dengue. This leaves a substantial unmet need for an effective vaccine with a good safety profile in both dengue-naive and seropositive individuals, including those dengue-naive populations residing in endemic areas, younger individuals who may never have developed any seropositivity to or been exposed to dengue, and travelers and individuals from non-endemic areas. There is also a need for outbreak control or travel vaccination that provides a reduction in 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 an unknown serostatus for dengue cannot be vaccinated with Dengvaxia®.

[0006] Therefore, a dengue vaccine and corresponding vaccines that stimulate an immune response to all dengue serotypes, preferably a balanced immune response to all serotypes, in both seronegative and seropositive populations, that are safe for a wider age group, especially for subjects under 9 years of age, and that protect against dengue disease of any severity (including DSS, DHF) are desirable. There is a need for a safe and effective vaccine that can protect all populations, including both seronegative and seropositive populations, particularly children and young adults, and elderly subjects, in epidemic situations and for travel purposes, representing 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 is administered in fewer doses than the current Dengvaxia® dosing schedule of three doses spaced six months apart, for example, a vaccine that can be administered in two doses or only one dose to be effective.

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

[0010] Yellow fever (YF) is an acute viral hemorrhagic disease transmitted by infected mosquitoes of the Aedes aegypti species. Symptoms of yellow fever take 3 to 6 days to develop and include fever, headache, jaundice, muscle pain, nausea, vomiting, and fatigue. A small percentage of patients (approximately 15% of people) who become infected with the virus develop severe illness that can lead to bleeding, shock, organ failure, and sometimes death. The virus is endemic in tropical regions of Africa and Central and South America.

[0011] Dengue and yellow fever (YF) viruses belong to the same Flaviviridae family and share antigenic determinants, which can lead to cross-reactive antibodies. They are both transmitted by mosquitoes (mainly Aedes aegypti) and are endemic in tropical regions of Africa and Central and South America with major public health impacts.

[0012] Currently, there is only one commercially available YF vaccine, the Yellow Fever Vaccine (YF-17D vaccine), which is based on a live-attenuated virus strain and is administered as a single subcutaneous injection. The YF-17D vaccine is highly effective (close to 100%) and generally safe, except for extremely rare cases of vaccine-associated neurotropic and viscerotropic disease. Vaccination against YF is also required for travelers to certain countries according to the International Health Regulations and is recommended by the WHO for all subjects traveling to areas with evidence of persistent or periodic YF virus transmission. The YF-17D vaccine is available from Sanofi under the product name YF-VAX®.

[0013] Yellow fever vaccine is recommended for people 9 months of age and older who reside in or travel to endemic areas and for people traveling to or through countries in need. A single dose of subcutaneously administered yellow fever vaccine is usually sufficient to confer lasting, lifelong protective immunity against yellow fever. However, booster doses are recommended every 10 years for people who remain at risk.

[0014] Therefore, there is a need for a safe and effective method for simultaneously preventing dengue and yellow fever diseases.

[0015] 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.

[0016] 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. People who recover from the illness may take weeks to months 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 hygiene standards, most children (90%) have been infected with hepatitis A virus before the age of 10.

[0017] 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.

[0018] For conventional hepatitis A vaccination, a two-dose schedule is recommended, particularly in travelers at substantial risk of contracting hepatitis A and in immunocompromised individuals. 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 of age) consists of a primary dose administered intramuscularly, followed by a further booster dose administered intramuscularly 6 to 18 months later.

[0019] Available hepatitis A vaccines include HAVRIX® and VAQTA®.

[0020] Therefore, there is a need for a safe and effective method for simultaneously preventing dengue disease and hepatitis A.

[0021] Human papillomavirus (HPV) is a common virus that is transmitted from one person to another through direct skin-to-skin contact during sexual activity. Most sexually active people will be infected with HPV at some point in their lives, with infections most common in people in their late teens and early twenties. There are approximately 40 types of HPV that can infect the genital areas of men and women. While most HPV types do not cause symptoms and are eliminated by the body's immune system, some HPV types are persistent and can cause cervical cancer in women and other less common cancers, such as cancer of the anus, penis, vagina, vulva, and oropharynx, or warts in the genital areas of men and women (called genital warts).

[0022] HPV vaccination prevents HPV-associated cervical cancer and HPV-related cancers of the anus, vulva, vagina, and oropharynx. HPV vaccination may prevent HPV-related cancers. Vaccination may also prevent HPV-related genital warts. HPV vaccination is particularly recommended for girls ages 11 and 12. It is also recommended for girls and women ages 13 to 26 who have not yet been vaccinated or have not completed the vaccine series. The HPV vaccine may also be offered to girls starting at age 9. The CDC recommends that girls ages 11 to 12 receive two doses of the HPV vaccine to protect against cancers caused by HPV. More recently, vaccination of boys in the same age range has also been recommended.

[0023] The conventional HPV vaccination schedule for young adults who begin their vaccination series before their 15th birthday includes two doses of HPV vaccine. The two doses are usually separated by 6 to 12 months. The minimum interval between doses is 5 calendar months. A three-dose schedule is recommended for subjects who begin their series after their 15th birthday and for subjects with certain immunocompromising conditions (such as cancer, HIV infection, or taking immunosuppressive medications). The second dose is usually given 1 to 2 months after the first dose, and the third dose is given 6 months after the first dose. The minimum interval between the first and second doses of the vaccine is usually 4 weeks. The minimum interval between the second and third doses of the vaccine is usually 12 weeks. The minimum interval between the first and third doses is usually 5 calendar months. If the vaccination series is interrupted, there is no need to restart the series.

[0024] Available HPV vaccines include Gardasil® 9, a recombinant 9-valent HPV (9vHPV) vaccine for protection against HPV serotypes 6, 11, 16, 18, 31, 33, 45, 52, and 58. The HPV vaccine does not contain any live or inactivated HPV, but does contain the L1 protein of each HPV serotype.

[0025] Therefore, there is a need for a safe and effective method to simultaneously prevent dengue disease and HPV-associated cancers or genital warts.

[0026] Measles is a highly contagious infectious disease caused by the measles virus, a single-stranded, negative-sense, non-enveloped RNA virus of the genus Morbillivirus in the family Paramyxoviridae. Complications occur in approximately 30% of cases and can include diarrhea, blindness, brain inflammation, and pneumonia, among others. Encephalitis occurs in approximately 1 in every 2,000 reported cases; survivors often have permanent brain damage and mental retardation. Death, primarily from respiratory and neurological causes, occurs in 1 in every 3,000 reported measles cases. The risk of death is higher in infants and adults than in children and young adults. Measles contraction during pregnancy increases the risk to the fetus. Most commonly, this risk is associated with preterm birth, with moderate increases in the rate of spontaneous abortion and low birth weight. Subacute sclerosing panencephalitis, a slow-onset viral infection of the central nervous system, has been associated with the measles virus. Measles is an airborne disease that spreads easily through the coughing and sneezing of infected people and can also be spread through contact with saliva or nasal secretions.

[0027] Mumps is an acute illness of children and young adults caused by the mumps virus, a single-stranded negative-stranded RNA virus of the genus Rubulavirus in the family Paramyxoviridae. The mumps virus causes no symptoms in approximately one-third of infected people. In those who have a clinical reaction, glandular and nervous tissue are most often affected, and the most common symptoms include fever and swelling of the parotid glands. Complications may include meningitis (15%), pancreatitis (4%), cardiac inflammation, or permanent hearing loss. Frequent viruria and abnormal renal function suggest that the mumps virus may affect the kidneys. Mumps is highly contagious and spreads rapidly among people living closely together via respiratory droplets or direct contact with infected individuals.

[0028] Rubella (German measles) is an infectious disease caused by the rubella virus, a single-stranded, positive-stranded RNA virus of the genus Rubivirus in the family Togaviridae. The virus usually causes a mild illness with few constitutional symptoms and most commonly occurs in childhood. However, if the infection occurs in a woman during early pregnancy, the virus can cross the placenta and reach the fetus, in which case the infection can induce birth defects. These defects can be severe and permanent, including congenital heart disease, cataract formation, hearing loss, and mental retardation. Rubella is usually spread through the air by coughing in infected people.

[0029] Combination vaccines for measles, mumps, and rubella (MMR) are widely used to immunize children in certain parts of the world because of their advantages over the individual vaccines: the combination vaccine simultaneously induces an adequate immune response in children against the three infectious diseases.

[0030] The MMR vaccine is indicated for simultaneous vaccination against measles, mumps, and rubella in individuals 12 months of age and older. Individuals initially vaccinated at 12 months of age and older should be revaccinated before entering elementary school. Revaccination is intended to seroconvert those who do not respond to the first dose. The Advisory Committee on Immunization Practices (ACIP) recommends administering the first dose at 12 to 15 months of age and the second dose at 4 to 6 years of age.

[0031] Therefore, there is a need for a safe and effective method to simultaneously prevent dengue fever disease as well as measles, mumps and rubella.

[0032] Tetanus is caused by infection with the bacterium Clostridium tetani, which is commonly found in soil, saliva, dust, and manure. The bacterium typically enters the body through a break in the skin, such as a cut or puncture by a contaminated object. The bacterium produces a toxin that interferes with muscle contractions, resulting in the classic symptom of muscle cramps. It affects the brain and nervous system, causing extremely painful muscle spasms, usually throughout the body. Jaw spasms can make it impossible to open the mouth (a condition called "trismus"). Tetanus kills one in ten people who contract the disease.

[0033] Diphtheria is an infectious disease caused by the bacterium Corynebacterium diphtheriae, which primarily infects the throat and upper respiratory tract and produces a toxin that can affect other organs. Diphtheria develops acutely and is primarily characterized by sore throat, mild fever, and swelling in the glands of the neck. In severe cases, the toxin can cause myocarditis or peripheral neuropathy. Diphtheria toxin causes a membrane of dead tissue to build up over the throat and tonsils, making breathing and swallowing difficult. Diphtheria is a highly contagious infection, and the bacteria usually spread between people by direct contact or through the air, but can also be spread by contaminated objects.

[0034] Whooping cough, or whooping cough, caused by the bacterium Bordetella pertussis, is an airborne disease that causes an extremely contagious respiratory infection that can lead to severe breathing problems, especially in young children. Whooping cough initially appears like a common cold but then causes violent, uncontrollable coughing fits that can cause difficulty breathing, vomiting, and sleep problems. A person may cough so hard that they vomit, break ribs, or become extremely exhausted from trying to exhale. Children under one year of age may have little or no coughing, instead spending periods not breathing. A high-pitched "whistle" sound can be heard when a person tries to breathe after coughing. Complications include pneumonia or death. While whooping cough can affect people of all ages, it is extremely serious and can be fatal in newborns under one year of age. It is even possible that...

[0035] Tdap is a combination vaccine that protects against three potentially life-threatening bacterial diseases: tetanus, diphtheria, and whooping cough (whooping cough). Tdap stands for tetanus and diphtheria toxoid and acellular pertussis. Tdap is an inactivated vaccine produced using killed cells. Vaccination against tetanus, diphtheria, and pertussis during childhood (children under 7 years of age) is recommended using specific diphtheria toxoid, tetanus toxoid, and acellular pertussis adsorbed vaccines, such as INFANRIX® manufactured by GlaxoSmithKline. A further booster Tdap vaccine is recommended for children over 10 years of age to ensure that immunity against tetanus, diphtheria, and pertussis is maintained into adulthood. A booster Tdap vaccine based on combined tetanus toxoid, attenuated diphtheria toxoid, and acellular pertussis (adsorbed) is available from GlaxoSmithKline under the brand name BOOSTRIX®. Tdap vaccination is administered intramuscularly as a single dose.

[0036] Therefore, there is a need for a safe and effective method of simultaneously preventing dengue disease as well as tetanus, diphtheria, and whooping cough.

[0037] In addition to diphtheria, tetanus, and whooping cough, acute poliomyelitis, often called polio or infantile paralysis, is a highly contagious viral disease caused by the poliovirus. It invades the nervous system and can cause total paralysis within hours. The virus is primarily transmitted by person-to-person spread via the fecal-oral route or, less frequently, through common vectors (e.g., contaminated water or food) and replicates in the intestines. Early symptoms include fever, fatigue, headache, vomiting, stiff neck, and pain in the hands and feet. One in 200 infections leads to irreversible paralysis (usually in the legs). Of those paralyzed, 5% to 10% die when the respiratory muscles become rigid. Polio primarily affects children under the age of five. There is no cure for polio; it can only be prevented.

[0038] Haemophilus influenzae type b (Hib) is a bacterium that causes severe pneumonia, meningitis, and other invasive diseases almost exclusively in children under 5 years of age. It is transmitted via the respiratory tract from infected individuals to susceptible individuals. Hib also causes potentially severe inflammatory infections of the face, mouth, blood, epiglottis, joints, heart, bones, peritoneum, and trachea. Although this problem occurs worldwide, the burden of Hib disease was significantly greater in resource-poor countries prior to the introduction of vaccines into national immunization programs. In 2000, Hib was estimated to have caused 2 to 3 million cases of severe illness, particularly pneumonia and meningitis, and 386,000 deaths in young children. Hib disease is observed in all regions of the world but is difficult to confirm because it requires rapid laboratory testing in patients who have not previously received antibiotic treatment.

[0039] Commercially available DTaP / IPV / Hib combination vaccines include Pentacel®, which is based on a combination of diphtheria toxoid, tetanus toxoid, acellular pertussis (adsorbed), and inactivated poliovirus combined with a Hib conjugate vaccine.

[0040] Therefore, there is a need for a safe and effective method of simultaneously preventing dengue fever disease as well as diphtheria, tetanus, whooping cough, poliomyelitis, and disease caused by Haemophilus influenzae type b.

[0041] Furthermore, elderly subjects infected with dengue virus often present with atypical symptoms, In some cases, fever may be the only symptom, and older adults are less likely to present with typical symptoms and signs of dengue fever, which can be defined as fever plus at least one of the following: bone pain, myalgia, arthralgia, retroorbital pain, headache, and papular rash (Lee et al. (2013) Am. J. Emerg. Med. 31(5):783-787). However, older adults are at higher risk of developing severe dengue fever and DHF / DSS compared with younger subjects (Liu et al. (2008) Am. J. Infect. Dis. 4(1):10-17; Lin et al. (2012) Emerg. Infect. Dis. 18(10):2003-1009; Rowe et al. (2014) PloS Negl. Trop. Dis. 8(4)). Several reasons that may contribute to severe illness in the elderly have been discussed, such as decreased physiological and immune function, increased likelihood of contracting secondary dengue fever, and increased prevalence of chronic diseases and other comorbidities in the elderly, i.e., over 60 years of age (Lin et al. (2017) Expert (See discussion in Review of Anti-infective Therapy 15(8):729-735). Summary of the Invention

[0042] The object of the present invention is to provide a safe and effective vaccine against all serotypes of dengue virus for dengue endemic and non-dengue endemic populations and for subjects over a wide age range, particularly between 2 months and 60 years of age, independent of previous exposure to dengue virus and the corresponding seropositive or seronegative status before vaccination, and a corresponding vaccination method.

[0043] The objective of the present invention is to minimize the risk of DHF and DSS caused by infection with DENV-1, DENV-2, DENV-3 or DENV-4 after vaccination, particularly in young children and people of any age who have not previously been exposed to dengue or who are seronegative for dengue prior to vaccination.

[0044] The object of the present invention is to provide a vaccine and a corresponding vaccination method for controlling the dengue fever pandemic situation.

[0045] It is an object of the present invention to provide a vaccine and corresponding method of administration that is useful in settings less familiar with or less resourced for the clinical management of dengue fever.

[0046] The object of the present invention is to provide a vaccine and a corresponding method of vaccination that avoids testing of individual serostatus before each vaccination or analysis of seroprevalence in mass-vaccinated areas.

[0047] It is an object of the present invention to provide a vaccine and a corresponding vaccination method that reduces and / or avoids the risk of subsequent antibody-dependent enhancement.

[0048] It is an object of the present invention to provide vaccines and corresponding methods that reduce and / or avoid dengue NS1 toxicosis.

[0049] It is an object of the present invention to provide a vaccine and corresponding vaccination method that induces cross-reactive multitype antibodies and / or cell-mediated immunity.

[0050] One object of the present invention is to provide safe and effective protection against dengue and yellow fever diseases.

[0051] Another object of the present invention is to provide safe and effective protection against dengue disease and hepatitis A.

[0052] A further object of the present invention is to provide safe and effective protection against dengue disease and HPV-associated cancers or genital warts.

[0053] A stated object of the present invention is to provide safe and effective protection against dengue fever disease as well as measles, mumps and rubella.

[0054] Another object of the present invention is to provide safe and effective protection against dengue fever disease as well as tetanus, diphtheria, and whooping cough.

[0055] Another object of the present invention is to provide safe and effective protection against dengue fever disease and diseases caused by diphtheria, tetanus, whooping cough, poliomyelitis and Haemophilus influenzae type b.

[0056] Another object of the present invention is to provide safe and effective protection against any combination of the above diseases.

[0057] Another object of the present invention is to provide a vaccine that stimulates a balanced immune response against all four dengue serotypes in subjects, particularly elderly subjects (ie, over 60 years of age).

[0058] Thus, the present invention is directed, in part, to a dengue vaccine for methods of mass vaccination without testing individuals for serostatus or prior assessment of seroprevalence, including in endemic areas, including pandemic situations, and in non-endemic areas for travelers.

[0059] Thus, the present invention provides a tetravalent dengue virus composition comprising four live attenuated dengue virus strains representing serotype 1, serotype 2, serotype 3 and serotype 4, preferably the tetravalent dengue virus composition comprises a chimeric dengue serotype 2 / 1 strain and a dengue serotype 2 strain and a chimeric dengue serotype 2 / 3 strain and a chimeric dengue serotype 2 / 4 strain, preferably the dengue serotype 2 strain is derived from the wild-type virus strain DEN-2 16681 (represented by SEQ ID NO: 11) and 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) c) NS3-250 Glu to Val (nt-5270 A to T); 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 chimera The present invention relates to a tetravalent dengue virus composition derived from a serotype 2 strain by providing:

[0060] The present invention particularly relates to a composition or lyophilized unit dose which, 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) Dengue serotype 4 at a concentration of at least 4.5 log pfu / 0.5 ml The present invention is directed to a composition or lyophilized unit dose comprising:

[0061] The present invention is also particularly directed to such compositions or unit doses, which, upon reconstitution with a pharmaceutically acceptable diluent, provide a total concentration of (i), (ii), (iii), and (iv) in pfu / 0.5 mL, wherein, based on said 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 8%, or preferably at least 10%, or at least 12%, or at least 14%, or at least 16%, or at least 18%.

[0062] The present invention is also particularly directed to such methods and uses, wherein, upon reconstitution with a pharmaceutically acceptable diluent, (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL, wherein, 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%.

[0063] Thus, the present invention provides a method and corresponding use comprising the use of a tetravalent dengue virus composition comprising four live attenuated dengue virus strains representing serotype 1, serotype 2, serotype 3 and serotype 4, the method being directed to inoculating a subject against virologically confirmable dengue disease, in particular 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, in particular the dengue serotype 2 strains are derived from the wild-type virus strain DEN-2 16681 (represented by SEQ ID NO: 11) and differ from its wild-type counterpart in at least three nucleotides as follows: d) 5'-noncoding region (NCR)-57 (nt-57 C to T) e) NS1-53 Gly to Asp (nt-2579 G to A) f) NS3-250 Glu to Val (nt-5270 A to T); 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 chimera The present invention relates to a method and corresponding uses of a serotype 2 strain derived from the serotype 2 strain by providing:

[0064] The present invention particularly relates to such methods and uses, wherein the unit dose 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) Dengue serotype 4 at a concentration of at least 4.5 log pfu / 0.5 ml The present invention is directed to methods and uses, including

[0065] The invention also relates to such methods and uses, in particular, wherein, upon reconstitution with a pharmaceutically acceptable diluent, (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL, and based on said total concentration, the concentration of (ii) in pfu / 0.5 mL is: the concentration of (i) in pfu / 0.5mL is at least 1%, or the concentration of (iii) in pfu / 0.5mL is at least 8%, or at least 10%, or at least 12%, or at least 14%, or at least 16%, or at least 18%, and the subject is preferably 2 to 17 years of age or 4 to 16 years of age.

[0066] The invention is also particularly directed to such methods and uses, wherein, upon reconstitution with a pharmaceutically acceptable diluent, (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL, wherein, based on said total concentration, the concentration of (ii) at pfu / 0.5 mL is less than 2%, the concentration of (iv) at pfu / 0.5 mL is at least 50%, the concentration of (i) at pfu / 0.5 mL is at least 1%, and the concentration of (iii) at pfu / 0.5 mL is at least 6%, and the subject is 18 to 60 years of age.

[0067] The present invention therefore relates to a method and a corresponding use, the method comprising: (A) administering to a subject a first unit dose of a tetravalent dengue virus composition; and (B) administering to the subject a second unit dose of the tetravalent dengue virus composition within three months of administering the first unit dose. a primary vaccination with only two administrations of a unit dose, comprising Preferably, the methods and uses do not include a step of determining whether there has been a previous dengue infection in the subject prior to administration of the unit dose, or the serostatus of the subject is unknown prior to administration of the unit dose, or the methods and uses are directed to methods and corresponding uses which preferably do not include a step of determining whether there has been a previous dengue infection in the subject, or the serostatus of the subject is preferably unknown at any time before, during or after the step of administration.

[0068] According to one embodiment, such methods and uses do not include active monitoring of subjects 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 facility for dengue evaluation. Subjects / guardians are contacted at least weekly to ensure positive identification of febrile illness by reminding the subject / guardian of their obligation to return to the testing facility 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 facility.

[0069] According to one embodiment, such methods and uses do not include vaccine immunogenicity assays involving GMT for dengue neutralizing antibodies.

[0070] According to one embodiment, such methods and uses do not comprise a reactogenicity analysis, e.g., relating to solitary local AEs (injection site pain, injection site erythema, and injection site swelling) and solitary systemic AEs (children <6 years: fever, irritability / tantrums, drowsiness, and loss of appetite; children ≥6 years: asthenia, fever, headache, malaise, and muscle pain), assessed respectively for 7 days and 14 days (including the day of vaccination) after each vaccination, e.g., by collecting diary cards.

[0071] The methods according to the invention do not require testing of serostatus before vaccination, thus allowing for immediate treatment and outbreak control. According to certain embodiments, the invention is directed to methods and uses wherein the subject is exposed to a dengue outbreak. In certain such embodiments, the outbreak is caused by dengue serotype 2 and / or is caused by serotype 1.

[0072] According to certain embodiments, the method of inoculation against virologically confirmable dengue disease is caused by dengue serotype 2 and / or is caused by dengue serotype 1.

[0073] According to one embodiment, the method comprises: (A) selecting a subject eligible for administration of a unit dose of a tetravalent dengue virus composition in need of protection against dengue infection without a determination of past dengue infection; and (B) administering to the subject a first unit dose of a tetravalent dengue virus composition; and (C) administering to the subject a second unit dose of the tetravalent dengue virus composition within three months of administering the first unit dose. This includes a primary vaccination consisting of: Thus, the method of inoculation is completed without determining past dengue infection. The method further optionally includes a booster dose of the unit dose at least one year after administration of the second unit dose.

[0074] According to one embodiment, the method and use is 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 serotypes 1, 2, 3 and 4, wherein the dengue serotype 2 strain is derived from the wild-type virus strain DEN-2 16681 (represented by SEQ ID NO: 11) and 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) c) NS3-250 Glu to Val (nt-5270 A to T); 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 chimera derived from serotype 2 strains by causing The method is: (A) selecting a subject eligible for administration of a unit dose of a tetravalent dengue virus composition in need of protection against dengue infection without determining whether the subject has had a previous dengue infection; and (B) administering to the subject a first unit dose of a tetravalent dengue virus composition; and (C) administering to the subject a second unit dose of the tetravalent dengue virus composition within three months of administering the first unit dose. The present invention relates to a method for administering a primary vaccination to a patient, the method comprising administering a primary vaccination to a patient in need thereof. Thus, the method of inoculation is completed without determining past dengue infection. The method further optionally includes a booster dose of the unit dose at least one year after administration of the second unit dose.

[0075] According to the present invention, the methods and uses are applicable to subjects of all ages, and the present invention is particularly directed to such methods and uses, in which 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 18-60 years old, or 18-45 years old, or 46-60 years old, or over 60 years old.

[0076] In particular, the present invention is directed to such uses where the method is safe.

[0077] In particular, the present invention relates to such a use, wherein the method provides a combined 95% confidence interval for virologically confirmed dengue fever with hospitalization for all four serotypes. The vaccine efficacy, the lower limit of which is intended for use, provides a combined vaccine efficacy of greater than 65% from the first dose of the dosing schedule to 12 to 18 months after the last dose of the dosing schedule, when measured against placebo in a target population of at least 5,000 healthy subjects aged 4 to 16 years, regardless of baseline serostatus.

[0078] In particular, the present invention is directed to such uses in which the method is effective.

[0079] In particular, the present invention is directed to such uses, wherein the method provides a combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease with a two-sided 95% confidence interval, the lower limit of which is greater than 60% from the first dose of the dosing schedule to 18 months after the last dose of the dosing schedule, when measured against placebo in a target population of at least 5,000 healthy subjects aged 14-16 years, regardless of serostatus at baseline.

[0080] The invention also provides, in part, a reconstituted dengue vaccine composition for use in a method of preventing virologically confirmable dengue disease in a subject, comprising sequentially administering to the subject at least a first and a second unit dose of a dengue vaccine composition, said first and second unit doses being administered subcutaneously within three months and at least four weeks apart, optionally at about day 1 and about day 90, wherein the dengue vaccine composition is a tetravalent dengue virus composition comprising four dengue virus strains representing dengue serotype 1, dengue serotype 2, dengue serotype 3 and dengue serotype 4, optionally wherein the dengue virus strains are live-attenuated, and which, upon reconstitution with 0.5 mL of a pharmaceutically acceptable diluent, (i) dengue serotype 1 has a concentration of at least 3.3 log pfu / 0.5 mL, optionally up to 5.0 log pfu / 0.5 mL; (ii) dengue serotype 2 has a concentration of at least 2.7 log pfu / 0.5 mL, optionally up to 4.9 log pfu / 0.5 mL; (iii) dengue serotype 3 has a concentration of at least 4.0 log pfu / 0.5 mL and optionally up to 5.7 log pfu / 0.5 mL; (iv) Dengue serotype 4 is directed to a reconstituted dengue vaccine composition having a concentration of at least 4.5 log10 pfu / 0.5 mL, and optionally up to 6.2 log10 pfu / 0.5 mL.

[0081] Thus, the present invention provides, in part, a dengue vaccine composition for use in a method of preventing virologically confirmable dengue disease (VCD) in a subject, comprising sequentially administering to the subject at least first and second unit doses of a dengue vaccine composition, said first and second unit doses being administered subcutaneously within three months and at least four weeks apart, optionally at about day 1 and about day 90, wherein the dengue vaccine composition is a tetravalent dengue virus comprising four live attenuated dengue virus strains representing dengue serotype 1, dengue serotype 2, dengue serotype 3, and dengue serotype 4. The present invention relates to a dengue vaccine composition, wherein the attenuated dengue virus strain comprises a chimeric dengue virus and preferably at least one non-chimeric dengue virus, and wherein dengue serotype 1 and dengue serotype 2 are each present at a concentration within 5 percentage points of each other, based on the total concentration in pfu / 0.5 mL, and / or together are less than about 10% of the total concentration in pfu / 0.5 mL, and particularly dengue serotype 3 is at least about 10% of the total concentration in pfu / 0.5 mL, and particularly dengue serotype 4 is at least about 70% of the total concentration in pfu / 0.5 mL.

[0082] Thus, the present invention relates, in part, to a unit dose of a dengue vaccine composition and uses thereof, the unit dose comprising: Four live attenuated dengue serotypes (e.g., virus strains): (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strains), (ii) dengue serotype 2 (e.g., dengue serotype 2 strains); (iii) dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strains); (iv) dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) a tetravalent dengue virus composition comprising: and one or more pharmaceutically acceptable excipients thereof. The present invention relates to a unit dose of a dengue vaccine composition comprising:

[0083] The invention further relates, in part, to a unit dose of a dengue vaccine composition and uses thereof, the unit dose comprising: Four live attenuated dengue virus strains: (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., a chimeric dengue serotype 2 / 3 strain) at a concentration of at least 4.0 log pfu / 0.5 ml; and (iv) dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) at a concentration of at least 4.5 log pfu / 0.5 ml a tetravalent virus composition comprising: and one or more pharmaceutically acceptable excipients The present invention relates to a unit dose of a dengue vaccine composition comprising:

[0084] The invention further relates, in part, to a unit dose of a dengue vaccine composition and uses thereof, the unit dose comprising: a tetravalent virus composition comprising four live attenuated dengue virus strains, the unit dose being lyophilized and, upon reconstitution with 0.5 mL of a 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 (iv) dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) at a concentration of at least 4.5 log pfu / 0.5 ml The present invention relates to a unit dose of a dengue vaccine composition comprising:

[0085] The invention further relates, in part, to a unit dose of a dengue vaccine composition and uses thereof, said unit dose being lyophilized; Four live attenuated dengue virus strains: (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 (iv) dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) at a concentration of at least 4.5 log pfu / 0.5 ml a tetravalent virus composition comprising: and one or more pharmaceutically acceptable excipients and uses thereof.

[0086] The invention is particularly directed to such a unit dose or composition which, upon reconstitution with a pharmaceutically acceptable diluent, provides a total concentration of (i), (ii), (iii), and (iv) at pfu / 0.5 mL, wherein, based on said 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%, or at least 10%, or at least 12%, or at least 14%, or at least 16%, or at least 18%, and particularly wherein the concentration of (iii) at pfu / 0.5 mL is at least 10%.

[0087] The present invention is particularly directed to such unit doses and compositions which, upon reconstitution with a pharmaceutically acceptable diluent, provide a total concentration of (i), (ii), (iii), and (iv) at pfu / 0.5 mL, wherein, 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%.

[0088] The invention further relates, in part, to a kit for preparing a reconstituted unit dose and its use, the kit comprising the following components: a) a lyophilized unit dose of the invention as described herein, and b) Concerning the kit and its use, including a pharmaceutically acceptable diluent for reconstitution.

[0089] The invention is further directed, in part, to a container, such as a vial, containing 1 to 10 unit doses of the invention described herein.

[0090] The present invention is further directed to a method of preventing dengue disease in a subject, comprising administering to the subject, e.g., by subcutaneous injection, a reconstituted unit dose of a dengue vaccine composition described herein.

[0091] The present invention is further directed to the use of a reconstituted unit dose of a dengue vaccine composition described herein for the manufacture of a medicament for preventing dengue disease in a subject, e.g., by subcutaneous injection.

[0092] The present invention is further directed to a reconstituted unit dose of a dengue vaccine composition described herein for use in a method of preventing dengue disease in a subject, for example, by subcutaneous injection.

[0093] The present invention is further directed to a method of preventing dengue disease in a population of subjects, comprising administering to the population a reconstituted unit dose of a vaccine composition described herein, wherein the population of subjects is seronegative to all dengue serotypes, wherein the geometric mean neutralizing antibody titers (GMTs) when tested in at least 40, or at least 50, or at least 60 subjects at least 180 days or 365 days after a first administration of said unit dose and, optionally, a second administration of said unit dose 90 days after said first administration, are 50 or less, or 40 or less, or 30 or less, or 20 or less, and optionally, a ratio of GMT for dengue serotype 2 to GMT for dengue serotype 1 (GMT DENV-2:GMT DENV-1) of 20 or less, and and optionally a ratio of GMT of dengue serotype 2 to GMT of dengue serotype 3 (GMT DENV-2:GMT DENV-3) of 20 or less.

[0094] The present invention is further directed to a method of preventing dengue disease in a subject comprising administering to the subject a reconstituted unit dose of a vaccine composition described herein, wherein the subject is seronegative to all dengue serotypes, wherein neutralizing antibody titers, when tested in the subject at least 180 days or 365 days after a first administration of said unit dose and, optionally, a second administration of said unit dose 90 days after said first administration, may provide a ratio of dengue serotype 2 neutralizing antibody titers to dengue serotype 4 neutralizing antibody titers of 50 or less, or 40 or less, or 30 or less, or 20 or less, and optionally a ratio of dengue serotype 2 neutralizing antibody titers to dengue serotype 1 neutralizing antibody titers of 20 or less, and optionally a ratio of dengue serotype 2 neutralizing antibody titers to dengue serotype 3 neutralizing antibody titers of 20 or less.

[0095] In one preferred embodiment, the methods of preventing dengue disease of the present invention are not associated with an increased likelihood of involuntary systemic adverse events, for example, in children under the age of 9 or in seronegative individuals.

[0096] Thus, the present invention is directed, in part, to methods for preventing dengue and yellow fever diseases.

[0097] Thus, the present invention is directed, in part, to methods of preventing dengue disease and hepatitis A.

[0098] Thus, the present invention is directed, in part, to methods of preventing dengue disease and HPV-associated cancers or genital warts.

[0099] Thus, the present invention is directed, in part, to methods of preventing dengue fever disease as well as measles, mumps, and rubella.

[0100] Thus, the present invention is directed, in part, to methods of preventing dengue fever disease as well as tetanus, diphtheria, and whooping cough.

[0101] Thus, the present invention is directed, in part, to methods of preventing dengue fever disease, as well as diphtheria, tetanus, whooping cough, poliomyelitis, and disease caused by Haemophilus influenzae type b.

[0102] As such, this invention is also directed, in part, to a method for preventing any combination of the above diseases.

[0103] The invention is further directed, in part, to the use of a reconstituted unit dose of a dengue vaccine composition / tetravalent dengue virus composition described herein for the manufacture of a medicament for preventing dengue disease in a subject, e.g., by subcutaneous injection.

[0104] The present invention is further directed, in part, to a reconstituted unit dose of the dengue vaccine composition / tetravalent dengue virus composition described herein for use in a method of preventing dengue disease in a subject, as described herein. definition

[0105] In describing the present invention, the following terminology shall be used as indicated below. As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0106] As used herein, the terms "unit dose of a dengue vaccine composition," "unit dose," and "unit dose of the invention described herein" refer to an amount of a dengue vaccine administered to a subject in a single dose. In one embodiment, one unit dose is present in a vial, and this unit dose is administered to a subject, e.g., optionally after reconstitution. In one embodiment, more than one unit dose of a dengue vaccine composition may be present in a vial, such that the contents of one vial can vaccinate more than one subject.

[0107] 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 comprising four dengue virus strains, preferably TDV-1 to TDV-4, is subjected to lyophilization to obtain a lyophilized unit dose.

[0108] 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.

[0109] As used herein, the term "upon reconstitution in 0.5 mL" refers to the concentration of dengue virus that would be present in a reconstituted unit dose if 0.5 mL of diluent were 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.

[0110] 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 has a volume of 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.

[0111] 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).

[0112] As used herein, the term "tetravalent dengue virus composition" refers to a composition that comprises four different immunogenic components, preferably four different live attenuated dengue viruses (each representing one dengue serotype), derived from four different dengue serotypes, DENV-1, DENV-2, DENV-3, and DENV-4, and that is capable of providing immunogenicity against all four dengue serotypes. It refers to a dengue virus composition that aims to stimulate an immune response.

[0113] As used herein, the term "live attenuated dengue virus" refers to a viable dengue virus that has been mutated to provide reduced pathogenicity. A live attenuated dengue virus can be a dengue virus in which all components are derived from the same dengue serotype, or it can be a chimeric dengue virus having portions from more than one dengue serotype.

[0114] A "virus strain," and particularly a "dengue virus strain," is a genetic subtype of a virus, particularly 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.

[0115] 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.

[0116] A "non-chimeric dengue virus" or "non-chimeric dengue serotype strain" or "non-chimeric dengue strain" contains portions from only one dengue serotype. In particular, a non-chimeric dengue virus does not contain portions from different flaviviruses, such as yellow fever virus, Zika virus, West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, or tick-borne encephalitis virus. TDV-2 is an example of a non-chimeric dengue virus.

[0117] 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, such as yellow fever virus, Zika virus, West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, or tick-borne encephalitis virus. Notably, 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. Notably, 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. In particular, 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. In particular, 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.

[0118] 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 by way of example), TDV-1 has a nucleotide sequence according to SEQ ID NO: 1 and / or an amino acid sequence according to SEQ ID NO: 2. In one embodiment, TDV-2 has a nucleotide sequence according to SEQ ID NO: 3 and / or an amino acid sequence according to SEQ ID NO: 4. In one embodiment, TDV-3 has a nucleotide sequence according to SEQ ID NO: 5 and / or an amino acid sequence according to SEQ ID NO: 6. In one embodiment, TDV-4 has a nucleotide sequence according to SEQ ID NO: 7 and / or an amino acid sequence according to SEQ ID NO: 8.

[0119] 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 dengue fever disease also includes more severe forms of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Symptoms of DHF include increased vascular permeability, hypovolemia, and abnormal blood clotting mechanisms. Subjects with DHF may show severe signs of plasma leakage and bleeding. If a subject with DHF goes into shock, 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, such as hepatomegaly and acute renal failure.

[0120] 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. In particular, 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.

[0121] 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" refers to a subject with a febrile illness or illness clinically suspected to be dengue fever disease, where, for example, testing the subject using RT-PCR will confirm the presence of at least one dengue serotype. Severe forms of VCD fever are identified as follows: dengue hemorrhagic fever (DHF) was defined according to WHO 1997 criteria. Severe dengue fever was defined through the evaluation of an independent Dengue Case Adjudication Committee, which evaluates all hospitalized VCD cases (severe / non-severe) based on criteria redefined in the Charter. All non-hospitalized cases are considered non-severe.

[0122] As used herein, the term "febrile illness" is defined as a body temperature of ≧38° C. on any two out of three consecutive days.

[0123] As used herein, the term "virologically confirmed dengue disease with hospitalization" is considered a surrogate for severe dengue, and "incidence of virologically confirmed dengue disease with hospitalization" is used as a safety parameter. As used herein, "relative risk for virologically confirmed dengue disease with hospitalization" means the number of virologically confirmed dengue disease events with hospitalization divided by the number of subjects treated with a unit dose disclosed herein divided by the number of subjects treated with a placebo. If the "relative risk for virologically confirmed dengue disease with hospitalization" is less than or equal to 1, the vaccine provides the same or lower risk of virologically confirmed dengue disease with hospitalization than placebo and is considered "safe." In this context, the risk of virologically confirmed dengue disease involving hospitalization may also 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 up 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.

[0124] Alternatively, as used herein, a vaccine is considered "safe" if its vaccine efficacy (VE) for virologically confirmed dengue disease with hospitalization is 0% or greater. This means that the vaccine provides the same or lower likelihood of virologically confirmed dengue disease with hospitalization as 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, with the lower bound measured against placebo in a subject population of at least 1,500 or at least 2,000 healthy subjects who are seronegative for all serotypes at baseline or seropositive for at least one serotype at baseline (particularly in the age group of subjects 4 to 16 years old, the age group of subjects 4 to <9 years old, and the age group of subjects 2 to <9 years old). and 12-16 year old subjects), particularly when the unit dose or the placebo is administered at least twice within less than six months, e.g., within three months, a combined vaccine efficacy of greater than 25% from about the first dose or 30 days after the second or last dose in the dosing schedule to at least 12 months, 12 to 18 months, 12 months, or 18 months after the second or last dose in the dosing schedule. In particular, 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%. In particular, the two-sided 95% confidence intervals for combined vaccine efficacy against virologically confirmed dengue fever with hospitalization for all four serotypes provide lower limits of two-sided confidence intervals within 10 percentage points, 15 percentage points, or 20 percentage points when comparing seropositive and seronegative subjects.In certain embodiments, "safe" means providing a combined vaccine efficacy against virologically confirmed dengue fever with hospitalization for all four serotypes with a two-sided 95% confidence interval, with the lower limit being greater than 65% from the first dose of the dosing schedule to 12 to 18 months after the last dose of the dosing schedule, when measured against placebo in a target population of at least 5,000 healthy subjects aged 4 to 16 years, regardless of serostatus at baseline.

[0125] A vaccine is considered safe within the meaning of the present invention if it satisfies one of the criteria defined above for the term "safety". In this context, safety particularly refers to a vaccine that is safe for all subjects, regardless of their serostatus at baseline. This means that the vaccine is safe for all subjects, regardless of the occurrence of past dengue infection in the subjects before administration. This means that the vaccine can be administered without the need for a pre-existing condition. Preferably, the vaccine is safe as defined above for all age groups starting from 4 years of age, particularly for ages 4-60 or 4-16 years, preferably regardless of serostatus. Relevant subgroups in this context are <9 years, 2 to <9 years, 4 to <9 years, 4-5 years, 6-11 years and 12-16 years or any age group within the range 4-16 years. For a further definition of VE for virologically confirmed dengue disease with hospitalization, reference is made to the disclosure below regarding certain treatment methods.

[0126] 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 among vaccinated and unvaccinated people and determining the percentage reduction in risk of disease among vaccinated people relative to unvaccinated people. The higher the percentage reduction in disease in the vaccinated group, the higher the vaccine efficacy. For example, a VE of 90% indicates a 90% reduction in disease incidence among the vaccinated group, or a 90% reduction from the number of cases that would be expected if not vaccinated. Vaccine efficacy is calculated by the formula: 100*(1-HR), where HR is the hazard ratio, defined as the hazard rate of the vaccine (λ) divided by the hazard rate of the placebo (λ), i.e., HR=λ / λ. λv denotes the hazard rate for subjects vaccinated with the tetravalent dengue vaccine composition disclosed herein, and λc denotes the hazard rate for unvaccinated subjects, i.e., subjects receiving a placebo. Hazard rate ratios (HR) are estimated from a Cox proportional hazards model with 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 serotypes causing virologically confirmed dengue disease and the subject's baseline serostatus. A vaccine is considered "effective" if the combined vaccine efficacy is greater than 30%. In this context, the combined vaccine efficacy may also be 40% or more, 50% or more, 60% or more, 70% or more, 72% or more, or 80% or more, particularly when determined in an age group selected from the age group of subjects 4 to 16 years old, the age group of subjects 4 to under 9 years old, the age group of subjects 2 to under 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, particularly when determined from 30 days after the second dose to 12 months after the second dose or 18 months after the second vaccination.In this context, efficacious particularly refers to a vaccine that is effective for all subjects, regardless of their serostatus at baseline. Preferably, the vaccine is effective for all age groups, preferably starting from 4 years old, regardless of serostatus, and particularly for 4-60 year olds or 4-16 year olds, regardless of serostatus. Relevant subgroups in this context are under 9 years old, 2 to under 9 years old, 4 to under 9 years old, 4 to 5 years old, 6 to 11 years old, and 12 to 16 years old, or any age group within the range of 4 to 16 years old. In certain embodiments, "efficacious" means providing a combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease with a two-sided 95% confidence interval, the lower limit being greater than 60% from the first dose of the dosing schedule to 18 months after the last dose of the dosing schedule, when measured against placebo in a target population of at least 5,000 healthy subjects aged 4 to 16 years, regardless of serostatus at baseline. Further specific efficacy 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.

[0127] 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 subjects treated with a placebo. As used herein, the term "combined relative risk for all four serotypes" is defined as the relative risk for the risk of dengue disease regardless of the serotype causing virologically confirmed dengue disease and the subject's baseline serostatus.

[0128] 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 the primary vaccination unless specified as a booster vaccination. Booster vaccination refers to an administration or administration schedule that occurs after the primary vaccination, e.g., at least one year or even five or ten years after the last administration, e.g., the second administration, of the primary vaccination schedule. Booster administration attempts to enhance or re-establish the immune response of the primary vaccination.

[0129] As used herein, the term "subject" or "subjects" refers to a human subject (e.g., an infant, child, or adult). The term "elderly subject" or "elderly subjects" refers to a subject having an age greater than 60 years old, such as 61-100 years old, 61-90 years old, 61-80 years old, 61-75 years old, or 61-70 years old.

[0130] As used herein, a "subject population" refers to a group of subjects. A subject population may 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 predetermined parameters. Parameters that may 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.

[0131] As used herein, "endemic area" refers to an area where a disease or infectious agent is constantly present and / or normally prevalent in the population within that area. As used herein, "non-endemic area" refers to an area where a disease is not present or normally prevalent. Thus, a "dengue-endemic area" refers to a geographic area where dengue virus infection is constantly maintained at a baseline level. A "non-dengue-endemic area" is a geographic area where dengue virus infection is not constantly 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 subject population is dengue-endemic or not can be determined by Bhatt et al. (2013) Nature 496(7446):504-507 and supplementary materials. The dengue burden can be determined by different calculation methods, such as those described in 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 infectiousness of an area is measured by seroprevalence surveys, which provide the seroprevalence rate. Areas with extremely high infectiousness are considered to have a seroprevalence rate above 80%. As used herein, the term "area," when referring to seroprevalence, refers to a geographic area where seroprevalence may be determined or is known, e.g., a village, town, city, region, county, state, district or part of any of the foregoing or an entire country.

[0132] As used herein, "serostatus" refers to the amount of antibodies a subject has against a given infectious agent, particularly 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 equal to or greater than 10 for at least one dengue serotype is defined as "seropositive" for that dengue serotype. Serostatus at baseline refers to the serostatus prior to administration of the dengue vaccine composition described herein.

[0133] 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, "a ratio of the neutralizing antibody titer of dengue serotype 2 to the neutralizing antibody titer of dengue serotype 4 of 20 or less" means that the neutralizing antibody titer of dengue serotype 2 is divided by the neutralizing antibody titer of dengue serotype 4, and the resulting ratio 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.

[0134] As used herein, the terms "geometric mean neutralizing antibody titer" and "GMT" refer to the geometric mean of the titers of neutralizing antibodies against 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 the GMT of dengue serotype 2 to the GMT of dengue serotype 4 of 20 or less" means that the geometric mean neutralizing antibody titer of dengue serotype 2 (GMT DENV-2) is divided by the geometric mean neutralizing antibody titer of dengue serotype 4 (GMT DENV-4), and the resulting ratio is 20 or less. That is, the geometric mean neutralizing antibody titer of dengue serotype 2 is 20-fold or less higher than the geometric mean neutralizing antibody titer of dengue serotype 4 in the subject population.

[0135] As used herein, an "immune response" refers to the response of a subject to administration of a dengue vaccine. In particular, an immune response includes the formation of neutralizing antibodies against one or more dengue serotypes. It may also include the stimulation of a cell-mediated response or the formation of antibodies against nonstructural proteins such as NS1. An immune response is stimulated by administration of a unit dose of the invention described herein if the titer of neutralizing antibodies against at least one dengue virus serotype, preferably all four dengue virus serotypes, increases after said administration of said unit dose. An immune response is stimulated by administration of a unit dose of the invention described herein if the secretion of interferon gamma by peripheral blood mononuclear cells stimulated with a peptide derived from a dengue virus protein increases after said administration of said unit dose. An immune response is stimulated by administration of a unit dose of the invention described herein if the titer of antibodies against nonstructural proteins such as NS1 increases after said administration of said unit dose. 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, a cell-mediated response, and the formation of antibodies against nonstructural proteins such as NS1.

[0136] As used herein, a "balanced immune response" means that the immune response to the four dengue serotypes is sufficient to provide protection against infection by all four dengue serotypes, and preferably the immune responses to the four dengue serotypes are of similar strength. In particular, 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.

[0137] 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.

[0138] 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 5pfu / 0.5ml) x 100 = 71%. The concentration percentage is rounded to the nearest whole number.

[0139] As used herein, "concomitant" administration of a vaccine refers to the combined administration of two or more vaccines. In the context of this part of the present invention, the combined administration of two or more vaccines refers to the combined administration of a dengue vaccine, such as a unit dose of the present invention, in combination with a yellow fever vaccine, e.g., YF-17D, and / or a hepatitis A vaccine, e.g., HAVRIX ( and / or a combination vaccine, such as a tetanus toxoid, attenuated diphtheria toxoid and acellular pertussis (adsorbed) combination vaccine, e.g., BOOSTRIX®, and / or a DTaP / IPV / HIB vaccine, particularly a combination DTaP / IPV / Hib vaccine, e.g., Pentacel®.

[0140] As used herein, "concurrent" administration means administration of at least two different vaccines on the same day, e.g., a dengue vaccine and a yellow fever vaccine, or a dengue vaccine and a hepatitis A vaccine, or a dengue vaccine and an HPV vaccine, or a dengue vaccine and an MMR vaccine, or a dengue vaccine and a Tdap vaccine, or a dengue vaccine and a DTaP / IPV / Hib vaccine. Concurrent administration can be administered by the same physician, e.g., during the same medical visit.

[0141] As used herein, "sequential" administration means administration of at least two different vaccines on subsequent days, e.g., within 90 days, but in a combined administration schedule that includes administration of a dengue vaccine and a yellow fever vaccine, e.g., a dengue vaccine and a yellow fever vaccine, or a dengue vaccine and a hepatitis A vaccine, or a dengue vaccine and an HPV vaccine, or a dengue vaccine and an MMR vaccine, or a dengue vaccine and a Tdap vaccine, or a dengue vaccine and a DTaP / IPV / Hib vaccine.

[0142] As used herein, "HPV-associated cancer or genital warts" refers to cancer and genital warts, respectively, caused by HPV infection. HPV serotypes 16, 18, 45, 31, 33, 52, and 58, also known as "high-risk" HPV serotypes, are the most common types of cervical cancer, with the two HPV serotypes 16 and 18 causing approximately 70% of cervical cancers worldwide. HPV serotypes 6 and 11, also known as "low-risk" HPV serotypes, cause genital warts.

[0143] As used herein, "9vHPV vaccine" refers to a 9-valent HPV vaccine that provides protection against HPV serotypes 6, 11, 16, 18, 31, 33, 45, 52, and 58. In particular, the 9vHPV vaccine is used to prevent cervical, vulvar, vaginal, and anal cancers, precancerous or dysplastic lesions, and genital warts caused by one or more of these HPV serotypes.

[0144] As used herein, "MMR vaccine" refers to a combination vaccine for measles, mumps, and rubella. Several MMR vaccines are known in the prior art, including MMR® II, Priorix®, Tresivac®, and Trimovac®.

[0145] As used herein, "Tdap vaccine" refers to a combination vaccine for tetanus, diphtheria, and whooping cough. Tdap vaccines known in the prior art include INFANRIX® (for vaccination of children 6 weeks to 7 years of age), as well as BOOSTRIX® manufactured by GlaxoSmithKline and Adacel® manufactured by Sanofi Pasteur (both for use in persons 10 years of age and older).

[0146] As used herein, "DTaP" refers to diphtheria, tetanus and acellular pertussis.

[0147] As used herein, "IPV" refers to inactivated poliovirus.

[0148] As used herein, "Hib" refers to Haemophilus influenzae type b.

[0149] As used herein, "DTaP / IPV / Hib vaccine" refers to a combination vaccine for diphtheria, tetanus, pertussis, poliomyelitis, and Haemophilus influenzae type b. DTaP / IPV / Hib vaccines known in the prior art include Pentacel® manufactured by Sanofi Pasteur.

[0150] As used herein, the term "chronic disease or condition" includes those diseases and conditions that persist for three months or longer in an elderly subject, including, among others, diabetes, hypertension, allergies, previous stroke, ischemic heart disease, chronic kidney disease, and chronic obstructive pulmonary disease.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] As used herein, a "spontaneous adverse event" is any adverse event (AE) that is not an involuntary local or systemic AE as defined above.

[0155] 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.

[0156] 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.

[0157] As used herein, a subject or subject population that is "2 to 17 years of age" refers to a subject or subject population that is 2 to 17 years of age on day 1 of administration of a dengue vaccine composition described herein.

[0158] As used herein, "percentage points" refers to the difference in percentage 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.

[0159] As used herein, the term "determination of previous dengue infection in a subject prior to dosing" means that previous dengue infection must be assessed prior to vaccination by an appropriately validated serological test, e.g., a laboratory-confirmed history of dengue by the methods disclosed herein, such as the MNT50 test described in Example 2, or any serological test with adequate performance in terms of specificity and cross-reactivity based on local disease epidemiology.

[0160] As used herein, % w / v refers to % mg / ml, for example, 150 mg / ml is 15% w / v. [Brief explanation of the drawings]

[0161] [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 3A] Percentage of subjects (±95% confidence interval) who were seropositive (reciprocal neutralization titer ≥ 10) for each of the dengue serotypes at different time points in the study in the HD-TDV group (dark, left bars) and the TDV group (light, right bars) throughout the study. Time points shown are baseline, day 15 (d15), day 30 (d30), day 90 (d90), day 180 (d180), and day 365 (d365). Part A shows results for the per-protocol set of seropositive (left set of graphs) and seronegative (right set of graphs) participants at baseline. [Figure 3B] Percentage of subjects (±95% confidence interval) who were seropositive (reciprocal neutralization titer ≥ 10) for each of the dengue serotypes at different time points in the study in the HD-TDV group (dark, left bars) and the TDV group (light, right bars) throughout the study. Time points shown are baseline, day 15 (d15), day 30 (d30), day 90 (d90), day 180 (d180), and day 365 (d365). Part B shows the results for the per-protocol set of the entire study population (All). [Figure 4A] Geometric mean titers (GMTs) (±95% confidence intervals) of neutralizing antibodies to each of the four dengue serotypes during the course of the study for recipients of HD-TDV (dark line with triangles) and TDV (light line with circles), the entire study population (Part B), and the per-protocol set of participants who were seropositive and seronegative at baseline (Part A). [Figure 4B]Geometric mean titers (GMTs) (±95% confidence intervals) of neutralizing antibodies to each of the four dengue serotypes during the course of the study for recipients of HD-TDV (dark line with triangles) and TDV (light line with circles), the entire study population (Part B), and the per-protocol set of participants who were seropositive and seronegative at baseline (Part A). [Figure 5] ELISpot analysis of IFNγ in peripheral blood mononuclear cells before vaccination (baseline) and at different time points after TDV administration. The frequency of responses to the entire DENV-2 proteome is shown. Subjects were considered to be responsive if they had a positive response (i.e., a mean of ≥4-fold over the negative control and ≥50 SFC / 10 PBMC) to multiple peptide pools derived from DENV-2. [Figure 6] ELISpot analysis of IFNγ in peripheral blood mononuclear cells (PBMCs) before vaccination (baseline) and at different time points after TDV administration. The frequency of responses to peptide pools corresponding to selected DENV-derived proteins is shown. Subjects were considered reactive if their responses to multiple DENV-2-derived peptide pools were positive (i.e., a mean of ≥4-fold higher than the negative control and ≥50 SFC / 10 PBMC). A = DENV-2 C; B = DENV-1 prM+E; C = DENV-2 prM+E; D = DENV-3 prM+E; E = DENV-4 prM+E; F = DENV-2 NS1; G = DENV-2 NS2; H = DENV-2 NS3; I = DENV-2 NS4; J = DENV-2 NS5. [Figure 7]Effect of serum from seronegative (A) and seropositive (B) subjects administered TDV on DENV-2 NS1-induced hyperpermeability as determined by TEER. HPMECs were grown on Transwell semipermeable membranes (0.4 μm pore size), and serum samples (30 μl) were added to the apical chamber in the presence or absence of DENV2 NS1 (5 μg / ml). DENV2 NS1 is shown as a square; day 0 serum alone is shown as a diamond; day 120 serum alone is shown as a triangle; day 0 serum + DENV2 NS1 is shown as an inverted triangle; and day 120 serum + DENV2 NS1 is shown as an X. (^) indicates medium change. Endothelial permeability was measured over 48 hours at the indicated time points. Relative TEER values ​​from one independent experiment performed in duplicate are plotted. Error bars indicate standard error of the mean (SEM). [Figure 8] Effect of serum from seronegative and seropositive subjects administered TDV on NS1-induced sialic acid and heparan sulfate degradation. Quantification of mean fluorescence intensity (MFI) of (A) sialic acid and (B) heparan sulfate after staining with sialic acid- and heparan sulfate-specific fluorescent antibodies visualized by confocal microscopy is shown. Values ​​are normalized to the MFI from the NS1 + positive control serum group (represented by the dotted line at 100%) and are expressed as a percentage of the control. Error bars indicate SEM. The left bar for each subject shows the results at day 0 (d0), and the right bar for each subject shows the results at day 120 (d120). [Figure 9] Flow diagram of the clinical trial of Example 6. [Figure 10] 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 11] Study design for the Phase III study described in Example 6 DETAILED DESCRIPTION OF THE INVENTION

[0162] 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.

[0163] [Table 1]

[0164] 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) Includes:

[0165] 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.

[0166] The unit dose of the invention described herein comprises a dengue virus composition comprising four live attenuated dengue virus strains (tetravalent dengue virus composition) representing dengue serotype 1, dengue serotype 2, dengue serotype 3 and dengue serotype 4. Preferably, the composition comprises a chimeric dengue virus and optionally at least one non-chimeric dengue virus, in particular a molecularly characterized and cloned dengue serotype 2 strain derived from the live attenuated DEN-2 PDK-53 virus strain (TDV-2), and the structural proteins prM and E from the TDV-2 strain are replaced with the corresponding structural proteins from other dengue serotypes to form the following chimeric dengue strains: -DENV-2 / 1 chimera (TDV-1), -DENV-2 / 3 chimera (TDV-3) and -DENV-2 / 4 chimera (TDV-4) The present invention includes three chimeric dengue strains derived from the TDV-2 strain by bringing about

[0167] 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 developed by the University of Mahidol, Bangkok, Thailand (Kinney University). It was generated by cDNA cloning of the laboratory-derived, attenuated DEN-2 PDK-53 virus strain originally isolated in (Bhamarapravati et al. (1987) Bull. World Health Organ. 65(2):189-195). 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).

[0168] 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 consists of nine nucleotides: differs from the wild type in (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

[0169] 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.

[0170] [Table 2]

[0171] In one embodiment, the TDV-2 contains, in addition to the three attenuating mutations: a) an adenine to thymidine mutation at nucleotide 524 in the prM gene, resulting in an amino acid change from asparagine to valine at position 143; and / or b) a silent mutation from cytosine to thymidine at nucleotide 2055 in the E gene, and / or c) a cytosine to thymidine mutation at nucleotide 4018 in the NS2A gene, resulting in an amino acid change from leucine to phenylalanine at position 1308; and / or d) a silent thymidine to cytosine mutation at nucleotide 5547 in the NS3 gene, and / or e) a guanine to cytosine mutation at nucleotide 6599 in the NS4A gene, resulting in an amino acid change from glycine to alanine at position 2168; and / or f) A silent thymidine to cytosine mutation at nucleotide 900 in the prM gene The present invention includes one or more mutations selected from the following: The silent cytosine-to-thymidine mutation at nucleotide 8571 in the NS5 gene of DEN-2 PDK-53 is absent in TDV-2 strains.

[0172] In another embodiment, the TDV-2 contains, in addition to the three attenuating mutations: g) an adenine to guanine mutation at nucleotide 592 in the prM gene, resulting in an amino acid change from lysine to glutamine at position 166; and / or h) an adenine to guanine mutation at nucleotide 8803 in the NS5 gene, resulting in an amino acid change from isoleucine to valine at position 2903 The present invention includes one or more mutations selected from the following:

[0173] 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.

[0174] The structural envelope (E) and premembrane (prM) proteins of dengue virus have been identified as the primary antigens that elicit neutralizing and protective antibody responses (Plotkin 2001). To generate a tetravalent dengue vaccine (TDV), the nucleic acid sequences encoding the prM and E glycoproteins of DENV-2 were cloned into the wild-type strains DENV-1, DENV-3, and DENV-4 using standard molecular genetic engineering methods (Huang et al. (2003) J. Virol. 77(21):11436-11447). TDV-2 was modified by replacing the nucleotide sequences encoding the corresponding wild-type prM and E glycoproteins from DENV-3 16007, DENV-3 16562, or DENV-4 1036 viruses, respectively (see Table 3).

[0175] [Table 3]

[0176] A diagram of the four TDV strains included in the dengue vaccine composition is shown in Figure 1 .

[0177] The 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.

[0178] In one embodiment, the TDV-1 contains, in addition to the three attenuating mutations: c) a cytosine to thymidine mutation at nucleotide 4018 in the NS2A gene, resulting in an amino acid change from leucine to phenylalanine at position 1308; and / or d) a silent thymidine to cytosine mutation at nucleotide 5547 in the NS3 gene, and / or e) a guanine to cytosine mutation at nucleotide 6599 in the NS4A gene, resulting in an amino acid change from glycine to alanine at position 2168; and / or i) a silent thymidine to cytosine mutation at nucleotide 1575 in the E gene, and / or j) a silent adenine to guanine mutation at nucleotide 453 at the junction between the prM-E gene and the DEN-2 PDK-53 backbone, and / or k) a thymidine-guanine to cytosine-cytosine mutation at nucleotides 2381 / 2382 at the junction between the prM-E gene and the DEN-2 PDK-53 backbone, resulting in an amino acid change from valine to alanine at position 762 The present invention includes one or more mutations selected from the following:

[0179] 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) an adenine to thymidine mutation at nucleotide 4407 in the NS2B gene, resulting in an amino acid change from glutamine to asparagine at position 1437; and / or n) A silent adenine to guanine mutation at nucleotide 7311 in the NS4B gene The present invention includes one or more mutations selected from the following:

[0180] 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), and 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.

[0181] In one embodiment, the TDV-3 contains, in addition to the three attenuating mutations: c) a cytosine to thymidine mutation at nucleotide 4012 in the NS2A gene, resulting in an amino acid change from leucine to phenylalanine at position 1306; and / or d) a silent thymidine to cytosine mutation at nucleotide 5541 in the NS3 gene, and / or e) a guanine to cytosine mutation at nucleotide 6593 in the NS4A gene, resulting in an amino acid change from glycine to alanine at position 2166; and / or j) a silent adenine to guanine mutation at nucleotide 453 at the junction between the prM-E gene and the DEN-2 PDK-53 backbone, and / or k) a thymidine-guanine to cytosine-cytosine mutation at nucleotides 2375 / 2376 at the junction between the prM-E gene and the DEN-2 PDK-53 backbone, resulting in an amino acid change from valine to alanine at position 760; and / or o) a silent mutation from cytosine to thymidine at nucleotide 552 in the prM gene, and / or p) an adenine to thymidine mutation at nucleotide 1970 in the E gene, resulting in an amino acid change from histidine to leucine at position 625 The present invention includes one or more mutations selected from the following:

[0182] In another embodiment, the TDV-3 contains, in addition to the three attenuating mutations: q) an adenine to thymidine mutation at nucleotide 1603 in the E gene, resulting in an amino acid change from threonine to serine at position 503, and / or r) a silent adenine to guanine mutation at nucleotide 7620 in the NS5 gene. The present invention includes one or more mutations selected from the following:

[0183] 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.

[0184] In one embodiment, the TDV-4 contains, in addition to the three attenuating mutations: c) a cytosine to thymidine mutation at nucleotide 4018 in the NS2A gene, resulting in an amino acid change from leucine to phenylalanine at position 1308; and / or d) a silent thymidine to cytosine mutation at nucleotide 5547 in the NS3 gene, and / or e) a guanine to cytosine mutation at nucleotide 6599 in the NS4A gene, resulting in an amino acid change from glycine to alanine at position 2168; and / or j) a silent adenine to guanine mutation at nucleotide 453 at the junction between the prM-E gene and the DEN-2 PDK-53 backbone, and / or k) a thymidine-guanine to cytosine-cytosine mutation at nucleotides 2381 / 2382 at the junction between the prM-E gene and the DEN-2 PDK-53 backbone, resulting in an amino acid change from valine to alanine at position 762; and / or s) an adenine to cytosine mutation at nucleotide 396 in the C gene, resulting in an amino acid change from arginine to serine at position 100; and / or t) a silent adenine to guanine mutation at nucleotide 1401 in the E gene, and / or u) a cytosine to thymidine mutation at nucleotide 2027 in the E gene, resulting in an amino acid change from alanine to valine at position 644; and / or v) an adenine to cytosine mutation at nucleotide 2275 in the E gene, resulting in an amino acid change from methionine to leucine at position 727 The present invention includes one or more mutations selected from the following:

[0185] In another embodiment, the TDV-4 contains, in addition to the three attenuating mutations: w) a silent adenine to thymidine mutation at nucleotide 225 in the C gene, and / or x) an adenine to guanine mutation at nucleotide 3674 in the NS2A gene, resulting in an amino acid change from asparagine to glycine at position 1193; and / or y) a mutation in the NS2A gene at nucleotide 3773 from adenine to adenine / guanine mix, resulting in an amino acid change at position 1226 from lysine to lysine / asparagine mix, and / or z) a silent mutation from cytosine to thymidine at nucleotide 5391 in the NS3 gene, and / or aa) a cytosine to thymidine mutation at nucleotide 6437 in the NS4A gene, resulting in an amino acid change from alanine to valine at position 2114; and / or bb) a silent mutation from thymidine to a thymidine / cytosine mix at nucleotide 7026 in the NS4B gene, and / or cc) Adenine to cytosine silencer at nucleotide 9750 in the NS5 gene nt mutation The present invention includes one or more mutations selected from the following:

[0186] 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.

[0187] 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.

[0188] [Table 4]

[0189] 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 certain preferred embodiment, TDV-1 is characterized by a nucleotide sequence according to SEQ ID NO: 1 and an amino acid sequence according to SEQ ID NO: 2, TDV-2 is characterized by a nucleotide sequence according to SEQ ID NO: 3 and an amino acid sequence according to SEQ ID NO: 4, TDV-3 is characterized by a nucleotide sequence according to SEQ ID NO: 5 and an amino acid sequence according to SEQ ID NO: 6, and TDV-4 is characterized by a nucleotide sequence according to SEQ ID NO: 7 and an amino acid sequence according to SEQ ID NO: 8.

[0190] The E protein of DENV-3 has two fewer amino acids than the E protein of DENV-2. Therefore, the nucleotides of TDV-2 starting after the E region of DENV-3 at nucleotide 2374 of SEQ ID NO:5 and amino acid 760 of SEQ ID NO:6 and the encoded The amino acid backbone has 6 fewer nucleotides and 2 fewer amino acids than the original TDV-2 nucleotide and amino acid positions, respectively.

[0191] Dengue vaccine composition The present invention is directed, in part, to a unit dose of the described dengue vaccine composition, which includes a tetravalent dengue virus composition, also referred to as a dengue virus composition, and a pharmaceutically acceptable excipient.

[0192] Dengue virus composition, virus concentration and % concentration The invention, in part, is 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) Dengue serotype 4 strain, preferably at a concentration of at least 4.5 log pfu / 0.5 mL The present invention is directed to a unit dose of a dengue vaccine composition, comprising a tetravalent dengue virus composition comprising:

[0193] 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) Dengue serotype 4, preferably at a concentration of at least 4.5 log pfu / 0.5 ml or 4.6 log pfu / 0.5 ml, optionally up to 6.2 log pfu / 0.5 ml and a tetravalent dengue virus composition comprising:

[0194] 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) a dengue serotype 2 strain 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 chimeric dengue serotype 2 / 4 strain at a concentration of at least 4.5 log pfu / 0.5 mL; The present invention is directed to a unit dose of a dengue vaccine composition, comprising a tetravalent dengue virus composition comprising:

[0195] 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 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 and a tetravalent dengue virus composition comprising:

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 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.

[0204] In another 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.

[0205] 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.

[0206] Preferably, in said embodiment, the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3 and the chimeric dengue serotype 2 / 4 strain is TDV-4. More preferably, TDV-1 is characterized by a nucleotide sequence according to SEQ ID NO: 1 and an amino acid sequence according to SEQ ID NO: 2, TDV-2 is characterized by a nucleotide sequence according to SEQ ID NO: 3 and an amino acid sequence according to SEQ ID NO: 4, TDV-3 is characterized by a nucleotide sequence according to SEQ ID NO: 5 and an amino acid sequence according to SEQ ID NO: 6 and TDV-4 is characterized by a nucleotide sequence according to SEQ ID NO: 7 and an amino acid sequence according to SEQ ID NO: 8.

[0207] The present invention relates, in part, to a 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) dengue serotype 4 at a concentration of at least 4.5 log pfu / dose (e.g., a chimeric dengue serotype 2 / 4 strain) The present invention is directed to a unit dose of a dengue vaccine composition, comprising a tetravalent dengue virus composition comprising:

[0208] 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.

[0209] 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) 4.0 lo having a concentration of 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.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] 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 / 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.

[0214] In a particularly preferred embodiment, the dengue vaccine composition comprises four live attenuated dengue viruses. a tetravalent dengue virus composition comprising a dengue virus strain, (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.

[0215] 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.

[0216] 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.

[0217] In one embodiment, in the composition, (i), (ii), (iii), and (iv) provide a total concentration in pfu / 0.5 mL, and based on said concentrations, the concentration of (iii) is at least 10% of the total concentration in pfu / 0.5 mL.

[0218] In one embodiment, in the composition, (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL, and based on said 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 8%, or at least 10%, or at least 12%, or at least 14%, or at least 16%, or at least 18%.

[0219] The concentration of (iii) in pfu / 0.5 mL in the reconstituted unit dose is preferably at least 10%.

[0220] In one embodiment, in the composition, (i), (ii), (iii), and (iv) provide 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%, and the concentration of (i) at pfu / 0.5 mL is at least 1%; (iii) The concentration in pfu / 0.5 mL is at least 6%.

[0221] Preferably, in said embodiment, the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3 and the chimeric dengue serotype 2 / 4 strain is TDV-4. More preferably, TDV-1 is characterized by a nucleotide sequence according to SEQ ID NO: 1 and an amino acid sequence according to SEQ ID NO: 2, TDV-2 is characterized by a nucleotide sequence according to SEQ ID NO: 3 and an amino acid sequence according to SEQ ID NO: 4, TDV-3 is characterized by a nucleotide sequence according to SEQ ID NO: 5 and an amino acid sequence according to SEQ ID NO: 6 and TDV-4 is characterized by a nucleotide sequence according to SEQ ID NO: 7 and an amino acid sequence according to SEQ ID NO: 8.

[0222] 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.

[0223] 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 providing a total concentration in 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.

[0224] 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 provided at a total concentration in pfu / 0.5 mL, wherein 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%, 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., The concentration of dengue serotype 2 (e.g., chimeric dengue serotype 2 / 4 strain) is 50%–90% or 60%–88% of the total concentration. This means that the concentration of dengue serotype 2 (e.g., dengue serotype 2 strain) is lower than the concentration of dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strain).

[0225] 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).

[0226] 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%.

[0227] 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, wherein the dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strains) measured in pfu / 0.5 mL are used. The concentration of dengue serotype 2 (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 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%. Dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) is particularly preferred, having the highest concentration of all four dengue serotypes.

[0228] 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 between 1% and 7% of the total concentration, the concentration of dengue serotype 2 (e.g., a dengue serotype 2 strain) measured in pfu / 0.5 mL is less than 8% of the total concentration, for example, 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 4 / 5 strain) measured in pfu / 0.5 mL is between 1% and 7% of the total concentration. The concentration of dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strain) 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.

[0229] 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, such as TDV-4 (e.g., a chimeric dengue serotype 2 / 4 strain), represents the highest concentration of all four serotypes in the composition, preferably having at least about 70% of the total concentration in pfu / 0.5 mL; dengue serotype 3, such as TDV-3 (e.g., a chimeric dengue serotype 2 / 3 strain), represents the second highest concentration of all four serotypes in the composition, preferably having at least about 10% of the total concentration in pfu / 0.5 mL; and 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), each represent a lower concentration than serotype 3, such as TDV-3 (e.g., a chimeric dengue serotype 2 / 3 strain), optionally together representing less than about 10% of the total concentration in pfu / 0.5 mL.

[0230] Preferably, in said embodiment, the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3 and the chimeric dengue serotype 2 / 4 strain is TDV-4. More preferably, TDV-1 is characterized by a nucleotide sequence according to SEQ ID NO: 1 and an amino acid sequence according to SEQ ID NO: 2, TDV-2 is characterized by a nucleotide sequence according to SEQ ID NO: 3 and an amino acid sequence according to SEQ ID NO: 4, TDV-3 is characterized by a nucleotide sequence according to SEQ ID NO: 5 and an amino acid sequence according to SEQ ID NO: 6 and TDV-4 is characterized by a nucleotide sequence according to SEQ ID NO: 7 and an amino acid sequence according to SEQ ID NO: 8.

[0231] 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.

[0232] 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.

[0233] 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 dengue vaccine composition comprising one or more pharmaceutically acceptable excipients. , 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.

[0234] 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 70mM to 140mM sodium chloride Includes:

[0235] In one embodiment, a unit dose of the dengue vaccine composition, when measured in 0.5 ml, contains 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.

[0236] In one embodiment, a unit dose of the dengue vaccine composition, when measured in 0.5 ml, contains 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.

[0237] 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 Approximately 100 mM sodium chloride Includes:

[0238] 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 Approximately 100 mM sodium chloride Includes:

[0239] 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 - approximately 50 μmoles of sodium chloride Includes:

[0240] In a preferred embodiment, the reconstituted 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 - approximately 137 mM sodium chloride, preferably Has a pH of -7 to 8.5

[0241] 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.

[0242] In a preferred embodiment, the reconstituted 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, 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.

[0243] The human serum albumin can be natural or recombinant human serum albumin (rHSA). The poloxamer 407 can be, for example, Pluronic F127.

[0244] 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.

[0245] Unit Dose The present invention is directed, in part, to a unit dose of a dengue vaccine composition comprising a tetravalent dengue virus composition described herein and a pharmaceutically acceptable excipient described herein.

[0246] The present invention is based, in part, on, 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) dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strains) with a concentration of at least 4.5 log pfu / 0.5 mL The present invention is directed to a unit dose of the above-mentioned dengue vaccine composition.

[0247] 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 a nucleotide sequence according to SEQ ID NO: 1 and an amino acid sequence according to SEQ ID NO: 2, TDV-2 is characterized by a nucleotide sequence according to SEQ ID NO: 3 and an amino acid sequence according to SEQ ID NO: 4, TDV-3 is characterized by a nucleotide sequence according to SEQ ID NO: 5 and an amino acid sequence according to SEQ ID NO: 6, and TDV-4 is characterized by a nucleotide sequence according to SEQ ID NO: 7 and an amino acid sequence according to SEQ ID NO: 8.

[0248] 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 and a dengue vaccine composition described herein comprising four dengue virus strains, particularly TDV-1 through TDV-4. In a preferred embodiment, the residual moisture content, as determined by Karl Fischer determination, is less than or equal to 5.0%, preferably less than or equal to 3%.

[0249] 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, an aqueous sodium chloride solution, such as a 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.

[0250] 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.

[0251] 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) 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 The present invention is directed to a unit dose of a dengue vaccine composition comprising:

[0252] In one embodiment, the reconstituted unit dose has a volume of, for example, 0.5 mL, and upon reconstitution with a pharmaceutically acceptable diluent, (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL, and based on said concentrations, the concentration of (iii) is at least 10% of the total concentration in pfu / 0.5 mL.

[0253] In another embodiment, the reconstituted unit dose has a volume of, for example, 0.5 mL, and upon reconstitution with a pharmaceutically acceptable diluent, (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL, where, based on said total concentration, the concentration of (ii) at pfu / 0.5 mL is less than 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%, or at least 10%, or at least 12%, or at least 14%, or at least 16%, or at least 18%.

[0254] The concentration of (iii) in pfu / 0.5 mL in the reconstituted unit dose is preferably at least 10%.

[0255] In one embodiment, the reconstituted unit dose has a volume of, for example, 0.5 mL, and upon reconstitution with a pharmaceutically acceptable diluent, (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL, where, based on said total concentration, the concentration of (ii) at pfu / 0.5 mL is less than 2%, the concentration of (iv) at pfu / 0.5 mL is at least 50%, the concentration of (i) at pfu / 0.5 mL is at least 1%, and the concentration of (iii) at pfu / 0.5 mL is at least 6%.

[0256] 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 a nucleotide sequence according to SEQ ID NO: 1 and an amino acid sequence according to SEQ ID NO: 2, TDV-2 is characterized by a nucleotide sequence according to SEQ ID NO: 3 and an amino acid sequence according to SEQ ID NO: 4, TDV-3 is characterized by a nucleotide sequence according to SEQ ID NO: 5 and an amino acid sequence according to SEQ ID NO: 6, and TDV-4 is characterized by a nucleotide sequence according to SEQ ID NO: 7 and an amino acid sequence according to SEQ ID NO: 8.

[0257] In one such embodiment, the lyophilized unit dose comprises dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strains) at a concentration of 3.3 log pfu / dose to 5.0 log pfu / 0.5 mL, dengue serotype 2 (e.g., dengue serotype 2 strains) at a concentration of 2.7 log pfu / dose to 4.9 log pfu / 0.5 mL, dengue serotype 3 (e.g., dengue serotype 3 strains) at a concentration of 4.0 log pfu / dose to 5.0 log pfu / 0.5 mL, and dengue serotype 4 (e.g., dengue serotype 5 strains) at a concentration of 4.0 log pfu / dose to 5.0 log pfu / 0.5 mL. The vaccine is obtained by lyophilizing 0.5 mL of a dengue vaccine composition comprising dengue serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain) at a concentration of 10 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.

[0258] In one such embodiment, the lyophilized unit dose contains dengue serotype 1 (e.g., 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, dengue serotype 2 (e.g., dengue serotype 2 strain) at a concentration of 2.7 log pfu / 0.5 mL to 4.0 log pfu / 0.5 mL, dengue serotype 3 (e.g., dengue serotype 3 strain) at a concentration of 4.0 log pfu / 0.5 mL to 4.6 log pfu / 0.5 mL, or dengue serotype 4 (e.g., dengue serotype 4 strain) at a concentration of 5.0 log pfu / 0.5 mL to 6.0 log pfu / 0.5 mL. The vaccine composition is obtained by lyophilizing 0.5 mL of a dengue vaccine composition comprising 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.

[0259] In certain embodiments, a lyophilized unit dose refers to 0.5 mL prior to lyophilization in which TDV-2 and TDV-4 are present in a predetermined relative amount 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%.

[0260] In certain embodiments, the reconstituted unit dose has a volume of 0.5 mL, and TDV-2 and TDV-4 are present in predetermined relative amounts based on the total concentration of TDV-1, TDV-2, TDV-3, and TDV-4 in pfu / 0.5 mL, where 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%.

[0261] 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 between 1% and 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, and 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., dengue serotype 5 strain) measured in pfu / 0.5 mL is between 1% and 7% of the total concentration. The concentration of dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strain) 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 log pfu / 0.5 mL to 6.7 log pfu / 0.5 mL, preferably in the range of 4.6 log pfu / 0.5 mL to 5.5 log pfu / 0.5 mL.

[0262] 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 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, such as TDV-4 (e.g., a chimeric dengue serotype 2 / 4 strain), represents the highest concentration of all four serotypes in the composition, preferably having at least about 70% of the total concentration in pfu / 0.5 mL; dengue serotype 3, such as TDV-3 (e.g., a chimeric dengue serotype 2 / 3 strain), represents the second highest concentration of all four serotypes in the composition, preferably having at least about 10% of the total concentration in pfu / 0.5 mL; and 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), each represent a lower concentration than serotype 3, such as TDV-3 (e.g., a chimeric dengue serotype 2 / 3 strain), optionally together representing less than about 10% of the total concentration in pfu / 0.5 mL.

[0263] A lyophilized unit dose reconstituted in 0.5 mL provides 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, without changing the absolute amount of virus administered or the ratio of viruses to each other.

[0264] 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.

[0265] 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.

[0266] 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.

[0267] 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.

[0268] In one embodiment, the solution from which the lyophilized unit dose is prepared further comprises a buffer. The buffer solution may be phosphate buffered saline (PBS). The buffer solution may contain 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 contain disodium hydrogen phosphate (Na2HPO4), potassium chloride (KCl), and potassium dihydrogen phosphate (KH2PO4). The buffer solution may have a pH in the range of about 7.0 to about 8.5 at 25°C, or a pH of about 6.8 to about 7.6 at 25°C, preferably about 7.2 at 25°C.

[0269] 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.

[0270] A unit dose of the invention described herein activates multiple levers of the immune system (neutralizing antibodies, cell-mediated immunity, and anti-NS1 antibodies) in both seronegative and seropositive subject populations or subjects, and thus protects both dengue-seronegative and dengue-seropositive subject populations or subjects against dengue disease.

[0271] In one embodiment, one unit dose is present in a container, preferably a vial, which is administered to a subject after reconstitution. In one embodiment, more than one unit dose of a dengue vaccine composition may be present in a container, preferably a vial, such that the contents of one container, preferably a vial, can vaccinate more than one subject. In one embodiment, a container containing more than one unit dose of the invention described herein is used to provide reconstituted unit doses for use in the methods of the invention described herein.

[0272] In certain embodiments, the container containing the unit dose of the invention is part of a kit. Thus, the invention is directed, in part, to a kit for preparing a reconstituted unit dose comprising a lyophilized unit dose of the invention described herein and a pharmaceutically acceptable reconstitution diluent.

[0273] 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.

[0274] In certain embodiments, the kit may further comprise a yellow fever vaccine, particularly YF-17D. In some embodiments, the yellow fever vaccine may be present in separate containers, e.g., vials. In other embodiments, the yellow fever 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 / yellow fever vaccine in which a unit dose of the invention described herein is combined with a yellow fever vaccine. Such a combined dengue / yellow fever vaccine comprises a unit dose of the invention described herein and a yellow fever vaccine, particularly YF-17D, in the same formulation. In certain embodiments, the present invention is directed to a kit comprising such a combined dengue / yellow fever vaccine and a unit dose of the invention described herein.

[0275] In certain embodiments, the kit may further comprise a Hepatitis A vaccine, such as HAVRIX® or VAQTA®. The vaccines may be present in separate containers, e.g., vials. In another embodiment, 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.

[0276] In certain embodiments, the kit may further comprise an HPV vaccine, particularly a 9vHPV vaccine, e.g., GARDASIL® 9. In some embodiments, the HPV vaccine may be present in separate containers, e.g., vials. In other embodiments, the HPV 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 / HPV vaccine in which a unit dose of the invention described herein is combined with an HPV vaccine. Such a combined dengue / HPV vaccine comprises a unit dose of the invention described herein and an HPV vaccine, particularly a 9vHPV vaccine, e.g., GARDASIL® 9, in the same formulation. In certain embodiments, the present invention is directed to a kit comprising such a combined dengue / HPV vaccine and a unit dose of the invention described herein.

[0277] In certain embodiments, the kit can further include an MMR vaccine, e.g., MMR® II. In some embodiments, the MMR vaccine can be present in separate containers, e.g., vials. In other embodiments, the MMR vaccine and the unit dose of the invention can be present in the same container. Thus, the present invention is directed, in part, to a combined dengue / MMR vaccine in which a unit dose of the invention described herein is combined with an MMR vaccine. Such a combined dengue / MMR vaccine comprises a unit dose of the invention described herein and an MMR vaccine, e.g., MMR® II, in the same formulation. In certain embodiments, the present invention is directed to a kit comprising such a combined dengue / MMR vaccine and a unit dose of the invention described herein.

[0278] In certain embodiments, the kit may further comprise a Tdap vaccine, particularly a tetanus toxoid, attenuated diphtheria toxoid, and acellular pertussis (adsorbed) combination vaccine, e.g., BOOSTRIX®. In some embodiments, the Tdap vaccine may be present in separate containers, e.g., vials. In other embodiments, the Tdap vaccine and a unit dose of the invention may be present in the same container. Thus, the present invention is directed, in part, to a combined dengue / Tdap vaccine in which a unit dose of the invention described herein is combined with a Tdap vaccine. Such a combined dengue / Tdap vaccine comprises a unit dose of the invention described herein and a Tdap vaccine, particularly a tetanus toxoid, attenuated diphtheria toxoid, and acellular pertussis (adsorbed) combination vaccine, e.g., BOOSTRIX®, in the same formulation. In certain embodiments, the present invention is directed to a kit comprising such a combined dengue / Tdap vaccine and a unit dose of the invention described herein.

[0279] In certain embodiments, the kit may further comprise a DTaP / IPV / Hib vaccine, particularly a combined DTaP / IPV / Hib vaccine, e.g., Pentacel®. In some embodiments, the DTaP / IPV / Hib vaccine may be present in separate containers, e.g., vials. In other embodiments, the DTaP / IPV / Hib 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 DTaP / IPV / Hib vaccine in which a unit dose of the invention described herein is combined with a DTaP / IPV / Hib vaccine. Such combinations A combined dengue / DTaP / IPV / Hib vaccine comprises a unit dose of the invention described herein and a DTaP / IPV / Hib vaccine, particularly a combined DTaP / IPV / Hib vaccine, such as Pentacel®, in the same formulation. In certain embodiments, the present invention is directed to a kit comprising such a combined dengue / DTaP / IPV / Hib vaccine and a unit dose of the invention described herein.

[0280] 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.

[0281] Hepatitis A vaccine HAVRIX®, a hepatitis A vaccine manufactured by GlaxoSmithKline, is a sterile suspension of inactivated virus for intramuscular administration. The virus (strain HM175) is grown 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). HAVRIX® is formulated without preservatives.

[0282] VAQTA®, a hepatitis A vaccine from Merck Sharp & Dohme Corp., is an inactivated whole virus vaccine derived from hepatitis A virus grown in cell culture in human MRC-5 diploid fibroblasts. It contains inactivated virus from a strain originally derived by further serial passage of a proven attenuated strain. The virus is propagated, 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 Contains less than 10 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.

[0283] HPV vaccine GARDASIL® 9, a non-infectious recombinant HPV vaccine manufactured by Merck, is a non-infectious, 9-valent vaccine prepared from purified virus-like particles (VLPs) of the major capsid (L1) proteins of HPV serotypes 6, 11, 16, 18, 31, 33, 45, 52, and 58. The L1 proteins are produced by separate fermentation using recombinant Saccharomyces cerevisiae and self-assemble into VLPs. The fermentation process involves growing S. cerevisiae in a chemically defined fermentation medium containing vitamins, amino acids, mineral salts, and carbohydrates. The VLPs are released from the yeast cells by cell disruption and purified by a series of chemical and physical methods. The purified VLPs are adsorbed to a preformed aluminum-containing adjuvant (amorphous aluminum hydroxyphosphate sulfate, or AAHS). The 9-valent HPV VLP vaccine is a sterile liquid suspension prepared by combining the adsorbed VLPs of each HPV serotype with additional amounts of aluminum-containing adjuvant and a final purification buffer. GARDASIL 9 is a sterile suspension for intramuscular administration. Each 0.5 mL dose contains approximately 30 μg of HPV serotype 6 L1 protein, 40 μg of HPV serotype 11 L1 protein, 60 μg of HPV serotype 16 L1 protein, 40 μg of HPV serotype 18 L1 protein, 20 μg of HPV serotype 31 L1 protein, 20 μg of HPV serotype 33 L1 protein, 20 μg of HPV serotype 45 L1 protein, 20 μg of HPV serotype 52 L1 protein, and 20 μg of HPV serotype 58 L1 protein. Each 0.5 mL dose of the vaccine also contains approximately 500 μg of aluminum (provided as AAHS), 9.56 mg of sodium chloride, 0.78 mg of L-histidine, 50 μg of polysorbate 80, and 35 μg of sodium borate.

[0284] MMR vaccine Several MMR vaccines are known in the prior art, including MMR® II, Priorix®, Tresivac®, and Trimovac®.

[0285] The MMR vaccine, MMR® II, manufactured by Merck Sharp & Dohme Corp., is a live virus vaccine for vaccination against measles, mumps, and rubella. MMR® II is a lyophilized, sterile preparation of (1) ATTENUVAX® (live measles virus vaccine), a more attenuated strain of measles virus derived from the Enders-attenuated Edmonston strain and grown in chicken embryo cell culture, (2) MUMPSVAX® (live mumps virus vaccine), the Jeryl Lynn™ (B level) strain of mumps virus grown in chicken embryo cell culture, and (3) MERUVAX® II (live rubella virus vaccine), the WistarRA27 / 3 strain of live attenuated rubella virus grown in WI-38 human diploid lung fibroblast cells. The growth medium for measles and mumps is 199 medium (a buffered salt solution containing vitamins and amino acids and supplemented with fetal bovine serum) containing SPGA (sucrose, phosphate, glutamate, and recombinant human albumin) and neomycin as stabilizers. The growth medium for rubella is minimum essential medium (MEM) (a buffered salt solution containing vitamins and amino acids and supplemented with fetal bovine serum) containing recombinant human albumin and neomycin. Sorbitol and hydrolyzed gelatin stabilizers are added to each virus harvest. Cells, virus pools, and fetal bovine serum are all screened for the absence of adventitious material. The reconstituted vaccine is for subcutaneous administration. Each 0.5 mL dose contains 1,000 T CID 50 (tissue culture infectious dose) or more of measles virus, 12,500 TCID 50 of mumps virus, and 1,000 TCID 50of rubella virus. Each dose of vaccine is calculated to contain sorbitol (14.5 mg), sodium phosphate, sucrose (1.9 mg), sodium chloride, hydrolyzed gelatin (14.5 mg), recombinant human albumin (≦0.3 mg), fetal bovine serum (<1 ppm), other buffer and media components, and approximately 25 μg of neomycin. The product does not contain preservatives. The lyophilized vaccine is reconstituted prior to administration.

[0286] Tetanus, diphtheria, and pertussis (Tdap) combined vaccine BOOSTRIX®, a tetanus toxoid, attenuated diphtheria toxoid, and acellular pertussis (adsorbed) (Tdap) combination vaccine, is a non-infectious, sterile vaccine for intramuscular administration. It contains tetanus toxoid, diphtheria toxoid, and pertussis antigens (inactivated pertussis toxin (iPT) and formaldehyde-treated filamentous hemagglutinin (FHA) and pertactin (PRN)). The antigens are the same as those in INFANRIX®, but BOOSTRIX® is formulated with reduced amounts of these antigens.

[0287] BOOSTRIX® is supplied as a 0.5 mL suspension for injection. Each 0.5 mL dose of BOOSTRIX® is formulated to contain 5 limit of flocculation (Lf) tetanus toxoid, 2.5 Lf diphtheria toxoid, 8 μg iPT, 8 μg formaldehyde-treated FHA, and 2.5 μg PRN (a 69 kilodalton outer membrane protein), with aluminum hydroxide (<0.39 mg aluminum by assay) as adjuvant, 4.5 mg sodium chloride, ≦100 μg residual formaldehyde, and ≦100 μg polysorbate 80 (Tween 80).

[0288] Tetanus toxin is produced by growing Clostridium tetani in modified Latham's medium derived from bovine casein. Diphtheria toxin is produced by growing Corynebacterium diphtheriae in Fenton's medium containing bovine extract. Both toxins are detoxified with formaldehyde, concentrated by ultrafiltration, and purified by precipitation, dialysis, and sterile filtration. The potency of tetanus and diphtheria toxoids is determined by measuring the amount of neutralizing antitoxin in pre-immunized guinea pigs.

[0289] Acellular pertussis antigens (iPT, FHA, and PRN) are isolated from Bordetella pertussis cultures grown in modified Stainer-Scholte liquid medium. iPT and FHA are isolated from the fermentation broth, and PRN is extracted from the cells by heat treatment and flocculation. The antigens are purified by sequential chromatography and precipitation steps. iPT is detoxified using glutaraldehyde and formaldehyde. FHA and PRN are treated with formaldehyde. Each antigen is individually adsorbed to aluminum hydroxide. The potency of the acellular pertussis components (inactivated iPT and formaldehyde-treated FHA and PRN) is determined by enzyme-linked immunosorbent assay (ELISA) on sera from pre-immunized mice.

[0290] Diphtheria, tetanus, pertussis, poliomyelitis, and Haemophilus influenzae type b (DTaP / IPV / Hib) combined vaccine Pentacel®, a DTaP / IPV / Hib combination vaccine, consists of adsorbed diphtheria and tetanus toxoid and acellular pertussis and inactivated poliovirus (DTaP-IPV) components and an ActHIB® component combined through reconstitution for intramuscular injection. ActHIB® (Haemophilus congenital anemia) The DTaP-IPV vaccine (tetanus toxoid conjugate) consists of Haemophilus influenzae type b capsular polysaccharide (polyribosyl-ribitol-phosphate (PRP)) covalently bound to tetanus toxoid (PRP-T). The DTaP-IPV component is supplied as a sterile liquid used to reconstitute the lyophilized ActHIB® component to form Pentacel®.

[0291] Each 0.5 mL dose contains 15 limit flocculation (Lf) of diphtheria toxoid, 5 Lf of tetanus toxoid, acellular pertussis antigens (20 μg of detoxified pertussis toxin (PT), 20 μg of filamentous hemagglutinin (FHA), 3 μg of pertactin (PRN), 5 μg of fimbriae types 2 and 3 (FIM)), inactivated poliovirus (40 D antigenic units (DU) of type 1 (Mahoney), 8 DU of type 2 (MEF-1), 32 DU of type 3 (Saukett)), and 10 μg of PRP (PRP-T) of Haemophilus influenzae type b covalently bound to 24 μg of tetanus toxoid.

[0292] Other components per 0.5 mL dose include 1.5 mg aluminum phosphate (0.33 mg aluminum) as an adjuvant, polysorbate 80 (calculated to be approximately 10 ppm), 42.5 mg sucrose, ≤5 μg residual formaldehyde, <50 ng residual glutaraldehyde, ≤50 ng residual bovine serum albumin, 3.3 mg (0.6% v / v) 2-phenoxyethanol (not as a preservative), <4 pg neomycin, and <4 pg polymyxin B sulfate.

[0293] Corynebacterium diphtheriae is grown in a modified Mueller growth medium. After purification by ammonium sulfate fractionation, diphtheria toxin is detoxified with formaldehyde and diafiltered.

[0294] Clostridium tetani is grown in modified Mueller-Miller casamino acids medium without bovine heart infusion. Tetanus toxin is detoxified with formaldehyde and purified by ammonium sulfate fractionation and ultrafiltration. Diphtheria and tetanus toxoids are individually adsorbed to aluminum phosphate.

[0295] Acellular pertussis vaccine antigens are produced from Bordetella pertussis cultures grown in Stainer-Scholte medium modified by the addition of casamino acids and dimethyl-beta-cyclodextrin. PT, FHA, and PRN are separately isolated from the supernatant culture medium. FIM is extracted from bacterial cells and co-purified. Pertussis antigens are purified by sequential filtration, salting out, ultrafiltration, and chromatography. PT is detoxified with glutaraldehyde. FHA is treated with formaldehyde, and residual aldehyde is removed by ultrafiltration. Individual antigens are separately adsorbed to aluminum phosphate.

[0296] Poliovirus types 1, 2, and 3 are each grown in separate cultures of MRC-5 cells, a normal human diploid cell line, using the microcarrier method. The cells are grown in CMRL (Connaught Medical Research Laboratories) 1969 medium supplemented with calf serum. For virus propagation, the culture medium is replaced with CMRL 1969 medium without calf serum. After clarification and filtration, the virus suspension is concentrated by ultrafiltration and purified by a liquid chromatography step. The monovalent virus suspension is inactivated with formaldehyde. The monovalent concentrates of each inactivated poliovirus are combined to produce a trivalent poliovirus concentrate.

[0297] Adsorbed diphtheria, tetanus, and acellular pertussis antigens are combined with aluminum phosphate (as an adjuvant), 2-phenoxyethanol (not as a preservative), and water for injection. The trivalent poliovirus concentrate is added and the DTaP-IPV component is diluted to its final concentration. The DTaP-IPV component does not contain a preservative.

[0298] Both diphtheria and tetanus toxoids induce at least 2 neutralizing units per mL in guinea pig efficacy tests. The efficacy of acellular pertussis antigens is assessed by the antibody response of immunized mice to inactivated PT, FHA, PRN, and FIM, as measured by enzyme-linked immunosorbent assay (ELISA). The efficacy of inactivated poliovirus antigens is determined by measuring antibody-mediated neutralization of poliovirus in serum from immunized rats.

[0299] PRP, a high molecular weight polymer, is prepared from Haemophilus influenzae type b strain 1482 grown in a semi-synthetic medium. Tetanus toxoid for conjugation to PRP is prepared by ammonium sulfate purification and formalin inactivation of the toxin from a culture of Clostridium tetani (Harvard strain) grown in modified Mueller and Miller medium. The toxoid is filter-sterilized before the conjugation process. ActHIB® components do not contain preservatives. The potency of ActHIB® components is specified for each lot by the PRP polysaccharide and protein content per dose and limits for the percentage of polysaccharides and proteins characterized as high molecular weight conjugates.

[0300] Prevention methods and uses, vaccination methods and uses How to prevent and vaccinate The present invention is directed, in part, to methods of preventing dengue disease (particularly virologically confirmed dengue, VCD) in a subject. Thus, in certain embodiments, the present invention is directed to methods of preventing dengue disease in a subject, comprising administering to the subject an inventive unit dose / tetravalent dengue virus composition described herein, particularly a reconstituted unit dose.

[0301] The present invention is directed, in part, to methods for preventing dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Thus, 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 an inventive reconstituted unit dose / tetravalent dengue virus composition described herein.

[0302] 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, in particular the tetravalent dengue virus composition comprises 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, in particular the dengue serotype 2 strains are derived from the wild-type virus strain DEN-2 16681 (SEQ ID NO: 11) and differ from the wild-type 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 chimera The present invention relates to a method for producing a serotype 2 strain comprising:

[0303] Further information regarding the serotypes of the tetravalent composition can be derived from the section "Dengue virus strains" above.

[0304] 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 log pfu / 0.5 ml It may be in the form of a unit dose comprising:

[0305] The present invention particularly relates to such a method, wherein the unit dose 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) Dengue serotype 4 at a concentration of at least 4.5 log pfu / 0.5 ml The present invention is directed to a method, including:

[0306] The invention is also particularly directed to such methods, wherein, upon reconstitution with a pharmaceutically acceptable diluent, (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL, wherein, based on said 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%, or at least 10%, or at least 12%, or at least 14%, or at least 16%, or at least 18%, and preferably the subject is aged 2-17 years or 4-16 years.

[0307] The invention is also particularly directed to such methods, wherein, upon reconstitution with a pharmaceutically acceptable diluent, (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL, wherein, based on said total concentration, the concentration of (ii) at pfu / 0.5 mL is less than 2%, the concentration of (iv) at pfu / 0.5 mL is at least 50%, the concentration of (i) at pfu / 0.5 mL is at least 1%, and the concentration of (iii) at pfu / 0.5 mL is at least 6%, and preferably the subject is 18 to 60 years of age.

[0308] Further information regarding tetravalent compositions or unit doses can be derived from the sections "Dengue vaccine compositions" and "Unit doses" above.

[0309] The present invention therefore relates to a method and a corresponding use, the method comprising: (A) administering to a subject a first unit dose of a tetravalent dengue virus composition; and (B) administering to the subject a second unit dose of the tetravalent dengue virus composition within three months of administering the first unit dose. The present invention is directed to methods and corresponding uses, including primary vaccination with only two administrations of a unit dose, comprising: According to this embodiment, administration of just two doses within three months is sufficient to provide effective protection against subsequent dengue infection.

[0310] Such methods preferably provide a combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease with a two-sided 95% confidence interval, the lower limit of which is greater than 60% from the first dose of the dosing schedule to 18 months after the second dose of the dosing schedule, when measured against placebo in a target population of at least 5,000 healthy subjects aged 14-16 years, regardless of serostatus at baseline.

[0311] Such methods also preferably provide a combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease within a two-sided 95% confidence interval, the lower limit of which is greater than 45% when measured against placebo in a target 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 in the dosing schedule to 18 months after the second dose in the dosing schedule.

[0312] According to certain embodiments, the method of vaccination against virologically confirmable dengue disease is caused by 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.

[0313] In certain embodiments, the invention is directed to a method having a vaccine efficacy against serotype 1 in preventing virologically confirmable dengue disease with a two-sided 95% confidence interval, the lower limit being greater than 25% from 30 days after the second dose through 12 to 18 months (e.g., at 12 or 18 months) after the second dose, when measured against placebo in a 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. 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, and the lower limit is greater than 35%. In certain such embodiments, the seronegative and seropositive populations each provide vaccine efficacy against serotype 1 with a two-sided 95% confidence interval, the lower limit of which is within 10 percentage points.

[0314] In certain embodiments, the invention is directed to the method, wherein the vaccine has vaccine efficacy against serotype 1 in preventing virologically confirmable dengue disease when measured against placebo in a subject population of at least 1,500, or at least 2,000, or at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4 to 16, regardless of baseline serostatus, from 30 days after the second dose through 12 to 18 months (e.g., at 12 or 18 months) after the second dose. In certain such embodiments, the vaccine efficacy is greater than 40%, greater than 50%, greater than 60%, or greater than 65%. In certain such embodiments, the subject population of at least 1,500 is seronegative to all serotypes at baseline. In certain such embodiments, the seronegative and seropositive populations, respectively, provide vaccine efficacy against serotype 1 within 5 percentage points.

[0315] In certain embodiments, the present invention provides a method for determining virologically confirmed dendritic cells with a two-sided 95% confidence interval. The method is directed to a vaccine having vaccine efficacy against serotype 2 in preventing Chlamydia fever disease, the lower limit of which is greater than 25% from 30 days after the second administration through 12 to 18 months (e.g., at 12 or 18 months) after the second administration, 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 to 16, regardless of baseline serostatus. 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 population of at least 1,500 subjects is seronegative to all serotypes. In certain such embodiments, the seronegative and seropositive populations each provide vaccine efficacy against serotype 2 with a two-sided 95% confidence interval, the lower limit of which is within 5 percentage points.

[0316] In certain embodiments, the invention is directed to the method, wherein the vaccine has vaccine efficacy against serotype 2 in preventing virologically confirmable dengue disease when measured against placebo in a subject population of at least 1,500, or at least 2,000, or at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4 to 16, regardless of baseline serostatus, from 30 days after the second dose through 12 to 18 months (e.g., at 12 or 18 months) after the second dose. In certain such embodiments, the vaccine efficacy is greater than 60%, greater than 70%, greater than 80%, or greater than 90%. In certain such embodiments, the subject population of at least 1,500 is seronegative to all serotypes at baseline. In certain such embodiments, the seronegative and seropositive populations, respectively, provide vaccine efficacy against serotype 2 within 5 percentage points.

[0317] The efficacy of the method is further described in more detail below in this section.

[0318] 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.

[0319] According to one embodiment, such a method does not include a step of determining whether the subject has had a previous dengue infection prior to administration of the unit dose, or the serostatus of the subject is unknown prior to administration of the unit dose.

[0320] According to one embodiment, such a method preferably does not include a step of determining past dengue infection in the subject at any time before, during or after the step of administering, or the serostatus of the subject is preferably unknown at any time before, during or after the step of administering.

[0321] 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 for a method wherein 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.

[0322] According to one embodiment of such a method, the subject is from an area where the seroprevalence is unknown and / or where the seroprevalence is less than 80%, or less than 70%, or less than 60%.

[0323] 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.

[0324] According to this embodiment, the vaccine and corresponding method are safe for both 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 a combined vaccine efficacy against virologically confirmed dengue with hospitalization against all four serotypes with a two-sided 95% confidence interval, the lower limit of which is greater than 65% from the first dose of the dosing schedule to 12-18 months after the second dose of the dosing schedule, when measured against placebo in a population of at least 5,000 healthy subjects aged 4-16 years, regardless of baseline serostatus, 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 against all four serotypes provides 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 therefore can be used even in pandemic situations with serotype 1 and / or serotype 2 or for seronegative subjects (e.g., travelers) or subjects with unknown serostatus in areas with very high seroprevalence (>80%) due to serotype 1 and / or serotype 2.

[0325] The safety of the method is further described in more detail in the section "Methods of prevention, methods of vaccination."

[0326] 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.

[0327] According to one embodiment, such methods do not include vaccine immunogenicity assays involving GMTs for dengue neutralizing antibodies.

[0328] According to one embodiment, such methods do not include reactogenicity analysis, e.g., regarding solitary local AEs (injection site pain, injection site erythema, and injection site swelling) and solitary systemic AEs (children <6 years: fever, irritability / tantrum, drowsiness, and loss of appetite; children ≥6 years: asthenia, fever, headache, malaise, and muscle pain), assessed for 7 days and 14 days (including the day of vaccination) after each vaccination, respectively, by collecting diary cards.

[0329] According to one embodiment, the method does not include active surveillance, immunogenicity analysis, and reactogenicity analysis.

[0330] According to such embodiments, the vaccine and corresponding administration method are safe and therefore do not require further steps of monitoring or analysis. In view of the above, a method according to one embodiment comprises: (A) selecting a subject eligible for administration of a unit dose of a tetravalent dengue virus composition in need of protection against dengue infection without a determination of past dengue infection; and (B) administering to the subject a first unit dose of a tetravalent dengue virus composition; and (C) administering to the subject a second unit dose of the tetravalent dengue virus composition within three months of administering the first unit dose. This includes a primary vaccination consisting of: Therefore, the vaccination method is completed without determining past dengue infection. The method further optionally includes a booster dose of one unit dose at least one year after administration of the second unit dose. Selecting the subject can include all types of considerations, but preferably does not include determining past dengue infection. Selection can include consideration of age, health status, and risk of infection. Risk of infection includes consideration of seroprevalence, serotype-specific seroprevalence, and pandemic status or serotype-specific pandemic status in the area where the subject normally resides or intends to travel. The subject can 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.

[0331] 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.

[0332] In particular, the present invention is directed to such methods, where the methods are safe.

[0333] In particular, the present invention is directed to such methods that provide a combined 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% from the first dose of the dosing schedule to 12 to 18 months after the last dose of the dosing schedule, when measured against placebo in a target population of at least 5,000 healthy subjects aged 4 to 16 years, regardless of serostatus at baseline.

[0334] In particular, the present invention is directed to such methods, where the methods are effective. In particular, the present invention is directed to such methods that provide a combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease with a two-sided 95% confidence interval, the lower limit of which is greater than 60% from the first dose of the dosing schedule to 18 months after the last dose of the dosing schedule, when measured against placebo in a target population of at least 5,000 healthy subjects aged 14-16 years, regardless of serostatus at baseline.

[0335] In certain embodiments, the invention is directed to the aforementioned methods, wherein the subject is seronegative to all dengue serotypes.

[0336] The present invention is directed, in part, to methods of preventing dengue disease (particularly virologically confirmed dengue, VCD) in a subject population. Thus, in certain embodiments, the present invention is directed to methods 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.

[0337] The present invention is directed, in part, to a method for preventing dengue disease (particularly virologically confirmable 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 predetermined ratio of geometric mean neutralizing antibody titers (GMT) is achieved at 180 days or 365 days after administration of the first unit dose to the subject population. According to some embodiments, the geometric mean neutralizing antibody titer for dengue serotype 2 (GMT DENV-2) and the dengue serotype 3 (VCD) are determined by the geometric mean neutralizing antibody titer (GMT DENV-3) and the dengue serotype 4 (VCD) at 180 days or 365 days after administration of the first unit dose to the subject population. The geometric mean neutralizing antibody titer for serotype 4 (GMT DENV-4), when tested in at least 40, or at least 50, or at least 60 subjects at least 180 days or 365 days after a first administration of the reconstituted unit dose of the invention described herein, and optionally a second administration of the reconstituted unit dose of the invention described herein 90 days after the first administration, provides 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. 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, and / or the ratio of GMT DENV-2:GMT DENV-3 is 20 or less, or 18 or less, or 15 or less, at 180 days or 365 days after administration of the first reconstituted unit dose.

[0338] The invention is directed, in part, to a method for preventing dengue disease (particularly virologically confirmable dengue, VCD) in a subject, comprising administering to the subject at least a first reconstituted unit dose of the invention described herein, wherein a predetermined ratio of neutralizing antibody titers is achieved 180 days or 365 days after administration of the first unit dose to the subject. According to some embodiments, the neutralizing antibody titers for dengue serotype 2 and the neutralizing antibody titers for dengue serotype 4 180 days or 365 days after administration of at least a first reconstituted unit dose of the invention described herein, and optionally a second reconstituted unit dose of the invention described herein 90 days after the first administration, provide a ratio of neutralizing antibody titers for 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 days 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 days or 365 days after administration of the first reconstituted unit dose.

[0339] The geometric mean neutralizing antibody titer (GMT) of a subject population, or the subject's neutralizing antibody titer, is determined according to the microneutralization test disclosed herein, for example, according to the method described in Example 2. Without wishing to be bound by any theory, it is now 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 a vaccinated subject or subject population in terms of a smaller difference between the geometric mean neutralizing antibody titers (GMT) or neutralizing antibody titers against the four dengue serotypes. In particular, it is understood that a much greater response against any one of the four serotypes, e.g., DENV-2, compared to the other serotypes is less beneficial.

[0340] The invention is directed, in part, to a method for preventing dengue disease (particularly virologically confirmable dengue, VCD) in a subject or subject population, the method providing a seropositivity rate in a subject population of at least 50 subjects comprising subcutaneous administration of two unit doses at days 1 and 90, 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% are seropositive for all four dengue serotypes at, for example, day 270 after administration of the second unit dose.

[0341] The invention is directed, in part, to a method for preventing dengue disease (particularly virologically confirmable dengue, VCD) in a subject or subject population, the method comprising subcutaneous administration of two unit doses at days 1 and 90 to provide a seropositivity rate in a subject population of at least 100 subjects, wherein the subjects in the subject population comprise 20%-40% subjects who are seronegative to all dengue serotypes and 60%-80% subjects who are seropositive to at least one dengue serotype at baseline, and wherein at day 120 and / or day 270, the seropositivity rates for all four dengue serotypes in the seronegative portion of the subject population and the seropositivity rates for all four dengue serotypes in the seropositive portion of the subject population do not deviate by more than 10 percentage points, and / or at day 120, the seropositivity rates for all four dengue serotypes in the seronegative portion of the subject population and the seropositivity rates for all four dengue serotypes in the seropositive portion of the subject population do not deviate by more than 5 percentage points.

[0342] 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.

[0343] 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.

[0344] 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, 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 (vi) 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 A reconstituted unit dose comprising:

[0345] 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) provide a total concentration of pfu / 0.5 mL, where, based on said total concentration, the concentration of (ii) at pfu / 0.5 mL is less than 2%, the concentration of (iv) at pfu / 0.5 mL is at least 50%, the concentration of (i) at pfu / 0.5 mL is at least 1%, and the concentration of (iii) at pfu / 0.5 mL is at least 6%, and the subject or subject population is preferably 18 to 60 years of age.

[0346] 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) provide a total concentration of pfu / 0.5 mL, wherein, based on said 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.

[0347] In certain embodiments, the invention provides a tetravalent dengue virus composition, wherein the unit dose comprises four live attenuated dengue serotypes, particularly the virus strains described herein, wherein the serotypes have predetermined concentrations as described herein for the virus composition and unit dose, e.g., (i) dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 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 log10 pfu / dose to 5.5 log10 pfu / 0.5 dose, or 4.5 log10 pfu / 0.5 mL to 5.5 log10 pfu / 0.5 mL. In preferred such embodiments, the subject or target population is between 2 and 17 years old, for example, between 4 and 16 years old, preferably under 9. In other preferred embodiments, the subject or target population is between 4 and 5 years old, between 6 and 11 years old, or between 12 and 16 years old.

[0348] In certain embodiments, the invention provides a method for producing a dengue virus having a concentration of 3.3 log pfu / 0.5 mL to 3.6 log pfu / 0.5 mL for dengue serotype 1 (e.g., chimeric dengue serotype 2 / 1 strains) and 2.7 log pfu / 0.5 mL to 4.0 log pfu / 0.5 mL for dengue serotype 2 (e.g., dengue serotype 2 strains) when reconstituted with 0.5 mL of a pharmaceutically acceptable diluent. The method is directed to a method comprising administering a dengue serotype 3 (e.g., chimeric dengue serotype 2 / 3 strain) vaccine to a subject having a concentration of 4.0 log pfu / 0.5 mL to 4.6 log pfu / 0.5 mL, and a dengue serotype 4 (e.g., chimeric dengue serotype 2 / 4 strain) vaccine to a subject having 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. In preferred such embodiments, the subject or subject population is between 2 and 17 years of age, e.g., between 4 and 16 years of age, preferably under 9 years of age. In other preferred embodiments, the subject or subject population is between 4 and 5 years of age, between 6 and 11 years of age, or between 12 and 16 years of age.

[0349] In certain embodiments, the invention provides a method for producing a dengue serotype 1 (e.g., a chimeric dengue serotype 2 / 1 strain) having a concentration of 1% to 7% of the total concentration, measured by pfu / 0.5 mL; a dengue serotype 2 (e.g., a dengue serotype 2 strain) having a concentration of less than 8% of the total concentration, e.g., in the range of 1% to 8% of the total concentration, measured by pfu / 0.5 mL; a dengue serotype 3 (e.g., a chimeric dengue serotype 2 / 3 strain) having a concentration of at least 10% of the total concentration, measured by pfu / 0.5 mL; and a dengue serotype 4 (e.g., a chimeric dengue serotype 2 / 4 strain) having a concentration of at least 65% of the total concentration, e.g., at least 6% of the total concentration. The method is directed to a method in which the arithmetic sum of all four serotypes is in the range of 5% 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, and 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.

[0350] In further preferred embodiments, the invention is directed to the 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, such as TDV-4 (e.g., a chimeric dengue serotype 2 / 4 strain), represents the highest concentration of all four serotypes in the composition, preferably having at least about 70% of the total concentration in pfu / 0.5 mL; dengue serotype 3, such as TDV-3 (e.g., a chimeric dengue serotype 2 / 3 strain), represents the second highest concentration of all four serotypes in the composition, preferably having at least about 10% of the total concentration in pfu / 0.5 mL; and 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), each represent a lower concentration than serotype 3, such as TDV-3 (e.g., a chimeric dengue serotype 2 / 3 strain), optionally together representing less than about 10% of the total concentration in pfu / 0.5 mL.

[0351] 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 a nucleotide sequence according to SEQ ID NO: 1 and an amino acid sequence according to SEQ ID NO: 2, TDV-2 is characterized by a nucleotide sequence according to SEQ ID NO: 3 and an amino acid sequence according to SEQ ID NO: 4, TDV-3 is characterized by a nucleotide sequence according to SEQ ID NO: 5 and an amino acid sequence according to SEQ ID NO: 6, and TDV-4 is characterized by a nucleotide sequence according to SEQ ID NO: 7 and an amino acid sequence according to SEQ ID NO: 8.

[0352] In certain embodiments, the present invention is directed to the method wherein a 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.

[0353] In certain embodiments, the invention is directed to the method wherein a reconstituted unit dose is administered to a subject of unknown serostatus, and / or wherein no testing to determine whether the subject is seropositive or seronegative has been performed (prior to) administering the unit dose described herein. In certain embodiments, the invention is directed to the method wherein the method does not include a step of determining past dengue infection in the subject or subjects. In certain embodiments, the invention is directed to the method wherein the method does not include analysis of seroprevalence in the area, or is performed in an area with less than 80%, less than 70%, or less than 60% seroprevalence. In certain embodiments, the invention is directed to the method wherein the serostatus of the subject is unknown. In such embodiments, the serostatus is not determined at any time before or after administration for this method. In certain embodiments of the invention, the method is used in a pandemic setting. In certain embodiments, The present invention is directed to such methods, which are carried out outside of clinical trials.

[0354] In certain embodiments, the invention is directed to the above-described methods, wherein the subject, or population of subjects, is seronegative to all dengue serotypes.

[0355] In certain embodiments, the present invention is directed to the 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 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.

[0356] In certain embodiments, the methods of the present invention comprise or consist of administering a single unit dose of the present invention.

[0357] In certain embodiments, the present invention is directed to the 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, hi other embodiments, the subject or population of subjects is seropositive for at least one dengue serotype.

[0358] In certain embodiments, the present invention is directed to the method, 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 present 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.

[0359] In certain embodiments, the present invention is directed to the 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 more 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.

[0360] In certain embodiments, the invention is directed to the method, 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 target population is under 9 years of age, or under 4 years of age. In some embodiments, the subject or target 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 target population is between 4 and 16 years of age. In some such embodiments, the subject or target population is between 4 and 5 years of age, 6 and 11 years of age, or 12 and 16 years of age. Optionally, the subject or target population is seronegative for all dengue serotypes.

[0361] In certain embodiments, the present invention is directed to the 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, or between 2 months and 1.5 years, or between 2 months and 1 year. According to some of these embodiments, the pediatric subject or population of pediatric subjects is seronegative and from a dengue-endemic area.

[0362] In certain embodiments, the present invention is directed to the 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, or between 2 months and 1.5 years, or between 2 months and 1 year. According to some of these embodiments, the pediatric subject or population of pediatric subjects is seronegative and from a dengue-endemic area.

[0363] In certain embodiments, the invention is directed to the method 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.

[0364] In certain embodiments, the invention is directed to the 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.

[0365] In certain embodiments, the invention is directed to the method, wherein the subject or subject population is from Asia-Pacific or Latin America and is seropositive for at least one dengue serotype at baseline, hi other embodiments, the subject or subject population is from Asia-Pacific or Latin America and is seronegative for all dengue serotypes at baseline.

[0366] In certain embodiments, the invention is directed to the 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.

[0367] In certain embodiments, the invention is directed to the above methods, 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, prior to 30 days after the second administration, such as within 4 months after the first administration. For example, 30 days after the second vaccination. For vaccine efficacy (VE) as determined in Examples 1 through 6, a prior yellow fever vaccination is defined as a yellow fever vaccination administered more than 30 days prior to a second vaccination. In certain embodiments, the subject or subject population received Dengvaxia® within the dosing regimen described herein or within 4.5 years after the first dose.

[0368] 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 demonstrates that seronegative subjects, in particular, 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 provide 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 needed to allow vaccination without prior serostatus analysis, which is a major advantage in vaccination programs and especially in pandemic situations.

[0369] 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.

[0370] 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, where the virus strains are optionally live-attenuated dengue virus strains.

[0371] In certain embodiments, the present invention is directed to methods that do not involve determining the subject's serostatus at baseline, i.e., the methods do not include determining the subject's past dengue infection at baseline prior to administration of the tetravalent dengue virus composition. In particular, such methods are safe and effective. Thus, in certain such embodiments, the subject has not been tested for the presence of a past dengue infection.

[0372] In certain embodiments, the present invention is directed to the methods wherein administration of the vaccine is safe regardless of whether there is a determination that the subject had a previous dengue infection prior to administration of the tetravalent dengue virus composition. Notably, such methods are also effective.

[0373] In certain embodiments, the present invention is directed to a method, wherein the method is safe and / or effective.

[0374] In certain embodiments, the invention is directed to the method wherein the composition comprises at least one chimeric dengue virus. In certain such embodiments, the invention is directed to the method wherein the composition comprises at least one non-chimeric dengue virus and at least one chimeric dengue virus, particularly chimeric dengue serotype 2 / 1 and dengue serotype 2 strains and chimeric dengue serotype 2 / 3 and dengue serotype 2 / 4 strains. Details of the compositions are provided above.

[0375] Thus, in certain embodiments, the present invention provides a vaccine efficacy, preferably a combined vaccine efficacy against all four serotypes, in preventing virologically confirmed dengue disease with a two-sided 95% confidence interval, with a lower bound of at least 5,000 healthy subjects (or at least 100) between the ages of 14 and 16, regardless of baseline serostatus.

[0023] The present invention relates to a method for administering a dengue virus vaccine comprising administering to a subject a vaccine comprising: a vaccine having a vaccine efficacy, preferably a combined vaccine efficacy against all four serotypes, of greater than 25% when measured against a placebo in a subject population of at least 10,000 healthy subjects, or at least 15,000 healthy subjects, after the first dose or 30 days after the second / final dose, and up to at least 12-18 months (e.g., at 12 or 18 months) after the second / final 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 embodiments, the invention is directed to a method in which a reconstituted unit dose or tetravalent dengue virus composition described herein or a placebo has a vaccine efficacy, preferably a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease within a two-sided 95% confidence interval, of greater than 25% by 15 to 21 months (e.g., 15 or 21 months) after the first dose of a dosing schedule, 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. 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 three months, eg, on days 0 and 90.

[0376] Thus, in certain embodiments, the invention is directed to methods having a combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease within a two-sided 95% confidence interval, where the lower limit is greater than 60% after the first administration through 18 months after the last administration, 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, and 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 these embodiments, the lower limit is, e.g., greater than 62%, greater than 64%, greater than 66%, greater than 68%, or greater than 69%.

[0377] In certain embodiments, the invention is directed to a method having a vaccine efficacy, preferably a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease of greater than 30% after the first dose or 30 days after the second / last dose, through at least 12 months or through 12 to 18 months (e.g., at 12 or 18 months) after the second / last dose, 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), e.g., between 14 and 16 years of age, wherein the reconstituted unit dose or tetravalent dengue virus composition described herein or placebo is administered at least twice within less than six months, e.g., within three months. In certain embodiments, the invention is directed to a method in which a reconstituted unit dose or tetravalent dengue virus composition described herein or a placebo has a vaccine efficacy, preferably a 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) regardless of baseline serostatus, of greater than 30% by 15 months after the first dose of a dosing schedule, wherein the reconstituted unit dose or tetravalent dengue virus composition described herein or a placebo is administered at least once. In certain such embodiments, the vaccine efficacy is greater than 40%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 78%, greater than 79%, or about 80%. Preferably, the reconstituted unit dose or tetravalent dengue virus composition described herein or a placebo has a vaccine efficacy, preferably a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease by 15 months after the first dose of a dosing schedule, 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. In certain such embodiments, the vaccine efficacy is greater than 40%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 78%, greater than 79%, or about 80%. The reconstituted unit dose or placebo is administered subcutaneously within about three months, for example, on days 0 and 90.

[0378] Thus, in certain embodiments, the invention is directed to methods having a combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease after the first administration through 18 months after the last administration, 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, of greater than 66%, 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 these embodiments, the vaccine efficacy is, e.g., greater than 68%, greater than 70%, greater than 72%, or greater than 74%.

[0379] In certain embodiments, the invention is directed to a method having a vaccine efficacy, preferably a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease with hospitalization, with a two-sided 95% confidence interval, the lower limit of which is greater than 0% 30 days after the second administration through at least 18 months after the second administration, 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, wherein the reconstituted unit dose or tetravalent dengue virus composition described herein or placebo is administered at least twice within less than six months, e.g., within three months. In certain such embodiments, the lower limit is greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70% or greater than 80%, or greater than 90%.

[0380] In certain embodiments, the invention is directed to a method having a combined vaccine efficacy against all four dengue serotypes in seronegative subjects, with a two-sided 95% confidence interval, of greater than 25% at about 30 days after the second dose of a dosing schedule, through at least 12 months or through 12 to 18 months (e.g., at 12 or 18 months) after the second dose of a dosing schedule, when measured against a 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, 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%.

[0381] In certain embodiments, the invention is directed to a method having a combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease within a two-sided 95% confidence interval, where the lower limit is greater than 25% from 30 days after the last dose through 12 to 18 months (e.g., at 12 or 18 months) after the last dose, 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 to 16 who are seronegative to all serotypes at baseline, 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%, or greater than 45%.

[0382] In certain embodiments, the invention is directed to a method having a combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease, with a two-sided 95% confidence interval, of greater than 25% against the dengue virus serotypes, measured against a placebo in a population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4-16 who are seropositive at baseline, from 30 days after the last dose through 12-18 months (e.g., at 12 or 18 months) after the last dose, wherein a reconstituted unit dose / tetravalent dengue virus composition described herein or a placebo is administered at least twice, e.g., within less than six months, e.g., within three months. In certain such embodiments, the lower limit is greater than 40%, greater than 45%, greater than 50%, greater than 60%, or greater than 65%.

[0383] In certain embodiments, the invention is directed to a method having a combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease at a two-sided 95% confidence interval, where the lower bound is greater than 25% from 30 days after the last dose through 12 to 18 months (e.g., at 12 or 18 months) after the last dose, 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 to 16 who are seropositive at baseline or seronegative to all serotypes at baseline, wherein a reconstituted unit dose / tetravalent dengue virus composition described herein or a placebo is administered at least twice, e.g., within less than six months, e.g., within three months. In certain such embodiments, the difference between the lower bound provided by subjects who are seropositive at baseline and subjects who are seronegative to all serotypes at baseline is 15 percentage points or less.

[0384] In certain embodiments, the invention is directed to a method having a combined vaccine efficacy against all four dengue serotypes in seronegative subjects of greater than 30% 30 days after the second administration, through at least 12 months, or through 12 to 18 months (e.g., at 12 or 18 months) after the second administration, when measured against a 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, 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 in seronegative subjects is greater than 40%, greater than 50%, greater than 60%, greater than 65%, or greater than 70%.

[0385] In certain embodiments, the invention is directed to a method of preventing virologically confirmable dengue disease against all four serotypes from 30 days after the last dose through 12 to 18 months (e.g., at 12 or 18 months) after the last dose, when measured against a placebo in a 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 who are seronegative to all serotypes at baseline, where the reconstituted unit dose / tetravalent dengue virus composition described herein or a placebo is administered at least twice, e.g., within less than six months, e.g., within three months. In certain such embodiments, the vaccine efficacy is greater than 30%, greater than 40%, greater than 50%, greater than 55%, greater than 60%, or greater than 65%.

[0386] In certain embodiments, the present invention provides a method for the treatment of children aged 4 to 16 years who are seropositive at baseline.

[0013] The method is directed to a method comprising administering a reconstituted unit dose / tetravalent dengue virus composition described herein or a placebo at least twice, e.g., within less than six months, e.g., within three months, that has a combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease, measured against a placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects), from 30 days after the last dose to 12 to 18 months (e.g., at 12 or 18 months) after the last dose. 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%.

[0387] In certain embodiments, the invention is directed to a method having a combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease at a two-sided 95% confidence interval, where the lower bound is greater than 25% from 30 days after the last dose through 12 to 18 months (e.g., at 12 or 18 months) after the last dose, 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 to 16 who are seropositive at baseline or seronegative to all serotypes at baseline, wherein a reconstituted unit dose / tetravalent dengue virus composition described herein or a placebo is administered at least twice, e.g., within less than six months, e.g., within three months. In certain such embodiments, the difference between the lower bound 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.

[0388] In certain embodiments, the invention is directed to a method having a combined vaccine efficacy against all four dengue serotypes at a two-sided 95% confidence interval, the lower limit of which is greater than 25% at about 30 days after the second dose of the dosing schedule, at least 12 months after the second dose of the dosing schedule, or at least 12 to 18 months (e.g., at 12 or 18 months) when measured against placebo in a population of at least 1,000 healthy subjects 4 to 5 years old at randomization, regardless of baseline serostatus, 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%, or greater than 45%.

[0389] In certain embodiments, the invention is directed to a method having a combined vaccine efficacy against all four dengue serotypes of greater than 30% 30 days after the second dose, through at least 12 months after the second dose, or through 12 to 18 months (e.g., at 12 or 18 months) after the second dose, when measured against placebo in a study population of at least 1,000 healthy subjects 4-5 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 65%, or greater than 70%.

[0390] In certain embodiments, the invention provides a vaccine having a combined vaccine efficacy against all four dengue serotypes with a two-sided 95% confidence interval, the lower limit of which is greater than 25% when measured against placebo in a study population of at least 1,000 healthy subjects aged 6-11 years at the time of randomization, regardless of baseline serostatus, about 30 days after the second dose of the dosing schedule, up to at least 12 months after the second dose of the dosing schedule, or up to 12-18 months (e.g., at 12 or 18 months). In some such embodiments, the lower limit is greater than 30%, greater than 40%, greater than 50%, greater than 60%, or greater than 70%.

[0391] In certain embodiments, the invention is directed to a method having a combined vaccine efficacy against all four dengue serotypes of greater than 30% 30 days after the second dose, through at least 12 months after the second dose, or through 12 to 18 months (e.g., at 12 or 18 months) after the second dose, when measured against placebo in a target 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%.

[0392] In certain embodiments, the invention is directed to a method having a combined vaccine efficacy against all four dengue serotypes at a two-sided 95% confidence interval, the lower limit of which is greater than 25% at about 30 days after the second dose of the dosing schedule, at least 12 months after the second dose of the dosing schedule, or at least 12 to 18 months after the second dose of the dosing schedule (e.g., at 12 or 18 months), when measured against placebo in a population of at least 1,000 healthy subjects aged 12 to 16 years at randomization, regardless of baseline serostatus, 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%, greater than 60%, greater than 65%, or greater than 68%.

[0393] In certain embodiments, the invention is directed to a method having a combined vaccine efficacy against all four dengue serotypes of greater than 30% 30 days after the second dose, through at least 12 months after the second dose, or through 12 to 18 months (e.g., at 12 or 18 months) after the second dose, when measured against placebo in a target population of at least 1,000 healthy subjects aged 12 to 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%.

[0394] In certain embodiments, the invention is directed to a method having a vaccine efficacy against serotype 1 with a two-sided 95% confidence interval, the lower limit of which is greater than 25% about 30 days after the second dose of a dosing schedule, through at least 12 months or through 12 to 18 months (e.g., at 12 or 18 months) after the second dose of a dosing schedule, 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, 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%, or greater than 50%.

[0395] In certain embodiments, the invention provides vaccine efficacy against serotype 1 in preventing virologically confirmed dengue disease with a two-sided 95% confidence interval, with a lower bound of at least 1,500 individuals aged 4 to 16 years, regardless of baseline serostatus, or at least The method also encompasses a vaccine having a vaccine efficacy of greater than 25% from 30 days after the last administration to 12 to 18 months (e.g., at 12 or 18 months) after the last administration, as measured against a placebo in a subject population of at least 2,000 or at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects), where the reconstituted unit dose / tetravalent dengue virus composition described herein or the 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, the subject population of at least 1,500 subjects is seronegative to all serotypes at baseline, and the lower limit is greater than 35%. In certain such embodiments, the seronegative and seropositive populations each provide vaccine efficacy against serotype 1 with a two-sided 95% confidence interval, the lower limit of which is within 10 percentage points.

[0396] In certain embodiments, the invention is directed to a method having a vaccine efficacy against dengue serotype 1 of greater than 30% 30 days after the second administration, through at least 12 months, or through 12 to 18 months (e.g., at 12 or 18 months) after the second administration, 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, 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 vaccine efficacy against dengue serotype 1 is greater than 40%, greater than 50%, greater than 60%, greater than 65%, or greater than 70%.

[0397] In certain embodiments, the invention is directed to a method having vaccine efficacy against serotype 1 in preventing virologically confirmable dengue disease from 30 days after the last dose through 12 to 18 months (e.g., at 12 or 18 months) after the last dose, when measured against a placebo in a subject population of at least 1,500, or at least 2,000, or at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4 to 16, regardless of baseline serostatus, wherein a reconstituted unit dose / tetravalent dengue virus composition described herein or a placebo is administered at least twice, e.g., within less than six months, e.g., within three months. In certain such embodiments, the vaccine efficacy is greater than 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 provide vaccine efficacy against serotype 1 within 5 percentage points, respectively.

[0398] In certain embodiments, the invention is directed to a method having a vaccine efficacy against serotype 2 with a two-sided 95% confidence interval, the lower limit of which is greater than 25% about 30 days after the second dose of a dosing schedule, through at least 12 months or through 12 to 18 months (e.g., at 12 or 18 months) after the second dose of a dosing schedule, 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, 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%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0399] In certain embodiments, the present invention provides a vaccine efficacy against serotype 2 in preventing virologically confirmed dengue disease with a two-sided 95% confidence interval, the lower bound of which is greater than or equal to the baseline. The present invention relates to a method for administering a dengue virus composition comprising a dengue virus vaccine comprising ... In certain such embodiments, the seronegative and seropositive populations each provide vaccine efficacy against serotype 2 with a two-sided 95% confidence interval, the lower limit of which is within 5 percentage points.

[0400] In certain embodiments, the invention is directed to a method having a vaccine efficacy against dengue serotype 2 of greater than 30% 30 days after the second administration, through at least 12 months, or through 12 to 18 months (e.g., at 12 or 18 months) after the second administration, 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, 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 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%.

[0401] In certain embodiments, the invention is directed to a method in which a reconstituted unit dose / tetravalent dengue virus composition described herein or a placebo has vaccine efficacy against serotype 2 in preventing virologically confirmable dengue disease from 30 days after the last dose through 12 to 18 months (e.g., at 12 or 18 months) after the last dose, as measured against a placebo in a subject population of at least 1,500, or at least 2,000, or at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) ages 4 to 16, regardless of baseline serostatus, wherein the reconstituted unit dose / tetravalent dengue virus composition described herein or a placebo is administered at least twice, e.g., within less than six months, e.g., within three months. In certain such embodiments, the vaccine efficacy is greater than 60%, greater than 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 provide vaccine efficacy against serotype 2 within 5 percentage points, respectively.

[0402] In certain embodiments, the invention is directed to a method having a vaccine efficacy against serotype 3 with a two-sided 95% confidence interval, the lower limit of which is greater than 25% about 30 days after the second dose of a dosing schedule through at least 12 months after the second dose of a dosing schedule, 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, 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%.

[0403] In certain embodiments, the present invention provides at least one antibody that is effective in treating a patient with HIV regardless of baseline serostatus.

[0013] In some embodiments, the vaccine efficacy against dengue serotype 3 is greater than 30%, measured against a placebo in a subject population of at least 5,000 healthy subjects, or at least 10,000 healthy subjects, or at least 15,000 healthy subjects, 30 days after the second administration, and at least 12 months after the second administration, 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 some such embodiments, the vaccine efficacy against dengue serotype 3 is greater than 40%, greater than 50%, greater than 55%, or greater than 60%.

[0404] In certain embodiments, the invention is directed to a method having a combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease with hospitalization, with a two-sided 95% confidence interval, the lower limit of which is greater than 25% from the first dose through 12 to 18 months (e.g., at 12 or 18 months) after the last dose, or from 30 days after the last dose through 12 to 18 months (e.g., at 12 or 18 months) after the last dose, 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 to 16 years, regardless of baseline serostatus, 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%.

[0405] In certain embodiments, the invention is directed to a method that has combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease with hospitalization from the first dose through 12 to 18 months (e.g., at 12 or 18 months) after the last dose, or from 30 days after the last dose through 12 to 18 months (e.g., at 12 or 18 months) after the last dose, 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 to 16, regardless of baseline serostatus, where a reconstituted unit dose / tetravalent dengue virus composition described herein or a placebo is administered at least twice, e.g., within less than six months, e.g., within three months. In certain such embodiments, the vaccine efficacy is greater than 70%, greater than 75%, greater than 80%, or greater than 82%, or greater than 85%, or greater than 88%.

[0406] In certain embodiments, the invention is directed to a method having a combined vaccine efficacy against virologically confirmed dengue fever with hospitalization for all four serotypes, with a two-sided 95% confidence interval, where the lower limit, 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, is greater than 25% about 30 days after the second dose of the dosing schedule, through at least 12 months or through 12 to 18 months (e.g., at 12 or 18 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%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, or greater than 75%.

[0407] In certain embodiments, the present invention provides a combined vaccine efficacy against all four serotypes in preventing virologically confirmed dengue disease with hospitalization with a two-sided 95% confidence interval, with the lower bound being seronegative to all serotypes at baseline.

[0013] Contemplated are methods having a combined vaccine efficacy of greater than 25% from 30 days after the last dose to 12 to 18 months (e.g., at 12 or 18 months) after the last dose, 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 to 16, wherein a reconstituted unit dose / tetravalent dengue virus composition described herein or a placebo is administered at least twice, e.g., within less than six months, e.g., within three months. In certain such embodiments, the 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%.

[0408] In certain embodiments, the invention is directed to a method having a combined vaccine efficacy against virologically confirmed dengue fever with hospitalization for all four serotypes of greater than 30% 30 days after the second administration, through at least 12 months after the second administration, or through 12 to 18 months (e.g., at 12 or 18 months) after the second administration, 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, 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 fever 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%.

[0409] In certain embodiments, the invention is directed to a method of preventing virologically confirmable dengue disease with hospitalization against all four serotypes from 30 days after the last dose through 12 to 18 months (e.g., at 12 or 18 months) after the last dose, 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 to 16 who are seronegative to all serotypes at baseline, wherein a reconstituted unit dose / tetravalent dengue virus composition described herein or a placebo is administered at least twice, e.g., within less than six months, e.g., within three months. In certain such embodiments, the vaccine efficacy is greater than 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%.

[0410] In certain embodiments, the invention is directed to a method having a combined vaccine efficacy against virologically confirmed dengue disease with hospitalization for all four serotypes, with a two-sided 95% confidence interval, of greater than 25% at about 30 days after the second dose of a dosing schedule, through at least 12 months after the second dose of a dosing schedule, or through 12 to 18 months (e.g., at 12 or 18 months) after the second dose of a dosing schedule, when measured against placebo in a target population of at least 1,500 or at least 2,000 healthy subjects who are seropositive at baseline, 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%, greater than 60%, greater than 70%, or greater than 80%.

[0411] In certain embodiments, the present invention provides a method for evaluating combined vaccine efficacy against all four serotypes in preventing virologically confirmed dengue disease with hospitalization, with a two-sided 95% confidence interval, with a lower bound of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000) ages 4-16 years who are seropositive at baseline.

[0010] The present invention relates to a method of administering a tetravalent dengue virus composition comprising a dengue virus vaccine comprising: a dengue virus vaccine comprising ...

[0412] In certain embodiments, the invention is directed to a method having a combined vaccine efficacy against virologically confirmed dengue with hospitalization for all four serotypes of greater than 30% 30 days after the second dose, through at least 12 months after the second dose, or through 12 to 18 months (e.g., at 12 or 18 months) after the second dose, when measured against placebo in a target population of at least 1,500 or at least 2,000 healthy subjects who are seropositive at baseline, 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%.

[0413] In certain embodiments, the invention is directed to a method having combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease with hospitalization from 30 days after the last dose through 12 to 18 months (e.g., at 12 or 18 months) after the last dose, 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 to 16 who are seropositive at baseline, wherein a reconstituted unit dose / tetravalent dengue virus composition described herein or a placebo is administered at least twice, e.g., within less than six months, e.g., within three months. In certain such embodiments, the vaccine efficacy is greater than 75%, greater than 70%, greater than 80%, greater than 85%, or greater than 90%.

[0414] In certain embodiments, the invention is directed to a method having a 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% from 30 days after the last dose through 12 to 18 months (e.g., at 12 or 18 months) after the last dose, 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 to 16 years who are seropositive at baseline or seronegative to all serotypes at baseline, 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 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.

[0415] In certain embodiments, the present invention provides a vaccine comprising a reconstituted unit dose / tetravalent vaccine as described herein that has combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue fever illness with hospitalization from 30 days after the last dose through 12 to 18 months (e.g., at 12 months or 18 months) after the last dose, 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 to 16 years who are seropositive at baseline or seronegative to all serotypes at baseline. 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.

[0416] In certain embodiments, the invention is directed to a method having a relative risk, preferably a combined relative risk for all four serotypes, with an upper bound, 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, of less than 0.75 at 30 days after the second administration and 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 upper bound 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.

[0417] In certain embodiments, the invention is directed to a method in which a reconstituted unit dose / tetravalent dengue virus composition described herein or placebo has a relative risk, preferably a combined relative risk for all four serotypes, of less than 0.70 when measured against a placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) regardless of baseline serostatus, 30 days after the second administration and at least 12 months after the second administration, and the 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, e.g., on days 0 and 90.

[0418] In certain embodiments, the invention is directed to a method in which virologically confirmable dengue disease occurs in less than 2.5% of subjects 30 days after the second administration, at least 12 months, or at least 18 months after the second administration, 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), regardless of baseline serostatus, and the 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, 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.

[0419] In certain embodiments, the present invention provides a combined vaccine efficacy against all four serotypes with a two-sided 95% confidence interval, the lower limit of which is 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 aged 4-16 years at randomization, regardless of baseline serostatus, at least 30 days after the last dose of the dosing schedule, at least 12 days after the last dose of the dosing schedule, or at least 14 days after the last dose of the dosing schedule. or greater than 61.0%, or greater than 65.0%, or greater than 70.0%, or greater than 72.0% after 13 months, wherein the unit dose / tetravalent dengue virus composition or the placebo is administered at least twice within six months or less.

[0420] In certain embodiments, the invention is directed to a method having a combined vaccine efficacy against all four serotypes of 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 randomization regardless of baseline serostatus, and up to about 30 days after the last dose of the dosing schedule, 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.

[0421] In certain embodiments, the invention is directed to the method, wherein the combined vaccine efficacy against all four serotypes is measured about 30 days after the last dose of the dosing schedule, up to 12 or 13 months after the last dose of the dosing schedule.

[0422] In certain embodiments, the invention is directed to the method, wherein the unit dose or the placebo is administered at least twice within three months, particularly at about day 1 and about day 90, and the combined vaccine efficacy against all four serotypes is measured 30 days after the second dose, up to 12 or 13 months after the second dose in the dosing schedule.

[0423] In certain embodiments, the invention is directed to the methods, wherein the methods are 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 subjects are seronegative for all dengue serotypes.

[0424] In certain embodiments, the invention is directed to methods wherein the method has a relative risk of virologically confirmed dengue fever with hospitalization of 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 2-9 years of age, or 2-5 years of age, or 4-9 years of age, or 6-9 years of age. Optionally, the subjects are seronegative for all dengue serotypes.

[0425] In certain embodiments, the invention is directed to the method, 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.

[0426] In certain embodiments, the present invention is directed to the method, wherein healthy subjects of the subject population are defined as being healthy in terms of the exclusion criteria set forth in Example 6.

[0427] 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.

[0428] In certain embodiments, the invention is directed to the method, wherein the healthy subjects of the subject population are seropositive for at least one serotype, hi other embodiments, the healthy subjects of the subject population are seronegative for all serotypes.

[0429] In certain embodiments, the invention is directed to the method, 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.

[0430] In certain embodiments, the invention is directed to the method, wherein healthy subjects of the target 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, healthy subjects of the target 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.

[0431] In certain embodiments, the invention is directed to the 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, hi 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.

[0432] In certain embodiments, the invention is directed to the 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.

[0433] In certain embodiments, the present invention is directed to the above methods, 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 vaccination 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 vaccination with yellow fever is defined as a yellow fever vaccination administered more than 30 days after the second vaccination.

[0434] In certain embodiments, the invention is directed to the aforementioned methods, wherein the healthy subjects of the target population have received a prior vaccination against Japanese encephalitis, hi other embodiments, the healthy subjects of the target population have not received a prior vaccination against Japanese encephalitis.

[0435] In certain embodiments, the invention is directed to the methods wherein healthy subjects in the subject population received Dengvaxia® within the dosing regimen described herein or within 4.5 years after administration of the first dose. In certain embodiments, the invention is directed to the methods wherein the incidence of vaccine-associated serious adverse events is less than 0.1%.

[0436] In certain embodiments, the present invention provides a vaccine-associated spontaneous adverse event that occurs within four weeks of administration. The method is directed to a method wherein the incidence of elephantiasis is less than 2%.

[0437] In certain embodiments, the invention is directed to the method, wherein the incidence of vaccine-associated unsolicited adverse events occurring within two weeks of administration is less than 35%.

[0438] In certain embodiments, the invention is directed to the method, wherein the incidence of vaccine-associated non-spontaneous local reactions occurring within one week of administration is less than 40%.

[0439] In certain embodiments, the invention is directed to methods, wherein the methods do not increase the risk of virologically confirmed dengue fever associated with hospitalization in an individual, eg, a seronegative individual.

[0440] In one embodiment, the unit dose is co-administered with a vaccine selected from the group of DTaP / IPV / Hib vaccines, particularly DTaP / IPV / Hib combination vaccines, MMR vaccines, yellow fever vaccines, particularly YF-17D, HPV vaccines, particularly 9vHPV vaccines, tetanus, diphtheria, and pertussis (Tdap) vaccines, particularly tetanus toxoid, attenuated diphtheria toxoid and acellular pertussis (adsorbed) combination vaccines, and hepatitis A vaccines, as described in more detail below.

[0441] Unit dosage for use in a method for preventing dengue disease The present invention is directed, in part, to compositions or unit doses of the invention described herein for use in methods of preventing dengue disease (particularly virologically confirmed dengue, VCD) in a subject.

[0442] The present invention is directed, in part, to compositions or unit doses of the invention described herein for use in methods of preventing dengue disease (particularly virologically confirmed dengue, VCD) in a subject population.

[0443] Inoculation against dengue disease, vaccination against dengue disease, and prevention of dengue disease have the same meaning. The present invention is directed, in part, to methods of preventing dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS).

[0444] Any method described herein above under the heading "Methods of preventing, methods of vaccinating," whether or not explicitly stated below, is also understood to be disclosed as a unit dose for use in such methods of preventing dengue disease in a subject or population of subjects.

[0445] According to certain embodiments, the composition or unit dose of the invention is a tetravalent dengue virus composition comprising chimeric dengue serotype 2 / 1 and dengue serotype 2 strains and chimeric dengue serotype 2 / 3 and dengue serotype 2 / 4 strains. According to certain such embodiments, the dengue serotype 2 strain is derived from the wild-type virus strain DEN-2 16681 (represented by SEQ ID NO: 11) and differs from the wild-type in at least three nucleotides as follows: a) 5'-noncoding region (NCR)-57 (nt-57 C to T) b) NS1-53 Gly to Asp (nt-2579 G to A) c) NS3-250 Glu to Val (nt-5270 A to T); 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 chimera It is derived from serotype 2 strains by causing

[0446] Further information regarding the serotypes of the tetravalent composition can be derived from the section "Dengue virus strains" above.

[0447] A tetravalent dengue virus composition for such use 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 log pfu / 0.5 ml It may be in the form of a unit dose comprising:

[0448] The tetravalent dengue virus composition for such use also has the following properties: when reconstituted 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) Dengue serotype 4 at a concentration of at least 4.5 log pfu / 0.5 ml The compound may be in the form of a lyophilized unit dose comprising:

[0449] In one embodiment, the tetravalent dengue virus composition for such use, when reconstituted with a pharmaceutically acceptable diluent, provides a total concentration of (i), (ii), (iii), and (iv) at pfu / 0.5 mL, where, based on said total concentration, the concentration of (ii) at pfu / 0.5 mL is preferably less than 10%, the concentration of (iv) at pfu / 0.5 mL is preferably at least 50%, the concentration of (i) at pfu / 0.5 mL is preferably at least 1%, and the concentration of (iii) at pfu / 0.5 mL is preferably at least 8%, or more preferably at least 10%, or at least 12%, or at least 14%, or at least 16%, or at least 18%, and the subject is preferably 2-17 years old or 4-16 years old.

[0450] In one embodiment, the tetravalent dengue virus composition for such use, when reconstituted with a pharmaceutically acceptable diluent, (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL, where, based on said total concentration, the concentration of (ii) at pfu / 0.5 mL is preferably less than 2%, the concentration of (iv) at pfu / 0.5 mL is preferably at least 50%, the concentration of (i) at pfu / 0.5 mL is preferably at least 1%, and the concentration of (iii) at pfu / 0.5 mL is preferably at least 6%, and the subject is preferably 18 to 60 years old.

[0451] Further information regarding tetravalent compositions or unit doses can be derived from the sections "Dengue vaccine compositions" and "Unit doses" above.

[0452] According to one embodiment, the unit dose for such use comprises: (A) administering to a subject a first unit dose of a tetravalent dengue virus composition; and (B) administering to the subject a second unit dose of the tetravalent dengue virus composition within three months of administering the first unit dose. The present invention is directed to a method comprising primary vaccination with only two administrations of a unit dose, comprising: According to this embodiment, administration of only two doses within three months provides protection against subsequent dengue infection. is sufficient to provide effective protection for

[0453] Such methods preferably provide a combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease with a two-sided 95% confidence interval, the lower limit of which is greater than 60% from the first dose of the dosing schedule to 18 months after the second dose of the dosing schedule, when measured against placebo in a target population of at least 5,000 healthy subjects aged 14-16 years, regardless of serostatus at baseline.

[0454] Such methods also preferably provide a combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease within a two-sided 95% confidence interval, the lower limit of which is greater than 45% when measured against placebo in a target 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 in the dosing schedule to 18 months after the second dose in the dosing schedule.

[0455] According to certain embodiments, the use is for a method of inoculation against virologically confirmable dengue disease caused by 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.

[0456] In certain ...

Claims

1. A dengue fever vaccine composition used to reduce the risk of virologically verifiable dengue fever disease requiring hospitalization in a target or target population, wherein the dengue fever vaccine composition is a tetravalent dengue fever vaccine composition containing four attenuated live dengue virus strains representing serotype 1, serotype 2, serotype 3, and serotype 4.

2. The dengue vaccine composition according to claim 1, wherein the reduction in the risk of virologically verifiable dengue fever requiring hospitalization is represented by a combined vaccine efficacy of at least 60%, at least 70%, at least 80%, or at least 90% against virologically verifiable dengue fever requiring hospitalization against all four dengue virus serotypes, regardless of baseline dengue serostatism.

3. The dengue vaccine composition according to claim 1, wherein the reduction in the risk of virologically verifiable dengue fever requiring hospitalization is represented by a combined vaccine efficacy of at least 60%, at least 70%, at least 80%, or at least 90% against virologically verifiable dengue fever requiring hospitalization for all four dengue virus serotypes in subjects who are seronegative for all dengue virus serotypes at baseline.

4. The dengue vaccine composition according to claim 1, wherein the reduction in the risk of virologically confirmed dengue fever disease requiring hospitalization is represented by a two-sided 95% confidence interval of the combined vaccine efficacy against virologically confirmed dengue fever disease requiring hospitalization for all four dengue virus serotypes, regardless of baseline dengue serostatism, wherein the lower limit of the confidence interval is greater than 60%, greater than 65%, greater than 70%, greater than 75%, or greater than 80%.

5. The dengue vaccine composition according to claim 1, wherein the reduction in the risk of virologically confirmed dengue fever disease requiring hospitalization is represented by a two-sided 95% confidence interval of the combined vaccine efficacy against virologically confirmed dengue fever disease requiring hospitalization for all four dengue virus serotypes in subjects that are seronegative for all dengue virus serotypes at baseline, wherein the lower limit of the confidence interval is greater than 60%, greater than 65%, greater than 70%, or greater than 75%.

6. The dengue fever vaccine composition according to any one of claims 1 to 5, wherein the reduction in the risk of virologically verifiable dengue fever disease requiring hospitalization is represented by a relative risk of 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, or 0.2 or less.

7. The dengue vaccine composition according to any one of claims 1 to 6, wherein the reduction in the risk of virologically verifiable dengue fever disease requiring hospitalization in the subject or target population is independent of the baseline serological status of the subject or target population.

8. The dengue vaccine composition according to any one of claims 1 to 6, wherein the reduction in the risk of virologically verifiable dengue fever disease requiring hospitalization in the subject or target population is independent of whether the subject or target population is seronegative at baseline or seropositive for at least one dengue serotype at baseline.

9. A dengue fever vaccine composition used to reduce the risk of dengue fever disease in a subject or target population, wherein the use of the dengue fever vaccine composition does not increase the risk of virologically verifiable dengue fever disease requiring hospitalization in the subject or target population, and the dengue fever vaccine composition is a tetravalent dengue fever vaccine composition containing four attenuated live dengue virus strains representing serotype 1, serotype 2, serotype 3, and serotype 4.

10. The dengue vaccine composition according to claim 9, wherein the use of the dengue vaccine composition does not increase the risk of virologically verifiable dengue disease requiring hospitalization in the subject or target population, regardless of the baseline serological status of the subject or target population.

11. The dengue vaccine composition according to claim 9, wherein the use of the dengue vaccine composition does not increase the risk of virologically verifiable dengue disease requiring hospitalization in the subject or target population, regardless of whether the subject or target population is seronegative at baseline or seropositive for at least one dengue serotype at baseline.

12. The dengue fever vaccine composition according to any one of claims 1 to 11, wherein the virologically verifiable dengue fever disease requiring hospitalization is caused by dengue serotype 1 and / or dengue serotype 2.

13. A dengue fever vaccine composition used to reduce the risk of severe dengue fever disease in a target or target population, wherein the dengue fever vaccine composition is a tetravalent dengue fever vaccine composition containing four attenuated live dengue virus strains representing serotype 1, serotype 2, serotype 3, and serotype 4.

14. The dengue fever vaccine composition according to claim 13, wherein the severe dengue fever disease is caused by dengue serotype 1 and / or dengue serotype 2.

15. A dengue fever vaccine composition used to reduce the risk of dengue hemorrhagic fever (DHF) and / or dengue shock syndrome (DSS) in a target or target population, wherein the dengue fever vaccine composition is a tetravalent dengue fever vaccine composition comprising four attenuated live dengue virus strains representing serotype 1, serotype 2, serotype 3, and serotype 4.

16. The dengue fever vaccine composition according to claim 15, wherein the DHF and / or DSS are due to dengue serotype 1 and / or dengue serotype 2.

17. The dengue fever vaccine composition according to any one of claims 1 to 16, wherein the subject or target population is seronegative for all dengue virus serotypes at baseline.

18. The dengue fever vaccine composition according to any one of claims 1 to 16, wherein the subject or target population is seropositive for at least one dengue virus serotype at baseline.

19. The dengue vaccine composition according to any one of claims 1 to 16, wherein the baseline serum status of the subject or target population is unknown, or the serum status of the subject or target population is unknown at any point before, during, or after administration of the dengue vaccine composition.

20. The dengue fever vaccine composition according to any one of claims 1 to 19, wherein the subject or target group is from a dengue-free area.

21. The dengue fever vaccine composition according to any one of claims 1 to 19, wherein the subject or target population is from a region where the seroprevalence of dengue is unknown, or from a region where the seroprevalence of dengue is less than 80%, less than 70%, or less than 60%.

22. The dengue fever vaccine composition according to any one of claims 1 to 19, wherein the subject or target group is from a dengue-endemic area.

23. The dengue fever vaccine composition according to any one of claims 1 to 19, wherein the subject or target group travels from a dengue-free area to a dengue-endemic area, and / or the subject or target group is selected based on the subject or target group's plan to travel from a dengue-free area to a dengue-endemic area.

24. The dengue fever vaccine composition according to claim 22 or 23, wherein the dengue-endemic area has a seroprevalence of at least 80% or at least 90% for dengue serotype 1 and / or dengue serotype 2.

25. The dengue fever vaccine composition according to any one of claims 22 to 24, wherein the dengue endemic area is located in Latin America, Southeast Asia and / or the Pacific Islands.

26. The dengue fever vaccine composition according to any one of claims 22 to 25, wherein the dengue endemic area is located in 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 / or Vietnam.

27. ​​The dengue fever vaccine composition according to any one of claims 1 to 26, wherein the subject or target group has received prior vaccination against yellow fever, where prior vaccination against yellow fever means vaccination prior to administration of the dengue fever vaccine composition.

28. The dengue fever vaccine composition according to any one of claims 1 to 27, wherein the subject or target group has received prior vaccination against Japanese encephalitis, where prior vaccination against Japanese encephalitis means vaccination prior to administration of the dengue fever vaccine composition.

29. The dengue fever vaccine composition according to any one of claims 1 to 28, wherein the subject or target group is under 9 years of age, 4 to 5 years of age, 6 to 11 years of age, 12 to 16 years of age, 6 to 16 years of age, 4 to 16 years of age, 2 to 17 years of age, 9 years of age, or over 9 years of age, 9 to 19 years of age, 18 to 60 years of age, 18 to 45 years of age, or 46 to 60 years of age.

30. The dengue fever vaccine composition according to any one of claims 1 to 28, wherein the subject or target group is over 60 years of age, for example, 61 to 100 years, 61 to 90 years, 61 to 80 years, 61 to 75 years, or 61 to 70 years.

31. The dengue fever vaccine composition according to claim 30, wherein the subject or target population has at least one chronic disease or condition, and optionally, the at least one chronic condition or disease is selected from diabetes, hypertension, allergy, past stroke, ischemic heart disease, chronic renal dysfunction and chronic obstructive pulmonary disease.

32. The dengue fever vaccine composition according to any one of claims 1 to 31, wherein each of the four attenuated live dengue virus serotypes is selected from the following: (i) Non-chimeric dengue viruses containing all components from the same dengue serotype, and (ii) A chimeric dengue virus having parts from two dengue serotypes.

33. The dengue fever vaccine composition according to claim 32, wherein the dengue fever vaccine composition comprises at least one chimeric dengue virus and at least one non-chimeric dengue virus.

34. The dengue fever vaccine composition according to any one of claims 1 to 33, wherein the dengue fever vaccine composition comprises a chimeric dengue serotype 2 / 1 strain, a non-chimeric dengue serotype 2 strain, a chimeric dengue serotype 2 / 3 strain, and a chimeric dengue serotype 2 / 4 strain.

35. A dengue fever vaccine composition according to any one of claims 1 to 34, wherein each of the four attenuated live dengue virus strains comprises a dengue structural protein including a capsid protein (C), a premembrane protein (prM), and an envelope protein (E), and a dengue non-structural protein including NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5.

36. The two dengue serotypes are non-chimeric dengue strains derived from the wild-type virus strain DEN-2 16681, and differ from the wild-type in at least the following a) to c): a) 5' - Non-coded area (NCR) - 57 b) NS1-53 Gly to Asp c) NS3-250 Glu to Val, Here, the following chimeric dengue strains are derived from serotype 2 by replacing the structural proteins prM and E from serotype 2 with corresponding structural proteins derived from other dengue serotypes: -DENV-2 / 1 Chimera, -DENV-2 / 3 chimera, and -DENV-2 / 4 Chimera, A dengue fever vaccine composition according to any one of claims 1 to 35.

37. The dengue serotype 1 is a chimeric dengue serotype 2 / 1 strain, the dengue serotype 2 is a non-chimeric serotype 2 strain, the dengue serotype 3 is a chimeric dengue serotype 2 / 3 strain, and the dengue serotype 4 is a chimeric dengue serotype 2 / 4 strain. Here, the three chimeric dengue strains are derived from the two serotype strains by replacing the structural proteins prM and E from the two serotype strains with corresponding structural proteins derived from the other dengue serotype: -DENV-2 / 1 Chimera, -DENV-2 / 3 chimera, and -DENV-2 / 4 Chimera, Furthermore, in the DENV-2 / 1 chimeric strain, the structural proteins prM and E from the two serotype strains are replaced with the corresponding structural proteins derived from DENV-1 16007. Furthermore, in the DENV-2 / 3 chimeric strain, the structural proteins prM and E from the two serotype strains are replaced with the corresponding structural proteins derived from DENV-3 16562. Furthermore, in the DENV-2 / 4 chimeric strain, the structural proteins prM and E from the two serotype strains are replaced with corresponding structural proteins derived from DENV-4 1036, according to any one of claims 1 to 36.

38. The dengue fever vaccine composition according to any one of claims 1 to 37, wherein the dengue serotype 1 is characterized by the amino acid sequence of SEQ ID NO: 2, and / or the dengue serotype 2 is characterized by the amino acid sequence of SEQ ID NO: 4, and / or the dengue serotype 3 is characterized by the amino acid sequence of SEQ ID NO: 6, and / or the dengue serotype 4 is characterized by the amino acid sequence of SEQ ID NO:

8.

39. The dengue fever vaccine composition according to any one of claims 1 to 38, wherein the dengue serotype 1 is TDV-1, and / or the dengue serotype 2 is TDV-2, and / or the dengue serotype 3 is TDV-3, and / or the dengue serotype 4 is TDV-4.

40. The dengue fever vaccine composition according to any one of claims 1 to 39, wherein the dengue fever vaccine composition is TAK-003.

41. The dengue fever vaccine composition is - Dengue serotype 1 at a concentration of at least 3.3 log 10 pfu / 0.5 ml, - Dengue serotype 2 at a concentration of at least 2.7 log 10 pfu / 0.5 ml, - Dengue serotype 3 at a concentration of at least 4.0 log 10 pfu / 0.5 ml, and - Dengue serotype 4 at a concentration of at least 4.5 log 10 pfu / 0.5 ml A dengue fever vaccine composition according to any one of claims 1 to 40, comprising:

42. The dengue fever vaccine composition according to any one of claims 1 to 41, wherein the arithmetic sum of the four serotypes in the dengue fever vaccine composition is less than 6.7 log 10 pfu / 0.5 ml, or the arithmetic sum of the four serotypes in the dengue fever vaccine composition is in the range of 4.6 log 10 pfu / 0.5 ml to 6.7 log 10 pfu / 0.5 ml.

43. The dengue vaccine composition according to any one of claims 1 to 42, further comprising a non-reducing sugar, a surfactant, a protein, and an inorganic salt, wherein optionally the non-reducing sugar is trehalose, the surfactant is poloxamer 407, the protein is human serum albumin, and the inorganic salt is sodium chloride.

44. The use of the dengue vaccine composition is not comprised of determining whether there has been a past dengue infection in the subject or target population prior to administration of the dengue vaccine composition, wherein the determination of a past dengue infection is based on a laboratory-confirmed history of dengue or a properly certified serological test, according to any one of claims 1 to 43.

45. The use of the dengue vaccine composition does not involve determining whether there has been a past dengue infection in the subject or target population at any point before, during, or after administration of the dengue vaccine composition, wherein the determination of a past dengue infection is based on a laboratory-confirmed history of dengue or a appropriately certified serological test, according to any one of claims 1 to 43.

46. The dengue vaccine composition according to any one of claims 1 to 45, wherein the use of the dengue vaccine composition comprises administering a first dose and a second dose of the dengue vaccine composition to the subject or target population.

47. The dengue fever vaccine composition according to claim 46, wherein the second administration is performed within 12 months, 6 months, or 3 months after the administration of the first administration, and preferably the second administration is performed at least 4 weeks after the administration of the first administration.

48. The dengue fever vaccine composition according to claim 46 or 47, wherein the second administration is performed 3 months or 90 days after the administration of the first administration.

49. The use of the dengue vaccine composition further comprises administering a booster dose of the dengue vaccine composition to the subject or target population after the initial vaccination, wherein the booster dose is optionally a third dose of the dengue vaccine composition administered to the subject or target population, according to any one of claims 1 to 48.

50. The dengue fever vaccine composition according to claim 49, wherein the booster dose is administered at least 12 months or at least 5 years after the administration of the first dose of the dengue fever vaccine composition, and optionally, the booster dose is administered 12 months after the administration of the first dose of the dengue fever vaccine composition.

51. The dengue fever vaccine composition according to any one of claims 1 to 50, wherein the dengue fever vaccine composition is administered by subcutaneous injection, preferably into the deltoid muscle region of the arm.

52. The dengue fever vaccine composition according to any one of claims 1 to 51, wherein the dengue fever vaccine composition is administered incidentally, simultaneously, or sequentially with other vaccines, and optionally, the dengue fever vaccine composition and the other vaccines are administered on the same day.

53. The dengue fever vaccine composition according to claim 52, wherein the other vaccine is administered by subcutaneous injection or intramuscular injection.

54. The dengue vaccine composition according to claim 52 or 53, wherein the dengue vaccine composition and the other vaccine are administered to different anatomical sites such as the opposite arm, or the arm and thigh.

55. The dengue fever vaccine composition is a yellow fever vaccine for preventing yellow fever, for example YF-17D, and / or a hepatitis A vaccine for preventing hepatitis A, for example HAVRIX® or VAQTA®, and / or an HPV vaccine for preventing HPV-related cancer or genital warts, preferably the HPV vaccine is a 9-valent HPV vaccine such as GARDASIL® 9, and / or another dengue fever vaccine composition, and / or an MMR vaccine for preventing measles, mumps and rubella, for example M-M-R® II, and / or a Tdap vaccine for preventing tetanus, diphtheria and pertussis, preferably the Tdap vaccine A dengue fever vaccine composition according to any one of claims 52 to 54, wherein the vaccine is a mixed vaccine of tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis (adsorbent), such as BOOSTRIX®, and / or a DTaP / IPV / Hib vaccine for preventing diphtheria, tetanus, pertussis, poliomyelitis, and diseases caused by Haemophilus influenzae type b, such as Pentacel®, and / or a cholera vaccine, and / or a hepatitis E vaccine, and / or a Japanese encephalitis vaccine, and / or a meningococcal disease vaccine, and / or a rabies vaccine, and / or a tick-borne encephalitis vaccine, and / or a typhoid vaccine.

56. The dengue fever vaccine composition according to any one of claims 1 to 55, wherein the use of the dengue fever vaccine composition comprises administering the dengue fever vaccine composition on the same day as administering a yellow fever vaccine, for example YF-17D, for the prevention of yellow fever.

57. The dengue vaccine composition according to any one of claims 1 to 55, wherein the use of the dengue vaccine composition comprises administering the dengue vaccine composition on the same day as administering a hepatitis A vaccine for the prevention of hepatitis A, such as HAVRIX® or VAQTA®.

58. The use of the dengue fever vaccine composition comprises administering the dengue fever vaccine composition on the same day as administering an HPV vaccine for the prevention of HPV-related cancer or genital warts, wherein the HPV vaccine is preferably a 9-valent HPV vaccine such as GARDASIL® 9, according to any one of claims 1 to 55.

59. A kit for preparing a reconstituted dengue vaccine composition, (a) A freeze-dried dengue vaccine composition, wherein the freeze-dried dengue vaccine composition is a quadrivalent dengue vaccine composition containing four attenuated live dengue virus strains representing serotype 1, serotype 2, serotype 3, and serotype 4, and (b) A pharmaceutically acceptable diluent for reconstitution, Includes, The reconstituted dengue vaccine composition is used in a kit to reduce the risk of virologically verifiable dengue disease requiring hospitalization in a target or target population.

60. A kit for preparing a reconstituted dengue vaccine composition, (a) A freeze-dried quadrivalent dengue fever vaccine composition containing four attenuated live dengue virus strains representing serotype 1, serotype 2, serotype 3, and serotype 4, and (b) pharmaceutically acceptable diluent for reconstitution Includes, The reconstituted dengue vaccine composition is used to reduce the risk of dengue disease in a subject or target population, and such use does not increase the risk of virologically confirmed dengue disease requiring hospitalization in the subject or target population, in a kit.

61. A kit for preparing a reconstituted dengue vaccine composition, (a) A freeze-dried quadrivalent dengue vaccine composition containing four attenuated live dengue virus strains representing serotype 1, serotype 2, serotype 3, and serotype 4, and (b) pharmaceutically acceptable diluent for reconstitution Includes, The reconstituted dengue vaccine composition is used to reduce the risk of severe dengue disease in a target or target population, in a kit.

62. A kit for preparing a reconstituted dengue vaccine composition, (a) A freeze-dried quadrivalent dengue vaccine composition containing four attenuated live dengue virus strains representing serotype 1, serotype 2, serotype 3, and serotype 4, and (b) pharmaceutically acceptable diluent for reconstitution Includes, The reconstituted dengue vaccine composition is used to reduce the risk of dengue hemorrhagic fever (DHF) and / or dengue shock syndrome (DSS) in a target or target population, in a kit.

63. The kit according to any one of claims 59 to 62, wherein the subject or target population is seronegative for all dengue virus serotypes at baseline.

64. The kit according to any one of claims 59 to 63, wherein the virologically verifiable dengue fever disease requiring hospitalization, the severe dengue fever disease, or the DHF and / or DSS is caused by dengue serotype 1 and / or dengue serotype 2.

65. The kit according to any one of claims 59 to 64, wherein the reconstitution diluent is provided in a vial or a pre-filled syringe.

66. The kit according to any one of claims 59 to 65, wherein the reconstitution diluent is selected from water for injection, phosphate-buffered saline, or an aqueous sodium chloride solution.

67. By reconstituting the freeze-dried quadrivalent dengue vaccine composition with the diluent, (i) Dengue serotype 1 at a concentration of at least 3.3 log 10 pfu / 0.5 ml, (ii) Dengue serotype 2 at a concentration of at least 2.7 log 10 pfu / 0.5 ml, (iii) Dengue serotype 3 at a concentration of at least 4.0 log 10 pfu / 0.5 ml, and (iv) Dengue serotype 4 at a concentration of at least 4.5 log 10 pfu / 0.5 ml A kit according to any one of claims 59 to 66, which can be obtained.

68. The kit according to any one of claims 59 to 67, wherein the reconstituted dengue vaccine composition is used in combination with a yellow fever vaccine.

69. The kit according to any one of claims 59 to 68, wherein the reconstituted dengue vaccine composition is used in combination with a hepatitis A vaccine.

70. The kit according to any one of claims 59 to 69, wherein the reconstituted dengue vaccine composition is used in combination with an HPV vaccine.

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