KR20220049023A - How to Prevent Dengue Fever and Hepatitis A - Google Patents

Abstract

The present invention relates to a method for preventing dengue disease and hepatitis A in a subject or population of subjects by concurrently administering a unit dose of a dengue vaccine composition and hepatitis A vaccine on the same day. A unit dose of a dengue vaccine composition includes constructs of each dengue serotype, such as TDV-1, TDV-2, TDV-3 and TDV-4, at various concentrations to improve protection against dengue infection.

Description

How to Prevent Dengue Fever and Hepatitis A

The present invention relates to a method for administering a unit dose of a dengue vaccine composition to a subject or population of subjects simultaneously on the same day as the hepatitis A vaccine. A unit dose according to the invention provides an immune response against all serotypes of dengue virus, namely DENV-1, DENV-2, DENV-3 and DENV-4 and against hepatitis A virus.

Vaccines for protection against viral infection have been used effectively to reduce the incidence of human disease. One of the most successful techniques for viral vaccines is to immunize animals or humans with an attenuated or attenuated virus strain (“live attenuated virus”). Due to limited replication after immunization, the attenuated virus strain does not cause disease. However, limited viral replication is sufficient to express a complete repertoire of viral antigens and can generate a robust and long-lasting immune response against the virus. Upon subsequent exposure to the pathogenic viral strain, the immunized individual is protected from the disease. These live attenuated virus vaccines are among the most successful vaccines used in public health.

Dengue fever is a mosquito-borne disease caused by infection with the dengue virus. Dengue virus infection can cause debilitating and painful symptoms, including a sudden high fever, headache, joint and muscle aches, nausea, vomiting, and a 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 1-4 can also cause dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). In most severe cases, DHF and DSS can be life-threatening. Dengue virus causes between 50 and 100 million debilitating cases of dengue each year, 500,000 cases of DHF/DSS, and more than 20,000 deaths, most of them in children. All four dengue virus serotypes are endemic to tropical regions of the world, where they constitute the most important mosquito-borne virus threat to humans. Dengue virus is transmitted to humans mainly by the Aedes aegypti mosquito but also by the Aedes albopictus mosquito. Infection with one dengue virus serotype induces lifetime protection from re-infection with that serotype but does not prevent secondary infection with one of the other three dengue virus serotypes. In fact, prior infection with one dengue virus serotype may increase the risk of severe disease (DHF/DSS) upon secondary infection with another serotype.

To date, CYD-TDV (Dengvaxia®, Sanofi Pasteur, Lyon, France), a tetravalent dengue vaccine based on the yellow fever backbone, has been shown to have full vaccine efficacy against 56-61% of virologically confirmed dengue fever (VCD) in children in Asia and Latin America. (VE) has been approved in several countries based on clinical validation (Capeding MR, etc. 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® can enhance rather than reduce the risk of severe disease due to dengue infection in individuals (seronegative population) not previously infected with dengue virus. Therefore, Dengvaxia® is only recommended for individuals who have previously been infected with at least one dengue virus serotype (seropositive population). More specifically, according to the European Medicine Agency's European Public Assessment Report (EPAR) for the product, Dengvaxia® is used in people 9 to 45 years of age who have previously been infected with the dengue virus and who live in areas where the infection is endemic. used only for Endemic areas are areas where 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 report - September 2018. Wkly. Epidemiol Rec. 2018, 93:457-476. Report by S.R. Hadinegoro et al., New England Journal of Medicine, Vol. 373, page 1195, “Efficacy and Long-Term Safety of a Dengue Vaccine in Regions of Endemic Disease” < 9 of those infected with dengue fever, the sum of the risk of hospitalization due to dengue disease confirmed as a virus is 1.58. This includes dengue-naïve populations living in endemic areas, young individuals who have not tested seropositive for dengue or have not tested any seropositive for dengue fever or have been exposed to dengue fever, and travelers from non-endemic areas and Effective vaccines with good safety profiles in both dengue pure and seropositive individuals, including individuals, are still largely unmet. There is also a need to provide a reduction in the risk of dengue fever after only one dose for outbreak control or travel vaccination.

Another disadvantage of Dengvaxia®, currently the only approved dengue vaccine, is that it should be vaccinated only to people who have previously tested positive for dengue virus (EPAR) infection, i.e. individuals with a known serologic status for dengue. Therefore, individuals with an unknown serum status for dengue cannot be vaccinated with Dengvaxia®.

Thus, in both seronegative and seropositive populations, it stimulates an immune response against all dengue serotypes, preferably a balanced immune response against all serotypes, and against dengue disease of all severities (including DSS, DHF). There is a need for a dengue vaccine and corresponding method of vaccination that protects and is safe for subjects of a greater age, particularly those under 9 years of age. The development of a safe and effective vaccine capable of protecting children and young adults and geriatric subjects in all populations, especially in endemic settings and for travel purposes, including seronegative and seropositive populations, is an important factor for the prevention and control of these diseases worldwide. indicates the approach.

Therefore, there is a medical need for a dengue vaccine and corresponding inoculation methods, which are safe and efficacious regardless of serum status and in a wide range of age groups. There is a need for a dengue vaccine and corresponding vaccination method that avoids costly and time-consuming serostat tests or seroprevalence considerations. There is a need for dengue vaccines and corresponding vaccination methods that can be used in outbreak situations. In addition, there is also a medical need for a dengue vaccine that is not only safe and effective, but also can be administered to individuals of unknown dengue serostat, children under 9 years of age, and seronegative individuals.

There is also a need for a vaccine that is administered at a lower dose than the current Dengvaxia® dosing schedule of three doses 6 months apart, eg a vaccine that can be administered in two doses or in only one dose.

This objective corresponds to the research priorities provided by WHO in the September 2018 Dengue Vaccines: WHO Position Paper (Wkly. Epidemiol. Rec. 2018, 93:457-476).

Hepatitis A is a liver disease caused by the hepatitis A virus (HAV). The virus is spread primarily when an uninfected (and unvaccinated) person ingests food or water contaminated with the feces of an infected person. The disease is closely related to unsafe water and food, inadequate hygiene and poor personal hygiene. The virus can also be transmitted through close physical contact with an infected person. Unlike hepatitis B and C, hepatitis A infection does not cause chronic liver disease, and although rarely fatal, it can cause debilitating and often fatal hepatitis fulminant (acute liver failure). Hepatitis A occurs sporadically, is prevalent worldwide, and tends to recur periodically.

Hepatitis A virus is one of the most common causes of food-borne infections. An epidemic related to contaminated food or water could erupt as an epidemic in Shanghai in 1988, affecting about 300,000 people. Hepatitis A virus persists in the environment and can withstand food production processes routinely used to inactivate and/or control bacterial pathogens. The disease can lead to significant economic and social consequences in a community. It can take weeks or months for people who have recovered from illness to return to work, school, or daily activities. The impact on food establishments identified as viruses and on local productivity in general could be significant. In developing countries with poor sanitation and hygiene practices, most children (90%) became infected with hepatitis A virus before the age of 10 years.

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 abroad in 2014. The risk of contracting an illness during international travel depends on many factors, such as the region visited, the duration of the trip, and the variety of activities planned. Vaccine recommendations are an important part of a pre-international health formulation. Vaccination against hepatitis A virus is generally recommended for travelers traveling to risk areas around the world, including Asia, Africa and Latin America.

For routine hepatitis A vaccination, a two-dose schedule is recommended, especially in travelers and immunocompromised individuals at significant risk of coming into contact with hepatitis A. However, in healthy subjects, similar effects were achieved with a single dose. Vaccination schedules for children/adolescents (ages 12 to 18 years) as well as for adults (age > 19 years) include a first dose administered intramuscularly, and an additional booster dose administered intramuscularly after 6 to 18 months is made of

Available hepatitis A vaccines include HAVRIX® and VAQTA®.

Therefore, there is a need for a safe and effective method for simultaneously preventing dengue fever and hepatitis A. In particular, there is a need for hepatitis A and dengue vaccines that, when administered simultaneously to a subject or population of subjects, provide suitable dosing schedules to achieve non-inferiority and synergy.

Additionally, there is a need for effective and safe prevention of dengue disease and hepatitis A in subjects unaware of hepatitis A and/or dengue fever serum status, particularly in subjects from non-endemic countries traveling to countries endemic to dengue and hepatitis A. there is.

Purpose and overview

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

It is an object of the present invention that hepatitis A and dengue fever useful in typical vaccination settings in which subjects are unaware of their serum status for dengue and/or hepatitis A and corresponding serologic tests are not available, practicable or reliable. To provide an administration method for preventing disease.

It is an object of the present invention to provide safe and effective protection against dengue disease and hepatitis A for travelers from non-endemic countries with hepatitis A and dengue, in particular for travelers vaccinated in travel clinics. In this regard, it is advantageous to avoid multiple appointments during the same appointment and to be able to vaccinate more than one disease at the same time.

It is an object of the present invention to provide a safe and effective vaccine for preventing hepatitis A and dengue disease in a subject or population of subjects, and in a subject or population of subjects of a wide age range from dengue endemic and non-endemic areas, in particular 2 to 60 years old. , preferably for subjects between the ages of 18 and 60 years and irrespective of previous exposure to any dengue virus serotype and/or hepatitis A virus and prior to vaccination with dengue and/or hepatitis A In this regard, it is to provide a corresponding method for preventing hepatitis A and dengue disease, whether seropositive or seronegative.

It is an object of the present invention to provide vaccines and corresponding methods for preventing hepatitis A and dengue disease that avoid testing for individual dengue and/or hepatitis A serum status prior to individual administration of the hepatitis A and dengue vaccine to a subject or population of subjects. , or to provide an analysis of the seroprevalence of dengue fever and/or hepatitis A in a subject or population to be vaccinated.

It is an object of the present invention to provide a dengue vaccine and hepatitis A vaccine, and methods of safely administering these vaccines, which can be safely co-administered with TDV as a travel vaccine prior to international travel of a subject to HAV and dengue-endemic countries.

Accordingly, the present invention relates to a method for preventing hepatitis A as well as dengue disease.

The present invention further relates to a method for preventing hepatitis A and dengue disease in a subject or population of subjects, said method comprising administering unit doses of hepatitis A vaccine and dengue vaccine composition simultaneously on the same day, wherein the unit dose comprises a tetravalent dengue virus composition comprising four wildly attenuated dengue virus strains.

Justice

In describing the present invention, the following terms should be used as indicated below. As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.

As used herein, the terms “unit dose of a dengue vaccine composition”, “unit dose” and “unit dose of the invention as used herein” refer to the amount of dengue vaccine administered to a subject in a single dose. In one embodiment, one unit dose is in a vial and the unit dose is administered to a subject, eg, after reconstitution. In one embodiment, more than one unit dose of the dengue vaccine composition may be present in a vial such that more than one subject can be vaccinated, in the content of one vial.

"Lyophilized unit dose" or "unit dose in lyophilized form" refers to a unit dose obtained by subjecting a predetermined volume, eg, 0.5 mL, of a liquid dengue vaccine composition to lyophilization. Accordingly, an aqueous formulation of a dengue vaccine composition produced by combining a dengue virus composition comprising a pharmaceutically acceptable excipient and four dengue virus strains, preferably TDV-1 to TDV-4, is subjected to lyophilization and lyophilized. unit capacity is obtained.

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. A reconstituted unit dose is a liquid that can be administered to a subject by injection, eg, subcutaneous injection.

As used herein, the term “on reconstitution with 0.5 mL” does not limit the reconstitution to be performed using 0.5 mL of diluent, but does not limit the dengue virus present in the reconstituted unit dose when 0.5 mL of diluent is used for reconstitution. refers to the concentration of The use of different volumes for reconstitution (eg, 0.8 mL) leads to different concentrations of dengue virus in the reconstituted unit dose, whereas administration of the total volume of the unit dose (eg, 0.8 mL) is the administered dengue virus. to derive the same total amount of

As used herein, “concentration of at least X log10 pfu/0.5 ml” refers to the concentration of dengue serotype in 0.5 ml, but does not limit the unit dose of 0.5 ml. When a unit dose has a volume different from 0.5 ml, or is reconstituted to a volume different from 0.5 mL, said concentration is different from "concentration of at least X log10 pfu/0.5 mL". However, when a unit dose has a volume of 0.5 ml, or when lyophilized from a volume of 0.5 mL or reconstituted to a volume of 0.5 mL, the concentration is "at least X log10 pfu/0.5 mL of concentration". Thus, the concentrations may be different, but the total amount of virus in a unit dose remains the same.

The term “dengue fever serotype” as used herein refers to a species of dengue virus defined by its cell surface antigen and thus can be distinguished by serological methods known in the art. Currently, there are four serotypes of dengue virus: dengue serotype 1 (DENV-1), dengue serotype 2 (DENV-2), dengue serotype 3 (DENV-3) and dengue serotype 4 (DENV-4). ) is known as

The term "tetravalent dengue virus composition" as used herein comprises four different immunogenic components from four different dengue serotypes DENV-1, DENV-2, DENV-3 and DENV-4, preferably Reference is made to a dengue virus composition comprising four different wildly attenuated dengue viruses, each representing one dengue serotype and aimed at stimulating an immune response against all four dengue serotypes.

The term “herbal attenuated dengue virus” as used herein refers to a viable dengue virus that has been mutated to provide reduced toxicity. A wild-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 with parts from two or more dengue serotypes or mixed with parts from different flaviviruses. It may be a chimeric dengue virus.

A “viral strain” and in particular a “dengue virus strain” is a virus, a genetic subtype of dengue virus characterized by specific nucleic acid sequences. Dengue serotypes may include different strains with different nucleic acid sequences with the same cell surface antigen. The dengue virus strain may be a dengue virus in which all components are derived from the same dengue serotype or it may be a chimeric dengue virus having portions from two or more dengue serotypes.

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

A “non-chimeric dengue virus” or “non-chimeric dengue serotype strain” or “non-chimeric dengue strain” includes only portions from one dengue serotype. In particular, the non-chimeric dengue virus includes different flaviviruses such as yellow fever virus, Zika virus, West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, tick-borne encephalitis virus. does not include parts from TDV-2 is one example of a non-chimeric dengue virus.

A “chimeric dengue virus” or “chimeric dengue serotype strain” or “chimeric dengue strain” includes portions from at least two different dengue serotypes. As used herein, chimeric dengue virus includes different types of virus such as yellow fever virus, Zika virus, West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, tick-borne encephalitis virus. Does not contain parts from flaviviruses. In particular, the chimeric dengue virus described herein does not contain portions from yellow fever virus. As used herein, “chimeric dengue serotype 2/1 strain” or “DENV-2/1 chimera” or “TDV-1” is a dengue virus chimera comprising portions from both DENV-2 and DENV-1. refer to the work. In particular, in the chimeric dengue serotype 2/1 strain, the prM and E proteins from DENV-1 replace the prM and E proteins from DENV-2 as detailed below. As used herein, “chimeric dengue serotype 2/3 strain” or “DENV-2/3 chimera” or “TDV-3” is a dengue virus chimera comprising portions from both DENV-2 and DENV-3. refer to the work. In particular, in the chimeric dengue serotype 2/3 strain, the prM and E proteins from DENV-3 replace the prM and E proteins from DENV-2 as detailed below. As used herein, “chimeric dengue serotype 2/4 strain” or “DENV-2/4 chimera” or “TDV-4” is a dengue virus chimera comprising portions from both DENV-2 and DENV-4. refer to the work. In particular, in the chimeric dengue serotype 2/4 strain, the prM and E proteins from DENV-4 replace the prM and E proteins from DENV-2 as detailed below. The mixed chimeric dengue virus has portions from other flaviviruses.

"TDV" as used herein refers to four wild-attenuated dengue virus strains TDV-1, each expressing surface antigens from the four dengue serotypes DENV-1, DENV-2, DENV-3 and DENV-4; Reference is made to a tetravalent, live attenuated dengue vaccine comprising a mixture of TDV-2, TDV-3 and TDV-4. In one embodiment (eg, also in the Examples), 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.

As used herein, the term “dengue disease” refers to a disease caused by infection with the dengue virus. Symptoms of dengue disease include sudden high fever, headache, joint and muscle aches, nausea, vomiting and skin rash. The term dengue disease also includes the 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 exhibit severe signs of plasma leakage and bleeding. When a subject with DHF experiences shock, it is classified as having DSS. Symptoms of DSS include bleeding, which can appear as small blood spots on the skin and larger patches of blood under the skin. Organ shock is a major factor associated with complications including massive gastrointestinal bleeding that can lead to death. DHF cases as used herein are defined as the VCD Cases Conference WHO 1997 DHF Criteria. With respect to preventing dengue disease in elderly subjects, the term “preventing dengue disease” preferably includes preventing DHF and/or DSS. With respect to preventing dengue disease in elderly subjects, the term “preventing dengue disease” preferably includes preventing severe end-organ symptoms of dengue, such as hepatomegaly and acute renal failure.

As used herein, “preventing dengue disease” refers to preventing a subject from developing one or more symptoms of dengue disease due to infection by the dengue virus. In particular, prevention of 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 comprises preventing DHS and/or DSS.

As used herein, the term “virologically-confirmed dengue disease,” “VCD case,” or “VCD fever” refers to a febrile disease or benign serotype-specific reverse transcriptase polymerase chain reaction (RT-PCR). It refers to a febrile disease clinically suspected to be a dengue disease with The term "virologically identifiable dengue" disease refers to a febrile disease or disease clinically suspected to be a dengue disease, wherein testing of a subject using, for example, RT-PCR, is performed with at least one dengue serum confirms the brother's existence. The severe form of VCD fever is 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 Board that will evaluate all hospitalized VCD cases (severe/severe) according to the criteria redefined in the Charter. All non-hospitalized cases are considered non-severe.

As used herein, the term “febrile illness” is defined as a temperature of ≧38° C. on any 2 of 3 consecutive days.

As used herein, the term "virally confirmed dengue disease with hospitalization" is considered a surrogate for severe dengue, and the incidence of virologically confirmed dengue disease with hospitalization" is used as a safety parameter. As used herein, "relative risk associated with virologically confirmed dengue disease with hospitalization" is defined as hospitalization for the number of events of virologically confirmed dengue disease with hospitalization divided by the number of subjects treated with placebo. together means the number of events of virologically confirmed dengue disease divided by the number of subjects treated with a unit dose as described herein, if "relative risk associated with virologically confirmed dengue disease with hospitalization" is 1 or less In this case, the vaccine provides less than the same risk for virologically confirmed dengue disease with hospitalization as placebo and is considered "safe." The risk is also particularly when determined from 30 days after the second administration to 12 months after the second administration, in particular in the age group of subjects 4 to 16 years old, in the age group of subjects under 4 to 9 years old, in the age group of 2 to less than 9 years old. 0.9 or less, 0.8 or less, 0.7 or less, as determined from an age group selected from an age group of a subject, an age group of a subject 4-5 years old, an age group of a 6-11 year old subject, and an age group of a 12-16 year old subject , 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.

As used herein, a vaccine is alternatively considered "safe" if the vaccine efficacy (VE) associated with virologically confirmed dengue disease with hospitalization is 0% or greater. This means that the vaccine offers the same or less potential for virologically confirmed dengue disease with hospitalization as a placebo. In particular, considered "safe" is particularly when measured versus 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. (in particular, age group of subjects 4 to 16 years old, age group of subjects 4 to less than 9 years old, age group of subjects 2 to less than 9 years old, age group of 4 to 5 years old subject, age group of 6 to 11 years old subject age group, and when measured in an age group selected from the age group of 12 to 16 year old subjects), in particular, said unit dose or said placebo within less than 6 months, eg, within 3 months, of the first administration; Two-sided 95% confidence intervals from 30 days after 2 doses or last dose to at least 12 months, 12 to 18 months, up to 12 months or up to 18 months after the second or last dose of the dosing schedule, when administered at least twice; Combined vaccine efficacy against virologically confirmed dengue with hospitalization for all four serotypes together, wherein the lower binding is greater than 25%. In particular, the lower binding may be greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 65%, greater than 66%, greater than 67%, greater than 68%, greater than 70%, or greater than 75%. Specifically, when comparing seropositive and seronegative subjects, the two-sided 95% confidence interval of combined vaccine efficacy against virologically confirmed dengue with hospitalization for all four serotypes was within 10 percentage points or 15 percentage points. It gives a lower combination of two-sided confidence intervals that are within or within 20 percentage points. In certain embodiments, "safe" is compared to placebo in a subject population of at least 5,000 healthy 4 to 16 years of age subjects regardless of serum status at baseline from the first administration of the dosing schedule to 12 to 18 months after the last administration of the dosing schedule. means to provide combined vaccine efficacy against virologically confirmed dengue with hospitalization for all four serotypes with a two-sided 95% confidence interval when measured for

A vaccine is considered safe within the meaning of the present invention if one of the criteria as defined above for the term "safe" is met. In this regard, particularly safe refers to a vaccine that is safe for all subjects irrespective of their serum status at baseline. This means that the vaccine can be administered without the need to determine the occurrence of a previous dengue infection in a subject prior to administration. Preferably, the vaccine starts from the age of 4 and is preferably safe as defined above with respect to all age groups, irrespective of serum status, in particular between 4 and 60 years of age, or between 4 and 16 years of age. Do. Relevant subgroups in this regard include: Ages less than 9 years, ages 2 to less than 9 years, ages 4 to less than 9 years, ages 4 to 5 years, ages 6 to 11 years, and ages 12 to 16 years. age or any age group within the age of 4 to 16 years. For further definitions of VE for virologically confirmed dengue disease with hospitalization, see the disclosure below in connection with specific treatment modalities.

As used herein, “vaccine efficacy” or “VE” measures a proportional decrease in cases among vaccinated persons. Vaccine efficacy (VE) is measured by calculating the risk of disease among vaccinated and unvaccinated people and determining the percent reduction in risk of disease among vaccinated people compared to unvaccinated people. The greater the percent 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 groups or a 90% reduction in the number of events expected if they were not vaccinated. Vaccine efficacy is calculated by the formula: 100 * (1 - HR), where HR is the risk ratio defined as the risk ratio of vaccine (λv) divided by the risk ratio of placebo (λc), i.e., HR = λv/λc. λv refers to the risk ratio for subjects vaccinated with the tetravalent dengue vaccine composition as described herein, and λc refers to the risk ratio for unvaccinated subjects, i.e., subjects receiving placebo. Hazard ratio HR is assessed from the Cox proportional hazards model with study vaccine as a factor, adjusted for age and stratified by region. As used herein, the term "combined vaccine efficacy against all four serotypes" refers to vaccine efficacy associated with virologically confirmed dengue disease and risk of dengue disease irrespective of the serotypes involved in the subject's baseline serotype status. is defined A vaccine is considered "effective" if the combined vaccine efficacy is greater than 30%. In this regard, combined vaccine efficacy is also particularly when determined from 30 days after the second administration up to 12 months after the second administration or up to 18 months after the second vaccination, in particular the age group of subjects 4 to 16 years old, 4 selected from an age group of subjects to less than 9 years of age, an age group of subjects 2 to less than 9 years old, an age group of subjects 4 to 5 years old, an age group of 6 to 11 years old subjects, and an age group of 12 to 16 years old subjects It may be 40% or more, 50% or more, 60% or more, 70% or more, 72% or more, or 80% or more. In this regard, particularly effective refers to a vaccine that is effective for all subjects irrespective of their serum status at baseline. Preferably, the vaccine starts from the age of 4 years and is preferably effective with respect to all age groups, irrespective of serum status, in particular from 4 to 60 years of age, or from 4 to 16 years of age. Relevant subgroups in this regard include: Ages less than 9 years, ages 2 to less than 9 years, ages 4 to less than 9 years, ages 4 to 5 years, ages 6 to 11 years, and ages 12 to 16 years. age or any age group within the age of 4 to 16 years. In certain embodiments, “effective” means placebo in a subject population of at least 5,000 healthy subjects irrespective of serum status at baseline and ages 4 to 16 years from the first administration of the dosing schedule to 18 months after the last administration of the dosing schedule. When measured for % is exceeded. Additional specific potencies 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 specific serotype, eg, serotype 1" refers to efficacy associated with a specific serotype involved in a virologically identified dengue disease. As used herein, "combined vaccine efficacy against all four serotypes against virologically confirmed dengue fever with hospitalization" refers only to efficacy for which virologically confirmed dengue cases with hospitalization are considered. . The vaccine efficacy can be determined with respect to subjects who are seronegative or seropositive at baseline and for different age groups.

"Relative risk" as used herein is the number of virologically confirmed dengue disease events divided by the number of subjects treated with placebo divided by the number of virologically confirmed dengue disease events as described herein. means divided by the number of subjects treated in unit dose. As used herein, the term "combined relative risk for all four serotypes" is defined as the relative risk associated with the risk of dengue disease regardless of the serotypes involved in the virologically identified dengue disease and the subject's baseline serotype status. is defined

"Vaccination" or "vaccination" as used herein refers to administration of a vaccine to a subject for the purpose of preventing the subject from developing one or more symptoms of a disease. As used herein, “vaccination against dengue disease” or “vaccination against dengue disease” refers to administration of a dengue vaccine composition to a subject for the purpose of preventing the subject from developing one or more symptoms of dengue disease. In principle, the method comprises a primary vaccination and optionally one or more booster vaccinations. Primary vaccination is defined as the primary dosing schedule for administration of a composition or unit dose as described herein to establish a protective immune response, eg consisting of two administrations within 3 months. Whenever reference is made to administration within this disclosure, such administration refers to primary vaccination unless specified as a booster vaccination. The booster vaccination is a dosing or dosing schedule that occurs after the primary vaccination, for example at least 1 year, or 4 to 4.5 years, or even 5 years or 10 years, eg the first vaccination schedule, after the last dose. Second dose is mentioned. Booster administration is an attempt to enhance or re-establish the immune response of primary vaccination.

As used herein, the term “subject” or “subjects” is limited to human subjects (eg, infants, children, or adults). The term "senior subject" or "geriatric subjects" refers to a subject greater than 60 years of age, e.g., 61 to 100 years old, 61 to 90 years old, 61 to 80 years old, 61 to 75 years old or 61 to 70 years old. refers to a subject having an age of

As used herein, “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. Parameters that may be used to define a subject population include, but are not limited to, the age of the subject, whether the subject is from an endemic area of dengue or a non-endemic area of dengue, and the serum status of the subject.

"Endemic area" as used herein refers to an area in which a disease or infectious agent continues to exist and/or is usually prevalent in a population within the area. "Non-endemic area" as used herein refers to an area where the disease is absent or not generally prevalent. Thus, "dengue fever endemic area" refers to a geographic area in which infection by the dengue virus is persistently maintained at baseline levels. "Non-endemic area of dengue" refers to a geographic area in which infection by the dengue virus is not consistently maintained at baseline levels. Thus, a subject population or subjects “from an area endemic to dengue” or “from an area non-endemic to dengue” refers to a population of subjects or subjects residing in a geographic area as described above. Whether a geographic region or subject population is endemic to dengue is determined by Bhatt et al. (2013) Nature 496 (7446): 504-507 and supplementary material and in Stanaway et al. (2016) Lancet Infect Dis. 16(6): 712-723 ) and can be determined by different calculation methods such as those described in the Supplementary Material. Overviews of dengue endemic areas and dengue epidemiology are published regularly, for example by WHO or CDC. Typical dengue-endemic regions are in Latin America, Southeast Asia, and Pacific islands, while dengue-endemic countries are Australia, Brazil, Bangladesh, Colombia, China, Dominican Republic, Indonesia, India, Mexico, Malaysia, Nicaragua, Nigeria, Pakistan, and Panama. , Philippines, Puerto Rico, Singapore, Sri Lanka, Thailand and Vietnam. The infectivity of an area is measured by a seroprevalence survey given as seroprevalence. Areas with very high infectivity are considered to have a seroprevalence of greater than 80%. As used herein, the term "region" when it is relevant refers to a geographic area in which serum prevalence is determined or known, e.g., a village, town, city, region, county ( county), a state, a province, or a part thereof or the whole country.

As used herein, “serum status” refers to the amount of antibody that a subject has with respect to a particular infectious agent, particularly the dengue virus. "Seronegative" or "no seronegative response" as used herein means that the subject does not have neutralizing antibodies to any of the dengue serotypes DENV-1, DENV-2, DENV-3 and DENV-4 in serum means A seronegative or seronegative subject or population of subjects is defined by a neutralizing antibody titer of less than 10 against each one of the four dengue serotypes. A subject or population of subjects having a neutralizing antibody titer of at least 10 against at least one dengue serotype is defined as “seropositive” for that dengue serotype. Serum status at baseline refers to serum status prior to administration of a dengue vaccine composition as described herein.

As used herein, “neutralizing antibody titer” refers to the amount of antibody in the serum of a subject that neutralizes each dengue serotype. The neutralizing antibody titers against DENV-1, DENV-2, DENV-3 and DENV-4 are determined in the 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)) in a serum sample of a subject using known methods such as the Plaque Reduction Neutralization Test (PRNT) as described herein or the Microneutralization (MNT50) assay as described herein. As used herein, "ratio of the neutralizing antibody titer of dengue serotype 2 to the neutralizing antibody titer of dengue serotype 4 to 20 or less" is the neutralizing antibody titer of dengue serotype 2 as the neutralizing antibody titer of dengue serotype 4 division means that the ratio obtained thereby is 20 or less. In other words, the neutralizing antibody titer of dengue serotype 2 is up to 20-fold higher than the neutralizing antibody titer of dengue serotype 4 in the subject.

As used herein, the terms "geometric mean neutralizing antibody titer" and "GMT" refer to the geometric mean value of neutralizing antibody titers for the corresponding dengue serotype in the serum of a subject in a subject population. The geometric mean value is calculated by a well-known formula. As used herein, “ratio of GMT of dengue serotype 2 to GMT of dengue serotype 4 of 20 or less” refers to the geometric mean neutralizing antibody titer of dengue serotype 2 (GMT DENV-2) to dengue serotype 4 ( It means that the ratio obtained by dividing by the geometric mean neutralizing antibody titer of GMT DENV-4) is 20 or less. In other words, the geometric mean neutralizing antibody titer of dengue serotype 2 is no more than 20-fold higher than the geometric mean neutralizing antibody titer of dengue serotype 4 in the subject population.

As used herein, “immune response” refers to the response of a subject to administration of a dengue vaccine. In particular, the immune response involves the formation of neutralizing antibodies to one or more dengue serotypes. This may also include stimulation of cell mediated responses or the formation of antibodies to non-structural proteins, such as NS1. The immune response is a unit dose of the invention as described herein when the titers of neutralizing antibodies against at least one dengue virus serotype and preferably against all four dengue virus serotypes are increased after said administration of said unit dose. stimulated by administration. The immune response is stimulated by administration of a unit dose of the invention as described herein when secretion of interferon gamma by peripheral blood mononuclear cells stimulated with a peptide from dengue virus protein is increased after said administration of said unit dose. . An immune response is stimulated by administration of a unit dose of the invention as described herein when the titer of an antibody to a non-structural protein such as NS1 is increased after said administration of said unit dose. In certain embodiments, administration of a reconstituted unit dose of the invention as described herein results in the formation of neutralizing antibodies to one or more dengue serotypes, cell mediated responses and formation of antibodies to non-structural proteins such as NS1. stimulate

A “balanced immune response” as used herein means that an immune response against all four dengue serotypes is sufficient to provide protection against infection by all four dengue serotypes, preferably against all four dengue serotypes. It means that the immune response to it has a similar strength. In particular, neutralizing antibody titers against the four dengue serotypes at 180 days or 365 days after administration of the first reconstituted unit dose of the invention as described herein are similar, i.e. they are less than 30-fold, less than 25-fold or differ by less than 20 times.

The "total concentration in pfu/0.5 ml" used as a base value for the calculation of the percent concentration for each individual component of the tetravalent dengue vaccine is dengue serotype 1, 4.00 log 10 pfu/0.5 at a concentration of 3.60 log 10 pfu/0.5 ml. One exemplary tetravalent vaccine composition comprising dengue serotype 2 at a concentration of ml, dengue serotype 3 at a concentration of 4.60 log 10 pfu/0.5 ml and dengue serotype 4 at a concentration of 5.11 log 10 pfu/0.5 ml is shown for one exemplary tetravalent vaccine composition.

Often, logarithmic values of concentrations are converted to numerical values. The conversion result is 4x10 ³ pfu/0.5 ml for serotype 1, 1x10 ⁴ pfu/0.5 ml for serotype 2, 4x10 ⁴ pfu/0.5 ml for serotype 3, and 1.3x10 ⁵ for serotype 4 pfu/0.5 ml. The total concentration in pfu/0.5 ml is the sum of previous numerical values resulting in 1.84 x 10 ⁵ pfu/0.5 ml.

The “percent concentration” for each of serotypes 1, 2, 3 and 4 is calculated by dividing the numerical concentration value of the individual serotype (expressed as pfu/0.5 ml) by the total concentration (expressed as pfu/0.5 ml) and adding 100 to the result. It is obtained by multiplying by

Percent concentration of serotype 1 = (4x10 ³ pfu/0.5 ml ÷ 1.84 x10 ⁵ pfu/0.5 ml) x 100 = 2%

Percent concentration of serotype 2 = (1x10 ⁴ pfu/0.5 ml ÷ 1.84 x10 ⁵ pfu/0.5 ml) x 100 = 5%

Percent concentration of serotype 3 = (4x10 ⁴ pfu/0.5 ml ÷ 1.84 x10 ⁵ pfu/0.5 ml) x 100 = 22%

Percent concentration of serotype 4 = (1.3x10 ⁵ pfu/0.5 ml ÷ 1.84 x10 ⁵ pfu/0.5 ml) x 100 = 71%.

Percent concentrations are rounded to integers.

"Simultaneous" administration as used herein refers to administration of at least two different vaccines, such as a dengue fever vaccine and a hepatitis A vaccine, on the same day. "Same day" has its ordinary meaning within 24 hours, such as within 1 day, for example. Simultaneous administration may be administered, for example, by the same practitioner during the same medical appointment.

"Sequential" administration as used herein means administration of at least two different vaccines, such as dengue vaccine and yellow fever vaccine, or dengue fever vaccine and hepatitis A vaccine, on different or subsequent dates, for example within 90 days. However, there is a combined dosing schedule.

As used herein, the term “chronic disease or condition” includes diseases and conditions that persist in a geriatric subject for at least 3 months. In particular, this includes diabetes, hypertension, allergies, previous strokes, ischemic heart disease, chronic kidney injury and chronic obstructive pulmonary disease.

The term “compromised immune system” as used herein means that at least one function of at least one component of the immune system is weaker in younger subjects, ie in subjects under 60 years of age. These functions include a lower antioxidant response of monocytes to oxidative stress induced by dengue virus and a lower T cell response and cytokine production in response to dengue virus infection.

As used herein, "induced systemic adverse events" in children younger than 6 years of age are defined as fever, irritability/fussiness, drowsiness and loss of appetite that occur within 14 days after each vaccination, and in children 6 years and older, respectively It is defined as fever, headache, asthenia, malaise, and myalgia within 14 days of vaccination.

As used herein, "induced local adverse events" are injection site pain, injection site erythema, and injection site swelling that occur within 7 days of each vaccination.

As used herein, an “uninduced adverse event” is any AE that is not an induced local or systemic adverse event (AE) as defined above.

"Severe adverse event" or "SAE" as used herein is a term that, at any dose, induces death, is life-threatening, requires hospitalization or prolongation of an existing hospitalization, or is persistent or significant disability/incapacity is any refractory medical occurrence or effect that is medically significant for reasons other than those mentioned above, or is a congenital anomaly/birth defect.

The relationship of attracted systemic AEs (induced local AEs are considered relevant) to the test vaccine(s) is assessed using the following categories: “IP-related AE” or “vaccine related” as used herein AE" is suspected of a relationship between the vaccine and the AE (without determining the degree of probability); means that there is a reasonable possibility that the vaccine contributes to the AE. "Non-IP related" or "non-vaccine related" as used herein means that there is no suspicion that there is a relationship between the vaccine and the AE, there is another more probable cause and that administration of the vaccine is not suspected of contributing to the AE. do.

As used herein, a subject or population of subjects who are “aged 2 to 60 years” or “aged 18 to 60 years” is between 2 and 60 years of age or 18 to 60 years of age on the first day of administration of a dengue vaccine composition as described herein. Refers to a subject or population of subjects that are three years of age.

"%-point" as used herein refers to the difference between two %-values in a %-value. For example, two values in % that are within 5%-point refer to, for example, one value in 1% and a second value in 6%.

As used herein, the term “determination of prior dengue infection in a subject prior to administration” refers to whether a prior dengue infection has a laboratory-confirmed history of dengue or via an appropriately confirmed serological test, e.g., Example 2 This means that pre-vaccination should be assessed by seroclassification using methods as described herein, such as the MNT50 test described in

% w/v as used herein refers to % mg/ml, eg 150 mg/ml is 15% w/v.

The term “hepatitis A virus” as used herein may be abbreviated as “HAV”.

As used herein, the term “placebo” may be abbreviated as “Pbo”.

As used herein, "hepatitis A seronegative at baseline" or "no hepatitis A (at baseline)" respectively means that the subject does not have a predefined amount of anti-hepatitis A antibody in serum. Quantitatively, a subject's hepatitis A seronegativeness is defined as an anti-hepatitis A antibody level of <10 mIU/ml. When anti-hepatitis A antibody levels are determined by ELISA, the lower level of quantitation is 12.5 mIU/ml, which is a lower anti-HAV antibody level to effectively determine seronegative. A subject with an anti-hepatitis A antibody level of ≧12.5 mIU/ml is defined as hepatitis A seropositive. An ELISA for determining anti-hepatitis A antibodies is described, for example, in Beck et al. J Travel Med 2004; 11:201-207.

As used herein, “at baseline” refers to the time of the last measurement of a subject's serum status prior to the first vaccination.

As used herein, the unit “mIU/ml” refers to milli-international units per milliliter. The concentration unit refers to the amount of anti-hepatitis A antibody in the serum of a subject (eg, if measured before or after vaccination). As used herein, the "viral antigen activity of the hepatitis A vaccine" of the present invention is expressed in terms of standard recommendations of the WHO using an enzyme linked immunosorbent assay (ELISA). According to the above recommendation of the WHO (see: WHO Information Sheet "Observed Rate of Vaccine Reactions - Hepatitis A Vaccine", published June 2012), the viral antigen activity of the hepatitis A vaccine is expressed in ELISA units (EL.U.). The viral antigen activity of hepatitis A vaccine is described, for example, in Andre FE., Hepburn A. D'Hondt E., "Inactivated candidate vaccines for hepatitis", A. Prog Med Virol 1990; 37:72-95). can be determined by ELISA.

The term “CCID” as used herein refers to the amount of virus (eg, vaccine virus) that infects 50% of a cell culture. The CCID50 test is a limiting dilution assay using statistical titer calculations (Morrison D et al., J Infect Dis. 2010; 201(3):370-7).

As used herein, "non-inferiority" associated with concurrent administration of hepatitis A vaccine and tetravalent dengue vaccine on the same day is particularly defined as a group of subjects receiving HAV and placebo (simultaneous on the same day, i.e. control subjects). population) and the upper limit of the two-sided 95% confidence interval in which the difference in seroprotection (SPR) between the SPR of a group of subjects receiving HAV and TDV (simultaneously on the same day) is less than the non-inferiority margin established at 10% , and seroprotection is based on measurements at 30 days following co-administration on day 1, using the Newcomb score method. A non-inferiority clinical study is a study designed to provide a comparison between at least two treatment modalities, in this case, concurrent administration of dengue vaccine and hepatitis A vaccine and administration of dengue vaccine or hepatitis A vaccine alone.

As used herein, the term “seroprotection rate”, the abbreviation “SPR” refers to either HAV or DEN-naive at baseline, seroprotected against HAV or DENV, respectively, 30 days (1 month) after the first vaccination. It is defined by the proportion/percentage of the subject.

As used herein, the term "control subject population" refers to those who have not received concurrent administration of unit doses of hepatitis A vaccine and dengue vaccine composition but on the same day in a clinical study setting, eg, as in a non-inferiority clinical study. refers to a group of subjects receiving a single verum (such as a unit dose of a hepatitis A vaccine or a dengue vaccine composition) and a placebo.

As used herein, the term “synergy” or “synergy” is defined as the effect of administering unit doses of a hepatitis A vaccine and a dengue vaccine composition to a subject or population of subjects simultaneously on the same day, said administration taking place on the same day Higher anti-neutralizing antibodies to each of the dengue virus serotypes than the corresponding simultaneous administration of hepatitis A vaccine and placebo on the same day and/or simultaneous administration of unit doses of dengue vaccine composition and placebo on the same day (administration alone) Hepatitis A antibody concentrations and/or higher mean titers. This higher antibody concentration after simultaneous administration compared to single administration is indicative of favoring simultaneous administration.

Figure 1: Genetic structure of four dengue strains contained in TDV. Solid red triangles indicate three attenuating mutations present in the 5' NCR, NS1 and NS3 proteins. The TDV-1, TDV-3 and TDV-4 strains are chimeric viruses in which the prM and E genes from dengue serotypes 1, 3 and 4, respectively, are inserted into the TDV-2 backbone.
Figure 2: Schematic diagram illustrating the microneutralization test (MNT) used to determine the titer of neutralizing antibodies.
Figure 3: Flowchart of the clinical trial of Example 3.
Figure 4: Cumulative incidence of A) virologically confirmed dengue cases and B) hospitalized virologically confirmed dengue cases over time during the Part 1 study period by baseline serum status (safety set data, truncated at Month 18) data display). The table shows the number of participants under various follow-up time points until the end of the Part 1 study period.
Figure 5: Study design of the Phase III study described in Example 3.
Figure 6: Schematic of the trial design of the concurrent HAV and TDV dosing study described in Example 4.

dengue virus strain

Dengue virus is a single-stranded, positive sense RNA virus of the family Flaviviridae. The taxonomy is summarized in Table 1. The Flaviviridae family includes three genera, flaviviruses, hepaciviruses and pestiviruses. The flavivirus genus includes 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. virus), and many less pathogenic viruses.

[graph One] Classification of GMO parental strains of dengue virus

The flavivirus genome comprises in the 5' to 3' direction (see FIG . 1 ):

- 5'-non-coding region (5'-NCR),

- capsid protein (C) coding region,

- pro-membrane protein (prM) coding region,

- the envelope protein (E) coding region,

-regions encoding nonstructural proteins (NSl, NS2A, NS2B, NS3, NS4A, NS4B, NS5) and

- 3' non-coding region (3'-NCR).

The viral structural proteins are C, prM and E, and the nonstructural proteins are NS1 to NS5. Structural and nonstructural proteins are translated as single polyproteins and processed by cellular and viral proteases.

The unit dose of the invention as described herein contains dengue fever comprising four wildly attenuated dengue virus strains (tetravalent dengue virus composition) exhibiting dengue serotype 1, dengue serotype 2, dengue serotype 3 and dengue serotype 4. a viral composition. Preferably, the composition is a molecularly characterized and cloned dengue serotype derived from a chimeric dengue virus and optionally at least one non-chimeric dengue virus, in particular a wild-attenuated DEN-2 PDK-53 virus strain (TDV-2). Two strains, and three chimeric dengue strains derived from the TDV-2 strain by replacing the structural proteins prM and E from TDV-2 with the corresponding structural proteins from other dengue serotypes to induce the following chimeric dengue strains Includes:

- DENV-2/1 Chimera (TDV-1),

- DENV-2/3 Chimera (TDV-3) and

- DENV-2/4 Chimera (TDV-4).

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

The attenuated DEN-2 PDK-53 strain (precursor of TDV-2) was derived from the wild-type virus strain DEN-2 16681 (SEQ ID NO: 11), which differs from the wild-type by 9 nucleotides as follows (Kinney et al. (Kinney et al.) 1997) Virology 230(2): 300-308):

(i) 5'-noncoding region (NCR)-57 (nt-57 C-to-T): major attenuated 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 attenuated loci

(v) NS2A-181 Leu to Phe (nt-4018 C to T)

(vi) NS3-250 Glu to Val (nt-5270 A to T): major attenuated locus

(vii) nt-5547 (NS3 gene) T to C silent mutation

(viii) NS4A-75 Gly to Ala (nt-6599 G to C)

* Silent mutation of nt-8571 C to T (NS5 gene)

5' non-coding region (NCR) (nucleotide 57) (mutation (i)), NS-1 (amino acid 828 of SEQ ID NO: 4) (mutation (iv)) and NS-3 gene (amino acid 1725 of SEQ ID NO: 4) ( The three nucleotide changes located in the mutation (vi)) form the criterion 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 included in TDV-1, TDV-2, TDV-3 and TDV-4.

Table 2: Attenuating Mutations in the Common Genetic Backbone of All TDV Variants

In one embodiment, TDV-2 comprises, in addition to the three attenuating mutations, one or more mutations selected from:

a) an adenine to thymidine mutation at nucleotide 524 in the prM gene leading to an asparagine to valine amino acid change at position 143, and/or

b) a cytosine to thymidine silent mutation at nucleotide 2055 in the E gene, and/or

c) a cytosine to thymidine mutation at nucleotide 4018 in the NS2A gene leading to an amino acid change from leucine to phenylalanine at position 1308, and/or

d) a silent mutation from thymidine to cytosine at nucleotide 5547 in the NS3 gene, and/or

e) a guanine to cytosine mutation at nucleotide 6599 in the NS4A gene leading to an amino acid change from glycine to alanine at position 2168 and/or

f) Silent mutation from thymidine to cytosine at nucleotide 900 in the prM gene.

The cytosine to thymidine silent mutation of DEN-2 PDK-53 at nucleotide 8571 in the NS5 gene is not present in the TDV-2 strain.

In another embodiment, TDV-2 comprises, in addition to the three attenuating mutations, one or more mutations selected from:

g) an adenine to guanine mutation at nucleotide 592 in the prM gene leading to an amino acid change from lysine to glutamine at position 166, and/or

h) Adenine to thymidine mutation at nucleotide 8803 in the NS5 gene leading to an isoleucine to valine amino acid change at position 2903.

In another embodiment, 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 ) to h). The nucleotide position and amino acid position of TDV-2 refer to the nucleotide sequence as shown in SEQ ID NO:3 and the amino acid sequence as shown in SEQ ID NO:4.

Dengue virus structural envelope (E) protein and precursor membrane (prM) protein were identified as primary antigens that elicited neutralizing protective antibody responses (Plotkin 2001). For the production of tetravalent dengue vaccine (TDV), TDV-2 was prepared using nucleic acids encoding DENV-2 prM and E glycoproteins, DENV-1, DENV-3, and DENV-4 wild-type strain DENV-1 16007, DENV. -3 16562 or DENV-4 1036 virus, respectively (see Table 3), by substituting nucleic acid sequences encoding the corresponding wild-type prM and E glycoproteins using standard molecular genetic engineering methods (Huang et al. (2003) ) J. Virol. 77(21): 11436-11447).

[Table 3] Viral origin of the prM/E gene region of the TDV virus strain.

A diagram of the four TDV strains included in the dengue vaccine composition is shown in FIG. 1 .

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, as shown in Table 3, and attenuation of TDV-2 carry genetic alterations that are involved in Thus, the TDV-1, TDV-3 and TDV-4 strains each contain the attenuating mutations listed in Table 2.

In one embodiment, TDV-1 comprises, in addition to the three attenuating mutations, one or more mutations selected from:

c) a cytosine to thymidine mutation at nucleotide 4018 in the NS2A gene leading to an amino acid change from leucine to phenylalanine at position 1308, and/or

d) a silent mutation from thymidine to cytosine at nucleotide 5547 in the NS3 gene, and/or

e) a guanine to cytosine mutation at nucleotide 6599 in the NS4A gene leading to an amino acid change from glycine to alanine at position 2168, and/or

i) a silent mutation from thymidine to cytosine at nucleotide 1575 in the E gene, and/or

j) a silent mutation from an adenine to a guamine at nucleotide 453 in the junction site between the prM-E gene and the DEN-2 PDK-53 backbone, and/or

k) thymidine-guanine to cytosine-cytosine mutation at nucleotides 2381/2382 in the junction site between the prM-E gene and the DEN-2 PDK-53 backbone, leading to an amino acid change from balanine to alanine at position 762 .

In another embodiment, TDV-1 comprises, in addition to the three attenuating mutations, one or more mutations selected from:

l) an adenine to cytosine mutation at nucleotide 3823 in the NS2A gene leading to an isoleucine to leucine amino acid change at position 1243, and/or

m) an adenine to thymidine mutation at nucleotide 4407 in the NS2B gene leading to an amino acid change from glutamine to asparagine at position 1437, and/or

n) Silent mutation from adenine to guanine at nucleotide 7311 in the NS4B gene.

In another embodiment, the TDV-1 variant 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 nucleotide position and amino acid position of TDV-1 refer to the nucleotide sequence as shown in SEQ ID NO: 1 and the amino acid sequence as shown in SEQ ID NO: 2.

In one embodiment, TDV-3 comprises, in addition to the three attenuating mutations, one or more mutations selected from:

c) a cytosine to thymidine mutation at nucleotide 4012 in the NS2A gene leading to an amino acid change from leucine to phenylalanine at position 1306, and/or

d) a silent mutation from thymidine to cytosine at nucleotide 5541 in the NS3 gene, and/or

e) a guanine to cytosine mutation at nucleotide 6593 in the NS4A gene leading to an amino acid change from a glycine to alanine at position 2166, and/or

j) a silent mutation from an adenine to a guamine at nucleotide 453 in the junction site 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 in the junction site between the prM-E gene and the DEN-2 PDK-53 backbone, leading to 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) Adenine to thymidine mutation at nucleotide 1970 in the E gene, leading to an amino acid change from histidine to leucine at position 625.

In another embodiment, TDV-3 comprises, in addition to the three attenuating mutations, one or more mutations selected from:

q) an adenine to thymidine mutation at nucleotide 1603 in the E gene leading to an amino acid change from a threonine to a serine at position 503, and/or

r) Silent mutation from adenine to guanine at nucleotide 7620 in the NS5 gene.

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

In one embodiment, TDV-4 comprises, in addition to the three attenuating mutations, one or more mutations selected from:

c) a cytosine to thymidine mutation at nucleotide 4018 in the NS2A gene leading to an amino acid change from leucine to phenylalanine at position 1308, and/or

d) a silent mutation from thymidine to cytosine at nucleotide 5547 in the NS3 gene, and/or

e) a guanine to cytosine mutation at nucleotide 6599 in the NS4A gene leading to an amino acid change from glycine to alanine at position 2168, and/or

j) a silent mutation from an adenine to a guamine at nucleotide 453 in the junction site 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 in the junction site between the prM-E gene and the DEN-2 PDK-53 backbone, leading to an amino acid change from valine to alanine at position 762; and/or

s) an adenine to guanine mutation at nucleotide 396 in the C gene leading to an arginine to serine amino acid change at position 100, and/or

t) a silent mutation from an adenine to guanine at nucleotide 1401 in the E gene, and/or

u) a cytosine to thymidine mutation at nucleotide 2027 in the E gene leading to an alanine to valine amino acid change at position 644, and/or

v) adenine to cytosine mutation at nucleotide 2275 in the E gene, leading to an amino acid change from methionine to leucine at position 727.

In another embodiment, TDV-4 comprises, in addition to the three attenuating mutations, one or more mutations selected from:

w) a silent mutation from adenine to thymidine at nucleotide 225 in the C gene, and/or

x) an adenine to guanine mutation at nucleotide 3674 in the NS2A gene leading to an asparagine to glycine amino acid change at position 1193, and/or

y) an adenine to adenine/guanine mixture at nucleotide 3773 in the NS2A gene leading to an amino acid change at position 1226 from a lysine to a lysine/asparagine mixture, and/or

z) a cytosine to thymidine silent mutation at nucleotide 5391 in NS3, and/or

aa) a cytosine to thymidine mutation at nucleotide 6437 in the NS4A gene leading to an alanine to valine amino acid change at position 2114, and/or

bb) a silent mutation from thymidine to thymidine/cytosine mixture at nucleotide 7026 in NS4B, and/or

cc) Silent mutation from adenine to cytosine at nucleotide 9750 in the NS5 gene.

In another embodiment, 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 position and amino acid position of TDV-4 refer to the nucleotide sequence as shown in SEQ ID NO:7 and the amino acid sequence as shown in SEQ ID NO:8.

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 It 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 is It has the SEQ ID NO 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, and TDV-3 has a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 6 has a nucleotide sequence encoding the amino acid sequence of , and TDV-4 has a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 8.

[Table 4] Sequences of TDV virus strains

Accordingly, in a particularly preferred embodiment, the unit dose of the invention as described herein comprises the wild-attenuated dengue virus strains TDV-1, TDV-2, TDV-3 and TDV-4, wherein TDV-1, TDV -3 and TDV-4 are based on TDV-2 and contain the prM and E regions of DENV-1, -3 and -4, respectively. In another particularly preferred embodiment, TDV-1 is characterized by the nucleotide sequence according to SEQ ID NO: 1 and the amino acid sequence according to SEQ ID NO: 2, and TDV-2 is the nucleotide sequence according to SEQ ID NO: 3 and the amino acid according to SEQ ID NO: 4 characterized by the sequence, 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 different from that of SEQ ID NO: 8 is characterized by

The E protein of DENV-3 has two fewer amino acids than the E protein of DENV-2. Thus, the nucleotide and encoded amino acid backbone 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 are 6 nucleotides longer than the original TDV-2 nucleotide and amino acid position, respectively less and two amino acids less.

Dengue Fever Vaccine Composition

The present disclosure relates in part to a unit dose of a dengue vaccine composition as described. The dengue vaccine composition also comprises a tetravalent dengue virus composition, also referred to as a dengue virus composition, and a pharmaceutically acceptable excipient.

Dengue virus composition, virus concentration and %-concentration

The present invention relates in part to a unit dose of a dengue vaccine composition, wherein the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four wild, attenuated dengue virus strains:

(i) dengue serotype 1, preferably at a concentration of at least 3.3 log10 pfu/0.5 mL,

(ii) dengue serotype 2, preferably at a concentration of at least 2.7 log10 pfu/0.5 mL;

(iii) dengue serotype 3, preferably at a concentration of at least 4.0 log10 pfu/0.5 mL, and

(iv) Dengue serotype 4 strain, preferably at a concentration of at least 4.5 log10 pfu/0.5 mL.

In one embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four wildly attenuated dengue virus strains:

(i) dengue fever serotype 1, preferably at a concentration of at least 3.3 log10 pfu/0.5 mL to 3.8 log10 pfu/0.5 mL,

(ii) dengue serotype 2, preferably at a concentration of at least 2.7 log10 pfu/0.5 mL;

(iii) dengue serotype 3, preferably at a concentration of at least 4.0 log10 pfu/0.5 mL, and

(iv) Dengue serotype 4. preferably at a concentration of at least 4.5 log10 pfu/0.5 ml or 4.6 log10 pfu/0.5 ml, optionally up to 6.2 log10 pfu/0.5 ml.

The present invention further relates, in part, to a unit dose of a dengue vaccine composition, said dengue vaccine composition comprising a tetravalent dengue virus composition comprising four wild-attenuated dengue virus strains:

(i) a chimeric dengue serotype 2/1 strain at a concentration of at least 3.3 log10 pfu/0.5 mL,

(ii) a dengue serotype 2 strain at a concentration of at least 2.7 log10 pfu/0.5 mL;

(iii) a chimeric dengue serotype 2/3 strain at a concentration of at least 4.0 log10 pfu/0.5 mL, and

(iv) a chimeric dengue serotype 2/4 strain at a concentration of at least 4.5 log10 pfu/0.5 mL.

In one embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four wildly attenuated dengue virus strains:

(i) a chimeric dengue serotype 2/1 strain at a concentration of at least 3.3 log10 pfu/0.5 mL to 3.8 log10 pfu/0.5 ml,

(ii) a dengue serotype 2 strain at a concentration of at least 2.7 log10 pfu/0.5 mL;

(iii) a chimeric dengue serotype 2/3 strain at a concentration of at least 4.0 log10 pfu/0.5 mL, and

(iv) a chimeric dengue serotype 2/4 strain at a concentration of at least 4.5 log10 pfu/0.5 mL or at least 4.6 log10 pfu/0.5 mL to optionally 6.2 log10 pfu/0.5 ml.

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 It is TDV-4.

In one embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four wild-attenuated dengue virus strains, wherein:

(i) said dengue fever serotype 1 (eg, chimeric dengue serotype 2/1 strain) has a concentration of 3.3 log10 pfu/0.5 mL to 5.3 log10 pfu/0.5 mL;

(ii) said dengue fever serotype 2 (eg, dengue serotype 2 strain) has a concentration of 2.7 log10 pfu/0.5 mL to 5.0 log10 pfu/0.5 mL;

(iii) said dengue serotype 3 (eg, chimeric dengue serotype 2/3 strain) has a concentration of 4.0 log10 pfu/0.5 mL to 6.0 log10 pfu/0.5 mL;

(iv) said dengue fever serotype 4 (eg, chimeric dengue serotype 2/4 strain) has a concentration of 4.5 log10 pfu/0.5 mL to 6.5 log10 pfu/0.5 mL;

In one such embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four wild-attenuated dengue virus strains, wherein:

(i) said dengue fever serotype 1 (eg, chimeric dengue serotype 2/1 strain) has a concentration of 3.3 log10 pfu/0.5 mL to 5.0 log10 pfu/0.5 mL;

(ii) said dengue fever serotype 2 (eg, dengue serotype 2 strain) has a concentration of 2.7 log10 pfu/0.5 mL to 4.9 log10 pfu/0.5 mL;

(iii) said dengue serotype 3 (eg, chimeric dengue serotype 2/3 strain) has a concentration of 4.0 log10 pfu/0.5 mL to 5.7 log10 pfu/0.5 mL;

(iv) said dengue fever serotype 4 (eg, chimeric dengue serotype 2/4 strain) has a concentration of 4.5 log10 pfu/0.5 mL to 6.2 log10 pfu/0.5 mL;

In further said embodiments, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four wildly attenuated dengue virus strains, wherein:

(i) dengue fever serotype 1 (eg, chimeric dengue serotype 2/1 strain) has a concentration of 3.3 log10 pfu/dose to 5.0 log10 pfu/dose,

(ii) dengue serotype 2 (eg, dengue serotype 2 strain) has a concentration of 2.7 log10 pfu/dose to 4.9 log10 pfu/dose,

(iii) dengue fever serotype 3 (eg, a chimeric dengue serotype 2/3 strain) has a concentration of 4.0 log10 pfu/dose to 5.7 log10 pfu/dose;

(iv) Dengue fever serotype 4 (eg, chimeric dengue serotype 2/4 strain) has a concentration of 4.5 log10 pfu/dose to 5.5 log10 pfu/dose.

In further said embodiments, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four wildly attenuated dengue virus strains, wherein:

(i) dengue fever serotype 1 (eg, chimeric dengue serotype 2/1 strain) has a concentration of 3.3 log10 pfu/dose to 4.1 log10 pfu/dose,

(ii) dengue fever serotype 2 (eg, dengue serotype 2 strain) has a concentration of 2.7 log10 pfu/dose to 3.6 log10 pfu/dose;

(iii) dengue fever serotype 3 (eg, chimeric dengue serotype 2/3 strain) has a concentration of 4.0 log10 pfu/dose to 4.7 log10 pfu/dose;

(iv) Dengue serotype 4 (eg, chimeric dengue serotype 2/4 strain) has a concentration of 4.5 log10 pfu/dose to 5.3 log10 pfu/dose.

In further said embodiments, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four wildly attenuated dengue virus strains, wherein:

(i) dengue fever serotype 1 (eg, chimeric dengue serotype 2/1 strain) has a concentration of 3.3 log10 pfu/0.5 mL to 3.6 log10 pfu/0.5 mL;

(ii) dengue serotype 2 (eg, dengue serotype 2 strain) has a concentration of 2.7 log10 pfu/0.5 mL to 4.0 log10 pfu/0.5 mL;

(iii) dengue fever serotype 3 (eg, chimeric dengue serotype 2/3 strain) has a concentration of 4.0 log10 pfu/0.5 mL to 4.6 log10 pfu/0.5 mL;

(iv) Dengue fever serotype 4 (eg, chimeric dengue serotype 2/4 strain) has a concentration of 4.5 log 10 pfu/0.5 ml or 4.6 log 10 pfu/0.5 mL to 5.1 log 10 pfu/0.5 mL.

In another embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four wild-attenuated dengue virus strains, wherein:

(i) said dengue fever serotype 1 (eg, chimeric dengue serotype 2/1 strain) has a concentration of 4.3 log10 pfu/0.5 mL to 4.4 log10 pfu/0.5 mL;

(ii) said dengue fever serotype 2 (eg, dengue serotype 2 strain) has a concentration of 3.7 log10 pfu/0.5 mL to 3.8 log10 pfu/0.5 mL;

(iii) said dengue fever serotype 3 (eg, chimeric dengue serotype 2/3 strain) has a concentration of 4.5 log10 pfu/0.5 mL to 5.0 log10 pfu/0.5 mL;

(iv) said dengue fever serotype 4 (eg, chimeric dengue serotype 2/4 strain) has a concentration of 5.5 log10 pfu/0.5 mL to 5.6 log10 pfu/0.5 mL;

In a particularly preferred embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four wild-attenuated dengue virus strains, wherein:

(i) said dengue fever serotype 1 (eg, chimeric dengue serotype 2/1 strain) has a concentration of 4.4 log10 pfu/0.5 mL;

(ii) said dengue fever serotype 2 (eg, dengue serotype 2 strain) has a concentration of 3.8 log10 pfu/0.5 mL;

(iii) said dengue serotype 3 (eg, chimeric dengue serotype 2/3 strain) has a concentration of 4.5 log10 pfu/0.5 mL;

(iv) the dengue serotype 4 (eg, chimeric dengue serotype 2/4 strain) has a concentration of 5.6 log10 pfu/0.5 mL.

In another particularly preferred embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four wildly attenuated dengue virus strains, wherein:

(i) said dengue fever serotype 1 (eg, chimeric dengue serotype 2/1 strain) has a concentration of 3.6 log10 pfu/0.5 mL;

(ii) said dengue fever serotype 2 (eg, dengue serotype 2 strain) has a concentration of 4.0 log10 pfu/0.5 mL;

(iii) said dengue serotype 3 (eg, chimeric dengue serotype 2/3 strain) has a concentration of 4.6 log10 pfu/0.5 mL;

(iv) the dengue serotype 4 (eg, chimeric dengue serotype 2/4 strain) has a concentration of 5.1 log10 pfu/0.5 mL.

In another preferred embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising the following four wild attenuated dengue virus strains, wherein the arithmetic sum of all four serotypes is 6.7 log10 pfu/0.5 mL less than, preferably less than 5.5 log10 pfu/0.5 mL. In certain such embodiments, the arithmetic sum of all four serotypes is at least 4.6 log10 pfu/0.5 mL. In a preferred embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising the following four wild attenuated dengue virus strains, wherein the arithmetic sum of all four serotypes is 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 this 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 serotype The 2/4 variant 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, and 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 and 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.

The present invention relates in part to a unit dose of a dengue vaccine composition, wherein the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four wild, attenuated dengue virus strains:

(i) dengue fever serotype 1 (eg, chimeric dengue serotype 2/1 strain) at a concentration of at least 3.3 log10 pfu/dose;

(ii) dengue serotype 2 (eg, dengue serotype 2 strain) at a concentration of at least 2.7 log10 pfu/dose;

(iii) dengue fever serotype 3 (eg, chimeric dengue serotype 2/3 strains) at a concentration of at least 4.0 log10 pfu/dose, and

(iv) Dengue serotype 4 (eg, chimeric dengue serotype 2/4 strains) at a concentration of at least 4.5 log10 pfu/dose.

In one embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four wild-attenuated dengue virus strains, wherein:

(i) said dengue fever serotype 1 (eg, a chimeric dengue serotype 2/1 strain) has a concentration of 3.3 log10 pfu/dose to 5.3 log10 pfu/dose;

(ii) said dengue fever serotype 2 (eg, dengue serotype 2 strain) has a concentration of 2.7 log10 pfu/dose to 5.0 log10 pfu/dose;

(iii) said dengue fever serotype 3 (eg, a chimeric dengue serotype 2/3 strain) has a concentration of 4.0 log10 pfu/dose to 6.0 log10 pfu/dose;

(iv) said dengue fever serotype 4 (eg, chimeric dengue serotype 2/4 strain) has a concentration of 4.5 log10 pfu/dose to 6.5 log10 pfu/dose.

In one such embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four wild-attenuated dengue virus strains, wherein:

(i) said dengue fever serotype 1 (eg, chimeric dengue serotype 2/1 strain) has a concentration of 3.3 log10 pfu/dose to 5.0 log10 pfu/dose,

(ii) said dengue fever serotype 2 (eg, dengue serotype 2 strain) has a concentration of 2.7 log10 pfu/dose to 4.9 log10 pfu/dose,

(iii) said dengue fever serotype 3 (eg, chimeric dengue serotype 2/3 strain) has a concentration of 4.0 log10 pfu/dose to 5.7 log10 pfu/dose,

(iv) said dengue fever serotype 4 (eg, chimeric dengue serotype 2/4 strain) has a concentration of 4.5 log10 pfu/dose to 6.2 log10 pfu/dose.

In further said embodiments, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four wildly attenuated dengue virus strains, wherein:

(i) dengue fever serotype 1 (eg, chimeric dengue serotype 2/1 strain) has a concentration of 3.3 log10 pfu/dose to 5.0 log10 pfu/dose,

(ii) dengue serotype 2 (eg, dengue serotype 2 strain) has a concentration of 2.7 log10 pfu/dose to 4.9 log10 pfu/dose,

(iii) dengue fever serotype 3 (eg, a chimeric dengue serotype 2/3 strain) has a concentration of 4.0 log10 pfu/dose to 5.7 log10 pfu/dose;

(iv) Dengue fever serotype 4 (eg, chimeric dengue serotype 2/4 strain) has a concentration of 4.5 log10 pfu/dose to 5.5 log10 pfu/dose.

In further said embodiments, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four wildly attenuated dengue virus strains, wherein:

(i) dengue fever serotype 1 (eg, chimeric dengue serotype 2/1 strain) has a concentration of 3.3 log10 pfu/dose to 4.1 log10 pfu/dose,

(ii) dengue fever serotype 2 (eg, dengue serotype 2 strain) has a concentration of 2.7 log10 pfu/dose to 3.6 log10 pfu/dose;

(iii) dengue fever serotype 3 (eg, chimeric dengue serotype 2/3 strain) has a concentration of 4.0 log10 pfu/dose to 4.7 log10 pfu/dose;

(iv) Dengue serotype 4 (eg, chimeric dengue serotype 2/4 strain) has a concentration of 4.5 log10 pfu/dose to 5.3 log10 pfu/dose.

In further said embodiments, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four wildly attenuated dengue virus strains, wherein:

(i) dengue fever serotype 1 (eg, chimeric dengue serotype 2/1 strain) has a concentration of 3.3 log10 pfu/dose to 3.6 log10 pfu/dose,

(ii) dengue fever serotype 2 (eg, dengue serotype 2 strain) has a concentration of 2.7 log10 pfu/dose to 4.0 log10 pfu/dose;

(iii) dengue fever serotype 3 (eg, a chimeric dengue serotype 2/3 strain) has a concentration of 4.0 log10 pfu/dose to 4.6 log10 pfu/dose;

(iv) Dengue fever serotype 4 (eg, chimeric dengue serotype 2/4 strain) has a concentration of 4.5 log10 pfu/dose 4.6log10 pfu/dose to 5.1 log10 pfu/dose.

In another embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four wild-attenuated dengue virus strains, wherein:

(i) said dengue fever serotype 1 (eg, chimeric dengue serotype 2/1 strain) has a concentration of 4.3 log10 pfu/dose to 4.4 log10 pfu/dose;

(ii) said dengue fever serotype 2 (eg, a dengue serotype 2 strain) has a concentration of 3.7 log10 pfu/dose to 3.8 log10 pfu/dose,

(iii) said dengue fever serotype 3 (eg, a chimeric dengue serotype 2/3 strain) has a concentration of 4.5 log10 pfu/dose to 5.0 log10 pfu/dose;

(iv) said dengue fever serotype 4 (eg, chimeric dengue serotype 2/4 strain) has a concentration of 5.5 log10 pfu/dose to 5.6 log10 pfu/dose.

In a particularly preferred embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four wild-attenuated dengue virus strains, wherein:

(i) said dengue fever serotype 1 (eg, chimeric dengue serotype 2/1 strain) has a concentration of 4.4 log10 pfu/dose,

(ii) said dengue fever serotype 2 (eg, dengue serotype 2 strain) has a concentration of 3.8 log10 pfu/dose,

(iii) said dengue fever serotype 3 (eg, a chimeric dengue serotype 2/3 strain) has a concentration of 4.5 log10 pfu/dose;

(iv) said dengue fever serotype 4 (eg, chimeric dengue serotype 2/4 strain) has a concentration of 5.6 log10 pfu/dose.

In another particularly preferred embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four wildly attenuated dengue virus strains, wherein:

(i) said dengue fever serotype 1 (eg, chimeric dengue serotype 2/1 strain) has a concentration of 3.6 log10 pfu/dose,

(ii) said dengue serotype 2 (eg, dengue serotype 2 strain) has a concentration of 4.0 log10 pfu/dose,

(iii) said dengue fever serotype 3 (eg, a chimeric dengue serotype 2/3 strain) has a concentration of 4.6 log10 pfu/dose;

(iv) said dengue fever serotype 4 (eg, chimeric dengue serotype 2/4 strain) has a concentration of 5.1 log10 pfu/dose.

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

In one embodiment, compositions (i), (ii), (iii), and (iv) provide a total concentration of pfu/0.5 mL in one embodiment, and based on said concentration, The concentration is at least 10% of the total concentration of pfu/0.5 ml.

In one embodiment, compositions (i), (ii), (iii), and (iv) provide a total concentration of pfu/0.5 mL, and based on the total concentration, pfu/0.5 mL of (ii) the concentration of 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 1% is at least 6%, or at least 8%, or at least 10%, or at least 12%, or at least 14%, or at least 16%, or at least 18%.

It is preferred that the concentration in the reconstituted unit dose of (iii) of pfu/0.5 mL is at least 10%.

In one embodiment, compositions (i), (ii), (iii), and (iv) provide a total concentration of pfu/0.5 mL, and based on the total concentration, pfu/0.5 mL of (ii) the concentration of is less than 2%, 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 1% is at least 6%.

Preferably, in this 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 serotype The 2/4 variant is TDV-4. More preferably, TDV-1 is characterized by the nucleotide sequence according to SEQ ID NO: 1 and the amino acid sequence according to SEQ ID NO: 2, and TDV-2 is characterized by the nucleotide sequence according to SEQ ID NO: 3 and the amino acid sequence according to SEQ ID NO: 4 and 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.

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

The dengue vaccine composition comprises a tetravalent dengue virus composition comprising four wild-attenuated dengue virus strains, a chimeric dengue fever serotype 2/1 strain, a dengue fever serotype 2 strain, a chimeric dengue fever serotype 2/3 strain, and chimeric dengue serotype 2/4 strains give a total concentration of pfu/0.5 ml. The term "total concentration of pfu/0.5 mL" or "total concentration of pfu/dose" refers to dengue serotype 1 (eg, chimeric dengue serotype 2/1 strain), dengue serotype 2 (eg, dengue sero Sum of concentrations of type 2 strain), dengue serotype 3 (eg chimeric dengue serotype 2/3 strain) and dengue serotype 4 (eg chimeric dengue serotype 2/4 strain), preferably TDV is the sum of the concentrations of -1, TDV-2, TDV-3 and TDV-4 and is defined as 100% of the concentration of dengue virus as determined by 0.5 mL or pfu (plaque forming units) of a dose.

In one embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four wildly attenuated dengue virus strains, wherein the dengue serotype 1 (eg, chimeric dengue serotype 2/1 strain) , dengue fever serotype 2 (eg, dengue serotype 2 strain), dengue fever serotype 3 (eg, chimeric dengue serotype 2/3 strain) and dengue fever serotype 4 (eg, chimeric dengue serotype 2) /4 strain) provides a total concentration of pfu/0.5 mL, wherein, based on the total concentration, the concentration of dengue serotype 2 (eg, 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, wherein the concentration of dengue serotype 4 (eg, 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 (eg, dengue serotype 2 strain) measured in pfu/0.5 mL, based on the total concentration, is 0.3-10% or 0.5-8% of the total concentration , the concentration of dengue serotype 4 (eg, chimeric dengue serotype 2/4 strain) measured in pfu/0.5 mL is 50% to 90% or 60% to 88% of the total concentration. This means that the concentration of dengue serotype 2 (eg, dengue serotype 2 strain) is less than the concentration of dengue serotype 4 (eg, chimeric dengue serotype 2/4 strain).

In one such embodiment, the concentration of dengue serotype 1 (eg, chimeric dengue serotype 2/1 strain) measured as pfu/0.5 mL is at least 1% of the total concentration and/or measured as pfu/0.5 mL The concentration of dengue serotype 3 (eg, chimeric dengue serotype 2/3 strain) is at least 6% of the total concentration, or at least 7% or 8%, 10%, 12%, 14%, 16% of the total concentration. % or 18%. In one such embodiment, the concentration of dengue serotype 2 (eg, chimeric dengue serotype 2/1 strain) measured in pfu/0.5 mL is between 1% and 7% or between 2% and 6% of the total concentration or and/or the concentration of dengue serotype 3 (eg, chimeric dengue serotype 2/3 strain) measured in pfu/0.5 mL is between 6% and 25% or between 7% and 25% of the total concentration between 2.0% and 5.0%; % or 10% to 25% or 18% to 25%. This means that the concentration of dengue serotype 1 (eg, chimeric dengue serotype 2/1 strain) is less than the concentration of dengue serotype 3 (eg, chimeric dengue serotype 2/3 strain).

In a preferred embodiment, the concentration of a dengue serotype 2 strain, such as TDV-2, measured in pfu/0.5 mL is less than 10%, preferably less than 6% or less than 2% of the total concentration, measured in pfu/0.5 mL The concentration of dengue serotype 4 (eg chimeric dengue serotype 2/4 strain), such as TDV-4, is at least 50%, preferably at least 65% of the total concentration, and TDV-1 measured in pfu/0.5 mL The concentration of dengue serotype 1 (e.g., chimeric dengue serotype 2/1 strain) as Measured concentration of dengue serotype 3 (eg chimeric dengue serotype 2/3 strain) such as TDV-3 At least 6%, preferably 6% to 25% or 10% to 25% or 18% of the total concentration to 25%.

In a further preferred embodiment, such as TDV-1, TDV-2, TDV-3 and TDV-4, dengue fever serotype 1 (eg chimeric dengue serotype 2/1 variant), dengue serotype 2 (eg For example, dengue fever serotype 2 strain), dengue fever serotype 1 (eg chimeric dengue serotype 2/3 strain) and dengue fever serotype 1 (eg chimeric dengue serotype 2/3 strain) and dengue fever sera A dengue virus composition is provided comprising type 4 (eg, chimeric dengue serotype 2/4 strain), wherein the dengue serotype 1 (eg, chimeric dengue serotype 2/) measured pfu/0.5 ml The concentration of strain 1) is at least 1%, preferably between 1% and 7% or between 2.0% and 5.0% of the total concentration of dengue serotype 2 (eg, dengue serotype 2 strain) measured in pfu/0.5 ml. The concentration of dengue serotype 3 (eg, chimeric dengue serotype 2/3 species) measured in pfu/0.5 mL is less than 10%, preferably less than 6% or less than 2% of the total concentration of the total concentration. at least 6% of the concentration, preferably between 6% and 25% or between 10% and 25% or between 18% and 25%. In particular, dengue serotype 4 (eg, chimeric dengue serotype 2/4 strains) has the highest concentration of all four dengue serotypes.

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

In a further preferred embodiment, dengue fever serotype 1 (eg chimeric dengue serotype 2/1 strain), such as TDV-1 and dengue serotype 2 (eg dengue serotype 2 strain), For example, TDV-2 is present in respective concentrations based on a total concentration of each pfu/0.5 mL, which are within 5%-points of each other and/or together less than about 10% of the total concentration of pfu/0.5 mL . In certain of the above embodiments, dengue fever serotype 3 (eg chimeric dengue serotype 2/3 strain), eg TDV-3, is preferably at least about 10% of the total concentration of pfu/0.5 mL, and more Preferably the dengue fever serotype 4 (eg chimeric dengue serotype 2/4 strain), eg TDV-4, is at least about 70% of the total concentration of pfu/0.5 mL. In certain of the above embodiments, dengue fever serotype 4 (eg chimeric dengue serotype 2/4 species) eg TDV-4 exhibits the highest concentration in the composition of all four serotypes, preferably pfu/ At least about 70% of the total concentration of 0.5 mL, dengue serotype 3 (eg, chimeric dengue serotype 2/3 strain), eg, TDV-3, is the second highest in the composition of all four serotypes. concentration, preferably at least about 10% of the total concentration of pfu/0.5 mL, dengue serotype 1 (eg, chimeric dengue serotype 2/1 strain), such as TDV-1 and dengue serotype 2 (eg, dengue serotype 2 strain), eg, TDV-2, each has a higher concentration of serotype 3 (eg, chimeric dengue serotype 2/3 strain), eg, TDV-3 low concentrations, optionally together representing less than about 10% of the total concentration of pfu/0.5 mL.

Preferably, in this 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 serotype The 2/4 variant is TDV-4. More preferably, TDV-1 is characterized by the nucleotide sequence according to SEQ ID NO: 1 and the amino acid sequence according to SEQ ID NO: 2, and TDV-2 is characterized by the nucleotide sequence according to SEQ ID NO: 3 and the amino acid sequence according to SEQ ID NO: 4 and 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.

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 a chimeric dengue serotype 2/3 strain, preferably TDV-3 followed by a chimeric Dengue 2/1 strain, preferably TDV-1 followed by Dengue serotype 2 strain, preferably TDV-2. In particular, it is preferred that the dengue serotype 2 strain has the lowest concentration of the four strains present in the dengue vaccine composition.

Whenever reference is made to concentration/0.5 ml, this does not limit the volume of the unit dose described herein to 0.5 ml. 0.5 ml is the reference volume for determination of the concentration of the virus strain in the composition at pfu/ml. Volumes and/or amounts per unit dose are described in each chapter.

Pharmaceutically Acceptable Excipients

The present invention relates in part to a unit dose of a dengue vaccine composition, wherein the dengue vaccine composition comprises one or more pharmaceutically acceptable excipients. In one embodiment, the dengue vaccine composition comprises a non-reducing sugar, a surfactant, a protein and an inorganic salt. Preferably, the non-reducing sugar is trehalose, the surfactant is poloxamer 407, the protein is human serum albumin and the inorganic salt is sodium chloride.

In one embodiment, the unit dose of the dengue vaccine composition comprises the following pharmaceutically acceptable excipients:

- from about 10% w/v to about 20% w/v of α,α-trehalose dihydrate or an equimolar amount of another form of α,α-trehalose,

- from about 0.5% w/v to about 1.5% w/v of poloxamer 407,

- about 0.05% w/v to about 2% w/v of human serum albumin, and

- about 70 mM to 140 mM sodium chloride.

In one embodiment, the unit dose of the dengue vaccine composition comprises the following pharmaceutically acceptable excipients when measured to be 0.5 ml:

- about 10% w/v to about 20% w/v of α,α-trehalose or an equimolar amount of another form of α,α-trehalose,

- from about 0.5% w/v to about 1.5% w/v of poloxamer 407,

- about 0.05% w/v to about 2% w/v of human serum albumin, and

- about 70 mM to 140 mM sodium chloride, and preferably

- pH 7 to 8.5.

In one embodiment, the unit dose of the dengue vaccine composition comprises the following pharmaceutically acceptable excipients when measured to be 0.5 ml:

- from about 143 mg/ml to about 185 mg/ml of α,α-trehalose dihydrate or an equimolar amount of another form of α,α-trehalose,

- from about 9.1 mg/ml to about 12.4 mg/ml of poloxamer 407,

- about 0.88% mg/ml to about 1.32 mg/ml of human serum albumin, and

- about 70 mM to 140 mM sodium chloride, and preferably

- pH 7 to 8.5.

In a preferred embodiment, the lyophilized unit dose of the invention as described herein comprises the following pharmaceutically acceptable excipients:

- 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.

In a preferred embodiment, the lyophilized unit dose of the invention as described herein comprises the following pharmaceutically acceptable excipients when measured to be 0.5 ml:

- about 15% w/v α,α-trehalose,

- about 1% w/v poloxamer 407,

- about 0.1% w/v human serum albumin, and

- about 100 mM sodium chloride.

In a preferred embodiment, the lyophilized unit dose of the invention as described herein comprises the following pharmaceutically acceptable excipients:

- about 82.9 mg of α,α-trehalose dihydrate,

- about 5 mg poloxamer 407,

- about 0.5 mg of human serum albumin, and

- About 50 μmole of sodium chloride.

In a preferred embodiment, the reconstituted unit dose of the invention as described herein comprises the following pharmaceutically acceptable excipients:

- 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, and preferably

- pH 7 to 8.5

In a preferred embodiment, the reconstituted unit dose of the invention as described herein comprises the following pharmaceutically acceptable excipients when measured to 0.5 ml:

- about 15% w/v α,α-trehalose,

- about 1% w/v poloxamer 407,

- about 0.1% w/v of human serum albumin, and preferably

- about 137 mM sodium chloride and preferably

- pH 7 to 8.5.

In a preferred embodiment, the reconstituted unit dose of the invention as described herein comprises the following pharmaceutically acceptable excipients:

- about 82.9 mg of α,α-trehalose dihydrate,

- about 5 mg poloxamer 407,

- about 0.5 mg of human serum albumin, and preferably

- about 68.5 μmole sodium chloride, and preferably

- pH 7 to 8.5.

Human serum albumin may be native or recombinant human serum albumin (rHSA). Poloxamer 407 can be, for example, Pluronic F127.

In one embodiment, the unit dose further comprises a buffer. The buffer may be phosphate buffered saline (PBS). The buffer comprises at least one of sodium chloride (NaCl), monosodium dihydrogen phosphate (NaH ₂ PO ₄ ), disodium hydrogen phosphate (Na ₂ HPO ₄ ), potassium chloride (KCl), and potassium dihydrogen phosphate (KH ₂ PO ₄ ) can do. In a preferred embodiment, the buffer may comprise disodium hydrogen phosphate (Na ₂ HPO ₄ ), potassium chloride (KCl), and potassium dihydrogen phosphate (KH ₂ PO ₄ ). The buffer may have a pH ranging from 7.0 to 8.5 at 25°C.

unit capacity

The present invention relates in part to a unit dose of a dengue vaccine composition comprising a tetravalent dengue virus composition as described herein and a pharmaceutically acceptable excipient as described herein.

The present invention relates in part to a unit dose of a dengue vaccine composition, for example as described above:

(i) dengue fever serotype 1 (eg, chimeric dengue serotype 2/1 strain) at a concentration of at least 3.3 log10 pfu/0.5 ml;

(ii) dengue serotype 2 (eg, dengue serotype 2 strain) at a concentration of at least 2.7 log10 pfu/0.5 ml;

(iii) dengue fever serotype 3 (eg, chimeric dengue serotype 2/3 strain) at a concentration of at least 4.0 log10 pfu/0.5 ml, and

(iv) dengue fever serotype 4 (eg, chimeric dengue serotype 2/4 strain) at a concentration of at least 4.5 log10 pfu/0.5 ml;

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 It is TDV-4. More preferably, TDV-1 is characterized by the nucleotide sequence according to SEQ ID NO: 1 and the amino acid sequence according to SEQ ID NO: 2, and TDV-2 is characterized by the nucleotide sequence according to SEQ ID NO: 3 and the amino acid sequence according to SEQ ID NO: 4 and 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.

In one embodiment, the unit dose is lyophilized. In one such embodiment, the lyophilized unit dose is a dengue vaccine composition as described herein comprising a pharmaceutically acceptable excipient as described herein and four dengue virus strains, in particular TDV-1 to TDV-4. It is obtained by subjecting a 0.5 mL volume of an aqueous dengue vaccine composition produced by blending to freeze-drying. In a preferred embodiment, the residual moisture content as determined by Karl Fisher crystals is 5.0% or less, preferably 3% or less.

In another implementation, the unit capacity is reconfigured. 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 accomplished 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, eg, 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, or 0.4 to 0.7 mL, or 0.5 mL of diluent. In a preferred embodiment, the lyophilized unit dose is reconstituted with 0.3-0.8 mL, 0.4-0.7 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 may subsequently be administered subcutaneously.

The unit dose in lyophilized form is preferably in the form of storage of the unit dose and the final product after preparation of the unit dose, wherein the unit dose in reconstituted form is prepared prior to administration of said unit dose to a subject.

The present invention further relates in part to a unit dose of a dengue vaccine composition comprising:

A tetravalent virus composition comprising four wild-attenuated dengue virus strains, wherein the unit dose is lyophilized and upon reconstitution of 0.5 mL of a pharmaceutically acceptable diluent comprising:

(i) dengue serotype 1, such as chimeric dengue serotype 2/1 strain, at a concentration of at least 3.3 log10 pfu/0.5 ml;

(ii) dengue serotype 2, such as the dengue serotype 2 strain at a concentration of at least 2.7 log10 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 log10 pfu/0.5 ml, and

(iv) a dengue serotype 4. such as a chimeric dengue serotype 2/4 strain at a concentration of at least 4.5 log10 pfu/0.5 ml.

In one embodiment, the reconstituted unit dose has a volume, e.g., of 0.5 mL, and upon reconstitution with a pharmaceutically acceptable diluent, (i), (ii), (iii), and (iv) providing a total concentration of pfu/0.5 mL, and based on said concentration, the concentration of (iii) is at least 10% of the total concentration of pfu/0.5 mL.

In another embodiment, the reconstituted unit dose has a volume, e.g., of 0.5 mL, and upon reconstitution with a pharmaceutically acceptable diluent, (i), (ii), (iii), and (iv) providing a total concentration of pfu/0.5 mL, wherein, based on the total concentration, the concentration of (ii) in pfu/0.5 mL is less than 10%, and the concentration of (iv) in pfu/0.5 mL is at least 50%; the concentration of (i) in pfu/0.5 mL is at least 1%, and the concentration of (iii) in pfu/0.5 mL is at least 6%, or at least 8%, or at least 10%, or at least 12%, or at least 14% , or at least 16%, or at least 18%.

It is preferred that the concentration in the reconstituted unit dose of (iii) of pfu/0.5 mL is at least 10%.

In one embodiment, the reconstituted unit dose has a volume, e.g., of 0.5 mL, and upon reconstitution with a pharmaceutically acceptable diluent, (i), (ii), (iii), and (iv) providing a total concentration of pfu/0.5 mL, wherein, based on the total concentration, the concentration of (ii) in pfu/0.5 mL is less than 2%, and the concentration of (iv) in pfu/0.5 mL is at least 50%; The concentration of (i) in pfu/0.5 mL is at least 1%, and the concentration of (iii) in pfu/0.5 mL is at least 6%.

In one embodiment, the present invention provides a concentration of dengue serotype 1 (e.g., chimeric dengue serotype 2/1 strain) of at least 3.3 log10 pfu/0.5 mL upon reconstitution with 0.5 mL of a pharmaceutically acceptable diluent. ), dengue fever serotype 2 (eg, dengue serotype 2 strain) at a concentration of at least 2.7 log10 pfu/0.5 mL, dengue serotype 3 (eg, chimeric dengue serum at a concentration of at least 4.0 log10 pfu/0.5 mL) type 2/3 strain), and dengue serotype 4 (eg, chimeric dengue serotype 2/4 strain) at a concentration of at least 4.5 log10 pfu/0.5 mL and a pharmaceutically acceptable excipient as described herein. It relates to a unit dose of a lyophilized dengue vaccine composition, 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 It is TDV-4. More preferably, TDV-1 is characterized by the nucleotide sequence according to SEQ ID NO: 1 and the amino acid sequence according to SEQ ID NO: 2, and TDV-2 is characterized by the nucleotide sequence according to SEQ ID NO: 3 and the amino acid sequence according to SEQ ID NO: 4 and 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.

In one such embodiment, the lyophilized unit dose is dengue serotype 1 (eg, chimeric dengue serotype 2/1 strain), 2.7 log10 pfu at a concentration of 3.3 log10 pfu/dose to 5.0 log10 pfu/0.5 mL. Dengue fever serotype 2 (e.g., dengue serotype 2 strain) at a concentration of /dose to 4.9 log10 pfu/0.5 mL, Dengue serotype 3 (e.g., at a concentration of 4.0 log10 pfu/dose to 5.7 log10 pfu/0.5 mL) For example, chimeric dengue serotype 2/3 strain), and dengue serotype 4 (eg, chimeric dengue serotype 2/4 strain) at a concentration of 4.5 log10 pfu/dose to 5.5 log10 pfu/0.5 mL and described herein It is obtained by lyophilizing 0.5 ml of a dengue vaccine composition comprising a pharmaceutically acceptable excipient as described above. 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 It is TDV-4.

In one such embodiment, the lyophilized unit dose is dengue serotype 1 (eg, chimeric dengue serotype 2/1 strain) at a concentration of 3.3 log10 pfu/0.5 mL to 3.6 log10 pfu/0.5 mL, 2.7 log10 Dengue serotype 2 (eg, dengue serotype 2 strain) at a concentration of pfu/0.5 mL to 4.0 log10 pfu/0.5 mL, dengue serotype 3 at a concentration of 4.0 log10 pfu/0.5 mL to 4.6 log10 pfu/0.5 mL (eg, chimeric dengue serotype 2/3 strain), and dengue serotype 4 (eg, chimeric dengue fever) 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 serotype 2/4 strain) and lyophilizing 0.5 ml of a dengue vaccine composition comprising 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 It is TDV-4.

In certain embodiments, the lyophilized unit dose refers to 0.5 mL prior to lyophilization, and TDV-2 and TDV-4 are pfu/0.5 mL of a total of TDV-1, TDV-2, TDV-3 and TDV-4. present in a certain relative amount based on concentration, 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%.

In certain embodiments, the reconstituted unit dose has a volume of 0.5 mL, and TDV-2 and TDV-4 are based on a total concentration of TDV-1, TDV-2, TDV-3 and TDV-4 of pfu/0.5 mL , wherein the concentration of TDV-2 measured as pfu/0.5 mL is less than 10% or less than 8% or less than 6%, and the concentration of TDV-4 measured as 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%.

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 wildly attenuated dengue virus strains, wherein dengue serum measured as pfu/0.5 mL The concentration of type 1 (eg, dengue serotype 2/1 strain) is between 1% and 7% of the total concentration, and dengue serotype 2 (eg, dengue serotype 2 strain) measured in pfu/0.5 mL The concentration of dengue serotype 3 (eg, dengue serotype 2/3 strains) measured in pfu/0.5 mL is less than 8% of the total concentration, eg, in the range of 1% to 8% of the total concentration. ) is at least 10% of the total concentration, and the concentration of dengue serotype 4 (e.g., dengue serotype 2/4 strain) measured in pfu/0.5 mL is at least 65% of the total concentration, e.g., in the range of 65% to 80%. In certain said embodiments, the arithmetic sum of all four serotypes is in the range of 4.6 log 10 pfu/0.5 mL to 6.7 log 10 pfu/0.5 mL, preferably in the range of 4.6 log 10 pfu/0.5 mL to 5.5 log 10 pfu/0.5 mL there is.

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 wildly attenuated dengue virus strains, said dengue serotype 1 (e.g., chimeric Dengue serotype 2/1 strains), e.g., TDV-1 and dengue serotype 2 (e.g., dengue serotype 2 strains), e.g. TDV-2, each have a total concentration of pfu/0.5 mL. are present at each concentration as a basis, which are within 5%-points of each other and/or together less than about 10% of the total concentration of pfu/0.5 mL. In certain of the above embodiments, dengue fever serotype 3 (eg chimeric dengue serotype 2/3 strain), eg TDV-3, is preferably at least about 10% of the total concentration of pfu/0.5 mL, and more Preferably the dengue fever serotype 4 (eg chimeric dengue serotype 2/4 strain), eg TDV-4, is at least about 70% of the total concentration of pfu/0.5 mL. In certain of the above embodiments, dengue fever serotype 4 (eg chimeric dengue serotype 2/4 species) eg TDV-4 exhibits the highest concentration in the composition of all four serotypes, preferably pfu/ At least about 70% of the total concentration of 0.5 mL, dengue serotype 3 (eg, chimeric dengue serotype 2/3 strain), eg, TDV-3, is the second highest in the composition of all four serotypes. concentration, preferably at least about 10% of the total concentration of pfu/0.5 mL, dengue serotype 1 (eg, chimeric dengue serotype 2/1 strain), such as TDV-1 and dengue serotype 2 (eg, dengue serotype 2 strain), eg, TDV-2, each has a higher concentration of serotype 3 (eg, chimeric dengue serotype 2/3 strain), eg, TDV-3 low concentrations, optionally together representing less than about 10% of the total concentration of pfu/0.5 mL.

A unit dose of lyophilized reconstituted in 0.5 mL provides these concentrations for four dengue serotypes. While the unit dose of a dengue vaccine composition as described herein refers to a concentration of dengue serotype of 0.5 mL, the lyophilized unit dose is pharmaceutically administered without changing the absolute amount of virus administered or the ratio of virus to each other. It may be reconstituted with another volume of an acceptable diluent, for example, aqueous sodium chloride solution.

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

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

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

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

In one embodiment, the solution from which the lyophilized unit dose is prepared further comprises a buffer. The buffer may be phosphate buffered saline (PBS). The buffer comprises at least one of sodium chloride (NaCl), monosodium dihydrogen phosphate (NaH ₂ PO ₄ ), disodium hydrogen phosphate (Na ₂ HPO ₄ ), potassium chloride (KCl), and potassium dihydrogen phosphate (KH ₂ PO ₄ ) can do. In a preferred embodiment, the buffer may comprise disodium hydrogen phosphate (Na ₂ HPO ₄ ), potassium chloride (KCl), and potassium dihydrogen phosphate (KH ₂ PO ₄ ). The buffer may have a pH in the range of about 7.0 to about 8.5 at 25°C or a pH of about 6.8 to about 7.6 at 25°C, preferably a pH of about 7.2 at 25°C.

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

The unit doses of the invention as described herein activate multiple arms of immune system-neutralizing antibodies, cellular immunity and anti-NS1 antibodies in both seronegative and seropositive subject populations or in both seronegative and seropositive subjects. Thus, the unit dose of the invention as described herein protects both a population or subjects of dengue seronegative and dengue seropositive subjects from dengue disease.

In one embodiment, one unit dose is presented in a container, preferably a vial and said unit dose is administered to the subject after reconstitution. In one embodiment, more than one unit dose of the dengue vaccine composition may be present in a container, preferably a vial, such that more than one subject can be vaccinated, together with the contents of one container, preferably a vial. In one dose, a container comprising more than one unit dose of the invention as described herein is used to provide a reconstituted unit dose for use in the method of the invention as described herein.

In certain embodiments, a container comprising a unit dose of the invention is part of a kit. Accordingly, the present invention relates, in part, to a kit for preparing a reconstituted unit dose comprising a lyophilized unit dose of the invention as described herein and a pharmaceutically acceptable diluent for reconstitution.

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

In certain embodiments, the kit may further comprise a hepatitis A vaccine, such as HAVRIX® or VAQTA®. In some embodiments, the hepatitis A vaccine may be in a separate container, eg, a vial. In another embodiment, the hepatitis A vaccine and unit dose of the present invention may be in the same container. Accordingly, the present invention relates in part to a combined dengue/hepatitis A vaccine, wherein a unit dose of the invention as described herein is combined with a hepatitis A vaccine. The combined dengue/hepatitis A vaccine comprises a unit dose of the invention as described herein and a hepatitis A vaccine, eg, HAVRIX® or VAQTA®, in the same formulation. In certain embodiments, the invention relates to a kit comprising a combined dengue/hepatitis A vaccine as described herein and a unit dose of the invention.

hepatitis A vaccine

In certain embodiments, the hepatitis A vaccine is an inactivated hepatitis A vaccine.

In certain embodiments, the hepatitis A vaccine comprises a hepatitis A virus derived from the hepatitis A virus strain HM-175.

In certain embodiments, the hepatitis A vaccine comprises inactivated hepatitis A virus and said inactivated hepatitis A virus is from wild-type hepatitis A virus strain HM-175.

In certain embodiments, the inactivated hepatitis A virus is absorbed on carrier aluminum. In some of these embodiments, the aluminum is aluminum hydroxide or aluminum hydroxyphosphate sulfate.

In certain embodiments, the hepatitis A vaccine comprises a phosphate buffered saline solution dissolved therein in the form of amino acids and in the form of polysorbate and an excipient. In some embodiments, the amino acid is present at a concentration of 0.2 to 0.8% w/v and/or the polysorbate is present at a concentration of 0.01 to 0.09 mg/ml.

In a specific embodiment, the hepatitis A vaccine is administered between 500 ELISA Units (EL.U.) and 2000 ELISA Units (EL.U.), preferably 700 EL.U. Hepatitis A virus expressing the viral antigen at a concentration ranging from 1300 to 1550 EL.U., most preferably from 1300 to 1550 EL.U. Alternatively, the concentration is 500 EL.U. to 900 EL.U. is the range In a further embodiment, the concentration is between 200 and 400 EL.U. is the range

In certain embodiments, the hepatitis A vaccine is included in a liquid 1 ml dose or 0.5 ml dose.

An example of such a hepatitis A virus is HAVRIX® from the manufacturer (GlaxoSmithKline), which is a sterile suspension of inactivated virus for intramuscular administration. HAVRIX® was obtained from wild-type hepatitis A virus (HAV) HM-175 whose complete nucleotide sequence was described in the literature (Cohen et al., Journal of Virology, Vol. 61, No. 1, published Jan. 1987, p. 50 to 59). The derived hepatitis A virus strain HM-175 is utilized (in particular, the full sequence of wild-type hepatitis A virus HM-175 is provided in FIG. 1 of this publication).

The virus (variant HM175) is propagated in MRC-5 human diploid cells. After removal of the cell culture medium, the cells are lysed to form a suspension. The suspension is purified through ultracentrifugation and gel permeation chromatography procedures. Treatment of the lysate with formalin ensures virus inactivation. Viral antigen activity is referred to as a standard using enzyme linked immunosorbent assay (ELISA) and is therefore expressed in ELISA units (EL.U.). Each 1 mL dose for adults (≥ 18 years of age) of vaccine contains 1440 EL.U of viral antigen adsorbed on 0.5 mg of aluminum as aluminum hydroxide. Each 0.5-mL dose for children and adolescents (ages 12 months to 18 years) of the vaccine contains 720 EL.U of viral antigen adsorbed on 0.25 mg of aluminum as aluminum hydroxide. HAVRIX® contains the following excipients: amino acid supplement (0.3% w/v) in phosphate-buffered saline solution and polysorbate 20 (0.05 mg/mL). From the manufacturing process, HAVRIX® also contains residual MRC-5 cellular protein (5 μg/mL or less), formalin (0.1 mg/mL or less), and neomycin sulfate (40 ng/mL or less), contained in the cell growth medium. Contains aminoglycoside antibiotics. HAVRIX® is formulated without preservatives.

Another useful hepatitis A vaccine is VAQTA® from Merck Sharp & Dohme Corp, which is an inactivated whole virus vaccine derived from hepatitis A virus grown in cell culture on human MRC-5 diploid fibroblasts. It contains an inactivated virus of a strain derived by further serial passages of the originally validated attenuated strain. Viruses are grown, harvested by a combination of physical and high performance liquid chromatography techniques developed by the manufacturer (Merck Research Laboratories), purified, formalin inactivated, and then adsorbed onto amorphous aluminum hydroxyphosphate sulfate. VAQTA® is a sterile suspension for intramuscular injection. 1 ml of vaccine contains approximately 50U of hepatitis A antigen, purified and formulated without preservatives. Within the limits of current assay variability, a 50U dose of VAQTA® contains less than 0.1 µg of non-viral protein, less than 4 x 10 ^-6 µg of DNA, less than 10 ^-4 µg of bovine albumin, and less than 0.8 µg of formaldehyde. contains Chemical residues from other processes are less than 10 parts per billion (ppb) and contain neomycin. Each 0.5 mL pediatric dose contains 25 U of hepatitis A virus antigen, adsorbed to approximately 0.225 mg aluminum provided as amorphous aluminum hydroxyphosphate sulfate in 0.9% sodium chloride and 35 μg sodium borate provided as a pH stabilizer. . Each 1 mL adult dose contains 50 U of hepatitis A virus antigen, adsorbed to approximately 0.45 mg aluminum provided as amorphous aluminum hydroxyphosphate sulfate in 0.9% sodium chloride and 70 µg sodium borate provided as a pH stabilizer. .

yellow fever vaccine

YF-VAX®, a yellow fever vaccine from the manufacturer (Sanofi) for subcutaneous use, is prepared by culturing the YF-17D strain of yellow fever virus in live avian leukemia virus-free (ALV-free) chicken embryos. The vaccine contains sorbitol and gelatin as stabilizers and is lyophilized. No preservatives are added. YF-VAX is formulated to contain at least 4.74 log ₁₀ pfu per 0.5 ml dose throughout product life.

Combination vaccine composition

The invention also provides, in part, a combination comprising a hepatitis A antigen as in HAVRIX® or VAQTA®, and a dengue antigen such as a tetravalent dengue vaccine as described herein or any other suitable tetravalent wild attenuated dengue virus vaccine. It relates to a vaccine composition.

In certain embodiments, the present invention relates to a combined vaccine composition, wherein the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four wildly attenuated dengue virus strains:

(i) dengue serotype 1, preferably at a concentration of at least 3.3 log10 pfu/0.5 mL,

(ii) dengue serotype 2, preferably at a concentration of at least 2.7 log10 pfu/0.5 mL;

(iii) dengue serotype 3, preferably at a concentration of at least 4.0 log10 pfu/0.5 mL, and

(iv) Dengue serotype 4 strain, preferably at a concentration of at least 4.5 log10 pfu/0.5 mL.

In certain embodiments, the present invention relates to a combined vaccine composition, wherein the dengue vaccine composition comprises a tetravalent dengue virus composition comprising four wildly attenuated dengue virus strains:

(i) a chimeric dengue serotype 2/1 strain at a concentration of at least 3.3 log10 pfu/0.5 mL to 3.8 log10 pfu/0.5 ml,

(ii) a dengue serotype 2 strain at a concentration of at least 2.7 log10 pfu/0.5 mL;

(iii) a chimeric dengue serotype 2/3 strain at a concentration of at least 4.0 log10 pfu/0.5 mL, and

(iv) a chimeric dengue serotype 2/4 strain at a concentration of at least 4.5 log10 pfu/0.5 mL or at least 4.6 log10 pfu/0.5 mL to optionally 6.2 log10 pfu/0.5 ml.

Preferably, in this 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 serotype The 2/4 variant 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, and 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 and 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.

In certain embodiments, the present invention relates to a combined vaccine composition, wherein upon reconstitution of the dengue vaccine composition with a pharmaceutically acceptable diluent, (i), (ii), (iii), and (iv) giving a total concentration of pfu/0.5 mL, based on the total concentration of pfu/0.5 mL, the concentration of (ii) in pfu/0.5 mL is less than 10%, and the concentration of (iv) in pfu/0.5 mL is at least 50%, the concentration of (i) in pfu/0.5 mL is at least 1%, and the concentration of (iii) in pfu/0.5 mL is at least 6%, or at least 8%, or at least 10%, or at least 12% , or at least 14%, or at least 16%, or at least 18%.

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

In addition, any vaccine excipient known to the person skilled in the art, or a combination thereof, described, for example, in WO 2018/027075 A1 may be used for the combination vaccine composition.

In one embodiment, the unit dose of the dengue vaccine composition comprises 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,

- from about 0.5% w/v to about 1.5% w/v of poloxamer 407,

- about 0.05% w/v to about 2% w/v of human serum albumin, and

- about 70 mM to 140 mM sodium chloride.

In certain embodiments, the present invention relates to a combined vaccine composition, wherein the dengue vaccine composition comprises another dengue vaccine, such as Dengvaxia®. Dengvaxia® is CYD-TDV (Dengvaxia®, Sanofi Pasteur, Lyon, France), a tetravalent dengue vaccine with a mixed chimeric dengue virus based on a yellow fever backbone, which is a virologically confirmed dengue fever in Asian and Latin American children ( VCD) has been approved in several countries based on clinical demonstration of overall vaccine efficacy (VE) for 56-61% (Capeding MR, etc. Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in Asia: a phase 3, randomized , observer-masked, placebo-controlled trial, Lancet 2014, 384:1358-65; Villar LA et al. Safety and immunogenicity of a recombinant tetravalent dengue vaccine in 9-16 year olds: a randomized, controlled, phase II trial in Latin America. Pediatr Infect Dis J 2013, 32:1102-9). The preparation of these specific variants CYD1, CYD2, CYD3 and CYD4 is described in international patent applications WO 98/37911, WO 03/101397, WO07/021672, WO 08/007021, the description of the exact process for their preparation may be referred to. WO 08/047023 and WO 08/065315 are described in detail. The corresponding nucleotide sequences of the prM-E regions of CYD1, CYD2, CYD3 and CYD4 are provided in WO2016034629 and the SEQ ID NOs are given in Table 16 of this reference.

In certain embodiments, the present invention relates to a combined vaccine composition, wherein the amount of chimeric dengue virus in the CYD-TDV comprised in the vaccine composition of the present invention is in the range of about 10 ⁵ CCID50 to about 10 ⁶ CCID50. The amount of the wild attenuated chimeric dengue virus of each of serotypes 1 to 4 contained in the CYD dosage form, Dengvaxia®, is preferably the same.

In this embodiment, CYD-TDV can be dissolved/dissolved in a solution containing 0.4% NaCl.

In certain embodiments, the present invention relates to a combined vaccine composition, wherein said dengue vaccine composition comprises another dengue vaccine such as TV003 or TV005. TV003, developed by the US National Institute of Allergy and Infectious Diseases, comprises vaccine components rDEN1Δ30, rDEN2/4Δ30, rDEN3Δ30/31 and rDEN4Δ30, wherein each of these components is present at a concentration of 3 log ₁₀ PFU. . TV005 is similar to TV003, the difference is that the concentration of rDEN2/4Δ30 in TV005 is 4 log ₁₀ PFU. Vaccines TV003 and TV005 and their vaccine components and their preparation are described in more detail in WO 2008/022196 A2 and SS Whitehead, Expert Rev Vaccines, 2016, 15(4): 509-517. Using recombinant DNA technology, two attenuation strategies were used for the structural gene chimerization and the deletion in the 3' untranslated region of the vaccine composition of TV003 or TV005. For example, component rDEN4Δ30 contains all structural and non-structural proteins of wild-type DENV-4, but is attenuated by a 30-nucleotide deletion in the 3′ untranslated region (referred to as “Δ30”). Other vaccine components are also attenuated due to a 30-nucleotide deletion in the 3' untranslated region. Additionally, rDEN3Δ30/31 contains a 31 nucleotide deletion in the 3' untranslated region (detailed in FIGS. 1c and 13 of WO 2008/022196 A2). The rDEN2/4Δ30 component was created by replacing the prM and E genes of DENV-2 with the rDEN4Δ30 genome. The complete genomic sequence of the dengue strain that can be used to generate TV003 or TV005 is available under Genbank accession number in Table A of WO 2008/022196 A1.

In certain embodiments, the present invention relates to a combined vaccine composition, wherein said hepatitis A vaccine is an inactivated hepatitis A vaccine.

In certain embodiments, the hepatitis A vaccine comprises a hepatitis A virus derived from the hepatitis A virus strain HM-175.

In certain embodiments, the present invention relates to a combined vaccine composition, wherein said hepatitis A vaccine comprises inactivated hepatitis A virus and said inactivated hepatitis A virus is wild-type hepatitis A virus strain HM. It is derived from -175.

In certain embodiments, the present invention relates to a combined vaccine composition, wherein the inactivated hepatitis A virus is adsorbed onto a carrier aluminum. In some of these embodiments, the aluminum is aluminum hydroxide or aluminum hydroxyphosphate sulfate.

In a specific embodiment, the present invention relates to a combined vaccine composition, wherein said hepatitis A vaccine comprises a phosphate buffered saline solution dissolved therein in the form of amino acids and in the form of polysorbate and an excipient. In some embodiments, the amino acid is present at a concentration of 0.2 to 0.8% w/v and/or the polysorbate is present at a concentration of 0.01 to 0.09 mg/ml.

In a specific embodiment, the present invention relates to a combined vaccine composition, wherein said hepatitis A vaccine comprises from 500 ELISA Units (EL.U.) to 2000 ELISA Units (EL.U.), preferably 700 EL.U. Hepatitis A virus expressing the viral antigen at a concentration ranging from 1300 to 1550 EL.U., most preferably from 1300 to 1550 EL.U. Alternatively, the concentration is 500 EL.U. to 900 EL.U. is the range In a further embodiment, the concentration is between 200 and 400 EL.U. is the range

In certain embodiments, the present invention relates to a combined vaccine composition, wherein the combined vaccine is comprised in a dose comprising a liquid, wherein the liquid has a volume of 0.5 ml, 1 ml, or 1.5 ml.

In certain embodiments, the combined vaccine compositions are provided in one single vial in liquid form or in dehydrated form, eg, lyophilized form.

In certain embodiments, the combined vaccine composition is obtained by mixing in a syringe a unit dose of the dengue vaccine composition and a dose of the hepatitis A vaccine.

The invention also relates, in part, to methods of administering to a subject or population of subjects any of the above combined vaccine compositions.

In certain embodiments, the present invention relates to a method in which the combined vaccine composition is administered subcutaneously or intramuscularly.

Methods for preventing dengue fever and hepatitis A, corresponding uses and corresponding kits

The present invention relates to a method for preventing hepatitis A and dengue fever.

The present invention relates in part to a method of preventing hepatitis A and dengue disease in a subject or population of subjects, said method comprising administering the same unit dose of a hepatitis A vaccine, e.g., HAVRIX® or VAQTA® and a dengue vaccine composition Simultaneously administering on the same day, wherein the unit dose comprises a tetravalent dengue virus composition comprising four wildly attenuated dengue virus strains.

In certain embodiments, the present invention provides a hepatitis A vaccine, eg, HAVRIX®, comprising an inactivated virus. Preferably, the hepatitis A vaccine comprises inactivated hepatitis A virus and said inactivated hepatitis A virus is from hepatitis A virus strain HM-175.

In certain embodiments, the hepatitis A vaccine, eg, HAVRIX®, is from hepatitis A virus strain HM-175.

In a specific embodiment, the present invention relates to a method in which a hepatitis A vaccine, eg HAVRIX®, preferably derived from the hepatitis A virus strain HM-175, is adsorbed onto aluminum. According to some of these embodiments, the aluminum is aluminum hydroxide or aluminum hydroxyphosphate sulfate.

In a specific embodiment, the present invention provides a hepatitis A vaccine derived from hepatitis A virus strain HM-175, e.g., HAVRIX® dissolved therein in the form of amino acids and in the form of polysorbate, a phosphate buffered saline solution and excipients It relates to a method comprising

In a specific embodiment, the present invention provides that a hepatitis A vaccine, eg, HAVRIX®, is administered in an amount of 500 ELISA Units (EL.U.) to 2000 ELISA Units (EL.U.), preferably 700 EL.U. hepatitis A virus expressing the viral antigen at a concentration ranging from 1300 to 1600 EL.U., most preferably 1300 to 1550 EL.U. Alternatively, the concentration is 500 EL.U. to 900 EL.U. is the range In a further embodiment, the concentration is between 200 and 400 EL.U. is the range

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

In certain embodiments, the present invention relates to a method, wherein the dengue vaccine composition, upon reconstitution with 0.5 mL of a pharmaceutically acceptable diluent, comprises:

(i) a chimeric dengue serotype 2/1 strain at a concentration of at least 3.3 log10 pfu/0.5 mL,

(ii) a dengue serotype 2 strain at a concentration of at least 2.7 log10 pfu/0.5 mL;

(iii) a chimeric dengue serotype 2/3 strain at a concentration of at least 4.0 log10 pfu/0.5 mL, and

(iv) a chimeric dengue serotype 2/4 strain at a concentration of at least 4.5 log10 pfu/0.5 mL.

According to some of these embodiments, upon reconstitution of the dengue vaccine composition with a pharmaceutically acceptable diluent, (i), (ii), (iii), and (iv) provide a total concentration of pfu/0.5 mL, Based on the above total concentration of pfu/0.5 ml, the concentration of (ii) of pfu/0.5 mL is less than 10%, the concentration of (iv) of pfu/0.5 mL is at least 50%, and the concentration of (ii) of pfu/0.5 mL is at least 50%, the concentration of i) is at least 1%, and the concentration of (iii) of pfu/0.5 mL is at least 6%, or at least 8%, or at least 10%, or at least 12%, or at least 14%, or at least 16%, or at least 18%.

In certain embodiments, the invention relates to a method, wherein the subject population or subject is seronegative with respect to all dengue serotypes. According to some of these embodiments, the subject population or subject is seronegative for hepatitis A at baseline.

In certain embodiments, the invention relates to a method, wherein a unit dose of the invention as described herein and a hepatitis A vaccine, eg, HAVRIX® or VAQTA®, are administered on Day 0/1.

In certain embodiments, the invention provides a method wherein the unit dose of the invention as described herein is administered by subcutaneous injection and the hepatitis A vaccine, eg, HAVRIX® or VAQTA®, is administered by intramuscular injection. is about According to some embodiments, the injection is administered to the arm, preferably in the deltoid region of the arm. According to some of these embodiments, a subcutaneous injection of a unit dose of the invention as described herein and an intramuscular injection of a hepatitis A vaccine, for example HAVRIX® or VAQTA®, are administered at different anatomical sites, for example, It is administered to the opposite arm.

In certain embodiments, the invention relates to the above method in which two unit doses of the dengue vaccine composition of the invention as described herein are administered. In some embodiments, the two unit doses of the invention as described herein are administered within at least 12 months or within 6 months or within 3 months, eg on Day 0/1 and Day 90. According to some of these embodiments, a further third unit dose of the invention as described herein is administered after the second administration. wherein said third administration is 6 to 12 months after the first administration, eg, 12 months after the first administration, or more than 12 months after the first administration, eg, 12 months (1 year) after the second administration; or It can even be administered more than 5 years after the first or second administration and can act as a booster.

In a specific embodiment, the present invention relates to a method in which two unit doses of the invention as described herein and one dose of a hepatitis A vaccine, for example HAVRIX® or VAQTA®, are administered in particular according to the schedule below is about:

- a first unit dose and a first concurrent administration of said hepatitis A vaccine on day 0/1, and

- a second administration of a second unit dose after said first simultaneous administration, eg about a month later, eg on day 90.

In certain embodiments, the invention provides that a unit dose of the invention as described herein is administered to a subject or population of subjects, and a hepatitis A vaccine, eg, HAVRIX® or VAQTA®, is administered intramuscularly to the subject or population of subjects. and wherein the subject or population of subjects is seronegative for all dengue serotypes. In other embodiments, the subject or population of subjects is seropositive for at least one dengue serotype.

In certain embodiments, the invention relates to a method, wherein a unit dose of the invention as described herein and a hepatitis A vaccine, e.g., HAVRIX® or VAQTA®, are administered to a subject or population of subjects from an area endemic to dengue. will be. In certain embodiments, a unit dose of the invention as described herein is administered subcutaneously and the hepatitis A vaccine, e.g., HAVRIX® or VAQTA®, is administered intramuscularly to a subject or population of subjects from an area endemic to dengue. .

In certain embodiments, the invention relates to a method wherein the subject or population of subjects originates from a dengue non-endemic area, preferably from a dengue non-endemic and hepatitis A non-endemic area.

According to some embodiments, a second dose of a hepatitis A vaccine, eg, HAVRIX® or VAQTA®, is administered. The second dose of the hepatitis A vaccine may be administered after the first administration of the hepatitis A vaccine. Said second administration may act as a booster and may be administered 6 to 12 months or 6 to 18 months after the first administration of the hepatitis A vaccine, eg 9 months, eg 270 days. .

In certain embodiments, the invention provides that the unit dose of the invention as described herein is administered subcutaneously, and the hepatitis A vaccine, e.g., HAVRIX® or VAQTA®, is over 17 years old, or over 18 years old, or 18-60 years old. administered intramuscularly to a subject or population of three years of age. In a further embodiment, the subject or population of subjects is an adult greater than 21 years of age, or between 21 and 60 years of age, or between 21 and 45 years of age. In some embodiments, the subject or population of subjects is from an area endemic to dengue. In another embodiment, the subject or population of subjects is from a dengue non-endemic area, preferably from a dengue non-endemic and hepatitis A non-endemic area. According to certain embodiments, the subject or population of subjects is seronegative for all four dengue fever serotypes.

In certain embodiments, the present invention provides a method for determining whether a subject has had a prior dengue infection and/or a prior hepatitis A infection or in a subject population prior to administration of the hepatitis A vaccine and prior to administration of a unit dose of a dengue vaccine composition. wherein the hepatitis A serum status and/or dengue fever serum status of a subject population or of a subject is not known prior to administration of the hepatitis A vaccine or prior to administration of a unit dose of a dengue vaccine composition will be. According to a particular embodiment, the method comprises prior to, during, and at any time after the administering step of administering a unit dose of the hepatitis A vaccine and the dengue vaccine composition in a subject population or prior to a previous dengue infection and/or prior to the administering step. Hepatitis A serum status and/or dengue fever serum status and/or dengue fever serum status of or of a subject population or subject population does not include a step of determining whether hepatitis A infection has been is not known at the time of

In certain embodiments, the present invention relates to a method, wherein said method comprises a primary vaccination comprising the steps of:

(A) a unit dose of a tetravalent dengue virus composition and type A requiring protection against dengue infection and hepatitis A infection without prior determination of whether there was a prior dengue infection and/or prior hepatitis A infection. selecting a subject for administration of a hepatitis vaccine, and

(B) concurrently administering to said subject a first unit dose of a tetravalent dengue virus composition and hepatitis A vaccine on the same day, and optionally

(C) administering at least one additional unit dose of the tetravalent dengue virus composition to the subject within 3 to 12 months of administration of the first unit dose, and optionally

(D) administering at least one additional dose of hepatitis A vaccine to the subject within 6 to 18 months of administration of the first unit dose.

In certain embodiments, the present invention relates to a method, wherein said method comprises a primary vaccination comprising the steps of:

(A) selecting a subject for administration of a unit dose of a tetravalent dengue virus composition and hepatitis A vaccine in need of protection against dengue infection and hepatitis A infection, and

(B) concurrently administering to the subject a first unit dose of a tetravalent dengue virus composition and a hepatitis A vaccine on the same day, and

(C) administering two additional unit doses of the tetravalent dengue virus composition to the subject at about 6 and about 12 months of administration of the first unit dose and an additional dose at about 6 or about 12 months of administration of the first unit dose. administering a hepatitis A vaccine to the subject. In some of these embodiments, the selection of the subject in step (A) is performed without determining whether there is a prior hepatitis A infection.

In certain embodiments, the present invention provides, upon reconstitution of said unit dose of a pharmaceutically acceptable diluent, wherein (i), (ii), (iii), and (iv) provide a total concentration of pfu/0.5 mL; , based on the total concentration of pfu/0.5 ml, the concentration of (ii) of pfu/0.5 mL is less than 10%, the concentration of (iv) of pfu/0.5 mL is at least 50%, and the concentration of (iv) of pfu/0.5 mL is at least 50%, the concentration of (i) is at least 1%, and the concentration of (iii) of pfu/0.5 mL is at least 6%, at least 8%, or at least 10%, or at least 12%, or at least 14%, or at least 16%, or at least 18%.

In certain embodiments, the method provides for compatibility between the dengue vaccine composition and the hepatitis A vaccine. Compatibility means that the immune response, especially after simultaneous administration, is not inferior compared to administration of these vaccines alone.

In certain embodiments, the method provides for synergy between the dengue vaccine composition and the hepatitis A vaccine. Synergy means that the immune response is better for one or both vaccines compared to administration of these vaccines alone, especially after simultaneous administration.

In certain embodiments, the invention provides for non-inferiority in a non-inferiority clinical study comprising at least 60 or at least 120 healthy subjects divided into one subject population and one control subject population, wherein: wherein the subject population is administered unit doses of the hepatitis A vaccine and the dengue vaccine composition simultaneously on the same day and the control subject population is concurrently administered the hepatitis A vaccine and the placebo administration on the same day.

In a specific embodiment, the present invention provides that the hepatitis A vaccine is seronegative with respect to hepatitis A at baseline, administered unit doses of the hepatitis A vaccine and the dengue vaccine composition simultaneously on the same day, and at baseline all dengue virus serum providing a hepatitis A seroprotection rate of at least 95% or at least 98% on Day 30 (on Day 0/1) after administration to a subject population of at least 30 or at least 50 healthy subjects who are seronegative with respect to type; to the method.

In certain embodiments, the present invention provides a difference in hepatitis A seroprotection rate with respect to hepatitis A mono-administration, wherein the difference is seronegative with respect to hepatitis A at baseline and with all dengue virus serotypes at baseline as determined in a non-inferiority clinical study comprising at least 60 or at least 120 healthy subjects who are relatedly seronegative,

healthy subjects

a) a subject population of at least 30 or at least 50 healthy subjects who received administration of a unit dose of a hepatitis A vaccine and a dengue vaccine composition (on Day 0/1) simultaneously on the same day, and

b) divided into a control subject population of at least 30 or at least 50 healthy subjects who received administration of hepatitis A vaccine and placebo (on Day 0/1) simultaneously on the same day,

the difference is determined between the hepatitis A seroprotection rate of the control subject population at Day 30 post-dose (on Day 0/1) and the hepatitis A seroprotection rate of the subject population at Day 30 post-dose (on Day 0/1) and ,

The difference has a joint upper bound within the two-sided 95% confidence interval of less than 10%.

In certain embodiments, the present invention provides that the hepatitis A vaccine is administered to at least 30 or at least 50 patients who are seronegative with respect to hepatitis A at baseline, receiving unit doses of the hepatitis A vaccine and the dengue vaccine composition simultaneously on the same day. provide a hepatitis A seroprotection rate of at least 95% or at least 98% or at least 99% at 30 days (on Day 0/1) following administration to a subject population of healthy subjects, wherein the healthy subject has at least one dengue fever at baseline including healthy subject(s) seropositive with respect to viral serotype and healthy subject(s) seronegative for all dengue virus serotypes at baseline.

In certain embodiments, the present invention provides a difference in hepatitis A seroprotection rate with respect to hepatitis A mono-administration, wherein the difference is at least 60 or at least 120 healthy subjects with hepatitis A seronegative values at baseline. A healthy subject(s) determined in a non-inferiority clinical study comprising subjects, said healthy subject being seropositive for at least one dengue virus serotype at baseline and seronegative for all dengue virus serotypes at baseline to the method, comprising healthy subject(s),

healthy subjects

a) a subject population of at least 30 or at least 50 healthy subjects receiving administration of unit doses of a hepatitis A vaccine and a dengue vaccine composition simultaneously on the same day (Day 0/1), wherein the subject population is at least at baseline a population of subjects, comprising healthy subject(s) seropositive for one dengue virus serotype and healthy subject(s) seronegative for all dengue virus serotypes at baseline; and

b) a control subject population of at least 30 or at least 50 healthy subjects receiving concurrent administration of hepatitis A vaccine and placebo on the same day (Day 0/1), wherein the control subject population has at least one dengue fever at baseline divided into a control subject population, comprising healthy subject(s) seropositive with respect to viral serotype and healthy subject(s) seronegative for all dengue virus serotypes at baseline;

the difference is determined between the hepatitis A seroprotection rate of the control subject population at Day 30 post-dose (on Day 0/1) and the hepatitis A seroprotection rate of the subject population at Day 30 post-dose (on Day 0/1) and ,

The difference has a joint upper bound within the two-sided 95% confidence interval of less than 10%.

In certain embodiments, the invention relates to the method, wherein the subject or population of subjects is exposed to a hepatitis A virus outbreak and/or a dengue virus outbreak.

In certain embodiments, the invention provides that unit doses of hepatitis A vaccine and dengue vaccine composition are administered simultaneously on the same day and are seronegative for hepatitis A vaccine at baseline and seronegative for all dengue virus serotypes at baseline at least 70 mIU/ml or at least 80 mIU/ml or at least 90 mIU/ml of anti-hepatitis A virus on Day 30 (on Day 0/1) after administration to a subject population of at least 30 or at least 50 healthy subjects and providing a geometric mean concentration (GMC) of an antibody.

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

In certain embodiments, the present invention relates to the method, wherein the simultaneous administration to a subject or population of subjects of a unit dose of the hepatitis A vaccine and the dengue vaccine composition does not provide the severe side effects associated with the simultaneous administration. In addition, there are no deaths related to concurrent administration.

In certain embodiments, the invention provides a geometric mean titer (GMT) of a neutralizing antibody as measured by MNT50, wherein the titer is

- at least 110 or at least 140 or at least 150 for dengue serotype 1,

- at least 3000 or at least 3500 or at least 3900 for dengue serotype 2,

- at least 100 or at least 120 or at least 140 for dengue serotype 3;

- at least 80 or at least 110 or at least 140 for dengue serotype 4,

At least 30 patients whose titers received unit doses of hepatitis A vaccine and dengue vaccine composition simultaneously on the same day and were seronegative for all hepatitis A at baseline and seronegative for all dengue virus serotypes at baseline or at Day 30 (on Day 0/1) after administration to a subject population of at least 50 healthy subjects.

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

The present invention relates in part to a method of preventing hepatitis A and dengue disease in a subject or population of subjects, the method comprising administering unit doses of a hepatitis A vaccine and a dengue vaccine composition simultaneously on the same day, wherein the unit dose comprises a tetravalent dengue virus composition comprising four wild-attenuated dengue virus strains, wherein the four wild-attenuated dengue virus strains are different from those used in the unit dose as defined above.

In one embodiment of the present invention, the method relates to concurrent administration of the hepatitis A vaccine together with another dengue vaccine, such as Dengvaxia®, on the same day. Dengvaxia® is CYD-TDV (Dengvaxia®, Sanofi Pasteur, Lyon, France), a tetravalent dengue vaccine based on a yellow fever backbone, which accounts for 56-61% of virologically confirmed dengue fever (VCD) in children in Asia and Latin America. Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in Asia: a phase 3, randomized, observer-masked, placebo -controlled trial Lancet 2014, 384:1358-65; Villar LA et al. Safety and immunogenicity of a recombinant tetravalent dengue vaccine in 9-16 year olds: a randomized, controlled, phase II trial in Latin America. Pediatr Infect Dis J 2013, 32:1102-9). The preparation of these specific variants CYD1, CYD2, CYD3 and CYD4 is described in international patent applications WO 98/37911, WO 03/101397, WO07/021672, WO 08/007021, the description of the exact process for their preparation may be referred to. WO 08/047023 and WO 08/065315 are described in detail. The corresponding nucleotide sequences of the prM-E regions of CYD1, CYD2, CYD3 and CYD4 are provided in WO2016034629 and the SEQ ID NOs are given in Table 16 of this reference.

In one such embodiment, the method comprises vaccination consisting of:

(A) selecting a subject for administration of the same dose of a CYD-TDV composition such as Dengvaxia® and a hepatitis A vaccine such as HAVRIX® or VAQTA® in need of protection against dengue infection and hepatitis A infection; and

(B) administering to the subject at month 0 a first dose of a CYD-TDV composition such as Dengvaxia® and a hepatitis A vaccine such as HAVRIX® or VAQTA®;

(C) an additional dose of a CYD-TDV composition such as Dengvaxia® and optionally a hepatitis A vaccine such as HAVRIX® or VAQTA® within 3 to 11 months, in particular at about 6 months of administration of said first CYD-TDV dose; administering to the subject, and

(D) administering to the subject at about 12 months a final dose of a CYD-TDV such as Dengvaxia® and optionally a hepatitis A vaccine such as HAVRIX® or VAQTA®.

In certain embodiments, the subject is between 2 and 60 years of age.

In certain embodiments, the subject is between 2 and 18 years of age, or between 4 and 16 years of age or between 18 and 60 years of age.

Preferably, the exact amount of each component of the CYD-TDV to be administered may vary depending on the age and weight of the vaccinated subject, the frequency of administration and other components in the composition. The amount of chimeric dengue virus in CYD-TDV included in the dose of the vaccine composition is in the range of about 10 ⁵ CCID50 to about 10 ⁶ CCID50. The amount of the wild attenuated chimeric dengue virus of each of serotypes 1 to 4 contained in the CYD dosage form, Dengvaxia®, is preferably the same. Advantageously, a vaccine composition as described in this section comprises an effective amount of a dengue antigen as described herein.

In certain embodiments, the present invention relates to said method, wherein said dengue vaccine composition comprises another dengue vaccine such as TV003 or TV005. TV003, developed by the US National Institute of Allergy and Infectious Diseases, comprises vaccine components rDEN1Δ30, rDEN2/4Δ30, rDEN3Δ30/31 and rDEN4Δ30, wherein each of these components is present at a concentration of 3 log ₁₀ PFU. . TV005 is similar to TV003, the difference is that the concentration of rDEN2/4Δ30 in TV005 is 4 log ₁₀ PFU. Vaccines TV003 and TV005 and their vaccine components and their preparation are described in WO 2008/022196 A2 and SS Whitehead, Expert Rev. Vaccines, 2016, 15(4): 509 to 517). Using recombinant DNA technology, two attenuation strategies were used for the structural gene chimerization and the deletion in the 3' untranslated region of the vaccine composition of TV003 or TV005. For example, component rDEN4Δ30 contains all structural and non-structural proteins of wild-type DENV-4, but is attenuated by a 30-nucleotide deletion in the 3' untranslated region (referred to as "Δ30"). Other vaccine components are also attenuated due to a 30-nucleotide deletion in the 3' untranslated region. Additionally, rDEN3Δ30/31 contains a 31 nucleotide deletion in the 3′ untranslated region (detailed in FIGS. 1c and 13 of WO 2008/022196 A2). The rDEN2/4Δ30 component was created by replacing the prM and E genes of DENV-2 with the rDEN4Δ30 genome. The complete genomic sequence of the dengue strain that can be used to generate TV003 or TV005 is available under Genbank accession number in Table A of WO 2008/022196 A1.

In certain embodiments, the present invention relates to, inter alia, a chimeric dengue fever serotype 2/1 strain, a wild-attenuated dengue fever serotype 2 strain, a chimeric dengue fever serotype 2/3 strain and a chimeric dengue serotype 2/4 strain, and the Dengvaxia described herein. ® and a unit dose described herein comprising a hepatitis A vaccine described herein are administered to a subject or a population of subjects simultaneously on the same day.

In certain embodiments, the present invention relates to, inter alia, chimeric dengue serotype 2/1 strains, wild-attenuated dengue serotype 2 strains, chimeric dengue serotype 2/3 strains and chimeric dengue serotype 2/4 strains and type A described herein. A unit dose described herein comprising the hepatitis vaccine is administered concurrently to a subject or to a population of subjects on Day 0/1 as a first dose or concurrently on the same day as a second dose of Dengvaxia® described herein followed by 3 doses from the first dose as a second dose. to the subject or to the population of subjects within a month, eg, on day 90 from the first administration. Alternatively, the Dengvaxia® described herein and the hepatitis A vaccine described herein are administered concurrently to the subject on Day 0/1 or to a population of subjects on the same day as the first dose, in particular, the chimeric dengue serotype 2/1 strain , a unit dose described herein comprising a wild-attenuated dengue serotype 2 strain, a chimeric dengue serotype 2/3 strain and a chimeric dengue serotype 2/4 strain is subsequently administered as a second administration within 3 months of the first administration. , eg, to the subject or population of subjects on day 90 from the first administration.

In certain embodiments, the present invention relates to, inter alia, a chimeric dengue serotype 2/1 strain, a wild-attenuated dengue serotype 2 strain, a chimeric dengue serotype 2/3 strain and a chimeric dengue serotype 2/4 strain, and TV003 described herein. or the unit doses described herein comprising TV005 and the hepatitis A vaccine described herein are administered to the subject or population of subjects simultaneously on the same day.

In certain embodiments, the present invention relates to, inter alia, chimeric dengue serotype 2/1 strains, wild-attenuated dengue serotype 2 strains, chimeric dengue serotype 2/3 strains and chimeric dengue serotype 2/4 strains and type A described herein. A unit dose described herein comprising the hepatitis vaccine is administered to a subject or to a population of subjects simultaneously on the same day on Day 0/1 as a first administration and a TV003 or TV005 described herein is subsequently administered from the first administration as a second administration. administered to the subject or population of subjects within 3 months, eg, 90 days from the first administration. Alternatively, TV003 or TV005 described herein and the hepatitis A vaccine described herein are administered concurrently to a subject or to a population of subjects on Day 0/1 as a first dose or concurrently to a population of subjects on the same day, in particular, chimeric dengue serotype 2/1 Unit doses described herein comprising strains, wild-attenuated dengue fever serotype 2 strains, chimeric dengue fever serotype 2/3 strains and chimeric dengue fever serotype 2/4 strains are subsequently administered as a second dose 3 months from the first dose. within, eg, 90 days from the first administration to the subject or population of subjects.

Said method also relates to the use of a unit dose of a dengue vaccine as described herein for said method of preventing dengue disease and hepatitis A or for the preparation of a medicament for said method of preventing dengue disease and hepatitis A should be considered in relation to the use of unit doses of dengue vaccine for

Further, the present invention relates to a kit for hepatitis A and dengue disease, said kit comprising:

At least

(a) a first container holding a hepatitis A vaccine as defined above, such as HAVRIX®, and

(b) a second container holding a unit dose of a dengue vaccine composition as defined above, wherein the unit dose comprises a second container comprising a tetravalent dengue virus composition comprising four live attenuated dengue virus strains includes a box that says

Methods and uses for preventing dengue disease and yellow fever

The present invention relates in part to methods of preventing yellow fever as well as dengue disease in a subject. Accordingly, in certain embodiments, the present invention relates to a method of preventing dengue disease in a subject, said method comprising administering to said subject a reconstituted unit dose of the present invention as described herein, wherein: The method further comprises preventing yellow fever in said subject by concomitant administration to said subject of a yellow fever vaccine, particularly YF-17D.

The present invention relates in part to methods of preventing yellow fever as well as dengue disease in a population of subjects. Accordingly, in certain embodiments, the present invention relates to a method of preventing dengue disease in a subject population, said method comprising administering to said subject population a reconstituted unit dose of the present invention as described herein, wherein the method further comprises preventing yellow fever in said subject population by concomitant administration to said subject population of a yellow fever vaccine, in particular YF-17D.

In certain embodiments, the present invention relates to the above method wherein a unit dose of the present invention as described herein and a yellow fever vaccine, in particular YF-17D, are administered simultaneously. In some of these embodiments, the simultaneous administration is administered on Day 0 or on Day 90, preferably on Day 0. In another embodiment, a unit dose of the present invention as described herein and a yellow fever vaccine, in particular F-17D, are administered sequentially, eg, wherein said yellow fever vaccine is administered prior to a unit dose of a dengue vaccine as described herein or Thereafter, eg, within about 6 weeks, or eg, within about 4 weeks, or eg, within about 2 weeks or eg, within about 1 week.

In certain embodiments, the invention relates to the above method, wherein the reconstituted unit dose of the invention as described herein is administered and the yellow fever vaccine, in particular YF-17D, is administered by simultaneous injection. According to some embodiments, the subcutaneous injection is administered to the arm, preferably to the deltoid region of the arm. According to some of these embodiments, a unit dose of the invention as described herein and a subcutaneous injection of a yellow fever vaccine, in particular YF-17D, are administered to different anatomical sites, e.g., the opposite arm, especially when the vaccines are administered simultaneously. .

In certain embodiments, the invention relates to the above method in which two unit doses of the invention as described herein are administered. In some embodiments, the two unit doses of the invention as described herein are administered within at least 12 months or within 6 months or within 3 months, eg on Day 0/1 and Day 90. According to some of these embodiments a further third unit dose of the invention as described herein is administered after the second administration. Said third administration may act as a booster and may be 6 to 12 months after the first administration, eg, 12 months after the first administration, or more than 12 months after the first administration, eg, 12 months after the second administration. (1 year) or even at least 5 years after the first or second administration.

In a specific embodiment, the present invention relates to the above method wherein two reconstituted unit doses of the invention as described herein and one dose of a yellow fever vaccine, in particular YF-17D, are administered in particular according to the following schedule:

- administration of the yellow fever vaccine on day 0,

- a first administration of a first reconstituted unit dose after administration of said yellow fever vaccine, for example after 3 months and preferably on day 90, and

- a second administration of a second reconstituted unit dose after said first administration of the reconstituted unit dose, for example after 3 months and preferably at day 180.

In a specific embodiment, the present invention relates to the above method wherein two reconstituted unit doses of the invention as described herein and one dose of a yellow fever vaccine, in particular YF-17D, are administered in particular according to the following schedule:

- a first administration of a first reconstituted unit dose on day 0,

- a second administration of a second reconstituted unit dose after said first administration of the reconstituted unit dose, for example after 3 months and preferably on day 90,

- administration of said yellow fever vaccine after said second administration of a reconstituted unit dose, for example after 3 months and preferably on day 180.

In a specific embodiment, the present invention relates to the above method wherein two reconstituted unit doses of the invention as described herein and one dose of a yellow fever vaccine, in particular YF-17D, are administered in particular according to the following schedule:

- simultaneous administration of the first reconstituted unit dose and said yellow fever vaccine on day 0, and

- a second administration of a second reconstituted unit dose after said simultaneous administration, for example after 3 months and preferably on day 90.

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

In certain embodiments, the invention relates to the above method, wherein the subject or population of subjects is seronegative for all dengue serotypes. In a specific embodiment, the present invention provides that a unit dose of the present invention as described herein is administered to a subject or population of subjects, wherein the yellow fever vaccine, in particular the YF-17D vaccine, is administered subcutaneously to the subject or population of subjects, and wherein the subject or population of subjects is administered subcutaneously. seronegative for all these dengue fever serotypes. In other embodiments, the subject or population of subjects is seropositive for at least one dengue serotype.

In certain embodiments, the invention relates to a method, wherein a unit dose of the invention as described herein and a yellow fever vaccine, in particular YF-17D, are administered to a subject or population of subjects from an area endemic to dengue. In certain embodiments, the reconstituted unit dose of the invention as described herein and the yellow fever vaccine, in particular YF-17D, are administered subcutaneously to a subject or population of subjects from an area endemic to dengue. In other embodiments, the subject or population of subjects is from a non-endemic area of dengue. Said subject population or said subject may be vaccinated according to the present invention in connection with travel to dengue fever endemic areas and yellow fever endemic areas.

In a specific embodiment, the present invention provides that the reconstituted unit dose of the present invention as described herein and the reconstituted unit dose of the yellow fever vaccine, in particular YF-17D, are greater than 17 years of age, or greater than 18 years of age or between 18 and 60 years of age in subjects or subcutaneously administered to a population of subjects. In a further embodiment, the subject or population of subjects is an adult greater than 21 years of age, or between 21 and 60 years of age, or between 21 and 45 years of age. In some embodiments, the subject or population of subjects is from an area endemic to dengue. In another embodiment, the subject or population of subjects is from a dengue fever non-endemic area, preferably from a dengue fever non-endemic and yellow fever non-endemic area. According to some of these embodiments, the subject or population of subjects is seronegative for all four dengue fever serotypes.

The method should also be considered in relation to the use of a unit dose of a dengue vaccine as described herein for the method or in relation to the use of a unit dose of a dengue vaccine for the manufacture of a medicament for the method.

Methods and uses for prevention, methods and uses for vaccination against dengue fever

The present invention relates, in part, to methods of preventing dengue disease (particularly, virally ascertainable dengue fever, VCD) in a subject. Accordingly, in certain embodiments, the present invention relates to a method of preventing dengue disease in a subject, said method comprising administering to said subject a unit dose / 4 multivalent dengue virus composition, in particular a reconstituted unit dose of the present invention as described herein. including administration to

The present invention relates in part to methods of preventing dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Accordingly, in certain embodiments, the present invention relates to a method of preventing dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), said method comprising a reconstituted unit dose/4 valent dengue virus of the invention as described herein. and administering the composition to the subject.

The present invention relates to a tetravalent dengue virus composition comprising four wildly attenuated dengue virus strains to isolate serotype 1, serotype 2, serotype 3 and serotype 4 in a subject for virologically identifiable dengue disease. to a method of inoculating a virus, wherein in particular the tetravalent dengue virus composition comprises a chimeric dengue fever serotype 2/1 strain and a dengue fever serotype 2 strain and a chimeric dengue fever serotype 2/3 strain and a chimeric dengue fever serotype 2/4 strain wherein the dengue serotype 2 strain is derived from the wild-type strain DEN-2 16681 (SEQ ID NO: 11) and is at least 3 nucleotides from the wild-type strain:

a) 5'-noncoding region (NCR)-57 (nt-57 C to T): major attenuated locus

b) NS1-53 Gly to Asp (nt-2579 G to A): a major attenuated locus and

c) NS3-250 Glu to Val (nt-5270 A to T): the major attenuation loci are different;

wherein the three chimeric dengue strains are derived from serotype 2 strains by replacing the structural proteins prM and E from serotype 2 with the corresponding structural proteins of another dengue blue-type origin to obtain the following chimeric dengue strains:

- DENV-2/1 Chimera,

- DENV-2/3 Chimera and

- DENV-2/4 Chimera.

Additional information regarding the serotypes of the tetravalent composition can be derived from the "Dengue virus strains" section above.

The tetravalent dengue virus composition for the method may be in unit dose form comprising:

(i) dengue serotype 1 at a concentration of at least 3.3 log10 pfu/0.5 ml,

(ii) dengue serotype 2 at a concentration of at least 2.7 log10 pfu/0.5 ml,

(iii) dengue serotype 3 at a concentration of at least 4.0 log10 pfu/0.5 ml,

(iv) Dengue fever serotype at a concentration of at least 4.5 log10 pfu/0.5 ml 4.

The present invention particularly relates to a method wherein said unit dose, when lyophilized and reconstituted with 0.5 ml of a pharmaceutically acceptable diluent, comprises:

(i) dengue serotype 1 at a concentration of at least 3.3 log10 pfu/0.5 ml,

(ii) dengue serotype 2 at a concentration of at least 2.7 log10 pfu/0.5 ml,

(iii) dengue serotype 3 at a concentration of at least 4.0 log10 pfu/0.5 ml,

(iv) Dengue fever serotype at a concentration of at least 4.5 log10 pfu/0.5 ml 4.

Additional information regarding tetravalent compositions or unit doses may be derived from the "Dengue Fever Vaccine Compositions" and "Unit Doses" sections above.

The present invention thus relates to a method and a corresponding use, said method comprising primary vaccination with only two administrations of a unit dose comprising the steps of:

(A) administering to the subject a first unit dose of the tetravalent dengue virus composition, and

(B) administering a second unit dose of the tetravalent dengue virus composition to the subject within 3 months of administration of the first unit dose.

According to this embodiment, administration of only two doses within 3 months is sufficient to provide effective protection against subsequent dengue infection.

The method preferably comprises a measurement versus placebo measured versus placebo in a subject population of at least 5,000 healthy subjects irrespective of serum status at baseline and ages 14 to 16 years from the first administration of the dosing schedule to 18 months after the second administration of the dosing schedule. case, provides a combination vaccine efficacy against all four serotypes in preventing virally identifiable dengue disease with a two-sided 95% confidence interval, wherein the lower binding is greater than 60%.

The method also preferably comprises a subject of at least 1,500 or at least 2,000 healthy subjects who are seronegative for all serotypes at baseline and ages 14 to 16 years by 18 months after the second administration of the dosing schedule after the second administration of the dosing schedule. Provides combination vaccine efficacy against all four serotypes in preventing virally identifiable dengue disease with a two-sided 95% confidence interval when measured versus placebo in a population, wherein lower binding over 45%.

According to a particular embodiment, the method of inoculation against a virologically identifiable dengue disease is due to dengue serotype 2 and/or is due to dengue serotype 1. This method has very high efficacy against dengue serotype 2 and dengue serotype 1 and best efficacy against dengue serotype 2.

In certain embodiments, the present invention provides serum levels at baseline and from 30 days after the second administration to 12 to 18 months (eg, at 12 months or 18 months) after the second administration at the age of 4 to 16 years. A virus having a two-sided 95% confidence interval 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) irrespective of to said method having vaccine efficacy against serotype 1 in preventing scientifically identifiable dengue disease, wherein the lower binding is greater than 25%. In certain such embodiments, the lower binding 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 population of at least 1,500 subjects is seronegative for all serotypes at baseline, and the lower binding is greater than 35%. In certain such embodiments, each of the seronegative and seropositive populations provides vaccine efficacy against serotype 1, with a two-sided 95% confidence interval, wherein the lower binding is within 10%-points.

In certain embodiments, the present invention provides serum levels at baseline and from 30 days after the second administration to 12 to 18 months (eg, at 12 months or 18 months) after the second administration at the age of 4 to 16 years. In preventing virologically identifiable 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) regardless of to the method having vaccine efficacy against serotype 1 in 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 population of at least 1,500 subjects is seronegative for all serotypes at baseline. In certain such embodiments, each of the seronegative and seropositive populations provides vaccine efficacy against serotype 1 that is within 5 percentage-points.

In certain embodiments, the present invention provides serum levels at baseline and from 30 days after the second administration to 12 to 18 months (eg, at 12 months or 18 months) after the second administration at the age of 4 to 16 years. A virus having a two-sided 95% confidence interval 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) irrespective of to said method having vaccine efficacy against serotype 2 in preventing scientifically identifiable dengue disease, wherein the lower binding is greater than 25%. In certain such embodiments, the lower binding 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 for all serotypes at baseline. In certain such embodiments, each of the seronegative and seropositive populations provides vaccine efficacy against serotype 2, with a two-sided 95% confidence interval, wherein the lower binding is within 5 percentage-points.

In certain embodiments, the present invention provides serum levels at baseline and from 30 days after the second administration to 12 to 18 months (eg, at 12 months or 18 months) after the second administration at the age of 4 to 16 years. In preventing virologically identifiable 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) regardless of to the method having vaccine efficacy against serotype 2 in 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 population of at least 1,500 subjects is seronegative for all serotypes at baseline. In certain such embodiments, each of the seronegative and seropositive populations provides vaccine efficacy against serotype 2 that is within 5 percentage-points.

The efficacy of the method is further described in more detail below in the section above.

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

According to one embodiment, the method does not comprise a step of determining whether the subject has prior dengue infection prior to administration of the unit dose or whether the subject's serum status is not known prior to administration of the unit dose.

According to one embodiment, the method preferably comprises prior to, during or after the administering step, the determining step of a previous dengue infection in the subject or the serum status of the subject at any time after the administering step, preferably before, during the administering step, It does not include an unknown determination step after the administering step.

The method according to the invention does not require testing of serum status prior to vaccination and thus allows immediate treatment and disease control. According to a particular embodiment, the use is for a method in which a subject is exposed to an outbreak of dengue fever. In certain such embodiments, the pathogenesis is due to dengue serotype 2 and/or due to serotype 1.

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

According to one embodiment of the method, the subject is seronegative at baseline and originates from or regionally from an area where the prevalence of serotype is high with respect to serotype 1 and/or serotype 2, i.e., greater than 80%, or 90%. travel

According to this embodiment, the vaccine and corresponding methods are safe for seronegative and seropositive and thus do not require analysis of serostats or determination of previous dengue infection in the area or determination of high seroprevalence. The method preferably comprises a subject population of at least 5,000 healthy 4 to 16 years of age subjects irrespective of serum status at baseline, preferably at baseline, from the first administration of the dosing schedule to 12 to 18 months after the second administration of the dosing schedule. Provides combined vaccine efficacy against virologically confirmed dengue with hospitalization for all four serotypes with a two-sided 95% confidence interval when measured on placebo in at least 1,500 healthy 4 to 16-year-old seronegative subjects, , where the lower binding is greater than 65%. Preferably, when comparing seropositive and seronegative subjects, the two-sided 95% confidence interval of combined vaccine efficacy against virologically confirmed dengue with hospitalization for all four serotypes is within 10 percentage points or 15% It gives a lower combination of two-sided confidence intervals that are within points or within 20 percentage points. The method may thus preferably be used in an outbreak situation due to serotype 1 and/or serotype 2 or very high seroprevalence rates due to serotype 1 and/or serotype 2 or seronegative subjects (eg travelers) or serotype 1 and/or serotype 2 It is safe with respect to serotype 1 and serotype 2 even for subjects with unknown serostat in regions with (>80%).

The safety of this method is further described in more detail in the section "Methods of prophylaxis, methods of inoculation".

According to one embodiment, the method does not include active monitoring with respect to the subject's febrile illness after administration of the first and second unit doses. During active monitoring, any subject with fever (defined as a fever >38° C. on any 2 of 3 consecutive days) is required to return to the site for dengue evaluation by the investigator. The subject/caregiver ensures conclusive confirmation of the fever by contacting at least weekly to remind the subject/caregiver of their obligation to return to the scene in case of fever. The contact is conducted via an appropriate method (eg, phone call, text message, home visit, school-based monitoring), which may be different at each test site.

According to one embodiment, the method does not comprise a vaccine immunogenicity assay comprising GMT against dengue neutralizing antibodies.

According to one embodiment, the method does not comprise a reactogenicity assay. These reactogenicity assays included attracted local AEs (injection site pain, injection site erythema and injection site swelling) and attracted systemic AEs (children <6 years of age: fever, irritability/disturbing, drowsiness and loss of appetite; children 6 years of age and older: asthenia , fever, headache, malaise and myalgia), which is assessed for 7 and 14 days, respectively, after each vaccination (including the day of inoculation), for example, through diary card collection.

According to one embodiment, the method does not include active monitoring, immunogenicity assays and reactogenicity assays.

According to this embodiment, the vaccine and the corresponding inoculation method are safe and thus do not require additional monitoring or analysis steps.

In view of the above, the method according to one embodiment comprises a primary vaccination comprising the steps of:

(A) selecting a subject for administration of a unit dose of a tetravalent dengue virus composition in need of protection against dengue infection without prior determination of dengue infection, and

(B) administering to the subject a first unit dose of the tetravalent dengue virus composition, and

(C) administering a second unit dose of the tetravalent dengue virus composition to the subject within 3 months of administration of the first unit dose.

Thus, the inoculation method is established without prior determination of dengue infection. The method further comprises at least one year after administration of a second unit dose, optionally a booster dose of the unit dose.

The selection of a subject may include all types of considerations, but preferably does not include a determination of a previous dengue infection. The selection may include consideration of age, health conditions and infection threat. Infection threats include consideration of seroprevalence rates, serotype-specific seroprevalence rates, and outbreak situations or serotype-specific outbreak situations in areas where the subject is intended to normally live or travel. Subjects may be selected due to their exposure to serotype 1 and/or serotype 2 or due to the fact that they require protection against a specific dengue serotype, ie, serotype 1 and/or serotype 2.

According to the present invention, the method can be applied to subjects of all types of age. According to one embodiment, the subject is less than 9 years old, or 4 to 5 years old, or 6 to 11 years old or 12 to 16 years old, or 6 to 16 years old or 4 to 16 years old, or 2 to 17 years old. age or age 9 or over 9 years of age, or between 9 and 17 years of age, or between 18 and 45 years of age, or between 46 and 60 years of age, or over 60 years of age.

In particular, the present invention relates to said method which is safe.

In particular, the present invention relates to when measured versus placebo in a subject population of at least 5,000 healthy 4 to 16 years of age subjects irrespective of serum status at baseline from the first dose of the dosing schedule to 12 to 18 months after the last dose of the dosing schedule. A method of providing combined vaccine efficacy against virologically confirmed dengue fever with hospitalization for all four serotypes with a two-sided 95% confidence interval, wherein the lower binding is greater than 65%.

In particular, the present invention relates to said method effective.

In particular, the present invention relates to when measured versus placebo in a subject population of at least 5,000 healthy subjects irrespective of serum status at baseline and ages 14 to 16 years from the first administration of the dosing schedule to 18 months after the last administration of the dosing schedule. , to a method of providing combination vaccine efficacy against all four serotypes in preventing virally identifiable dengue disease with a two-sided 95% confidence interval, wherein the lower binding is greater than 60% .

In certain embodiments, the invention relates to the method, wherein the subject is seronegative for all dengue serotypes.

The present invention relates, in part, to methods of preventing dengue disease (particularly virally identifiable dengue fever, VCD) in a population of subjects. Accordingly, in certain embodiments, the present invention relates to a method for preventing dengue disease in a subject population, said method comprising administering to said subject population a unit dose, in particular a reconstituted unit dose of the present invention as described herein. include

The present invention relates in part to a method of preventing dengue disease (particularly virally ascertainable dengue fever, VCD) in a population of subjects, said method comprising administering at least a first reconstituted unit dose of the present invention as described herein; administering to said population of subjects, wherein the specified proportion of geometric mean neutralizing antibody titers (GMTs) is achieved at 180 days or 365 days after administration of said first unit dose to said subjects. According to some embodiments, the geometric mean neutralizing antibody titer to dengue serotype 2 (GMT DENV-2) and the geometric mean neutralizing antibody titer to dengue serotype 4 (GMT DENV-4) are of the invention as described herein. at least 40 people, or at least at 180 days or 365 days after at least a first administration of the reconstituted unit dose and optionally after a second administration of a reconstituted unit dose of the invention as described herein, and at 90 days after said first administration A ratio of GMT DENV-2:GMT DENV-4 of 50 or less or 40 or less or 30 or less or 20 or less when tested in 50 or at least 60 subjects is provided. 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 at 180 days or 365 days after administration of said first reconstituted unit dose and/or GMT DENV-2 The ratio of :GMT DENV-3 is 20 or less, or 18 or less, or 15 or less at 180 days or 365 days after administration of said first reconstituted unit dose.

The present invention relates in part to a method for preventing dengue disease (particularly virally ascertainable dengue fever, VCD) in a subject, said method comprising administering at least a first reconstituted unit dose of the invention as described herein; administering to a population of subjects, wherein a specified proportion of neutralizing antibody titers is achieved at 180 days or 365 days after administration of said first unit dose to said subjects. According to some embodiments, after at least a first administration of a reconstituted unit dose of the invention as described herein and optionally 90 days after said first administration, 180 days after a second administration of a reconstituted unit dose of the invention as described herein Neutralizing antibody titers to dengue serotype 2 and neutralizing antibody titers to dengue serotype 4 at day or 365 days are 50 or less, or 40 or less, or 30 or less, or 20 or less neutralizing antibody to DENV-2:GMT DENV It gives the ratio of neutralizing antibody to -4. In some of these embodiments, the ratio of neutralizing antibody titers of DENV-2:DENV-1 is 20 or less, or 18 or less or 15 or less, and/or DENV- The ratio of the neutralizing antibody titers of 2:DENV-3 is no more than 20, or no more than 18, or no more than 15 at 180 days or 365 days after administration of said first reconstituted unit dose.

The geometric mean neutralizing antibody titer (GMT) of a subject population or neutralizing antibody titer of a subject is determined according to the microneutralization test described herein, eg, according to the method described in Example 2. Without wishing to be bound by any theory, the invention as described herein with respect to small differences between the current geometric mean neutralizing antibody titers (GMT) for the four dengue serotypes or the neutralizing antibody titers for the four dengue serotypes It is understood that a method of inducing a more balanced immune response resulting from administration of a reconstituted unit dose of In particular, it is understood that a greater response to any one of the four serotypes, for example to DENV-2, is less favorable compared to the other serotypes.

The present invention relates in part to said method for preventing dengue disease (particularly virologically identifiable dengue, VCD) in a subject or population of subjects, said method comprising two unit doses subcutaneously on days 1 and 90 provide a seropositive rate in a subject population of at least 50 subjects comprising administration of 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., 30 days, and/or at least one of the subject population 80% are seropositive for all four dengue serotypes, eg, before or at the time of administration of the second unit dose on Day 90, and/or at least 80%, or at least 85% of the subject population, or at least 90%, or at least 95%, are seropositive for all four dengue serotypes, eg, at day 120 after administration of the second unit dose, and/or at least 80%, or at least 85%, or at least of the subject population 90% are seropositive for all four dengue serotypes, eg, after administration of the second unit dose on day 270.

The present invention relates in part to said method for preventing dengue disease (particularly virally identifiable dengue, VCD) in a subject or population of subjects, said method comprising 2 units subcutaneously on day 1 and day 90 providing a seropositive rate in a subject population of at least 100 subjects comprising administration of a dose, wherein the subjects in the subject population have 20% to 40% of subjects seronegative for all dengue serotypes and at least one dengue sera at baseline seropositive rates for all four dengue serotypes in the seronegative portion of the subject population and all 4 in the seropositive portion of the subject population at Days 120 and/or 270, including between 60% and 80% of subjects seropositive for the type The seropositive rates for dengue serotypes in dogs did not deviate by more than 10%-points and/or at Day 120, the seropositive rates for all 4 dengue serotypes in the seronegative portion of the subject population and all 4 in the seropositive portion of the subject population. Seropositive rates for dengue serotypes in dogs do not deviate by more than 5 percentage points.

The present invention relates, in part, to a method for preventing a virologically identifiable dengue disease in a subject or population of subjects, said method comprising four wildly attenuated dengue serotypes, in particular virus strains as described herein. and administering to the subject or population of subjects a reconstituted unit dose of a tetravalent dengue virus composition.

The present invention relates in part to a method for preventing a virologically identifiable dengue disease with hospitalization in a subject or population of subjects, said method comprising four herbal, attenuated dengue serotypes, in particular a virus strain as described herein. administering to the subject or population of subjects a reconstituted unit dose of a tetravalent dengue virus composition comprising

In certain embodiments, the method comprises a reconstituted unit dose/4 valent 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 4 herbally attenuated A tetravalent virus composition comprising a dengue virus strain, wherein the unit dose is lyophilized and upon reconstitution with 0.5 ml of a pharmaceutically acceptable diluent, a reconstituted unit dose comprising:

(i) dengue serotype 1, such as chimeric dengue serotype 2/1 strain, at a concentration of at least 3.3 log10 pfu/0.5 ml;

(ii) dengue serotype 2, such as the dengue serotype 2 strain at a concentration of at least 2.7 log10 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 log10 pfu/0.5 ml, and

(iv) a dengue serotype 4. such as a chimeric dengue serotype 2/4 strain at a concentration of at least 4.5 log10 pfu/0.5 ml.

Preferably, the reconstituted unit dose/4-valent dengue virus composition is used in the method for preventing dengue fever of the present invention, and upon reconstitution of the unit dose using a pharmaceutically acceptable diluent, (i), (ii), ( iii), and (iv) provide a total concentration of pfu/0.5 mL, and based on the total concentration, the concentration of (ii) of pfu/0.5 mL is less than 2%, and pfu/0.5 mL of (iv) wherein the concentration of is at least 50%, the concentration of (i) in pfu/0.5 mL is at least 1%, and the concentration of (iii) in pfu/0.5 mL is at least 6%, and wherein the subject or population of subjects is between 18 and 60 years of age. is the age

In another preferred embodiment, the reconstituted unit dose/4-valent dengue virus composition is used in the method for preventing dengue disease of the present invention, upon reconstitution with a pharmaceutically acceptable diluent, (i), (ii), ( iii), and (iv) provide a total concentration of pfu/0.5 mL, and based on the total concentration, the concentration of (ii) of pfu/0.5 mL is less than 10%, and pfu/0.5 mL of (iv) wherein the concentration of (i) in pfu/0.5 mL is at least 1%, the concentration of (iii) in pfu/0.5 mL is at least 8%, and wherein the subject or population of subjects is between 2 and 17 years of age. is the age

In certain embodiments, the present invention comprises a tetravalent dengue virus composition wherein said unit dose comprises four wildly attenuated dengue serotypes, in particular the virus strains described herein, said serotypes comprising: It has the specific concentrations described herein with respect to dose:

(i) Dengue fever serotype 1 (eg, chimeric dengue serotype 2/1 strain) has a concentration of 3.3 log10 pfu/dose to 5.0 log10 pfu dose or 3.3 log10 pfu/0.5 mL to 5.0 log10 pfu/0.5 mL .

(ii) dengue fever serotype 2 (eg, chimeric dengue serotype 2 strain) has a concentration of 2.7 log 10 pfu/dose to 4.9 log 10 pfu/0.5 dose or 2.7 log 10 pfu/0.5 ml to 4.9 log 10 pfu/0.5 ml .

(iii) Dengue serotype 3 (eg, chimeric dengue serotype 2/3 strain) has a concentration of 4.0 log10 pfu/dose to 5.7 log10 pfu/0.5 dose or 4.0 log10 pfu/0.5 mL to 5.7 log10 pfu/0.5 mL has

(iv) dengue fever serotype 4 (eg, chimeric dengue serotype 2/4 strain) at a concentration of 4.5 log 10 pfu/dose to 5.5 log 10 pfu/0.5 dose or 4.5 log 10 pfu/0.5 mL to 5.5 log 10 pfu/0.5 mL has

In this preferred embodiment, the subject or population of subjects is between 2 and 17 years of age, eg between 4 and 16 years of age, preferably under 9 years of age. In other preferred embodiments, the subject or population of subjects is 4-5 years old, 6-11 years old or 12-16 years old.

In certain embodiments, the present invention provides that, upon reconstitution with 0.5 mL of a pharmaceutically acceptable diluent, the unit dose is from 3.3 log10 pfu/0.5 mL to dengue serum 1 (eg, chimeric dengue serotype 2/1 strain). a concentration of 3.6 log10 pfu/0.5 mL, a concentration of 2.7 log10 pfu/0.5 mL to 4.0 log10 pfu/0.5 mL for dengue serotype 2 (eg, dengue serotype 2 strain), a concentration of dengue serotype 3 (eg, dengue serotype 2 strain) , a concentration of 4.0 log10 pfu/0.5 mL to 4.6 log10 pfu/0.5 mL for chimeric dengue serotype 2/3 strain) and 4.5 log10 for dengue serotype 4 (eg, chimeric dengue serotype 2/4 strain) pfu/0.5 mL or a concentration of 4.6 log 10 pfu/0.5 mL to 5.1 log 10 pfu/0.5 mL. In this preferred embodiment, the subject or population of subjects is between 2 and 17 years of age, eg between 4 and 16 years of age, preferably under 9 years of age. In other preferred embodiments, the subject or population of subjects is 4-5 years old, 6-11 years old or 12-16 years old.

In certain embodiments, the present invention provides that the concentration of dengue serotype 1 (eg, chimeric dengue serotype 2/1 strain) measured in pfu/0.5 mL is 1% to 7% of the total concentration, and pfu/0.5 mL The concentration of dengue serotype 2 (eg, dengue serotype 2 strain) measured as The measured concentration of dengue serotype 3 (eg, chimeric dengue serotype 2/3 strain) is at least 10% of the total concentration, and the measured concentration of dengue serotype 4 (eg, chimeric dengue serotype) in pfu/0.5 mL The concentration of the type 2/4 variant) is at least 65% of the total concentration, for example in the range of 65% to 80%. In certain said embodiments, the arithmetic sum of all four serotypes is in the range of 4.6 log 10 pfu/0.5 mL to 6.7 log 10 pfu/0.5 mL, preferably in the range of 4.6 log 10 pfu/0.5 mL to 5.5 log 10 pfu/0.5 mL there is. Preferably, in this embodiment, the subject or population of subjects is between 2 and 17 years of age, eg between 4 and 16 years of age, and even more preferably under 9 years of age. In other preferred embodiments, the subject or population of subjects is 4-5 years old, 6-11 years old or 12-16 years old.

In a further preferred embodiment, the present invention relates to dengue fever serotype 1 (eg chimeric dengue serotype 2/1 strain), eg TDV-1 and dengue serotype 2 (eg dengue serotype 2) variants), for example, TDV-2 is present in respective concentrations based on a total concentration of each pfu/0.5 mL, which are within 5%-points of each other and/or together about 10 of a total concentration of pfu/0.5 mL. % or less. In certain of the above embodiments, dengue fever serotype 3 (eg chimeric dengue serotype 2/3 strain), eg TDV-3, is preferably at least about 10% of the total concentration of pfu/0.5 mL, and more Preferably the dengue fever serotype 4 (eg chimeric dengue serotype 2/4 strain), eg TDV-4, is at least about 70% of the total concentration of pfu/0.5 mL. In certain of the above embodiments, dengue fever serotype 4 (eg chimeric dengue serotype 2/4 species) eg TDV-4 exhibits the highest concentration in the composition of all four serotypes, preferably pfu/ At least about 70% of the total concentration of 0.5 mL, dengue serotype 3 (eg, chimeric dengue serotype 2/3 strain), eg, TDV-3, is the second highest in the composition of all four serotypes. concentration, preferably at least about 10% of the total concentration of pfu/0.5 mL, dengue serotype 1 (eg, chimeric dengue serotype 2/1 strain), such as TDV-1 and dengue serotype 2 (eg, dengue serotype 2 strain), eg, TDV-2, each has a higher concentration of serotype 3 (eg, chimeric dengue serotype 2/3 strain), eg, TDV-3 low concentrations, optionally together representing less than about 10% of the total concentration of pfu/0.5 mL.

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 It 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, and 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 and 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.

In certain embodiments, the invention relates to the above method, wherein the reconstituted unit dose of the invention as described herein is administered by subcutaneous injection. According to some of these embodiments, the subcutaneous injection is administered to the arm, preferably to the deltoid region of the arm.

In certain embodiments, the present invention provides that said reconstituted unit dose is administered to a subject of an unknown serostate and/or which test is performed to determine whether the subject is seropositive or seronegative (before) the unit dose as described herein is administered. was not performed to determine the above method. In certain embodiments, the present invention relates to the method above, which does not include the step of determining a prior dengue infection in the subject or subjects. In certain embodiments, the present invention relates to said method comprising no analysis of seroprevalence in said region or being carried out in an area having a seroprevalence rate of less than 80%, less than 70% or less than 60%. In certain embodiments, the invention relates to a method wherein the subject's serum status is unknown. In this embodiment, the serum status is not determined at any time before and after administration in connection with the method. In a specific embodiment of the present invention, the method is used in an outbreak situation. In certain embodiments, the present invention relates to said method being performed outside of a clinical trial.

In certain embodiments, the present invention relates to the above method, wherein the subject or population of subjects is seronegative for all dengue serotypes.

In certain embodiments, the invention relates to the above method in which two unit doses of the invention as 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, optionally at least 4 weeks apart, for example, on days 0 and 90 or on days 1 and 90. is administered as According to some of these embodiments, a further third unit dose of the invention as described herein is administered after the second administration. Said third administration may act as a booster and may be 6 to 12 months after the first administration, eg, 12 months after the first administration, or more than 12 months after the first administration, eg, 12 months after the second administration. (1 year) or even more than 5 years after the first or second administration.

In certain embodiments, the methods of the invention comprise or consist of a single unit dose of the invention being administered.

In certain embodiments, the invention relates to a method, wherein said reconstituted unit dose of the invention is administered subcutaneously to a subject or population of subjects that are seronegative for all dengue serotypes. In other embodiments, the subject or population of subjects is seropositive for at least one dengue serotype.

In certain embodiments, the invention relates to a method, wherein a unit dose of the invention as described herein is administered to a subject or population of subjects from an area endemic to dengue. 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 from Singapore, the Dominican Republic, Panama or the Philippines. In a preferred embodiment, the subject or population of subjects is from Asia Pacific or Latin America. In some other of these 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 non-endemic area of dengue. Said subject population or said subject may be vaccinated according to the present invention with a dengue endemic area. In certain embodiments, a reconstituted unit dose of the invention as described herein is administered subcutaneously to a subject or population of subjects from an area endemic to dengue or non-endemic to dengue.

In certain embodiments, the invention relates to the above method, wherein a reconstituted unit dose of the invention as described herein is administered subcutaneously to a subject or population of subjects between the ages of 2 to 60 years. In some embodiments, the subject or population of subjects is an adult greater than 17 years of age, or greater than 18 years of age, or between 18 and 60 years of age. In a further specific embodiment, the subject or population of subjects is an adult greater than 21 years of age, or between 21 and 60 years of age, or between 21 and 45 years of age.

In certain embodiments, the invention relates to the above method, wherein the reconstituted unit dose of the invention as described herein is administered subcutaneously to a child or adolescent between the ages of 2 to 17 years. In some embodiments, the subject or population of subjects is less than 9 years of age or less than 4 years of age. In some embodiments, the subject or population of subjects 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 population of subjects is between 4 and 16 years of age. In some such embodiments, the subject or population of subjects is 4-5 years old, 6-11 years old, or 12-16 years old. Optionally, the subject or population of subjects is seronegative for all dengue serotypes.

In a specific embodiment, the present invention relates to a pediatric subject or pediatric subject, wherein the unit dose of the invention as described herein is less than 2 years of age, preferably between 2 months and 2 years or between 2 months and 1.5 years or between 2 months and 1 years. to said method of administering to a population. According to some of these embodiments, the pediatric subject or population of pediatric subjects is seronegative and from an area endemic to dengue.

In certain embodiments, the present invention provides that the reconstituted unit dose of the present invention as described herein is administered to a pediatric subject of less than 2 years of age, preferably between 2 months and 2 years or between 2 months and 1.5 years or between 2 months and 1 year, or to a population of pediatric subjects, preferably by subcutaneous injection. According to some of these embodiments, the pediatric subject or population of pediatric subjects is seronegative and from an area endemic to dengue.

In certain embodiments, the invention relates to said method, wherein said subject or population of subjects is 4 to 5 years old from Asia Pacific, 6 to 11 years old from Asia Pacific or 12 to 16 years old from Asia Pacific. In other embodiments, the subject or population of subjects is 4 to 5 years old from Latin America, 6 to 11 years old from Latin America or 12 to 16 years old from Latin America.

In certain embodiments, the invention provides that said subject or population of subjects is 4 to 5 years of age and seropositive for at least one dengue serotype, or is 6 to 11 years of age and seropositive for dengue serotype at least 1 month, or 12 wherein said method is between the ages of 16 and 16 years and is seropositive for at least one dengue serotype. In other embodiments, the subject or population of subjects is 4 to 5 years old and seronegative for all dengue serotypes, 6 to 11 years old and seronegative for all dengue serotypes, 12 to 16 years of age and all It is seronegative for the dengue fever serotype.

In certain embodiments, the invention relates to the above method, wherein the subject or population of subjects is from Asia Pacific or Latin America and is seropositive for at least one dengue serotype at baseline. In other embodiments, the subject or population of subjects is from Asia Pacific or Latin America and is seronegative for all dengue serotypes at baseline.

In certain embodiments, the present invention provides a method wherein the subject or population of subjects is from Asia Pacific and is seropositive for at least one dengue serotype at baseline and at the age of 4 to 5 years, 6 to 11 years of age, or 12 to 16 years of age. is about In other embodiments, the subject or population of subjects is from Asia Pacific and is seronegative for all dengue serotypes at baseline and at age 4-5, age 6-11, or age 12-16. In another embodiment, the subject or population of subjects is from Latin America and is seropositive for at least one dengue serotype at baseline and at age 4-5, age 6-11, or age 12-16. In another embodiment, the invention provides that the subject or population of subjects is from the Americas and is seronegative for all dengue serotypes at baseline and at age 4-5, age 6-11, or age 12-16.

In certain embodiments, the present invention relates to the above method, wherein the subject or population of subjects had previously been vaccinated against yellow fever. In other embodiments, the subject or population of subjects has previously been vaccinated against Japanese encephalitis. In another embodiment, the subject or population of subjects has not had prior vaccination against yellow fever. In other embodiments, the subject or population of subjects has not had prior vaccination against Japanese encephalitis. Prior vaccination refers to vaccination with a reconstituted unit dose as described herein, eg within 4 months after the first dose, and 30 days after the second dose. For example, for vaccine efficacy (VE) determined in Example 6 from 30 days after the second vaccination, a prior vaccination of yellow fever is defined as a yellow fever vaccination that occurs 30 days before the second vaccination. In certain embodiments, the subject or population of subjects received Dengvaxia® within 4.5 years after administration of the first dose or within a dosing regimen as described herein.

In particular, disproportionate titers of neutralizing antibodies to the four dengue serotypes are observed in seronegative populations or subjects following administration of a commercially available dengue vaccine. The present invention particularly shows that seronegative subjects exhibit a more balanced immune response against the four dengue serotypes after administration of the reconstituted unit dose of the present invention as described herein. Accordingly, it is contemplated that the unit doses of the invention as described herein and the methods of the invention as described herein may provide a stronger immune response in subjects, including both seropositive and seronegative subjects. Such a balanced response and balanced efficacy and safety are required to enable vaccination without prior serologic status analysis, which is a major advantage in vaccination programs and particularly in outbreak situations.

The present invention relates in part to a method for preventing a virologically identifiable dengue disease in a subject, said method comprising four strains of dengue virus exhibiting serotype 1, serotype 2, serotype 3 and serotype 4 administering to the subject a tetravalent dengue virus composition, wherein the strain of virus is optionally a wild-attenuated strain of dengue virus.

The present invention relates in part to a method for preventing a virologically identifiable dengue disease in a subject, said method comprising four strains of dengue virus exhibiting serotype 1, serotype 2, serotype 3 and serotype 4 comprising administering to said subject a tetravalent dengue virus composition, wherein said strain of virus is optionally a wild-attenuated strain of dengue virus.

In certain embodiments, the present invention relates to a method wherein there is no step of determining the serum status of the subject at baseline and in other words, the method does not include determining a prior dengue infection of the subject prior to administration of a tetravalent dengue virus composition. . In particular, the method is safe and effective. Thus, in certain of the above embodiments, the subject has not been tested for the presence of a previous dengue fever infection.

In certain embodiments, the present invention relates to said method wherein administration is safe regardless of whether the subject has a determination prior to administration of the tetravalent dengue virus composition whether the subject has a previous dengue infection. In particular, the method is also effective.

In certain embodiments, the present invention relates to said method, wherein said method is safe and/or effective.

In certain embodiments, the present invention relates to the above method, wherein the composition comprises at least one chimeric dengue virus. In certain such embodiments, the present invention provides that the composition comprises at least one non-chimeric dengue virus and at least one chimeric dengue virus, in particular chimeric dengue serotype 2/1 strain and dengue fever serotype 2 strain and chimeric dengue serotype 2/3 Variants and chimeric dengue serotype 2/4 strains. Details of the composition are described above.

Accordingly, in certain embodiments, the present invention relates to a subject population of at least 5,000 healthy subjects (or at least 10,000 or at least 15,000 healthy subjects) irrespective of serum status at baseline and, e.g., at 14 to 16 years of age, in a subject population. to the method having vaccine efficacy in preventing virally identifiable dengue disease with a two-sided 95% confidence interval when measured against placebo, wherein the lower binding is greater than 25% and as described herein The same reconstituted unit dose/4 valent dengue virus composition or placebo is administered, e.g., less than 6 months after the first administration, e.g. within 3 months, or at least 12 to 18 months after the second/last administration (e.g. , at 12 months or 18 months) at least twice 30 days after the second/last dose. In an embodiment, the invention provides virology with a two-sided 95% confidence interval 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) irrespective of serum status at baseline. to the above method having vaccine efficacy, preferably combined vaccine efficacy, against all four serotypes in preventing identifiable dengue disease, the lower binding is greater than 25%, and the reconstituted unit as described herein The dose or tetravalent dengue virus composition or placebo is administered at least once, up to 15 to 21 months (eg, 15 or 21 months) after the first administration of the dosing schedule. In certain such embodiments, the lower binding 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 said reconstituted unit dose or placebo is administered simultaneously within about 3 months, eg, on days 0 and 90.

Accordingly, in certain embodiments, the present invention provides a measure 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) irrespective of serum status at baseline and at ages 4 to 16 years of age. to the method of having combined vaccine efficacy against all four serotypes in preventing virally identifiable dengue disease with a two-sided 95% confidence interval when provided, wherein the lower binding is greater than 60% and , the reconstituted unit dose/4 dose dengue virus composition or placebo as described herein is administered at least twice, e.g., within 6 months, e.g., within 3 months of the first administration, until 18 months after the last administration . In these embodiments, lower binding is, for example, greater than 62%, greater than 64%, greater than 66%, greater than 68%, or greater than 69%.

In certain embodiments the present invention provides for placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000 or at least 15,000 healthy subjects) irrespective of serum status at baseline and, for example, at age 14-16 years to the method having, when measured, vaccinia efficacy against all four serotypes, preferably combined vaccine efficacy, in preventing more than 30% of virologically identifiable dengue disease, wherein If the reconstituted unit dose/4 valent dengue virus composition is less than 6 months, e.g., within 3 months after the first administration or by at least 12 months or 12 to 18 months after the second administration/last administration (e.g., 12 month or 18 months) at least twice 30 days after the second dose/last dose. In certain embodiments, the present invention provides greater than 30% virologically confirmed, when measured versus placebo, in a subject population of at least 5,000 healthy subjects (or at least 10,000 or at least 15,000 healthy subjects) irrespective of serum status at baseline It relates to the above method having vaccine efficacy, preferably combined vaccine efficacy, against all four serotypes in preventing possible dengue disease, wherein a reconstituted unit dose as described herein or a tetravalent dengue virus composition or placebo is administered. At least once up to 15 months after the first administration of the schedule. 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 said reconstituted unit dose or placebo is administered subcutaneously within about 3 months, eg, on days 0 and 90.

Accordingly, in certain embodiments, the present invention provides a measure 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) irrespective of serum status at baseline and at ages 14-16 years of age. to the method having combined vaccine efficacy against all four serotypes in preventing greater than 66% of virally identifiable dengue disease when administered, wherein the reconstituted unit dose/4 dose as described herein is The dengue virus composition or placebo is administered at least twice, eg, within 6 months, eg, within 3 months, after the first administration, eg, up to 18 months after the last administration. In these embodiments, the vaccine efficacy is, for example, greater than 68%, greater than 70%, greater than 72%, or greater than 74%.

In certain embodiments, the invention provides a two-sided 95% confidence interval 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) irrespective of serum status at baseline. to said method having vaccine efficacy, preferably combination vaccine efficacy, in preventing virally identifiable dengue disease with hospitalization, wherein said lower binding is greater than 0% and the reconstituted unit as described herein The dose/4-valent dengue virus composition or placebo is administered at least twice in less than 6 months, eg, within 3 months, 30 days after the second administration by at least 18 months after the second administration. In certain of the above embodiments, the lower binding 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%.

In certain embodiments, the present invention provides for a two-sided 95% confidence interval in all 4 seronegative subjects when measured against placebo in a subject population of at least 1,500 or at least 2,000 healthy subjects who are seronegative for all serotypes at baseline. to the above method having combination vaccine efficacy against dengue serotypes in dogs, wherein said unit dose / 4 dengue virus composition or placebo is administered for at least 12 months or 12 to 18 months after the second administration of the dosing schedule (e.g. eg at 12 months or 18 months) at least twice less than 6 months, eg, within 3 months after the second administration of the dosing schedule, about 30 days. In certain such embodiments, the lower binding is greater than 30%, greater than 40%, greater than 50%, or greater than 55%.

In certain embodiments, the present invention relates to 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) who are seronegative for all serotypes at baseline and at ages 4 to 16 years of age. to the method having combined vaccine efficacy against all four serotypes in preventing virally identifiable dengue disease with a two-sided 95% confidence interval when measured versus placebo in a population of subjects, wherein The low binding is greater than 25%, and the reconstituted unit dose/4-valent dengue virus composition or placebo as described herein is administered, for example, up to 12 to 18 months after the last dose (eg at 12 months or 18 months). day) at least twice in less than 6 months, eg within 3 months, 30 days after the last administration. In certain such embodiments, the lower binding is greater than 30%, greater than 35%, greater than 40%, or greater than 45%.

In certain embodiments, the present invention provides two-sided 95% 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) who are seropositive at baseline and at ages 4 to 16 years old. to the method having combined vaccine efficacy against all four serotypes in preventing virally identifiable dengue disease with confidence intervals, wherein the lower binding is greater than 25% and as described herein. If the reconstituted unit dose/4-valent dengue virus composition or placebo is administered, e.g., by 12 to 18 months after last administration (eg at 12 months or 18 months) less than 6 months after last administration, e.g. , within 3 months, at least twice in 30 days. In certain such embodiments, the lower binding is greater than 40%, greater than 45%, greater than 50%, greater than 60%, or greater than 65%.

In certain embodiments, the invention provides a subject population of at least 5,000 healthy subjects (or at least 10,000 or at least 15,000 healthy subjects) who are seropositive at baseline or seronegative for all serotypes at baseline and ages 4 to 16 years of age. to the method having combined vaccine efficacy against all four serotypes in preventing virally identifiable dengue disease with a two-sided 95% confidence interval when measured against placebo in is greater than 25%, and the reconstituted unit dose / 4 dengue virus composition as described herein or placebo is administered by, for example, 12 to 18 months after the last dose (e.g., at 12 months or at 18 months). ) are administered at least twice in less than 6 months, eg within 3 months, 30 days after the last dose. In certain such embodiments, the difference between the lower binding provided by the seropositive subject at baseline and the seronegative subject for all serotypes at baseline is no more than 15%-points.

In certain embodiments, the invention provides sera of all four dengue fevers in greater than 30% of seronegative subjects when measured against placebo in a subject population of at least 1,500 or at least 2,000 healthy subjects who are seronegative for all serotypes at baseline. to the above method of having combination vaccine efficacy against a type, wherein said unit dose / 4 dengue virus composition or said placebo is at least 12 months or 12 to 18 months (e.g., 12 months) after the second administration. or at 18 months) less than 6 months, eg, within 3 months after the second administration, at least twice at about 30 days. 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%.

In certain embodiments, the invention provides for 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) who are seronegative for all serotypes at baseline and at ages 4 to 16 years of age. to the method having combined vaccine efficacy against all four serotypes in preventing virologically identifiable dengue disease when measured against placebo in a subject population of If the dose/4-valent dengue virus composition or placebo is administered, e.g., by 12 to 18 months after the last dose (e.g., at 12 months or at 18 months) less than 6 months after the last dose, e.g., 3 months within, at least twice in 30 days. 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%.

In certain embodiments, the present invention is virologically confirmed 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) who are seropositive at baseline and at ages 4 to 16 years of age. It relates to the above method having combined vaccine efficacy against all four serotypes in preventing possible dengue disease, wherein the reconstituted unit dose/4 valent dengue virus composition as described herein or placebo, for example, 12 to 18 months after the last dose (eg at 12 months or 18 months) at least twice in less than 6 months, eg within 3 months, 30 days 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%.

In certain embodiments, the invention provides a subject population of at least 5,000 healthy subjects (or at least 10,000 or at least 15,000 healthy subjects) who are seropositive at baseline or seronegative for all serotypes at baseline and ages 4 to 16 years of age. to the method having combined vaccine efficacy against all four serotypes in preventing virally identifiable dengue disease with a two-sided 95% confidence interval when measured against placebo in is greater than 25%, and the reconstituted unit dose / 4 dengue virus composition as described herein or placebo is administered by, for example, 12 to 18 months after the last dose (e.g., at 12 months or at 18 months). ) are administered at least twice in less than 6 months, eg within 3 months, 30 days after the last dose. In certain such embodiments, the difference between the lower binding provided by the seropositive subject at baseline and the seronegative subject for all serotypes at baseline is no more than 15%-points, or no more than 10%-points.

In certain embodiments, the present invention provides coverage for all four dengue fevers with a two-sided 95% confidence interval when measured against placebo at the time of randomization and at baseline and in a subject population of at least 1,000 healthy subjects irrespective of serum status at baseline. to the above method of having combination vaccine efficacy against a serotype, wherein said unit dose / 4 dengue virus composition or said placebo is administered for at least 12 months or 12 to 18 months after the second administration of the dosing schedule (e.g. eg at 12 months or 18 months) at least twice in less than 6 months, eg, within 3 months after the second administration of the dosing schedule, about 30 days. In certain such embodiments, the lower binding is greater than 30%, greater than 40%, or greater than 45%.

In certain embodiments, the present invention provides a combination vaccine for greater than 30% of all four dengue serotypes when measured versus placebo in a subject population of at least 1,000 healthy subjects irrespective of serum status at randomization and at baseline. to said method having efficacy, wherein said unit dose/4 dose dengue virus composition or said placebo is administered for at least 12 months or 12 to 18 months (eg, at 12 months or 18 months) after the second administration. ) at least twice in less than 6 months, eg, within 3 months after the second administration, at about 30 days. 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%.

In certain embodiments, the present invention provides a two-sided 95%, when measured against placebo, in a subject population of at least 1,000 healthy subjects ages 6-11 years of age, regardless of serum status at randomization and at baseline and at baseline, as measured against placebo. to said method having combination vaccine efficacy against all four dengue serotypes with confidence intervals, wherein said unit dose / 4 dengue virus composition or said placebo is at least 12 months or 12 months after the second administration of the dosing schedule to 18 months (eg, 12 months or 18 months) less than 6 months, eg, within 3 months after the second administration of the dosing schedule, at least twice at about 30 days. In certain such embodiments, the lower binding is greater than 30%, greater than 40%, greater than 50%, greater than 60%, or greater than 70%.

In certain embodiments, the present invention provides sera of all six dengue fever greater than 30% when measured versus placebo in a subject population of at least 1,000 healthy subjects 6-11 years of age, irrespective of serum status at randomization and at baseline. to the above method of having combination vaccine efficacy against a type, wherein said unit dose / 4 dengue virus composition or said placebo is at least 12 months or 12 to 18 months (e.g., 12 months) after the second administration. or at 18 months) less than 6 months, eg, within 3 months after the second administration, at least twice at about 30 days. 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%.

In certain embodiments, the present invention provides two-sided 95%, when measured versus placebo, in a subject population of at least 1,000 healthy subjects aged 12 to 16 years of age at the time of randomization and at baseline and irrespective of serum status at baseline. to said method having combination vaccine efficacy against all four dengue serotypes with confidence intervals, wherein said unit dose / 4 dengue virus composition or said placebo is at least 12 months or 12 months after the second administration of the dosing schedule to 18 months (eg, 12 months or 18 months) less than 6 months, eg, within 3 months after the second administration of the dosing schedule, at least twice at about 30 days. In certain such embodiments, the lower binding is greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 65%, or greater than 68%.

In certain embodiments, the present invention provides for greater than 30% of all 4 > 30% when measured versus placebo in a subject population of at least 1,000 healthy subjects aged 12 to 16 years of age irrespective of serum status at randomization and at baseline. to the method of having combination vaccine efficacy against dengue serotypes, wherein said unit dose / 4 dengue virus composition or said placebo is administered for at least 12 months or 12 to 18 months (e.g., at 12 months or 18 months) at least twice less than 6 months, eg, within 3 months after the second administration, at about 30 days. 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%.

In certain embodiments, the present invention relates to a two-sided 95%, when measured versus placebo, subject population of at least 5,000 healthy subjects, or at least 10,000 healthy subjects, or at least 15,000 healthy subjects, irrespective of serum status at baseline, as measured against placebo. to the method of having vaccine efficacy against dengue serotype 1 with a confidence interval, wherein said unit dose / 4 dengue virus composition or said placebo is at least 12 months or 12 to 18 months after the second administration of the dosing schedule up to (eg, at 12 months or 18 months) less than 6 months, eg, within 3 months after the second administration of the dosing schedule, at least twice on about 30 days. In certain such embodiments, the lower binding is greater than 30%, greater than 40%, or greater than 50%.

In certain embodiments, the present invention provides a subject 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) irrespective of serum status at baseline and at ages 4 to 16 years of age. to said method having vaccine efficacy against serotype 1 in preventing virally identifiable dengue disease with a two-sided 95% confidence interval when measured against placebo in a population, wherein said lower binding is 25% is greater than, and the reconstituted unit dose/4 dose dengue virus composition as described herein or placebo is administered, for example, until 12 to 18 months (eg, at 12 months or at 18 months) after the last administration. at least twice in less than 6 months, eg, within 3 months, at 30 days. In certain such embodiments, the lower binding 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 population of at least 1,500 subjects is seronegative for all serotypes at baseline, and the lower binding is greater than 35%. In certain such embodiments, each of the seronegative and seropositive populations provides vaccine efficacy against serotype 1, with a two-sided 95% confidence interval, wherein the lower binding is within 10%-points.

In certain embodiments, the invention provides for a dengue serotype greater than 30% when measured versus placebo in a subject population of at least 5,000 healthy subjects, or 10,000 healthy subjects, or 15,000 healthy subjects, irrespective of serum status at baseline. 1 , wherein said unit dose / 4 dengue virus composition or said placebo is at least 12 months or 12 to 18 months (eg, 12 months) after the second administration. or at 18 months) less than 6 months, eg, within 3 months after the second administration, at least twice at about 30 days. 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%.

In certain embodiments, the present invention provides a subject 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) irrespective of serum status at baseline and at ages 4 to 16 years of age. to the method having vaccine efficacy against serotype 1 in preventing virologically identifiable dengue disease when measured against placebo in a population, wherein the reconstituted unit dose / 4 dengue virus as described herein The composition or placebo is administered at least in less than 6 months, e.g., within 3 months, at 30 days after the last administration, e.g., by 12 to 18 months (e.g., at 12 months or at 18 months) after last administration It is administered twice. 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 population of at least 1,500 subjects is seronegative for all serotypes at baseline. In certain such embodiments, each of the seronegative and seropositive populations provides vaccine efficacy against serotype 1 that is within 5 percentage-points.

In certain embodiments, the invention provides for Dengue Serotype 2 with a two-sided 95% confidence interval when measured versus placebo in a subject population of at least 5,000 healthy subjects, or at least 10,000 healthy subjects, irrespective of serum status at baseline. to the method having vaccine efficacy against or at 18 months) at least twice in less than 6 months, eg, within 3 months after the second administration of the dosing schedule, at about 30 days. In certain such embodiments, the lower binding is greater than 30%, greater than 40%, greater than 50, greater than 60, greater than 70, greater than 80% or greater than 90.

In certain embodiments, the present invention provides a subject 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) irrespective of serum status at baseline and at ages 4 to 16 years of age. to the method having vaccine efficacy against serotype 2 in preventing virally identifiable dengue disease with a two-sided 95% confidence interval when measured versus placebo in a population, wherein the lower binding is 25% is greater than, and the reconstituted unit dose/4 dose dengue virus composition as described herein or placebo is administered, for example, until 12 to 18 months (eg, at 12 months or at 18 months) after the last administration. at least twice in less than 6 months, eg, within 3 months, at 30 days. In certain such embodiments, the lower binding 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 for all serotypes at baseline. In certain such embodiments, each of the seronegative and seropositive populations provides vaccine efficacy against serotype 2, with a two-sided 95% confidence interval, wherein the lower binding is within 5 percentage-points.

In certain embodiments, the invention provides for a dengue serotype greater than 30% when measured versus placebo in a subject population of at least 5,000 healthy subjects, or 10,000 healthy subjects, or 15,000 healthy subjects, irrespective of serum status at baseline. to the above method having vaccine efficacy against 2, wherein said unit dose / 4 dengue virus composition or said placebo is at least 12 months or 12 to 18 months (e.g., at 12 months) after the second administration. or at 18 months) less than 6 months, eg, within 3 months after the second administration, at least twice at about 30 days. 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%.

In certain embodiments, the present invention provides a subject 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) irrespective of serum status at baseline and at ages 4 to 16 years of age. to the method having vaccine efficacy against serotype 2 in preventing virologically identifiable dengue disease when measured against placebo in a population, wherein the reconstituted unit dose / 4 dengue virus as described herein The composition or placebo is administered at least in less than 6 months, e.g., within 3 months, at 30 days after the last administration, e.g., by 12 to 18 months (e.g., at 12 months or at 18 months) after last administration It is administered twice. 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 population of at least 1,500 subjects is seronegative for all serotypes at baseline. In certain such embodiments, each of the seronegative and seropositive populations provides vaccine efficacy against serotype 2 that is within 5 percentage-points.

In certain embodiments, the present invention relates to a two-sided 95%, when measured versus placebo, subject population of at least 5,000 healthy subjects, or at least 10,000 healthy subjects, or at least 15,000 healthy subjects, irrespective of serum status at baseline, as measured against placebo. to said method having vaccine efficacy against dengue serotype 3 with a confidence interval, wherein said lower value is greater than 25%, and wherein said unit dose / 4 dengue virus composition or said placebo is a second administration of the dosing schedule up to at least 12 months thereafter, less than 6 months, eg, within 3 months after the second administration of the dosing schedule, at least twice at about 30 days. In certain such embodiments, the lower binding is greater than 30% or greater than 40%.

In certain embodiments, the invention provides for a dengue serotype greater than 30% when measured versus placebo in a subject population of at least 5,000 healthy subjects, or 10,000 healthy subjects, or 15,000 healthy subjects, irrespective of serum status at baseline. to said method having vaccine efficacy against 3, wherein said unit dose / 4 dengue virus composition or said placebo is less than 6 months to at least 12 months after the second administration, eg, 3 months after the second administration. within, at least twice in about 30 days. In certain such embodiments, the vaccine efficacy against dengue serotype 3 is greater than 40%, greater than 50%, greater than 55%, or greater than 60%.

In certain embodiments, the invention provides for placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000 or at least 15,000 healthy subjects) irrespective of serum status at baseline and at ages 4 to 16 years versus placebo. to the method having combined vaccine efficacy against all four serotypes in preventing virologically identifiable dengue disease with hospitalization with a two-sided 95% confidence interval, wherein the lower binding is greater than 25% and wherein the reconstituted unit dose/4-valent dengue virus composition or placebo as described herein is administered after the last administration, e.g., by 12 to 18 months (e.g., at 12 months or at 18 months) after the last administration. at least twice in 30 days less than 6 months, eg, within 3 months, or from 12 to 18 months after the last administration (eg, at 12 months or 18 months). In certain such embodiments, the lower binding 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%.

In certain embodiments, the invention provides for placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000 or at least 15,000 healthy subjects) irrespective of serum status at baseline and at ages 4 to 16 years versus placebo. to said method having combined vaccine efficacy against all four serotypes in preventing virologically identifiable dengue disease with hospitalization, wherein the reconstituted unit dose/4 valent dengue virus composition as described herein or placebo is less than 6 months, e.g., within 3 months, from administration of the first dose, e.g., by 12 to 18 months after the last dose (e.g., at 12 months or 18 months), or the last dose up to 12 to 18 months after (eg, 12 months or 18 months) at least twice 30 days after the last dose. In certain such embodiments, the vaccine efficacy is greater than 70%, greater than 75%, greater than 80%, greater than 82%, greater than 85%, or greater than 88%.

In certain embodiments, the present invention relates to all four serotypes with a two-sided 95% confidence interval when measured against placebo in a subject population of at least 1,500 or at least 2,000 healthy subjects who are seronegative for all serotypes at baseline. to the method of having a combination vaccine efficacy against virologically confirmed dengue fever with hospitalization for 12 to 18 months (eg, 12 months or 18 months) less than 6 months, eg, within 3 months after the second administration of the dosing schedule, at least twice at about 30 days. In certain such embodiments, the lower binding is greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, or greater than 75%.

In certain embodiments, the present invention measures placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000 or at least 15,000 healthy subjects) who are seronegative for all serotypes at baseline and at ages 4 to 16 years of age to the method having combined vaccine efficacy against all four serotypes in preventing virologically identifiable dengue disease with hospitalization with a two-sided 95% confidence interval when provided, wherein the lower binding is 25 % of the reconstituted unit dose/4 dengue virus composition as described herein or placebo, e.g., by 12 to 18 months (e.g., at 12 months or at 18 months) after the last administration less than 6 months after administration, eg, within 3 months, at least twice in 30 days. In certain such embodiments, the lower binding 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%.

In certain embodiments, the invention provides hospitalizations for all four dengue serotypes greater than 30% when measured versus 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. to said method having combined vaccine efficacy against virologically confirmed dengue fever together with said unit dose/4 dengue virus composition or said placebo for at least 12 months or 12 to 18 months after the second administration up to (eg, at 12 months or 18 months) less than 6 months, eg, within 3 months after the second administration, at least twice at about 30 days. In certain of the above embodiments, the combined vaccine efficacy against virologically confirmed dengue with hospitalization for all four dengue serotypes is greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

In certain embodiments, the present invention measures placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000 or at least 15,000 healthy subjects) who are seronegative for all serotypes at baseline and at ages 4 to 16 years of age to the above method having combined vaccine efficacy against all four serotypes in preventing virally identifiable dengue disease with hospitalization if present, wherein the reconstituted unit dose/4 dose dengue as described herein If the viral composition or placebo is administered, e.g., by 12 to 18 months (e.g., at 12 or 18 months) after last administration, less than 6 months, e.g., within 3 months, at 30 days after last administration It is administered at least twice. 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%, over 85%.

In certain embodiments, the invention provides virology with hospitalization for all four serotypes with a two-sided 95% confidence interval when measured against placebo in a subject population of at least 1,500 or at least 2,000 healthy subjects who are seropositive at baseline. to the method having combination vaccine efficacy against dengue fever identified as at least twice in less than 6 months, eg, within 3 months, about 30 days after the second administration of the dosing schedule (eg, at 12 months or 18 months). In certain such embodiments, the lower binding is greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, or greater than 80%.

In certain embodiments, the present invention provides two-sided 95% 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) who are seropositive at baseline and at ages 4 to 16 years old. to the method having combined vaccine efficacy against all four serotypes in preventing virally identifiable dengue disease with hospitalization with confidence intervals, wherein the lower binding is greater than 25%, If the reconstituted unit dose / 4 dose dengue virus composition as described or placebo is administered, e.g., by 12 to 18 months after last administration (e.g., at 12 months or 18 months) less than 6 months after last administration; For example, within 3 months, at least twice in 30 days. In certain such embodiments, the lower binding is greater than 60%, greater than 65%, greater than 70%, greater than 75%, or greater than 80%.

In certain embodiments, the present invention provides virologically confirmed with hospitalization for >30% of all 4 serotypes when measured against placebo in a subject population of at least 1,500 or at least 2,000 healthy subjects who are seropositive at baseline. to the above method with combined vaccine efficacy against dengue, wherein said unit dose / 4 dengue virus composition or said placebo is administered for at least 12 months or 12 to 18 months after the second administration (e.g., 12 or at 18 months) at least twice in less than 6 months, eg, within 3 months, about 30 days after the second administration. In certain of the above embodiments, the combined vaccine efficacy against virologically confirmed dengue with hospitalization for all four dengue serotypes is greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

In certain embodiments, the present invention provides a method for treating the virus with hospitalization when measured for placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000 or at least 15,000 healthy subjects) who are seropositive at baseline and at ages 4 to 16 years of age. to the above method having combined vaccine efficacy against all four serotypes in preventing scientifically identifiable dengue disease, wherein the reconstituted unit dose/4 valent dengue virus composition or placebo as described herein is exemplified For example, from 12 to 18 months after the last administration (eg, at 12 months or 18 months) less than 6 months after the last administration, eg, within 3 months, at least twice on 30 days. In certain such embodiments, the vaccine efficacy is greater than 75%, greater than 70%, greater than 80%, greater than 85%, or greater than 90%.

In certain embodiments, the invention provides a subject population of at least 5,000 healthy subjects (or at least 10,000 or at least 15,000 healthy subjects) who are seropositive at baseline or seronegative for all serotypes at baseline and ages 4 to 16 years of age. to the method having combined vaccine efficacy against all four serotypes in preventing virologically identifiable dengue disease with hospitalization with a two-sided 95% confidence interval when measured against placebo in The lower binding is greater than 25%, and the reconstituted unit dose/4-valent dengue virus composition or placebo as described herein is administered, for example, by 12 to 18 months after last administration (eg at 12 months or 18 months). month) at least twice in less than 6 months, eg within 3 months, 30 days after the last administration. In certain such embodiments, the difference between the lower binding provided by the seropositive subject at baseline and the seronegative subject for all serotypes at baseline is no more than 15%-points.

In certain embodiments, the invention provides a subject population of at least 5,000 healthy subjects (or at least 10,000 or at least 15,000 healthy subjects) who are seropositive at baseline or seronegative for all serotypes at baseline and ages 4 to 16 years of age. to the method having combined vaccine efficacy against all four serotypes in preventing virologically identifiable dengue disease with hospitalization when measured against placebo in, wherein the reconstituted unit as described herein If the dose/4-valent dengue virus composition or placebo is administered, e.g., by 12 to 18 months after the last dose (e.g., at 12 months or at 18 months) less than 6 months after the last dose, e.g., 3 months within, at least twice in 30 days. In certain such embodiments, the difference between vaccine efficacy provided by a seropositive subject at baseline and a seronegative subject for all serotypes at baseline is no more than 10 percentage points, or no more than 5 percentage points.

In certain embodiments, the invention provides a two-sided 95% confidence interval 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) irrespective of serum status at baseline. to said method having a relative risk, preferably a combined relative risk, for all four serotypes, wherein said higher binding is less than 0.75 and a reconstituted unit dose / 4 dengue virus composition as described herein or the placebo is administered at least twice, up to at least 12 months after the second administration, and less than 6 months after the second administration, eg, within 3 months, 30 days. In certain such embodiments, the higher binding is less than 0.70, less than 0.65, less than 0.60, less than 0.55, less than 0.50, less than 0.45, less than 0.40, less than 0.35, less than 0.30 or less than 0.28. Preferably said reconstituted unit dose or placebo is administered subcutaneously within about 3 months, eg, on days 0 and 90.

In certain embodiments, the present invention provides sera of less than 0.70 when measured versus placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000 or at least 15,000 healthy subjects) irrespective of serum status at baseline. to the above method having a relative risk, preferably a combined relative risk, for the type, wherein the reconstituted unit dose/4 dose dengue virus composition or placebo as described herein is administered a second time by at least 12 months after the second administration. It is administered at least twice in less than 6 months, eg, within 3 months, about 30 days after administration. In certain such embodiments, the relative risk is less than 0.65, less than 0.60, less than 0.55, less than 0.50, less than 0.45, less than 0.40, less than 0.35, less than 0.30, less than 0.25 or less than 0.23. Preferably said reconstituted unit dose or placebo is administered subcutaneously within about 3 months, eg, on days 0 and 90.

In certain embodiments, the present invention provides a method for determining a virologically identifiable dengue disease versus placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000 or at least 15,000 healthy subjects) irrespective of serum status at baseline. cases occur in less than 2.5% of subjects, wherein the reconstituted unit dose/4 dose dengue virus composition as described herein or placebo is a second administration at least 12 months or at least 18 months after the second administration at least twice in less than 6 months, eg, within 3 months, about 30 days. In certain such embodiments, the virally identifiable dengue disease develops in less than 2.0% of subjects, less than 1.5% of subjects, less than 1.0% of subjects, less than 0.8% of subjects, or less than 0.6% of subjects. Preferably said reconstituted unit dose or placebo is administered subcutaneously within about 3 months, eg, on days 0 and 90.

In certain embodiments, the present invention provides at least 5,000 healthy subjects (or at least 10,000 or at least 15,000 healthy subjects) with a combination vaccine efficacy against all four serotypes with a two-sided 95% confidence interval when measured versus placebo in a subject population, wherein the lower value is greater than 61.0%; or greater than 65.0%, or greater than 70.0%, or greater than 72.0%, wherein said unit dose / 4 dengue virus composition or said placebo is at least 12 or 13 months after the last dose of the dosing schedule 6 months or less after the last dose of the dosing schedule , at least twice in about 30 days.

In certain embodiments, the present invention provides at least 5,000 healthy subjects (or at least 10,000 or at least 15,000 In the method having combination vaccine efficacy against all four serotypes greater than 66%, or greater than 70% or greater than 75% or greater than 77% or greater than 80.0%, when measured versus placebo in a subject population of healthy subjects) wherein said unit dose/4 dose dengue virus composition or said placebo is administered at least twice, up to 6 months, about 30 days after the last administration of the dosing schedule, up to at least 12 or 13 months after the last administration of the dosing schedule.

In certain embodiments, the present invention relates to the method, wherein the combined vaccine efficacy for all four serotypes is measured about 30 days after the last administration of the dosing schedule until 12 or 13 months after the last administration of the dosing schedule.

In certain embodiments, the invention provides that said unit dose or said placebo is administered at least twice within 3 months, particularly on about 1 and about 90 days, and the efficacy of the combination vaccine for all four serotypes is the second of the dosing schedule. to 12 or 13 months post-dose, measured 30 days after the second dose.

In certain embodiments, the present invention relates to said method, which is effective and safe. In some of these embodiments, the subject or population of subjects is less than 9 years old, less than 4 years old, or less than 2 years old or 2 to 9 years old, or 2 to 5 years old, or 4 to 9 years old. of age, or the age of 6 to 9 years. Optionally, the subject is seronegative for all dengue serotypes.

In certain embodiments, the present invention provides a method of treating viruses with hospitalizations of 1 or less, or 0.8 or less, or 0.6 or less when measured versus placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000 or at least 15,000 healthy subjects). to said method having a relative risk for scientifically confirmed dengue fever. In some of these embodiments, the subject or population of subjects is less than 9 years old, less than 4 years old, or less than 2 years old or 2 to 9 years old, or 2 to 5 years old, or 4 to 9 years old. of age, or the age of 6 to 9 years. Optionally the subject is seronegative for all dengue serotypes.

In certain embodiments, the present invention relates to said method, wherein said healthy subject of said subject population is between 4 and 16 years of age. In some of the above embodiments, healthy subjects of the subject population are between 4 and 5 years of age, between 6 and 11 years of age, or between 12 and 16 years of age.

In certain embodiments, the present invention relates to said method wherein a healthy subject of a subject population is defined as healthy in terms of the exclusion criteria specified in Table 6.

In certain embodiments, the present invention relates to said method, wherein said healthy subject of said subject population is from Asia Pacific or Latin America.

In certain embodiments, the invention relates to said method, wherein said healthy subject of said subject population is seropositive with respect to at least one serotype. In other embodiments, healthy subjects of the subject population are seronegative with respect to all serotypes.

In certain embodiments, the invention relates to said method, wherein said healthy subject of said subject population is 4-5 years old from Asia Pacific, 6-11 years old from Asia Pacific or 12-16 years old from Asia Pacific. will be. In other embodiments, healthy subjects of the subject population are 4-5 years old from Latin America, 6-11 years old from Latin America or 12-16 years old from Latin America.

In certain embodiments, the invention provides that a healthy subject of said population of subjects is 4-5 years old and seropositive for at least one dengue serotype, or is 6-11 years old and seropositive for a dengue serotype at least 1 month old, or 12 to 16 years of age and seropositive for at least one dengue serotype. In other embodiments, healthy subjects of the subject population are 4-5 years old and seronegative for all dengue serotypes, 6-11 years old and seronegative for all dengue serotypes, or 12-16 years old and It is seronegative for all dengue serotypes.

In certain embodiments, the present invention relates to said method, wherein healthy subjects of the subject population are from Asia Pacific or Latin America and are seropositive for at least one dengue serotype at baseline. In other embodiments, healthy subjects of the subject population are from Asia Pacific or Latin America and are seronegative for all dengue serotypes at baseline.

In certain embodiments, the invention provides that healthy subjects of a population of subjects are from Asia Pacific and are seropositive for at least one dengue serotype at baseline and at the age of 4 to 5, 6 to 11 or 12 to 16 years of age. it's about how In other embodiments, healthy subjects of the subject population are from Asia Pacific and are seronegative for all dengue serotypes at baseline and at age 4-5, age 6-11, or age 12-16. In another embodiment, healthy subjects of the subject population are from Latin America and are seropositive for at least one dengue serotype at baseline and at age 4-5, age 6-11, or age 12-16. In other embodiments, healthy subjects in the subject population are from the Americas and are seronegative for all dengue serotypes at baseline and at age 4-5, age 6-11, or age 12-16.

In certain embodiments, the present invention relates to the above method, wherein healthy subjects of a population of subjects have had a previous vaccination against yellow fever. In other embodiments, healthy subjects in the subject population have not had prior vaccination against yellow fever. Prior vaccination refers to vaccination prior to the first vaccination in unit doses reconstituted as described herein. For example, for vaccine efficacy (VE) determined in Example 6 from 30 days after the second vaccination, a prior vaccination of yellow fever is defined as a yellow fever vaccination that occurs 30 days before the second vaccination.

In certain embodiments, the present invention relates to said method, wherein healthy subjects of the subject population had a prior vaccination against Japanese encephalitis. In another embodiment, healthy subjects in the subject population have not had prior vaccination against Japanese encephalitis.

In certain embodiments, the invention relates to said method wherein healthy subjects of a subject population are administered Dengvaxia ^® within 4.5 years after administration of the first dose or within a dosing regimen as described herein. In certain embodiments, the invention relates to the above method, wherein the incidence of vaccine-related severe adverse events is less than 0.1%.

In certain embodiments, the present invention relates to the above method, wherein the incidence of vaccine-related unattractive adverse events occurring within 4 weeks of administration is less than 2%.

In certain embodiments, the invention relates to the above method, wherein the incidence of vaccine-related induced severe adverse events occurring within 2 weeks of administration is less than 35%.

In certain embodiments, the invention relates to the above method wherein the incidence of vaccine-related induced local reactions occurring within one week of administration is less than 40%.

In certain embodiments, the present invention relates to said method, wherein said method does not increase the risk of virologically confirmed dengue fever with hospitalization in a subject, eg, in a seronegative subject.

The method should also be considered with respect to the unit dose for use in the method or with respect to the use of the unit dose for use in the manufacture of a medicament for the method.

In certain embodiments, a tetravalent dengue vaccine, eg, Dengvaxia®, is used to vaccinate against dengue disease. Dengvaxia® is CYD-TDV (Dengvaxia®, Sanofi Pasteur, Lyon, France), a tetravalent dengue vaccine based on a yellow fever backbone, which accounts for 56-61% of virologically confirmed dengue fever (VCD) in children in Asia and Latin America. Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in Asia: a phase 3, randomized, observer-masked, placebo -controlled trial Lancet 2014, 384:1358-65; Villar LA et al. Safety and immunogenicity of a recombinant tetravalent dengue vaccine in 9-16 year olds: a randomized, controlled, phase II trial in Latin America. Pediatr Infect Dis J 2013, 32:1102-9). The preparation of these specific variants CYD1, CYD2, CYD3 and CYD4 is described in international patent applications WO 98/37911, WO 03/101397, WO07/021672, WO 08/007021, the description of the exact process for their preparation may be referred to. WO 08/047023 and WO 08/065315 are described in detail. The corresponding nucleotide sequences of the prM-E regions of CYD1, CYD2, CYD3 and CYD4 are provided in WO2016034629 and the SEQ ID NOs are given in Table 16 of this reference.

In certain embodiments, the present invention relates to, inter alia, a chimeric dengue fever serotype 2/1 strain, a wild-attenuated dengue fever serotype 2 strain, a chimeric dengue fever serotype 2/3 strain and a chimeric dengue serotype 2/4 strain, and the Dengvaxia described herein. to the method, wherein the unit doses described herein comprising ® are administered to a subject or to a population of subjects simultaneously on the same day.

In certain embodiments, the present invention relates to the disclosed herein, inter alia, a chimeric dengue fever serotype 2/1 strain, a wild-attenuated dengue fever serotype 2 strain, a chimeric dengue fever serotype 2/3 strain and a chimeric dengue serotype 2/4 strain. A unit dose is administered to a subject or a population of subjects on Day 0/1 as a first dose and Dengvaxia® described herein is subsequently administered as a second dose within 3 months of the first dose, e.g., 90 from the first dose to a subject or a population of subjects in a primary manner. Alternatively, the Dengvaxia® described herein is administered to a subject or a population of subjects on Day 0/1 as a first dose, in particular, chimeric dengue serotype 2/1 strain, herbal attenuated dengue serotype 2 strain, chimeric dengue fever The unit doses described herein comprising the serotype 2/3 strain and the chimeric dengue serotype 2/4 strain are subsequently administered as a second dose within 3 months of the first dose, eg, at day 90 from the first dose. administered to a subject or a population of subjects.

In certain embodiments, the present invention relates to said method, wherein said dengue vaccine composition comprises another dengue vaccine such as TV003 or TV005. TV003, developed by the US National Institute of Allergy and Infectious Diseases, comprises vaccine components rDEN1Δ30, rDEN2/4Δ30, rDEN3Δ30/31 and rDEN4Δ30, wherein each of these components is present at a concentration of 3 log ₁₀ PFU. . TV005 is similar to TV003, the difference is that the concentration of rDEN2/4Δ30 in TV005 is 4 log ₁₀ PFU. The vaccines TV003 and TV005 and their vaccine components and their preparation are described in more detail in WO 2008/022196 A2 and SS Whitehead, Expert Rev Vaccines, 2016, 15(4): 509 to 517. Using recombinant DNA technology, two attenuation strategies were used for the structural gene chimerization and the deletion in the 3' untranslated region of the vaccine composition of TV003 or TV005. For example, component rDEN4Δ30 contains all structural and non-structural proteins of wild-type DENV-4, but is attenuated by a 30-nucleotide deletion in the 3' untranslated region (referred to as "Δ30"). Other vaccine components are also attenuated due to a 30-nucleotide deletion in the 3' untranslated region. Additionally, rDEN3Δ30/31 contains a 31 nucleotide deletion in the 3' untranslated region (detailed in FIGS. 1c and 13 of WO 2008/022196 A2). The rDEN2/4Δ30 component was created by replacing the prM and E genes of DENV-2 with the rDEN4Δ30 genome. The complete genomic sequence of the dengue strain that can be used to generate TV003 or TV005 is available under Genbank accession number in Table A of WO 2008/022196 A1.

In certain embodiments, the present invention relates to, inter alia, a chimeric dengue serotype 2/1 strain, a wild-attenuated dengue serotype 2 strain, a chimeric dengue serotype 2/3 strain and a chimeric dengue serotype 2/4 strain, and TV003 described herein. or the unit doses described herein comprising TV005 are administered to the subject or to a population of subjects simultaneously on the same day.

In certain embodiments, the present invention relates to the disclosed herein, inter alia, a chimeric dengue fever serotype 2/1 strain, a wild-attenuated dengue fever serotype 2 strain, a chimeric dengue fever serotype 2/3 strain and a chimeric dengue serotype 2/4 strain. The unit dose is administered to a subject or population of subjects on Day 0/1 as the first administration and a TV003 or TV005 described herein is subsequently administered as the second administration within 3 months of the first administration, e.g., from the first administration. and administered to the subject or population of subjects on day 90. Alternatively, TV003 or TV005 described herein is administered to a subject or a population of subjects on day 0/1 as a first administration, in particular a chimeric dengue serotype 2/1 strain, a wild attenuated dengue serotype 2 strain, a chimeric A unit dose described herein comprising a dengue fever serotype 2/3 strain and a chimeric dengue fever serotype 2/4 strain is subsequently administered as a second administration within 3 months of the first administration, e.g., 90 days from the first administration. administered to a subject or a population of subjects.

Example

The following examples are included to demonstrate specific aspects and embodiments of the invention as set forth in the claims. It is to be understood that the following description is for illustrative purposes only and is not to be taken as a limitation of the present invention in any way.

Example 1: Preparation of strains of dengue virus.

The methods used to generate the chimeric dengue strains TDV-1, -3 and -4 are standard molecular cloning and DNA processing methods and are described in (2003) J. Virology 77(21): 11436-11447, et al. there is. The following well-known methods were used to construct the prM-E gene of dengue fever serotypes 1, 3 and 4 and introduce it into the TDV-2 backbone: reverse transcriptase PCR (RT-PCR), PCR, restriction enzyme digestion, DNA Fragment ligation, bacterial transformation by electroporation, plasmid DNA preparation, in vitro transcription with T7 RNA polymerase, and transfection of Vero cells by electroporation.

After growing and purifying the different dengue serotypes separately as described in Huang et al. (2013) PLOS Neglected Dis, 7(5):e2243, they are mixed at the specific concentrations given in Example 4. A mixture of dengue fever serotypes is present in the dengue vaccine composition and contains 15% w/v α,α trehalose dihydrate, 1% w/v poloxamer 407, 0.1% w/v human serum albumin and 100 mM sodium chloride. It is combined with a composition of a pharmaceutically acceptable excipient to induce a dengue vaccine composition comprising The dengue vaccine composition is lyophilized and represents a lyophilized unit dose of TDV. The lyophilized unit dose is reconstituted with 37 mM aqueous sodium chloride solution, and the reconstituted unit dose is 15% w/v α,α trehalose dihydrate, 1% w/v poloxamer 407, 0.1% w/v human serum albumin and 137 mM sodium chloride.

Example 2: Microneutralization test

Immunogenicity was determined by microneutralization assay in each one of the four dengue serotypes and titers were defined as the dilution that resulted in a 50% reduction in plaque value (MNT50). Briefly, on day 1, 1 Vero cells were seeded onto 96-well assay plates in DMEM and 10% FBS at a density of 2.5×10 ⁵ cells/ml and incubated at 37° C. for 24 h. Serial dilutions (dilution range 1:10 to 1:20480) of heat-inactivated antibody-containing test and control serum samples were prepared on day 2 and a concentration of dengue virus, especially DENV-1 strain 16007, DENV-2 Viruses caused by antibodies present in serum by mixing strain 16681, DENV-3 strain 16562 and DENV-4 strain 1036 (target 60-80 pfu/well) in 96-well microtiter plates and incubating overnight at 2-8°C. neutralization was possible. After incubation, the mixture of virus and antibody was transferred onto 96-well plates with Vero cells, and the plates were incubated at 37°C for 90-120 min to infect Vero cells. An overlay of 1% methylcellulose in DMEM was applied to the plates to limit the spread of progeny viruses and the plates were incubated at 34°C for 46-70 hours at 34°C depending on the dengue serotype:

DENV1 - 66±2 hours

DENV2 - 70±2 hours

DENV3 - 66±2 hours

DENV4 - 46±2 hours

After incubation, cells were washed twice with PBS and fixed by addition of cold methanol and incubation for 60 min at a temperature of ≤ -20 °C. After immobilization, the plates are dried and wash buffer (0.5% Tween and 0.5% Tween) before addition of 50 μl of serotype-specific anti-dengue monoclonal antibody in blocking buffer (2.5% non-fat dry milk in PBST) per well. together, washed 3 times with 1x PBS, pH 7.4) and incubated with cells at 2-8° C. for 18±4 hours.

Monoclonal antibodies are described in Gentry et al. (1982) Am. J. Trop. Med. Hyg. 31, 548-555; Henchal et al. (1985) Am. J. Trop. Med. Hyg. 34, 162-169; and (1982) Am. J. Trop. Med. Hyg. 31(4):830-6). Briefly, anti-DENV-1 HBD was prepared against Dengue 1 strain Hawaii, integument, and anti-DENV-2 was prepared against Dengue 2 strain New Guinea seed, incubation, isotype 1, anti-DENV -3 HBD was prepared against Dengue 3 strain H87, isotype 2A, and anti-DENV-4 HBD was prepared against Dengue 4 strain H241, envelope, isotype 2A.

After incubation, the plates were washed 3 times with wash buffer and 50 μl of secondary peroxidase labeled goat anti-mouse IgG (H + L) (KPL Cat#074-1806) in blocking buffer was added, 37 Incubated for 90-120 min at &lt;RTI ID=0.0&gt; The plate is then washed 3 times with wash buffer and 50 μl of precipitant substrate (2-amino-9-ethyl carbazole (AEC) purification in 2.5 ml DMSO, 47.5 ml 50 mM acetate buffer and 250 μl hydrogen peroxide) is added. and the mixture was incubated at room temperature for 20 min. Finally, the substrate was removed and the plate was washed with dH ₂ O and dried.

Sample titers were calculated using a linear regression method and reported as MNT50 titers for each sample. Clinical data are reported as geometric mean titers for all individual MNT50 titers in each treatment group. Briefly, the number of infectious lesions in each well was counted and the titer of neutralizing antibody was determined by comparing the percent reduction in the centers of infectious lesions in wells containing antibody (test sample) versus wells containing virus alone. It was decided. MNT50 was calculated using the following linear regression equation:

MNT50 = 10^[(50-c)/m]), where c = the y-intercept of the regression line and m = the slope of the regression line.

Each test sample was tested in triplicate and titers were calculated from the average of triplicate values. A schematic of the steps performed in the test is provided in FIG. 2 .

Example 3: Phase III clinical trial in children

A Phase III, double-blind, randomized, and placebo-controlled trial was conducted in 20100 subjects aged 4 to 16 years residing in Thailand, Sri Lanka, Philippines, Panama, Nicaragua, Dominican Republic, Colombia or Brazil for efficacy, safety and sub-TDV The immunogenicity of a tetravalent dengue vaccine, referred to as (TDV-1, TDV-2, TDV-3 and TDV-4 described herein), was evaluated. The test includes three parts. Part 1 evaluates vaccine efficacy (VE) and continues until both of the following two criteria are met: (i) 120 cases of dengue are identified, and (ii) the duration of immunity of the subject is determined by the second vaccine Followed by 12 months after vaccination. Part 2 assessed VE for a secondary efficacy analysis for an additional 6 months. Part 3 evaluates long-term safety by the following participants for adverse events and continues for an additional 3 years.

Part 1: Active monitoring for primary evaluation of efficacy in all subjects. During this period, subjects were contacted at least weekly to ensure identification of a fever potentially due to dengue fever. This part started on the day of vaccination and ended when both of the following two criteria were met: (i) 120 cases of dengue were identified, and (ii) the duration of immunity of the subject was 12 months after the second vaccination. goes on The end of Part 1 was defined for each subject such that the follow-up period after the second vaccination was approximately the same for all subjects. Virologically-confirmed events in Part 1 are counted towards the primary efficacy objective if they occur at least 30 days after the second vaccination. Part 1 ended 12 months after the second vaccination

Part 2: Active monitoring for an additional 6 months for each subject after completion of Part 1, I, ie 18 months after second vaccination. During this period, subjects were contacted at least weekly to ensure identification of a fever potentially due to dengue fever. Virologically confirmed cases in parts 1 and 2 serve towards secondary efficacy objectives.

Part 3: Modified Active Surveillance for Assessment of Safety in All Subjects after Completion of Part 2 and 3 Years Duration for Each Subject. Surveillance modified during Part 3 maintains contact at least weekly through Part 3 of the trial, but irradiation intensity is modified based on need for hospitalization. Surveillance primarily identifies fevers of any severity that may be due to dengue fever.

Inclusion criteria include:

· Subjects 4 to 16 years of age were included at randomization.

Subjects in good health at trial entry as determined by medical history , physical examination (including vital signs) and the investigator's clinical judgment.

After the subject and / or the subject's parent/guardian, if applicable, sign and date the informed consent form, the nature of the trial has been explained in accordance with local regulations, and all necessary Privacy Approval Requirements.

· Subjects capable of complying with the testing procedure and available for a subsequent period.

Exclusion criteria include:

1. Fever (temperature ≥38°C) or moderate or severe acute illness or infection at randomization.

2. Any medical history or any disease in the investigator's opinion that participation in the trial could interfere with trial results or pose an additional risk to the trial subject, including, but not limited to:

a. Known hypersensitivity or allergy to vaccine components.

b. Female subjects who are pregnant or breastfeeding (post menarche).

c. Subjects with any serious chronic or progressive disease (eg, neoplasia, insulin dependent diabetes mellitus, heart, kidney or liver disease, neurological or seizure disorder, or Guillain-Barré syndrome) at the investigator's judgment.

d. Impairment/alteration of known or suspected immune function, including:

i. Chronic use (inhaled, intranasal or topical corticosteroids) of oral steroids (20 mg/day prednisone ≥12 weeks/≥2 mg/kg body weight/day equivalent to prednisone ≥2 weeks) within 60 days prior to Day 1 (0 months) use allowed).

ii. Inoculation of parenteral steroids (20 mg/day prednisone ≥12 weeks/≥2 mg/kg body weight/day equivalent to prednisone ≥2 weeks) within 60 days prior to Day 1 (month 0).

iii. Administration of immunoglobulin and/or any blood product within 3 months prior to Day 1 (Month 0) or scheduled dosing during the study period.

iv. Immunostimulant vaccination within 60 days prior to day 1 (month 0).

v. Immunosuppressive therapy, such as chemotherapy or radiation therapy, within 6 months prior to Day 1 (Month 0).

vi. Human Immunodeficiency Virus (HIV) infection or HIV-associated disease.

vii. genetic immunodeficiency.

3. Receive another vaccine within 14 days (for inactivated vaccine) or 28 days (for live vaccine) before day 1 (month 0), or plan to receive another vaccine within 28 days after day 1 (month 0).

4. Willingness to participate in a clinical trial with another trial product prior to 30 days prior to Day 1 (Month 0), or to participate in another trial at any time while conducting this trial.

5. Previously participating in a clinical trial of a dengue candidate vaccine or previously vaccinated with a dengue vaccine.

6. First degree relative values of individuals involved in conducting the trial.

7. Women of childbearing potential who are sexually active and have not used any acceptable method of contraception for at least 2 months prior to Day 1 (Month 0).

8. Women of childbearing potential who have an active sexual life and refuse to use an acceptable method of contraception for up to 6 weeks after the second dose of vaccination.

9. Deprived of liberty by administrative or court order, or hospitalized in an emergency or involuntarily.

10. Current alcohol abuse or drug addiction that could interfere with the subject's ability to comply with the testing process.

11. Identified as an investigator or an employee of the test center directly involved in a proposed trial or other trial under the direction of the investigator or test center.

Eligible subjects were randomized (2:1) into two treatment groups: Group 1 received one subcutaneous (SC) dose of TDV in the upper arm on Days 1 and 90, and Group 2 received on Days 1 and 90. Received one subcutaneous dose of placebo in the upper arm. Randomization was stratified by region (Asia Pacific and Latin America) and age group (children ages 4-5, 6-11 and 12-16 years) to ensure that each region had an appropriate TDV to placebo ratio. Dropouts after randomization were not replaced. Study Day 1 is defined as the date of administration of the first dose of TDV or placebo. TDV was prepared as described in Example 1. Each subcutaneous dose of TDV was 0.5 mL, and the concentrations of the four dengue serotypes in the TDV vaccine at each dose were 3.6 log ₁₀ PFU/dose, 4.0 log ₁₀ PFU/dose, 4.6 log ₁₀ PFU/dose and 5.1 log, respectively. TDV-1, TDV-2, TDV-3 and TDV-4 at ₁₀ PFU/dose.

The “total concentration in pfu/0.5 ml” used as the base value for the calculation of the percent concentration for each individual component of the tetravalent dengue vaccine is 3.60 log ₁₀ pfu/0.5 ml of dengue serotype 1, 4.00 log One exemplary 4 comprising dengue serotype 2 at a concentration of ₁₀ pfu/0.5 ml, dengue serotype 3 at a concentration of 4.60 log ₁₀ pfu/0.5 ml and dengue serotype 4 at a concentration of 5.11 log ₁₀ pfu/0.5 ml. is shown for the vaccine composition.

Often, logarithmic values of concentrations are converted to numerical values. The conversion result is 4x10 ³ pfu/0.5 ml for serotype 1, 1x10 ⁴ pfu/0.5 ml for serotype 2, 4x10 ⁴ pfu/0.5 ml for serotype 3, and 1.3x10 ⁵ for serotype 4 pfu/0.5 ml. The total concentration in pfu/0.5 ml is the sum of previous numerical values resulting in 1.84 x 10 ⁵ pfu/0.5 ml.

The “percent concentration” for each of serotypes 1, 2, 3 and 4 is calculated by dividing the numerical concentration value of the individual serotype (expressed as pfu/0.5 ml) by the total concentration (expressed as pfu/0.5 ml) and the result is obtained by multiplying by 100, i.e.:

Percent concentration of serotype 1 = (4x10 ³ pfu/0.5 ml ÷ 1.84 x10 ⁵ pfu/0.5 ml) x 100 = 2%

Percent concentration of serotype 2 = (1x10 ⁴ pfu/0.5 ml ÷ 1.84 x10 ⁵ pfu/0.5 ml) x 100 = 5%

Percent concentration of serotype 3 = (4x10 ⁴ pfu/0.5 ml ÷ 1.84 x10 ⁵ pfu/0.5 ml) x 100 = 22%

Percent concentration of serotype 4 = (1.3x10 ⁵ pfu/0.5 ml ÷ 1.84 x10 ⁵ pfu/0.5 ml) x 100 = 71%.

Percent concentrations are rounded to integers.

Primary outcome measures included vaccine efficacy (VE) of two doses of TDV in preventing virologically confirmed dengue (VCD) caused by any dengue serotype [Timetable: 1st until end of part 1 2 30 days (120 days) after vaccination]. VE is defined as 1 - (λv/λc), where λv and λc represent the risk rates for the TDV and placebo groups, respectively. Virologically confirmed cases of dengue fever (defined as a temperature ≥ 38°C on any 2 of 3 consecutive days) or positive serotype-specific reverse transcriptase polymerase chain reaction (RT-PCR) by investigators with It is defined as a disease clinically suspected to be dengue fever. Fever requires a minimum of 14 days from the previous fever to avoid overlapping acute and convalescent visits from one episode with visits from the second episode.

Secondary outcome measures include:

1) VE of 2 doses of TDV to prevent virologically confirmed dengue fever induced by each dengue serotype [Timetable: 30 days (120 days) after second vaccination until end of part 2].

2) VE of 2 doses of TDV to prevent virologically confirmed dengue induced by any dengue serotype in participants who were dengue seronegative at baseline [Timetable: 30 after second vaccination until end of part 2: days (120 days) (up to 21 months)].

3) VE of 2 doses of TDV to prevent virologically confirmed dengue induced by any dengue serotype in participants who are dengue seropositive at baseline [Timetable: 30 days after second vaccination until end of part 2 (120 days)].

4) VE of 2 doses of TDV to prevent hospitalization for virologically confirmed dengue induced by any dengue serotype [Timetable: 30 days (120 days) after second vaccination until end of part 2 ].

5) VE of 2 doses of TDV to prevent virally confirmed severe dengue fever induced by any dengue fever serotype [Timetable: 30 days (120 days) after second vaccination until end of part 2] .

6) Percentage of participants with induced local injection site adverse events (AEs) in the safety subset [time frame: days 1-7 after each vaccination] and severity of induced local injection AEs. Local AEs induced at the injection site were defined as pain, erythema and swelling that occurred within 7 days of each vaccination.

7) Percentage of participants with induced systemic adverse events (AEs) in the safety subset [time frame: days 1-14 after each vaccination] and severity of induced systemic AEs. Induced systemic AEs in children (<6 years of age) were defined as fever, irritability/turbulence, drowsiness and loss of appetite that occurred within 14 days after each vaccination. Induced systemic AEs in children (≥ 6 years of age) are defined as fever, headache, asthenia, malaise and myalgia that developed within 14 days after each vaccination.

8) Percentage of participants with any induced adverse events (AEs) in the safety subset [time frame: days 1 to 28 after each vaccination]. A non-attractive AE is any AE that is not an attracted local or systemic AE as defined above.

9) Percentage of Participants with Serious Adverse Events (SAE) during Parts 1 and 2 [Timetable: Day 1 to End of Parts 1 and 2]. Serious adverse events (SAEs) at any dose are death-threatening, life-threatening, requiring hospitalization or prolongation of existing hospitalization, leading to persistent or significant disability/disability, or birth defects/birth defects. or any intractable medical occurrence or effect that is medically important for reasons other than the criteria mentioned above.

10) Percentage of Participants with Fatal SAEs and Study Drug-related SAEs during the First and Second Half of Part 3 [Timetable: 3 years (18 months after first vaccination) starting at the end of Part 2 (approximately 21 months after first vaccination) half) while].

11) Percentage of participants with a seropositive response for each of the four dengue serotypes in the immunogenic subset [Timetable: 1 day and 1, 3, 4, 9, 15 months and then yearly (up to 3 years)]. A seropositive response is defined as a reciprocal neutralizing titer of ≧10. The four DENV serotypes are DEN-1, DEN-2, DEN-3 and DEN-4.

12) Percentage of participants with seropositive responses for multiple dengue serotypes in the immunogenic subset [Timetable: 1 day and 1, 3, 4, 9, 15 months and then yearly (up to 3 years)].

13) Geometric mean titers (GMTs) of neutralizing antibodies to each of the four dengue serotypes in the immunogenic subset [timetable: 1 day and 1, 3, 4, 9, 15 months and then yearly (up to 3 years)]. The GMT of neutralizing antibodies is determined via the microneutralization test (MNT) as described in Example 2.

a) study population

After screening, 20,099 participants were randomized and 20,071 received at least one injection. Overall, 97.4% of placebo participants (n/N: 6,521/6,698) and 97.3% of vaccinated (n/N: 13,038/13,401) completed Study Part 1 ( FIG. 3 ). Reasons for study withdrawal included AEs, participants who failed to follow-up, pregnancy, protocol violations, and withdrawal of participants (or parent/guardian). Baseline characteristics were similar across both treatment groups (Table 5). The mean age of study participants was 9.6 years, baseline seronegative 27.7%, and enrollment was widely balanced by region (46.5% in Asia, 53.5% in Latin America). The highest seronegative rates were found in Panama (62.2%), followed by Sri Lanka (38.5%), Thailand (34.4%), Brazil (28.8%), Nicaragua (22.3%), Colombia (15.4%) and the Philippines (12.4%). , and in the Dominican Republic (2.8%).

[Table 5] Baseline characteristics of study population (number, %)

^a seronegative for all serotypes; Mutual neutralizing antibody titer ≥10; SD, seropositive defined as standard deviation.

^b The number of participants in the TVD + placebo group is not equal to the total number shown because misassigned participants (ie, those who received both TVD and placebo due to dosing error) were not included in the TDV and placebo group data.

b) fever and VCD

During Part 1, 5,754 and 4,663 episodes of fever were reported in the Asian and Latin American regions, respectively. Acute samples were obtained in 99.5% and 96.6% of these cases, and 98.3% and 85.1% of samples were obtained within 5 days in Asia and Latin America, respectively. There were 278 VCD cases (76 hospitalizations) in the safety set for the entire Part 1 period, of which 210 (58 hospitalizations) were 30 days after the second vaccination in PPS (Table 6; Table 8), the primary endpoint included in the analysis.

c) Distribution of VCDs included in primary endpoint analysis

DENV-1 was reported in all countries with VCD and included all 21 cases in Panama. In Sri Lanka, 54 out of 60 VCD cases were DENV-2, and in the Philippines 87 out of 109 VCD cases were DENV-3. All seven cases of DENV-4 VCD were reported in the Philippines. No VCD was reported in Nicaragua or the Dominican Republic. Of the associated 58 hospitalized VCD cases, 43 were reported in Sri Lanka. A total of 2 severe dengue fever (both DENV-3) and 5 dengue hemorrhagic fever (DHF; 3 DENV-2; 2 DENV-3) cases were reported (Table 7). These 7 events were also the only such events in the full Part 1 safety set.

d) vaccine efficacy

The VE for VCD of any serotype was 80.2% (95% CI: 73.3-85.3; P<0.001). Similar efficacy of 81% (95% CI: 64.1-90.0) between doses and from after the first dose in the set of safety lines (Table 6) suggests that the vaccine is effective after the first dose. Exploratory analysis of secondary efficacy endpoints showed a trend of differential efficacy according to serotype, with the highest efficacy for DENV-2 (97.7%) followed by DENV-1 (73.7%) and DENV-4 (63.2). %, with a CI of 0) and DENV-3 (62.6%; Table 7). Overall, efficacy was similar at baseline seronegative (74.9%) and seropositive (82.2%, FIG. 4 supra); However, this differed according to serotype. Efficacy against DENV-2 was not affected by serum status; Efficacy for DENV-1 was slightly higher in baseline seropositive (79.8%; 95% CI: 51.3-91.6) than in baseline seronegative (67.2%; 95% CI: 23.2-86.0). No efficacy was observed for DENV-3 in baseline seronegative (-38.7%; 95% CI: -335.7-55.8) compared to baseline seropositive (71.3%; 95% CI: 54.2-82.0). Efficacy by serostat cannot be calculated for DENV-4 as no events were observed at baseline seronegative. In the primary endpoint timeline for PPS, the only 5 VCDs requiring hospitalization were reported in the vaccine group compared to 53 cases in the placebo group, and VE was 95.4% (95% CI: 88.4-98.2; baseline seronegative). 97.2% for and 94.4% for baseline seropositivity; Table 7;

The primary vaccine efficacy (VE) of two doses of TDV in virologically confirmed dengue (VCD) induced by any dengue serotype is shown in Table 6.

Table 6 End of Set Part 1 (PPS) per protocol, i.e. to prevent virologically confirmed dengue (VCD) for any serotype from 30 days after the second vaccination until 12 months after the second vaccination In the vaccine efficacy of TDV. Safety set analysis from the first dose to the end of the Part 1 study period, i.e., 12 months after the second vaccination

Note 1: Percentage of virologically confirmed dengue fever (VCD) is based on the number of subjects evaluated.

Note 2: Individual-risk years are defined as the cumulative time in years until the onset of the VCD fever or until the onset of the VCD fever, whichever comes first, or the end or date of discontinuation of the Part 1 study period. The incidence density is defined as the number of cases per 100 individual-risk years. Percentages are based on the total number of participants in the analysis set evaluated (the denominator) and cannot be equated to the total number of participants in the analysis set per protocol.

* One participant had 2 cases of VCD during Part 1, only the first VCD was included in efficacy calculations.

Note 3: Vaccine efficacy (VE) and two-sided 95% CI are assessed from the Cox proportional hazards model with TDV as a factor adjusted by age and stratified by region.

Note 4: Statistical significance is concluded when the lower binding of 95% CI to VE is >25%. Because the hypothesis is tested in a confirmatory manner at a two-sided significance level of 5%, the calculated p-value should be compared to 0.025.

Note 5: Relative risk is calculated as the number of events divided by the number of subjects evaluated in the TDV group divided by the number of subjects evaluated in the placebo group.

For efficacy assessments shown in Table 6, cases of VCD were defined as fever using positive serotype-specific RT-PCR (ie, positive dengue detection RT-PCR) (fever >38 on any 2 of 3 consecutive days). °C), occurring at any time point starting from 30 days (120 days [4 months]) after the second vaccination through the end of Part 1. Analyzes were performed on per protocol (PPS) and safety sets.

As used herein, “set per protocol (PPS)” refers to all subjects in a complete analysis set (FAS) consisting of all randomized subjects who received at least one dose of TDV or placebo and did not have any major protocol violations. is done The major protocol violations were not receiving both doses of TDV or placebo administration, not receiving both doses at the correct intervals, not receiving correct administration of TDV or placebo, subject using a prohibited drug/vaccine, and subject being meet any exclusion criteria of 2d, 3, 4 or 5 defined above or product manufacturing error.

The p-value is obtained by solving the threshold Z in the following equation:

, 여기서,

는 처리를 정의하고

는 관련 표준 오차를 정의함.A top combination of 1-sided (1-p%) CI of HR=0.75, where HR is the hazard ratio and defined as HR=λV/λC.

, here,

defines the processing and

defines the relevant standard error.

The 1-sided p-value is 1-(area to the left of the threshold Z from the standard normal distribution). Because the hypothesis is tested in a two-sided confirmatory manner at a significance level of 5%, the calculated one-sided p-value should be compared to 0.025.

In summary, in Part 1 of this study, a high vaccine efficacy of 80.2% was found against virologically confirmed dengue of any serotype in children aged 4 to 16 years. These included an efficacy of 74.9% at baseline seronegative and a strong 95.4% reduction in hospitalization. Onset of protection may occur after the first dose with an efficacy of 81% between doses. Altogether, these results suggest a potential benefit for each vaccine recipient irrespective of previous dengue exposure or age. The above findings are significant as developing a vaccine against dengue fever has been particularly challenging for individuals with no experience with dengue and dengue remains one of the WHO's top 10 threats to global health in 2019.19. Initiation of protection after the first dose also has potential utility in relation to outbreak control or travel vaccination, providing a reduction in dengue risk after only one dose.

The severe form of dengue was assessed as follows: Dengue hemorrhagic fever (DHF) as defined in the 1997 WHO definition. Severe dengue fever through the Dengue Case Trial Committee. The Dengue Case Trial Committee (DCAC) was made up of one voting chair, two voting members, and four independent non-voting statisticians. All three DCAC voting members are physicians and clinical dengue experts. DCAC members are not research investigators and there are no conflicts of interest that could bias the review of trial data. All non-hospitalized cases are considered non-severe. The criteria for DCAC severe dengue cases blinded for virologically confirmed hospitalized dengue cases are: 1) bleeding abnormalities, necessary for response to a bleeding episode such as blood transfusion, nasal packing, or hormone therapy to be considered serious If significant intervention is required or bleeding has occurred in a vital organ, such as the brain; 2) Plasma leakage, if considered serious, there must be evidence of both plasma leakage and functional impairment (plasma leakage includes clinical evidence, radiological evidence, or elevated hematocrit greater than 20% above normal or baseline; functional impairment is shock or as dyspnea); 3) Liver, if considered severe, evidence of both hepatitis and functional impairment is required (hepatitis is defined as aspartate aminotransferase [AST] or alanine aminotransferase [ALT]) > 10 upper limit of normal [ ULN]; Disorder defined as prothrombin [PT] >1.5 ULN or hypoalbuminemia; 4) renal, serum creatinine > 2.5 fold ULN or require dialysis; 5) Heart, heart-specific abnormalities (i.e. not due to intravascular volume depletion) and evidence of functional impairment (internal abnormalities such as myocarditis, pericarditis and myocarditis; functional impairments such as new conduction abnormalities resulting in irregular heart rhythms) [i.e., not a temporary first degree heart block]); 6) any abnormalities except central nervous system, simple febrile convulsions or brief delirium; 7) Shock, all shock cases are considered serious. At least one functional impairment (criteria 3,4,5,6) needs to be present, but the full data were considered by the member in their evaluation.

Additional results of Part 1 and Part 2 are provided in Tables 7a-c.

Table 7a Distribution of cases contributing to the primary endpoint by set subgroups per protocol (30 days after second vaccination, i.e. 12 months after second vaccination until end of part 1)

VCD, virologically-confirmed dengue; DHF, dengue, hemorrhagic fever

^a seronegative for all serotypes; Baseline seropositive, defined as a mutual neutralizing antibody titer ≧10 for one or more serotypes.

^b VCD cases meeting the WHO 1997 DHF criteria; incidence density, defined as the number of cases per 100 individual-risk years; Percentages are based on the total number of evaluated participants (the denominator) per protocol set.

^c Two severe VCDs were not classified as DHF.

^d The number of cases identified was sufficient to provide a reasonably accurate assessment of vaccine efficacy for all individual serotypes except DENV-4.

Table 7b Distribution of cases contributing to secondary endpoints by set subgroups per protocol (30 days after second vaccination, i.e. 18 months after second vaccination until end of part 2)

VCD, virologically-confirmed dengue;

^a seronegative for all serotypes; Baseline seropositive, defined as a mutual neutralizing antibody titer ≧10 for one or more serotypes.

Table 7c Distribution of events contributing to secondary endpoints by safety set (1st vaccination until end of part 2, i.e. 21 months after 1st vaccination)

VCD, virologically-confirmed dengue;

Between ^a : VCD after the first vaccination and before the second vaccination.

^b seronegative for all serotypes; Baseline seropositive, defined as a mutual neutralizing antibody titer ≧10 for one or more serotypes.

[Table 7d] Dengvaxia® VCD (first vaccination up to 25 months after first vaccination (ie, 13 months after third vaccination), ITT from CYD15, ages 9-16 years) ^a

^a Luis Villar et al. Efficacy of tetravalent dengue vaccine in children in Latin America: N Engl J of Med 2015 Vol. 372 No2, 113-123

Clinical signs and symptoms of virologically confirmed dengue cases during the Part 1 study period in the safety set data are presented in Table 8 .

TABLE 8 Clinical Signs and Symptoms of Virologically Confirmed Dengue Cases During the Part 1 Study Period (Safety Set Data)

VCD, virologically-confirmed dengue; ALT, alanine aminotransferase; AST, aspartate aminotransferase

^a Duration of fever defined as the date of onset of earliest symptoms vs. most recent symptoms plus end of day (symptoms considered include fever and any general symptoms).

^b An increase in hematocrit, defined as the maximum hematocrit from 3 to 7 days after the onset of the fever, is >20% greater than the minimum hematocrit before 3 days or 7 days after the onset of the fever.

^c For platelet, ALT and AST data, evaluation within 14 days of onset of fever was considered.

N refers to the number of VCD cases with data available for specific parameters

e) immunogenicity

The highest geometric mean titer (GMT) was observed for DENV-2 irrespective of baseline serum status (Table 10). A very high tetravalent seropositive rate (99.5%) was observed at baseline seronegative 1 month after the second dose (Tables 9 and 10).

The seropositive rates (% of seropositive subjects) for each of the four dengue serotypes were: pre-vaccination at day 1 (0 months), 30 days (1 month) after the first vaccination, and 90 days (3 months) vaccine Before vaccination, at Day 120 (4 months), at Day 270 (9 months), at Day 450 (15 months), and then after the annual second vaccination. Seropositive rates by dengue serotype (% participants, 95% CI) per protocol set for immunogenicity data for days 0, 30, 90, 120 and 270 are shown in Table 9.

Dengue fever serotypes for 3 or more serotypes (trivalent) and for all 4 serotypes (tetravalent) per protocol set for immunogenicity data for days 0, 30, 90, 120 and 270 days Seropositive rates by (% participants, 95% CI) are shown in Table 9. The quadrivalent seropositive rate is high (>91%) at baseline seronegative 6 months after the second dose.

TABLE 9 Seropositive Rate (% Participants, 95% CI) by Dengue Serotype (set per protocol for immunogenicity data)

Seropositive rate by dengue serotype (% participants, 95% CI) (per protocol set for immunogenicity data; seropositive defined as mutual neutralizing antibody titers ≥10; baseline seronegative defined as seronegative for all serotypes) ; baseline seropositivity, defined as seropositive for one or more serotypes; N represents the number of participants in the analysis set; the number of participants assessed at each time point may vary)

Geometric mean titers (GMTs) of neutralizing antibodies (microneutralization test [MNT]) for each dengue serotype were measured on day 1 (month 0) before vaccination, on day 30 (month 1) after the first vaccination, pre-vaccination at day 90 (3 months), at day 120 (4 months), at day 270 (9 months), at day 450 (15 months) and then after the annual second vaccination. Geometric mean titers (95% CI) by dengue serotype mean per protocol set for immunogenicity data for days 0, 30, 90, 120 and 270 are shown in Table 10.

Table 10: Geometric mean titers (95% CI) by dengue serotype (set per protocol for immunogenicity data)

Vaccine viremia is assessed by three PCRs: dengue detection RT-PCR, vaccine screening PCR and TDV sequencing in subjects with fever within 30 days of each vaccination.

f) safety

The rates of severe adverse events (SAEs) were similar in the vaccine and placebo groups (3.1% and 3.8% of participants, respectively, Table 11). 1 vaccinated and 4 placebo recipients experienced SAEs considered to be related to receiving blinded study product by the investigator (2 experienced hypersensitivity, 2 diagnosed dengue, 1 diagnosed DHF) . There were 5 deaths during Part 1 and all were considered unrelated to the study product or study procedure. The total rates of unattractive AEs were similar between the vaccine and placebo groups. The most commonly (≥1% of vaccine recipients) reported unattractive AEs within 4 weeks of administration of any dose, depending on duration of preference, were fever (1.5% in vaccine group; 1.4% placebo), nasopharyngitis (2.7% in vaccine; placebo). 3.0%), upper respiratory tract infections (2.6% vaccine; 2.9% placebo) and viral infections (1.1% vaccine; 0.9% placebo). Induced local reactions were reported more frequently in the vaccine group.

[Table 11a] Summary of safety data. Subjects having at least one side effect after administration of any vaccine dose. Data provided as number of events (percent of subjects; subjects/total [n] number of subjects[N]), unless otherwise stated (safety set data)

AEs, adverse events; SAE, severe adverse events; IP, Research Product/TDV

^a IP-related, defined as related to the study product as assessed by the Investigator

^b Only participants with available routine data were evaluated

^c All injection site (attracted local) reactions considered IP-related

Table 11b Number of participants with severe adverse events after any vaccination during Part 1 by MedDRA (Medical Dictionary of Modulatory Activity) systemic organ class in order of decreasing frequency (%) (due to risk of unblinding) Safety set data provided by TDV and placebo groups for events that occurred in >3 participants).

* Total column includes participants receiving both TAK-003 and placebo due to dosing error and excluded from TAK-003 and placebo groups. In the column headings, N represents the number of participants in the safety set.

[Table 11c] Number of participants with any serious unattractive adverse events after any vaccination during Part 28 by MedDRA systemic organ class in order of decreasing frequency (%) of participants (>6 participants due to risk of unblinding) a subset of the safety set data provided by the TDV and placebo groups for events that occurred in

* Total column includes participants receiving both TAK-003 and placebo due to dosing error and excluded from TAK-003 and placebo groups. In the column headings N refers to the number of participants in a subset of the safety set.

Table 11d Summary of routines reporting injection site reactions up to 7 days and systemic adverse events up to 14 days after any vaccination (a subset of safety-setting data). Data provided as number of participants with event/number of participants rated in the analysis set (% of participants assessed with event).

Additionally, studies are possible to evaluate the efficacy of a booster dose as a follow-up study of the Phase III study such that the booster is given 4 to 4.5 years after the second dose in a large sufficient subset of the Phase III studies described above, wherein: The subset includes, for example, at least 20 subjects or at least 200 subjects.

Example 4: Concomitant administration of hepatitis A vaccine and dengue vaccine

4.1 Introduction, purpose and goals of the study

A randomized, observer-blind, Phase 3 trial was conducted in 900 healthy adult subjects aged 18 to 60 years (distributed across the entire age range) in countries non-endemic for Dengue Fever Disease and Hepatitis A Virus (HAV). Two doses of tetravalent dengue vaccine TDV (subcutaneous (SC) injection) and a single dose of HAV vaccine (containing inactivated HAV: intramuscular (IM) injection) and simultaneous administration of TDV (SC injection) on the same day. Immunogenicity and safety were investigated.

The purpose of the study was to evaluate whether the HAV vaccine can be safely administered on the same day as TDV as a travel vaccine prior to international travel of subjects to HAV and dengue fever (DENV)-endemic countries.

The primary objective of this study was in DENV/HAV-naive subjects 1 month post-vaccination compared to 1 dose of TDV equivalent on the same day compared to 1 dose of HAV vaccine administered concurrently with placebo on the same day and administered on the same day. To demonstrate non-inferiority (NI) of immune response to one dose of HAV vaccine administered simultaneously on the same day.

The secondary objective of this study was to describe TDV-induced immunogenicity following a single dose of DENV/HAV-naive subjects; To describe TDV-induced immunogenicity following two doses of TDV administered 90 days apart in DENV/HAV-naive subjects; Describe HAV vaccine induced immunogenicity in DENV/HAV-naive subjects; To evaluate the safety profile after each vaccination in all test groups.

4.2 Eligibility Criteria

Criteria for inclusion include:

1. Participants are inclusively 18 to 60 years of age.

2. Participants in good health at trial entry as determined by medical history, physical examination (including vital signs) and the investigator's clinical judgment.

3. Consent/Written Notice Participants who have signed and dated the consent form and, after the nature of the examination has been explained in accordance with local regulations, all necessary privacy authorization requirements prior to commencement of any examination procedure.

4. Participants who are able to comply with the exam procedure and are available for the subsequent period.

Exclusion criteria include:

1. Participants with an elevated oral temperature (≥38°C or 100.4°F) within 3 days of the intended vaccination day.

2. Known hypersensitivity or allergy to any vaccine component (including excipients in study vaccine or placebo).

3. Participants with a behavioral or cognitive impairment or mental illness that, in the opinion of the Investigator, could interfere with the participant's ability to participate in the trial;

) 증후군)의 병력이 있는 참여자.4. Progressive or severe neurological disorders, seizure disorders or neuroinflammatory disorders (e.g., Guillain-Barré)

) syndrome).

5. Participants with medical history or any disease in the opinion of the Investigator that participation in the trial could interfere with trial results or pose additional risks to the participant.

6. Impairment/alteration of known or suspected immune function, including:

1. Chronic use (inhalation, intranasal or topical corticosteroids) of oral steroids (20 mg/day prednisone ≥12 weeks/≥2 mg/kg body weight/day equivalent to prednisone ≥2 weeks) within 60 days prior to Day 1 (M0) use of steroids allowed).

2. Inoculation of parenteral steroids (20 mg/day prednisone ≥12 weeks/≥2 mg/kg body weight/day equivalent to prednisone ≥2 weeks) within 60 days before Day 1 (M0).

3. Scheduled dosing during the trial period of immunoglobulins and/or any blood products within 3 months prior to Day 1 (M0).

4. Immunostimulatory inoculation within 60 days prior to Day 1 (M0).

5. Immunosuppressive therapy, such as chemotherapy or radiation therapy, within 6 months prior to Day 1 (M0).

6. Human Immunodeficiency Virus (HIV) infection or HIV-associated disease.

7. Hepatitis A virus (HAV) infection.

8. Hepatitis C virus infection.

9. Genetic Immunodeficiency.

7. Abnormalities in spleen or thymus function.

8. Participants with known bleeding predisposition or conditions that may be associated with prolonged bleeding time.

9. Participants with any severe chronic or progressive disease (eg, neoplasia, insulin dependent diabetes mellitus, heart, kidney or liver disease) at the discretion of the Investigator.

10. Participants with a body mass index (BMI) of 35 kg/m^2 (=weight in kg/[height in meters ² ])).

11. Participants who are willing to participate in a clinical trial with another study product prior to Day 1 (M0) 30 days before or at any point during the conduct of the study in another clinical trial.

12. Participants who receive any other vaccine within 14 days (for inactivated vaccine) or 28 days (for live vaccine) prior to admission to the trial, or who plan to receive any vaccine within 28 days of trial vaccine administration .

13. Previous HAV vaccination (in clinical trials or with approved products).

14. Participants involved in the conduct of the trial or their immediate family

15. Participants with a history of substance or alcohol abuse within the past 2 years.

16. Female participants who are pregnant or breastfeeding.

17. Women of childbearing potential who are sexually active and have not used any acceptable method of contraception for at least 2 months prior to Day 1 (M0).

1. Fertility is defined as the condition after the onset of menarche and none of the conditions of bilateral fallopian tube ligation (at least 1 year ago), bilateral oophorectomy (at least 1 year ago), or hysterectomy are met.

2. Acceptable methods of contraception are defined as one or more of the following:

i. Hormonal contraceptives (eg, oral, injection, transdermal patches, implants, cervical rings).

ii. Blocking each and every time during intercourse (condom with spermicide or diaphragm with spermicide).

iii. Intrauterine device (IUD). iv. A monogamous relationship with a partner who has had a vasectomy (partner must have had a vasectomy for at least 6 months prior to Day 1 [M0]).

Other methods of contraception may be considered in agreement with the sponsor and may only be implemented after substantive amendments have been approved by regulatory authorities and appropriate ethics committees.

18. Females of childbearing potential who are sexually active and refuse to use an acceptable method of contraception for up to 6 weeks after the last dose of study vaccine (Day 90 [M3]). They should also be advised not to donate eggs during this period.

19. Any positive or uncertain pregnancy test.

20. Prior and planned vaccination (during trial conduct) against any flavivirus, including dengue, yellow fever (YF), Japanese encephalitis (JE) virus or tick-borne encephalitis.

21. Previous participation in any clinical trial of a dengue or other flavivirus (eg, West Nile [WN] virus) candidate vaccine, excluding participants who received placebo in that trial.

22. Participants who are currently or previously infected with flaviviruses such as dengue, Zika virus, YF, JE, WN fever, tick-borne encephalitis or Murray Valley encephalitis and have a history of long-term (≥1 year) residence in areas endemic to dengue fever; participants who are there.

23. Participants with contraindications, warnings and/or precautions for vaccination with the HAV vaccine specified in the product information.

4.3 Study design and vaccination

Eligible subjects were randomized equally (1:1:1 ratio) into one of the following 3 trial groups (300 subjects per group).

- Group 1: HAV vaccine (IM) and placebo (SC) concurrently on the same day administered on Day 1 (Month 0); Placebo (SC) administered on Day 90 (Month 3).

-Group 2: TDV (SC) and placebo (IM) simultaneously on the same day administered on Day 1 (Month 0); TDV (SC) administered at day 90 (3 months).

-Group 3: TDV (SC) and HAV vaccine (IM) administered simultaneously on the same day on day 1 (month 0); TDV (SC) administered at day 90 (3 months).

A more detailed scheme of the study design is shown in FIG. 6 . Up to 28 days prior to the first vaccination, admissions were performed and blood samples were obtained to screen for anti-HAV antibodies. On Day 1, pre-vaccinated blood samples were obtained. 3 At 30 days post-vaccination (after the first vaccination on Day 1), blood samples were obtained. Another blood sample was obtained on day 120 (after the first vaccination on day 1). A safety follow-up is performed on Day 270 (after the first vaccination on Day 1).

TDV was prepared as described in Example 1. Each subcutaneous dose of TDV had a volume of 0.5 ml, and the concentrations of the four dengue serotypes in TDV at each dose were 5.1 log ₁₀ pfu/0.5 ml, 4.5 log ₁₀ pfu/0.5 ml, 5.4 log ₁₀ pfu/, respectively. TDV-1, TDV-2, TDV-3 and TDV-4 at 0.5 ml and 5.9 log ₁₀ pfu/0.5 ml. Each subcutaneous dose contains TDV dispersed in 0.5 ml of an aqueous solution containing Pluronic F127 (10.6 mg/ml), trehalose dihydrate (170 mg/ml) and human serum albumin (1.08 mg/ml).

The HAV vaccine contains an inactivated hepatitis A virus derived from hepatitis A virus strain HM-175 (see definition above) and is commercially available under the trade designation HAVRIX® as described above. The intramuscular dose of the HAV vaccine administered to groups 1) and 3) was 1 ml and each 1 ml dose had a viral antigen activity of about 1440 EL.U., wherein the viral antigen was 0.5 mg aluminum in the form of aluminum hydroxide. adsorbed to the phase. Hepatitis A vaccine contains excipients in the form of amino acid supplement (about 0.3% w/v) and polysorbate (about 0.05 mg/ml) dissolved in phosphate buffered saline solution.

The test vaccine, co-administered on the same day, was injected into the contralateral arm. Normal saline solution for injection (0.9% NaCl) was used as placebo. Blood samples for anti-HAV antibody testing were collected at screening from all subjects to exclude subjects positive for anti-HAV antibody for up to 28 days prior to vaccination (see FIG. 6 ). Because all subjects were followed up for 6 months after the second vaccine at Day 90 (Month 3), the study duration was either 270 days or 9 months (excluding the screening period) for each subject. Outside the context of this trial, subjects in Groups 1 and 3 will receive a HAV vaccine booster dose after completion of the trial procedure on Day 270 (Month 9).

Dengue fever neutralizing antibody (micro-neutralization test (MNT50)) was administered before the first test vaccination (1 day (0 months)), after the first test vaccination 1 month (30 days (1 month)) and after the second test vaccination Measurements were made using blood samples collected at 1 month (120 days (4 months)). Blood samples for determination of anti-HAV antibodies (enzyme-linked immunosorbent assay (ELISA)) were collected before the first test vaccination (day 1 (0 months) and 1 month after the first test vaccination (30 days (1 month)) ) were collected.

4.4 Primary endpoints

Primary endpoints included the ratio of HAV/DENV naive subjects at baseline that were serum protected against HAV at baseline to Day 30 (Month 1) as determined by ELISA (Enzyme Linked Immunosorbent Assay). In other words, the primary endpoint includes seroprotection rate (SPR). Seroprotection is defined as serum anti-HAV antibody levels ≧10 mIU/mL. Immunological purity for HAV/DENV is defined as anti-HAV antibody levels <10 mIU/mL and mutual neutralizing titers <10 against all four dengue serotypes.

4.5 Secondary endpoints

a) secondary immunogenicity endpoints

The secondary endpoint was the geometric mean titer (GMT) of neutralizing antibodies to each of the four dengue serotypes at Day 30 (Month 1) and at Day 120 (Month 4) as determined in HAV/DENV-naive subjects at baseline (microneutralization test) (MNT50)); In each of the four dengue serotypes at Day 30 (Month 1) and Day 120 (Month 4) ?? proportion of HAV/DENV-naive subjects at baseline who are seropositive (seroprotection rate); and geometric mean concentrations (GMC) of anti-HAV antibody at Day 30 (Month 1) in HAV/DENV-naive subjects at baseline.

Seropositive for dengue virus is defined as a reciprocal neutralizing titer ≧10 for any four dengue serotypes in the secondary immunogenicity endpoint.

b) secondary safety endpoints

Secondary safety endpoints included the frequency and severity of induced local adverse events (AEs) for 7 days after each trial vaccination; frequency and severity of induced systemic AEs for 14 days after each test vaccination; percentage of subjects with any unattractive AEs for 28 days after each test vaccination; percentage of subjects with severe adverse events (SAEs) throughout the trial; and the percentage of subjects with medically attended adverse events (MAAE) throughout the trial.

4.6 Analysis set of studies

Table 12 below shows each assay set in this study. Collectively, 1199 subjects belonged to the “all screened” group and included all subjects who signed the written informed consent regardless of whether or not screening failed. After initial screening, 900 subjects were included in the “randomized set”, which included all randomized subjects whether or not they received any IP dose. The safety set of 897 subjects included all randomized subjects who received >1 dose of IP. The immunogenic subjects included a total of 359 subjects and are subdivided into the following four subsets. The HAV-Complete Assay Set (HAV-FAS) includes all randomized subjects in the immunogenic subset who received >1 dose of test vaccine with available 1-day and 30-day anti-HAV antibody measurements. The HAV-Protocol Per Set (HAV-PPS) includes all HAV- and DENV-naive subjects from HAV-FAS who did not have a major protocol violation. The TDV-Complete Assay Set (TDV-FAS) includes all randomized subjects in the immunogenic subset who received ≧1 dose of test vaccine with available daily and ≧1 post-dose measurements. The TDV-Protocol Per Set (TDV-PPS) of 197 subjects included all HAV- and DENV-naive subjects from TDV-FAS with no major protocol violations.

[Table 12] Analysis set of studies.

¹ All screened: All subjects who signed written informed consent regardless of whether the subject failed screening

² Randomized Set: All randomized subjects regardless of whether they received any dose of test vaccine.

³ Safety Set: All randomized subjects who received > 1 dose of study vaccine.

⁴ HAV-FAS: All randomized subjects in the immunogenic subset who received ≧1 dose of test vaccine with available 1-day and 30-day HAV measurements.

⁵ HAV-PPS: All HAV & DENV-naive subjects from HAV-FAS, without any major protocol violations.

⁶ TDV-FAS: All randomized subjects in immunogenic subjects who received ≧1 dose of test vaccine and available 1 day and ≧1 post-dose measurements.

⁷ TDV-PPS: All HAV & DENV-naive subjects from TDV-FAS, without any major protocol violations.

⁸ Subjects excluded from TDV-PPS but included in the analysis for the primary non-inferiority goal had their Day 30 measurements outside the protocol definition visit window

From a total of 359 subjects in the immunogenicity subset (including all subjects vaccinated ≧1), 13 subjects were excluded from HAV-FAS because they did not provide valid baseline and post-dose measures (Day 30) for HAV. . Additionally, HAV-PPS includes all HAV- and DENV-naive subjects from HAV-FAS who did not have major protocol violations. Subjects analyzed for the primary non-inferiority goal were based on HAV-PPS subjects in the HAV/Pbo and HAV/TDV groups, where. Based on the HAV-PPS of these two groups (HAV/Pbo and HAV//TDV), some subjects were not included in the 30-day analysis because these subjects had their 30-day measurements outside the visit window defined in the protocol. (6 subjects in the HAV/Pbo group and 2 subjects in the HAV/TDV group). Therefore, a total of 69 subjects were included in the TDV/Pbo group and a total of 79 subjects were included in the HAV/TDV group to analyze the primary non-inferiority goal.

From a total of 359 subjects in the immunogenicity subset (including all subjects vaccinated ≧1), a total of 11 subjects had baseline and at least one post-dose measurement (days 30 and/or 120) for which they were effective for TDV. Because it was not provided, it was excluded from the TDV-FAS. In addition, a total of 151 if not HAV and DENV-naive at baseline, did not receive both vaccinations 1 and 2, or if vaccination 2 (usually Day 90) was outside window -15/+25, or if a major protocol violation occurred 3 subjects were excluded from TDV-PPS.

4.7 HAV Baseline Serum Status and Demographics and Baseline Characteristics

The safety set assessed for baseline HAV antibody levels included a total of 362 subjects, of which 27.3% were HAV seropositive at baseline (see Table 13).

HAS-FAS included a total of 346 subjects assessed for baseline HAV antibody levels (see Table 13). HAV-purity was defined as an anti-HAV antibody (ab) level of <10 mIU/ml. However, the ELISA used for serological analysis cannot be confirmed below the level of 12.5 mIU/ml. Qualitative screening tests had specifications that virtually reached a lower limit of quantitation of 70 mIU/ml. In view of these criteria, 72.5% of subjects with the HAS-FAS assessed for baseline HAV antibody levels were HAV naive at baseline (see Table 13).

Table 13: HAV baseline serostats in the safety set and in HAV-FAS

The HAV-PPS included a total of 227 subjects (see Table 14). The mean age of the total number of DENV- and HAV-naive HAV-PPS was 34.8 years. 31.3% of the total number of subjects on HAV-PPS were female (see Table 14). Overall, 97.8% of all subjects in HAV-PPS were of non-Hispanic or Latino ethnicity, notably 89.9% of HAV-PPS participants were racial “white European”, particularly those of travelers from HAV- and dengue non-endemic countries. reflect (see Table 14).

The safety set included a total of 897 subjects (see Table 14). The mean age of the total number of subjects in the safety set was 35.4 years, of which 31.3% were female. Collectively, 97.7% of all subjects in HAV-PPS were of non-Hispanic or Latino ethnicity, notably 87.1% of HAV-PPS participants were racial “white European”, particularly the situation of travelers from HAV- and dengue non-endemic countries. reflect (see Table 14).

[Table 14] Demographics and Baseline Characteristics (HAV-PPS and Safety Set)

4.8 Study Results

a) Primary endpoint and sensitivity analysis

This study was successful in meeting the primary goal of non-inferiority for concurrent administration of HAV and TDV on the same day. Table 15 shows the seroprotection rates (SPR) of groups 1 (administered with HAV/Pbo) and 3 (administered with HAV/TDV) at 30 days after the first vaccination (Day 1), at 30 days, in the HAV/Pbo group. SPR differences between the and HAV/TDV groups, and the confidence intervals (CIs) of these SPR differences for HAV and DENV-baseline naive subjects are shown. These values (SPR, SPR difference; CI) were used to evaluate the primary endpoint of the study. Table 15 further shows these values for the results of three sensitivity analyzes (also used for non-inferiority assessments), wherein the subjects had different, ie, mixed, HAV/TDV serum status at baseline. The non-inferiority between hepatitis A vaccine and tetravalent dengue vaccine when administered concurrently on the same day in this study was calculated using the Newcomb score method with a non-inferiority limit of less than 10% for group 1 (HAV and Conclude if the seroprotection rate (SPR) between placebo administered on the same day 1) and group 3 (HAV and TDV received on the same day 1) had an upper bound of two-sided 95% confidence intervals. Criteria are met for each group in Table 15. In the primary endpoint group, the upper bound of 95% CI of SPR difference was 4.31% , which is below the non-inferiority limit of 10% (see second line from Singhi in Table 15).

As noted above, sensitization assays 1 to 3 are used to evaluate a population comprising subjects seropositive for dengue and/or hepatitis A at baseline, particularly for "real life" travel in non-endemic countries. Reflecting the clinical setting, where subjects, i.e. travelers planning to go to countries endemic to dengue and HAV, are not always aware of their HAV and/or dengue serum status prior to requiring pre-travel vaccination.

The goal of non-inferiority of concurrent dosing on the same day was also sensitized assays 1 to 3 (upper bound CI of 95% difference in SPR: 3.21%; 2.93%; and 2.55%, which were below the non-inferiority limit of 10%). ) was satisfied in In addition, the SPR of the HAV/TDV group (98.8%; 99.0%; 99.1%, respectively, see Table 15) was higher than the SPR of the HAV/Pbo group, respectively (96.2%; 96.9%; 97.2%, respectively, see Table 15).

Therefore, due to the non-inferiority of concurrent administration of HAV vaccine and TDV to subjects with mixed baselines with respect to HAV and all dengue serostats (and baseline naivety) on the same day of HAV vaccine and TDV, prior to concurrent administration of HAV vaccine and TDV on the same day , there is no need to determine or know the baseline serum status of a subject with respect to each of the two diseases.

[Table 15] Primary endpoints: non-inferiority (NI) assessment and sensitivity analysis

b) secondary immunogenicity endpoints

Table 16 shows that Day 1 before vaccination and Day 30 after primary vaccination (Day 1) for subjects of DENV-PPS, including groups receiving HAV and placebo (Pbo) as well as groups receiving HAV/TDV, TDV and placebo, respectively. , and GMT (for each of the four dengue virus serotypes DENV-1 to DENV-4) as measured on day 120 after the first day vaccination (Day 1). Table 16 shows the positive trend in favor of the concurrent dosing group (administered with HAV/TDV) on the same day for all dengue GMTs.

This positive trend in favor of co-administration on the same day and Day 30 synergy of the same co-administered vaccine is also confirmed in Table 17.

Table 16: Immunogenicity of DENV-PPS: GMT of DENV MNT50. In particular, the DENV GMT for each serotype is shown with respect to the mean titer of neutralizing antibody measured by MNT50 for each dengue serotype by test visit.

¹ SD = standard deviation.

[Table 17] Secondary endpoint: HAV geometric mean concentration (GMC)

c) secondary safety endpoints

The safety set was investigated for induced adverse events, induced systemic adverse events, unattractive adverse events, and severe adverse events throughout the study. Tables 18a-21c show the results of the study in the safety set with respect to each secondary safety endpoint.

Table 18a Frequency of Attracted Local AEs and Attracted Systemic AEs after First Vaccination - Safety Set

Table 18b Frequency of Attracted Local AEs and Attracted Systemic AEs after Second Vaccination - Safety Set

Table 18c Frequency of Attracted Local AEs and Attracted Systemic AEs after Any Vaccination - Safety Set

Table 19a Overview of Unattracted AEs by Day 28 Post Vaccination (Post 1st Vaccination) - Safety Set

Table 19b Summary of Unattracted AEs by Day 28 Post Vaccination (Post Second Vaccination) - Safety Set

Table 20: Most Common Unattracted AE* - Safety Set by Day 28 Post Vaccination (Post any Vaccination)

Table 21a Safety: Overview of Serious Adverse Events (SAE) after Any Dose - Safety Set

Table 21b Safety: Severe Adverse Events by Systemic Organ Class and Preferred Duration - Safety Set after 1st and 2nd Dosing

Table 21c Safety: Serious Adverse Events by Systemic Organ Class and Preferred Duration - Safety Set - Continued after Dosing 1 and 2

List of first items of the present invention

1. A method of preventing hepatitis A and dengue disease in a subject or population of subjects, the method comprising administering unit doses of a hepatitis A vaccine and a dengue vaccine composition simultaneously on the same day, wherein the unit dose A method comprising a tetravalent dengue virus composition comprising the four wildly attenuated dengue virus strains.

2. The method according to item 1, wherein the hepatitis A vaccine is an inactivated virus vaccine.

3. The method according to item 1 or 2, wherein the dengue vaccine composition comprises upon reconstitution with 0.5 mL of a pharmaceutically acceptable diluent:

(i) a chimeric dengue serotype 2/1 strain at a concentration of at least 3.3 log10 pfu/0.5 mL,

(ii) a dengue serotype 2 strain at a concentration of at least 2.7 log10 pfu/0.5 mL;

(iii) a chimeric dengue serotype 2/3 strain at a concentration of at least 4.0 log10 pfu/0.5 mL, and

(iv) a chimeric dengue serotype 2/4 strain at a concentration of at least 4.5 log10 pfu/0.5 mL.

4. The method according to any one of the preceding items, wherein the subject population or subjects are seronegative for all dengue fever serotypes.

5. The method according to any one of the preceding items, wherein said unit dose of said dengue vaccine composition is administered by subcutaneous injection, and said hepatitis A vaccine is administered by intramuscular injection, said injection preferably in the arm, more preferably administered to the deltoid region of the arm.

6. The method according to item 5, wherein the unit dose of the dengue vaccine composition and the hepatitis A vaccine are administered at different anatomical sites, for example the contralateral arm.

7. The method according to any one of the preceding items, wherein the two unit doses of the dengue vaccine composition are administered within at least 12 months or within 6 months or within 3 months.

8. The method according to item 7, which comprises, in particular, administration of two said unit doses of said dengue vaccine composition and one dose of said hepatitis A vaccine according to the following schedule:

- a first concurrent administration of a first unit dose of said dengue vaccine composition and said hepatitis A vaccine on day 0, and

- a second administration of a second unit dose of said dengue vaccine composition after said first simultaneous administration, eg about 3 months.

9. The method according to any one of the preceding items, wherein the subject population or subjects are between 2 and 60 years of age.

10. The method according to any one of the preceding items, wherein the subject population or subjects are from an area endemic to dengue.

11. The method according to any one of items 1 to 9, wherein said subject population or subjects are from a dengue non-endemic area, preferably from a dengue fever non-endemic and hepatitis A non-endemic area.

12. The method according to any one of the preceding items, wherein the hepatitis A vaccine comprises hepatitis A virus derived from hepatitis A virus strain HM-175.

13. The method according to any one of the preceding items, wherein the hepatitis A vaccine comprises an inactivated hepatitis A virus and the inactivated hepatitis A virus is from hepatitis A virus strain HM-175.

14. The method according to any one of the preceding items, wherein the hepatitis A vaccine comprises an inactivated hepatitis A virus, and the inactivated hepatitis A virus is adsorbed on aluminum.

15. The method according to item 14, wherein the aluminum is aluminum hydroxide or aluminum hydroxyphosphate sulfate.

16. A phosphate buffered saline solution according to any one of the preceding items, wherein said hepatitis A virus comprises an inactivated hepatitis A virus, and wherein said hepatitis A vaccine is dissolved therein in amino acid form and in polysorbate form. and an excipient.

17. The hepatitis A virus according to any one of the preceding items, wherein the hepatitis A vaccine expresses the viral antigen at a concentration ranging from 500 ELISA Units (EL.U.) to 2000 ELISA Units (EL.U.). Including method.

18. The method according to any one of the preceding clauses, wherein the method comprises prior to hepatitis A vaccine administration and prior to administration of the unit dose of the dengue vaccine composition, prior dengue infection and/or prior hepatitis A infection in the subject population or in the subject. does not include the step of determining whether or not

wherein the hepatitis A serum status and/or the dengue fever serum status of or of the subject population is unknown prior to administration of the hepatitis A vaccine and prior to administration of a unit dose of the dengue vaccine composition.

19. A previous dengue infection in or in said subject according to item 18, wherein said method comprises prior to, during, and any time after the administering step of administering the unit dose of the hepatitis A vaccine and the dengue vaccine composition in the subject population or at any time after the administering step. and/or determining whether there is a previous hepatitis A infection;

wherein the hepatitis A serum status and/or the dengue fever serum status of the subject population or of the subjects are determined prior to, during, and at any time after the administering step of the hepatitis A vaccine and a unit dose of the dengue vaccine composition. unknown, how.

20. The method according to any one of the preceding items, wherein the method comprises primary vaccination consisting of:

(A) a unit dose of a tetravalent dengue virus composition and type A requiring protection against dengue infection and hepatitis A infection without prior determination of whether there was a prior dengue infection and/or prior hepatitis A infection. selecting a subject for administration of a hepatitis vaccine, and

(B) administering to said subject a first unit dose of a tetravalent dengue virus composition and hepatitis A vaccine, and optionally

(C) administering at least one additional unit dose of a tetravalent dengue virus composition to the subject within 3 to 12 months of administration of the first unit dose, and optionally

(D) administering at least one additional dose of hepatitis A vaccine to the subject within 6 to 18 months of administration of the first unit dose.

21. The method according to any one of the preceding items, wherein the method comprises primary vaccination consisting of:

(A) selecting a subject for administration of a unit dose of a tetravalent dengue virus composition and hepatitis A vaccine in need of protection against dengue infection and hepatitis A infection, and

(B) administering to the subject a first unit dose of a tetravalent dengue virus composition and a hepatitis A vaccine, and

(C) administering two additional unit doses of the tetravalent dengue virus composition to the subject at about 6 and about 12 months of administration of the first unit dose and Form A at about 6 or about 12 months of administration of the first unit dose administering a hepatitis vaccine to the subject.

22. The method according to item 21, wherein step (A) is performed without determining whether there is a previous hepatitis A infection.

23. The composition according to any one of items 3 to 22, wherein upon reconstitution of the dengue vaccine composition with a pharmaceutically acceptable diluent, (i), (ii), (iii), and (iv) is pfu/0.5 mL of providing a total concentration, wherein, based on the total concentration of pfu/0.5 ml, the concentration of (ii) in pfu/0.5 mL is less than 10%, and the concentration of (iv) in pfu/0.5 mL is at least 50%; the concentration of (i) in pfu/0.5 mL is at least 1%, and the concentration of (iii) in pfu/0.5 mL is at least 6%, at least 8%, or at least 10%, or at least 12%, or at least 14%, or at least 16%, or at least 18%.

24. The method according to any one of the preceding items, wherein the method provides compatibility between the dengue vaccine composition and the hepatitis A vaccine.

25. The method according to any one of the preceding items, wherein the method provides for synergy between the dengue vaccine composition and the hepatitis A vaccine.

26. Non-inferiority in a non-inferiority clinical study according to any one of the preceding items, wherein the method comprises at least 60 or at least 120 healthy subjects divided into one subject population and one control subject population. wherein the subject population receives unit doses of hepatitis A vaccine and dengue vaccine composition on the same day simultaneously and the control subject population receives hepatitis A vaccine and placebo administration simultaneously on the same day.

27. The hepatitis A vaccine according to any one of the preceding items, wherein the hepatitis A vaccine is administered concurrently on the same day at unit doses of the hepatitis A vaccine and the dengue vaccine composition and is at least 30 seronegative with respect to all dengue virus serotypes at baseline. providing a hepatitis A seroprotection rate of at least 95% or at least 98% on Day 30 (on Day 0/1) after administration to a subject population of persons or at least 50 healthy subjects.

28. The method according to any one of the preceding items, wherein the method provides a difference in hepatitis A seroprotection rate with respect to hepatitis A mono-administration, wherein the difference is seronegative with respect to hepatitis A at baseline and at baseline. determined in a non-inferiority clinical study comprising at least 60 or at least 120 healthy subjects who were seronegative for all dengue virus serotypes,

the healthy subject

a) a subject population of at least 30 or at least 50 healthy subjects who received administration of a unit dose of a hepatitis A vaccine and a dengue vaccine composition (on Day 0/1) simultaneously on the same day, and

b) divided into a control subject population of at least 30 or at least 50 healthy subjects who received administration of hepatitis A vaccine and placebo (on Day 0/1) simultaneously on the same day,

wherein the difference is determined between the hepatitis A seroprotection rate of the control subject population at Day 30 post-dose (on Day 0/1) and the Hepatitis A seroprotection rate of the subject population at Day 30 post-dose (on Day 0/1), and ,

wherein the difference has a joint upper bound within a two-sided 95% confidence interval that is less than 10%.

29. At least 30 patients according to any one of items 1 to 26, wherein the hepatitis A vaccine receives unit doses of the hepatitis A vaccine and the dengue vaccine composition simultaneously on the same day and is seronegative with respect to hepatitis A at baseline or at least 95% or at least 98% or at least 99% hepatitis A seroprotection on Day 30 (on Day 0/1) after administration to a subject population of at least 50 healthy subjects, wherein said healthy subject is at baseline A method comprising healthy subject(s) seropositive for at least one dengue virus serotype and healthy subject(s) seronegative for all dengue virus serotypes at baseline.

30. The method according to any one of items 1-26, wherein the method provides a difference in hepatitis A seroprotection rate with respect to hepatitis A mono-administration, wherein the difference is at least seronegative with respect to hepatitis A at baseline. Healthy subject(s) determined in a non-inferior clinical study comprising 60 or at least 120 healthy subjects, said healthy subjects being seropositive for at least one dengue virus serotype at baseline and all dengue virus at baseline healthy subject(s) that are seronegative with respect to the serotype;

the healthy subject

a) a subject population of at least 30 or at least 50 healthy subjects receiving administration of unit doses of a hepatitis A vaccine and a dengue vaccine composition simultaneously on the same day (Day 0/1), wherein the subject population is at least at baseline a population of subjects, comprising healthy subject(s) seropositive for one dengue virus serotype and healthy subject(s) seronegative for all dengue virus serotypes at baseline; and

b) a control subject population of at least 30 or at least 50 healthy subjects who simultaneously received administration of hepatitis A vaccine and placebo on the same day (Day 0/1), wherein the control subject population has at least one dengue fever at baseline divided into a control subject population, comprising healthy subject(s) seropositive with respect to viral serotype and healthy subject(s) seronegative for all dengue virus serotypes at baseline;

wherein the difference is determined between the hepatitis A seroprotection rate of the control subject population at Day 30 post-dose (on Day 0/1) and the Hepatitis A seroprotection rate of the subject population at Day 30 post-dose (on Day 0/1), and ,

wherein the difference has a joint upper bound within a two-sided 95% confidence interval that is less than 10%.

31. The method according to any one of the preceding items, wherein the subject or population of subjects is exposed to a hepatitis A virus outbreak and/or a dengue virus outbreak.

32. The method according to any one of the preceding items, wherein the method is administered simultaneously with unit doses of hepatitis A vaccine and dengue vaccine composition on the same day and is seronegative with respect to hepatitis A vaccine at baseline and all dengue virus serum at baseline At least 70 mIU/ml or at least 80 mIU/ml or at least 90 mIU/ml on Day 30 (on Day 0/1) after administration to a subject population of at least 30 or at least 50 healthy subjects who are seronegative with respect to type A method of providing an anti-hepatitis A virus antibody Geometric Mean Concentration (GMC).

33. The method according to any one of the preceding items, wherein it is safe to concurrently administer unit doses of the hepatitis A vaccine and the dengue vaccine composition to the subject or population of subjects on the same day.

34. The method according to any one of the preceding items, wherein there are no severe side effects associated with simultaneous administration on the same day.

35. The method according to any one of the preceding items, wherein the method provides a geometric mean titer (GMT) of neutralizing antibody measured by MNT50, wherein the titer is

- at least 110 or at least 140 or at least 150 for dengue serotype 1,

- at least 3000 or at least 3500 or at least 3900 for dengue serotype 2,

- at least 100 or at least 120 or at least 140 for dengue serotype 3;

- at least 80 or at least 110 or at least 140 for dengue serotype 4,

At least 30 patients whose titers received unit doses of hepatitis A vaccine and dengue vaccine composition simultaneously on the same day and were seronegative for all hepatitis A at baseline and seronegative for all dengue virus serotypes at baseline or at Day 30 (on Day 0/1) after administration to a subject population of at least 50 healthy subjects.

36. The method according to any one of the preceding items, wherein the subject or population of subjects is between 18 and 60 years of age.

37. A kit for hepatitis A and dengue fever comprising:

At least

(a) a first container holding the hepatitis A vaccine, and

(b) a second container holding a unit dose of a dengue vaccine composition, wherein the unit dose comprises a second container comprising a tetravalent dengue virus composition comprising four wildly attenuated dengue virus strains.

Second item list of the present invention

1. Use of a hepatitis A vaccine for the manufacture of a medicament for preventing hepatitis A in a subject or population of subjects and as a unit dose of a dengue vaccine composition for the manufacture of a medicament for dengue disease in said subject or population of subjects. , wherein the prevention of hepatitis A and dengue disease comprises administering unit doses of the hepatitis A vaccine and the dengue vaccine composition simultaneously on the same day, wherein the unit dose comprises four wildly attenuated dengue virus strains. A use comprising a dengue virus composition.

2. Use according to item 1, wherein the hepatitis A vaccine is an inactivated virus vaccine.

3. Use according to item 1 or 2, wherein the dengue vaccine composition comprises, upon reconstitution with 0.5 mL of a pharmaceutically acceptable diluent:

(i) a chimeric dengue serotype 2/1 strain at a concentration of at least 3.3 log10 pfu/0.5 mL,

(ii) a dengue serotype 2 strain at a concentration of at least 2.7 log10 pfu/0.5 mL;

(iii) a chimeric dengue serotype 2/3 strain at a concentration of at least 4.0 log10 pfu/0.5 mL, and

(iv) a chimeric dengue serotype 2/4 strain at a concentration of at least 4.5 log10 pfu/0.5 mL.

4. Use according to any one of the preceding items, wherein the subject population or subjects are seronegative for all dengue serotypes.

5. The method according to any one of the preceding items, wherein said unit dose of said dengue vaccine composition is administered by subcutaneous injection, and said hepatitis A vaccine is administered by intramuscular injection, said injection preferably in the arm, more Preferably administered to the deltoid region of the arm.

6. Use according to item 5, wherein the unit dose of the dengue vaccine composition and the hepatitis A vaccine are administered at different anatomical sites, for example the contralateral arm.

7. Use according to any one of the preceding items, wherein the two unit doses of the dengue vaccine composition are administered within at least 12 months or within 6 months or within 3 months.

8. Use according to item 7, which comprises, in particular, administration of two said unit doses of said dengue vaccine composition and one dose of said hepatitis A vaccine according to the following schedule:

- a first concurrent administration of a first unit dose of said dengue vaccine composition and said hepatitis A vaccine on day 0, and

- a second administration of a second unit dose of said dengue vaccine composition after said first simultaneous administration, eg about 3 months.

9. Use according to any one of the preceding items, wherein the subject population or subjects are between 2 and 60 years of age.

10. Use according to any one of the preceding items, wherein the subject population or subjects are from an area endemic to dengue.

11. Use according to any one of items 1 to 9, wherein said subject population or subjects are from a dengue non-endemic area, preferably from a dengue fever non-endemic and hepatitis A non-endemic area.

12. Use according to any one of the preceding items, wherein the hepatitis A vaccine comprises hepatitis A virus derived from hepatitis A virus strain HM-175.

13. Use according to any one of the preceding items, wherein the hepatitis A vaccine comprises an inactivated hepatitis A virus and the inactivated hepatitis A virus is derived from the hepatitis A virus strain HM-175.

14. Use according to any one of the preceding items, wherein the hepatitis A vaccine comprises an inactivated hepatitis A virus, wherein the inactivated hepatitis A virus is adsorbed on aluminum.

15. Use according to item 14, wherein the aluminum is aluminum hydroxide or aluminum hydroxyphosphate sulfate.

16. A phosphate buffered saline solution according to any one of the preceding items, wherein said hepatitis A virus comprises an inactivated hepatitis A virus, and wherein said hepatitis A vaccine is dissolved therein in amino acid form and in polysorbate form. and excipients.

17. The hepatitis A virus according to any one of the preceding items, wherein the hepatitis A vaccine expresses the viral antigen at a concentration ranging from 500 ELISA Units (EL.U.) to 2000 ELISA Units (EL.U.). Including uses.

18. The method according to any one of the preceding clauses, wherein the method comprises prior to hepatitis A vaccine administration and prior to administration of the unit dose of the dengue vaccine composition, prior dengue infection and/or prior hepatitis A infection in the subject population or in the subject. does not include the step of determining whether or not

wherein the hepatitis A serum status and/or the dengue fever serum status of or of the subject population is unknown prior to administration of the hepatitis A vaccine and prior to administration of a unit dose of the dengue vaccine composition.

19. A previous dengue infection in or in said subject according to item 18, wherein said method comprises prior to, during, and any time after the administering step of administering the unit dose of the hepatitis A vaccine and the dengue vaccine composition in the subject population or at any time after the administering step. and/or determining whether there is a previous hepatitis A infection;

wherein the hepatitis A serum status and/or the dengue fever serum status of the subject population or of the subjects are determined prior to, during, and at any time after the administering step of the hepatitis A vaccine and a unit dose of the dengue vaccine composition. unknown, use.

20. Use according to any one of the preceding items, wherein the method comprises a primary vaccination consisting of:

(A) a unit dose of a tetravalent dengue virus composition and hepatitis A in need of protection against dengue infection and hepatitis A infection without determining if there is a prior dengue infection and/or prior hepatitis A infection selecting a subject for administration of the vaccine, and

(B) administering to said subject a first unit dose of a tetravalent dengue virus composition and hepatitis A vaccine, and optionally

(C) administering at least one additional unit dose of a tetravalent dengue virus composition to the subject within 3 to 12 months of administration of the first unit dose, and optionally

(D) administering at least one additional dose of hepatitis A vaccine to the subject within 6 to 18 months of administration of the first unit dose.

21. Use according to any one of the preceding items, wherein the method comprises a primary vaccination consisting of:

(A) selecting a subject for administration of a unit dose of a tetravalent dengue virus composition and hepatitis A vaccine in need of protection against dengue infection and hepatitis A infection, and

(B) administering to the subject a first unit dose of a tetravalent dengue virus composition and a hepatitis A vaccine, and

(C) administering two additional unit doses of the tetravalent dengue virus composition to the subject at about 6 and about 12 months of administration of the first unit dose and Form A at about 6 or about 12 months of administration of the first unit dose administering a hepatitis vaccine to the subject.

22. Use according to item 21, wherein step (A) is carried out without determining whether there is a previous hepatitis A infection.

23. The method according to any one of items 3 to 22, wherein upon reconstitution of the dengue vaccine composition with a pharmaceutically acceptable diluent, (i), (ii), (iii), and (iv) is pfu/0.5 mL of providing a total concentration, wherein, based on the total concentration of pfu/0.5 mL, the concentration of (ii) in pfu/0.5 mL is less than 10%, and the concentration of (iv) in pfu/0.5 mL is at least 50%; the concentration of (i) in pfu/0.5 mL is at least 1%, and the concentration of (iii) in pfu/0.5 mL is at least 6%, at least 8%, or at least 10%, or at least 12%, or at least 14%, or at least 16%, or at least 18%.

24. Use according to any one of the preceding items, wherein the method provides compatibility between the dengue vaccine composition and the hepatitis A vaccine.

25. Use according to any one of the preceding items, wherein the method provides synergy between the dengue vaccine composition and the hepatitis A vaccine.

26. Non-inferiority in a non-inferiority clinical study according to any one of the preceding items, wherein the method comprises at least 60 or at least 120 healthy subjects divided into one subject population and one control subject population. wherein the subject population receives unit doses of hepatitis A vaccine and dengue vaccine composition on the same day simultaneously and the control subject population receives hepatitis A vaccine and placebo administration simultaneously on the same day.

27. The hepatitis A vaccine according to any one of the preceding items, wherein the hepatitis A vaccine and the dengue vaccine composition are administered simultaneously on the same day and are seronegative with respect to hepatitis A at baseline, and at baseline all Hepatitis A seroprotection of at least 95% or at least 98% at Day 30 (on Day 0/1) after administration to a subject population of at least 30 or at least 50 healthy subjects seronegative with respect to the dengue virus serotype; provided, use.

28. The method according to any one of the preceding items, wherein the method provides a difference in hepatitis A seroprotection rate with respect to hepatitis A mono-administration, wherein the difference is seronegative with respect to hepatitis A at baseline and at baseline. determined in a non-inferiority clinical study comprising at least 60 or at least 120 healthy subjects who were seronegative for all dengue virus serotypes,

the healthy subject

c) a subject population of at least 30 or at least 50 healthy subjects receiving administration of unit doses of hepatitis A vaccine and dengue vaccine composition (on Day 0/1) simultaneously on the same day, and

d) divided into a control subject population of at least 30 or at least 50 healthy subjects receiving administration of hepatitis A vaccine and placebo (on Day 0/1) simultaneously on the same day,

the difference is determined between the hepatitis A seroprotection rate of the control subject population at Day 30 post-dose (on Day 0/1) and the hepatitis A seroprotection rate of the subject population at Day 30 post-dose (on Day 0/1), and ,

wherein the difference has an upper binding limit within a two-sided 95% confidence interval of less than 10%.

29. At least 30 patients according to any one of items 1 to 26, wherein the hepatitis A vaccine receives unit doses of the hepatitis A vaccine and the dengue vaccine composition simultaneously on the same day and is seronegative with respect to hepatitis A at baseline or at least 95% or at least 98% or at least 99% hepatitis A seroprotection on Day 30 (on Day 0/1) after administration to a subject population of at least 50 healthy subjects, wherein said healthy subject is at baseline Use, comprising healthy subject(s) seropositive for at least one dengue virus serotype and healthy subject(s) seronegative for all dengue virus serotypes at baseline.

30. The method according to any one of items 1-26, wherein the method provides a difference in hepatitis A seroprotection rate with respect to hepatitis A mono-administration, wherein the difference is at least seronegative with respect to hepatitis A at baseline. Healthy subject(s) determined in a non-inferior clinical study comprising 60 or at least 120 healthy subjects, said healthy subjects being seropositive for at least one dengue virus serotype at baseline and all dengue virus at baseline healthy subject(s) that are seronegative with respect to the serotype;

the healthy subject

c) a subject population of at least 30 or at least 50 healthy subjects receiving administration of unit doses of a hepatitis A vaccine and a dengue vaccine composition simultaneously on the same day (Day 0/1), wherein the subject population is at least at baseline a population of subjects, comprising healthy subject(s) seropositive for one dengue virus serotype and healthy subject(s) seronegative for all dengue virus serotypes at baseline; and

d) a control subject population of at least 30 or at least 50 healthy subjects receiving concurrent administration of hepatitis A vaccine and placebo on the same day (Day 0/1), said control subject population having at least one dengue fever at baseline divided into a control subject population, comprising healthy subject(s) seropositive with respect to viral serotype and healthy subject(s) seronegative for all dengue virus serotypes at baseline;

the difference is determined between the hepatitis A seroprotection rate of the control subject population at Day 30 post-dose (on Day 0/1) and the hepatitis A seroprotection rate of the subject population at Day 30 post-dose (on Day 0/1), and ,

wherein the difference has an upper binding limit within a two-sided 95% confidence interval of less than 10%.

31. Use according to any one of the preceding items, wherein the subject or population of subjects is exposed to hepatitis A virus outbreak and/or dengue virus outbreak.

32. The method according to any one of the preceding items, wherein the method is administered simultaneously with unit doses of hepatitis A vaccine and dengue vaccine composition on the same day and is seronegative with respect to hepatitis A vaccine at baseline and all dengue virus serum at baseline At least 70 mIU/ml or at least 80 mIU/ml or at least 90 mIU/ml on Day 30 (on Day 0/1) after administration to a subject population of at least 30 or at least 50 healthy subjects who are seronegative with respect to type Use of providing an anti-hepatitis A virus antibody geometric mean concentration (GMC).

33. Use according to any one of the preceding items, wherein the simultaneous administration of unit doses of said hepatitis A vaccine and said dengue vaccine composition to said subject or population of subjects on the same day is safe.

34. Use according to any one of the preceding items, wherein there are no serious side effects associated with simultaneous administration on the same day.

35. The method according to any one of the preceding items, wherein the method provides a geometric mean titer (GMT) of neutralizing antibody measured by MNT50, wherein the titer is

- at least 110 or at least 140 or at least 150 for dengue serotype 1,

- at least 3000 or at least 3500 or at least 3900 for dengue serotype 2,

- at least 100 or at least 120 or at least 140 for dengue serotype 3;

- at least 80 or at least 110 or at least 140 for dengue serotype 4,

At least 30 patients whose titers received unit doses of hepatitis A vaccine and dengue vaccine composition simultaneously on the same day and were seronegative for all hepatitis A at baseline and seronegative for all dengue virus serotypes at baseline or at Day 30 (on Day 0/1) after administration to a subject population of at least 50 healthy subjects.

36. Use according to any one of the preceding items, wherein the subject or population of subjects is between 18 and 60 years of age.

37. A kit for hepatitis A and dengue fever comprising:

At least

(a) a first container holding the hepatitis A vaccine, and

(b) a second container holding a unit dose of a dengue vaccine composition, wherein the unit dose comprises a second container comprising a tetravalent dengue virus composition comprising four wildly attenuated dengue virus strains.

<110> Takeda Vaccines, Inc. <120> METHODS FOR PREVENTING DENGUE AND HEPATITIS A <130> 2022-FPA-1270 <150> EP19 195 692.9 <151> 2019-09-05 <150> US16/561,953 <151> 2019-09-05 <150> PCT/US2019/049749 <151> 2019-09-05 <150> PID201907241 <151> 2019-08-16 <160> 16 <170> PatentIn version 3.5 <210> 1 <211> 10723 <212> DNA <213> chimeric dengue seroytpe 2/1 (MVS) <400> 1 agttgttagt ctacgtggac cgacaaagac agattctttg agggagctaa gctcaatgta 60 gttctaacag ttttttaatt agagagcaga tctctgatga ataaccaacg gaaaaaggcg 120 aaaaacacgc ctttcaatat gctgaaacgc gagagaaacc gcgtgtcgac tgtgcaacag 180 ctgacaaaga gattctcact tggaatgctg cagggacgag gaccattaaa actgttcatg 240 gccctggtgg cgttccttcg tttcctaaca atcccaccaa cagcagggat attgaagaga 300 tggggaacaa ttaaaaaatc aaaagctatt aatgttttga gagggttcag gaaagagatt 360 ggaaggatgc tgaacatctt gaataggaga cgcagatctg caggcatgat cattatgctg 420 attccaacag tgatggcgtt ccatttaacc acgcgtgggg gagagccgca tatgatagtt 480 agcaagcagg aaagaggaaa gtcacttttg ttcaagacct ctgcaggtgt caacatgtgc 540 accctcattg cgatggattt gggagagttg tgtgaggaca cgatgaccta caaatgcccc 600 cggatcactg aggcggaacc agatgacgtt gactgttggt gcaatgccac ggacacatgg 660 gtgacctatg gaacgtgctc tcaaactggc gaacaccgac gagacaaacg ttccgtcgca 720 ttggccccac acgtggggct tggcctagaa acaagagccg aaacgtggat gtcctctgaa 780 ggtgcttgga aacagataca aaaagtagag acttgggctc tgagacatcc aggattcacg 840 gtgatagccc tttttctagc acatgccata ggaacatcca tcacccagaa agggatcatt 900 ttcattttgc tgatgctggt aacaccatct atggccatgc gatgcgtggg aataggcaac 960 agagacttcg tggaaggact gtcaggagca acatgggtgg atgtggtact ggagcatgga 1020 agttgcgtca ccaccatggc aaaaaacaaa ccaacactgg acattgaact cttgaagacg 1080 gaggtcacaa accctgcagt tctgcgtaaa ttgtgcattg aagctaaaat atcaaacacc 1140 accaccgatt cgagatgtcc aacacaagga gaagccacac tggtggaaga acaagacgcg 1200 aactttgtgt gccgacgaac gttcgtggac agaggctggg gcaatggctg tgggctattc 1260 ggaaaaggta gtctaataac gtgtgccaag tttaagtgtg tgacaaaact agaaggaaag 1320 atagttcaat atgaaaacct aaaatattca gtgatagtca ccgtccacac tggagatcag 1380 caccaggtgg gaaatgagac tacagaacat ggaacaactg caaccataac acctcaagct 1440 cctacgtcgg aaatacagct gaccgactac ggaaccctta cattagattg ttcacctagg 1500 acagggctag attttaacga gatggtgttg ctgacaatga aagaaagatc atggcttgtc 1560 cacaaacaat ggttcctaga cttaccactg ccttggacct ctggggcttc aacatcccaa 1620 gagacttgga acagacaaga tttactggtc acatttaaga cagctcatgc aaagaagcag 1680 gaagtagtcg tactaggatc acaagaagga gcaatgcaca ctgcgctgac tggagcgaca 1740 gaaatccaaa cgtcaggaac gacaacaatt ttcgcaggac acctaaaatg cagactaaaa 1800 atggacaaac taactttaaa agggatgtca tatgtgatgt gcacaggctc attcaagtta 1860 gagaaagaag tggctgagac ccagcatgga actgttctgg tgcaggttaa atatgaagga 1920 acagacgcac catgcaagat tcccttttcg acccaagatg agaaaggagc aacccagaat 1980 gggagattaa taacagccaa ccccatagtc actgacaaag aaaaaccagt caatattgag 2040 gcagaaccac cctttggtga gagctacatc gtggtaggag caggtgaaaa agctttgaaa 2100 ctaagctggt tcaagaaagg aagcagcata gggaaaatgt ttgaagcaac tgcccgagga 2160 gcacgaagga tggccattct gggagacacc gcatgggact tcggttctat aggaggagtg 2220 ttcacgtcta tgggaaaact ggtacaccag gtttttggaa ctgcatatgg agttttgttt 2280 agcggagttt cttggaccat gaaaatagga atagggattc tgctgacatg gctaggatta 2340 aattcaagga acacgtccct ttcgatgatg tgcatcgcag ccggcattgt gacactgtat 2400 ttgggagtca tggtgcaggc cgatagtggt tgcgttgtga gctggaaaaa caaagaactg 2460 aaatgtggca gtgggatttt catcacagac aacgtgcaca catggacaga acaatacaag 2520 ttccaaccag aatccccttc aaaactagct tcagctatcc agaaagccca tgaagaggac 2580 atttgtggaa tccgctcagt aacaagactg gagaatctga tgtggaaaca aataacacca 2640 gaattgaatc acattctatc agaaaatgag gtgaagttaa ctattatgac aggagacatc 2700 aaaggaatca tgcaggcagg aaaacgatct ctgcggcctc agcccactga gctgaagtat 2760 tcatggaaaa catggggcaa agcaaaaatg ctctctacag agtctcataa ccagaccttt 2820 ctcattgatg gccccgaaac agcagaatgc cccaacacaa atagagcttg gaattcgttg 2880 gaagttgaag actatggctt tggagtattc accaccaata tatggctaaa attgaaagaa 2940 aaacaggatg tattctgcga ctcaaaactc atgtcagcgg ccataaaaga caacagagcc 3000 gtccatgccg atatgggtta ttggatagaa agtgcactca atgacacatg gaagatagag 3060 aaagcctctt tcattgaagt taaaaactgc cactggccaa aatcacacac cctctggagc 3120 aatggagtgc tagaaagtga gatgataatt ccaaagaatc tcgctggacc agtgtctcaa 3180 cacaactata gaccaggcta ccatacacaa ataacaggac catggcatct aggtaagctt 3240 gagatggact ttgatttctg tgatggaaca acagtggtag tgactgagga ctgcggaaat 3300 agaggaccct ctttgagaac aaccactgcc tctggaaaac tcataacaga atggtgctgc 3360 cgatcttgca cattaccacc gctaagatac agaggtgagg atgggtgctg gtacgggatg 3420 gaaatcagac cattgaagga gaaagaagag aatttggtca actccttggt cacagctgga 3480 catgggcagg tcgacaactt ttcactagga gtcttgggaa tggcattgtt cctggaggaa 3540 atgcttagga cccgagtagg aacgaaacat gcaatactac tagttgcagt ttcttttgtg 3600 acattgatca cagggaacat gtcctttaga gacctgggaa gagtgatggt tatggtaggc 3660 gccactatga cggatgacat aggtatgggc gtgacttatc ttgccctact agcagccttc 3720 aaagtcagac caacttttgc agctggacta ctcttgagaa agctgacctc caaggaattg 3780 atgatgacta ctataggaat tgtactcctc tcccagagca ccctaccaga gaccattctt 3840 gagttgactg atgcgttagc cttaggcatg atggtcctca aaatggtgag aaatatggaa 3900 aagtatcaat tggcagtgac tatcatggct atcttgtgcg tcccaaacgc agtgatatta 3960 caaaacgcat ggaaagtgag ttgcacaata ttggcagtgg tgtccgtttc cccactgttc 4020 ttaacatcct cacagcaaaa aacagattgg ataccattag cattgacgat caaaggtctc 4080 aatccaacag ctatttttct aacaaccctc tcaagaacca gcaagaaaag gagctggcca 4140 ttaaatgagg ctatcatggc agtcgggatg gtgagcattt tagccagttc tctcctaaaa 4200 aatgatattc ccatgacagg accattagtg gctggagggc tcctcactgt gtgctacgtg 4260 ctcactggac gatcggccga tttggaactg gagagagcag ccgatgtcaa atgggaagac 4320 caggcagaga tatcaggaag cagtccaatc ctgtcaataa caatatcaga agatggtagc 4380 atgtcgataa aaaatgaaga ggaagatcaa acactgacca tactcattag aacaggattg 4440 ctggtgatct caggactttt tcctgtatca ataccaatca cggcagcagc atggtacctg 4500 tgggaagtga agaaacaacg ggccggagta ttgtgggatg ttccttcacc cccacccatg 4560 ggaaaggctg aactggaaga tggagcctat agaattaagc aaaaagggat tcttggatat 4620 tcccagatcg gagccggagt ttacaaagaa ggaacattcc atacaatgtg gcatgtcaca 4680 cgtggcgctg ttctaatgca taaaggaaag aggattgaac catcatgggc ggacgtcaag 4740 aaagacctaa tatcatatgg aggaggctgg aagttagaag gagaatggaa ggaaggagaa 4800 gaagtccagg tattggcact ggagcctgga aaaaatccaa gagccgtcca aacgaaacct 4860 ggtcttttca aaaccaacgc cggaacaata ggtgctgtat ctctggactt ttctcctgga 4920 acgtcaggat ctccaattat cgacaaaaaa ggaaaagttg tgggtcttta tggtaatggt 4980 gttgttacaa ggagtggagc atatgtgagt gctatagccc agactgaaaa aagcattgaa 5040 gacaacccag agatcgaaga tgacattttc cgaaagagaa gactgaccat catggacctc 5100 cacccaggag cgggaaagac gaagagatac cttccggcca tagtcagaga agctataaaa 5160 cggggtttga gaacattaat cttggccccc actagagttg tggcagctga aatggaggaa 5220 gcccttagag gacttccaat aagataccag accccagcca tcagagctgt gcacaccggg 5280 cgggagattg tggacctaat gtgtcatgcc acatttacca tgaggctgct atcaccagtt 5340 agagtgccaa actacaacct gattatcatg gacgaagccc atttcacaga cccagcaagt 5400 atagcagcta gaggatacat ctcaactcga gtggagatgg gtgaggcagc tgggattttt 5460 atgacagcca ctccccccggg aagcagagac ccatttcctc agagcaatgc accaatcata 5520 gatgaagaaa gagaaatccc tgaacgctcg tggaattccg gacatgaatg ggtcacggat 5580 tttaaaggga agactgtttg gttcgttcca agtataaaag caggaaatga tatagcagct 5640 tgcctgagga aaaatggaaa gaaagtgata caactcagta ggaagacctt tgattctgag 5700 tatgtcaaga ctagaaccaa tgattgggac ttcgtggtta caactgacat ttcagaaatg 5760 ggtgccaatt tcaaggctga gagggttata gaccccagac gctgcatgaa accagtcata 5820 ctaacagatg gtgaagagcg ggtgattctg gcaggaccta tgccagtgac ccactctagt 5880 gcagcacaaa gaagagggag aataggaaga aatccaaaaa atgagaatga ccagtacata 5940 tacatggggg aacctctgga aaatgatgaa gactgtgcac actggaaaga agctaaaatg 6000 ctcctagata acatcaacac gccagaagga atcattccta gcatgttcga accagagcgt 6060 gaaaaggtgg atgccattga tggcgaatac cgcttgagag gagaagcaag gaaaaccttt 6120 gtagacttaa tgagaagagg agacctacca gtctggttgg cctacagagt ggcagctgaa 6180 ggcatcaact acgcagacag aaggtggtgt tttgatggag tcaagaacaa ccaaatccta 6240 gaagaaaacg tggaagttga aatctggaca aaagaagggg aaaggaagaa attgaaaccc 6300 agatggttgg atgctaggat ctattctgac ccactggcgc taaaagaatt taaggaattt 6360 gcagccggaa gaaagtctct gaccctgaac ctaatcacag aaatgggtag gctcccaacc 6420 ttcatgactc agaaggcaag agacgcactg gacaacttag cagtgctgca cacggctgag 6480 gcaggtggaa gggcgtacaa ccatgctctc agtgaactgc cggagaccct ggagacattg 6540 cttttactga cacttctggc tacagtcacg ggagggatct ttttattctt gatgagcgca 6600 aggggcatag ggaagatgac cctgggaatg tgctgcataa tcacggctag catcctccta 6660 tggtacgcac aaatacagcc acactggata gcagcttcaa taatactgga gttttttctc 6720 atagttttgc ttattccaga acctgaaaaa cagagaacac cccaagacaa ccaactgacc 6780 tacgttgtca tagccatcct cacagtggtg gccgcaacca tggcaaacga gatgggtttc 6840 ctagaaaaaa cgaagaaaga tctcggattg ggaagcattg caacccagca acccgagagc 6900 aacatcctgg acatagatct acgtcctgca tcagcatgga cgctgtatgc cgtggccaca 6960 acatttgtta caccaatgtt gagacatagc attgaaaatt cctcagtgaa tgtgtcccta 7020 acagctatag ccaaccaagc cacagtgtta atgggtctcg ggaaaggatg gccattgtca 7080 aagatggaca tcggagttcc ccttctcgcc attggatgct actcacaagt caaccccata 7140 actctcacag cagctctttt cttattggta gcacattatg ccatcatagg gccaggactc 7200 caagcaaaag caaccagaga agctcagaaa agagcagcgg cgggcatcat gaaaaaccca 7260 actgtcgatg gaataacagt gattgaccta gatccaatac cttatgatcc gaagtttgaa 7320 aagcagttgg gacaagtaat gctcctagtc ctctgcgtga ctcaagtatt gatgatgagg 7380 actacatggg ctctgtgtga ggctttaacc ttagctaccg ggcccatctc cacatgtgg 7440 gaaggaaatc cagggaggtt ttggaacact accattgcgg tgtcaatggc taacattttt 7500 agagggagtt acttggccgg agctggactt ctcttttcta ttatgaagaa cacaaccaac 7560 acaagaaggg gaactggcaa cataggagag acgcttggag agaaatggaa aagccgattg 7620 aacgcattgg gaaaaagtga attccagatc tacaagaaaa gtggaatcca ggaagtggat 7680 agaaccttag caaaagaagg cattaaaaga ggagaaacgg accatcacgc tgtgtcgcga 7740 ggctcagcaa aactgagatg gttcgttgag agaaacatgg tcacaccaga agggaaagta 7800 gtggacctcg gttgtggcag aggaggctgg tcatactatt gtggaggact aaagaatgta 7860 agagaagtca aaggcctaac aaaaggagga ccaggacacg aagaacccat ccccatgtca 7920 acatatgggt ggaatctagt gcgtcttcaa agtggagttg acgttttctt catcccgcca 7980 gaaaagtgtg acacattatt gtgtgacata ggggagtcat caccaaatcc cacagtggaa 8040 gcaggacgaa cactcagagt ccttaactta gtagaaaatt ggttgaacaa caacactcaa 8100 ttttgcataa aggttctcaa cccatatatg ccctcagtca tagaaaaaat ggaagcacta 8160 caaaggaaat atggaggagc cttagtgagg aatccactct cacgaaactc cacacatgag 8220 atgtactggg tatccaatgc ttccgggaac atagtgtcat cagtgaacat gatttcaagg 8280 atgttgatca acagattac aatgagatac aagaaagcca cttacgagcc ggatgttgac 8340 ctcggaagcg gaacccgtaa catcgggatt gaaagtgaga taccaaacct agatataatt 8400 gggaaaagaa tagaaaaaat aaagcaagag catgaaacat catggcacta tgaccaagac 8460 cacccataca aaacgtgggc ataccatggt agctatgaaa caaaacagac tggatcagca 8520 tcatccatgg tcaacggagt ggtcaggctg ctgacaaaac cttgggacgt cgtccccatg 8580 gtgacacaga tggcaatgac agacacgact ccatttggac aacagcgcgt ttttaaagag 8640 aaagtggaca cgagaaccca agaaccgaaa gaaggcacga agaaactaat gaaaataaca 8700 gcagagtggc tttggaaaga attagggaag aaaaagacac ccaggatgtg caccagagaa 8760 gaattcacaa gaaaggtgag aagcaatgca gccttggggg ccatattcac tgatgagaac 8820 aagtggaagt cggcacgtga ggctgttgaa gatagtaggt tttgggagct ggttgacaag 8880 gaaaggaatc tccatcttga aggaaagtgt gaaacatgtg tgtacaacat gatgggaaaa 8940 agagagaaga agctagggga attcggcaag gcaaaaggca gcagagccat atggtacat 9000 tggcttggag cacgcttctt agagtttgaa gccctaggat tcttaaatga agatcactgg 9060 ttctccagag agaactccct gagtggagtg gaaggagaag ggctgcacaa gctaggttac 9120 attctaagag acgtgagcaa gaaagaggga ggagcaatgt atgccgatga caccgcagga 9180 tgggatacaa gaatcacact agaagaccta aaaaatgaag aaatggtaac aaaccacatg 9240 gaaggagaac acaagaaact agccgaggcc attttcaaac taacgtacca aaacaaggtg 9300 gtgcgtgtgc aaagaccaac accaagaggc acagtaatgg acatcatatc gagaagagac 9360 caaagaggta gtggacaagt tggcacctat ggactcaata ctttcaccaa tatggaagcc 9420 caactaatca gacagatgga gggagaagga gtctttaaaa gcattcagca cctaacaatc 9480 acagaagaaa tcgctgtgca aaactggtta gcaagagtgg ggcgcgaaag gttatcaaga 9540 atggccatca gtggagatga ttgtgttgtg aaacctttag atgacaggtt cgcaagcgct 9600 ttaacagctc taaatgacat gggaaagatt aggaaagaca tacaacaatg ggaaccttca 9660 agaggatgga atgattggac acaagtgccc ttctgttcac accatttcca tgagttaatc 9720 atgaaagacg gtcgcgtact cgttgttcca tgtagaaacc aagatgaact gattggcaga 9780 gcccgaatct cccaaggagc agggtggtct ttgcgggaga cggcctgttt ggggaagtct 9840 tacgcccaaa tgtggagctt gatgtacttc cacagacgcg acctcaggct ggcggcaaat 9900 gctatttgct cggcagtacc atcacattgg gttccaacaa gtcgaacaac ctggtccata 9960 catgctaaac atgaatggat gacaacggaa gacatgctga cagtctggaa cagggtgtgg 10020 attcaagaaa acccatggat ggaagacaaa actccagtgg aatcatggga ggaaatccca 10080 tacttgggga aaagagaaga ccaatggtgc ggctcattga ttgggttaac aagcagggcc 10140 acctgggcaa agaacatcca agcagcaata aatcaagtta gatcccttat aggcaatgaa 10200 gaatacacag attacatgcc atccatgaaa agattcagaa gagaagagga agaagcagga 10260 gttctgtggt agaaagcaaa actaacatga aacaaggcta gaagtcaggt cggattaagc 10320 catagtacgg aaaaaactat gctacctgtg agccccgtcc aaggacgtta aaagaagtca 10380 ggccatcata aatgccatag cttgagtaaa ctatgcagcc tgtagctcca cctgagaagg 10440 tgtaaaaaat ccgggaggcc acaaaccatg gaagctgtac gcatggcgta gtggactagc 10500 ggttagagga gacccctccc ttacaaatcg cagcaacaat gggggcccaa ggcgagatga 10560 agctgtagtc tcgctggaag gactagaggt tagaggagac ccccccgaaa caaaaaacag 10620 catattgacg ctgggaaaga ccagagatcc tgctgtctcc tcagcatcat tccaggcaca 10680 gaacgccaga aaatggaatg gtgctgttga atcaacaggt tct 10723 <210> 2 <211> 3391 <212> PRT <213> chimeric dengue serotype 2/1 (MVS) <400> 2 Met Asn Asn Gln Arg Lys Lys Ala Lys Asn Thr Pro Phe Asn Met Leu 1 5 10 15 Lys Arg Glu Arg Asn Arg Val Ser Thr Val Gln Gln Leu Thr Lys Arg 20 25 30 Phe Ser Leu Gly Met Leu Gln Gly Arg Gly Pro Leu Lys Leu Phe Met 35 40 45 Ala Leu Val Ala Phe Leu Arg Phe Leu Thr Ile Pro Pro Thr Ala Gly 50 55 60 Ile Leu Lys Arg Trp Gly Thr Ile Lys Lys Ser Lys Ala Ile Asn Val 65 70 75 80 Leu Arg Gly Phe Arg Lys Glu Ile Gly Arg Met Leu Asn Ile Leu Asn 85 90 95 Arg Arg Arg Arg Ser Ala Gly Met Ile Ile Met Leu Ile Pro Thr Val 100 105 110 Met Ala Phe His Leu Thr Thr Arg Gly Gly Glu Pro His Met Ile Val 115 120 125 Ser Lys Gin Glu Arg Gly Lys Ser Leu Leu Phe Lys Thr Ser Ala Gly 130 135 140 Val Asn Met Cys Thr Leu Ile Ala Met Asp Leu Gly Glu Leu Cys Glu 145 150 155 160 Asp Thr Met Thr Tyr Lys Cys Pro Arg Ile Thr Glu Ala Glu Pro Asp 165 170 175 Asp Val Asp Cys Trp Cys Asn Ala Thr Asp Thr Trp Val Thr Tyr Gly 180 185 190 Thr Cys Ser Gln Thr Gly Glu His Arg Arg Asp Lys Arg Ser Val Ala 195 200 205 Leu Ala Pro His Val Gly Leu Gly Leu Glu Thr Arg Ala Glu Thr Trp 210 215 220 Met Ser Ser Glu Gly Ala Trp Lys Gln Ile Gln Lys Val Glu Thr Trp 225 230 235 240 Ala Leu Arg His Pro Gly Phe Thr Val Ile Ala Leu Phe Leu Ala His 245 250 255 Ala Ile Gly Thr Ser Ile Thr Gln Lys Gly Ile Ile Phe Ile Leu Leu 260 265 270 Met Leu Val Thr Pro Ser Met Ala Met Arg Cys Val Gly Ile Gly Asn 275 280 285 Arg Asp Phe Val Glu Gly Leu Ser Gly Ala Thr Trp Val Asp Val Val 290 295 300 Leu Glu His Gly Ser Cys Val Thr Thr Met Ala Lys Asn Lys Pro Thr 305 310 315 320 Leu Asp Ile Glu Leu Leu Lys Thr Glu Val Thr Asn Pro Ala Val Leu 325 330 335 Arg Lys Leu Cys Ile Glu Ala Lys Ile Ser Asn Thr Thr Thr Asp Ser 340 345 350 Arg Cys Pro Thr Gln Gly Glu Ala Thr Leu Val Glu Glu Gln Asp Ala 355 360 365 Asn Phe Val Cys Arg Arg Thr Phe Val Asp Arg Gly Trp Gly Asn Gly 370 375 380 Cys Gly Leu Phe Gly Lys Gly Ser Leu Ile Thr Cys Ala Lys Phe Lys 385 390 395 400 Cys Val Thr Lys Leu Glu Gly Lys Ile Val Gln Tyr Glu Asn Leu Lys 405 410 415 Tyr Ser Val Ile Val Thr Val His Thr Gly Asp Gln His Gln Val Gly 420 425 430 Asn Glu Thr Thr Glu His Gly Thr Thr Ala Thr Ile Thr Pro Gln Ala 435 440 445 Pro Thr Ser Glu Ile Gln Leu Thr Asp Tyr Gly Thr Leu Thr Leu Asp 450 455 460 Cys Ser Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Val Leu Leu Thr 465 470 475 480 Met Lys Glu Arg Ser Trp Leu Val His Lys Gln Trp Phe Leu Asp Leu 485 490 495 Pro Leu Pro Trp Thr Ser Gly Ala Ser Thr Ser Gln Glu Thr Trp Asn 500 505 510 Arg Gln Asp Leu Leu Val Thr Phe Lys Thr Ala His Ala Lys Lys Gln 515 520 525 Glu Val Val Val Leu Gly Ser Gln Glu Gly Ala Met His Thr Ala Leu 530 535 540 Thr Gly Ala Thr Glu Ile Gln Thr Ser Gly Thr Thr Thr Ile Phe Ala 545 550 555 560 Gly His Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Thr Leu Lys Gly 565 570 575 Met Ser Tyr Val Met Cys Thr Gly Ser Phe Lys Leu Glu Lys Glu Val 580 585 590 Ala Glu Thr Gln His Gly Thr Val Leu Val Gln Val Lys Tyr Glu Gly 595 600 605 Thr Asp Ala Pro Cys Lys Ile Pro Phe Ser Thr Gln Asp Glu Lys Gly 610 615 620 Ala Thr Gln Asn Gly Arg Leu Ile Thr Ala Asn Pro Ile Val Thr Asp 625 630 635 640 Lys Glu Lys Pro Val Asn Ile Glu Ala Glu Pro Phe Gly Glu Ser 645 650 655 Tyr Ile Val Val Gly Ala Gly Glu Lys Ala Leu Lys Leu Ser Trp Phe 660 665 670 Lys Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ala Thr Ala Arg Gly 675 680 685 Ala Arg Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser 690 695 700 Ile Gly Gly Val Phe Thr Ser Met Gly Lys Leu Val His Gln Val Phe 705 710 715 720 Gly Thr Ala Tyr Gly Val Leu Phe Ser Gly Val Ser Trp Thr Met Lys 725 730 735 Ile Gly Ile Gly Ile Leu Leu Thr Trp Leu Gly Leu Asn Ser Arg Asn 740 745 750 Thr Ser Leu Ser Met Met Cys Ile Ala Ala Gly Ile Val Thr Leu Tyr 755 760 765 Leu Gly Val Met Val Gln Ala Asp Ser Gly Cys Val Val Ser Trp Lys 770 775 780 Asn Lys Glu Leu Lys Cys Gly Ser Gly Ile Phe Ile Thr Asp Asn Val 785 790 795 800 His Thr Trp Thr Glu Gln Tyr Lys Phe Gln Pro Glu Ser Pro Ser Lys 805 810 815 Leu Ala Ser Ala Ile Gln Lys Ala His Glu Glu Asp Ile Cys Gly Ile 820 825 830 Arg Ser Val Thr Arg Leu Glu Asn Leu Met Trp Lys Gln Ile Thr Pro 835 840 845 Glu Leu Asn His Ile Leu Ser Glu Asn Glu Val Lys Leu Thr Ile Met 850 855 860 Thr Gly Asp Ile Lys Gly Ile Met Gln Ala Gly Lys Arg Ser Leu Arg 865 870 875 880 Pro Gln Pro Thr Glu Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala 885 890 895 Lys Met Leu Ser Thr Glu Ser His Asn Gln Thr Phe Leu Ile Asp Gly 900 905 910 Pro Glu Thr Ala Glu Cys Pro Asn Thr Asn Arg Ala Trp Asn Ser Leu 915 920 925 Glu Val Glu Asp Tyr Gly Phe Gly Val Phe Thr Thr Asn Ile Trp Leu 930 935 940 Lys Leu Lys Glu Lys Gln Asp Val Phe Cys Asp Ser Lys Leu Met Ser 945 950 955 960 Ala Ala Ile Lys Asp Asn Arg Ala Val His Ala Asp Met Gly Tyr Trp 965 970 975 Ile Glu Ser Ala Leu Asn Asp Thr Trp Lys Ile Glu Lys Ala Ser Phe 980 985 990 Ile Glu Val Lys Asn Cys His Trp Pro Lys Ser His Thr Leu Trp Ser 995 1000 1005 Asn Gly Val Leu Glu Ser Glu Met Ile Ile Pro Lys Asn Leu Ala Gly 1010 1015 1020 Pro Val Ser Gln His Asn Tyr Arg Pro Gly Tyr His Thr Gln Ile Thr 1025 1030 1035 1040 Gly Pro Trp His Leu Gly Lys Leu Glu Met Asp Phe Asp Phe Cys Asp 1045 1050 1055 Gly Thr Thr Val Val Val Thr Glu Asp Cys Gly Asn Arg Gly Pro Ser 1060 1065 1070 Leu Arg Thr Thr Thr Ala Ser Gly Lys Leu Ile Thr Glu Trp Cys Cys 1075 1080 1085 Arg Ser Cys Thr Leu Pro Leu Arg Tyr Arg Gly Glu Asp Gly Cys 1090 1095 1100 Trp Tyr Gly Met Glu Ile Arg Pro Leu Lys Glu Lys Glu Glu Asn Leu 1105 1110 1115 1120 Val Asn Ser Leu Val Thr Ala Gly His Gly Gln Val Asp Asn Phe Ser 1125 1130 1135 Leu Gly Val Leu Gly Met Ala Leu Phe Leu Glu Glu Met Leu Arg Thr 1140 1145 1150 Arg Val Gly Thr Lys His Ala Ile Leu Leu Val Ala Val Ser Phe Val 1155 1160 1165 Thr Leu Ile Thr Gly Asn Met Ser Phe Arg Asp Leu Gly Arg Val Met 1170 1175 1180 Val Met Val Gly Ala Thr Met Thr Asp Asp Ile Gly Met Gly Val Thr 1185 1190 1195 1200 Tyr Leu Ala Leu Leu Ala Ala Phe Lys Val Arg Pro Thr Phe Ala Ala 1205 1210 1215 Gly Leu Leu Leu Arg Lys Leu Thr Ser Lys Glu Leu Met Met Thr Thr 1220 1225 1230 Ile Gly Ile Val Leu Leu Ser Gln Ser Thr Leu Pro Glu Thr Ile Leu 1235 1240 1245 Glu Leu Thr Asp Ala Leu Ala Leu Gly Met Met Val Leu Lys Met Val 1250 1255 1260 Arg Asn Met Glu Lys Tyr Gln Leu Ala Val Thr Ile Met Ala Ile Leu 1265 1270 1275 1280 Cys Val Pro Asn Ala Val Ile Leu Gln Asn Ala Trp Lys Val Ser Cys 1285 1290 1295 Thr Ile Leu Ala Val Val Ser Val Ser Pro Leu Phe Leu Thr Ser Ser 1300 1305 1310 Gln Gln Lys Thr Asp Trp Ile Pro Leu Ala Leu Thr Ile Lys Gly Leu 1315 1320 1325 Asn Pro Thr Ala Ile Phe Leu Thr Thr Leu Ser Arg Thr Ser Lys Lys 1330 1335 1340 Arg Ser Trp Pro Leu Asn Glu Ala Ile Met Ala Val Gly Met Val Ser 1345 1350 1355 1360 Ile Leu Ala Ser Leu Leu Lys Asn Asp Ile Pro Met Thr Gly Pro 1365 1370 1375 Leu Val Ala Gly Gly Leu Leu Thr Val Cys Tyr Val Leu Thr Gly Arg 1380 1385 1390 Ser Ala Asp Leu Glu Leu Glu Arg Ala Ala Asp Val Lys Trp Glu Asp 1395 1400 1405 Gln Ala Glu Ile Ser Gly Ser Ser Pro Ile Leu Ser Ile Thr Ile Ser 1410 1415 1420 Glu Asp Gly Ser Met Ser Ile Lys Asn Glu Glu Glu Asp Gln Thr Leu 1425 1430 1435 1440 Thr Ile Leu Ile Arg Thr Gly Leu Leu Val Ile Ser Gly Leu Phe Pro 1445 1450 1455 Val Ser Ile Pro Ile Thr Ala Ala Ala Trp Tyr Leu Trp Glu Val Lys 1460 1465 1470 Lys Gln Arg Ala Gly Val Leu Trp Asp Val Pro Ser Pro Pro Met 1475 1480 1485 Gly Lys Ala Glu Leu Glu Asp Gly Ala Tyr Arg Ile Lys Gln Lys Gly 1490 1495 1500 Ile Leu Gly Tyr Ser Gln Ile Gly Ala Gly Val Tyr Lys Glu Gly Thr 1505 1510 1515 1520 Phe His Thr Met Trp His Val Thr Arg Gly Ala Val Leu Met His Lys 1525 1530 1535 Gly Lys Arg Ile Glu Pro Ser Trp Ala Asp Val Lys Lys Asp Leu Ile 1540 1545 1550 Ser Tyr Gly Gly Gly Trp Lys Leu Glu Gly Glu Trp Lys Glu Gly Glu 1555 1560 1565 Glu Val Gln Val Leu Ala Leu Glu Pro Gly Lys Asn Pro Arg Ala Val 1570 1575 1580 Gln Thr Lys Pro Gly Leu Phe Lys Thr Asn Ala Gly Thr Ile Gly Ala 1585 1590 1595 1600 Val Ser Leu Asp Phe Ser Pro Gly Thr Ser Gly Ser Pro Ile Ile Asp 1605 1610 1615 Lys Lys Gly Lys Val Val Gly Leu Tyr Gly Asn Gly Val Val Thr Arg 1620 1625 1630 Ser Gly Ala Tyr Val Ser Ala Ile Ala Gln Thr Glu Lys Ser Ile Glu 1635 1640 1645 Asp Asn Pro Glu Ile Glu Asp Asp Ile Phe Arg Lys Arg Arg Leu Thr 1650 1655 1660 Ile Met Asp Leu His Pro Gly Ala Gly Lys Thr Lys Arg Tyr Leu Pro 1665 1670 1675 1680 Ala Ile Val Arg Glu Ala Ile Lys Arg Gly Leu Arg Thr Leu Ile Leu 1685 1690 1695 Ala Pro Thr Arg Val Val Ala Ala Glu Met Glu Glu Ala Leu Arg Gly 1700 1705 1710 Leu Pro Ile Arg Tyr Gln Thr Pro Ala Ile Arg Ala Val His Thr Gly 1715 1720 1725 Arg Glu Ile Val Asp Leu Met Cys His Ala Thr Phe Thr Met Arg Leu 1730 1735 1740 Leu Ser Pro Val Arg Val Pro Asn Tyr Asn Leu Ile Ile Met Asp Glu 1745 1750 1755 1760 Ala His Phe Thr Asp Pro Ala Ser Ile Ala Ala Arg Gly Tyr Ile Ser 1765 1770 1775 Thr Arg Val Glu Met Gly Glu Ala Ala Gly Ile Phe Met Thr Ala Thr 1780 1785 1790 Pro Pro Gly Ser Arg Asp Pro Phe Pro Gln Ser Asn Ala Pro Ile Ile 1795 1800 1805 Asp Glu Glu Arg Glu Ile Pro Glu Arg Ser Trp Asn Ser Gly His Glu 1810 1815 1820 Trp Val Thr Asp Phe Lys Gly Lys Thr Val Trp Phe Val Pro Ser Ile 1825 1830 1835 1840 Lys Ala Gly Asn Asp Ile Ala Ala Cys Leu Arg Lys Asn Gly Lys Lys 1845 1850 1855 Val Ile Gln Leu Ser Arg Lys Thr Phe Asp Ser Glu Tyr Val Lys Thr 1860 1865 1870 Arg Thr Asn Asp Trp Asp Phe Val Val Thr Asp Ile Ser Glu Met 1875 1880 1885 Gly Ala Asn Phe Lys Ala Glu Arg Val Ile Asp Pro Arg Arg Cys Met 1890 1895 1900 Lys Pro Val Ile Leu Thr Asp Gly Glu Glu Arg Val Ile Leu Ala Gly 1905 1910 1915 1920 Pro Met Pro Val Thr His Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile 1925 1930 1935 Gly Arg Asn Pro Lys Asn Glu Asn Asp Gln Tyr Ile Tyr Met Gly Glu 1940 1945 1950 Pro Leu Glu Asn Asp Glu Asp Cys Ala His Trp Lys Glu Ala Lys Met 1955 1960 1965 Leu Leu Asp Asn Ile Asn Thr Pro Glu Gly Ile Ile Pro Ser Met Phe 1970 1975 1980 Glu Pro Glu Arg Glu Lys Val Asp Ala Ile Asp Gly Glu Tyr Arg Leu 1985 1990 1995 2000 Arg Gly Glu Ala Arg Lys Thr Phe Val Asp Leu Met Arg Arg Gly Asp 2005 2010 2015 Leu Pro Val Trp Leu Ala Tyr Arg Val Ala Ala Glu Gly Ile Asn Tyr 2020 2025 2030 Ala Asp Arg Arg Trp Cys Phe Asp Gly Val Lys Asn Asn Gln Ile Leu 2035 2040 2045 Glu Glu Asn Val Glu Val Glu Ile Trp Thr Lys Glu Gly Glu Arg Lys 2050 2055 2060 Lys Leu Lys Pro Arg Trp Leu Asp Ala Arg Ile Tyr Ser Asp Pro Leu 2065 2070 2075 2080 Ala Leu Lys Glu Phe Lys Glu Phe Ala Ala Gly Arg Lys Ser Leu Thr 2085 2090 2095 Leu Asn Leu Ile Thr Glu Met Gly Arg Leu Pro Thr Phe Met Thr Gln 2100 2105 2110 Lys Ala Arg Asp Ala Leu Asp Asn Leu Ala Val Leu His Thr Ala Glu 2115 2120 2125 Ala Gly Gly Arg Ala Tyr Asn His Ala Leu Ser Glu Leu Pro Glu Thr 2130 2135 2140 Leu Glu Thr Leu Leu Leu Leu Thr Leu Leu Ala Thr Val Thr Gly Gly 2145 2150 2155 2160 Ile Phe Leu Phe Leu Met Ser Ala Arg Gly Ile Gly Lys Met Thr Leu 2165 2170 2175 Gly Met Cys Cys Ile Ile Thr Ala Ser Ile Leu Leu Trp Tyr Ala Gln 2180 2185 2190 Ile Gln Pro His Trp Ile Ala Ala Ser Ile Ile Leu Glu Phe Phe Leu 2195 2200 2205 Ile Val Leu Leu Ile Pro Glu Pro Glu Lys Gln Arg Thr Pro Gln Asp 2210 2215 2220 Asn Gln Leu Thr Tyr Val Val Ile Ala Ile Leu Thr Val Val Ala Ala 2225 2230 2235 2240 Thr Met Ala Asn Glu Met Gly Phe Leu Glu Lys Thr Lys Lys Asp Leu 2245 2250 2255 Gly Leu Gly Ser Ile Ala Thr Gln Gln Pro Glu Ser Asn Ile Leu Asp 2260 2265 2270 Ile Asp Leu Arg Pro Ala Ser Ala Trp Thr Leu Tyr Ala Val Ala Thr 2275 2280 2285 Thr Phe Val Thr Pro Met Leu Arg His Ser Ile Glu Asn Ser Ser Val 2290 2295 2300 Asn Val Ser Leu Thr Ala Ile Ala Asn Gln Ala Thr Val Leu Met Gly 2305 2310 2315 2320 Leu Gly Lys Gly Trp Pro Leu Ser Lys Met Asp Ile Gly Val Pro Leu 2325 2330 2335 Leu Ala Ile Gly Cys Tyr Ser Gln Val Asn Pro Ile Thr Leu Thr Ala 2340 2345 2350 Ala Leu Phe Leu Leu Val Ala His Tyr Ala Ile Ile Gly Pro Gly Leu 2355 2360 2365 Gln Ala Lys Ala Thr Arg Glu Ala Gln Lys Arg Ala Ala Ala Gly Ile 2370 2375 2380 Met Lys Asn Pro Thr Val Asp Gly Ile Thr Val Ile Asp Leu Asp Pro 2385 2390 2395 2400 Ile Pro Tyr Asp Pro Lys Phe Glu Lys Gln Leu Gly Gln Val Met Leu 2405 2410 2415 Leu Val Leu Cys Val Thr Gln Val Leu Met Met Arg Thr Thr Trp Ala 2420 2425 2430 Leu Cys Glu Ala Leu Thr Leu Ala Thr Gly Pro Ile Ser Thr Leu Trp 2435 2440 2445 Glu Gly Asn Pro Gly Arg Phe Trp Asn Thr Thr Ile Ala Val Ser Met 2450 2455 2460 Ala Asn Ile Phe Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu Leu Phe 2465 2470 2475 2480 Ser Ile Met Lys Asn Thr Thr Asn Thr Arg Arg Gly Thr Gly Asn Ile 2485 2490 2495 Gly Glu Thr Leu Gly Glu Lys Trp Lys Ser Arg Leu Asn Ala Leu Gly 2500 2505 2510 Lys Ser Glu Phe Gln Ile Tyr Lys Lys Ser Gly Ile Gln Glu Val Asp 2515 2520 2525 Arg Thr Leu Ala Lys Glu Gly Ile Lys Arg Gly Glu Thr Asp His His 2530 2535 2540 Ala Val Ser Arg Gly Ser Ala Lys Leu Arg Trp Phe Val Glu Arg Asn 2545 2550 2555 2560 Met Val Thr Pro Glu Gly Lys Val Val Asp Leu Gly Cys Gly Arg Gly 2565 2570 2575 Gly Trp Ser Tyr Tyr Cys Gly Gly Leu Lys Asn Val Arg Glu Val Lys 2580 2585 2590 Gly Leu Thr Lys Gly Gly Pro Gly His Glu Glu Pro Ile Pro Met Ser 2595 2600 2605 Thr Tyr Gly Trp Asn Leu Val Arg Leu Gln Ser Gly Val Asp Val Phe 2610 2615 2620 Phe Ile Pro Pro Glu Lys Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu 2625 2630 2635 2640 Ser Ser Pro Asn Pro Thr Val Glu Ala Gly Arg Thr Leu Arg Val Leu 2645 2650 2655 Asn Leu Val Glu Asn Trp Leu Asn Asn Asn Thr Gln Phe Cys Ile Lys 2660 2665 2670 Val Leu Asn Pro Tyr Met Pro Ser Val Ile Glu Lys Met Glu Ala Leu 2675 2680 2685 Gln Arg Lys Tyr Gly Gly Ala Leu Val Arg Asn Pro Leu Ser Arg Asn 2690 2695 2700 Ser Thr His Glu Met Tyr Trp Val Ser Asn Ala Ser Gly Asn Ile Val 2705 2710 2715 2720 Ser Ser Val Asn Met Ile Ser Arg Met Leu Ile Asn Arg Phe Thr Met 2725 2730 2735 Arg Tyr Lys Lys Ala Thr Tyr Glu Pro Asp Val Asp Leu Gly Ser Gly 2740 2745 2750 Thr Arg Asn Ile Gly Ile Glu Ser Glu Ile Pro Asn Leu Asp Ile Ile 2755 2760 2765 Gly Lys Arg Ile Glu Lys Ile Lys Gln Glu His Glu Thr Ser Trp His 2770 2775 2780 Tyr Asp Gln Asp His Pro Tyr Lys Thr Trp Ala Tyr His Gly Ser Tyr 2785 2790 2795 2800 Glu Thr Lys Gln Thr Gly Ser Ala Ser Met Val Asn Gly Val Val 2805 2810 2815 Arg Leu Leu Thr Lys Pro Trp Asp Val Val Pro Met Val Thr Gln Met 2820 2825 2830 Ala Met Thr Asp Thr Thr Pro Phe Gly Gln Gln Arg Val Phe Lys Glu 2835 2840 2845 Lys Val Asp Thr Arg Thr Gln Glu Pro Lys Glu Gly Thr Lys Lys Leu 2850 2855 2860 Met Lys Ile Thr Ala Glu Trp Leu Trp Lys Glu Leu Gly Lys Lys Lys Lys 2865 2870 2875 2880 Thr Pro Arg Met Cys Thr Arg Glu Glu Phe Thr Arg Lys Val Arg Ser 2885 2890 2895 Asn Ala Ala Leu Gly Ala Ile Phe Thr Asp Glu Asn Lys Trp Lys Ser 2900 2905 2910 Ala Arg Glu Ala Val Glu Asp Ser Arg Phe Trp Glu Leu Val Asp Lys 2915 2920 2925 Glu Arg Asn Leu His Leu Glu Gly Lys Cys Glu Thr Cys Val Tyr Asn 2930 2935 2940 Met Met Gly Lys Arg Glu Lys Lys Leu Gly Glu Phe Gly Lys Ala Lys 2945 2950 2955 2960 Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg Phe Leu Glu 2965 2970 2975 Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp Phe Ser Arg Glu 2980 2985 2990 Asn Ser Leu Ser Gly Val Glu Gly Glu Gly Leu His Lys Leu Gly Tyr 2995 3000 3005 Ile Leu Arg Asp Val Ser Lys Lys Glu Gly Gly Ala Met Tyr Ala Asp 3010 3015 3020 Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr Leu Glu Asp Leu Lys Asn 3025 3030 3035 3040 Glu Glu Met Val Thr Asn His Met Glu Gly Glu His Lys Lys Lys Leu Ala 3045 3050 3055 Glu Ala Ile Phe Lys Leu Thr Tyr Gln Asn Lys Val Val Arg Val Gln 3060 3065 3070 Arg Pro Thr Pro Arg Gly Thr Val Met Asp Ile Ile Ser Arg Arg Asp 3075 3080 3085 Gln Arg Gly Ser Gly Gln Val Gly Thr Tyr Gl y Leu Asn Thr Phe Thr 3090 3095 3100 Asn Met Glu Ala Gln Leu Ile Arg Gln Met Glu Gly Glu Gly Val Phe 3105 3110 3115 3120 Lys Ser Ile Gln His Leu Thr Ile Thr Glu Glu Ile Ala Val Gln Asn 3125 3130 3135 Trp Leu Ala Arg Val Gly Arg Glu Arg Leu Ser Arg Met Ala Ile Ser 3140 3145 3150 Gly Asp Asp Cys Val Val Lys Pro Leu Asp Asp Arg Phe Ala Ser Ala 3155 3160 3165 Leu Thr Ala Leu Asn Asp Met Gly Lys Ile Arg Lys Asp Ile Gln Gln 3170 3175 3180 Trp Glu Pro Ser Arg Gly Trp Asn Asp Trp Thr Gln Val Pro Phe Cys 3185 3190 3195 3200 Ser His His Phe His Glu Leu Ile Met Lys Asp Gly Arg Val Leu Val 3205 3210 3215 Val Pro Cys Arg Asn Gln Asp Glu Leu Ile Gly Arg Ala Arg Ile Ser 3220 3225 3230 Gln Gly Ala Gly Trp Ser Leu Arg Glu Thr Ala Cys Leu Gly Lys Ser 3235 3240 3245 Tyr Ala Gln Met Trp Ser Leu Met Tyr Phe His Arg Arg Asp Leu Arg 3250 3255 3260 Leu Ala Ala Asn Ala Ile Cys Ser Ala Val Pro Ser His Trp Val Pro 3265 3270 3275 3280 Thr Ser Arg Thr Thr Trp Ser Ile His Ala Lys His Glu Trp Met Thr 3285 3290 3295 Thr Glu Asp Met Leu Thr Val Trp Asn Arg Val Trp Ile Gln Glu Asn 3300 3305 3310 Pro Trp Met Glu Asp Lys Thr Pro Val Glu Ser Trp Glu Glu Ile Pro 3315 3320 3325 Tyr Leu Gly Lys Arg Glu Asp Gln Trp Cys Gly Ser Leu Ile Gly Leu 3330 3335 3340 Thr Ser Arg Ala Thr Trp Ala Lys Asn Ile Gln Ala Ala Ile Asn Gln 3345 3350 3355 3360 Val Arg Ser Leu Ile Gly Asn Glu Glu Tyr Thr Asp Tyr Met Pro Ser 3365 3370 3375Met Lys Arg Phe Arg Arg Glu Glu Glu Glu Ala Gly Val Leu Trp 3380 3385 3390 <210> 3 <211> 10723 <212> DNA <213> dengue serotype 2 (MVS) <400> 3 agttgttagt ctacgtggac cgacaaagac agattctttg agggagctaa gctcaatgta 60 gttctaacag ttttttaatt agagagcaga tctctgatga ataaccaacg gaaaaaggcg 120 aaaaacacgc ctttcaatat gctgaaacgc gagagaaacc gcgtgtcgac tgtgcaacag 180 ctgacaaaga gattctcact tggaatgctg cagggacgag gaccattaaa actgttcatg 240 gccctggtgg cgttccttcg tttcctaaca atcccaccaa cagcagggat attgaagaga 300 tggggaacaa ttaaaaaatc aaaagctatt aatgttttga gagggttcag gaaagagatt 360 ggaaggatgc tgaacatctt gaataggaga cgcagatctg caggcatgat cattatgctg 420 attccaacag tgatggcgtt ccatttaacc acacgtaacg gagaaccaca catgatcgtc 480 agcagacaag agaaagggaa aagtcttctg tttaaaacag aggttggcgt gaacatgtgt 540 accctcatgg ccatggacct tggtgaattg tgtgaagaca caatcacgta cgagtgtccc 600 cttctcaggc agaatgagcc agaagacata gactgttggt gcaactctac gtccacgtgg 660 gtaacttatg ggacgtgtac caccatggga gaacatagaa gagaaaaaag atcagtggca 720 ctcgttccac atgtgggaat gggactggag acacgaactg aaacatggat gtcatcagaa 780 ggggcctgga aacatgtcca gagaattgaa acttggatct tgagacatcc aggcttcacc 840 atgatggcag caatcctggc atacaccata ggaacgacac atttccaaag agccctgatc 900 ttcatcttac tgacagctgt cactccttca atgacaatgc gttgcatagg aatgtcaaat 960 agagactttg tggaaggggt ttcaggagga agctgggttg acatagtctt agaacatgga 1020 agctgtgtga cgacgatggc aaaaaacaaa ccaacattgg attttgaact gataaaaaca 1080 gaagccaaac agcctgccac cctaaggaag tactgtatag aggcaaagct aaccaacaca 1140 acaacagaat ctcgctgccc aacacaaggg gaacccagcc taaatgaaga gcaggacaaa 1200 aggttcgtct gcaaacactc catggtagac agaggatggg gaaatggatg tggactattt 1260 ggaaagggag gcattgtgac ctgtgctatg ttcagatgca aaaagaacat ggaaggaaaa 1320 gttgtgcaac cagaaaactt ggaatacacc attgtgataa cacctcactc aggggaagag 1380 catgcagtcg gaaatgacac aggaaaacat ggcaaggaaa tcaaaataac accacagagt 1440 tccatcacag aagcagaatt gacaggttat ggcactgtca caatggagtg ctctccaaga 1500 acgggcctcg acttcaatga gatggtgttg ctgcagatgg aaaataaagc ttggctggtg 1560 cacaggcaat ggttcctaga cctgccgtta ccatggttgc ccggagcgga cacacaaggg 1620 tcaaattgga tacagaaaga gacattggtc actttcaaaa atccccatgc gaagaaacag 1680 gatgttgttg ttttaggatc ccaagaaggg gccatgcaca cagcacttac aggggccaca 1740 gaaatccaaa tgtcatcagg aaacttactc ttcacaggac atctcaagtg caggctgaga 1800 atggacaagc tacagctcaa aggaatgtca tactctatgt gcacaggaaa gtttaaagtt 1860 gtgaaggaaa tagcagaaac acaacatgga acaatagtta tcagagtgca atatgaaggg 1920 gacggctctc catgcaagat cccttttgag ataatggatt tggaaaaaag acatgtctta 1980 ggtcgcctga ttacagtcaa cccaattgtg acagaaaaag atagcccagt caacatagaa 2040 gcagaacctc catttggaga cagctacatc atcataggag tagagccggg acaactgaag 2100 ctcaactggt ttaagaaagg aagttctatc ggccaaatgt ttgagacaac aatgagggg 2160 gcgaagagaa tggccatttt aggtgacaca gcctgggatt ttggatcctt gggaggagtg 2220 tttacatcta taggaaaggc tctccaccaa gtctttggag caatctatgg agctgccttc 2280 agtggggttt catggactat gaaaatcctc ataggagtca ttatcacatg gataggaatg 2340 aattcacgca gcacctcact gtctgtgaca ctagtattgg tgggaattgt gacactgtat 2400 ttgggagtca tggtgcaggc cgatagtggt tgcgttgtga gctggaaaaa caaagaactg 2460 aaatgtggca gtgggatttt catcacagac aacgtgcaca catggacaga acaatacaag 2520 ttccaaccag aatccccttc aaaactagct tcagctatcc agaaagccca tgaagaggac 2580 atttgtggaa tccgctcagt aacaagactg gagaatctga tgtggaaaca aataacacca 2640 gaattgaatc acattctatc agaaaatgag gtgaagttaa ctattatgac aggagacatc 2700 aaaggaatca tgcaggcagg aaaacgatct ctgcggcctc agcccactga gctgaagtat 2760 tcatggaaaa catggggcaa agcaaaaatg ctctctacag agtctcataa ccagaccttt 2820 ctcattgatg gccccgaaac agcagaatgc cccaacacaa atagagcttg gaattcgttg 2880 gaagttgaag actatggctt tggagtattc accaccaata tatggctaaa attgaaagaa 2940 aaacaggatg tattctgcga ctcaaaactc atgtcagcgg ccataaaaga caacagagcc 3000 gtccatgccg atatgggtta ttggatagaa agtgcactca atgacacatg gaagatagag 3060 aaagcctctt tcattgaagt taaaaactgc cactggccaa aatcacacac cctctggagc 3120 aatggagtgc tagaaagtga gatgataatt ccaaagaatc tcgctggacc agtgtctcaa 3180 cacaactata gaccaggcta ccatacacaa ataacaggac catggcatct aggtaagctt 3240 gagatggact ttgatttctg tgatggaaca acagtggtag tgactgagga ctgcggaaat 3300 agaggaccct ctttgagaac aaccactgcc tctggaaaac tcataacaga atggtgctgc 3360 cgatcttgca cattaccacc gctaagatac agaggtgagg atgggtgctg gtacgggatg 3420 gaaatcagac cattgaagga gaaagaagag aatttggtca actccttggt cacagctgga 3480 catgggcagg tcgacaactt ttcactagga gtcttgggaa tggcattgtt cctggaggaa 3540 atgcttagga cccgagtagg aacgaaacat gcaatactac tagttgcagt ttcttttgtg 3600 acattgatca cagggaacat gtcctttaga gacctgggaa gagtgatggt tatggtaggc 3660 gccactatga cggatgacat aggtatgggc gtgacttatc ttgccctact agcagccttc 3720 aaagtcagac caacttttgc agctggacta ctcttgagaa agctgacctc caaggaattg 3780 atgatgacta ctataggaat tgtactcctc tcccagagca ccataccaga gaccattctt 3840 gagttgactg atgcgttagc cttaggcatg atggtcctca aaatggtgag aaatatggaa 3900 aagtatcaat tggcagtgac tatcatggct atcttgtgcg tcccaaacgc agtgatatta 3960 caaaacgcat ggaaagtgag ttgcacaata ttggcagtgg tgtccgtttc cccactgttc 4020 ttaacatcct cacagcaaaa aacagattgg ataccattag cattgacgat caaaggtctc 4080 aatccaacag ctatttttct aacaaccctc tcaagaacca gcaagaaaag gagctggcca 4140 ttaaatgagg ctatcatggc agtcgggatg gtgagcattt tagccagttc tctcctaaaa 4200 aatgatattc ccatgacagg accattagtg gctggagggc tcctcactgt gtgctacgtg 4260 ctcactggac gatcggccga tttggaactg gagagagcag ccgatgtcaa atgggaagac 4320 caggcagaga tatcaggaag cagtccaatc ctgtcaataa caatatcaga agatggtagc 4380 atgtcgataa aaaatgaaga ggaagaacaa acactgacca tactcattag aacaggattg 4440 ctggtgatct caggactttt tcctgtatca ataccaatca cggcagcagc atggtacctg 4500 tgggaagtga agaaacaacg ggccggagta ttgtgggatg ttccttcacc cccacccatg 4560 ggaaaggctg aactggaaga tggagcctat agaattaagc aaaaagggat tcttggatat 4620 tcccagatcg gagccggagt ttacaaagaa ggaacattcc atacaatgtg gcatgtcaca 4680 cgtggcgctg ttctaatgca taaaggaaag aggattgaac catcatgggc ggacgtcaag 4740 aaagacctaa tatcatatgg aggaggctgg aagttagaag gagaatggaa ggaaggagaa 4800 gaagtccagg tattggcact ggagcctgga aaaaatccaa gagccgtcca aacgaaacct 4860 ggtcttttca aaaccaacgc cggaacaata ggtgctgtat ctctggactt ttctcctgga 4920 acgtcaggat ctccaattat cgacaaaaaa ggaaaagttg tgggtcttta tggtaatggt 4980 gttgttacaa ggagtggagc atatgtgagt gctatagccc agactgaaaa aagcattgaa 5040 gacaacccag agatcgaaga tgacattttc cgaaagagaa gactgaccat catggacctc 5100 cacccaggag cgggaaagac gaagagatac cttccggcca tagtcagaga agctataaaa 5160 cggggtttga gaacattaat cttggccccc actagagttg tggcagctga aatggaggaa 5220 gcccttagag gacttccaat aagataccag accccagcca tcagagctgt gcacaccggg 5280 cgggagattg tggacctaat gtgtcatgcc acatttacca tgaggctgct atcaccagtt 5340 agagtgccaa actacaacct gattatcatg gacgaagccc atttcacaga cccagcaagt 5400 atagcagcta gaggatacat ctcaactcga gtggagatgg gtgaggcagc tgggattttt 5460 atgacagcca ctccccccggg aagcagagac ccatttcctc agagcaatgc accaatcata 5520 gatgaagaaa gagaaatccc tgaacgctcg tggaattccg gacatgaatg ggtcacggat 5580 tttaaaggga agactgtttg gttcgttcca agtataaaag caggaaatga tatagcagct 5640 tgcctgagga aaaatggaaa gaaagtgata caactcagta ggaagacctt tgattctgag 5700 tatgtcaaga ctagaaccaa tgattgggac ttcgtggtta caactgacat ttcagaaatg 5760 ggtgccaatt tcaaggctga gagggttata gaccccagac gctgcatgaa accagtcata 5820 ctaacagatg gtgaagagcg ggtgattctg gcaggaccta tgccagtgac ccactctagt 5880 gcagcacaaa gaagagggag aataggaaga aatccaaaaa atgagaatga ccagtacata 5940 tacatggggg aacctctgga aaatgatgaa gactgtgcac actggaaaga agctaaaatg 6000 ctcctagata acatcaacac gccagaagga atcattccta gcatgttcga accagagcgt 6060 gaaaaggtgg atgccattga tggcgaatac cgcttgagag gagaagcaag gaaaaccttt 6120 gtagacttaa tgagaagagg agacctacca gtctggttgg cctacagagt ggcagctgaa 6180 ggcatcaact acgcagacag aaggtggtgt tttgatggag tcaagaacaa ccaaatccta 6240 gaagaaaacg tggaagttga aatctggaca aaagaagggg aaaggaagaa attgaaaccc 6300 agatggttgg atgctaggat ctattctgac ccactggcgc taaaagaatt taaggaattt 6360 gcagccggaa gaaagtctct gaccctgaac ctaatcacag aaatgggtag gctcccaacc 6420 ttcatgactc agaaggcaag agacgcactg gacaacttag cagtgctgca cacggctgag 6480 gcaggtggaa gggcgtacaa ccatgctctc agtgaactgc cggagaccct ggagacattg 6540 cttttactga cacttctggc tacagtcacg ggagggatct ttttattctt gatgagcgca 6600 aggggcatag ggaagatgac cctgggaatg tgctgcataa tcacggctag catcctccta 6660 tggtacgcac aaatacagcc acactggata gcagcttcaa taatactgga gttttttctc 6720 atagttttgc ttattccaga acctgaaaaa cagagaacac cccaagacaa ccaactgacc 6780 tacgttgtca tagccatcct cacagtggtg gccgcaacca tggcaaacga gatgggtttc 6840 ctagaaaaaa cgaagaaaga tctcggattg ggaagcattg caacccagca acccgagagc 6900 aacatcctgg acatagatct acgtcctgca tcagcatgga cgctgtatgc cgtggccaca 6960 acatttgtta caccaatgtt gagacatagc attgaaaatt cctcagtgaa tgtgtcccta 7020 acagctatag ccaaccaagc cacagtgtta atgggtctcg ggaaaggatg gccattgtca 7080 aagatggaca tcggagttcc ccttctcgcc attggatgct actcacaagt caaccccata 7140 actctcacag cagctctttt cttattggta gcacattatg ccatcatagg gccaggactc 7200 caagcaaaag caaccagaga agctcagaaa agagcagcgg cgggcatcat gaaaaaccca 7260 actgtcgatg gaataacagt gattgaccta gatccaatac cttatgatcc aaagtttgaa 7320 aagcagttgg gacaagtaat gctcctagtc ctctgcgtga ctcaagtatt gatgatgagg 7380 actacatggg ctctgtgtga ggctttaacc ttagctaccg ggcccatctc cacatgtgg 7440 gaaggaaatc cagggaggtt ttggaacact accattgcgg tgtcaatggc taacattttt 7500 agagggagtt acttggccgg agctggactt ctcttttcta ttatgaagaa cacaaccaac 7560 acaagaaggg gaactggcaa cataggagag acgcttggag agaaatggaa aagccgattg 7620 aacgcattgg gaaaaagtga attccagatc tacaagaaaa gtggaatcca ggaagtggat 7680 agaaccttag caaaagaagg cattaaaaga ggagaaacgg accatcacgc tgtgtcgcga 7740 ggctcagcaa aactgagatg gttcgttgag agaaacatgg tcacaccaga agggaaagta 7800 gtggacctcg gttgtggcag aggaggctgg tcatactatt gtggaggact aaagaatgta 7860 agagaagtca aaggcctaac aaaaggagga ccaggacacg aagaacccat ccccatgtca 7920 acatatgggt ggaatctagt gcgtcttcaa agtggagttg acgttttctt catcccgcca 7980 gaaaagtgtg acacattatt gtgtgacata ggggagtcat caccaaatcc cacagtggaa 8040 gcaggacgaa cactcagagt ccttaactta gtagaaaatt ggttgaacaa caacactcaa 8100 ttttgcataa aggttctcaa cccatatatg ccctcagtca tagaaaaaat ggaagcacta 8160 caaaggaaat atggaggagc cttagtgagg aatccactct cacgaaactc cacacatgag 8220 atgtactggg tatccaatgc ttccgggaac atagtgtcat cagtgaacat gatttcaagg 8280 atgttgatca acagattac aatgagatac aagaaagcca cttacgagcc ggatgttgac 8340 ctcggaagcg gaacccgtaa catcgggatt gaaagtgaga taccaaacct agatataatt 8400 gggaaaagaa tagaaaaaat aaagcaagag catgaaacat catggcacta tgaccaagac 8460 cacccataca aaacgtgggc ataccatggt agctatgaaa caaaacagac tggatcagca 8520 tcatccatgg tcaacggagt ggtcaggctg ctgacaaaac cttgggacgt cgtccccatg 8580 gtgacacaga tggcaatgac agacacgact ccatttggac aacagcgcgt ttttaaagag 8640 aaagtggaca cgagaaccca agaaccgaaa gaaggcacga agaaactaat gaaaataaca 8700 gcagagtggc tttggaaaga attagggaag aaaaagacac ccaggatgtg caccagagaa 8760 gaattcacaa gaaaggtgag aagcaatgca gccttggggg ccgtattcac tgatgagaac 8820 aagtggaagt cggcacgtga ggctgttgaa gatagtaggt tttgggagct ggttgacaag 8880 gaaaggaatc tccatcttga aggaaagtgt gaaacatgtg tgtacaacat gatgggaaaa 8940 agagagaaga agctagggga attcggcaag gcaaaaggca gcagagccat atggtacatg 9000 tggcttggag cacgcttctt agagtttgaa gccctaggat tcttaaatga agatcactgg 9060 ttctccagag agaactccct gagtggagtg gaaggagaag ggctgcacaa gctaggttac 9120 attctaagag acgtgagcaa gaaagaggga ggagcaatgt atgccgatga caccgcagga 9180 tgggatacaa gaatcacact agaagaccta aaaaatgaag aaatggtaac aaaccacatg 9240 gaaggagaac acaagaaact agccgaggcc attttcaaac taacgtacca aaacaaggtg 9300 gtgcgtgtgc aaagaccaac accaagaggc acagtaatgg acatcatatc gagaagagac 9360 caaagaggta gtggacaagt tggcacctat ggactcaata ctttcaccaa tatggaagcc 9420 caactaatca gacagatgga gggagaagga gtctttaaaa gcattcagca cctaacaatc 9480 acagaagaaa tcgctgtgca aaactggtta gcaagagtgg ggcgcgaaag gttatcaaga 9540 atggccatca gtggagatga ttgtgttgtg aaacctttag atgacaggtt cgcaagcgct 9600 ttaacagctc taaatgacat gggaaagatt aggaaagaca tacaacaatg ggaaccttca 9660 agaggatgga atgattggac acaagtgccc ttctgttcac accatttcca tgagttaatc 9720 atgaaagacg gtcgcgtact cgttgttcca tgtagaaacc aagatgaact gattggcaga 9780 gcccgaatct cccaaggagc agggtggtct ttgcgggaga cggcctgttt ggggaagtct 9840 tacgcccaaa tgtggagctt gatgtacttc cacagacgcg acctcaggct ggcggcaaat 9900 gctatttgct cggcagtacc atcacattgg gttccaacaa gtcgaacaac ctggtccata 9960 catgctaaac atgaatggat gacaacggaa gacatgctga cagtctggaa cagggtgtgg 10020 attcaagaaa acccatggat ggaagacaaa actccagtgg aatcatggga ggaaatccca 10080 tacttgggga aaagagaaga ccaatggtgc ggctcattga ttgggttaac aagcagggcc 10140 acctgggcaa agaacatcca agcagcaata aatcaagtta gatcccttat aggcaatgaa 10200 gaatacacag attacatgcc atccatgaaa agattcagaa gagaagagga agaagcagga 10260 gttctgtggt agaaagcaaa actaacatga aacaaggcta gaagtcaggt cggattaagc 10320 catagtacgg aaaaaactat gctacctgtg agccccgtcc aaggacgtta aaagaagtca 10380 ggccatcata aatgccatag cttgagtaaa ctatgcagcc tgtagctcca cctgagaagg 10440 tgtaaaaaat ccgggaggcc acaaaccatg gaagctgtac gcatggcgta gtggactagc 10500 ggttagagga gacccctccc ttacaaatcg cagcaacaat gggggcccaa ggcgagatga 10560 agctgtagtc tcgctggaag gactagaggt tagaggagac ccccccgaaa caaaaaacag 10620 catattgacg ctgggaaaga ccagagatcc tgctgtctcc tcagcatcat tccaggcaca 10680 gaacgccaga aaatggaatg gtgctgttga atcaacaggt tct 10723 <210> 4 <211> 3391 <212> PRT <213> dengue seroytpe 2 (MVS) <400> 4 Met Asn Asn Gln Arg Lys Lys Ala Lys Asn Thr Pro Phe Asn Met Leu 1 5 10 15 Lys Arg Glu Arg Asn Arg Val Ser Thr Val Gln Gln Leu Thr Lys Arg 20 25 30 Phe Ser Leu Gly Met Leu Gln Gly Arg Gly Pro Leu Lys Leu Phe Met 35 40 45 Ala Leu Val Ala Phe Leu Arg Phe Leu Thr Ile Pro Pro Thr Ala Gly 50 55 60 Ile Leu Lys Arg Trp Gly Thr Ile Lys Lys Ser Lys Ala Ile Asn Val 65 70 75 80 Leu Arg Gly Phe Arg Lys Glu Ile Gly Arg Met Leu Asn Ile Leu Asn 85 90 95 Arg Arg Arg Arg Ser Ala Gly Met Ile Ile Met Leu Ile Pro Thr Val 100 105 110 Met Ala Phe His Leu Thr Thr Arg Asn Gly Glu Pro His Met Ile Val 115 120 125 Ser Arg Gin Glu Lys Gly Lys Ser Leu Leu Phe Lys Thr Glu Val Gly 130 135 140 Val Asn Met Cys Thr Leu Met Ala Met Asp Leu Gly Glu Leu Cys Glu 145 150 155 160 Asp Thr Ile Thr Tyr Glu Cys Pro Leu Leu Arg Gln Asn Glu Pro Glu 165 170 175 Asp Ile Asp Cys Trp Cys Asn Ser Thr Ser Thr Trp Val Thr Tyr Gly 180 185 190 Thr Cys Thr Thr Met Gly Glu His Arg Arg Glu Lys Arg Ser Val Ala 195 200 205 Leu Val Pro His Val Gly Met Gly Leu Glu Thr Arg Thr Glu Thr Trp 210 215 220 Met Ser Ser Glu Gly Ala Trp Lys His Val Gln Arg Ile Glu Thr Trp 225 230 235 240 Ile Leu Arg His Pro Gly Phe Thr Met Met Ala Ala Ile Leu Ala Tyr 245 250 255 Thr Ile Gly Thr Thr His Phe Gln Arg Ala Leu Ile Phe Ile Leu Leu 260 265 270 Thr Ala Val Thr Pro Ser Met Thr Met Arg Cys Ile Gly Met Ser Asn 275 280 285 Arg Asp Phe Val Glu Gly Val Ser Gly Gly Ser Trp Val Asp Ile Val 290 295 300 Leu Glu His Gly Ser Cys Val Thr Thr Met Ala Lys Asn Lys Pro Thr 305 310 315 320 Leu Asp Phe Glu Leu Ile Lys Thr Glu Ala Lys Gln Pro Ala Thr Leu 325 330 335 Arg Lys Tyr Cys Ile Glu Ala Lys Leu Thr Asn Thr Thr Thr Glu Ser 340 345 350 Arg Cys Pro Thr Gln Gly Glu Pro Ser Leu Asn Glu Glu Gln Asp Lys 355 360 365 Arg Phe Val Cys Lys His Ser Met Val Asp Arg Gly Trp Gly Asn Gly 370 375 380 Cys Gly Leu Phe Gly Lys Gly Gly Ile Val Thr Cys Ala Met Phe Arg 385 390 395 400 Cys Lys Lys Asn Met Glu Gly Lys Val Val Gin Pro Glu Asn Leu Glu 405 410 415 Tyr Thr Ile Val Ile Thr Pro His Ser Gly Glu Glu His Ala Val Gly 420 425 430 Asn Asp Thr Gly Lys His Gly Lys Glu Ile Lys Ile Thr Pro Gln Ser 435 440 445 Ser Ile Thr Glu Ala Glu Leu Thr Gly Tyr Gly Thr Val Thr Met Glu 450 455 460 Cys Ser Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Val Leu Leu Gln 465 470 475 480 Met Glu Asn Lys Ala Trp Leu Val His Arg Gln Trp Phe Leu Asp Leu 485 490 495 Pro Leu Pro Trp Leu Pro Gly Ala Asp Thr Gln Gly Ser Asn Trp Ile 500 505 510 Gln Lys Glu Thr Leu Val Thr Phe Lys Asn Pro His Ala Lys Lys Gln 515 520 525 Asp Val Val Val Leu Gly Ser Gln Glu Gly Ala Met His Thr Ala Leu 530 535 540 Thr Gly Ala Thr Glu Ile Gln Met Ser Ser Gly Asn Leu Leu Phe Thr 545 550 555 560 Gly His Leu Lys Cys Arg Leu Arg Met Asp Lys Leu Gln Leu Lys Gly 565 570 575 Met Ser Tyr Ser Met Cys Thr Gly Lys Phe Lys Val Val Lys Glu Ile 580 585 590 Ala Glu Thr Gln His Gly Thr Ile Val Ile Arg Val Gln Tyr Glu Gly 595 600 605 Asp Gly Ser Pro Cys Lys Ile Pro Phe Glu Ile Met Asp Leu Glu Lys 610 615 620 Arg His Val Leu Gly Arg Leu Ile Thr Val Asn Pro Ile Val Thr Glu 625 630 635 640 Lys Asp Ser Pro Val Asn Ile Glu Ala Glu Pro Phe Gly Asp Ser 645 650 655 Tyr Ile Ile Ile Gly Val Glu Pro Gly Gln Leu Lys Leu Asn Trp Phe 660 665 670 Lys Lys Gly Ser Ser Ile Gly Gln Met Phe Glu Thr Thr Met Arg Gly 675 680 685 Ala Lys Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser 690 695 700 Leu Gly Gly Val Phe Thr Ser Ile Gly Lys Ala Leu His Gln Val Phe 705 710 715 720 Gly Ala Ile Tyr Gly Ala Ala Phe Ser Gly Val Ser Trp Thr Met Lys 725 730 735 Ile Leu Ile Gly Val Ile Ile Thr Trp Ile Gly Met Asn Ser Arg Ser 740 745 750 Thr Ser Leu Ser Val Thr Leu Val Leu Val Gly Ile Val Thr Leu Tyr 755 760 765 Leu Gly Val Met Val Gln Ala Asp Ser Gly Cys Val Val Ser Trp Lys 770 775 780 Asn Lys Glu Leu Lys Cys Gly Ser Gly Ile Phe Ile Thr Asp Asn Val 785 790 795 800 His Thr Trp Thr Glu Gln Tyr Lys Phe Gln Pro Glu Ser Pro Ser Lys 805 810 815 Leu Ala Ser Ala Ile Gln Lys Ala His Glu Glu Asp Ile Cys Gly Ile 820 825 830 Arg Ser Val Thr Arg Leu Glu Asn Leu Met Trp Lys Gln Ile Thr Pro 835 840 845 Glu Leu Asn His Ile Leu Ser Glu Asn Glu Val Lys Leu Thr Ile Met 850 855 860 Thr Gly Asp Ile Lys Gly Ile Met Gln Ala Gly Lys Arg Ser Leu Arg 865 870 875 880 Pro Gln Pro Thr Glu Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala 885 890 895 Lys Met Leu Ser Thr Glu Ser His Asn Gln Thr Phe Leu Ile Asp Gly 900 905 910 Pro Glu Thr Ala Glu Cys Pro Asn Thr Asn Arg Ala Trp Asn Ser Leu 915 920 925 Glu Val Glu Asp Tyr Gly Phe Gly Val Phe Thr Thr Asn Ile Trp Leu 930 935 940 Lys Leu Lys Glu Lys Gln Asp Val Phe Cys Asp Ser Lys Leu Met Ser 945 950 955 960 Ala Ala Ile Lys Asp Asn Arg Ala Val His Ala Asp Met Gly Tyr Trp 965 970 975 Ile Glu Ser Ala Leu Asn Asp Thr Trp Lys Ile Glu Lys Ala Ser Phe 980 985 990 Ile Glu Val Lys Asn Cys His Trp Pro Lys Ser His Thr Leu Trp Ser 995 1000 1005 Asn Gly Val Leu Glu Ser Glu Met Ile Ile Pro Lys Asn Leu Ala Gly 1010 1015 1020 Pro Val Ser Gln His Asn Tyr Arg Pro Gly Tyr His Thr Gln Ile Thr 1025 1030 1035 1040 Gly Pro Trp His Leu Gly Lys Leu Glu Met Asp Phe Asp Phe Cys Asp 1045 1050 1055 Gly Thr Thr Val Val Val Thr Glu Asp Cys Gly Asn Arg Gly Pro Ser 1060 1065 1070 Leu Arg Thr Thr Thr Ala Ser Gly Lys Leu Ile Thr Glu Trp Cys Cys 1075 1080 1085 Arg Ser Cys Thr Leu Pro Leu Arg Tyr Arg Gly Glu Asp Gly Cys 1090 1095 1100 Trp Tyr Gly Met Glu Ile Arg Pro Leu Lys Glu Lys Glu Glu Asn Leu 1105 1110 1115 1120 Val Asn Ser Leu Val Thr Ala Gly His Gly Gln Val Asp Asn Phe Ser 1125 1130 1135 Leu Gly Val Leu Gly Met Ala Leu Phe Leu Glu Glu Met Leu Arg Thr 1140 1145 1150 Arg Val Gly Thr Lys His Ala Ile Leu Leu Val Ala Val Ser Phe Val 1155 1160 1165 Thr Leu Ile Thr Gly Asn Met Ser Phe Arg Asp Leu Gly Arg Val Met 1170 1175 1180 Val Met Val Gly Ala Thr Met Thr Asp Asp Ile Gly Met Gly Val Thr 1185 1190 1195 1200 Tyr Leu Ala Leu Leu Ala Ala Phe Lys Val Arg Pro Thr Phe Ala Ala 1205 1210 1215 Gly Leu Leu Leu Arg Lys Leu Thr Ser Lys Glu Leu Met Met Thr Thr 1220 1225 1230 Ile Gly Ile Val Leu Leu Ser Gln Ser Thr Ile Pro Glu Thr Ile Leu 1235 1240 1245 Glu Leu Thr Asp Ala Leu Ala Leu Gly Met Met Val Leu Lys Met Val 1250 1255 1260 Arg Asn Met Glu Lys Tyr Gln Leu Ala Val Thr Ile Met Ala Ile Leu 1265 1270 1275 1280 Cys Val Pro Asn Ala Val Ile Leu Gln Asn Ala Trp Lys Val Ser Cys 1285 1290 1295 Thr Ile Leu Ala Val Val Ser Val Ser Pro Leu Phe Leu Thr Ser Ser 1300 1305 1310 Gln Gln Lys Thr Asp Trp Ile Pro Leu Ala Leu Thr Ile Lys Gly Leu 1315 1320 1325 Asn Pro Thr Ala Ile Phe Leu Thr Thr Leu Ser Arg Thr Ser Lys Lys 1330 1335 1340 Arg Ser Trp Pro Leu Asn Glu Ala Ile Met Ala Val Gly Met Val Ser 1345 1350 1355 1360 Ile Leu Ala Ser Leu Leu Lys Asn Asp Ile Pro Met Thr Gly Pro 1365 1370 1375 Leu Val Ala Gly Gly Leu Leu Thr Val Cys Tyr Val Leu Thr Gly Arg 1380 1385 1390 Ser Ala Asp Leu Glu Leu Glu Arg Ala Ala Asp Val Lys Trp Glu Asp 1395 1400 1405 Gln Ala Glu Ile Ser Gly Ser Ser Pro Ile Leu Ser Ile Thr Ile Ser 1410 1415 1420 Glu Asp Gly Ser Met Ser Ile Lys Asn Glu Glu Glu Glu Gln Thr Leu 1425 1430 1435 1440 Thr Ile Leu Ile Arg Thr Gly Leu Leu Val Ile Ser Gly Leu Phe Pro 1445 1450 1455 Val Ser Ile Pro Ile Thr Ala Ala Ala Trp Tyr Leu Trp Glu Val Lys 1460 1465 1470 Lys Gln Arg Ala Gly Val Leu Trp Asp Val Pro Ser Pro Pro Met 1475 1480 1485 Gly Lys Ala Glu Leu Glu Asp Gly Ala Tyr Arg Ile Lys Gln Lys Gly 1490 1495 1500 Ile Leu Gly Tyr Ser Gln Ile Gly Ala Gly Val Tyr Lys Glu Gly Thr 1505 1510 1515 1520 Phe His Thr Met Trp His Val Thr Arg Gly Ala Val Leu Met His Lys 1525 1530 1535 Gly Lys Arg Ile Glu Pro Ser Trp Ala Asp Val Lys Lys Asp Leu Ile 1540 1545 1550 Ser Tyr Gly Gly Gly Trp Lys Leu Glu Gly Glu Trp Lys Glu Gly Glu 1555 1560 1565 Glu Val Gln Val Leu Ala Leu Glu Pro Gly Lys Asn Pro Arg Ala Val 1570 1575 1580 Gln Thr Lys Pro Gly Leu Phe Lys Thr Asn Ala Gly Thr Ile Gly Ala 1585 1590 1595 1600 Val Ser Leu Asp Phe Ser Pro Gly Thr Ser Gly Ser Pro Ile Ile Asp 1605 1610 1615 Lys Lys Gly Lys Val Val Gly Leu Tyr Gly Asn Gly Val Val Thr Arg 1620 1625 1630 Ser Gly Ala Tyr Val Ser Ala Ile Ala Gln Thr Glu Lys Ser Ile Glu 1635 1640 1645 Asp Asn Pro Glu Ile Glu Asp Asp Ile Phe Arg Lys Arg Arg Leu Thr 1650 1655 1660 Ile Met Asp Leu His Pro Gly Ala Gly Lys Thr Lys Arg Tyr Leu Pro 1665 1670 1675 1680 Ala Ile Val Arg Glu Ala Ile Lys Arg Gly Leu Arg Thr Leu Ile Leu 1685 1690 1695 Ala Pro Thr Arg Val Val Ala Ala Glu Met Glu Glu Ala Leu Arg Gly 1700 1705 1710 Leu Pro Ile Arg Tyr Gln Thr Pro Ala Ile Arg Ala Val His Thr Gly 1715 1720 1725 Arg Glu Ile Val Asp Leu Met Cys His Ala Thr Phe Thr Met Arg Leu 1730 1735 1740 Leu Ser Pro Val Arg Val Pro Asn Tyr Asn Leu Ile Ile Met Asp Glu 1745 1750 1755 1760 Ala His Phe Thr Asp Pro Ala Ser Ile Ala Ala Arg Gly Tyr Ile Ser 1765 1770 1775 Thr Arg Val Glu Met Gly Glu Ala Ala Gly Ile Phe Met Thr Ala Thr 1780 1785 1790 Pro Pro Gly Ser Arg Asp Pro Phe Pro Gln Ser Asn Ala Pro Ile Ile 1795 1800 1805 Asp Glu Glu Arg Glu Ile Pro Glu Arg Ser Trp Asn Ser Gly His Glu 1810 1815 1820 Trp Val Thr Asp Phe Lys Gly Lys Thr Val Trp Phe Val Pro Ser Ile 1825 1830 1835 1840 Lys Ala Gly Asn Asp Ile Ala Ala Cys Leu Arg Lys Asn Gly Lys Lys 1845 1850 1855 Val Ile Gln Leu Ser Arg Lys Thr Phe Asp Ser Glu Tyr Val Lys Thr 1860 1865 1870 Arg Thr Asn Asp Trp Asp Phe Val Val Thr Asp Ile Ser Glu Met 1875 1880 1885 Gly Ala Asn Phe Lys Ala Glu Arg Val Ile Asp Pro Arg Arg Cys Met 1890 1895 1900 Lys Pro Val Ile Leu Thr Asp Gly Glu Glu Arg Val Ile Leu Ala Gly 1905 1910 1915 1920 Pro Met Pro Val Thr His Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile 1925 1930 1935 Gly Arg Asn Pro Lys Asn Glu Asn Asp Gln Tyr Ile Tyr Met Gly Glu 1940 1945 1950 Pro Leu Glu Asn Asp Glu Asp Cys Ala His Trp Lys Glu Ala Lys Met 1955 1960 1965 Leu Leu Asp Asn Ile Asn Thr Pro Glu Gly Ile Ile Pro Ser Met Phe 1970 1975 1980 Glu Pro Glu Arg Glu Lys Val Asp Ala Ile Asp Gly Glu Tyr Arg Leu 1985 1990 1995 2000 Arg Gly Glu Ala Arg Lys Thr Phe Val Asp Leu Met Arg Arg Gly Asp 2005 2010 2015 Leu Pro Val Trp Leu Ala Tyr Arg Val Ala Ala Glu Gly Ile Asn Tyr 2020 2025 2030 Ala Asp Arg Arg Trp Cys Phe Asp Gly Val Lys Asn Asn Gln Ile Leu 2035 2040 2045 Glu Glu Asn Val Glu Val Glu Ile Trp Thr Lys Glu Gly Glu Arg Lys 2050 2055 2060 Lys Leu Lys Pro Arg Trp Leu Asp Ala Arg Ile Tyr Ser Asp Pro Leu 2065 2070 2075 2080 Ala Leu Lys Glu Phe Lys Glu Phe Ala Ala Gly Arg Lys Ser Leu Thr 2085 2090 2095 Leu Asn Leu Ile Thr Glu Met Gly Arg Leu Pro Thr Phe Met Thr Gln 2100 2105 2110 Lys Ala Arg Asp Ala Leu Asp Asn Leu Ala Val Leu His Thr Ala Glu 2115 2120 2125 Ala Gly Gly Arg Ala Tyr Asn His Ala Leu Ser Glu Leu Pro Glu Thr 2130 2135 2140 Leu Glu Thr Leu Leu Leu Leu Thr Leu Leu Ala Thr Val Thr Gly Gly 2145 2150 2155 2160 Ile Phe Leu Phe Leu Met Ser Ala Arg Gly Ile Gly Lys Met Thr Leu 2165 2170 2175 Gly Met Cys Cys Ile Ile Thr Ala Ser Ile Leu Leu Trp Tyr Ala Gln 2180 2185 2190 Ile Gln Pro His Trp Ile Ala Ala Ser Ile Ile Leu Glu Phe Phe Leu 2195 2200 2205 Ile Val Leu Leu Ile Pro Glu Pro Glu Lys Gln Arg Thr Pro Gln Asp 2210 2215 2220 Asn Gln Leu Thr Tyr Val Val Ile Ala Ile Leu Thr Val Val Ala Ala 2225 2230 2235 2240 Thr Met Ala Asn Glu Met Gly Phe Leu Glu Lys Thr Lys Lys Asp Leu 2245 2250 2255 Gly Leu Gly Ser Ile Ala Thr Gln Gln Pro Glu Ser Asn Ile Leu Asp 2260 2265 2270 Ile Asp Leu Arg Pro Ala Ser Ala Trp Thr Leu Tyr Ala Val Ala Thr 2275 2280 2285 Thr Phe Val Thr Pro Met Leu Arg His Ser Ile Glu Asn Ser Ser Val 2290 2295 2300 Asn Val Ser Leu Thr Ala Ile Ala Asn Gln Ala Thr Val Leu Met Gly 2305 2310 2315 2320 Leu Gly Lys Gly Trp Pro Leu Ser Lys Met Asp Ile Gly Val Pro Leu 2325 2330 2335 Leu Ala Ile Gly Cys Tyr Ser Gln Val Asn Pro Ile Thr Leu Thr Ala 2340 2345 2350 Ala Leu Phe Leu Leu Val Ala His Tyr Ala Ile Ile Gly Pro Gly Leu 2355 2360 2365 Gln Ala Lys Ala Thr Arg Glu Ala Gln Lys Arg Ala Ala Ala Gly Ile 2370 2375 2380 Met Lys Asn Pro Thr Val Asp Gly Ile Thr Val Ile Asp Leu Asp Pro 2385 2390 2395 2400 Ile Pro Tyr Asp Pro Lys Phe Glu Lys Gln Leu Gly Gln Val Met Leu 2405 2410 2415 Leu Val Leu Cys Val Thr Gln Val Leu Met Met Arg Thr Thr Trp Ala 2420 2425 2430 Leu Cys Glu Ala Leu Thr Leu Ala Thr Gly Pro Ile Ser Thr Leu Trp 2435 2440 2445 Glu Gly Asn Pro Gly Arg Phe Trp Asn Thr Thr Ile Ala Val Ser Met 2450 2455 2460 Ala Asn Ile Phe Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu Leu Phe 2465 2470 2475 2480 Ser Ile Met Lys Asn Thr Thr Asn Thr Arg Arg Gly Thr Gly Asn Ile 2485 2490 2495 Gly Glu Thr Leu Gly Glu Lys Trp Lys Ser Arg Leu Asn Ala Leu Gly 2500 2505 2510 Lys Ser Glu Phe Gln Ile Tyr Lys Lys Ser Gly Ile Gln Glu Val Asp 2515 2520 2525 Arg Thr Leu Ala Lys Glu Gly Ile Lys Arg Gly Glu Thr Asp His His 2530 2535 2540 Ala Val Ser Arg Gly Ser Ala Lys Leu Arg Trp Phe Val Glu Arg Asn 2545 2550 2555 2560 Met Val Thr Pro Glu Gly Lys Val Val Asp Leu Gly Cys Gly Arg Gly 2565 2570 2575 Gly Trp Ser Tyr Tyr Cys Gly Gly Leu Lys Asn Val Arg Glu Val Lys 2580 2585 2590 Gly Leu Thr Lys Gly Gly Pro Gly His Glu Glu Pro Ile Pro Met Ser 2595 2600 2605 Thr Tyr Gly Trp Asn Leu Val Arg Leu Gln Ser Gly Val Asp Val Phe 2610 2615 2620 Phe Ile Pro Pro Glu Lys Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu 2625 2630 2635 2640 Ser Ser Pro Asn Pro Thr Val Glu Ala Gly Arg Thr Leu Arg Val Leu 2645 2650 2655 Asn Leu Val Glu Asn Trp Leu Asn Asn Asn Thr Gln Phe Cys Ile Lys 2660 2665 2670 Val Leu Asn Pro Tyr Met Pro Ser Val Ile Glu Lys Met Glu Ala Leu 2675 2680 2685 Gln Arg Lys Tyr Gly Gly Ala Leu Val Arg Asn Pro Leu Ser Arg Asn 2690 2695 2700 Ser Thr His Glu Met Tyr Trp Val Ser Asn Ala Ser Gly Asn Ile Val 2705 2710 2715 2720 Ser Ser Val Asn Met Ile Ser Arg Met Leu Ile Asn Arg Phe Thr Met 2725 2730 2735 Arg Tyr Lys Lys Ala Thr Tyr Glu Pro Asp Val Asp Leu Gly Ser Gly 2740 2745 2750 Thr Arg Asn Ile Gly Ile Glu Ser Glu Ile Pro Asn Leu Asp Ile Ile 2755 2760 2765 Gly Lys Arg Ile Glu Lys Ile Lys Gln Glu His Glu Thr Ser Trp His 2770 2775 2780 Tyr Asp Gln Asp His Pro Tyr Lys Thr Trp Ala Tyr His Gly Ser Tyr 2785 2790 2795 2800 Glu Thr Lys Gln Thr Gly Ser Ala Ser Met Val Asn Gly Val Val 2805 2810 2815 Arg Leu Leu Thr Lys Pro Trp Asp Val Val Pro Met Val Thr Gln Met 2820 2825 2830 Ala Met Thr Asp Thr Thr Pro Phe Gly Gln Gln Arg Val Phe Lys Glu 2835 2840 2845 Lys Val Asp Thr Arg Thr Gln Glu Pro Lys Glu Gly Thr Lys Lys Leu 2850 2855 2860 Met Lys Ile Thr Ala Glu Trp Leu Trp Lys Glu Leu Gly Lys Lys Lys Lys 2865 2870 2875 2880 Thr Pro Arg Met Cys Thr Arg Glu Glu Phe Thr Arg Lys Val Arg Ser 2885 2890 2895 Asn Ala Ala Leu Gly Ala Val Phe Thr Asp Glu Asn Lys Trp Lys Ser 2900 2905 2910 Ala Arg Glu Ala Val Glu Asp Ser Arg Phe Trp Glu Leu Val Asp Lys 2915 2920 2925 Glu Arg Asn Leu His Leu Glu Gly Lys Cys Glu Thr Cys Val Tyr Asn 2930 2935 2940 Met Met Gly Lys Arg Glu Lys Lys Leu Gly Glu Phe Gly Lys Ala Lys 2945 2950 2955 2960 Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg Phe Leu Glu 2965 2970 2975 Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp Phe Ser Arg Glu 2980 2985 2990 Asn Ser Leu Ser Gly Val Glu Gly Glu Gly Leu His Lys Leu Gly Tyr 2995 3000 3005 Ile Leu Arg Asp Val Ser Lys Lys Glu Gly Gly Ala Met Tyr Ala Asp 3010 3015 3020 Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr Leu Glu Asp Leu Lys Asn 3025 3030 3035 3040 Glu Glu Met Val Thr Asn His Met Glu Gly Glu His Lys Lys Lys Leu Ala 3045 3050 3055 Glu Ala Ile Phe Lys Leu Thr Tyr Gln Asn Lys Val Val Arg Val Gln 3060 3065 3070 Arg Pro Thr Pro Arg Gly Thr Val Met Asp Ile Ile Ser Arg Arg Asp 3075 3080 3085 Gln Arg Gly Ser Gly Gln Val Gly Thr Tyr Gl y Leu Asn Thr Phe Thr 3090 3095 3100 Asn Met Glu Ala Gln Leu Ile Arg Gln Met Glu Gly Glu Gly Val Phe 3105 3110 3115 3120 Lys Ser Ile Gln His Leu Thr Ile Thr Glu Glu Ile Ala Val Gln Asn 3125 3130 3135 Trp Leu Ala Arg Val Gly Arg Glu Arg Leu Ser Arg Met Ala Ile Ser 3140 3145 3150 Gly Asp Asp Cys Val Val Lys Pro Leu Asp Asp Arg Phe Ala Ser Ala 3155 3160 3165 Leu Thr Ala Leu Asn Asp Met Gly Lys Ile Arg Lys Asp Ile Gln Gln 3170 3175 3180 Trp Glu Pro Ser Arg Gly Trp Asn Asp Trp Thr Gln Val Pro Phe Cys 3185 3190 3195 3200 Ser His His Phe His Glu Leu Ile Met Lys Asp Gly Arg Val Leu Val 3205 3210 3215 Val Pro Cys Arg Asn Gln Asp Glu Leu Ile Gly Arg Ala Arg Ile Ser 3220 3225 3230 Gln Gly Ala Gly Trp Ser Leu Arg Glu Thr Ala Cys Leu Gly Lys Ser 3235 3240 3245 Tyr Ala Gln Met Trp Ser Leu Met Tyr Phe His Arg Arg Asp Leu Arg 3250 3255 3260 Leu Ala Ala Asn Ala Ile Cys Ser Ala Val Pro Ser His Trp Val Pro 3265 3270 3275 3280 Thr Ser Arg Thr Thr Trp Ser Ile His Ala Lys His Glu Trp Met Thr 3285 3290 3295 Thr Glu Asp Met Leu Thr Val Trp Asn Arg Val Trp Ile Gln Glu Asn 3300 3305 3310 Pro Trp Met Glu Asp Lys Thr Pro Val Glu Ser Trp Glu Glu Ile Pro 3315 3320 3325 Tyr Leu Gly Lys Arg Glu Asp Gln Trp Cys Gly Ser Leu Ile Gly Leu 3330 3335 3340 Thr Ser Arg Ala Thr Trp Ala Lys Asn Ile Gln Ala Ala Ile Asn Gln 3345 3350 3355 3360 Val Arg Ser Leu Ile Gly Asn Glu Glu Tyr Thr Asp Tyr Met Pro Ser 3365 3370 3375Met Lys Arg Phe Arg Arg Glu Glu Glu Glu Ala Gly Val Leu Trp 3380 3385 3390 <210> 5 <211> 10717 <212> DNA <213> chimeric dengue serotype 2/3 (MVS) <400> 5 agttgttagt ctacgtggac cgacaaagac agattctttg agggagctaa gctcaatgta 60 gttctaacag ttttttaatt agagagcaga tctctgatga ataaccaacg gaaaaaggcg 120 aaaaacacgc ctttcaatat gctgaaacgc gagagaaacc gcgtgtcgac tgtgcaacag 180 ctgacaaaga gattctcact tggaatgctg cagggacgag gaccattaaa actgttcatg 240 gccctggtgg cgttccttcg tttcctaaca atcccaccaa cagcagggat attgaagaga 300 tggggaacaa ttaaaaaatc aaaagctatt aatgttttga gagggttcag gaaagagatt 360 ggaaggatgc tgaacatctt gaataggaga cgcagatctg caggcatgat cattatgctg 420 attccaacag tgatggcgtt ccatttaacc acgcgtgatg gagagccgcg catgattgtg 480 gggaagaatg aaagaggaaa atccctactt ttcaagacag cctctggaat caacatgtgc 540 acactcatag ctatggatct gggagagatg tgtgatgaca cggtcactta caaatgcccc 600 cacattaccg aagtggagcc tgaagacatt gactgctggt gcaaccttac atcgacatgg 660 gtgacttatg gaacatgcaa tcaagctgga gagcatagac gcgataagag atcagtggcg 720 ttagctcccc atgttggcat gggactggac acacgcactc aaacctggat gtcggctgaa 780 ggagcttgga gacaagtcga gaaggtagag acatgggccc ttaggcaccc agggtttacc 840 atactagccc tatttcttgc ccattacata ggcacttcct tgacccagaa agtggttatt 900 tttatactat taatgctggt taccccatcc atgacaatga gatgtgtagg agtaggaaac 960 agagattttg tggaaggcct atcgggagct acgtgggttg acgtggtgct cgagcacggt 1020 gggtgtgtga ctaccatggc taagaacaag cccacgctgg acatagagct tcagaagacc 1080 gaggccaccc aactggcgac cctaaggaag ctatgcattg agggaaaaat taccaacata 1140 acaaccgact caagatgtcc cacccaaggg gaagcgattt tacctgagga gcaggaccag 1200 aactacgtgt gtaagcatac atacgtggac agaggctggg gaaacggttg tggtttgttt 1260 ggcaagggaa gcttggtgac atgcgcgaaa tttcaatgtt tagaatcaat agagggaaaa 1320 gtggtgcaac atgagaacct caaatacacc gtcatcatca cagtgcacac aggagaccaa 1380 caccaggtgg gaaatgaaac gcagggagtc acggctgaga taacacccca ggcatcaacc 1440 gctgaagcca ttttacctga atatggaacc ctcgggctag aatgctcacc acggacaggt 1500 ttggatttca atgaaatgat ctcattgaca atgaagaaca aagcatggat ggtacataga 1560 caatggttct ttgacttacc cctaccatgg acatcaggag cttcagcaga aacaccaact 1620 tggaacagga aagagcttct tgtgacattt aaaaatgcac atgcaaaaaa gcaagaagta 1680 gttgttcttg gatcacaaga gggagcaatg catacagcac tgacaggagc tacagagatc 1740 caaacctcag gaggcacaag tatctttgcg gggcacttaa aatgtagact caagatggac 1800 aaattggaac tcaaggggat gagctatgca atgtgcttga gtagctttgt gttgaagaaa 1860 gaagtctccg aaacgcagca tgggacaata ctcattaagg ttgagtacaa aggggaagat 1920 gcaccctgca agattccttt ctccacggag gatggacaag gaaaagctct caatggcaga 1980 ctgatcacag ccaatccagt ggtgaccaag aaggaggagc ctgtcaacat tgaggctgaa 2040 cctccttttg gagaaagtaa catagtaatt ggaattggag acaaagccct gaaaatcaac 2100 tggtacaaga agggaagctc gattgggaag atgttcgagg ccactgccag aggtgcaagg 2160 cgcatggcca tcttgggaga cacagcctgg gactttggat cagtgggtgg tgttttgaat 2220 tcattaggga aaatggtcca ccaaatattt gggagtgctt acacagccct atttggtgga 2280 gtctcctgga tgatgaaaat tggaataggt gtcctcttaa cctggatagg gttgaactca 2340 aaaaatactt ctatgtcatt ttcatgcatc gcggccggca ttgtgacact gtatttggga 2400 gtcatggtgc aggccgatag tggttgcgtt gtgagctgga aaaacaaaga actgaaatgt 2460 ggcagtggga ttttcatcac agacaacgtg cacacatgga cagaacaata caagttccaa 2520 ccagaatccc cttcaaaact agcttcagct atccagaaag cccatgaaga ggacatttgt 2580 ggaatccgct cagtaacaag actggagaat ctgatgtgga aacaaataac accagaattg 2640 aatcacattc tatcagaaaa tgaggtgaag ttaactatta tgacaggaga catcaaagga 2700 atcatgcagg caggaaaacg atctctgcgg cctcagccca ctgagctgaa gtattcatgg 2760 aaaacatggg gcaaagcaaa aatgctctct acagagtctc ataaccagac ctttctcatt 2820 gatggccccg aaacagcaga atgccccaac acaaatagag cttggaattc gttggaagtt 2880 gaagactatg gctttggagt attcaccacc aatatatggc taaaattgaa agaaaaacag 2940 gatgtattct gcgactcaaa actcatgtca gcggccataa aagacaacag agccgtccat 3000 gccgatatgg gttattggat agaaagtgca ctcaatgaca catggaagat agagaaagcc 3060 tctttcattg aagttaaaaa ctgccactgg ccaaaatcac acaccctctg gagcaatgga 3120 gtgctagaaa gtgagatgat aattccaaag aatctcgctg gaccagtgtc tcaacacaac 3180 tatagaccag gctaccatac acaaataaca ggaccatggc atctaggtaa gcttgagatg 3240 gactttgatt tctgtgatgg aacaacagtg gtagtgactg aggactgcgg aaatagagga 3300 ccctctttga gaacaaccac tgcctctgga aaactcataa cagaatggtg ctgccgatct 3360 tgcacattac caccgctaag atacagaggt gaggatgggt gctggtacgg gatggaaatc 3420 agaccattga aggagaaaga agagaatttg gtcaactcct tggtcacagc tggacatggg 3480 caggtcgaca acttttcact aggagtcttg ggaatggcat tgttcctgga ggaaatgctt 3540 aggacccgag taggaacgaa acatgcaata ctactagttg cagtttcttt tgtgacattg 3600 atcacaggga acatgtcctt tagagacctg ggaagagtga tggttatggt aggcgccact 3660 atgacggatg acataggtat gggcgtgact tatcttgccc tactagcagc cttcaaagtc 3720 agaccaactt ttgcagctgg actactcttg agaaagctga cctccaagga attgatgatg 3780 actactatag gaattgtact cctctcccag agcaccatac cagagaccat tcttgagttg 3840 actgatgcgt tagccttagg catgatggtc ctcaaaatgg tgagaaatat ggaaaagtat 3900 caattggcag tgactatcat ggctatcttg tgcgtcccaa acgcagtgat attacaaaac 3960 gcatggaaag tgagttgcac aatattggca gtggtgtccg tttccccact gttcttaaca 4020 tcctcacagc aaaaaacaga ttggatacca ttagcattga cgatcaaagg tctcaatcca 4080 acagctattt ttctaacaac cctctcaaga accagcaaga aaaggagctg gccattaaat 4140 gaggctatca tggcagtcgg gatggtgagc attttagcca gttctctcct aaaaaatgat 4200 attcccatga caggaccatt agtggctgga gggctcctca ctgtgtgcta cgtgctcact 4260 ggacgatcgg ccgatttgga actggagaga gcagccgatg tcaaatggga agaccaggca 4320 gagatatcag gaagcagtcc aatcctgtca ataacaatat cagaagatgg tagcatgtcg 4380 ataaaaaatg aagaggaaga acaaacactg accatactca ttagaacagg attgctggtg 4440 atctcaggac tttttcctgt atcaatacca atcacggcag cagcatggta cctgtgggaa 4500 gtgaagaaac aacgggccgg agtattgtgg gatgttcctt cacccccacc catgggaaag 4560 gctgaactgg aagatggagc ctatagaatt aagcaaaaag ggattcttgg atattcccag 4620 atcggagccg gagtttacaa agaaggaaca ttccatacaa tgtggcatgt cacacgtggc 4680 gctgttctaa tgcataaagg aaagaggatt gaaccatcat gggcggacgt caagaaagac 4740 ctaatatcat atggaggagg ctggaagtta gaaggagaat ggaaggaagg agaagaagtc 4800 caggtattgg cactggagcc tggaaaaaat ccaagagccg tccaaacgaa acctggtctt 4860 ttcaaaacca acgccggaac aataggtgct gtatctctgg acttttctcc tggaacgtca 4920 ggatctccaa ttatcgacaa aaaaggaaaa gttgtgggtc tttatggtaa tggtgttgtt 4980 acaaggagtg gagcatatgt gagtgctata gcccagactg aaaaaagcat tgaagacaac 5040 ccagagatcg aagatgacat tttccgaaag agaagactga ccatcatgga cctccaccca 5100 ggagcgggaa agacgaagag ataccttccg gccatagtca gagaagctat aaaacggggt 5160 ttgagaacat taatcttggc ccccactaga gttgtggcag ctgaaatgga ggaagccctt 5220 agaggacttc caataagata ccagacccca gccatcagag ctgtgcacac cgggcgggag 5280 attgtggacc taatgtgtca tgccacattt accatgaggc tgctatcacc agttagagtg 5340 ccaaactaca acctgattat catggacgaa gcccatttca cagacccagc aagtatagca 5400 gctagaggat acatctcaac tcgagtggag atgggtgagg cagctgggat ttttatgaca 5460 gccactcccc cgggaagcag agacccattt cctcagagca atgcaccaat catagatgaa 5520 gaaagagaaa tccctgaacg ctcgtggaat tccggacatg aatgggtcac ggattttaaa 5580 gggaagactg tttggttcgt tccaagtata aaagcaggaa atgatatagc agcttgcctg 5640 aggaaaaatg gaaagaaagt gatacaactc agtaggaaga cctttgattc tgagtatgtc 5700 aagactagaa ccaatgattg ggacttcgtg gttacaactg acatttcaga aatgggtgcc 5760 aatttcaagg ctgagagggt tatagacccc agacgctgca tgaaaccagt catactaaca 5820 gatggtgaag agcgggtgat tctggcagga cctatgccag tgacccactc tagtgcagca 5880 caaagaagag ggagaatagg aagaaatcca aaaaatgaga atgaccagta catatacatg 5940 ggggaacctc tggaaaatga tgaagactgt gcacactgga aagaagctaa aatgctccta 6000 gataacatca acacgccaga aggaatcatt cctagcatgt tcgaaccaga gcgtgaaaag 6060 gtggatgcca ttgatggcga ataccgcttg agaggagaag caaggaaaac ctttgtagac 6120 ttaatgagaa gaggagacct accagtctgg ttggcctaca gagtggcagc tgaaggcatc 6180 aactacgcag acagaaggtg gtgttttgat ggatcaaga acaaccaaat cctagaagaa 6240 aacgtggaag ttgaaatctg gacaaaagaa ggggaaagga agaaattgaa acccagatgg 6300 ttggatgcta ggatctattc tgacccactg gcgctaaaag aatttaagga atttgcagcc 6360 ggaagaaagt ctctgaccct gaacctaatc acagaaatgg gtaggctccc aaccttcatg 6420 actcagaagg caagagacgc actggacaac ttagcagtgc tgcacacggc tgaggcaggt 6480 ggaagggcgt acaaccatgc tctcagtgaa ctgccggaga ccctggagac attgctttta 6540 ctgacacttc tggctacagt cacgggaggg atctttttat tcttgatgag cgcaaggggc 6600 atagggaaga tgaccctggg aatgtgctgc ataatcacgg ctagcatcct cctatggtac 6660 gcacaaatac agccacactg gatagcagct tcaataatac tggagttttt tctcatagtt 6720 ttgcttattc cagaacctga aaaacagaga acaccccaag acaaccaact gacctacgtt 6780 gtcatagcca tcctcacagt ggtggccgca accatggcaa acgagatggg tttcctagaa 6840 aaaacgaaga aagatctcgg attgggaagc attgcaaccc agcaacccga gagcaacatc 6900 ctggacatag atctacgtcc tgcatcagca tggacgctgt atgccgtggc cacaacattt 6960 gttacaccaa tgttgagaca tagcattgaa aattcctcag tgaatgtgtc cctaacagct 7020 atagccaacc aagccacagt gttaatgggt ctcgggaaag gatggccatt gtcaaagatg 7080 gacatcggag ttccccttct cgccattgga tgctactcac aagtcaaccc cataactctc 7140 acagcagctc ttttcttatt ggtagcacat tatgccatca tagggccagg actccaagca 7200 aaagcaacca gagaagctca gaaaagagca gcggcgggca tcatgaaaaa cccaactgtc 7260 gatggaataa cagtgattga cctagatcca ataccttatg atccaaagtt tgaaaagcag 7320 ttgggacaag taatgctcct agtcctctgc gtgactcaag tattgatgat gaggactaca 7380 tgggctctgt gtgaggcttt aaccttagct accgggccca tctccacatt gtgggaagga 7440 aatccaggga ggttttggaa cactaccatt gcggtgtcaa tggctaacat ttttagaggg 7500 agttacttgg ccggagctgg acttctcttt tctattatga agaacacaac caacacaaga 7560 aggggaactg gcaacatagg agagacgctt ggagagaaat ggaaaagccg attgaacgcg 7620 ttgggaaaaa gtgaattcca gatctacaag aaaagtggaa tccaggaagt ggatagaacc 7680 ttagcaaaag aaggcattaa aagaggagaa acggaccatc acgctgtgtc gcgaggctca 7740 gcaaaactga gatggttcgt tgagagaaac atggtcacac cagaagggaa agtagtggac 7800 ctcggttgtg gcagaggagg ctggtcatac tattgtggag gactaaagaa tgtaagagaa 7860 gtcaaaggcc taacaaaagg aggaccagga cacgaagaac ccatccccat gtcaacatat 7920 gggtggaatc tagtgcgtct tcaaagtgga gttgacgttt tcttcatccc gccagaaaag 7980 tgtgacacat tattgtgtga cataggggag tcatcaccaa atcccacagt ggaagcagga 8040 cgaacactca gagtccttaa cttagtagaa aattggttga acaacaacac tcaattttgc 8100 ataaaggttc tcaacccata tatgccctca gtcatagaaa aaatggaagc actacaaagg 8160 aaatatggag gagccttagt gaggaatcca ctctcacgaa actccacaca tgagatgtac 8220 tgggtatcca atgcttccgg gaacatagtg tcatcagtga acatgatttc aaggatgttg 8280 atcaacagat ttacaatgag atacaagaaa gccacttacg agccggatgt tgacctcgga 8340 agcggaaccc gtaacatcgg gattgaaagt gagataccaa acctagatat aattgggaaa 8400 agaatagaaa aaataaagca agagcatgaa acatcatggc actatgacca agaccaccca 8460 tacaaaacgt gggcatacca tggtagctat gaaacaaaac agactggatc agcatcatcc 8520 atggtcaacg gagtggtcag gctgctgaca aaaccttggg acgtcgtccc catggtgaca 8580 cagatggcaa tgacagacac gactccattt ggacaacagc gcgtttttaa agagaaagtg 8640 gacacgagaa cccaagaacc gaaagaaggc acgaagaaac taatgaaaat aacagcagag 8700 tggctttgga aagaattagg gaagaaaaag accacccagga tgtgcaccag agaagaattc 8760 acaagaaagg tgagaagcaa tgcagccttg ggggccatat tcactgatga gaacaagtgg 8820 aagtcggcac gtgaggctgt tgaagatagt aggttttggg agctggttga caaggaaagg 8880 aatctccatc ttgaaggaaa gtgtgaaaca tgtgtgtaca acatgatggg aaaaagagag 8940 aagaagctag gggaattcgg caaggcaaaa ggcagcagag ccatatggta catgtggctt 9000 ggagcacgct tcttagagtt tgaagcccta ggattcttaa atgaagatca ctggttctcc 9060 agagagaact ccctgagtgg agtggaagga gaagggctgc acaagctagg ttacattcta 9120 agagacgtga gcaagaaaga gggaggagca atgtatgccg atgacaccgc aggatgggat 9180 acaagaatca cactagaaga cctaaaaaat gaagaaatgg taacaaacca catggaagga 9240 gaacacaaga aactagccga ggccattttc aaactaacgt accaaaacaa ggtggtgcgt 9300 gtgcaaagac caacaccaag aggcacagta atggacatca tatcgagaag agaccaaaga 9360 ggtagtggac aagttggcac ctatggactc aatactttca ccaatatgga agcccaacta 9420 atcagacaga tggagggaga aggagtcttt aaaagcattc agcacctaac aatcacagaa 9480 gaaatcgctg tgcaaaactg gttagcaaga gtggggcgcg aaaggttatc aagaatggcc 9540 atcagtggag atgattgtgt tgtgaaacct tagatgaca ggttcgcaag cgctttaaca 9600 gctctaaatg acatgggaaa gattaggaaa gacatacaac aatgggaacc ttcaagagga 9660 tggaatgatt ggacacaagt gcccttctgt tcacaccatt tccatgagtt aatcatgaaa 9720 gacggtcgcg tactcgttgt tccatgtaga aaccaagatg aactgattgg cagagcccga 9780 atctcccaag gagcagggtg gtctttgcgg gagacggcct gtttggggaa gtcttacgcc 9840 caaatgtgga gcttgatgta cttccacaga cgcgacctca ggctggcggc aaatgctatt 9900 tgctcggcag taccatcaca ttgggttcca acaagtcgaa caacctggtc catacatgct 9960 aaacatgaat ggatgacaac ggaagacatg ctgacagtct ggaacagggt gtggattcaa 10020 gaaaacccat ggatggaaga caaaactcca gtggaatcat gggaggaaat cccatacttg 10080 gggaaaagag aagaccaatg gtgcggctca ttgattgggt taacaagcag ggccacctgg 10140 gcaaagaaca tccaagcagc aataaatcaa gttagatccc ttataggcaa tgaagaatac 10200 acagattaca tgccatccat gaaaagattc agaagagaag aggaagaagc aggagttctg 10260 tggtagaaag caaaactaac atgaaacaag gctagaagtc aggtcggatt aagccatagt 10320 acggaaaaaa ctatgctacc tgtgagcccc gtccaaggac gttaaaagaa gtcaggccat 10380 cataaatgcc atagcttgag taaactatgc agcctgtagc tccacctgag aaggtgtaaa 10440 aaatccggga ggccacaaac catggaagct gtacgcatgg cgtagtggac tagcggttag 10500 aggagacccc tcccttacaa atcgcagcaa caatgggggc ccaaggcgag atgaagctgt 10560 agtctcgctg gaaggactag aggttagagg agaccccccc gaaacaaaaa acagcatatt 10620 gacgctggga aagaccagag atcctgctgt ctcctcagca tcattccagg cacagaacgc 10680 cagaaaatgg aatggtgctg ttgaatcaac aggttct 10717 <210> 6 <211> 3389 <212> PRT <213> chimeric dengue serotype 2/3 (MVS) <400> 6 Met Asn Asn Gln Arg Lys Lys Ala Lys Asn Thr Pro Phe Asn Met Leu 1 5 10 15 Lys Arg Glu Arg Asn Arg Val Ser Thr Val Gln Gln Leu Thr Lys Arg 20 25 30 Phe Ser Leu Gly Met Leu Gln Gly Arg Gly Pro Leu Lys Leu Phe Met 35 40 45 Ala Leu Val Ala Phe Leu Arg Phe Leu Thr Ile Pro Pro Thr Ala Gly 50 55 60 Ile Leu Lys Arg Trp Gly Thr Ile Lys Lys Ser Lys Ala Ile Asn Val 65 70 75 80 Leu Arg Gly Phe Arg Lys Glu Ile Gly Arg Met Leu Asn Ile Leu Asn 85 90 95 Arg Arg Arg Arg Ser Ala Gly Met Ile Ile Met Leu Ile Pro Thr Val 100 105 110 Met Ala Phe His Leu Thr Thr Arg Asp Gly Glu Pro Arg Met Ile Val 115 120 125 Gly Lys Asn Glu Arg Gly Lys Ser Leu Leu Phe Lys Thr Ala Ser Gly 130 135 140 Ile Asn Met Cys Thr Leu Ile Ala Met Asp Leu Gly Glu Met Cys Asp 145 150 155 160 Asp Thr Val Thr Tyr Lys Cys Pro His Ile Thr Glu Val Glu Pro Glu 165 170 175 Asp Ile Asp Cys Trp Cys Asn Leu Thr Ser Thr Trp Val Thr Tyr Gly 180 185 190 Thr Cys Asn Gln Ala Gly Glu His Arg Arg Asp Lys Arg Ser Val Ala 195 200 205 Leu Ala Pro His Val Gly Met Gly Leu Asp Thr Arg Thr Gln Thr Trp 210 215 220 Met Ser Ala Glu Gly Ala Trp Arg Gln Val Glu Lys Val Glu Thr Trp 225 230 235 240 Ala Leu Arg His Pro Gly Phe Thr Ile Leu Ala Leu Phe Leu Ala His 245 250 255 Tyr Ile Gly Thr Ser Leu Thr Gln Lys Val Val Ile Phe Ile Leu Leu 260 265 270 Met Leu Val Thr Pro Ser Met Thr Met Arg Cys Val Gly Val Gly Asn 275 280 285 Arg Asp Phe Val Glu Gly Leu Ser Gly Ala Thr Trp Val Asp Val Val 290 295 300 Leu Glu His Gly Gly Cys Val Thr Thr Met Ala Lys Asn Lys Pro Thr 305 310 315 320 Leu Asp Ile Glu Leu Gln Lys Thr Glu Ala Thr Gln Leu Ala Thr Leu 325 330 335 Arg Lys Leu Cys Ile Glu Gly Lys Ile Thr Asn Ile Thr Thr Asp Ser 340 345 350 Arg Cys Pro Thr Gln Gly Glu Ala Ile Leu Pro Glu Glu Gln Asp Gln 355 360 365 Asn Tyr Val Cys Lys His Thr Tyr Val Asp Arg Gly Trp Gly Asn Gly 370 375 380 Cys Gly Leu Phe Gly Lys Gly Ser Leu Val Thr Cys Ala Lys Phe Gln 385 390 395 400 Cys Leu Glu Ser Ile Glu Gly Lys Val Val Gln His Glu Asn Leu Lys 405 410 415 Tyr Thr Val Ile Ile Thr Val His Thr Gly Asp Gln His Gln Val Gly 420 425 430 Asn Glu Thr Gln Gly Val Thr Ala Glu Ile Thr Pro Gln Ala Ser Thr 435 440 445 Ala Glu Ala Ile Leu Pro Glu Tyr Gly Thr Leu Gly Leu Glu Cys Ser 450 455 460 Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Ile Ser Leu Thr Met Lys 465 470 475 480 Asn Lys Ala Trp Met Val His Arg Gln Trp Phe Phe Asp Leu Pro Leu 485 490 495 Pro Trp Thr Ser Gly Ala Ser Ala Glu Thr Pro Thr Trp Asn Arg Lys 500 505 510 Glu Leu Leu Val Thr Phe Lys Asn Ala His Ala Lys Lys Gln Glu Val 515 520 525 Val Val Leu Gly Ser Gln Glu Gly Ala Met His Thr Ala Leu Thr Gly 530 535 540 Ala Thr Glu Ile Gln Thr Ser Gly Gly Thr Ser Ile Phe Ala Gly His 545 550 555 560 Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Glu Leu Lys Gly Met Ser 565 570 575 Tyr Ala Met Cys Leu Ser Ser Phe Val Leu Lys Lys Glu Val Ser Glu 580 585 590 Thr Gln His Gly Thr Ile Leu Ile Lys Val Glu Tyr Lys Gly Glu Asp 595 600 605 Ala Pro Cys Lys Ile Pro Phe Ser Thr Glu Asp Gly Gln Gly Lys Ala 610 615 620 Leu Asn Gly Arg Leu Ile Thr Ala Asn Pro Val Val Thr Lys Lys Glu 625 630 635 640 Glu Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Glu Ser Asn Ile 645 650 655 Val Ile Gly Ile Gly Asp Lys Ala Leu Lys Ile Asn Trp Tyr Lys Lys 660 665 670 Gly Ser Ser Ile Gly Lys Met Phe Glu Ala Thr Ala Arg Gly Ala Arg 675 680 685 Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Val Gly 690 695 700 Gly Val Leu Asn Ser Leu Gly Lys Met Val His Gln Ile Phe Gly Ser 705 710 715 720 Ala Tyr Thr Ala Leu Phe Gly Gly Val Ser Trp Met Met Lys Ile Gly 725 730 735 Ile Gly Val Leu Leu Thr Trp Ile Gly Leu Asn Ser Lys Asn Thr Ser 740 745 750 Met Ser Phe Ser Cys Ile Ala Ala Gly Ile Val Thr Leu Tyr Leu Gly 755 760 765 Val Met Val Gln Ala Asp Ser Gly Cys Val Ser Trp Lys Asn Lys 770 775 780 Glu Leu Lys Cys Gly Ser Gly Ile Phe Ile Thr Asp Asn Val His Thr 785 790 795 800 Trp Thr Glu Gln Tyr Lys Phe Gln Pro Glu Ser Pro Ser Lys Leu Ala 805 810 815 Ser Ala Ile Gln Lys Ala His Glu Glu Asp Ile Cys Gly Ile Arg Ser 820 825 830 Val Thr Arg Leu Glu Asn Leu Met Trp Lys Gln Ile Thr Pro Glu Leu 835 840 845 Asn His Ile Leu Ser Glu Asn Glu Val Lys Leu Thr Ile Met Thr Gly 850 855 860 Asp Ile Lys Gly Ile Met Gln Ala Gly Lys Arg Ser Leu Arg Pro Gln 865 870 875 880 Pro Thr Glu Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala Lys Met 885 890 895 Leu Ser Thr Glu Ser His Asn Gln Thr Phe Leu Ile Asp Gly Pro Glu 900 905 910 Thr Ala Glu Cys Pro Asn Thr Asn Arg Ala Trp Asn Ser Leu Glu Val 915 920 925 Glu Asp Tyr Gly Phe Gly Val Phe Thr Thr Asn Ile Trp Leu Lys Leu 930 935 940 Lys Glu Lys Gln Asp Val Phe Cys Asp Ser Lys Leu Met Ser Ala Ala 945 950 955 960 Ile Lys Asp Asn Arg Ala Val His Ala Asp Met Gly Tyr Trp Ile Glu 965 970 975 Ser Ala Leu Asn Asp Thr Trp Lys Ile Glu Lys Ala Ser Phe Ile Glu 980 985 990 Val Lys Asn Cys His Trp Pro Lys Ser His Thr Leu Trp Ser Asn Gly 995 1000 1005 Val Leu Glu Ser Glu Met Ile Ile Pro Lys Asn Leu Ala Gly Pro Val 1010 1015 1020 Ser Gln His Asn Tyr Arg Pro Gly Tyr His Thr Gln Ile Thr Gly Pro 1025 1030 1035 1040 Trp His Leu Gly Lys Leu Glu Met Asp Phe Asp Phe Cys Asp Gly Thr 1045 1050 1055 Thr Val Val Val Thr Glu Asp Cys Gly Asn Arg Gly Pro Ser Leu Arg 1060 1065 1070 Thr Thr Thr Ala Ser Gly Lys Leu Ile Thr Glu Trp Cys Cys Arg Ser 1075 1080 1085 Cys Thr Leu Pro Pro Leu Arg Tyr Arg Gly Glu Asp Gly Cys Trp Tyr 1090 1095 1100 Gly Met Glu Ile Arg Pro Leu Lys Glu Lys Glu Glu Asn Leu Val Asn 1105 1110 1115 1120 Ser Leu Val Thr Ala Gly His Gly Gln Val Asp Asn Phe Ser Leu Gly 1125 1130 1135 Val Leu Gly Met Ala Leu Phe Leu Glu Glu Met Leu Arg Thr Arg Val 1140 1145 1150 Gly Thr Lys His Ala Ile Leu Leu Val Ala Val Ser Phe Val Thr Leu 1155 1160 1165 Ile Thr Gly Asn Met Ser Phe Arg Asp Leu Gly Arg Val Met Val Met 1170 1175 1180 Val Gly Ala Thr Met Thr Asp Asp Ile Gly Met Gly Val Thr Tyr Leu 1185 1190 1195 1200 Ala Leu Leu Ala Ala Phe Lys Val Arg Pro Thr Phe Ala Ala Gly Leu 1205 1210 1215 Leu Leu Arg Lys Leu Thr Ser Lys Glu Leu Met Met Thr Thr Ile Gly 1220 1225 1230 Ile Val Leu Leu Ser Gln Ser Thr Ile Pro Glu Thr Ile Leu Glu Leu 1235 1240 1245 Thr Asp Ala Leu Ala Leu Gly Met Met Val Leu Lys Met Val Arg Asn 1250 1255 1260 Met Glu Lys Tyr Gln Leu Ala Val Thr Ile Met Ala Ile Leu Cys Val 1265 1270 1275 1280 Pro Asn Ala Val Ile Leu Gln Asn Ala Trp Lys Val Ser Cys Thr Ile 1285 1290 1295 Leu Ala Val Val Ser Val Ser Pro Leu Phe Leu Thr Ser Ser Gln Gln 1300 1305 1310 Lys Thr Asp Trp Ile Pro Leu Ala Leu Thr Ile Lys Gly Leu Asn Pro 1315 1320 1325 Thr Ala Ile Phe Leu Thr Thr Leu Ser Arg Thr Ser Lys Lys Arg Ser 1330 1335 1340 Trp Pro Leu Asn Glu Ala Ile Met Ala Val Gly Met Val Ser Ile Leu 1345 1350 1355 1360 Ala Ser Ser Leu Leu Lys Asn Asp Ile Pro Met Thr Gly Pro Leu Val 1365 1370 1375 Ala Gly Gly Leu Leu Thr Val Cys Tyr Val Leu Thr Gly Arg Ser Ala 1380 1385 1390 Asp Leu Glu Leu Glu Arg Ala Ala Asp Val Lys Trp Glu Asp Gln Ala 1395 1400 1405 Glu Ile Ser Gly Ser Ser Pro Ile Leu Ser Ile Thr Ile Ser Glu Asp 1410 1415 1420 Gly Ser Met Ser Ile Lys Asn Glu Glu Glu Glu Gln Thr Leu Thr Ile 1425 1430 1435 1440 Leu Ile Arg Thr Gly Leu Leu Val Ile Ser Gly Leu Phe Pro Val Ser 1445 1450 1455 Ile Pro Ile Thr Ala Ala Ala Trp Tyr Leu Trp Glu Val Lys Lys Gln 1460 1465 1470 Arg Ala Gly Val Leu Trp Asp Val Pro Ser Pro Pro Pro Met Gly Lys 1475 1480 1485 Ala Glu Leu Glu Asp Gly Ala Tyr Arg Ile Lys Gln Lys Gly Ile Leu 1490 1495 1500 Gly Tyr Ser Gln Ile Gly Ala Gly Val Tyr Lys Glu Gly Thr Phe His 1505 1510 1515 1520 Thr Met Trp His Val Thr Arg Gly Ala Val Leu Met His Lys Gly Lys 1525 1530 1535 Arg Ile Glu Pro Ser Trp Ala Asp Val Lys Lys Asp Leu Ile Ser Tyr 1540 1545 1550 Gly Gly Gly Trp Lys Leu Glu Gly Glu Trp Lys Glu Gly Glu Glu Val 1555 1560 1565 Gln Val Leu Ala Leu Glu Pro Gly Lys Asn Pro Arg Ala Val Gln Thr 1570 1575 1580 Lys Pro Gly Leu Phe Lys Thr Asn Ala Gly Thr Ile Gly Ala Val Ser 1585 1590 1595 1600 Leu Asp Phe Ser Pro Gly Thr Ser Gly Ser Pro Ile Ile Asp Lys Lys 1605 1610 1615 Gly Lys Val Val Gly Leu Tyr Gly Asn Gly Val Val Thr Arg Ser Gly 1620 1625 1630 Ala Tyr Val Ser Ala Ile Ala Gln Thr Glu Lys Ser Ile Glu Asp Asn 1635 1640 1645 Pro Glu Ile Glu Asp Asp Ile Phe Arg Lys Arg Arg Leu Thr Ile Met 1650 1655 1660 Asp Leu His Pro Gly Ala Gly Lys Thr Lys Arg Tyr Leu Pro Ala Ile 1665 1670 1675 1680 Val Arg Glu Ala Ile Lys Arg Gly Leu Arg Thr Leu Ile Leu Ala Pro 1685 1690 1695 Thr Arg Val Val Ala Ala Glu Met Glu Glu Ala Leu Arg Gly Leu Pro 1700 1705 1710 Ile Arg Tyr Gln Thr Pro Ala Ile Arg Ala Val His Thr Gly Arg Glu 1715 1720 1725 Ile Val Asp Leu Met Cys His Ala Thr Phe Thr Met Arg Leu Leu Ser 1730 1735 1740 Pro Val Arg Val Pro Asn Tyr Asn Leu Ile Ile Met Asp Glu Ala His 1745 1750 1755 1760 Phe Thr Asp Pro Ala Ser Ile Ala Ala Arg Gly Tyr Ile Ser Thr Arg 1765 1770 1775 Val Glu Met Gly Glu Ala Ala Gly Ile Phe Met Thr Ala Thr Pro Pro 1780 1785 1790 Gly Ser Arg Asp Pro Phe Pro Gln Ser Asn Ala Pro Ile Ile Asp Glu 1795 1800 1805 Glu Arg Glu Ile Pro Glu Arg Ser Trp Asn Ser Gly His Glu Trp Val 1810 1815 1820 Thr Asp Phe Lys Gly Lys Thr Val Trp Phe Val Pro Ser Ile Lys Ala 1825 1830 1835 1840 Gly Asn Asp Ile Ala Ala Cys Leu Arg Lys Asn Gly Lys Lys Val Ile 1845 1850 1855 Gln Leu Ser Arg Lys Thr Phe Asp Ser Glu Tyr Val Lys Thr Arg Thr 1860 1865 1870 Asn Asp Trp Asp Phe Val Val Thr Thr Asp Ile Ser Glu Met Gly Ala 1875 1880 1885 Asn Phe Lys Ala Glu Arg Val Ile Asp Pro Arg Arg Cys Met Lys Pro 1890 1895 1900 Val Ile Leu Thr Asp Gly Glu Glu Arg Val Ile Leu Ala Gly Pro Met 1905 1910 1915 1920 Pro Val Thr His Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly Arg 1925 1930 1935 Asn Pro Lys Asn Glu Asn Asp Gln Tyr Ile Tyr Met Gly Glu Pro Leu 1940 1945 1950 Glu Asn Asp Glu Asp Cys Ala His Trp Lys Glu Ala Lys Met Leu Leu 1955 1960 1965 Asp Asn Ile Asn Thr Pro Glu Gly Ile Ile Pro Ser Met Phe Glu Pro 1970 1975 1980 Glu Arg Glu Lys Val Asp Ala Ile Asp Gly Glu Tyr Arg Leu Arg Gly 1985 1990 1995 2000 Glu Ala Arg Lys Thr Phe Val Asp Leu Met Arg Arg Gly Asp Leu Pro 2005 2010 2015 Val Trp Leu Ala Tyr Arg Val Ala Ala Glu Gly Ile Asn Tyr Ala Asp 2020 2025 2030 Arg Arg Trp Cys Phe Asp Gly Val Lys Asn Asn Gln Ile Leu Glu Glu 2035 2040 2045 Asn Val Glu Val Glu Ile Trp Thr Lys Glu Gly Glu Arg Lys Lys Leu 2050 2055 2060 Lys Pro Arg Trp Leu Asp Ala Arg Ile Tyr Ser Asp Pro Leu Ala Leu 2065 2070 2075 2080 Lys Glu Phe Lys Glu Phe Ala Ala Gly Arg Lys Ser Leu Thr Leu Asn 2085 2090 2095 Leu Ile Thr Glu Met Gly Arg Leu Pro Thr Phe Met Thr Gln Lys Ala 2100 2105 2110 Arg Asp Ala Leu Asp Asn Leu Ala Val Leu His Thr Ala Glu Ala Gly 2115 2120 2125 Gly Arg Ala Tyr Asn His Ala Leu Ser Glu Leu Pro Glu Thr Leu Glu 2130 2135 2140 Thr Leu Leu Leu Leu Thr Leu Leu Ala Thr Val Thr Gly Gly Ile Phe 2145 2150 2155 2160 Leu Phe Leu Met Ser Ala Arg Gly Ile Gly Lys Met Thr Leu Gly Met 2165 2170 2175 Cys Cys Ile Ile Ile Thr Ala Ser Ile Leu Leu Trp Tyr Ala Gln Ile Gln 2180 2185 2190 Pro His Trp Ile Ala Ala Ser Ile Ile Leu Glu Phe Phe Leu Ile Val 2195 2200 2205 Leu Leu Ile Pro Glu Pro Glu Lys Gln Arg Thr Pro Gln Asp Asn Gln 2210 2215 2220 Leu Thr Tyr Val Val Ile Ala Ile Leu Thr Val Val Ala Ala Thr Met 2225 2230 2235 2240 Ala Asn Glu Met Gly Phe Leu Glu Lys Thr Lys Lys Asp Leu Gly Leu 2245 2250 2255 Gly Ser Ile Ala Thr Gln Gln Pro Glu Ser Asn Ile Leu Asp Ile Asp 2260 2265 2270 Leu Arg Pro Ala Ser Ala Trp Thr Leu Tyr Ala Val Ala Thr Thr Phe 2275 2280 2285 Val Thr Pro Met Leu Arg His Ser Ile Glu Asn Ser Ser Val Asn Val 2290 2295 2300 Ser Leu Thr Ala Ile Ala Asn Gln Ala Thr Val Leu Met Gly Leu Gly 2305 2310 2315 2320 Lys Gly Trp Pro Leu Ser Lys Met Asp Ile Gly Val Pro Leu Leu Ala 2325 2330 2335 Ile Gly Cys Tyr Ser Gln Val Asn Pro Ile Thr Leu Thr Ala Ala Leu 2340 2345 2350 Phe Leu Leu Val Ala His Tyr Ala Ile Ile Gly Pro Gly Leu Gln Ala 2355 2360 2365 Lys Ala Thr Arg Glu Ala Gln Lys Arg Ala Ala Ala Gly Ile Met Lys 2370 2375 2380 Asn Pro Thr Val Asp Gly Ile Thr Val Ile Asp Leu Asp Pro Ile Pro 2385 2390 2395 2400 Tyr Asp Pro Lys Phe Glu Lys Gln Leu Gly Gln Val Met Leu Leu Val 2405 2410 2415 Leu Cys Val Thr Gln Val Leu Met Met Arg Thr Thr Trp Ala Leu Cys 2420 2425 2430 Glu Ala Leu Thr Leu Ala Thr Gly Pro Ile Ser Thr Leu Trp Glu Gly 2435 2440 2445 Asn Pro Gly Arg Phe Trp Asn Thr Thr Ile Ala Val Ser Met Ala Asn 2450 2455 2460 Ile Phe Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu Leu Phe Ser Ile 2465 2470 2475 2480 Met Lys Asn Thr Thr Asn Thr Arg Arg Gly Thr Gly Asn Ile Gly Glu 2485 2490 2495 Thr Leu Gly Glu Lys Trp Lys Ser Arg Leu Asn Ala Leu Gly Lys Ser 2500 2505 2510 Glu Phe Gln Ile Tyr Lys Lys Ser Gly Ile Gln Glu Val Asp Arg Thr 2515 2520 2525 Leu Ala Lys Glu Gly Ile Lys Arg Gly Glu Thr Asp His His Ala Val 2530 2535 2540 Ser Arg Gly Ser Ala Lys Leu Arg Trp Phe Val Glu Arg Asn Met Val 2545 2550 2555 2560 Thr Pro Glu Gly Lys Val Val Asp Leu Gly Cys Gly Arg Gly Gly Trp 2565 2570 2575 Ser Tyr Tyr Cys Gly Gly Leu Lys Asn Val Arg Glu Val Lys Gly Leu 2580 2585 2590 Thr Lys Gly Gly Pro Gly His Glu Glu Pro Ile Pro Met Ser Thr Tyr 2595 2600 2605 Gly Trp Asn Leu Val Arg Leu Gln Ser Gly Val Asp Val Phe Phe Ile 2610 2615 2620 Pro Pro Glu Lys Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser 2625 2630 2635 2640 Pro Asn Pro Thr Val Glu Ala Gly Arg Thr Leu Arg Val Leu Asn Leu 2645 2650 2655 Val Glu Asn Trp Leu Asn Asn Asn Thr Gln Phe Cys Ile Lys Val Leu 2660 2665 2670 Asn Pro Tyr Met Pro Ser Val Ile Glu Lys Met Glu Ala Leu Gln Arg 2675 2680 2685 Lys Tyr Gly Gly Ala Leu Val Arg Asn Pro Leu Ser Arg Asn Ser Thr 2690 2695 2700 His Glu Met Tyr Trp Val Ser Asn Ala Ser Gly Asn Ile Val Ser Ser 2705 2710 2715 2720 Val Asn Met Ile Ser Arg Met Leu Ile Asn Arg Phe Thr Met Arg Tyr 2725 2730 2735 Lys Lys Ala Thr Tyr Glu Pro Asp Val Asp Leu Gly Ser Gly Thr Arg 2740 2745 2750 Asn Ile Gly Ile Glu Ser Glu Ile Pro Asn Leu Asp Ile Ile Gly Lys 2755 2760 2765 Arg Ile Glu Lys Ile Lys Gln Glu His Glu Thr Ser Trp His Tyr Asp 2770 2775 2780 Gln Asp His Pro Tyr Lys Thr Trp Ala Tyr His Gly Ser Tyr Glu Thr 2785 2790 2795 2800 Lys Gln Thr Gly Ser Ala Ser Ser Met Val Asn Gly Val Val Arg Leu 2805 2810 2815 Leu Thr Lys Pro Trp Asp Val Val Pro Met Val Thr Gln Met Ala Met 2820 2825 2830 Thr Asp Thr Thr Pro Phe Gly Gln Gln Arg Val Phe Lys Glu Lys Val 2835 2840 2845 Asp Thr Arg Thr Gln Glu Pro Lys Glu Gly Thr Lys Lys Leu Met Lys 2850 2855 2860 Ile Thr Ala Glu Trp Leu Trp Lys Glu Leu Gly Lys Lys Lys Lys Thr Pro 2865 2870 2875 2880 Arg Met Cys Thr Arg Thr Glu Glu Phe Thr Arg Lys Val Arg Ser Asn Ala 2885 2890 2895 Ala Leu Gly Ala Ile Phe Thr Asp Glu Asn Lys Trp Lys Ser Ala Arg 2900 2905 2910 Glu Ala Val Glu Asp Ser Arg Phe Trp Glu Leu Val Asp Lys Glu Arg 2915 2920 2925 Asn Leu His Leu Glu Gly Lys Cys Glu Thr Cys Val Tyr Asn Met Met 2930 2935 2940 Gly Lys Arg Glu Lys Lys Leu Gly Glu Phe Gly Lys Ala Lys Gly Ser 2945 2950 2955 2960 Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg Phe Leu Glu Phe Glu 2965 2970 2975 Ala Leu Gly Leu Asn Glu Asp His Trp Phe Ser Arg Glu Asn Ser 2980 2985 2990 Leu Ser Gly Val Glu Gly Glu Gly Leu His Lys Leu Gly Tyr Ile Leu 2995 3000 3005 Arg Asp Val Ser Lys Lys Glu Gly Gly Ala Met Tyr Ala Asp Asp Thr 3010 3015 3020 Ala Gly Trp Asp Thr Arg Ile Thr Leu Glu Asp Leu Lys Asn Glu Glu 3025 3030 3035 3040 Met Val Thr Asn His Met Glu Gly Glu His Lys Lys Leu Ala Glu Ala 3045 3050 3055 Ile Phe Lys Leu Thr Tyr Gln Asn Lys Val Val Arg Val Gln Arg Pro 3060 3065 3070 Thr Pro Arg Gly Thr Val Met Asp Ile Ile Ser Arg Arg Asp Gln Arg 3075 3080 3085 Gly Ser Gly Gln Val Gly Thr Tyr Gly Leu As n Thr Phe Thr Asn Met 3090 3095 3100 Glu Ala Gln Leu Ile Arg Gln Met Glu Gly Glu Gly Val Phe Lys Ser 3105 3110 3115 3120 Ile Gln His Leu Thr Ile Thr Glu Glu Ile Ala Val Gln Asn Trp Leu 3125 3130 3135 Ala Arg Val Gly Arg Glu Arg Leu Ser Arg Met Ala Ile Ser Gly Asp 3140 3145 3150 Asp Cys Val Val Lys Pro Leu Asp Asp Arg Phe Ala Ser Ala Leu Thr 3155 3160 3165 Ala Leu Asn Asp Met Gly Lys Ile Arg Lys Asp Ile Gln Gln Trp Glu 3170 3175 3180 Pro Ser Arg Gly Trp Asn Asp Trp Thr Gln Val Pro Phe Cys Ser His 3185 3190 3195 3200 His Phe His Glu Leu Ile Met Lys Asp Gly Arg Val Leu Val Val Pro 3205 3210 3215 Cys Arg Asn Gln Asp Glu Leu Ile Gly Arg Ala Arg Ile Ser Gln Gly 3220 3225 3230 Ala Gly Trp Ser Leu Arg Glu Thr Ala Cys Leu Gly Lys Ser Tyr Ala 3235 3240 3245 Gln Met Trp Ser Leu Met Tyr Phe His Arg Arg Asp Leu Arg Leu Ala 3250 3255 3260 Ala Asn Ala Ile Cys Ser Ala Val Pro Ser His Trp Val Pro Thr Ser 3265 3270 3275 3280 Arg Thr Thr Trp Ser Ile His Ala Lys His Glu Trp Met Thr Thr Glu 3285 3290 3295 Asp Met Leu Thr Val Trp Asn Arg Val Trp Ile Gln Glu Asn Pro Trp 3300 3305 3310 Met Glu Asp Lys Thr Pro Val Glu Ser Trp Glu Glu Ile Pro Tyr Leu 3315 3320 3325 Gly Lys Arg Glu Asp Gln Trp Cys Gly Ser Leu Ile Gly Leu Thr Ser 3330 3335 3340 Arg Ala Thr Trp Ala Lys Asn Ile Gln Ala Ala Ile Asn Gln Val Arg 3345 3350 3355 3360 Ser Leu Ile Gly Asn Glu Glu Tyr Thr Asp Tyr Met Pro Ser Met Lys 3365 3370 3375Arg Phe Arg Arg Glu Glu Glu Glu Ala Gly Val Leu Trp 3380 3385 <210> 7 <211> 10723 <212> DNA <213> chimeric dengue seroytpe 2/4 (MVS) <220> <221> misc_feature <222> (3773) <223> r is a or g <400> 7 agttgttagt ctacgtggac cgacaaagac agattctttg agggagctaa gctcaatgta 60 gttctaacag ttttttaatt agagagcaga tctctgatga ataaccaacg gaaaaaggcg 120 aaaaacacgc ctttcaatat gctgaaacgc gagagaaacc gcgtgtcgac tgtgcaacag 180 ctgacaaaga gattctcact tggaatgctg cagggacgag gacctttaaa actgttcatg 240 gccctggtgg cgttccttcg tttcctaaca atcccaccaa cagcagggat attgaagaga 300 tggggaacaa ttaaaaaatc aaaagctatt aatgttttga gagggttcag gaaagagatt 360 ggaaggatgc tgaacatctt gaataggaga cgcagctctg caggcatgat cattatgctg 420 attccaacag tgatggcgtt ccatttaacc acgcgtgatg gcgaacccct catgatagtg 480 gcaaaacatg aaagggggag acctctcttg tttaagacaa cagaggggat caacaaatgc 540 actctcattg ccatggactt gggtgaaatg tgtgaggaca ctgtcacgta taaatgcccc 600 ttactggtca ataccgaacc tgaagacatt gattgctggt gcaatctcac gtctacctgg 660 gtcatgtatg ggacatgcac ccagagcgga gaacggagac gagagaagcg ctcagtagct 720 ttaacaccac attcaggaat gggattggaa acaagagctg agacatggat gtcatcggaa 780 ggggcttgga agcatgctca gagagtagag agctggatac tcagaaaccc aggattcgcg 840 ctcttggcag gattatggc ttatatgatt gggcaaacag gaatccagcg aactgtcttc 900 tttgtcctaa tgatgctggt cgccccatcc tacggaatgc gatgcgtagg agtaggaaac 960 agagactttg tggaaggagt ctcaggtgga gcatgggtcg atctggtgct agaacatgga 1020 ggatgcgtca caaccatggc ccagggaaaa ccaaccttgg attttgaact gactaagaca 1080 acagccaagg aagtggctct gttaagaacc tattgcattg aagcctcaat atcaaacata 1140 accacggcaa caagatgtcc aacgcaagga gagccttatc taaaagagga acaagaccaa 1200 cagtacattt gccggagaga tgtggtagac agagggtggg gcaatggctg tggcttgttt 1260 ggaaaaggag gagttgtgac atgtgcgaag ttttcatgtt cggggaagat aacaggcaat 1320 ttggtccaaa ttgagaacct tgaatacaca gtggttgtaa cagtccacaa tggagacacc 1380 catgcagtag gaaatgacac gtccaatcat ggagttacag ccacgataac tcccaggtca 1440 ccatcggtgg aagtcaaatt gccggactat ggagaactaa cactcgattg tgaacccagg 1500 tctggaattg actttaatga gatgattctg atgaaaatga aaaagaaaac atggcttgtg 1560 cataagcaat ggtttttgga tctacctcta ccatggacag caggagcaga cacatcagag 1620 gttcactgga attacaaaga gagaatggtg acatttaagg ttcctcatgc caagagacag 1680 gatgtgacag tgctgggatc tcaggaagga gccatgcatt ctgccctcgc tggagccaca 1740 gaagtggact ccggtgatgg aaatcacatg tttgcaggac atctcaagtg caaagtccgt 1800 atggagaaat tgagaatcaa gggaatgtca tacacgatgt gttcaggaaa gttctcaatt 1860 gacaaagaga tggcagaaac acagcatggg acaacagtgg tgaaagtcaa gtatgaaggt 1920 gctggagctc cgtgtaaagt ccccatagag ataagagatg tgaacaagga aaaagtggtt 1980 gggcgtatca tctcatccac ccctttggct gagaatacca acagtgtaac caacatagag 2040 ttagaacccc cctttgggga cagctacata gtgataggtg ttggaaacag tgcattaaca 2100 ctccattggt tcaggaaagg gagttccatt ggcaagatgt ttgagtccac atacagaggt 2160 gcaaaacgaa tggccattct aggtgaaaca gcttgggatt ttggttccgt tggtggactg 2220 ttcacatcat tgggaaaggc tgtgcaccag gtttttggaa gtgtgtatac aaccctgttt 2280 ggaggagtct catggatgat tagaatccta attgggttcc tagtgttgtg gattggcacg 2340 aactcaagga acacttcaat ggctatgacg tgcatagctg ccggcattgt gacactgtat 2400 ttgggggtca tggtgcaggc cgatagtggt tgcgttgtga gctggaaaaa caaagaactg 2460 aaatgtggca gtgggatttt catcacagac aacgtgcaca catggacaga acaatacaag 2520 ttccaaccag aatccccttc aaaactagct tcagctatcc agaaagccca tgaagaggac 2580 atttgtggaa tccgctcagt aacaagactg gagaatctga tgtggaaaca aataacacca 2640 gaattgaatc acattctatc agaaaatgag gtgaagttaa ctattatgac aggagacatc 2700 aaaggaatca tgcaggcagg aaaacgatct ctgcggcctc agcccactga gctgaagtat 2760 tcatggaaaa catggggcaa agcaaaaatg ctctctacag agtctcataa ccagaccttt 2820 ctcattgatg gccccgaaac agcagaatgc cccaacacaa atagagcttg gaattcgttg 2880 gaagttgaag actatggctt tggagtattc accaccaata tatggctaaa attgaaagaa 2940 aaacaggatg tattctgcga ctcaaaactc atgtcagcgg ccataaaaga caacagagcc 3000 gtccatgccg atatgggtta ttggatagaa agtgcactca atgacacatg gaagatagag 3060 aaagcctctt tcattgaagt taaaaactgc cactggccaa aatcacacac cctctggagc 3120 aatggagtgc tagaaagtga gatgataatt ccaaagaatc tcgctggacc agtgtctcaa 3180 cacaactata gaccaggcta ccatacacaa ataacaggac catggcatct aggtaagctt 3240 gagatggact ttgatttctg tgatggaaca acagtggtag tgactgagga ctgcggaaat 3300 agaggaccct ctttgagaac aaccactgcc tctggaaaac tcataacaga atggtgctgc 3360 cgatcttgca cattaccacc gctaagatac agaggtgagg atgggtgctg gtacgggatg 3420 gaaatcagac cattgaagga gaaagaagag aatttggtca actccttggt cacagctgga 3480 catgggcagg tcgacaactt ttcactagga gtcttgggaa tggcattgtt cctggaggaa 3540 atgcttagga cccgagtagg aacgaaacat gcaatactac tagttgcagt ttcttttgtg 3600 acattgatca cagggaacat gtcctttaga gacctgggaa gagtgatggt tatggtaggc 3660 gccactatga cgggtgacat aggtatgggc gtgacttatc ttgccctact agcagccttc 3720 aaagtcagac caacttttgc agctggacta ctcttgagaa agctgacctc carggaattg 3780 atgatgacta ctataggaat tgtactcctc tcccagagca ccataccaga gaccattctt 3840 gagttgactg atgcgttagc cttaggcatg atggtcctca aaatggtgag aaatatggaa 3900 aagtatcaat tggcagtgac tatcatggct atcttgtgcg tcccaaacgc agtgatatta 3960 caaaacgcat ggaaagtgag ttgcacaata ttggcagtgg tgtccgtttc cccactgttc 4020 ttaacatcct cacagcaaaa aacagattgg ataccattag cattgacgat caaaggtctc 4080 aatccaacag ctatttttct aacaaccctc tcaagaacca gcaagaaaag gagctggcca 4140 ttaaatgagg ctatcatggc agtcgggatg gtgagcattt tagccagttc tctcctaaaa 4200 aatgatattc ccatgacagg accattagtg gctggagggc tcctcactgt gtgctacgtg 4260 ctcactggac gatcggccga tttggaactg gagagagcag ccgatgtcaa atgggaagac 4320 caggcagaga tatcaggaag cagtccaatc ctgtcaataa caatatcaga agatggtagc 4380 atgtcgataa aaaatgaaga ggaagaacaa acactgacca tactcattag aacaggattg 4440 ctggtgatct caggactttt tcctgtatca ataccaatca cggcagcagc atggtacctg 4500 tgggaagtga agaaacaacg ggccggagta ttgtgggatg ttccttcacc cccacccatg 4560 ggaaaggctg aactggaaga tggagcctat agaattaagc aaaaagggat tcttggatat 4620 tcccagatcg gagccggagt ttacaaagaa ggaacattcc atacaatgtg gcatgtcaca 4680 cgtggcgctg ttctaatgca taaaggaaag aggattgaac catcatgggc ggacgtcaag 4740 aaagacctaa tatcatatgg aggaggctgg aagttagaag gagaatggaa ggaaggagaa 4800 gaagtccagg tattggcact ggagcctgga aaaaatccaa gagccgtcca aacgaaacct 4860 ggtcttttca aaaccaacgc cggaacaata ggtgctgtat ctctggactt ttctcctgga 4920 acgtcaggat ctccaattat cgacaaaaaa ggaaaagttg tgggtcttta tggtaatggt 4980 gttgttacaa ggagtggagc atatgtgagt gctatagccc agactgaaaa aagcattgaa 5040 gacaacccag agatcgaaga tgacattttc cgaaagagaa gactgaccat catggacctc 5100 cacccaggag cgggaaagac gaagagatac cttccggcca tagtcagaga agctataaaa 5160 cggggtttga gaacattaat cttggccccc actagagttg tggcagctga aatggaggaa 5220 gcccttagag gacttccaat aagataccag accccagcca tcagagctgt gcacaccggg 5280 cgggagattg tggacctaat gtgtcatgcc acatttacca tgaggctgct atcaccagtt 5340 agagtgccaa actacaacct gattatcatg gacgaagccc atttcacaga tccagcaagt 5400 atagcagcta gaggatacat ctcaactcga gtggagatgg gtgaggcagc tgggattttt 5460 atgacagcca ctccccccggg aagcagagac ccatttcctc agagcaatgc accaatcata 5520 gatgaagaaa gagaaatccc tgaacgctcg tggaattccg gacatgaatg ggtcacggat 5580 tttaaaggga agactgtttg gttcgttcca agtataaaag caggaaatga tatagcagct 5640 tgcctgagga aaaatggaaa gaaagtgata caactcagta ggaagacctt tgattctgag 5700 tatgtcaaga ctagaaccaa tgattgggac ttcgtggtta caactgacat ttcagaaatg 5760 ggtgccaatt tcaaggctga gagggttata gaccccagac gctgcatgaa accagtcata 5820 ctaacagatg gtgaagagcg ggtgattctg gcaggaccta tgccagtgac ccactctagt 5880 gcagcacaaa gaagagggag aataggaaga aatccaaaaa atgagaatga ccagtacata 5940 tacatggggg aacctctgga aaatgatgaa gactgtgcac actggaaaga agctaaaatg 6000 ctcctagata acatcaacac gccagaagga atcattccta gcatgttcga accagagcgt 6060 gaaaaggtgg atgccattga tggcgaatac cgcttgagag gagaagcaag gaaaaccttt 6120 gtagacttaa tgagaagagg agacctacca gtctggttgg cctacagagt ggcagctgaa 6180 ggcatcaact acgcagacag aaggtggtgt tttgatggag tcaagaacaa ccaaatccta 6240 gaagaaaacg tggaagttga aatctggaca aaagaagggg aaaggaagaa attgaaaccc 6300 agatggttgg atgctaggat ctattctgac ccactggcgc taaaagaatt taaggaattt 6360 gcagccggaa gaaagtctct gaccctgaac ctaatcacag aaatgggtag gctcccaacc 6420 ttcatgactc agaaggtaag agacgcactg gacaacttag cagtgctgca cacggctgag 6480 gcaggtggaa gggcgtacaa ccatgctctc agtgaactgc cggagaccct ggagacattg 6540 cttttactga cacttctggc tacagtcacg ggagggatct ttttattctt gatgagcgca 6600 aggggcatag ggaagatgac cctgggaatg tgctgcataa tcacggctag catcctccta 6660 tggtacgcac aaatacagcc acactggata gcagcttcaa taatactgga gttttttctc 6720 atagttttgc ttattccaga acctgaaaaa cagagaacac cccaagacaa ccaactgacc 6780 tacgttgtca tagccatcct cacagtggtg gccgcaacca tggcaaacga gatgggtttc 6840 ctagaaaaaa cgaagaaaga tctcggattg ggaagcattg caacccagca acccgagagc 6900 aacatcctgg acatagatct acgtcctgca tcagcatgga cgctgtatgc cgtggccaca 6960 acatttgtta caccaatgtt gagacatagc attgaaaatt cctcagtgaa tgtgtcccta 7020 acagccatag ccaaccaagc cacagtgtta atgggtctcg ggaaaggatg gccattgtca 7080 aagatggaca tcggagttcc ccttctcgcc attggatgct actcacaagt caaccccata 7140 actctcacag cagctctttt cttattggta gcacattatg ccatcatagg gccaggactc 7200 caagcaaaag caaccagaga agctcagaaa agagcagcgg cgggcatcat gaaaaaccca 7260 actgtcgatg gaataacagt gattgaccta gatccaatac cttatgatcc aaagtttgaa 7320 aagcagttgg gacaagtaat gctcctagtc ctctgcgtga ctcaagtatt gatgatgagg 7380 actacatggg ctctgtgtga ggctttaacc ttagctaccg ggcccatctc cacatgtgg 7440 gaaggaaatc cagggaggtt ttggaacact accattgcgg tgtcaatggc taacattttt 7500 agagggagtt acttggccgg agctggactt ctcttttcta ttatgaagaa cacaaccaac 7560 acaagaaggg gaactggcaa cataggagag acgcttggag agaaatggaa aagccgattg 7620 aacgcattgg gaaaaagtga attccagatc tacaagaaaa gtggaatcca ggaagtggat 7680 agaaccttag caaaagaagg cattaaaaga ggagaaacgg accatcacgc tgtgtcgcga 7740 ggctcagcaa aactgagatg gttcgttgag agaaacatgg tcacaccaga agggaaagta 7800 gtggacctcg gttgtggcag aggaggctgg tcatactatt gtggaggact aaagaatgta 7860 agagaagtca aaggcctaac aaaaggagga ccaggacacg aagaacccat ccccatgtca 7920 acatatgggt ggaatctagt gcgtcttcaa agtggagttg acgttttctt catcccgcca 7980 gaaaagtgtg acacattatt gtgtgacata ggggagtcat caccaaatcc cacagtggaa 8040 gcaggacgaa cactcagagt ccttaactta gtagaaaatt ggttgaacaa caacactcaa 8100 ttttgcataa aggttctcaa cccatatatg ccctcagtca tagaaaaaat ggaagcacta 8160 caaaggaaat atggaggagc cttagtgagg aatccactct cacgaaactc cacacatgag 8220 atgtactggg tatccaatgc ttccgggaac atagtgtcat cagtgaacat gatttcaagg 8280 atgttgatca acagattac aatgagatac aagaaagcca cttacgagcc ggatgttgac 8340 ctcggaagcg gaacccgtaa catcgggatt gaaagtgaga taccaaacct agatataatt 8400 gggaaaagaa tagaaaaaat aaagcaagag catgaaacat catggcacta tgaccaagac 8460 cacccataca aaacgtgggc ataccatggt agctatgaaa caaaacagac tggatcagca 8520 tcatccatgg tcaacggagt ggtcaggctg ctgacaaaac cttgggacgt cgtccccatg 8580 gtgacacaga tggcaatgac agacacgact ccatttggac aacagcgcgt ttttaaagag 8640 aaagtggaca cgagaaccca agaaccgaaa gaaggcacga agaaactaat gaaaataaca 8700 gcagagtggc tttggaaaga attagggaag aaaaagacac ccaggatgtg caccagagaa 8760 gaattcacaa gaaaggtgag aagcaatgca gccttggggg ccatattcac tgatgagaac 8820 aagtggaagt cggcacgtga ggctgttgaa gatagtaggt tttgggagct ggttgacaag 8880 gaaaggaatc tccatcttga aggaaagtgt gaaacatgtg tgtacaacat gatgggaaaa 8940 agagagaaga agctagggga attcggcaag gcaaaaggca gcagagccat atggtacatg 9000 tggcttggag cacgcttctt agagtttgaa gccctaggat tcttaaatga agatcactgg 9060 ttctccagag agaactccct gagtggagtg gaaggagaag ggctgcacaa gctaggttac 9120 attctaagag acgtgagcaa gaaagaggga ggagcaatgt atgccgatga caccgcagga 9180 tgggatacaa gaatcacact agaagaccta aaaaatgaag aaatggtaac aaaccacatg 9240 gaaggagaac acaagaaact agccgaggcc attttcaaac taacgtacca aaacaaggtg 9300 gtgcgtgtgc aaagaccaac accaagaggc acagtaatgg acatcatatc gagaagagac 9360 caaagaggta gtggacaagt tggcacctat ggactcaata ctttcaccaa tatggaagcc 9420 caactaatca gacagatgga gggagaagga gtctttaaaa gcattcagca cctaacaatc 9480 acagaagaaa tcgctgtgca aaactggtta gcaagagtgg ggcgcgaaag gttatcaaga 9540 atggccatca gtggagatga ttgtgttgtg aaacctttag atgacaggtt cgcaagcgct 9600 ttaacagctc taaatgacat gggaaagatt aggaaagaca tacaacaatg ggaaccttca 9660 agaggatgga atgattggac acaagtgccc ttctgttcac accatttcca tgagttaatc 9720 atgaaagacg gtcgcgtact cgttgttccc tgtagaaacc aagatgaact gattggcaga 9780 gcccgaatct cccaaggagc agggtggtct ttgcgggaga cggcctgttt ggggaagtct 9840 tacgcccaaa tgtggagctt gatgtacttc cacagacgcg acctcaggct ggcggcaaat 9900 gctatttgct cggcagtacc atcacattgg gttccaacaa gtcgaacaac ctggtccata 9960 catgctaaac atgaatggat gacaacggaa gacatgctga cagtctggaa cagggtgtgg 10020 attcaagaaa acccatggat ggaagacaaa actccagtgg aatcatggga ggaaatccca 10080 tacttgggga aaagagaaga ccaatggtgc ggctcattga ttgggttaac aagcagggcc 10140 acctgggcaa agaacatcca agcagcaata aatcaagtta gatcccttat aggcaatgaa 10200 gaatacacag attacatgcc atccatgaaa agattcagaa gagaagagga agaagcagga 10260 gttctgtggt agaaagcaaa actaacatga aacaaggcta gaagtcaggt cggattaagc 10320 catagtacgg aaaaaactat gctacctgtg agccccgtcc aaggacgtta aaagaagtca 10380 ggccatcata aatgccatag cttgagtaaa ctatgcagcc tgtagctcca cctgagaagg 10440 tgtaaaaaat ccgggaggcc acaaaccatg gaagctgtac gcatggcgta gtggactagc 10500 ggttagagga gacccctccc ttacaaatcg cagcaacaat gggggcccaa ggcgagatga 10560 agctgtagtc tcgctggaag gactagaggt tagaggagac ccccccgaaa caaaaaacag 10620 catattgacg ctgggaaaga ccagagatcc tgctgtctcc tcagcatcat tccaggcaca 10680 gaacgccaga aaatggaatg gtgctgttga atcaacaggt tct 10723 <210> 8 <211> 3391 <212> PRT <213> chimeric dengue serotype 2/4 (MVS) <220> <221> MISC_FEATURE <222> (1226) <223> Xaa is Arg or Lys <400> 8 Met Asn Asn Gln Arg Lys Lys Ala Lys Asn Thr Pro Phe Asn Met Leu 1 5 10 15 Lys Arg Glu Arg Asn Arg Val Ser Thr Val Gln Gln Leu Thr Lys Arg 20 25 30 Phe Ser Leu Gly Met Leu Gln Gly Arg Gly Pro Leu Lys Leu Phe Met 35 40 45 Ala Leu Val Ala Phe Leu Arg Phe Leu Thr Ile Pro Pro Thr Ala Gly 50 55 60 Ile Leu Lys Arg Trp Gly Thr Ile Lys Lys Ser Lys Ala Ile Asn Val 65 70 75 80 Leu Arg Gly Phe Arg Lys Glu Ile Gly Arg Met Leu Asn Ile Leu Asn 85 90 95 Arg Arg Arg Ser Ser Ala Gly Met Ile Ile Met Leu Ile Pro Thr Val 100 105 110 Met Ala Phe His Leu Thr Thr Arg Asp Gly Glu Pro Leu Met Ile Val 115 120 125 Ala Lys His Glu Arg Gly Arg Pro Leu Leu Phe Lys Thr Thr Glu Gly 130 135 140 Ile Asn Lys Cys Thr Leu Ile Ala Met Asp Leu Gly Glu Met Cys Glu 145 150 155 160 Asp Thr Val Thr Tyr Lys Cys Pro Leu Leu Val Asn Thr Glu Pro Glu 165 170 175 Asp Ile Asp Cys Trp Cys Asn Leu Thr Ser Thr Trp Val Met Tyr Gly 180 185 190 Thr Cys Thr Gln Ser Gly Glu Arg Arg Arg Glu Lys Arg Ser Val Ala 195 200 205 Leu Thr Pro His Ser Gly Met Gly Leu Glu Thr Arg Ala Glu Thr Trp 210 215 220 Met Ser Ser Glu Gly Ala Trp Lys His Ala Gln Arg Val Glu Ser Trp 225 230 235 240 Ile Leu Arg Asn Pro Gly Phe Ala Leu Leu Ala Gly Phe Met Ala Tyr 245 250 255 Met Ile Gly Gln Thr Gly Ile Gln Arg Thr Val Phe Phe Val Leu Met 260 265 270 Met Leu Val Ala Pro Ser Tyr Gly Met Arg Cys Val Gly Val Gly Asn 275 280 285 Arg Asp Phe Val Glu Gly Val Ser Gly Gly Ala Trp Val Asp Leu Val 290 295 300 Leu Glu His Gly Gly Cys Val Thr Thr Met Ala Gln Gly Lys Pro Thr 305 310 315 320 Leu Asp Phe Glu Leu Thr Lys Thr Thr Ala Lys Glu Val Ala Leu Leu 325 330 335 Arg Thr Tyr Cys Ile Glu Ala Ser Ile Ser Asn Ile Thr Thr Ala Thr 340 345 350 Arg Cys Pro Thr Gln Gly Glu Pro Tyr Leu Lys Glu Glu Gln Asp Gln 355 360 365 Gln Tyr Ile Cys Arg Arg Asp Val Val Asp Arg Gly Trp Gly Asn Gly 370 375 380 Cys Gly Leu Phe Gly Lys Gly Gly Val Val Thr Cys Ala Lys Phe Ser 385 390 395 400 Cys Ser Gly Lys Ile Thr Gly Asn Leu Val Gln Ile Glu Asn Leu Glu 405 410 415 Tyr Thr Val Val Val Thr Val His Asn Gly Asp Thr His Ala Val Gly 420 425 430 Asn Asp Thr Ser Asn His Gly Val Thr Ala Thr Ile Thr Pro Arg Ser 435 440 445 Pro Ser Val Glu Val Lys Leu Pro Asp Tyr Gly Glu Leu Thr Leu Asp 450 455 460 Cys Glu Pro Arg Ser Gly Ile Asp Phe Asn Glu Met Ile Leu Met Lys 465 470 475 480 Met Lys Lys Lys Thr Trp Leu Val His Lys Gln Trp Phe Leu Asp Leu 485 490 495 Pro Leu Pro Trp Thr Ala Gly Ala Asp Thr Ser Glu Val His Trp Asn 500 505 510 Tyr Lys Glu Arg Met Val Thr Phe Lys Val Pro His Ala Lys Arg Gln 515 520 525 Asp Val Thr Val Leu Gly Ser Gln Glu Gly Ala Met His Ser Ala Leu 530 535 540 Ala Gly Ala Thr Glu Val Asp Ser Gly Asp Gly Asn His Met Phe Ala 545 550 555 560 Gly His Leu Lys Cys Lys Val Arg Met Glu Lys Leu Arg Ile Lys Gly 565 570 575 Met Ser Tyr Thr Met Cys Ser Gly Lys Phe Ser Ile Asp Lys Glu Met 580 585 590 Ala Glu Thr Gln His Gly Thr Thr Val Val Lys Val Lys Tyr Glu Gly 595 600 605 Ala Gly Ala Pro Cys Lys Val Pro Ile Glu Ile Arg Asp Val Asn Lys 610 615 620 Glu Lys Val Val Gly Arg Ile Ile Ser Ser Thr Pro Leu Ala Glu Asn 625 630 635 640 Thr Asn Ser Val Thr Asn Ile Glu Leu Glu Pro Phe Gly Asp Ser 645 650 655 Tyr Ile Val Ile Gly Val Gly Asn Ser Ala Leu Thr Leu His Trp Phe 660 665 670 Arg Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ser Thr Tyr Arg Gly 675 680 685 Ala Lys Arg Met Ala Ile Leu Gly Glu Thr Ala Trp Asp Phe Gly Ser 690 695 700 Val Gly Gly Leu Phe Thr Ser Leu Gly Lys Ala Val His Gln Val Phe 705 710 715 720 Gly Ser Val Tyr Thr Thr Leu Phe Gly Gly Val Ser Trp Met Ile Arg 725 730 735 Ile Leu Ile Gly Phe Leu Val Leu Trp Ile Gly Thr Asn Ser Arg Asn 740 745 750 Thr Ser Met Ala Met Thr Cys Ile Ala Ala Gly Ile Val Thr Leu Tyr 755 760 765 Leu Gly Val Met Val Gln Ala Asp Ser Gly Cys Val Val Ser Trp Lys 770 775 780 Asn Lys Glu Leu Lys Cys Gly Ser Gly Ile Phe Ile Thr Asp Asn Val 785 790 795 800 His Thr Trp Thr Glu Gln Tyr Lys Phe Gln Pro Glu Ser Pro Ser Lys 805 810 815 Leu Ala Ser Ala Ile Gln Lys Ala His Glu Glu Asp Ile Cys Gly Ile 820 825 830 Arg Ser Val Thr Arg Leu Glu Asn Leu Met Trp Lys Gln Ile Thr Pro 835 840 845 Glu Leu Asn His Ile Leu Ser Glu Asn Glu Val Lys Leu Thr Ile Met 850 855 860 Thr Gly Asp Ile Lys Gly Ile Met Gln Ala Gly Lys Arg Ser Leu Arg 865 870 875 880 Pro Gln Pro Thr Glu Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala 885 890 895 Lys Met Leu Ser Thr Glu Ser His Asn Gln Thr Phe Leu Ile Asp Gly 900 905 910 Pro Glu Thr Ala Glu Cys Pro Asn Thr Asn Arg Ala Trp Asn Ser Leu 915 920 925 Glu Val Glu Asp Tyr Gly Phe Gly Val Phe Thr Thr Asn Ile Trp Leu 930 935 940 Lys Leu Lys Glu Lys Gln Asp Val Phe Cys Asp Ser Lys Leu Met Ser 945 950 955 960 Ala Ala Ile Lys Asp Asn Arg Ala Val His Ala Asp Met Gly Tyr Trp 965 970 975 Ile Glu Ser Ala Leu Asn Asp Thr Trp Lys Ile Glu Lys Ala Ser Phe 980 985 990 Ile Glu Val Lys Asn Cys His Trp Pro Lys Ser His Thr Leu Trp Ser 995 1000 1005 Asn Gly Val Leu Glu Ser Glu Met Ile Ile Pro Lys Asn Leu Ala Gly 1010 1015 1020 Pro Val Ser Gln His Asn Tyr Arg Pro Gly Tyr His Thr Gln Ile Thr 1025 1030 1035 1040 Gly Pro Trp His Leu Gly Lys Leu Glu Met Asp Phe Asp Phe Cys Asp 1045 1050 1055 Gly Thr Thr Val Val Val Thr Glu Asp Cys Gly Asn Arg Gly Pro Ser 1060 1065 1070 Leu Arg Thr Thr Thr Ala Ser Gly Lys Leu Ile Thr Glu Trp Cys Cys 1075 1080 1085 Arg Ser Cys Thr Leu Pro Leu Arg Tyr Arg Gly Glu Asp Gly Cys 1090 1095 1100 Trp Tyr Gly Met Glu Ile Arg Pro Leu Lys Glu Lys Glu Glu Asn Leu 1105 1110 1115 1120 Val Asn Ser Leu Val Thr Ala Gly His Gly Gln Val Asp Asn Phe Ser 1125 1130 1135 Leu Gly Val Leu Gly Met Ala Leu Phe Leu Glu Glu Met Leu Arg Thr 1140 1145 1150 Arg Val Gly Thr Lys His Ala Ile Leu Leu Val Ala Val Ser Phe Val 1155 1160 1165 Thr Leu Ile Thr Gly Asn Met Ser Phe Arg Asp Leu Gly Arg Val Met 1170 1175 1180 Val Met Val Gly Ala Thr Met Thr Gly Asp Ile Gly Met Gly Val Thr 1185 1190 1195 1200 Tyr Leu Ala Leu Leu Ala Ala Phe Lys Val Arg Pro Thr Phe Ala Ala 1205 1210 1215 Gly Leu Leu Leu Arg Lys Leu Thr Ser Xaa Glu Leu Met Met Thr Thr 1220 1225 1230 Ile Gly Ile Val Leu Leu Ser Gln Ser Thr Ile Pro Glu Thr Ile Leu 1235 1240 1245 Glu Leu Thr Asp Ala Leu Ala Leu Gly Met Met Val Leu Lys Met Val 1250 1255 1260 Arg Asn Met Glu Lys Tyr Gln Leu Ala Val Thr Ile Met Ala Ile Leu 1265 1270 1275 1280 Cys Val Pro Asn Ala Val Ile Leu Gln Asn Ala Trp Lys Val Ser Cys 1285 1290 1295 Thr Ile Leu Ala Val Val Ser Val Ser Pro Leu Phe Leu Thr Ser Ser 1300 1305 1310 Gln Gln Lys Thr Asp Trp Ile Pro Leu Ala Leu Thr Ile Lys Gly Leu 1315 1320 1325 Asn Pro Thr Ala Ile Phe Leu Thr Thr Leu Ser Arg Thr Ser Lys Lys 1330 1335 1340 Arg Ser Trp Pro Leu Asn Glu Ala Ile Met Ala Val Gly Met Val Ser 1345 1350 1355 1360 Ile Leu Ala Ser Leu Leu Lys Asn Asp Ile Pro Met Thr Gly Pro 1365 1370 1375 Leu Val Ala Gly Gly Leu Leu Thr Val Cys Tyr Val Leu Thr Gly Arg 1380 1385 1390 Ser Ala Asp Leu Glu Leu Glu Arg Ala Ala Asp Val Lys Trp Glu Asp 1395 1400 1405 Gln Ala Glu Ile Ser Gly Ser Ser Pro Ile Leu Ser Ile Thr Ile Ser 1410 1415 1420 Glu Asp Gly Ser Met Ser Ile Lys Asn Glu Glu Glu Glu Gln Thr Leu 1425 1430 1435 1440 Thr Ile Leu Ile Arg Thr Gly Leu Leu Val Ile Ser Gly Leu Phe Pro 1445 1450 1455 Val Ser Ile Pro Ile Thr Ala Ala Ala Trp Tyr Leu Trp Glu Val Lys 1460 1465 1470 Lys Gln Arg Ala Gly Val Leu Trp Asp Val Pro Ser Pro Pro Met 1475 1480 1485 Gly Lys Ala Glu Leu Glu Asp Gly Ala Tyr Arg Ile Lys Gln Lys Gly 1490 1495 1500 Ile Leu Gly Tyr Ser Gln Ile Gly Ala Gly Val Tyr Lys Glu Gly Thr 1505 1510 1515 1520 Phe His Thr Met Trp His Val Thr Arg Gly Ala Val Leu Met His Lys 1525 1530 1535 Gly Lys Arg Ile Glu Pro Ser Trp Ala Asp Val Lys Lys Asp Leu Ile 1540 1545 1550 Ser Tyr Gly Gly Gly Trp Lys Leu Glu Gly Glu Trp Lys Glu Gly Glu 1555 1560 1565 Glu Val Gln Val Leu Ala Leu Glu Pro Gly Lys Asn Pro Arg Ala Val 1570 1575 1580 Gln Thr Lys Pro Gly Leu Phe Lys Thr Asn Ala Gly Thr Ile Gly Ala 1585 1590 1595 1600 Val Ser Leu Asp Phe Ser Pro Gly Thr Ser Gly Ser Pro Ile Ile Asp 1605 1610 1615 Lys Lys Gly Lys Val Val Gly Leu Tyr Gly Asn Gly Val Val Thr Arg 1620 1625 1630 Ser Gly Ala Tyr Val Ser Ala Ile Ala Gln Thr Glu Lys Ser Ile Glu 1635 1640 1645 Asp Asn Pro Glu Ile Glu Asp Asp Ile Phe Arg Lys Arg Arg Leu Thr 1650 1655 1660 Ile Met Asp Leu His Pro Gly Ala Gly Lys Thr Lys Arg Tyr Leu Pro 1665 1670 1675 1680 Ala Ile Val Arg Glu Ala Ile Lys Arg Gly Leu Arg Thr Leu Ile Leu 1685 1690 1695 Ala Pro Thr Arg Val Val Ala Ala Glu Met Glu Glu Ala Leu Arg Gly 1700 1705 1710 Leu Pro Ile Arg Tyr Gln Thr Pro Ala Ile Arg Ala Val His Thr Gly 1715 1720 1725 Arg Glu Ile Val Asp Leu Met Cys His Ala Thr Phe Thr Met Arg Leu 1730 1735 1740 Leu Ser Pro Val Arg Val Pro Asn Tyr Asn Leu Ile Ile Met Asp Glu 1745 1750 1755 1760 Ala His Phe Thr Asp Pro Ala Ser Ile Ala Ala Arg Gly Tyr Ile Ser 1765 1770 1775 Thr Arg Val Glu Met Gly Glu Ala Ala Gly Ile Phe Met Thr Ala Thr 1780 1785 1790 Pro Pro Gly Ser Arg Asp Pro Phe Pro Gln Ser Asn Ala Pro Ile Ile 1795 1800 1805 Asp Glu Glu Arg Glu Ile Pro Glu Arg Ser Trp Asn Ser Gly His Glu 1810 1815 1820 Trp Val Thr Asp Phe Lys Gly Lys Thr Val Trp Phe Val Pro Ser Ile 1825 1830 1835 1840 Lys Ala Gly Asn Asp Ile Ala Ala Cys Leu Arg Lys Asn Gly Lys Lys 1845 1850 1855 Val Ile Gln Leu Ser Arg Lys Thr Phe Asp Ser Glu Tyr Val Lys Thr 1860 1865 1870 Arg Thr Asn Asp Trp Asp Phe Val Val Thr Asp Ile Ser Glu Met 1875 1880 1885 Gly Ala Asn Phe Lys Ala Glu Arg Val Ile Asp Pro Arg Arg Cys Met 1890 1895 1900 Lys Pro Val Ile Leu Thr Asp Gly Glu Glu Arg Val Ile Leu Ala Gly 1905 1910 1915 1920 Pro Met Pro Val Thr His Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile 1925 1930 1935 Gly Arg Asn Pro Lys Asn Glu Asn Asp Gln Tyr Ile Tyr Met Gly Glu 1940 1945 1950 Pro Leu Glu Asn Asp Glu Asp Cys Ala His Trp Lys Glu Ala Lys Met 1955 1960 1965 Leu Leu Asp Asn Ile Asn Thr Pro Glu Gly Ile Ile Pro Ser Met Phe 1970 1975 1980 Glu Pro Glu Arg Glu Lys Val Asp Ala Ile Asp Gly Glu Tyr Arg Leu 1985 1990 1995 2000 Arg Gly Glu Ala Arg Lys Thr Phe Val Asp Leu Met Arg Arg Gly Asp 2005 2010 2015 Leu Pro Val Trp Leu Ala Tyr Arg Val Ala Ala Glu Gly Ile Asn Tyr 2020 2025 2030 Ala Asp Arg Arg Trp Cys Phe Asp Gly Val Lys Asn Asn Gln Ile Leu 2035 2040 2045 Glu Glu Asn Val Glu Val Glu Ile Trp Thr Lys Glu Gly Glu Arg Lys 2050 2055 2060 Lys Leu Lys Pro Arg Trp Leu Asp Ala Arg Ile Tyr Ser Asp Pro Leu 2065 2070 2075 2080 Ala Leu Lys Glu Phe Lys Glu Phe Ala Ala Gly Arg Lys Ser Leu Thr 2085 2090 2095 Leu Asn Leu Ile Thr Glu Met Gly Arg Leu Pro Thr Phe Met Thr Gln 2100 2105 2110 Lys Val Arg Asp Ala Leu Asp Asn Leu Ala Val Leu His Thr Ala Glu 2115 2120 2125 Ala Gly Gly Arg Ala Tyr Asn His Ala Leu Ser Glu Leu Pro Glu Thr 2130 2135 2140 Leu Glu Thr Leu Leu Leu Leu Thr Leu Leu Ala Thr Val Thr Gly Gly 2145 2150 2155 2160 Ile Phe Leu Phe Leu Met Ser Ala Arg Gly Ile Gly Lys Met Thr Leu 2165 2170 2175 Gly Met Cys Cys Ile Ile Thr Ala Ser Ile Leu Leu Trp Tyr Ala Gln 2180 2185 2190 Ile Gln Pro His Trp Ile Ala Ala Ser Ile Ile Leu Glu Phe Phe Leu 2195 2200 2205 Ile Val Leu Leu Ile Pro Glu Pro Glu Lys Gln Arg Thr Pro Gln Asp 2210 2215 2220 Asn Gln Leu Thr Tyr Val Val Ile Ala Ile Leu Thr Val Val Ala Ala 2225 2230 2235 2240 Thr Met Ala Asn Glu Met Gly Phe Leu Glu Lys Thr Lys Lys Asp Leu 2245 2250 2255 Gly Leu Gly Ser Ile Ala Thr Gln Gln Pro Glu Ser Asn Ile Leu Asp 2260 2265 2270 Ile Asp Leu Arg Pro Ala Ser Ala Trp Thr Leu Tyr Ala Val Ala Thr 2275 2280 2285 Thr Phe Val Thr Pro Met Leu Arg His Ser Ile Glu Asn Ser Ser Val 2290 2295 2300 Asn Val Ser Leu Thr Ala Ile Ala Asn Gln Ala Thr Val Leu Met Gly 2305 2310 2315 2320 Leu Gly Lys Gly Trp Pro Leu Ser Lys Met Asp Ile Gly Val Pro Leu 2325 2330 2335 Leu Ala Ile Gly Cys Tyr Ser Gln Val Asn Pro Ile Thr Leu Thr Ala 2340 2345 2350 Ala Leu Phe Leu Leu Val Ala His Tyr Ala Ile Ile Gly Pro Gly Leu 2355 2360 2365 Gln Ala Lys Ala Thr Arg Glu Ala Gln Lys Arg Ala Ala Ala Gly Ile 2370 2375 2380 Met Lys Asn Pro Thr Val Asp Gly Ile Thr Val Ile Asp Leu Asp Pro 2385 2390 2395 2400 Ile Pro Tyr Asp Pro Lys Phe Glu Lys Gln Leu Gly Gln Val Met Leu 2405 2410 2415 Leu Val Leu Cys Val Thr Gln Val Leu Met Met Arg Thr Thr Trp Ala 2420 2425 2430 Leu Cys Glu Ala Leu Thr Leu Ala Thr Gly Pro Ile Ser Thr Leu Trp 2435 2440 2445 Glu Gly Asn Pro Gly Arg Phe Trp Asn Thr Thr Ile Ala Val Ser Met 2450 2455 2460 Ala Asn Ile Phe Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu Leu Phe 2465 2470 2475 2480 Ser Ile Met Lys Asn Thr Thr Asn Thr Arg Arg Gly Thr Gly Asn Ile 2485 2490 2495 Gly Glu Thr Leu Gly Glu Lys Trp Lys Ser Arg Leu Asn Ala Leu Gly 2500 2505 2510 Lys Ser Glu Phe Gln Ile Tyr Lys Lys Ser Gly Ile Gln Glu Val Asp 2515 2520 2525 Arg Thr Leu Ala Lys Glu Gly Ile Lys Arg Gly Glu Thr Asp His His 2530 2535 2540 Ala Val Ser Arg Gly Ser Ala Lys Leu Arg Trp Phe Val Glu Arg Asn 2545 2550 2555 2560 Met Val Thr Pro Glu Gly Lys Val Val Asp Leu Gly Cys Gly Arg Gly 2565 2570 2575 Gly Trp Ser Tyr Tyr Cys Gly Gly Leu Lys Asn Val Arg Glu Val Lys 2580 2585 2590 Gly Leu Thr Lys Gly Gly Pro Gly His Glu Glu Pro Ile Pro Met Ser 2595 2600 2605 Thr Tyr Gly Trp Asn Leu Val Arg Leu Gln Ser Gly Val Asp Val Phe 2610 2615 2620 Phe Ile Pro Pro Glu Lys Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu 2625 2630 2635 2640 Ser Ser Pro Asn Pro Thr Val Glu Ala Gly Arg Thr Leu Arg Val Leu 2645 2650 2655 Asn Leu Val Glu Asn Trp Leu Asn Asn Asn Thr Gln Phe Cys Ile Lys 2660 2665 2670 Val Leu Asn Pro Tyr Met Pro Ser Val Ile Glu Lys Met Glu Ala Leu 2675 2680 2685 Gln Arg Lys Tyr Gly Gly Ala Leu Val Arg Asn Pro Leu Ser Arg Asn 2690 2695 2700 Ser Thr His Glu Met Tyr Trp Val Ser Asn Ala Ser Gly Asn Ile Val 2705 2710 2715 2720 Ser Ser Val Asn Met Ile Ser Arg Met Leu Ile Asn Arg Phe Thr Met 2725 2730 2735 Arg Tyr Lys Lys Ala Thr Tyr Glu Pro Asp Val Asp Leu Gly Ser Gly 2740 2745 2750 Thr Arg Asn Ile Gly Ile Glu Ser Glu Ile Pro Asn Leu Asp Ile Ile 2755 2760 2765 Gly Lys Arg Ile Glu Lys Ile Lys Gln Glu His Glu Thr Ser Trp His 2770 2775 2780 Tyr Asp Gln Asp His Pro Tyr Lys Thr Trp Ala Tyr His Gly Ser Tyr 2785 2790 2795 2800 Glu Thr Lys Gln Thr Gly Ser Ala Ser Met Val Asn Gly Val Val 2805 2810 2815 Arg Leu Leu Thr Lys Pro Trp Asp Val Val Pro Met Val Thr Gln Met 2820 2825 2830 Ala Met Thr Asp Thr Thr Pro Phe Gly Gln Gln Arg Val Phe Lys Glu 2835 2840 2845 Lys Val Asp Thr Arg Thr Gln Glu Pro Lys Glu Gly Thr Lys Lys Leu 2850 2855 2860 Met Lys Ile Thr Ala Glu Trp Leu Trp Lys Glu Leu Gly Lys Lys Lys Lys 2865 2870 2875 2880 Thr Pro Arg Met Cys Thr Arg Glu Glu Phe Thr Arg Lys Val Arg Ser 2885 2890 2895 Asn Ala Ala Leu Gly Ala Ile Phe Thr Asp Glu Asn Lys Trp Lys Ser 2900 2905 2910 Ala Arg Glu Ala Val Glu Asp Ser Arg Phe Trp Glu Leu Val Asp Lys 2915 2920 2925 Glu Arg Asn Leu His Leu Glu Gly Lys Cys Glu Thr Cys Val Tyr Asn 2930 2935 2940 Met Met Gly Lys Arg Glu Lys Lys Leu Gly Glu Phe Gly Lys Ala Lys 2945 2950 2955 2960 Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg Phe Leu Glu 2965 2970 2975 Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp Phe Ser Arg Glu 2980 2985 2990 Asn Ser Leu Ser Gly Val Glu Gly Glu Gly Leu His Lys Leu Gly Tyr 2995 3000 3005 Ile Leu Arg Asp Val Ser Lys Lys Glu Gly Gly Ala Met Tyr Ala Asp 3010 3015 3020 Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr Leu Glu Asp Leu Lys Asn 3025 3030 3035 3040 Glu Glu Met Val Thr Asn His Met Glu Gly Glu His Lys Lys Lys Leu Ala 3045 3050 3055 Glu Ala Ile Phe Lys Leu Thr Tyr Gln Asn Lys Val Val Arg Val Gln 3060 3065 3070 Arg Pro Thr Pro Arg Gly Thr Val Met Asp Ile Ile Ser Arg Arg Asp 3075 3080 3085 Gln Arg Gly Ser Gly Gln Val Gly Thr Tyr Gl y Leu Asn Thr Phe Thr 3090 3095 3100 Asn Met Glu Ala Gln Leu Ile Arg Gln Met Glu Gly Glu Gly Val Phe 3105 3110 3115 3120 Lys Ser Ile Gln His Leu Thr Ile Thr Glu Glu Ile Ala Val Gln Asn 3125 3130 3135 Trp Leu Ala Arg Val Gly Arg Glu Arg Leu Ser Arg Met Ala Ile Ser 3140 3145 3150 Gly Asp Asp Cys Val Val Lys Pro Leu Asp Asp Arg Phe Ala Ser Ala 3155 3160 3165 Leu Thr Ala Leu Asn Asp Met Gly Lys Ile Arg Lys Asp Ile Gln Gln 3170 3175 3180 Trp Glu Pro Ser Arg Gly Trp Asn Asp Trp Thr Gln Val Pro Phe Cys 3185 3190 3195 3200 Ser His His Phe His Glu Leu Ile Met Lys Asp Gly Arg Val Leu Val 3205 3210 3215 Val Pro Cys Arg Asn Gln Asp Glu Leu Ile Gly Arg Ala Arg Ile Ser 3220 3225 3230 Gln Gly Ala Gly Trp Ser Leu Arg Glu Thr Ala Cys Leu Gly Lys Ser 3235 3240 3245 Tyr Ala Gln Met Trp Ser Leu Met Tyr Phe His Arg Arg Asp Leu Arg 3250 3255 3260 Leu Ala Ala Asn Ala Ile Cys Ser Ala Val Pro Ser His Trp Val Pro 3265 3270 3275 3280 Thr Ser Arg Thr Thr Trp Ser Ile His Ala Lys His Glu Trp Met Thr 3285 3290 3295 Thr Glu Asp Met Leu Thr Val Trp Asn Arg Val Trp Ile Gln Glu Asn 3300 3305 3310 Pro Trp Met Glu Asp Lys Thr Pro Val Glu Ser Trp Glu Glu Ile Pro 3315 3320 3325 Tyr Leu Gly Lys Arg Glu Asp Gln Trp Cys Gly Ser Leu Ile Gly Leu 3330 3335 3340 Thr Ser Arg Ala Thr Trp Ala Lys Asn Ile Gln Ala Ala Ile Asn Gln 3345 3350 3355 3360 Val Arg Ser Leu Ile Gly Asn Glu Glu Tyr Thr Asp Tyr Met Pro Ser 3365 3370 3375Met Lys Arg Phe Arg Arg Glu Glu Glu Glu Ala Gly Val Leu Trp 3380 3385 3390 <210> 9 <211> 10735 <212> DNA <213> dengue virus <400> 9 agttgttagt ctacgtggac cgacaagaac agtttcgaat cggaagcttg cttaacgtag 60 ttctaacagt tttttattag agagcagatc tctgatgatc aaccaacgaa aaaagacggg 120 tcgaccgtct ttcaatatgc tgaaacgcgc gagaaaccgc gtgtcaactg tttcacagtt 180 ggcgaagaga ttctcaaaag gattgctctc aggccaagga cccatgaaat tggtgatggc 240 tttcatagca ttcttaagat ttctagccat acccccaaca gcaggaattt tggctagatg 300 gggctcattc aagaagaatg gagcgattaa agtgttacgg ggtttcaaga gagaaatctc 360 aaacatgcta aacataatga acaggaggaa aagatccgtg accatgctcc ttatgctgct 420 gcccacagcc ctggcgttcc atctgacgac acgaggggga gagccgcata tgatagttag 480 caagcaggaa agaggaaagt cacttttgtt caagacctct gcaggtgtca acatgtgcac 540 cctcattgcg atggatttgg gagagttgtg tgaggacacg atgacctaca aatgcccccg 600 gatcactgag gcggaaccag atgacgttga ctgttggtgc aatgccacgg acacatgggt 660 gacctatgga acgtgctctc aaactggcga acaccgacga gacaaacgtt ccgtcgcatt 720 ggccccacac gtggggcttg gcctagaaac aagagccgaa acgtggatgt cctctgaagg 780 tgcttggaaa cagatacaaa aagtagagac ttgggctctg agacatccag gattcacggt 840 gatagccctt tttctagcac atgccatagg aacatccatc acccagaaag ggatcatttt 900 cattttgctg atgctggtaa caccatctat ggccatgcga tgcgtgggaa taggcaacag 960 agacttcgtg gaaggactgt caggagcaac atgggtggat gtggtactgg agcatggaag 1020 ttgcgtcacc accatggcaa aaaacaaacc aacactggac attgaactct tgaagacgga 1080 ggtcacaaac cctgcagttc tgcgtaaatt gtgcattgaa gctaaaatat caaacaccac 1140 caccgattcg agatgtccaa cacaaggaga agccacactg gtggaagaac aagacgcgaa 1200 ctttgtgtgc cgacgaacgt tcgtggacag aggctggggc aatggctgtg ggctattcgg 1260 aaaaggtagt ctaataacgt gtgccaagtt taagtgtgtg acaaaactag aaggaaagat 1320 agttcaatat gaaaacctaa aatattcagt gatagtcacc gtccacactg gagatcagca 1380 ccaggtggga aatgagacta cagaacatgg aacaactgca accataacac ctcaagctcc 1440 tacgtcggaa atacagctga ccgactacgg aacccttaca ttagattgtt cacctaggac 1500 agggctagat tttaacgaga tggtgttgct gacaatgaaa gaaagatcat ggcttgtcca 1560 caaacaatgg tttctagact taccactgcc ttggacctct ggggcttcaa catcccaaga 1620 gacttggaac agacaagatt tactggtcac atttaagaca gctcatgcaa agaagcagga 1680 agtagtcgta ctaggatcac aagaaggagc aatgcacact gcgctgactg gagcgacaga 1740 aatccaaacg tcaggaacga caacaatttt cgcaggacac ctaaaatgca gactaaaaat 1800 ggacaaacta actttaaaag ggatgtcata tgtgatgtgc acaggctcat tcaagttaga 1860 gaaagaagtg gctgagaccc agcatggaac tgttctggtg caggttaaat atgaaggaac 1920 agacgcacca tgcaagattc ccttttcgac ccaagatgag aaaggagcaa cccagaatgg 1980 gagattaata acagccaacc ccatagtcac tgacaaagaa aaaccagtca atattgaggc 2040 agaaccaccc tttggtgaga gctacatcgt ggtaggagca ggtgaaaaag ctttgaaact 2100 aagctggttc aagaaaggaa gcagcatagg gaaaatgttt gaagcaactg cccgaggagc 2160 acgaaggatg gccattctgg gagacaccgc atgggacttc ggttctatag gaggagtgtt 2220 cacgtctatg ggaaaactgg tacaccaggt ttttggaact gcatatggag ttttgtttag 2280 cggagtttct tggaccatga aaataggaat agggattctg ctgacatggc taggattaaa 2340 ttcaaggaac acgtcccttt cgatgatgtg catcgcagtt ggcatggtca cactgtacct 2400 aggagtcatg gttcaggcag attcgggatg tgtaatcaac tggaaaggca gagaacttaa 2460 atgtggaagc ggcatttttg tcactaatga agttcacact tggacagagc aatacaaatt 2520 ccaggctgac tcccccaaga gactatcagc agccattggg aaggcatggg aggagggtgt 2580 gtgtggaatc cgatcagcca ctcgtctcga gaacatcatg tggaaacaaa tatcaaatga 2640 attgaaccac atcctacttg aaaatgacat gaaatttaca gtggtcgtgg gagatgttag 2700 tggaatcttg gcccaaggga aaaaaatgat taggccacaa cccatggaac acaaatactc 2760 gtggaaaagc tggggaaaag ccaaaatcat aggagcggat gtacagaaca ccaccttcat 2820 catcgacggc ccaaacaccc cagaatgccc tgacaatcaa agagcatgga atatttggga 2880 agtagaggac tatggatttg ggattttcac gacaaacata tggttgaaat tgcgtgactc 2940 ctacacccaa gtatgtgacc accggctgat gtcagctgcc attaaggaca gcaaggcagt 3000 ccatgctgac atggggtact ggatagaaag tgaaaagaac gagacatgga agttggcgag 3060 agcctccttt atagaagtta agacatgcat ctggccaaaa tcccacactc tatggagcaa 3120 tggagttctg gaaagtgaaa tgataattcc aaagatatat ggaggaccaa tatctcagca 3180 caactacaga ccaggatatt tcacacaaac agcagggccg tggcacctag gcaagttgga 3240 actagatttc gatttttgtg aaggtaccac agttgttgtg gatgaacatt gtggaaatcg 3300 aggaccatct ctcagaacca caacagtcac aggaaagata atccatgaat ggtgctgcag 3360 atcttgtacg ctaccccccc tacgtttcaa aggggaagac gggtgttggt acggcatgga 3420 aatcagacca gtgaaggaca aggaagagaa cctggtcaag tcaatggtct ctgcagggtc 3480 aggagaagtg gacagctttt cactaggact gctatgcata tcaataatga ttgaagaagt 3540 gatgagatcc agatggagca aaaaaatgct gatgactgga acactggctg tgttcctcct 3600 tcttataatg ggacaattga catggagtga tctgatcagg ttatgtatta tggttggagc 3660 caacgcttca gacaagatgg ggatgggaac aacgtaccta gctttaatgg ccactttcaa 3720 aatgagacca atgttcgccg tcgggctatt atttcgcaga ctaacatcta gagaagttct 3780 tcttcttaca attggcttga gcctggtggc atccgtggag ctaccaagtt ccctagagga 3840 gctgggggat ggacttgcaa taggcatcat gatgttgaaa ttattgactg attttcagtc 3900 acaccagcta tgggctactc tgctatcctt gacatttatt aaaacaactt tttcattgca 3960 ctatgcatgg aagacaatgg ctatggtact gtcaattgta tctctcttcc ctttatgcct 4020 gtccacgacc tctcaaaaaa caacatggct tccggtgctg ttgggatctc ttggatgcaa 4080 accactaccc atgtttctta taacagaaaa caaaatctgg ggaaggaaga gttggcccct 4140 caatgaagga attatggctg ttggaatagt tagtattcta ctaagttcac ttttaaaaaa 4200 tgatgtgccg ctagccggcc cattaatagc tggaggcatg ctaatagcat gttatgtcat 4260 atccggaagc tcagctgatt tatcactgga gaaagcggct gaggtctcct gggaggaaga 4320 agcagaacac tcaggcgcct cacacaacat actagtagag gttcaagatg atggaaccat 4380 gaagataaaa gatgaagaga gagatgacac gctcaccatt ctccttaaag caactctgct 4440 ggcagtctca ggggtgtacc caatgtcaat accagcgacc ctttttgtgt ggtatttttg 4500 gcagaaaaag aaacagagat caggagtgct atgggacaca cccagccccc cagaagtgga 4560 aagagcagtt cttgatgatg gcatctatag aattttgcaa agaggactgt tgggcaggtc 4620 ccaagtagga gtaggagttt tccaagaagg cgtgttccac acaatgtggc acgtcactag 4680 gggagctgtc ctcatgtatc aaggaaaaag gctggaacca agctgggcca gtgtcaaaaa 4740 agacttgatc tcatatggag gaggttggag gtttcaagga tcctggaaca cgggagaaga 4800 agtacaggtg attgctgttg aaccgggaaa aaaccccaaa aatgtacaaa caacgccggg 4860 taccttcaag acccctgaag gcgaagttgg agccatagcc ttagacttta aacctggcac 4920 atctggatct cccatcgtaa acagagaggg aaaaatagta ggtctttatg gaaatggagt 4980 ggtgacaaca agcggaactt acgttagtgc catagctcaa gctaaggcat cacaagaagg 5040 gcctctacca gagattgagg acgaggtgtt taggaaaaga aacttaacaa taatggacct 5100 acatccagga tcgggaaaaa caagaagata ccttccagcc atagtccgtg aggccataaa 5160 aaggaagctg cgcacgctaa tcctagctcc cacaagagtt gtcgcttctg aaatggcaga 5220 ggcactcaag ggagtgccaa taaggtatca gacaacagca gtgaagagtg aacacacagg 5280 aaaggagata gttgacctta tgtgccacgc cactttcacc atgcgcctcc tgtctcccgt 5340 gagagttccc aattataaca tgattatcat ggatgaagca cacttcaccg atccagccag 5400 catagcagcc agagggtaca tctcaacccg agtgggtatg ggtgaagcag ctgcgatctt 5460 tatgacagcc actcccccag gatcggtgga ggcctttcca cagagcaatg caattatcca 5520 agatgaggaa agagacattc ctgagagatc atggaactca ggctatgact ggatcactga 5580 ttttccaggt aaaacagtct ggtttgttcc aagcatcaaa tcaggaaatg acattgccaa 5640 ctgtttaaga aaaaacggga aacgggtgat ccaattgagc agaaaaacct ttgacactga 5700 gtaccagaaa acaaaaaaca acgactggga ctatgtcgtc acaacagaca tttccgaaat 5760 gggagcaaat ttccgggccg acagggtaat agacccaagg cggtgtctga aaccggtaat 5820 actaaaagat ggtccagagc gcgtcattct agccggaccg atgccagtga ctgtggccag 5880 tgccgcccag aggagaggaa gaattggaag gaaccaaaac aaggaaggtg atcagtatat 5940 ttacatggga cagcctttaa acaatgatga ggaccacgct cattggacag aagcaaagat 6000 gctccttgac aatataaaca caccagaagg gattatccca gccctctatg agccggagag 6060 agaaaagagt gcagctatag acggggaata cagactgcgg ggtgaagcaa ggaaaacgtt 6120 cgtggagctc atgagaagag gggatctacc agtctggcta tcctacaaag ttgcctcaga 6180 aggcttccag tactccgaca gaaggtggtg cttcgatggg gaaaggaaca accaggtgtt 6240 ggaggagaac atggacgtgg agatctggac aaaagaagga gaaagaaaga aactacgacc 6300 tcgctggttg gacgccagaa catactctga cccactggct ctgcgcgagt ttaaagagtt 6360 tgcagcagga agaagaagcg tctcaggtga cctaatatta gaaataggga aacttccaca 6420 acattgacg caaagggccc agaatgcttt ggacaacttg gtcatgttgc acaattccga 6480 acaaggagga aaagcctata gacatgctat ggaagaactg ccagacacaa tagaaacgtt 6540 gatgctccta gccttgatag ctgtgttgac tggtggagtg acgctgttct tcctatcagg 6600 aagaggtcta ggaaaaacat ctatcggctt actctgcgtg atggcctcaa gcgcactgtt 6660 atggatggcc agtgtggagc cccattggat agcggcctcc atcatactgg agttctttct 6720 gatggtactg cttattccag agccagacag acagcgcact ccacaggaca accagctagc 6780 atatgtggtg ataggtctgt tattcatgat attgacagtg gcagccaatg agatgggatt 6840 attggaaacc acaaagaaag acctggggat tggccatgta gctgctgaaa accaccacca 6900 tgctacaatg ctggacgtag acctacatcc agcttcagcc tggaccctct atgcagtggc 6960 cacaacaatc atcactccta tgatgagaca cacaattgaa aacacaacgg caaatatttc 7020 cctgacagcc atcgcaaacc aagcagctat attgatggga cttgacaagg gatggccaat 7080 atcgaagatg gacataggag ttccacttct cgccttgggg tgctattccc aagtgaatcc 7140 gctgacactg atagcggcag tattgatgct agtagctcat tacgccataa ttggacctgg 7200 actgcaagca aaagctacta gagaagctca aaaaagaaca gcggctggaa taatgaaaaa 7260 tccaactgtc gacgggattg ttgcaataga cttagatccc gtggtttacg atgcaaaatt 7320 tgaaaaacaa ctaggccaaa taatgttgtt gatactttgc acatcacaga ttcttttgat 7380 gcggactaca tgggccttgt gtgaatccat cacattggct actggacctc tgaccactct 7440 ttgggaggga tctccaggaa aattctggaa caccacaata gcggtatcca tggcaaacat 7500 tttcaggggg agttatctag caggagcagg tctggccttc tcattaatga aatctctagg 7560 aggaggtagg agaggcacgg gagcccaagg ggaaacactg ggagaaaaat ggaaaagaca 7620 actaaaccaa ctgagcaagt cagaattcaa tacttacaag aggagtggga ttatggaggt 7680 ggatagatcc gaagccaaag agggactgaa aagaggagaa acaaccaaac acgcagtatc 7740 gagaggaacg gccaaactga ggtggttcgt ggagaggaac cttgtgaaac cagaagggaa 7800 agtcatagac ctcggttgtg gaagaggtgg ctggtcatat tattgcgctg ggctgaagaa 7860 agtcacagaa gtgaaaggat acacaaaagg aggacctgga catgaggaac caatcccaat 7920 ggcgacctat ggatggaacc tagtaaagct gcactccgga aaagatgtat tttttatacc 7980 acctgagaaa tgtgacaccc ttttgtgtga tattggtgag tcctctccga acccaactat 8040 agaggaagga agaacgttac gtgttctgaa aatggtggaa ccatggctca gaggaaacca 8100 attttgcata aaaattctaa atccctatat gccgagcgtg gtagaaactc tggaacaaat 8160 gcaaagaaaa catggaggaa tgctagtgcg aaacccactc tcaagaaatt ccacccatga 8220 aatgtactgg gtttcatgtg gaacaggaaa cattgtgtca gcagtaaaca tgacatctag 8280 aatgttgcta aatcggttca caatggctca caggaagcca acatatgaaa gagacgtgga 8340 cttaggcgct ggaacaagac atgtggcagt agaaccagag gtagccaacc tagatatcat 8400 tggccagagg atagagaata taaaaaatga acataagtca acatggcatt atgatgagga 8460 caatccatac aaaacatggg cctatcatgg atcatatgag gttaagccat caggatcggc 8520 ctcatccatg gtcaatggcg tggtgagatt gctcaccaaa ccatgggatg ttatccccat 8580 ggtcacacaa atagccatga ctgataccac accctttgga caacagaggg tgtttaaaga 8640 gaaagttgac acgcgcacac caaaagcaaa acgtggcaca gcacaaatta tggaagtgac 8700 agccaggtgg ttatggggtt tcctttctag aaacaaaaaa cccagaattt gcacaagaga 8760 ggagtttaca agaaaagtta ggtcaaacgc agctattgga gcagtgttcg ttgatgaaaa 8820 tcaatggaac tcggcaaaag aagcagtgga agacgaacgg ttctgggaac ttgtccacag 8880 agagagggag cttcataaac aggggaaatg tgccacgtgt gtctacaata tgatggggaa 8940 gagagagaaa aaattaggag agttcggaaa ggcaaaagga agtcgtgcaa tatggtacat 9000 gtggttggga gcacgcttcc tagagtttga agcccttggt ttcatgaatg aagatcactg 9060 gttcagtaga gagaattcac tcagtggagt ggaaggagaa ggactccaca aacttggata 9120 catactcaga gacatatcaa ggattccagg ggggaacatg tatgcagatg acacagccgg 9180 atgggacaca agaataacag aggatgatct ccagaatgag gctaaaatca ctgacatcat 9240 ggagcccgaa catgccctgc tggctacgtc aatctttaag ctgacctacc aaaataaggt 9300 ggtaagggtg cagagaccag caaaaaatgg aaccgtgatg gatgttatat ccagacgtga 9360 ccagagaggc agtggacagg ttggaactta tggcttaaac actttcacca acatggaggc 9420 ccaactgata agacaaatgg agtccgaggg aatcttttta cccagcgaat tggaaacccc 9480 aaatctagcc ggaagagttc tcgactggtt ggaaaaatat ggtgtcgaaa ggctgaaaag 9540 aatggcaatc agcggagatg actgtgtggt gaaaccaatt gatgacaggt tcgcaacagc 9600 cttaacagct ttgaatgaca tgggaaaagt aagaaaagac ataccacaat gggaaccttc 9660 aaaaggatgg aatgattggc aacaagtgcc tttctgttca caccacttcc accagctaat 9720 tatgaaggat gggagggaga tagtggtgcc atgccgcaac caagatgaac ttgtggggag 9780 ggccagagta tcacaaggcg ccggatggag cctgagagaa accgcatgcc taggcaagtc 9840 atatgcacaa atgtggcagc tgatgtattt ccacaggaga gacctgagac tggcggctaa 9900 cgctatttgt tcagccgttc cagttgattg ggtcccaacc agccgcacca cctggtcgat 9960 ccatgcccat caccaatgga tgacaacaga agacatgtta tcagtatgga atagggtctg 10020 gatagaggaa aacccatgga tggaggataa gactcatgtg tccagttggg aagaagttcc 10080 atacctagga aagagggaag atcagtggtg tggatccctg ataggcttaa cagcaagggc 10140 cacctgggcc actaatatac aagtggccat aaaccaagtg agaaggctca ttgggaatga 10200 gaattatcta gattacatga catcaatgaa gagattcaag aatgagagtg atcccgaagg 10260 ggcactctgg taagtcaaca cattcacaaa ataaaggaaa ataaaaaatc aaatgaggca 10320 agaagtcagg ccagattaag ccatagtacg gtaagagcta tgctgcctgt gagccccgtc 10380 caaggacgta aaatgaagtc aggccgaaag ccacggtttg agcaagccgt gctgcctgtg 10440 gctccatcgt ggggatgtaa aaacccggga ggctgcaacc catggaagct gtacgcatgg 10500 ggtagcagac tagtggttag aggagacccc tcccaagaca caacgcagca gcggggccca 10560 acaccagggg aagctgtacc ctggtggtaa ggactagagg ttagaggaga ccccccgcgt 10620 aacaataaac agcatattga cgctgggaga gaccagagat cctgctgtct ctacagcatc 10680 attccaggca cagaacgcca gaaaatggaa tggtgctgtt gaatcaacag gttct 10735 <210> 10 <211> 3392 <212> PRT <213> dengue virus <400> 10 Met Ile Asn Gln Arg Lys Lys Thr Gly Arg Pro Ser Phe Asn Met Leu 1 5 10 15 Lys Arg Ala Arg Asn Arg Val Ser Thr Val Ser Gln Leu Ala Lys Arg 20 25 30 Phe Ser Lys Gly Leu Leu Ser Gly Gln Gly Pro Met Lys Leu Val Met 35 40 45 Ala Phe Ile Ala Phe Leu Arg Phe Leu Ala Ile Pro Pro Thr Ala Gly 50 55 60 Ile Leu Ala Arg Trp Gly Ser Phe Lys Lys Asn Gly Ala Ile Lys Val 65 70 75 80 Leu Arg Gly Phe Lys Arg Glu Ile Ser Asn Met Leu Asn Ile Met Asn 85 90 95 Arg Arg Lys Arg Ser Val Thr Met Leu Leu Met Leu Leu Pro Thr Ala 100 105 110 Leu Ala Phe His Leu Thr Thr Arg Gly Gly Glu Pro His Met Ile Val 115 120 125 Ser Lys Gin Glu Arg Gly Lys Ser Leu Leu Phe Lys Thr Ser Ala Gly 130 135 140 Val Asn Met Cys Thr Leu Ile Ala Met Asp Leu Gly Glu Leu Cys Glu 145 150 155 160 Asp Thr Met Thr Tyr Lys Cys Pro Arg Ile Thr Glu Ala Glu Pro Asp 165 170 175 Asp Val Asp Cys Trp Cys Asn Ala Thr Asp Thr Trp Val Thr Tyr Gly 180 185 190 Thr Cys Ser Gln Thr Gly Glu His Arg Arg Asp Lys Arg Ser Val Ala 195 200 205 Leu Ala Pro His Val Gly Leu Gly Leu Glu Thr Arg Ala Glu Thr Trp 210 215 220 Met Ser Ser Glu Gly Ala Trp Lys Gln Ile Gln Lys Val Glu Thr Trp 225 230 235 240 Ala Leu Arg His Pro Gly Phe Thr Val Ile Ala Leu Phe Leu Ala His 245 250 255 Ala Ile Gly Thr Ser Ile Thr Gln Lys Gly Ile Ile Phe Ile Leu Leu 260 265 270 Met Leu Val Thr Pro Ser Met Ala Met Arg Cys Val Gly Ile Gly Asn 275 280 285 Arg Asp Phe Val Glu Gly Leu Ser Gly Ala Thr Trp Val Asp Val Val 290 295 300 Leu Glu His Gly Ser Cys Val Thr Thr Met Ala Lys Asn Lys Pro Thr 305 310 315 320 Leu Asp Ile Glu Leu Leu Lys Thr Glu Val Thr Asn Pro Ala Val Leu 325 330 335 Arg Lys Leu Cys Ile Glu Ala Lys Ile Ser Asn Thr Thr Thr Asp Ser 340 345 350 Arg Cys Pro Thr Gln Gly Glu Ala Thr Leu Val Glu Glu Gln Asp Ala 355 360 365 Asn Phe Val Cys Arg Arg Thr Phe Val Asp Arg Gly Trp Gly Asn Gly 370 375 380 Cys Gly Leu Phe Gly Lys Gly Ser Leu Ile Thr Cys Ala Lys Phe Lys 385 390 395 400 Cys Val Thr Lys Leu Glu Gly Lys Ile Val Gln Tyr Glu Asn Leu Lys 405 410 415 Tyr Ser Val Ile Val Thr Val His Thr Gly Asp Gln His Gln Val Gly 420 425 430 Asn Glu Thr Thr Glu His Gly Thr Thr Ala Thr Ile Thr Pro Gln Ala 435 440 445 Pro Thr Ser Glu Ile Gln Leu Thr Asp Tyr Gly Thr Leu Thr Leu Asp 450 455 460 Cys Ser Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Val Leu Leu Thr 465 470 475 480 Met Lys Glu Arg Ser Trp Leu Val His Lys Gln Trp Phe Leu Asp Leu 485 490 495 Pro Leu Pro Trp Thr Ser Gly Ala Ser Thr Ser Gln Glu Thr Trp Asn 500 505 510 Arg Gln Asp Leu Leu Val Thr Phe Lys Thr Ala His Ala Lys Lys Gln 515 520 525 Glu Val Val Val Leu Gly Ser Gln Glu Gly Ala Met His Thr Ala Leu 530 535 540 Thr Gly Ala Thr Glu Ile Gln Thr Ser Gly Thr Thr Thr Ile Phe Ala 545 550 555 560 Gly His Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Thr Leu Lys Gly 565 570 575 Met Ser Tyr Val Met Cys Thr Gly Ser Phe Lys Leu Glu Lys Glu Val 580 585 590 Ala Glu Thr Gln His Gly Thr Val Leu Val Gln Val Lys Tyr Glu Gly 595 600 605 Thr Asp Ala Pro Cys Lys Ile Pro Phe Ser Thr Gln Asp Glu Lys Gly 610 615 620 Ala Thr Gln Asn Gly Arg Leu Ile Thr Ala Asn Pro Ile Val Thr Asp 625 630 635 640 Lys Glu Lys Pro Val Asn Ile Glu Ala Glu Pro Phe Gly Glu Ser 645 650 655 Tyr Ile Val Val Gly Ala Gly Glu Lys Ala Leu Lys Leu Ser Trp Phe 660 665 670 Lys Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ala Thr Ala Arg Gly 675 680 685 Ala Arg Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser 690 695 700 Ile Gly Gly Val Phe Thr Ser Met Gly Lys Leu Val His Gln Val Phe 705 710 715 720 Gly Thr Ala Tyr Gly Val Leu Phe Ser Gly Val Ser Trp Thr Met Lys 725 730 735 Ile Gly Ile Gly Ile Ile Leu Leu Thr Trp Leu Gly Leu Asn Ser Arg Asn 740 745 750 Thr Ser Leu Ser Met Met Cys Ile Ala Val Gly Met Val Thr Leu Tyr 755 760 765 Leu Gly Val Met Val Gln Ala Asp Ser Gly Cys Val Ile Asn Trp Lys 770 775 780 Gly Arg Glu Leu Lys Cys Gly Ser Gly Ile Phe Val Thr Asn Glu Val 785 790 795 800 His Thr Trp Thr Glu Gln Tyr Lys Phe Gln Ala Asp Ser Pro Lys Arg 805 810 815 Leu Ser Ala Ala Ile Gly Lys Ala Trp Glu Glu Gly Val Cys Gly Ile 820 825 830 Arg Ser Ala Thr Arg Leu Glu Asn Ile Met Trp Lys Gln Ile Ser Asn 835 840 845 Glu Leu Asn His Ile Leu Leu Glu Asn Asp Met Lys Phe Thr Val Val 850 855 860 Val Gly Asp Val Ser Gly Ile Leu Ala Gln Gly Lys Lys Met Ile Arg 865 870 875 880 Pro Gln Pro Met Glu His Lys Tyr Ser Trp Lys Ser Trp Gly Lys Ala 885 890 895 Lys Ile Ile Gly Ala Asp Val Gln Asn Thr Thr Phe Ile Ile Asp Gly 900 905 910 Pro Asn Thr Pro Glu Cys Pro Asp Asn Gln Arg Ala Trp Asn Ile Trp 915 920 925 Glu Val Glu Asp Tyr Gly Phe Gly Ile Phe Thr Thr Asn Ile Trp Leu 930 935 940 Lys Leu Arg Asp Ser Tyr Thr Gln Val Cys Asp His Arg Leu Met Ser 945 950 955 960 Ala Ala Ile Lys Asp Ser Lys Ala Val His Ala Asp Met Gly Tyr Trp 965 970 975 Ile Glu Ser Glu Lys Asn Glu Thr Trp Lys Leu Ala Arg Ala Ser Phe 980 985 990 Ile Glu Val Lys Thr Cys Ile Trp Pro Lys Ser His Thr Leu Trp Ser 995 1000 1005 Asn Gly Val Leu Glu Ser Glu Met Ile Ile Pro Lys Ile Tyr Gly Gly 1010 1015 1020 Pro Ile Ser Gln His Asn Tyr Arg Pro Gly Tyr Phe Thr Gln Thr Ala 1025 1030 1035 1040 Gly Pro Trp His Leu Gly Ly s Leu Glu Leu Asp Phe Asp Phe Cys Glu 1045 1050 1055 Gly Thr Thr Val Val Val Asp Glu His Cys Gly Asn Arg Gly Pro Ser 1060 1065 1070 Leu Arg Thr Thr Thr Val Thr Gly Lys Ile Ile His Glu Trp Cys Cys 1075 1080 1085 Arg Ser Cys Thr Leu Pro Leu Arg Phe Lys Gly Glu Asp Gly Cys 1090 1095 1100 Trp Tyr Gly Met Glu Ile Arg Pro Val Lys Asp Lys Glu Glu Asn Leu 1105 1110 1115 1120 Val Lys Ser Met Val Ser Ala Gly Ser Gly Glu Val Asp Ser Phe Ser 1125 1130 1135 Leu Gly Leu Leu Cys Ile Ser Ile Met Ile Glu Glu Val Met Arg Ser 1140 1145 1150 Arg Trp Ser Lys Lys Met Leu Met Thr Gly Thr Leu Ala Val Phe Leu 1155 1160 1165 Leu Leu Ile Met Gly Gly Thr Thr 1185 1190 1195 1200 Tyr Leu Ala Leu Met Ala Thr Phe Lys Met Arg Pro Met Phe Ala Val 1205 1210 1215 Gly Leu Leu Phe Arg Arg Leu Thr Ser Arg Glu Val Leu Leu Leu Thr 1220 1225 1230 Ile Gly Leu Ser Leu Val Ala Ser Val Glu Leu Pro Ser Ser Leu Glu 1235 1240 1245 Glu Leu Gly Asp Gly Leu Ala Ile Gly Ile Met Met Leu Lys Leu Leu 1250 1255 1260 Thr Asp Phe Gln Ser His Gln Leu Trp Ala Thr Leu Leu Ser Leu Thr 1265 1270 1275 1280 Phe Ile Lys Thr Thr Phe Ser Leu His Tyr Ala Trp Lys Thr Met Ala 1285 1290 1295 Met Val Leu Ser Ile Val Ser Leu Phe Pro Leu Cys Leu Ser Thr Thr 1300 1305 1310 Ser Gln Lys Thr Thr Trp Leu Pro Val Leu Leu Gly Ser Leu Gly Cys 1315 1320 1325 Lys Pro Leu Pro Met Phe Leu Ile Thr Glu Asn Lys Ile Trp Gly Arg 1330 1335 1340 Lys Ser Trp Pro Leu Asn Glu Gly Ile Met Ala Val Gly Ile Val Ser 1345 1350 1355 1360 Ile Leu Leu Ser Ser Leu Leu Lys Asn Asp Val Pro Leu Ala Gly Pro 1365 1370 1375 Leu Ile Ala Gly Gly Met Leu Ile Ala Cys Tyr Val Ile Ser Gly Ser 1380 1385 1390 Ser Ala Asp Leu Ser Leu Glu Lys Ala Ala Glu Val Ser Trp Glu Glu 1395 1400 1405 Glu Ala Glu His Ser Gly Ala Ser His Asn Ile Leu Val Glu Val Gln 1410 1415 1420 Asp Asp Gly Thr Met Lys Ile Lys Asp Glu Glu Arg Asp Asp Thr Leu 1425 1430 1435 1440 Thr Ile Leu Leu Lys Ala Th r Leu Leu Ala Val Ser Gly Val Tyr Pro 1445 1450 1455 Met Ser Ile Pro Ala Thr Leu Phe Val Trp Tyr Phe Trp Gln Lys Lys 1460 1465 1470 Lys Gln Arg Ser Gly Val Leu Trp Asp Thr Pro Ser Pro Glu Val 1475 1480 1485 Glu Arg Ala Val Leu Asp Asp Gly Ile Tyr Arg Ile Leu Gln Arg Gly 1490 1495 1500 Leu Leu Gly Arg Ser Gln Val Gly Val Gly Val Phe Gln Glu Gly Val 1505 1510 1515 1520 Phe His Thr Met Trp His Val Thr Arg Gly Ala Val Leu Met Tyr Gln 1525 1530 1535 Gly Lys Arg Leu Glu Pro Ser Trp Ala Ser Val Lys Lys Asp Leu Ile 1540 1545 1550 Ser Tyr Gly Gly Gly Trp Arg Phe Gln Gly Ser Trp Asn Thr Gly Glu 1555 1560 1565 Glu Val Gln Val Ile Ala Val Glu Pro Gly Lys Asn Pro Lys Asn Val 1570 1575 1580 Gln Thr Thr Pro Gly Thr Phe Lys Thr Pro Glu Gly Glu Val Gly Ala 1585 1590 1595 1600 Ile Ala Leu Asp Phe Lys Pro Gly Thr Ser Gly Ser Pro Ile Val Asn 1605 1610 1615 Arg Glu Gly Lys Ile Val Gly Leu Tyr Gly Asn Gly Val Val Thr Thr 1620 1625 1630 Ser Gly Thr Tyr Val Ser Ala Ile Ala Gln Ala Lys Ala Ser Gln Glu 1635 1640 1645 Gly Pro Leu Pro Glu Ile Glu Asp Glu Val Phe Arg Lys Arg Asn Leu 1650 1655 1660 Thr Ile Met Asp Leu His Pro Gly Ser Gly Lys Thr Arg Arg Tyr Leu 1665 1670 1675 1680 Pro Ala Ile Val Arg Glu Ala Ile Lys Arg Lys Leu Arg Thr Leu Ile 1685 1690 1695 Leu Ala Pro Thr Arg Val Val Ala Ser Glu Met Ala Glu Ala Leu Lys 1700 1705 1710 Gly Val Pro Ile Arg Tyr Gln Thr Thr Ala Val Lys Ser Glu His Thr 1715 1720 1725 Gly Lys Glu Ile Val Asp Leu Met Cys His Ala Thr Phe Thr Met Arg 1730 1735 1740 Leu Leu Ser Pro Val Arg Val Pro Asn Tyr Asn Met Ile Ile Met Asp 1745 1750 1755 1760 Glu Ala His Phe Thr Asp Pro Ala Ser Ile Ala Ala Arg Gly Tyr Ile 1765 1770 1775 Ser Thr Arg Val Gly Met Gly Glu Ala Ala Ala Ile Phe Met Thr Ala 1780 1785 1790 Thr Pro Pro Gly Ser Val Glu Ala Phe Pro Gln Ser Asn Ala Ile Ile 1795 1800 1805 Gln Asp Glu Glu Arg Asp Ile Pro Glu Arg Ser Trp Asn Ser Gly Tyr 1810 1815 1820 Asp Trp Ile Thr Asp Phe Pro Gly Lys Thr Val Trp Phe Val Pro Ser 1825 1830 1835 1840 Ile Lys Ser Gly Asn Asp Il e Ala Asn Cys Leu Arg Lys Asn Gly Lys 1845 1850 1855 Arg Val Ile Gln Leu Ser Arg Lys Thr Phe Asp Thr Glu Tyr Gln Lys 1860 1865 1870 Thr Lys Asn Asn Asp Trp Asp Tyr Val Val Thr Thr Asp Ile Ser Glu 1875 1880 1885 Met Gly Ala Asn Phe Arg Ala Asp Arg Val Ile Asp Pro Arg Arg Cys 1890 1895 1900 Leu Lys Pro Val Ile Leu Lys Asp Gly Pro Glu Arg Val Ile Leu Ala 1905 1910 1915 1920 Gly Pro Met Pro Val Thr Val Ala Ser Ala Ala Gln Arg Arg Gly Arg 1925 1930 1935 Ile Gly Arg Asn Gln Asn Lys Glu Gly Asp Gln Tyr Ile Tyr Met Gly 1940 1945 1950 Gln Pro Leu Asn Asn Asp Glu Asp His Ala His Trp Thr Glu Ala Lys 1955 1960 1965 Met Leu Leu Asp Asn Ile Asn Thr Pro Glu Gly Ile Ile Pro Ala Leu 1970 1975 1980 Tyr Glu Pro Glu Arg Glu Lys Ser Ala Ala Ile Asp Gly Glu Tyr Arg 1985 1990 1995 2000 Leu Arg Gly Glu Ala Arg Lys Thr Phe Val Glu Leu Met Arg Arg Gly 2005 2010 2015 Asp Leu Pro Val Trp Leu Ser Tyr Lys Val Ala Ser Glu Gly Phe Gln 2020 2025 2030 Tyr Ser Asp Arg Arg Trp Cys Phe Asp Gly Glu Arg Asn Asn Gln Val 2035 2040 2045 Leu Glu Glu Asn Met Asp Val Glu Ile Trp Thr Lys Glu Gly Glu Arg 2050 2055 2060 Lys Lys Leu Arg Pro Arg Trp Leu Asp Ala Arg Thr Tyr Ser Asp Pro 2065 2070 2075 2080 Leu Ala Leu Arg Glu Phe Lys Glu Phe Ala Ala Gly Arg Arg Ser Val 2085 2090 2095 Ser Gly Asp Leu Ile Leu Glu Ile Gly Lys Leu Pro Gln His Leu Thr 2100 2105 2110 Gln Arg Ala Gln Asn Ala Leu Asp Asn Leu Val Met Leu His Asn Ser 2115 2120 2125 Glu Gln Gly Gly Lys Ala Tyr Arg His Ala Met Glu Glu Leu Pro Asp 2130 2135 2140 Thr Ile Glu Thr Leu Met Leu Leu Ala Leu Ile Ala Val Leu Thr Gly 2145 2150 2155 2160 Gly Val Thr Leu Phe Phe Leu Ser Gly Arg Gly Leu Gly Lys Thr Ser 2165 2170 2175 Ile Gly Leu Leu Cys Val Met Ala Ser Ser Ala Leu Leu Trp Met Ala 2180 2185 2190 Ser Val Glu Pro His Trp Ile Ala Ala Ser Ile Ile Leu Glu Phe Phe 2195 2200 2205 Leu Met Val Leu Leu Ile Pro Glu Pro Asp Arg Gln Arg Thr Pro Gln 2210 2215 2220 Asp Asn Gln Leu Ala Tyr Val Val Ile Gly Leu Leu Phe Met Ile Leu 2225 2230 2235 2240 Thr Val Ala Ala Asn Glu Me t Gly Leu Leu Glu Thr Thr Lys Lys Asp 2245 2250 2255 Leu Gly Ile Gly His Val Ala Ala Glu Asn His His His Ala Thr Met 2260 2265 2270 Leu Asp Val Asp Leu His Pro Ala Ser Ala Trp Thr Leu Tyr Ala Val 2275 2280 2285 Ala Thr Thr Ile Ile Thr Pro Met Met Arg His Thr Ile Glu Asn Thr 2290 2295 2300 Thr Ala Asn Ile Ser Leu Thr Ala Ile Ala Asn Gln Ala Ala Ile Leu 2305 2310 2315 2320 Met Gly Leu Asp Lys Gly Trp Pro Ile Ser Lys Met Asp Ile Gly Val 2325 2330 2335 Pro Leu Leu Ala Leu Gly Cys Tyr Ser Gln Val Asn Pro Leu Thr Leu 2340 2345 2350 Ile Ala Ala Val Leu Met Leu Val Ala His Tyr Ala Ile Ile Gly Pro 2355 2360 2365 Gly Leu Gln Ala Lys Ala Thr Arg Glu Ala Gln Lys Arg Thr Ala Ala 2370 2375 2380 Gly Ile Met Lys Asn Pro Thr Val Asp Gly Ile Val Ala Ile Asp Leu 2385 2390 2395 2400 Asp Pro Val Val Tyr Asp Ala Lys Phe Glu Lys Gln Leu Gly Gln Ile 2405 2410 2415 Met Leu Leu Ile Leu Cys Thr Ser Gln Ile Leu Leu Met Arg Thr Thr 2420 2425 2430 Trp Ala Leu Cys Glu Ser Ile Thr Leu Ala Thr Gly Pro Leu Thr Thr 2435 2440 2445 Leu Trp Glu Gly Ser Pro Gly Lys Phe Trp Asn Thr Thr Ile Ala Val 2450 2455 2460 Ser Met Ala Asn Ile Phe Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu 2465 2470 2475 2480 Ala Phe Ser Leu Met Lys Ser Leu Gly Gly Gly Arg Arg Gly Thr Gly 2485 2490 2495 Ala Gln Gly Glu Thr Leu Gly Glu Lys Trp Lys Arg Gln Leu Asn Gln 2500 2505 2510 Leu Ser Lys Ser Glu Phe Asn Thr Tyr Lys Arg Ser Gly Ile Met Glu 2515 2520 2525 Val Asp Arg Ser Glu Ala Lys Glu Gly Leu Lys Arg Gly Glu Thr Thr 2530 2535 2540 Lys His Ala Val Ser Arg Gly Thr Ala Lys Leu Arg Trp Phe Val Glu 2545 2550 2555 2560 Arg Asn Leu Val Lys Pro Glu Gly Lys Val Ile Asp Leu Gly Cys Gly 2565 2570 2575 Arg Gly Gly Trp Ser Tyr Tyr Cys Ala Gly Leu Lys Lys Val Thr Glu 2580 2585 2590 Val Lys Gly Tyr Thr Lys Gly Gly Pro Gly His Glu Glu Pro Ile Pro 2595 2600 2605 Met Ala Thr Tyr Gly Trp Asn Leu Val Lys Leu His Ser Gly Lys Asp 2610 2615 2620 Val Phe Phe Ile Pro Glu Lys Cys Asp Thr Leu Leu Cys Asp Ile 2625 2630 2635 2640 Gly Glu Ser Ser Pro Asn Pr o Thr Ile Glu Glu Gly Arg Thr Leu Arg 2645 2650 2655 Val Leu Lys Met Val Glu Pro Trp Leu Arg Gly Asn Gln Phe Cys Ile 2660 2665 2670 Lys Ile Leu Asn Pro Tyr Met Pro Ser Val Val Glu Thr Leu Glu Gln 2675 2680 2685 Met Gln Arg Lys His Gly Gly Met Leu Val Arg Asn Pro Leu Ser Arg 2690 2695 2700 Asn Ser Thr His Glu Met Tyr Trp Val Ser Cys Gly Thr Gly Asn Ile 2705 2710 2715 2720 Val Ser Ala Val Asn Met Thr Ser Arg Met Leu Leu Asn Arg Phe Thr 2725 2730 2735 Met Ala His Arg Lys Pro Thr Tyr Glu Arg Asp Val Asp Leu Gly Ala 2740 2745 2750 Gly Thr Arg His Val Ala Val Glu Pro Glu Val Ala Asn Leu Asp Ile 2755 2760 2765 Ile Gly Gln Arg Ile Glu Asn Ile Lys Asn Glu His Lys Ser Thr Trp 2770 2775 2780 His Tyr Asp Glu Asp Asn Pro Tyr Lys Thr Trp Ala Tyr His Gly Ser 2785 2790 2795 2800 Tyr Glu Val Lys Pro Ser Gly Ser Ala Ser Ser Met Val Asn Gly Val 2805 2810 2815 Val Arg Leu Leu Thr Lys Pro Trp Asp Val Ile Pro Met Val Thr Gln 2820 2825 2830 Ile Ala Met Thr Asp Thr Thr Pro Phe Gly Gln Gln Arg Val Phe Lys 2835 2840 2845 Glu Lys Val Asp Thr Arg Thr Pro Lys Ala Lys Arg Gly Thr Ala Gln 2850 2855 2860 Ile Met Glu Val Thr Ala Arg Trp Leu Trp Gly Phe Leu Ser Arg Asn 2865 2870 2875 2880 Lys Lys Pro Arg Ile Cys Thr Arg Glu Glu Phe Thr Arg Lys Val Arg 2885 2890 2895 Ser Asn Ala Ala Ile Gly Ala Val Phe Val Asp Glu Asn Gln Trp Asn 2900 2905 2910 Ser Ala Lys Glu Ala Val Glu Asp Glu Arg Phe Trp Glu Leu Val His 2915 2920 2925 Arg Glu Arg Glu Leu His Lys Gln Gly Lys Cys Ala Thr Cys Val Tyr 2930 2935 2940 Asn Met Met Gly Lys Arg Glu Lys Lys Leu Gly Glu Phe Gly Lys Ala 2945 2950 2955 2960 Lys Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg Phe Leu 2965 2970 2975 Glu Phe Glu Ala Leu Gly Phe Met Asn Glu Asp His Trp Phe Ser Arg 2980 2985 2990 Glu Asn Ser Leu Ser Gly Val Glu Gly Glu Gly Leu His Lys Leu Gly 2995 3000 3005 Tyr Ile Leu Arg Asp Ile Ser Arg Ile Pro Gly Gly Asn Met Tyr Ala 3010 3015 3020 Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr Glu Asp Asp Leu Gln 3025 3030 3035 3040 Asn Glu Ala Lys Ile Thr Asp Ile Met Glu Pro Glu His Ala Leu Leu 3045 3050 3055 Ala Thr Ser Ile Phe Lys Leu Tyr Gln Asn Lys Val Val Arg Val 3060 3065 3070 Gln Arg Pro Ala Lys Asn Gly Thr Val Met Asp Val Ile Ser Arg Arg 3075 3080 3085 Asp Gln Arg Gly Ser Gly Gln Val Gly Thr Ty r Gly Leu Asn Thr Phe 3090 3095 3100 Thr Asn Met Glu Ala Gln Leu Ile Arg Gln Met Glu Ser Glu Gly Ile 3105 3110 3115 3120 Phe Leu Pro Ser Glu Leu Glu Thr Pro Asn Leu Ala Gly Arg Val Leu 3125 3130 3135 Asp Trp Leu Glu Lys Tyr Gly Val Glu Arg Leu Lys Arg Met Ala Ile 3140 3145 3150 Ser Gly Asp Asp Cys Val Val Lys Pro Ile Asp Asp Arg Phe Ala Thr 3155 3160 3165 Ala Leu Thr Ala Leu Asn Asp Met Gly Lys Val Arg Lys Asp Ile Pro 3170 3175 3180 Gln Trp Glu Pro Ser Lys Gly Trp Asn Asp Trp Gln Gln Val Pro Phe 3185 3190 3195 3200 Cys Ser His His Phe His Gln Leu Ile Met Lys Asp Gly Arg Glu Ile 3205 3210 3215 Val Val Pro Cys Arg Asn Gln Asp Glu Leu Val Gly Arg Ala Arg Val 3220 3225 3230 Ser Gln Gly Ala Gly Trp Ser Leu Arg Glu Thr Ala Cys Leu Gly Lys 3235 3240 3245 Ser Tyr Ala Gln Met Trp Gln Leu Met Tyr Phe His Arg Arg Asp Leu 3250 3255 3260 Arg Leu Ala Ala Asn Ala Ile Cys Ser Ala Val Pro Val Asp Trp Val 3265 3270 3275 3280 Pro Thr Ser Arg Thr Thr Trp Ser Ile His Ala His Gln Trp Met 3285 3290 3295 Thr Thr Glu Asp Met Leu Ser Val Trp Asn Arg Val Trp Ile Glu Glu 3300 3305 3310 Asn Pro Trp Met Glu Asp Lys Thr His Val Ser Ser Trp Glu Glu Val 3315 3320 3325 Pro Tyr Leu Gly Lys Arg Glu Asp Gln Trp Cys Gly Ser Leu Ile Gly 3330 3335 3340 Leu Thr Ala Arg Ala Thr Trp Ala Thr Asn Ile Gln Val Ala Ile Asn 3345 3350 3355 3360 Gln Val Arg Arg Leu Ile Gly Asn Glu Asn Tyr Leu Asp Tyr Met Thr 3365 3370 3375Ser Met Lys Arg Phe Lys Asn Glu Ser Asp Pro Glu Gly Ala Leu Trp 3380 3385 3390 <210> 11 <211> 10723 <212> DNA <213> dengue virus <400> 11 agttgttagt ctacgtggac cgacaaagac agattctttg agggagctaa gctcaacgta 60 gttctaacag ttttttaatt agagagcaga tctctgatga ataaccaacg gaaaaaggcg 120 aaaaacacgc ctttcaatat gctgaaacgc gagagaaacc gcgtgtcgac tgtgcaacag 180 ctgacaaaga gattctcact tggaatgctg cagggacgag gaccattaaa actgttcatg 240 gccctggtgg cgttccttcg tttcctaaca atcccaccaa cagcagggat attgaagaga 300 tggggaacaa ttaaaaaatc aaaagctatt aatgttttga gagggttcag gaaagagatt 360 ggaaggatgc tgaacatctt gaataggaga cgcagatctg caggcatgat cattatgctg 420 attccaacag tgatggcgtt ccatttaacc acacgtaacg gagaaccaca catgatcgtc 480 agcagacaag agaaagggaa aagtcttctg tttaaaacag aggatggcgt gaacatgtgt 540 accctcatgg ccatggacct tggtgaattg tgtgaagaca caatcacgta caagtgtccc 600 cttctcaggc agaatgagcc agaagacata gactgttggt gcaactctac gtccacgtgg 660 gtaacttatg ggacgtgtac caccatggga gaacatagaa gagaaaaaag atcagtggca 720 ctcgttccac atgtgggaat gggactggag acacgaactg aaacatggat gtcatcagaa 780 ggggcctgga aacatgtcca gagaattgaa acttggatct tgagacatcc aggcttcacc 840 atgatggcag caatcctggc atacaccata ggaacgacac atttccaaag agccctgatt 900 ttcatcttac tgacagctgt cactccttca atgacaatgc gttgcatagg aatgtcaaat 960 agagactttg tggaaggggt ttcaggagga agctgggttg acatagtctt agaacatgga 1020 agctgtgtga cgacgatggc aaaaaacaaa ccaacattgg attttgaact gataaaaaca 1080 gaagccaaac agcctgccac cctaaggaag tactgtatag aggcaaagct aaccaacaca 1140 acaacagaat ctcgctgccc aacacaaggg gaacccagcc taaatgaaga gcaggacaaa 1200 aggttcgtct gcaaacactc catggtagac agaggatggg gaaatggatg tggactattt 1260 ggaaagggag gcattgtgac ctgtgctatg ttcagatgca aaaagaacat ggaaggaaaa 1320 gttgtgcaac cagaaaactt ggaatacacc attgtgataa cacctcactc aggggaagag 1380 catgcagtcg gaaatgacac aggaaaacat ggcaaggaaa tcaaaataac accacagagt 1440 tccatcacag aagcagaatt gacaggttat ggcactgtca caatggagtg ctctccaaga 1500 acgggcctcg acttcaatga gatggtgttg ctgcagatgg aaaataaagc ttggctggtg 1560 cacaggcaat ggttcctaga cctgccgtta ccatggttgc ccggagcgga cacacaaggg 1620 tcaaattgga tacagaaaga gacattggtc actttcaaaa atccccatgc gaagaaacag 1680 gatgttgttg ttttaggatc ccaagaaggg gccatgcaca cagcacttac aggggccaca 1740 gaaatccaaa tgtcatcagg aaacttactc ttcacaggac atctcaagtg caggctgaga 1800 atggacaagc tacagctcaa aggaatgtca tactctatgt gcacaggaaa gtttaaagtt 1860 gtgaaggaaa tagcagaaac acaacatgga acaatagtta tcagagtgca atatgaaggg 1920 gacggctctc catgcaagat cccttttgag ataatggatt tggaaaaaag acatgtctta 1980 ggtcgcctga ttacagtcaa cccaattgtg acagaaaaag atagcccagt caacatagaa 2040 gcagaacctc cattcggaga cagctacatc atcataggag tagagccggg acaactgaag 2100 ctcaactggt ttaagaaagg aagttctatc ggccaaatgt ttgagacaac aatgagggg 2160 gcgaagagaa tggccatttt aggtgacaca gcctgggatt ttggatcctt gggaggagtg 2220 tttacatcta taggaaaggc tctccaccaa gtctttggag caatctatgg agctgccttc 2280 agtggggttt catggactat gaaaatcctc ataggagtca ttatcacatg gataggaatg 2340 aattcacgca gcacctcact gtctgtgaca ctagtattgg tgggaattgt gacactgtat 2400 ttgggagtca tggtgcaggc cgatagtggt tgcgttgtga gctggaaaaa caaagaactg 2460 aaatgtggca gtgggatttt catcacagac aacgtgcaca catggacaga acaatacaag 2520 ttccaaccag aatccccttc aaaactagct tcagctatcc agaaagccca tgaagagggc 2580 atttgtggaa tccgctcagt aacaagactg gagaatctga tgtggaaaca aataacacca 2640 gaattgaatc acattctatc agaaaatgag gtgaagttaa ctattatgac aggagacatc 2700 aaaggaatca tgcaggcagg aaaacgatct ctgcggcctc agcccactga gctgaagtat 2760 tcatggaaaa catggggcaa agcaaaaatg ctctctacag agtctcataa ccagaccttt 2820 ctcattgatg gccccgaaac agcagaatgc cccaacacaa atagagcttg gaattcgttg 2880 gaagttgaag actatggctt tggagtattc accaccaata tatggctaaa attgaaagaa 2940 aaacaggatg tattctgcga ctcaaaactc atgtcagcgg ccataaaaga caacagagcc 3000 gtccatgccg atatgggtta ttggatagaa agtgcactca atgacacatg gaagatagag 3060 aaagcctctt tcattgaagt taaaaactgc cactggccaa aatcacacac cctctggagc 3120 aatggagtgc tagaaagtga gatgataatt ccaaagaatc tcgctggacc agtgtctcaa 3180 cacaactata gaccaggcta ccatacacaa ataacaggac catggcatct aggtaagctt 3240 gagatggact ttgatttctg tgatggaaca acagtggtag tgactgagga ctgcggaaat 3300 agaggaccct ctttgagaac aaccactgcc tctggaaaac tcataacaga atggtgctgc 3360 cgatcttgca cattaccacc gctaagatac agaggtgagg atgggtgctg gtacgggatg 3420 gaaatcagac cattgaagga gaaagaagag aatttggtca actccttggt cacagctgga 3480 catgggcagg tcgacaactt ttcactagga gtcttgggaa tggcattgtt cctggaggaa 3540 atgcttagga cccgagtagg aacgaaacat gcaatactac tagttgcagt ttcttttgtg 3600 acattgatca cagggaacat gtcctttaga gacctgggaa gagtgatggt tatggtaggc 3660 gccactatga cggatgacat aggtatgggc gtgacttatc ttgccctact agcagccttc 3720 aaagtcagac caacttttgc agctggacta ctcttgagaa agctgacctc caaggaattg 3780 atgatgacta ctataggaat tgtactcctc tcccagagca ccataccaga gaccattctt 3840 gagttgactg atgcgttagc cttaggcatg atggtcctca aaatggtgag aaatatggaa 3900 aagtatcaat tggcagtgac tatcatggct atcttgtgcg tcccaaacgc agtgatatta 3960 caaaacgcat ggaaagtgag ttgcacaata ttggcagtgg tgtccgtttc cccactgctc 4020 ttaacatcct cacagcaaaa aacagattgg ataccattag cattgacgat caaaggtctc 4080 aatccaacag ctatttttct aacaaccctc tcaagaacca gcaagaaaag gagctggcca 4140 ttaaatgagg ctatcatggc agtcgggatg gtgagcattt tagccagttc tctcctaaaa 4200 aatgatattc ccatgacagg accattagtg gctggagggc tcctcactgt gtgctacgtg 4260 ctcactggac gatcggccga tttggaactg gagagagcag ccgatgtcaa atgggaagac 4320 caggcagaga tatcaggaag cagtccaatc ctgtcaataa caatatcaga agatggtagc 4380 atgtcgataa aaaatgaaga ggaagaacaa acactgacca tactcattag aacaggattg 4440 ctggtgatct caggactttt tcctgtatca ataccaatca cggcagcagc atggtacctg 4500 tgggaagtga agaaacaacg ggccggagta ttgtgggatg ttccttcacc cccacccatg 4560 ggaaaggctg aactggaaga tggagcctat agaattaagc aaaaagggat tcttggatat 4620 tcccagatcg gagccggagt ttacaaagaa ggaacattcc atacaatgtg gcatgtcaca 4680 cgtggcgctg ttctaatgca taaaggaaag aggattgaac catcatgggc ggacgtcaag 4740 aaagacctaa tatcatatgg aggaggctgg aagttagaag gagaatggaa ggaaggagaa 4800 gaagtccagg tattggcact ggagcctgga aaaaatccaa gagccgtcca aacgaaacct 4860 ggtcttttca aaaccaacgc cggaacaata ggtgctgtat ctctggactt ttctcctgga 4920 acgtcaggat ctccaattat cgacaaaaaa ggaaaagttg tgggtcttta tggtaatggt 4980 gttgttacaa ggagtggagc atatgtgagt gctatagccc agactgaaaa aagcattgaa 5040 gacaacccag agatcgaaga tgacattttc cgaaagagaa gactgaccat catggacctc 5100 cacccaggag cgggaaagac gaagagatac cttccggcca tagtcagaga agctataaaa 5160 cggggtttga gaacattaat cttggccccc actagagttg tggcagctga aatggaggaa 5220 gcccttagag gacttccaat aagataccag accccagcca tcagagctga gcacaccggg 5280 cgggagattg tggacctaat gtgtcatgcc acatttacca tgaggctgct atcaccagtt 5340 agagtgccaa actacaacct gattatcatg gacgaagccc atttcacaga cccagcaagt 5400 atagcagcta gaggatacat ctcaactcga gtggagatgg gtgaggcagc tgggattttt 5460 atgacagcca ctccccccggg aagcagagac ccatttcctc agagcaatgc accaatcata 5520 gatgaagaaa gagaaatccc tgaacgttcg tggaattccg gacatgaatg ggtcacggat 5580 tttaaaggga agactgtttg gttcgttcca agtataaaag caggaaatga tatagcagct 5640 tgcctgagga aaaatggaaa gaaagtgata caactcagta ggaagacctt tgattctgag 5700 tatgtcaaga ctagaaccaa tgattgggac ttcgtggtta caactgacat ttcagaaatg 5760 ggtgccaatt tcaaggctga gagggttata gaccccagac gctgcatgaa accagtcata 5820 ctaacagatg gtgaagagcg ggtgattctg gcaggaccta tgccagtgac ccactctagt 5880 gcagcacaaa gaagagggag aataggaaga aatccaaaaa atgagaatga ccagtacata 5940 tacatggggg aacctctgga aaatgatgaa gactgtgcac actggaaaga agctaaaatg 6000 ctcctagata acatcaacac gccagaagga atcattccta gcatgttcga accagagcgt 6060 gaaaaggtgg atgccattga tggcgaatac cgcttgagag gagaagcaag gaaaaccttt 6120 gtagacttaa tgagaagagg agacctacca gtctggttgg cctacagagt ggcagctgaa 6180 ggcatcaact acgcagacag aaggtggtgt tttgatggag tcaagaacaa ccaaatccta 6240 gaagaaaacg tggaagttga aatctggaca aaagaagggg aaaggaagaa attgaaaccc 6300 agatggttgg atgctaggat ctattctgac ccactggcgc taaaagaatt taaggaattt 6360 gcagccggaa gaaagtctct gaccctgaac ctaatcacag aaatgggtag gctcccaacc 6420 ttcatgactc agaaggcaag agacgcactg gacaacttag cagtgctgca cacggctgag 6480 gcaggtggaa gggcgtacaa ccatgctctc agtgaactgc cggagaccct ggagacattg 6540 cttttactga cacttctggc tacagtcacg ggagggatct ttttattctt gatgagcgga 6600 aggggcatag ggaagatgac cctgggaatg tgctgcataa tcacggctag catcctccta 6660 tggtacgcac aaatacagcc acactggata gcagcttcaa taatactgga gttttttctc 6720 atagttttgc ttattccaga acctgaaaaa cagagaacac cccaagacaa ccaactgacc 6780 tacgttgtca tagccatcct cacagtggtg gccgcaacca tggcaaacga gatgggtttc 6840 ctagaaaaaa cgaagaaaga tctcggattg ggaagcattg caacccagca acccgagagc 6900 aacatcctgg acatagatct acgtcctgca tcagcatgga cgctgtatgc cgtggccaca 6960 acatttgtta caccaatgtt gagacatagc attgaaaatt cctcagtgaa tgtgtcccta 7020 acagctatag ccaaccaagc cacagtgtta atgggtctcg ggaaaggatg gccattgtca 7080 aagatggaca tcggagttcc ccttctcgcc attggatgct actcacaagt caaccccata 7140 actctcacag cagctctttt cttattggta gcacattatg ccatcatagg gccaggactc 7200 caagcaaaag caaccagaga agctcagaaa agagcagcgg cgggcatcat gaaaaaccca 7260 actgtcgatg gaataacagt gattgaccta gatccaatac cttatgatcc aaagtttgaa 7320 aagcagttgg gacaagtaat gctcctagtc ctctgcgtga ctcaagtatt gatgatgagg 7380 actacatggg ctctgtgtga ggctttaacc ttagctaccg ggcccatctc cacatgtgg 7440 gaaggaaatc cagggaggtt ttggaacact accattgcgg tgtcaatggc taacattttt 7500 agagggagtt acttggccgg agctggactt ctcttttcta ttatgaagaa cacaaccaac 7560 acaagaaggg gaactggcaa cataggagag acgcttggag agaaatggaa aagccgattg 7620 aacgcattgg gaaaaagtga attccagatc tacaagaaaa gtggaatcca ggaagtggat 7680 agaaccttag caaaagaagg cattaaaaga ggagaaacgg accatcacgc tgtgtcgcga 7740 ggctcagcaa aactgagatg gttcgttgag agaaacatgg tcacaccaga agggaaagta 7800 gtggacctcg gttgtggcag aggaggctgg tcatactatt gtggaggact aaagaatgta 7860 agagaagtca aaggcctaac aaaaggagga ccaggacacg aagaacccat ccccatgtca 7920 acatatgggt ggaatctagt gcgtcttcaa agtggagttg acgttttctt catcccgcca 7980 gaaaagtgtg acacattatt gtgtgacata ggggagtcat caccaaatcc cacagtggaa 8040 gcaggacgaa cactcagagt ccttaactta gtagaaaatt ggttgaacaa caacactcaa 8100 ttttgcataa aggttctcaa cccatatatg ccctcagtca tagaaaaaat ggaagcacta 8160 caaaggaaat atggaggagc cttagtgagg aatccactct cacgaaactc cacacatgag 8220 atgtactggg tatccaatgc ttccgggaac atagtgtcat cagtgaacat gatttcaagg 8280 atgttgatca acagattac aatgagatac aagaaagcca cttacgagcc ggatgttgac 8340 ctcggaagcg gaacccgtaa catcgggatt gaaagtgaga taccaaacct agatataatt 8400 gggaaaagaa tagaaaaaat aaagcaagag catgaaacat catggcacta tgaccaagac 8460 cacccataca aaacgtgggc ataccatggt agctatgaaa caaaacagac tggatcagca 8520 tcatccatgg tcaacggagt ggtcaggctg ctgacaaaac cttgggacgt cgtccccatg 8580 gtgacacaga tggcaatgac agacacgact ccatttggac aacagcgcgt ttttaaagag 8640 aaagtggaca cgagaaccca agaaccgaaa gaaggcacga agaaactaat gaaaataaca 8700 gcagagtggc tttggaaaga attagggaag aaaaagacac ccaggatgtg caccagagaa 8760 gaattcacaa gaaaggtgag aagcaatgca gccttggggg ccatattcac tgatgagaac 8820 aagtggaagt cggcacgtga ggctgttgaa gatagtaggt tttgggagct ggttgacaag 8880 gaaaggaatc tccatcttga aggaaagtgt gaaacatgtg tgtacaacat gatgggaaaa 8940 agagagaaga agctagggga attcggcaag gcaaaaggca gcagagccat atggtacatg 9000 tggcttggag cacgcttctt agagtttgaa gccctaggat tcttaaatga agatcactgg 9060 ttctccagag agaactccct gagtggagtg gaaggagaag ggctgcacaa gctaggttac 9120 attctaagag acgtgagcaa gaaagaggga ggagcaatgt atgccgatga caccgcagga 9180 tgggatacaa gaatcacact agaagaccta aaaaatgaag aaatggtaac aaaccacatg 9240 gaaggagaac acaagaaact agccgaggcc attttcaaac taacgtacca aaacaaggtg 9300 gtgcgtgtgc aaagaccaac accaagaggc acagtaatgg acatcatatc gagaagagac 9360 caaagaggta gtggacaagt tggcacctat ggactcaata ctttcaccaa tatggaagcc 9420 caactaatca gacagatgga gggagaagga gtctttaaaa gcattcagca cctaacaatc 9480 acagaagaaa tcgctgtgca aaactggtta gcaagagtgg ggcgcgaaag gttatcaaga 9540 atggccatca gtggagatga ttgtgttgtg aaacctttag atgacaggtt cgcaagcgct 9600 ttaacagctc taaatgacat gggaaagatt aggaaagaca tacaacaatg ggaaccttca 9660 agaggatgga atgattggac acaagtgccc ttctgttcac accatttcca tgagttaatc 9720 atgaaagacg gtcgcgtact cgttgttcca tgtagaaacc aagatgaact gattggcaga 9780 gcccgaatct cccaaggagc agggtggtct ttgcgggaga cggcctgttt ggggaagtct 9840 tacgcccaaa tgtggagctt gatgtacttc cacagacgcg acctcaggct ggcggcaaat 9900 gctatttgct cggcagtacc atcacattgg gttccaacaa gtcgaacaac ctggtccata 9960 catgctaaac atgaatggat gacaacggaa gacatgctga cagtctggaa cagggtgtgg 10020 attcaagaaa acccatggat ggaagacaaa actccagtgg aatcatggga ggaaatccca 10080 tacttgggga aaagagaaga ccaatggtgc ggctcattga ttgggttaac aagcagggcc 10140 acctgggcaa agaacatcca agcagcaata aatcaagtta gatcccttat aggcaatgaa 10200 gaatacacag attacatgcc atccatgaaa agattcagaa gagaagagga agaagcagga 10260 gttctgtggt agaaagcaaa actaacatga aacaaggcta gaagtcaggt cggattaagc 10320 catagtacgg aaaaaactat gctacctgtg agccccgtcc aaggacgtta aaagaagtca 10380 ggccatcata aatgccatag ctggagtaaa ctatgcagcc tgtagctcca cctgagaagg 10440 tgtaaaaaat ccgggaggcc acaaaccatg gaagctgtac gcatggcgta gtggactagc 10500 ggttagagga gacccctccc ttacaaatcg cagcaacaat gggggcccaa ggcgagatga 10560 agctgtagtc tcgctggaag gactagaggt tagaggagac ccccccgaaa caaaaaacag 10620 catattgacg ctgggaaaga ccagagatcc tgctgtctcc tcagcatcat tccaggcaca 10680 gaacgccaga aaatggaatg gtgctgttga atcaacaggt tct 10723 <210> 12 <211> 3391 <212> PRT <213> dengue virus <400> 12 Met Asn Asn Gln Arg Lys Lys Ala Lys Asn Thr Pro Phe Asn Met Leu 1 5 10 15 Lys Arg Glu Arg Asn Arg Val Ser Thr Val Gln Gln Leu Thr Lys Arg 20 25 30 Phe Ser Leu Gly Met Leu Gln Gly Arg Gly Pro Leu Lys Leu Phe Met 35 40 45 Ala Leu Val Ala Phe Leu Arg Phe Leu Thr Ile Pro Pro Thr Ala Gly 50 55 60 Ile Leu Lys Arg Trp Gly Thr Ile Lys Lys Ser Lys Ala Ile Asn Val 65 70 75 80 Leu Arg Gly Phe Arg Lys Glu Ile Gly Arg Met Leu Asn Ile Leu Asn 85 90 95 Arg Arg Arg Arg Ser Ala Gly Met Ile Ile Met Leu Ile Pro Thr Val 100 105 110 Met Ala Phe His Leu Thr Thr Arg Asn Gly Glu Pro His Met Ile Val 115 120 125 Ser Arg Gln Glu Lys Gly Lys Ser Leu Leu Phe Lys Thr Glu Asp Gly 130 135 140 Val Asn Met Cys Thr Leu Met Ala Met Asp Leu Gly Glu Leu Cys Glu 145 150 155 160 Asp Thr Ile Thr Tyr Lys Cys Pro Leu Leu Arg Gln Asn Glu Pro Glu 165 170 175 Asp Ile Asp Cys Trp Cys Asn Ser Thr Ser Thr Trp Val Thr Tyr Gly 180 185 190 Thr Cys Thr Thr Met Gly Glu His Arg Arg Glu Lys Arg Ser Val Ala 195 200 205 Leu Val Pro His Val Gly Met Gly Leu Glu Thr Arg Thr Glu Thr Trp 210 215 220 Met Ser Ser Glu Gly Ala Trp Lys His Val Gln Arg Ile Glu Thr Trp 225 230 235 240 Ile Leu Arg His Pro Gly Phe Thr Met Met Ala Ala Ile Leu Ala Tyr 245 250 255 Thr Ile Gly Thr Thr His Phe Gln Arg Ala Leu Ile Phe Ile Leu Leu 260 265 270 Thr Ala Val Thr Pro Ser Met Thr Met Arg Cys Ile Gly Met Ser Asn 275 280 285 Arg Asp Phe Val Glu Gly Val Ser Gly Gly Ser Trp Val Asp Ile Val 290 295 300 Leu Glu His Gly Ser Cys Val Thr Thr Met Ala Lys Asn Lys Pro Thr 305 310 315 320 Leu Asp Phe Glu Leu Ile Lys Thr Glu Ala Lys Gln Pro Ala Thr Leu 325 330 335 Arg Lys Tyr Cys Ile Glu Ala Lys Leu Thr Asn Thr Thr Thr Glu Ser 340 345 350 Arg Cys Pro Thr Gln Gly Glu Pro Ser Leu Asn Glu Glu Gln Asp Lys 355 360 365 Arg Phe Val Cys Lys His Ser Met Val Asp Arg Gly Trp Gly Asn Gly 370 375 380 Cys Gly Leu Phe Gly Lys Gly Gly Ile Val Thr Cys Ala Met Phe Arg 385 390 395 400 Cys Lys Lys Asn Met Glu Gly Lys Val Val Gin Pro Glu Asn Leu Glu 405 410 415 Tyr Thr Ile Val Ile Thr Pro His Ser Gly Glu Glu His Ala Val Gly 420 425 430 Asn Asp Thr Gly Lys His Gly Lys Glu Ile Lys Ile Thr Pro Gln Ser 435 440 445 Ser Ile Thr Glu Ala Glu Leu Thr Gly Tyr Gly Thr Val Thr Met Glu 450 455 460 Cys Ser Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Val Leu Leu Gln 465 470 475 480 Met Glu Asn Lys Ala Trp Leu Val His Arg Gln Trp Phe Leu Asp Leu 485 490 495 Pro Leu Pro Trp Leu Pro Gly Ala Asp Thr Gln Gly Ser Asn Trp Ile 500 505 510 Gln Lys Glu Thr Leu Val Thr Phe Lys Asn Pro His Ala Lys Lys Gln 515 520 525 Asp Val Val Val Leu Gly Ser Gln Glu Gly Ala Met His Thr Ala Leu 530 535 540 Thr Gly Ala Thr Glu Ile Gln Met Ser Ser Gly Asn Leu Leu Phe Thr 545 550 555 560 Gly His Leu Lys Cys Arg Leu Arg Met Asp Lys Leu Gln Leu Lys Gly 565 570 575 Met Ser Tyr Ser Met Cys Thr Gly Lys Phe Lys Val Val Lys Glu Ile 580 585 590 Ala Glu Thr Gln His Gly Thr Ile Val Ile Arg Val Gln Tyr Glu Gly 595 600 605 Asp Gly Ser Pro Cys Lys Ile Pro Phe Glu Ile Met Asp Leu Glu Lys 610 615 620 Arg His Val Leu Gly Arg Leu Ile Thr Val Asn Pro Ile Val Thr Glu 625 630 635 640 Lys Asp Ser Pro Val Asn Ile Glu Ala Glu Pro Phe Gly Asp Ser 645 650 655 Tyr Ile Ile Ile Gly Val Glu Pro Gly Gln Leu Lys Leu Asn Trp Phe 660 665 670 Lys Lys Gly Ser Ser Ile Gly Gln Met Phe Glu Thr Thr Met Arg Gly 675 680 685 Ala Lys Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser 690 695 700 Leu Gly Gly Val Phe Thr Ser Ile Gly Lys Ala Leu His Gln Val Phe 705 710 715 720 Gly Ala Ile Tyr Gly Ala Ala Phe Ser Gly Val Ser Trp Thr Met Lys 725 730 735 Ile Leu Ile Gly Val Ile Ile Thr Trp Ile Gly Met Asn Ser Arg Ser 740 745 750 Thr Ser Leu Ser Val Thr Leu Val Leu Val Gly Ile Val Thr Leu Tyr 755 760 765 Leu Gly Val Met Val Gln Ala Asp Ser Gly Cys Val Val Ser Trp Lys 770 775 780 Asn Lys Glu Leu Lys Cys Gly Ser Gly Ile Phe Ile Thr Asp Asn Val 785 790 795 800 His Thr Trp Thr Glu Gln Tyr Lys Phe Gln Pro Glu Ser Pro Ser Lys 805 810 815 Leu Ala Ser Ala Ile Gln Lys Ala His Glu Glu Gly Ile Cys Gly Ile 820 825 830 Arg Ser Val Thr Arg Leu Glu Asn Leu Met Trp Lys Gln Ile Thr Pro 835 840 845 Glu Leu Asn His Ile Leu Ser Glu Asn Glu Val Lys Leu Thr Ile Met 850 855 860 Thr Gly Asp Ile Lys Gly Ile Met Gln Ala Gly Lys Arg Ser Leu Arg 865 870 875 880 Pro Gln Pro Thr Glu Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala 885 890 895 Lys Met Leu Ser Thr Glu Ser His Asn Gln Thr Phe Leu Ile Asp Gly 900 905 910 Pro Glu Thr Ala Glu Cys Pro Asn Thr Asn Arg Ala Trp Asn Ser Leu 915 920 925 Glu Val Glu Asp Tyr Gly Phe Gly Val Phe Thr Thr Asn Ile Trp Leu 930 935 940 Lys Leu Lys Glu Lys Gln Asp Val Phe Cys Asp Ser Lys Leu Met Ser 945 950 955 960 Ala Ala Ile Lys Asp Asn Arg Ala Val His Ala Asp Met Gly Tyr Trp 965 970 975 Ile Glu Ser Ala Leu Asn Asp Thr Trp Lys Ile Glu Lys Ala Ser Phe 980 985 990 Ile Glu Val Lys Asn Cys His Trp Pro Lys Ser His Thr Leu Trp Ser 995 1000 1005 Asn Gly Val Leu Glu Ser Glu Met Ile Ile Pro Lys Asn Leu Ala Gly 1010 1015 1020 Pro Val Ser Gln His Asn Tyr Arg Pro Gly Tyr His Thr Gln Ile Thr 1025 1030 1035 1040 Gly Pro Trp His Leu Gly Ly s Leu Glu Met Asp Phe Asp Phe Cys Asp 1045 1050 1055 Gly Thr Thr Val Val Val Thr Glu Asp Cys Gly Asn Arg Gly Pro Ser 1060 1065 1070 Leu Arg Thr Thr Thr Ala Ser Gly Lys Leu Ile Thr Glu Trp Cys Cys 1075 1080 1085 Arg Ser Cys Thr Leu Pro Leu Arg Tyr Arg Gly Glu Asp Gly Cys 1090 1095 1100 Trp Tyr Gly Met Glu Ile Arg Pro Leu Lys Glu Lys Glu Glu Asn Leu 1105 1110 1115 1120 Val Asn Ser Leu Val Thr Ala Gly His Gly Gln Val Asp Asn Phe Ser 1125 1130 1135 Leu Gly Val Leu Gly Met Ala Leu Phe Leu Glu Glu Met Leu Arg Thr 1140 1145 1150 Arg Val Gly Thr Lys His Ala Ile Leu Leu Val Ala Val Ser Phe Val 1155 1160 1165 Thr Leu Ile Thr Gly Asn Met Ser Phe Arg Asp Leu Gly Arg Val Met 1170 1175 1180 Val Met Val Gly Ala Thr Met Thr Asp Asp Ile Gly Met Gly Val Thr 1185 1190 1195 1200 Tyr Leu Ala Leu Leu Ala Ala Phe Lys Val Arg Pro Thr Phe Ala Ala 1205 1210 1215 Gly Leu Leu Leu Arg Lys Leu Thr Ser Lys Glu Leu Met Met Thr Thr 1220 1225 1230 Ile Gly Ile Val Leu Leu Ser Gln Ser Thr Ile Pro Glu Thr Ile Leu 1235 1240 1245 Glu Leu Thr Asp Ala Leu Ala Leu Gly Met Met Val Leu Lys Met Val 1250 1255 1260 Arg Asn Met Glu Lys Tyr Gln Leu Ala Val Thr Ile Met Ala Ile Leu 1265 1270 1275 1280 Cys Val Pro Asn Ala Val Ile Leu Gln Asn Ala Trp Lys Val Ser Cys 1285 1290 1295 Thr Ile Leu Ala Val Val Ser Val Ser Pro Leu Leu Leu Thr Ser Ser 1300 1305 1310 Gln Gln Lys Thr Asp Trp Ile Pro Leu Ala Leu Thr Ile Lys Gly Leu 1315 1320 1325 Asn Pro Thr Ala Ile Phe Leu Thr Thr Leu Ser Arg Thr Ser Lys Lys 1330 1335 1340 Arg Ser Trp Pro Leu Asn Glu Ala Ile Met Ala Val Gly Met Val Ser 1345 1350 1355 1360 Ile Leu Ala Ser Leu Leu Lys Asn Asp Ile Pro Met Thr Gly Pro 1365 1370 1375 Leu Val Ala Gly Gly Leu Leu Thr Val Cys Tyr Val Leu Thr Gly Arg 1380 1385 1390 Ser Ala Asp Leu Glu Leu Glu Arg Ala Ala Asp Val Lys Trp Glu Asp 1395 1400 1405 Gln Ala Glu Ile Ser Gly Ser Ser Pro Ile Leu Ser Ile Thr Ile Ser 1410 1415 1420 Glu Asp Gly Ser Met Ser Ile Lys Asn Glu Glu Glu Glu Gln Thr Leu 1425 1430 1435 1440 Thr Ile Leu Ile Arg Thr Gl y Leu Leu Val Ile Ser Gly Leu Phe Pro 1445 1450 1455 Val Ser Ile Pro Ile Thr Ala Ala Ala Trp Tyr Leu Trp Glu Val Lys 1460 1465 1470 Lys Gln Arg Ala Gly Val Leu Trp Asp Val Pro Ser Pro Pro Met 1475 1480 1485 Gly Lys Ala Glu Leu Glu Asp Gly Ala Tyr Arg Ile Lys Gln Lys Gly 1490 1495 1500 Ile Leu Gly Tyr Ser Gln Ile Gly Ala Gly Val Tyr Lys Glu Gly Thr 1505 1510 1515 1520 Phe His Thr Met Trp His Val Thr Arg Gly Ala Val Leu Met His Lys 1525 1530 1535 Gly Lys Arg Ile Glu Pro Ser Trp Ala Asp Val Lys Lys Asp Leu Ile 1540 1545 1550 Ser Tyr Gly Gly Gly Trp Lys Leu Glu Gly Glu Trp Lys Glu Gly Glu 1555 1560 1565 Glu Val Gln Val Leu Ala Leu Glu Pro Gly Lys Asn Pro Arg Ala Val 1570 1575 1580 Gln Thr Lys Pro Gly Leu Phe Lys Thr Asn Ala Gly Thr Ile Gly Ala 1585 1590 1595 1600 Val Ser Leu Asp Phe Ser Pro Gly Thr Ser Gly Ser Pro Ile Ile Asp 1605 1610 1615 Lys Lys Gly Lys Val Val Gly Leu Tyr Gly Asn Gly Val Val Thr Arg 1620 1625 1630 Ser Gly Ala Tyr Val Ser Ala Ile Ala Gln Thr Glu Lys Ser Ile Glu 1635 1640 1645 Asp Asn Pro Glu Ile Glu Asp Asp Ile Phe Arg Lys Arg Arg Leu Thr 1650 1655 1660 Ile Met Asp Leu His Pro Gly Ala Gly Lys Thr Lys Arg Tyr Leu Pro 1665 1670 1675 1680 Ala Ile Val Arg Glu Ala Ile Lys Arg Gly Leu Arg Thr Leu Ile Leu 1685 1690 1695 Ala Pro Thr Arg Val Val Ala Ala Glu Met Glu Glu Ala Leu Arg Gly 1700 1705 1710 Leu Pro Ile Arg Tyr Gln Thr Pro Ala Ile Arg Ala Glu His Thr Gly 1715 1720 1725 Arg Glu Ile Val Asp Leu Met Cys His Ala Thr Phe Thr Met Arg Leu 1730 1735 1740 Leu Ser Pro Val Arg Val Pro Asn Tyr Asn Leu Ile Ile Met Asp Glu 1745 1750 1755 1760 Ala His Phe Thr Asp Pro Ala Ser Ile Ala Ala Arg Gly Tyr Ile Ser 1765 1770 1775 Thr Arg Val Glu Met Gly Glu Ala Ala Gly Ile Phe Met Thr Ala Thr 1780 1785 1790 Pro Pro Gly Ser Arg Asp Pro Phe Pro Gln Ser Asn Ala Pro Ile Ile 1795 1800 1805 Asp Glu Glu Arg Glu Ile Pro Glu Arg Ser Trp Asn Ser Gly His Glu 1810 1815 1820 Trp Val Thr Asp Phe Lys Gly Lys Thr Val Trp Phe Val Pro Ser Ile 1825 1830 1835 1840 Lys Ala Gly Asn Asp Ile Al a Ala Cys Leu Arg Lys Asn Gly Lys Lys 1845 1850 1855 Val Ile Gln Leu Ser Arg Lys Thr Phe Asp Ser Glu Tyr Val Lys Thr 1860 1865 1870 Arg Thr Asn Asp Trp Asp Phe Val Val Thr Thr Asp Ile Ser Glu Met 1875 1880 1885 Gly Ala Asn Phe Lys Ala Glu Arg Val Ile Asp Pro Arg Arg Cys Met 1890 1895 1900 Lys Pro Val Ile Leu Thr Asp Gly Glu Glu Arg Val Ile Leu Ala Gly 1905 1910 1915 1920 Pro Met Pro Val Thr His Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile 1925 1930 1935 Gly Arg Asn Pro Lys Asn Glu Asn Asp Gln Tyr Ile Tyr Met Gly Glu 1940 1945 1950 Pro Leu Glu Asn Asp Glu Asp Cys Ala His Trp Lys Glu Ala Lys Met 1955 1960 1965 Leu Leu Asp Asn Ile Asn Thr Pro Glu Gly Ile Ile Pro Ser Met Phe 1970 1975 1980 Glu Pro Glu Arg Glu Lys Val Asp Ala Ile Asp Gly Glu Tyr Arg Leu 1985 1990 1995 2000 Arg Gly Glu Ala Arg Lys Thr Phe Val Asp Leu Met Arg Arg Gly Asp 2005 2010 2015 Leu Pro Val Trp Leu Ala Tyr Arg Val Ala Ala Glu Gly Ile Asn Tyr 2020 2025 2030 Ala Asp Arg Arg Trp Cys Phe Asp Gly Val Lys Asn Asn Gln Ile Leu 2035 2040 2045 Glu Glu Asn Val Glu Val Glu Ile Trp Thr Lys Glu Gly Glu Arg Lys 2050 2055 2060 Lys Leu Lys Pro Arg Trp Leu Asp Ala Arg Ile Tyr Ser Asp Pro Leu 2065 2070 2075 2080 Ala Leu Lys Glu Phe Lys Glu Phe Ala Ala Gly Arg Lys Ser Leu Thr 2085 2090 2095 Leu Asn Leu Ile Thr Glu Met Gly Arg Leu Pro Thr Phe Met Thr Gln 2100 2105 2110 Lys Ala Arg Asp Ala Leu Asp Asn Leu Ala Val Leu His Thr Ala Glu 2115 2120 2125 Ala Gly Gly Arg Ala Tyr Asn His Ala Leu Ser Glu Leu Pro Glu Thr 2130 2135 2140 Leu Glu Thr Leu Leu Leu Leu Thr Leu Leu Ala Thr Val Thr Gly Gly 2145 2150 2155 2160 Ile Phe Leu Phe Leu Met Ser Gly Arg Gly Ile Gly Lys Met Thr Leu 2165 2170 2175 Gly Met Cys Cys Ile Ile Thr Ala Ser Ile Leu Leu Trp Tyr Ala Gln 2180 2185 2190 Ile Gln Pro His Trp Ile Ala Ala Ser Ile Ile Leu Glu Phe Phe Leu 2195 2200 2205 Ile Val Leu Leu Ile Pro Glu Pro Glu Lys Gln Arg Thr Pro Gln Asp 2210 2215 2220 Asn Gln Leu Thr Tyr Val Val Ile Ala Ile Leu Thr Val Val Ala Ala 2225 2230 2235 2240 Thr Met Ala Asn Glu Met Gl y Phe Leu Glu Lys Thr Lys Lys Asp Leu 2245 2250 2255 Gly Leu Gly Ser Ile Ala Thr Gln Gln Pro Glu Ser Asn Ile Leu Asp 2260 2265 2270 Ile Asp Leu Arg Pro Ala Ser Ala Trp Thr Leu Tyr Ala Val Ala Thr 2275 2280 2285 Thr Phe Val Thr Pro Met Leu Arg His Ser Ile Glu Asn Ser Ser Val 2290 2295 2300 Asn Val Ser Leu Thr Ala Ile Ala Asn Gln Ala Thr Val Leu Met Gly 2305 2310 2315 2320 Leu Gly Lys Gly Trp Pro Leu Ser Lys Met Asp Ile Gly Val Pro Leu 2325 2330 2335 Leu Ala Ile Gly Cys Tyr Ser Gln Val Asn Pro Ile Thr Leu Thr Ala 2340 2345 2350 Ala Leu Phe Leu Leu Val Ala His Tyr Ala Ile Ile Gly Pro Gly Leu 2355 2360 2365 Gln Ala Lys Ala Thr Arg Glu Ala Gln Lys Arg Ala Ala Ala Gly Ile 2370 2375 2380 Met Lys Asn Pro Thr Val Asp Gly Ile Thr Val Ile Asp Leu Asp Pro 2385 2390 2395 2400 Ile Pro Tyr Asp Pro Lys Phe Glu Lys Gln Leu Gly Gln Val Met Leu 2405 2410 2415 Leu Val Leu Cys Val Thr Gln Val Leu Met Met Arg Thr Thr Trp Ala 2420 2425 2430 Leu Cys Glu Ala Leu Thr Leu Ala Thr Gly Pro Ile Ser Thr Leu Trp 2435 2440 2445 Glu Gly Asn Pro Gly Arg Phe Trp Asn Thr Thr Ile Ala Val Ser Met 2450 2455 2460 Ala Asn Ile Phe Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu Leu Phe 2465 2470 2475 2480 Ser Ile Met Lys Asn Thr Thr Asn Thr Arg Arg Gly Thr Gly Asn Ile 2485 2490 2495 Gly Glu Thr Leu Gly Glu Lys Trp Lys Ser Arg Leu Asn Ala Leu Gly 2500 2505 2510 Lys Ser Glu Phe Gln Ile Tyr Lys Lys Ser Gly Ile Gln Glu Val Asp 2515 2520 2525 Arg Thr Leu Ala Lys Glu Gly Ile Lys Arg Gly Glu Thr Asp His His 2530 2535 2540 Ala Val Ser Arg Gly Ser Ala Lys Leu Arg Trp Phe Val Glu Arg Asn 2545 2550 2555 2560 Met Val Thr Pro Glu Gly Lys Val Val Asp Leu Gly Cys Gly Arg Gly 2565 2570 2575 Gly Trp Ser Tyr Tyr Cys Gly Gly Leu Lys Asn Val Arg Glu Val Lys 2580 2585 2590 Gly Leu Thr Lys Gly Gly Pro Gly His Glu Glu Pro Ile Pro Met Ser 2595 2600 2605 Thr Tyr Gly Trp Asn Leu Val Arg Leu Gln Ser Gly Val Asp Val Phe 2610 2615 2620 Phe Ile Pro Pro Glu Lys Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu 2625 2630 2635 2640 Ser Ser Pro Asn Pro Thr Va l Glu Ala Gly Arg Thr Leu Arg Val Leu 2645 2650 2655 Asn Leu Val Glu Asn Trp Leu Asn Asn Asn Thr Gln Phe Cys Ile Lys 2660 2665 2670 Val Leu Asn Pro Tyr Met Pro Ser Val Ile Glu Lys Met Glu Ala Leu 2675 2680 2685 Gln Arg Lys Tyr Gly Gly Ala Leu Val Arg Asn Pro Leu Ser Arg Asn 2690 2695 2700 Ser Thr His Glu Met Tyr Trp Val Ser Asn Ala Ser Gly Asn Ile Val 2705 2710 2715 2720 Ser Ser Val Asn Met Ile Ser Arg Met Leu Ile Asn Arg Phe Thr Met 2725 2730 2735 Arg Tyr Lys Lys Ala Thr Tyr Glu Pro Asp Val Asp Leu Gly Ser Gly 2740 2745 2750 Thr Arg Asn Ile Gly Ile Glu Ser Glu Ile Pro Asn Leu Asp Ile Ile 2755 2760 2765 Gly Lys Arg Ile Glu Lys Ile Lys Gln Glu His Glu Thr Ser Trp His 2770 2775 2780 Tyr Asp Gln Asp His Pro Tyr Lys Thr Trp Ala Tyr His Gly Ser Tyr 2785 2790 2795 2800 Glu Thr Lys Gln Thr Gly Ser Ala Ser Ser Met Val Asn Gly Val Val 2805 2810 2815 Arg Leu Leu Thr Lys Pro Trp Asp Val Val Pro Met Val Thr Gln Met 2820 2825 2830 Ala Met Thr Asp Thr Thr Pro Phe Gly Gln Gln Arg Val Phe Lys Glu 2835 2840 2845 Lys Val Asp Thr Arg Thr Gln Glu Pro Lys Glu Gly Thr Lys Lys Leu 2850 2855 2860 Met Lys Ile Thr Ala Glu Trp Leu Trp Lys Glu Leu Gly Lys Lys Lys Lys 2865 2870 2875 2880 Thr Pro Arg Met Cys Thr Arg Glu Glu Phe Thr Arg Lys Val Arg Ser 2885 2890 2895 Asn Ala Ala Leu Gly Ala Ile Phe Thr Asp Glu Asn Lys Trp Lys Ser 2900 2905 2910 Ala Arg Glu Ala Val Glu Asp Ser Arg Phe Trp Glu Leu Val Asp Lys 2915 2920 2925 Glu Arg Asn Leu His Leu Glu Gly Lys Cys Glu Thr Cys Val Tyr Asn 2930 2935 2940 Met Met Gly Lys Arg Glu Lys Lys Leu Gly Glu Phe Gly Lys Ala Lys 2945 2950 2955 2960 Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg Phe Leu Glu 2965 2970 2975 Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp Phe Ser Arg Glu 2980 2985 2990 Asn Ser Leu Ser Gly Val Glu Gly Glu Gly Leu His Lys Leu Gly Tyr 2995 3000 3005 Ile Leu Arg Asp Val Ser Lys Lys Glu Gly Gly Ala Met Tyr Ala Asp 3010 3015 3020 Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr Leu Glu Asp Leu Lys Asn 3025 3030 3035 3040 Glu Glu Met Val Thr Asn His Met Glu Gly Glu His Lys Lys Lys Leu Ala 3045 3050 3055 Glu Ala Ile Phe Lys Leu Thr Tyr Gln Asn Lys Val Val Arg Val Gln 3060 3065 3070 Arg Pro Thr Pro Arg Gly Thr Val Met Asp Ile Ile Ser Arg Arg Asp 3075 3080 3085 Gln Arg Gly Ser Gly Gln Val Gly Thr Tyr Gl y Leu Asn Thr Phe Thr 3090 3095 3100 Asn Met Glu Ala Gln Leu Ile Arg Gln Met Glu Gly Glu Gly Val Phe 3105 3110 3115 3120 Lys Ser Ile Gln His Leu Thr Ile Thr Glu Glu Ile Ala Val Gln Asn 3125 3130 3135 Trp Leu Ala Arg Val Gly Arg Glu Arg Leu Ser Arg Met Ala Ile Ser 3140 3145 3150 Gly Asp Asp Cys Val Val Lys Pro Leu Asp Asp Arg Phe Ala Ser Ala 3155 3160 3165 Leu Thr Ala Leu Asn Asp Met Gly Lys Ile Arg Lys Asp Ile Gln Gln 3170 3175 3180 Trp Glu Pro Ser Arg Gly Trp Asn Asp Trp Thr Gln Val Pro Phe Cys 3185 3190 3195 3200 Ser His His Phe His Glu Leu Ile Met Lys Asp Gly Arg Val Leu Val 3205 3210 3215 Val Pro Cys Arg Asn Gln Asp Glu Leu Ile Gly Arg Ala Arg Ile Ser 3220 3225 3230 Gln Gly Ala Gly Trp Ser Leu Arg Glu Thr Ala Cys Leu Gly Lys Ser 3235 3240 3245 Tyr Ala Gln Met Trp Ser Leu Met Tyr Phe His Arg Arg Asp Leu Arg 3250 3255 3260 Leu Ala Ala Asn Ala Ile Cys Ser Ala Val Pro Ser His Trp Val Pro 3265 3270 3275 3280 Thr Ser Arg Thr Thr Trp Ser Ile His Ala Lys His Glu Trp Met Thr 3285 3290 3295 Thr Glu Asp Met Leu Thr Val Trp Asn Arg Val Trp Ile Gln Glu Asn 3300 3305 3310 Pro Trp Met Glu Asp Lys Thr Pro Val Glu Ser Trp Glu Glu Ile Pro 3315 3320 3325 Tyr Leu Gly Lys Arg Glu Asp Gln Trp Cys Gly Ser Leu Ile Gly Leu 3330 3335 3340 Thr Ser Arg Ala Thr Trp Ala Lys Asn Ile Gln Ala Ala Ile Asn Gln 3345 3350 3355 3360 Val Arg Ser Leu Ile Gly Asn Glu Glu Tyr Thr Asp Tyr Met Pro Ser 3365 3370 3375Met Lys Arg Phe Arg Arg Glu Glu Glu Glu Ala Gly Val Leu Trp 3380 3385 3390 <210> 13 <211> 10696 <212> DNA <213> dengue virus <400> 13 agttgttagt ctacgtggac cgacaagaac agtttcgact cggaagcttg cttaacgtag 60 tgctgacagt tttttattag agagcagatc tctgatgaac aaccaacgga aaaagacggg 120 aaaaccgtct atcaatatgc tgaaacgcgt gagaaaccgt gtgtcaactg gatcacagtt 180 ggcgaagaga ttctcaagag gattgctgaa cggccaagga ccaatgaaat tggttatggc 240 atttatagct ttcctcagat ttctagccat tccaccgaca gcaggagtct tggctagatg 300 gggtaccttt aagaagtcgg gggctattaa ggtcttaaaa ggcttcaaga aggagatctc 360 aaacatgctg agcattatca acaaacggaa aaagacatcg ctctgtctca tgatgatgtt 420 accagcaaca cttgctttcc acttaacttc acgagatgga gagccgcgca tgattgtggg 480 gaagaatgaa agaggaaaat ccctactttt caagacagcc tctggaatca acatgtgcac 540 actcatagcc atggatctgg gagagatgtg tgatgacacg gtcacttaca aatgccccca 600 cattaccgaa gtggagcctg aagacattga ctgctggtgc aaccttacat cgacatgggt 660 gacttatgga acatgcaatc aagctggaga gcatagacgc gataagagat cagtggcgtt 720 agctccccat gttggcatgg gactggacac acgcactcaa acctggatgt cggctgaagg 780 agcttggaga caagtcgaga aggtagagac atgggccctt aggcacccag ggtttaccat 840 actagcccta tttcttgccc attacatagg cacttccttg acccagaaag tggttatttt 900 tatactatta atgctggtta ccccatccat gacaatgaga tgtgtaggag taggaaacag 960 agattttgtg gaaggcctat cgggagctac gtgggttgac gtggtgctcg agcacggtgg 1020 gtgtgtgact accatggcta agaacaagcc cacgctggac atagagcttc agaagaccga 1080 ggccacccaa ctggcgaccc taaggaagct atgcattgag ggaaaaatta ccaacataac 1140 aaccgactca agatgtccca cccaagggga agcgatttta cctgaggagc aggaccagaa 1200 ctacgtgtgt aagcatacat acgtggacag aggctgggga aacggttgtg gtttgtttgg 1260 caagggaagc ttggtgacat gcgcgaaatt tcaatgttta gaatcaatag agggaaaagt 1320 ggtgcaacat gagaacctca aatacaccgt catcatcaca gtgcacacag gagaccaaca 1380 ccaggtggga aatgaaacgc agggagtcac ggctgagata acaccccagg catcaaccgc 1440 tgaagtcatt ttacctgaat atggaaccct cgggctagaa tgctcaccac ggacaggttt 1500 ggatttcaat gaaatgatct cattgacaat gaagaacaaa gcatggatgg tacatagaca 1560 atggttcttt gacttacccc taccatggac atcaggagct acagcagaaa caccaacttg 1620 gaacaggaaa gagcttcttg tgacatttaa aaatgcacat gcaaaaaagc aagaagtagt 1680 tgttcttgga tcacaagagg gagcaatgca tacagcactg acaggagcta cagagatcca 1740 aacctcagga ggcacaagta tctttgcggg gcacttaaaa tgtagactca agatggacaa 1800 attggaactc aaggggatga gctatgcaat gtgcttgagt agctttgtgt tgaagaaaga 1860 agtctccgaa acgcagcatg ggacaatact cattaaggtt gagtacaaag gggaagatgc 1920 accctgcaag attcctttct ccacggagga tggacaagga aaagctcaca atggcagact 1980 gatcacagcc aatccagtgg tgaccaagaa ggaggagcct gtcaacattg aggctgaacc 2040 tccttttgga gaaagtaaca tagtaattgg aattggagac aaagccctga aaatcaactg 2100 gtacaagaag ggaagctcga ttgggaagat gttcgaggcc actgccagag gtgcaaggcg 2160 catggccatc ttgggagaca cagcctggga ctttggatca gtgggtggtg ttttgaattc 2220 attagggaaa atggtccacc aaatatttgg gagtgcttac acagccctat ttggtggagt 2280 ctcctggatg atgaaaattg gaataggtgt cctcttaacc tggatagggt tgaactcaaa 2340 aaatacttct atgtcatttt catgcatcgc gataggaatc attacactct atctgggagc 2400 cgtggtgcaa gctgacatgg ggtgtgtcat aaactggaaa ggcaaagaac tcaaatgtgg 2460 aagtggaatt ttcgtcacta atgaggtcca cacctggaca gagcaataca aatttcaagc 2520 agactccccc aagagactgg caacagccat tgcaggcgct tgggaaaatg gagtgtgcgg 2580 aattaggtca acaaccagaa tggagaacct cttgtggaag caaatagcca atgaactgaa 2640 ttacatatta tgggaaaaca acattaaatt aacggtagtt gtaggcgaca taactggggt 2700 cttagagcaa gggaaaagaa cactaacacc acaacccatg gagctaaaat attcttggaa 2760 aacatgggga aaggcaaaaa tagtgacagc tgaaacacaa aattcctctt tcataataga 2820 tgggccaagc acaccggagt gtccaagtgc ctcaagagca tggaatgtgt gggaggtgga 2880 ggattacggg ttcggagttt tcacaaccaa catatggctg aaactccgag aggtgtacac 2940 ccaactatgt gaccataggc taatgtcggc agccgtcaag gatgagaggg ctgtacatgc 3000 cgacatgggc tattggatag aaagccaaaa gaatgggagt tggaagctag aaaaagcatc 3060 cttcatagag gtgaaaacct gcacatggcc aaaatcacac actctctgga gcaatggtgt 3120 gctagagagt gacatgatta tcccaaagag tctagctggt cccatttcgc aacacaacca 3180 caggcccggg taccacaccc aaacggcagg accctggcac ttaggaaaat tggagctgga 3240 cttcaactat tgtgaaggaa caacagttgt catctcagaa aactgtggga caagaggccc 3300 atcattgaga acaacaacgg tgtcagggaa gttgatacac gaatggtgct gccgctcgtg 3360 cacacttcct cccctacgat acatgggaga agacggctgc tggtatggca tggaaatcag 3420 acccattaat gagaaagaag agaatatggt aaagtctcta gcctcagcag ggagtggaaa 3480 ggtggacaac ttcacaatgg gtgtcttgtg tttggcaatc ctctttgaag aggtgatgag 3540 aggaaaattt gggaaaaaac acatgattgc aggggttctc ttcacgtttg tgctcctcct 3600 ctcagggcaa ataacatgga gagacatggc gcacacactc ataatgattg ggtccaacgc 3660 ctctgacaga atggggatgg gcgtcactta cctagctcta attgcaacat ttaaaattca 3720 gccattcctg gctttgggat tcttcctgag gaaactgaca tctagagaaa atttattgct 3780 gggagttggg ttggccatgg cagcaacgtt acgactgcca gaggacattg aacagatggc 3840 gaatggaatt gctttggggc tcatggctct taaactgata acacaatttg aaacatacca 3900 actatggacg gcattagttt ccctaacgtg ttcaaataca attttcacgt tgactgttgc 3960 ctggagaaca gccactctga ttttagccgg aatttcgctt ttgccagtgt gccagtcttc 4020 gagcatgagg aaaacagatt ggctcccaat gactgtggca gctatgggag ctcaacccct 4080 accacttttt attttcagtc tgaaagatac actcaaaagg agaagctggc cactgaatga 4140 gggggtgatg gcagttggac ttgtgagcat tctagctagt tctctcctta ggaatgatgt 4200 gcctatggct ggaccattag tggctggggg cttgctgata gcgtgctacg tcataactgg 4260 cacgtcagca gacctcactg tagaaaaagc agcagatgta acatgggagg aagaggccga 4320 gcaaacagga gtgtcccaca atttaatggt cacagttgat gatgatggaa caatgagaat 4380 aaaagatgac gagactgaga acatcttaac agtgctttta aaaacagcac tactaatagt 4440 atcaggcatc tttccatact ccatacccgc aacactgttg gtctggcata cttggcaaaa 4500 gcaaacccaa agatccggcg tcctatggga cgtacccagc cccccagaga cacagaaagc 4560 ggaactggaa gaaggggtct ataggatcaa acagcaagga atttttggga aaacccaagt 4620 gggggttgga gtacagaaag aaggagtttt ccacaccatg tggcatgtca caagaggggc 4680 agtgttgaca cacaatggga aaagactgga accaaactgg gctagcgtga aaaaagatct 4740 gatttcatac ggaggaggat ggagattgag tgcacaatgg caaaaggggg aggaggtgca 4800 ggttattgcc gtagagcctg ggaagaaccc aaagaacttt caaaccatgc caggcatttt 4860 tcagacaaca acaggggaaa taggagcaat tgcactggat ttcaagcctg gaacttcagg 4920 atctcccatc ataaacagag agggaaaggt agtgggactg tatggcaatg gagtggttac 4980 aaagaatgga ggctatgtca gtggaatagc gcaaacaaat gcagaaccag atggaccgac 5040 accagagttg gaagaagaga tgttcaaaaa gcgaaatcta accataatgg atctccatcc 5100 tgggtcagga aagacgcgga aatatcttcc agctattgtt agagaggcaa tcaagagacg 5160 cttaaggact ctaattttgg caccaacaag ggtagttgca gctgagatgg aagaagcatt 5220 gaaagggctc ccaataaggt atcaaacaac tgcaacaaaa tctgaacaca caggaagaga 5280 gattgttgat ctaatgtgtc acgcaacgtt cacaatgcgc ttgctgtcac cagtcagggt 5340 tccaaactac aacttgataa taatggatga ggcccatttc acagacccag ccagtatagc 5400 ggctagaggg tacatatcaa ctcgtgtagg aatgggagag gcagccgcaa ttttcatgac 5460 agcaacaccc cctggaacag ctgatgcctt tcctcagagc aacgctccaa ttcaagatga 5520 agagagagac ataccggaac gctcatggaa ttcaggcaat gaatggatta ctgactttgt 5580 tgggaagaca gtgtggtttg tccctagcat caaagccgga aatgacatag caaactgctt 5640 gcggaaaaat ggaaaaaagg ttattcaact cagcaggaag acctttgaca cagaatatca 5700 aaagaccaaa ctgaatgatt gggactttgt ggtgacaaca gacatttcag aaatgggagc 5760 caatttcaaa gcagatagag tgatcgaccc aagaagatgt ctcaagccgg tgattttgac 5820 agatggaccc gagcgggtga tcctggctgg accaatgcca gtcaccgtag cgagcgctgc 5880 gcaaaggaga gggagagttg gcaggaaccc acaaaaagaa aatgaccagt acatattcat 5940 gggccagcct ctcaacaatg atgaagacca tgctcactgg acagaagcaa aaatgctgct 6000 ggacaacatc aacacaccag aagggattat accagctctc tttgaaccag aaagggagaa 6060 gtcagccgcc atagacggcg aataccgcct gaagggtgag tccaggaaga ctttcgtgga 6120 actcatgagg aggggtgacc tcccagtttg gctagcccat aaagtagcat cagaagggat 6180 caaatataca gatagaaaat ggtgctttga tggagaacgt aataatcaaa ttttagagga 6240 gaatatggat gtggaaatct ggacaaagga aggagaaaag aaaaaactga gacctaggtg 6300 gcttgatgcc cgcacttatt cagatccttt agcactcaaa gaattcaagg attttgcagc 6360 tggcagaaag tcaatcgccc ttgatcttgt gacagaaata ggaagagtgc cttcacactt 6420 agccccacaga acgagaaacg ccctggataa tttggtgatg ctgcacacgt cagaacatgg 6480 cggtagggcc tacaggcatg cagtggagga actaccagaa acgatggaaa cactcttact 6540 cctgggactg atgatcttgt taacaggtgg agcaatgctc ttcttgatat caggtaaagg 6600 gattggaaag acttcaatag gactcatttg tgtaattgct tccagcggca tgttatggat 6660 ggctgatgtc ccactccaat ggatcgcatc ggctatagtc ctggagtttt ttatgatggt 6720 gttgctcata ccagaaccag aaaagcagag aactccccaa gacaaccaac tcgcatatgt 6780 cgtgataggc atacttacat tggctgcaat agtagcggcc aatgaaatgg gactgttgga 6840 aactacaaag agagatttag gaatgtctaa agaaccaggt gttgtttctc caaccagcta 6900 tttggatgtg gacttgcacc cagcatcagc ctggacattg tacgccgtgg ccacaacagt 6960 aataacacca atgttgagac acaccataga gaattccaca gcaaatgtgt ctctggcagc 7020 catagctaac caggcagtgg tcctgatggg tttagacaaa ggatggccga tatcgaaaat 7080 ggacttgggc gtaccactat tggcactggg ttgctattca caagtgaacc cactaactct 7140 tgcagcggca gtacttttgc tagtcacaca ttatgcaatt ataggtccag gattgcaggc 7200 aaaagccacc cgtgaagctc agaaaaggac agctgctgga ataatgaaga atccaacggt 7260 ggatggaata atgacaatag acctagatcc tgtaatatat gattcaaaat ttgaaaagca 7320 actaggacag gtcatgctcc tggttctgtg tgcagtccaa cttttattga tgagaacatc 7380 atgggccttg tgtgaagttc taaccctagc cacaggacca ataacaacac tctgggaagg 7440 atcacctggg aagttctgga acaccacgat agctgtttcc atggcgaaca tctttagagg 7500 gagctattta gcaggagctg ggcttgcttt ttctatcatg aaatcagttg gaacaggaaa 7560 gagaggaaca gggtcacaag gtgaaacctt aggagaaaag tggaaaaaga aattaaatca 7620 gttatcccgg aaagagtttg acctttacaa gaaatccgga atcaccgaag tggatagaac 7680 agaagccaaa gaagggttaa aaagaggaga aataacacac catgccgtgt ccagaggcag 7740 cgcaaaactt caatggttcg tggagagaaa catggtcatt cctgaaggaa gagtcataga 7800 cctaggctgt ggaagaggag gctggtcata ttactgtgca ggactgaaaa aagttacaga 7860 agtgcgagga tacacaaaag gcggcccagg acacgaagaa ccagtaccta tgtctacata 7920 cggatggaac atagtcaagt taatgagtgg aaaggatgtt ttttatctgc cacctgaaaa 7980 gtgtgatacc ctattgtgtg acattggaga atcttcacca agcccaacag tggaagaaag 8040 cagaaccata agagttttga agatggttga accatggcta aagaacaacc agttttgcat 8100 taaagtattg aacccataca tgccaactgt gattgagcac ttagaaagac tacaaaggaa 8160 acatggagga atgcttgtga gaaatccact ctcacgaaac tccacgcacg aaatgtattg 8220 gatatccaat ggtacaggca atatcgtctc ttcagtcaac atggtatcca gattgctact 8280 gaacagattc acaatgacac acaggagacc caccatagag aaagatgtgg atctaggagc 8340 aggaacccga catgtcaatg cggaaccaga aacacccaac atggatgtca ttggggaaag 8400 aataaaaagg atcaaagagg agcatagttc aacatggcac tatgatgatg aaaatcctta 8460 caaaacgtgg gcttaccatg gatcctatga agtaaaagcc acaggctcag cctcctccat 8520 gataaatgga gtcgtgaaac tcctcacaaa accatgggat gtggtgccca tggtgacaca 8580 gatggcaatg acagatacaa ctccattcgg ccagcaaaga gtttttaaag agaaagtgga 8640 caccaggaca cctaggccca tgccaggaac aagaaaggtt atggagatca cagcggagtg 8700 gctttggagg accctgggaa ggaacaaaag acccagatta tgcacaaggg aggaattcac 8760 aaagaaggtc agaaccaacg cagctatggg cgctgtcttc acagaagaga accaatggga 8820 cagtgcgaga gctgctgttg aggacgaaga attttggaaa cttgtggaca gagaacgtga 8880 actccacaaa ctgggcaagt gtggaagctg cgtttacaac atgatgggca agagagagaa 8940 aaaacttgga gagtttggta aagcaaaagg cagtagggct atatggtaca tgtggttggg 9000 agccaggtac cttgagttcg aggcgctcgg attcctcaat gaagaccact ggttctcgcg 9060 tgaaaactct tacagtggag tagaaggaga aggactgcac aagctgggat acatcttgag 9120 agatatttcc aagatacccg gaggagccat gtatgctgat gacacagccg gttgggacac 9180 aagaataaca gaagatgacc tgcacaatga ggaaaaaatc acacagcaga tggaccctga 9240 acacaggcag ctagcgaacg ctatattcaa gctcacatac caaaacaaag tggtcaaagt 9300 ccaacgacca actccaaagg gcacggtaat ggacatcata tctaggaaag accaaagagg 9360 cagtggacag gtgggaactt atggtctgaa cacattcacc aacatggaag cccagctaat 9420 cagacaaatg gaaggagaag gcgtgttgtc aaaggcagac ctcgagaacc cccatccgct 9480 agagaagaaa attacacaat ggttggaaac taaaggagtg gaaaggttaa aaagaatggc 9540 catcagcggg gatgattgcg ttgtgaaacc aatcgacgac agattcgcca atgccctgct 9600 tgccctgaac gatatgggaa aggttagaaa ggacatacct caatggcagc catcaaaggg 9660 atggcatgat tggcaacagg tccccttctg ctcccaccac tttcatgaat tgatcatgaa 9720 agatggaaga aagttggtag ttccctgcag accccaggac gaactaatag gaagagcgag 9780 aatctcccaa ggagcaggat ggagccttag agaaactgca tgtctaggga aagcctacgc 9840 tcaaatgtgg gctctcatgt attttcacag aagagatctt agactagcat ccaacgccat 9900 atgttcagca gtaccagtcc actgggtccc cacgagcaga acgacatggt ctattcatgc 9960 tcaccatcag tggatgacta cagaagacat gcttactgtc tggaacaggg tgtggataga 10020 ggacaatcca tggatggaag acaaaactcc agtcacaacg tgggaagatg ttccatatct 10080 agggaagaga gaagaccaat ggtgcggatc actcataggt ctcacttcca gagcaacctg 10140 ggcccagaac atactcacag caatccaaca ggtgagaagc ctcataggca atgaagagtt 10200 tctggactac atgccttcga tgaagagatt caggaaggag gaggagtcag agggagccat 10260 ttggtaaaag caggaggtaa actgtcaggc cacattaagc cacagtacgg aagaagctgt 10320 gcagcctgtg agccccgtcc aaggacgtta aaagaagaag tcaggcccaa aagccacggt 10380 ttgagcaaac cgtgctgcct gtagctccgt cgtggggacg taaagcctgg gaggctgcaa 10440 accgtggaag ctgtacgcac ggtgtagcag actagtggtt agaggagacc cctcccatga 10500 cacaacgcag cagcggggcc cgagcactga gggaagctgt acctccttgc aaaggactag 10560 aggttagagg agaccccccg caaacaaaaa cagcatattg acgctgggag agaccagaga 10620 tcctgctgtc tcctcagcat cattccaggc acagaacgcc agaaaatgga atggtgctgt 10680 tgaatcaaca ggttct 10696 <210> 14 <211> 3390 <212> PRT <213> dengue virus <400> 14 Met Asn Asn Gln Arg Lys Lys Thr Gly Lys Pro Ser Ile Asn Met Leu 1 5 10 15 Lys Arg Val Arg Asn Arg Val Ser Thr Gly Ser Gln Leu Ala Lys Arg 20 25 30 Phe Ser Arg Gly Leu Leu Asn Gly Gln Gly Pro Met Lys Leu Val Met 35 40 45 Ala Phe Ile Ala Phe Leu Arg Phe Leu Ala Ile Pro Pro Thr Ala Gly 50 55 60 Val Leu Ala Arg Trp Gly Thr Phe Lys Lys Ser Gly Ala Ile Lys Val 65 70 75 80 Leu Lys Gly Phe Lys Lys Glu Ile Ser Asn Met Leu Ser Ile Ile Asn 85 90 95 Lys Arg Lys Lys Thr Ser Leu Cys Leu Met Met Met Leu Pro Ala Thr 100 105 110 Leu Ala Phe His Leu Thr Ser Arg Asp Gly Glu Pro Arg Met Ile Val 115 120 125 Gly Lys Asn Glu Arg Gly Lys Ser Leu Leu Phe Lys Thr Ala Ser Gly 130 135 140 Ile Asn Met Cys Thr Leu Ile Ala Met Asp Leu Gly Glu Met Cys Asp 145 150 155 160 Asp Thr Val Thr Tyr Lys Cys Pro His Ile Thr Glu Val Glu Pro Glu 165 170 175 Asp Ile Asp Cys Trp Cys Asn Leu Thr Ser Thr Trp Val Thr Tyr Gly 180 185 190 Thr Cys Asn Gln Ala Gly Glu His Arg Arg Asp Lys Arg Ser Val Ala 195 200 205 Leu Ala Pro His Val Gly Met Gly Leu Asp Thr Arg Thr Gln Thr Trp 210 215 220 Met Ser Ala Glu Gly Ala Trp Arg Gln Val Glu Lys Val Glu Thr Trp 225 230 235 240 Ala Leu Arg His Pro Gly Phe Thr Ile Leu Ala Leu Phe Leu Ala His 245 250 255 Tyr Ile Gly Thr Ser Leu Thr Gln Lys Val Val Ile Phe Ile Leu Leu 260 265 270 Met Leu Val Thr Pro Ser Met Thr Met Arg Cys Val Gly Val Gly Asn 275 280 285 Arg Asp Phe Val Glu Gly Leu Ser Gly Ala Thr Trp Val Asp Val Val 290 295 300 Leu Glu His Gly Gly Cys Val Thr Thr Met Ala Lys Asn Lys Pro Thr 305 310 315 320 Leu Asp Ile Glu Leu Gln Lys Thr Glu Ala Thr Gln Leu Ala Thr Leu 325 330 335 Arg Lys Leu Cys Ile Glu Gly Lys Ile Thr Asn Ile Thr Thr Asp Ser 340 345 350 Arg Cys Pro Thr Gln Gly Glu Ala Ile Leu Pro Glu Glu Gln Asp Gln 355 360 365 Asn Tyr Val Cys Lys His Thr Tyr Val Asp Arg Gly Trp Gly Asn Gly 370 375 380 Cys Gly Leu Phe Gly Lys Gly Ser Leu Val Thr Cys Ala Lys Phe Gln 385 390 395 400 Cys Leu Glu Ser Ile Glu Gly Lys Val Val Gln His Glu Asn Leu Lys 405 410 415 Tyr Thr Val Ile Ile Thr Val His Thr Gly Asp Gln His Gln Val Gly 420 425 430 Asn Glu Thr Gln Gly Val Thr Ala Glu Ile Thr Pro Gln Ala Ser Thr 435 440 445 Ala Glu Val Ile Leu Pro Glu Tyr Gly Thr Leu Gly Leu Glu Cys Ser 450 455 460 Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Ile Ser Leu Thr Met Lys 465 470 475 480 Asn Lys Ala Trp Met Val His Arg Gln Trp Phe Phe Asp Leu Pro Leu 485 490 495 Pro Trp Thr Ser Gly Ala Thr Ala Glu Thr Pro Thr Trp Asn Arg Lys 500 505 510 Glu Leu Leu Val Thr Phe Lys Asn Ala His Ala Lys Lys Gln Glu Val 515 520 525 Val Val Leu Gly Ser Gln Glu Gly Ala Met His Thr Ala Leu Thr Gly 530 535 540 Ala Thr Glu Ile Gln Thr Ser Gly Gly Thr Ser Ile Phe Ala Gly His 545 550 555 560 Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Glu Leu Lys Gly Met Ser 565 570 575 Tyr Ala Met Cys Leu Ser Ser Phe Val Leu Lys Lys Glu Val Ser Glu 580 585 590 Thr Gln His Gly Thr Ile Leu Ile Lys Val Glu Tyr Lys Gly Glu Asp 595 600 605 Ala Pro Cys Lys Ile Pro Phe Ser Thr Glu Asp Gly Gln Gly Lys Ala 610 615 620 His Asn Gly Arg Leu Ile Thr Ala Asn Pro Val Val Thr Lys Lys Glu 625 630 635 640 Glu Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Glu Ser Asn Ile 645 650 655 Val Ile Gly Ile Gly Asp Lys Ala Leu Lys Ile Asn Trp Tyr Lys Lys 660 665 670 Gly Ser Ser Ile Gly Lys Met Phe Glu Ala Thr Ala Arg Gly Ala Arg 675 680 685 Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Val Gly 690 695 700 Gly Val Leu Asn Ser Leu Gly Lys Met Val His Gln Ile Phe Gly Ser 705 710 715 720 Ala Tyr Thr Ala Leu Phe Gly Gly Val Ser Trp Met Met Lys Ile Gly 725 730 735 Ile Gly Val Leu Leu Thr Trp Ile Gly Leu Asn Ser Lys Asn Thr Ser 740 745 750 Met Ser Phe Ser Cys Ile Ala Ile Gly Ile Ile Thr Leu Tyr Leu Gly 755 760 765 Ala Val Val Gln Ala Asp Met Gly Cys Val Ile Asn Trp Lys Gly Lys 770 775 780 Glu Leu Lys Cys Gly Ser Gly Ile Phe Val Thr Asn Glu Val His Thr 785 790 795 800 Trp Thr Glu Gln Tyr Lys Phe Gln Ala Asp Ser Pro Lys Arg Leu Ala 805 810 815 Thr Ala Ile Ala Gly Ala Trp Glu Asn Gly Val Cys Gly Ile Arg Ser 820 825 830 Thr Thr Arg Met Glu Asn Leu Leu Trp Lys Gln Ile Ala Asn Glu Leu 835 840 845 Asn Tyr Ile Leu Trp Glu Asn Asn Ile Lys Leu Thr Val Val Val Gly 850 855 860 Asp Ile Thr Gly Val Leu Glu Gln Gly Lys Arg Thr Leu Thr Pro Gln 865 870 875 880 Pro Met Glu Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala Lys Ile 885 890 895 Val Thr Ala Glu Thr Gln Asn Ser Ser Phe Ile Ile Asp Gly Pro Ser 900 905 910 Thr Pro Glu Cys Pro Ser Ala Ser Arg Ala Trp Asn Val Trp Glu Val 915 920 925 Glu Asp Tyr Gly Phe Gly Val Phe Thr Thr Asn Ile Trp Leu Lys Leu 930 935 940 Arg Glu Val Tyr Thr Gln Leu Cys Asp His Arg Leu Met Ser Ala Ala 945 950 955 960 Val Lys Asp Glu Arg Ala Val His Ala Asp Met Gly Tyr Trp Ile Glu 965 970 975 Ser Gln Lys Asn Gly Ser Trp Lys Leu Glu Lys Ala Ser Phe Ile Glu 980 985 990 Val Lys Thr Cys Thr Trp Pro Lys Ser His Thr Leu Trp Ser Asn Gly 995 1000 1005 Val Leu Glu Ser Asp Met Ile Ile Pro Lys Ser Leu Ala Gly Pro Ile 1010 1015 1020 Ser Gln His Asn His Arg Pro Gly Tyr His Thr Gln Thr Ala Gly Pro 1025 1030 1035 1040 Trp His Leu Gly Lys Leu Gl u Leu Asp Phe Asn Tyr Cys Glu Gly Thr 1045 1050 1055 Thr Val Val Ile Ser Glu Asn Cys Gly Thr Arg Gly Pro Ser Leu Arg 1060 1065 1070 Thr Thr Thr Val Ser Gly Lys Leu Ile His Glu Trp Cys Cys Arg Ser 1075 1080 1085 Cys Thr Leu Pro Pro Leu Arg Tyr Met Gly Glu Asp Gly Cys Trp Tyr 1090 1095 1100 Gly Met Glu Ile Arg Pro Ile Asn Glu Lys Glu Glu Asn Met Val Lys 1105 1110 1115 1120 Ser Leu Ala Ser Ala Gly Ser Gly Lys Val Asp Asn Phe Thr Met Gly 1125 1130 1135 Val Leu Cys Leu Ala Ile Leu Phe Glu Glu Val Met Arg Gly Lys Phe 1140 1145 1150 Gly Lys Lys His Met Ile Ala Gly Val Leu Phe Thr Phe Val Leu Leu 1155 1160 1165 Leu Ser Gly Gln Ile Thr Trp Arg Asp Met Ala His Thr Leu Ile Met 1170 1175 1180 Ile Gly Ser Asn Ala Ser Asp Arg Met Gly Met Gly Val Thr Tyr Leu 1185 1190 1195 1200 Ala Leu Ile Ala Thr Phe Lys Ile Gln Pro Phe Leu Ala Leu Gly Phe 1205 1210 1215 Phe Leu Arg Lys Leu Thr Ser Arg Glu Asn Leu Leu Leu Gly Val Gly 1220 1225 1230 Leu Ala Met Ala Ala Thr Leu Arg Leu Pro Glu Asp Ile Glu Gln Met 1235 1240 1245 Ala Asn Gly Ile Ala Leu Gly Leu Met Ala Leu Lys Leu Ile Thr Gln 1250 1255 1260 Phe Glu Thr Tyr Gln Leu Trp Thr Ala Leu Val Ser Leu Thr Cys Ser 1265 1270 1275 1280 Asn Thr Ile Phe Thr Leu Thr Val Ala Trp Arg Thr Ala Thr Leu Ile 1285 1290 1295 Leu Ala Gly Ile Ser Leu Leu Pro Val Cys Gln Ser Ser Ser Met Arg 1300 1305 1310 Lys Thr Asp Trp Leu Pro Met Thr Val Ala Ala Met Gly Ala Gln Pro 1315 1320 1325 Leu Pro Leu Phe Ile Phe Ser Leu Lys Asp Thr Leu Lys Arg Arg Ser 1330 1335 1340 Trp Pro Leu Asn Glu Gly Val Met Ala Val Gly Leu Val Ser Ile Leu 1345 1350 1355 1360 Ala Ser Ser Leu Leu Arg Asn Asp Val Pro Met Ala Gly Pro Leu Val 1365 1370 1375 Ala Gly Gly Leu Leu Ile Ala Cys Tyr Val Ile Thr Gly Thr Ser Ala 1380 1385 1390 Asp Leu Thr Val Glu Lys Ala Ala Asp Val Thr Trp Glu Glu Glu Ala 1395 1400 1405 Glu Gln Thr Gly Val Ser His Asn Leu Met Val Thr Val Asp Asp Asp 1410 1415 1420 Gly Thr Met Arg Ile Lys Asp Asp Glu Thr Glu Asn Ile Leu Thr Val 1425 1430 1435 1440 Leu Leu Lys Thr Ala Leu Le u Ile Val Ser Gly Ile Phe Pro Tyr Ser 1445 1450 1455 Ile Pro Ala Thr Leu Leu Val Trp His Thr Trp Gln Lys Gln Thr Gln 1460 1465 1470 Arg Ser Gly Val Leu Trp Asp Val Pro Ser Pro Pro Glu Thr Gln Lys 1475 1480 1485 Ala Glu Leu Glu Glu Gly Val Tyr Arg Ile Lys Gln Gln Gly Ile Phe 1490 1495 1500 Gly Lys Thr Gln Val Gly Val Gly Val Gln Lys Glu Gly Val Phe His 1505 1510 1515 1520 Thr Met Trp His Val Thr Arg Gly Ala Val Leu Thr His Asn Gly Lys 1525 1530 1535 Arg Leu Glu Pro Asn Trp Ala Ser Val Lys Lys Asp Leu Ile Ser Tyr 1540 1545 1550 Gly Gly Gly Trp Arg Leu Ser Ala Gln Trp Gln Lys Gly Glu Glu Val 1555 1560 1565 Gln Val Ile Ala Val Glu Pro Gly Lys Asn Pro Lys Asn Phe Gln Thr 1570 1575 1580 Met Pro Gly Ile Phe Gln Thr Thr Thr Gly Glu Ile Gly Ala Ile Ala 1585 1590 1595 1600 Leu Asp Phe Lys Pro Gly Thr Ser Gly Ser Pro Ile Ile Asn Arg Glu 1605 1610 1615 Gly Lys Val Val Gly Leu Tyr Gly Asn Gly Val Val Thr Lys Asn Gly 1620 1625 1630 Gly Tyr Val Ser Gly Ile Ala Gln Thr Asn Ala Glu Pro Asp Gly Pro 1635 1640 1645 Thr Pro Glu Leu Glu Glu Glu Met Phe Lys Lys Arg Asn Leu Thr Ile 1650 1655 1660 Met Asp Leu His Pro Gly Ser Gly Lys Thr Arg Lys Tyr Leu Pro Ala 1665 1670 1675 1680 Ile Val Arg Glu Ala Ile Lys Arg Arg Leu Arg Thr Leu Ile Leu Ala 1685 1690 1695 Pro Thr Arg Val Val Ala Ala Glu Met Glu Glu Ala Leu Lys Gly Leu 1700 1705 1710 Pro Ile Arg Tyr Gln Thr Thr Ala Thr Lys Ser Glu His Thr Gly Arg 1715 1720 1725 Glu Ile Val Asp Leu Met Cys His Ala Thr Phe Thr Met Arg Leu Leu 1730 1735 1740 Ser Pro Val Arg Val Pro Asn Tyr Asn Leu Ile Ile Met Asp Glu Ala 1745 1750 1755 1760 His Phe Thr Asp Pro Ala Ser Ile Ala Ala Arg Gly Tyr Ile Ser Thr 1765 1770 1775 Arg Val Gly Met Gly Glu Ala Ala Ala Ile Phe Met Thr Ala Thr Pro 1780 1785 1790 Pro Gly Thr Ala Asp Ala Phe Pro Gln Ser Asn Ala Pro Ile Gln Asp 1795 1800 1805 Glu Glu Arg Asp Ile Pro Glu Arg Ser Trp Asn Ser Gly Asn Glu Trp 1810 1815 1820 Ile Thr Asp Phe Val Gly Lys Thr Val Trp Phe Val Pro Ser Ile Lys 1825 1830 1835 1840 Ala Gly Asn Asp Ile Ala As n Cys Leu Arg Lys Asn Gly Lys Lys Val 1845 1850 1855 Ile Gln Leu Ser Arg Lys Thr Phe Asp Thr Glu Tyr Gln Lys Thr Lys 1860 1865 1870 Leu Asn Asp Trp Asp Phe Val Val Thr Thr Asp Ile Ser Glu Met Gly 1875 1880 1885 Ala Asn Phe Lys Ala Asp Arg Val Ile Asp Pro Arg Arg Cys Leu Lys 1890 1895 1900 Pro Val Ile Leu Thr Asp Gly Pro Glu Arg Val Ile Leu Ala Gly Pro 1905 1910 1915 1920 Met Pro Val Thr Val Ala Ser Ala Ala Gln Arg Arg Gly Arg Val Gly 1925 1930 1935 Arg Asn Pro Gln Lys Glu Asn Asp Gln Tyr Ile Phe Met Gly Gln Pro 1940 1945 1950 Leu Asn Asn Asp Glu Asp His Ala His Trp Thr Glu Ala Lys Met Leu 1955 1960 1965 Leu Asp Asn Ile Asn Thr Pro Glu Gly Ile Ile Pro Ala Leu Phe Glu 1970 1975 1980 Pro Glu Arg Glu Lys Ser Ala Ala Ile Asp Gly Glu Tyr Arg Leu Lys 1985 1990 1995 2000 Gly Glu Ser Arg Lys Thr Phe Val Glu Leu Met Arg Arg Gly Asp Leu 2005 2010 2015 Pro Val Trp Leu Ala His Lys Val Ala Ser Glu Gly Ile Lys Tyr Thr 2020 2025 2030 Asp Arg Lys Trp Cys Phe Asp Gly Glu Arg Asn Asn Gln Ile Leu Glu 2035 2040 2045 Glu Asn Met Asp Val Glu Ile Trp Thr Lys Glu Gly Glu Lys Lys Lys 2050 2055 2060 Leu Arg Pro Arg Trp Leu Asp Ala Arg Thr Tyr Ser Asp Pro Leu Ala 2065 2070 2075 2080 Leu Lys Glu Phe Lys Asp Phe Ala Ala Gly Arg Lys Ser Ile Ala Leu 2085 2090 2095 Asp Leu Val Thr Glu Ile Gly Arg Val Pro Ser His Leu Ala His Arg 2100 2105 2110 Thr Arg Asn Ala Leu Asp Asn Leu Val Met Leu His Thr Ser Glu His 2115 2120 2125 Gly Gly Arg Ala Tyr Arg His Ala Val Glu Glu Leu Pro Glu Thr Met 2130 2135 2140 Glu Thr Leu Leu Leu Leu Gly Leu Met Ile Leu Leu Thr Gly Gly Ala 2145 2150 2155 2160 Met Leu Phe Leu Ile Ser Gly Lys Gly Ile Gly Lys Thr Ser Ile Gly 2165 2170 2175 Leu Ile Cys Val Ile Ala Ser Ser Gly Met Leu Trp Met Ala Asp Val 2180 2185 2190 Pro Leu Gln Trp Ile Ala Ser Ala Ile Val Leu Glu Phe Phe Met Met 2195 2200 2205 Val Leu Leu Ile Pro Glu Pro Glu Lys Gln Arg Thr Pro Gln Asp Asn 2210 2215 2220 Gln Leu Ala Tyr Val Val Ile Gly Ile Leu Thr Leu Ala Ala Ile Val 2225 2230 2235 2240 Ala Ala Asn Glu Met Gly Le u Leu Glu Thr Thr Lys Arg Asp Leu Gly 2245 2250 2255 Met Ser Lys Glu Pro Gly Val Val Ser Pro Thr Ser Tyr Leu Asp Val 2260 2265 2270 Asp Leu His Pro Ala Ser Ala Trp Thr Leu Tyr Ala Val Ala Thr 2275 2280 2285 Val Ile Thr Pro Met Leu Arg His Thr Ile Glu Asn Ser Thr Ala Asn 2290 2295 2300 Val Ser Leu Ala Ala Ile Ala Asn Gln Ala Val Val Leu Met Gly Leu 2305 2310 2315 2320 Asp Lys Gly Trp Pro Ile Ser Lys Met Asp Leu Gly Val Pro Leu Leu 2325 2330 2335 Ala Leu Gly Cys Tyr Ser Gln Val Asn Pro Leu Thr Leu Ala Ala Ala 2340 2345 2350 Val Leu Leu Leu Val Thr His Tyr Ala Ile Ile Gly Pro Gly Leu Gln 2355 2360 2365 Ala Lys Ala Thr Arg Glu Ala Gln Lys Arg Thr Ala Ala Gly Ile Met 2370 2375 2380 Lys Asn Pro Thr Val Asp Gly Ile Met Thr Ile Asp Leu Asp Pro Val 2385 2390 2395 2400 Ile Tyr Asp Ser Lys Phe Glu Lys Gln Leu Gly Gln Val Met Leu Leu 2405 2410 2415 Val Leu Cys Ala Val Gln Leu Leu Leu Met Arg Thr Ser Trp Ala Leu 2420 2425 2430 Cys Glu Val Leu Thr Leu Ala Thr Gly Pro Ile Thr Thr Leu Trp Glu 2435 2440 2445 Gly Ser Pro Gly Lys Phe Trp Asn Thr Thr Ile Ala Val Ser Met Ala 2450 2455 2460 Asn Ile Phe Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu Ala Phe Ser 2465 2470 2475 2480 Ile Met Lys Ser Val Gly Thr Gly Lys Arg Gly Thr Gly Ser Gln Gly 2485 2490 2495 Glu Thr Leu Gly Glu Lys Trp Lys Lys Lys Leu Asn Gln Leu Ser Arg 2500 2505 2510 Lys Glu Phe Asp Leu Tyr Lys Lys Ser Gly Ile Thr Glu Val Asp Arg 2515 2520 2525 Thr Glu Ala Lys Glu Gly Leu Lys Arg Gly Glu Ile Thr His Ala 2530 2535 2540 Val Ser Arg Gly Ser Ala Lys Leu Gln Trp Phe Val Glu Arg Asn Met 2545 2550 2555 2560 Val Ile Pro Glu Gly Arg Val Ile Asp Leu Gly Cys Gly Arg Gly Gly 2565 2570 2575 Trp Ser Tyr Tyr Cys Ala Gly Leu Lys Lys Val Thr Glu Val Arg Gly 2580 2585 2590 Tyr Thr Lys Gly Gly Pro Gly His Glu Glu Pro Val Pro Met Ser Thr 2595 2600 2605 Tyr Gly Trp Asn Ile Val Lys Leu Met Ser Gly Lys Asp Val Phe Tyr 2610 2615 2620 Leu Pro Pro Glu Lys Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser 2625 2630 2635 2640 Ser Pro Ser Pro Thr Val Gl u Glu Ser Arg Thr Ile Arg Val Leu Lys 2645 2650 2655 Met Val Glu Pro Trp Leu Lys Asn Asn Gln Phe Cys Ile Lys Val Leu 2660 2665 2670 Asn Pro Tyr Met Pro Thr Val Ile Glu His Leu Glu Arg Leu Gln Arg 2675 2680 2685 Lys His Gly Gly Met Leu Val Arg Asn Pro Leu Ser Arg Asn Ser Thr 2690 2695 2700 His Glu Met Tyr Trp Ile Ser Asn Gly Thr Gly Asn Ile Val Ser 2705 2710 2715 2720 Val Asn Met Val Ser Arg Leu Leu Leu Asn Arg Phe Thr Met Thr His 2725 2730 2735 Arg Arg Pro Thr Ile Glu Lys Asp Val Asp Leu Gly Ala Gly Thr Arg 2740 2745 2750 His Val Asn Ala Glu Pro Glu Thr Pro Asn Met Asp Val Ile Gly Glu 2755 2760 2765 Arg Ile Lys Arg Ile Lys Glu Glu His Ser Ser Thr Trp His Tyr Asp 2770 2775 2780 Asp Glu Asn Pro Tyr Lys Thr Trp Ala Tyr His Gly Ser Tyr Glu Val 2785 2790 2795 2800 Lys Ala Thr Gly Ser Ala Ser Ser Met Ile Asn Gly Val Val Lys Leu 2805 2810 2815 Leu Thr Lys Pro Trp Asp Val Val Pro Met Val Thr Gln Met Ala Met 2820 2825 2830 Thr Asp Thr Thr Pro Phe Gly Gln Gln Arg Val Phe Lys Glu Lys Val 2835 2840 2845 Asp Thr Arg Thr Pro Arg Pro Met Pro Gly Thr Arg Lys Val Met Glu 2850 2855 2860 Ile Thr Ala Glu Trp Leu Trp Arg Thr Leu Gly Arg Asn Lys Arg Pro 2865 2870 2875 2880 Arg Leu Cys Thr Arg Glu Glu Phe Thr Lys Lys Val Arg Thr Asn Ala 2885 2890 2895 Ala Met Gly Ala Val Phe Thr Glu Glu Asn Gln Trp Asp Ser Ala Arg 2900 2905 2910 Ala Ala Val Glu Asp Glu Glu Phe Trp Lys Leu Val Asp Arg Glu Arg 2915 2920 2925 Glu Leu His Lys Leu Gly Lys Cys Gly Ser Cys Val Tyr Asn Met Met 2930 2935 2940 Gly Lys Arg Glu Lys Lys Leu Gly Glu Phe Gly Lys Ala Lys Gly Ser 2945 2950 2955 2960 Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg Tyr Leu Glu Phe Glu 2965 2970 2975 Ala Leu Gly Leu Asn Glu Asp His Trp Phe Ser Arg Glu Asn Ser 2980 2985 2990 Tyr Ser Gly Val Glu Gly Glu Gly Leu His Lys Leu Gly Tyr Ile Leu 2995 3000 3005 Arg Asp Ile Ser Lys Ile Pro Gly Gly Ala Met Tyr Ala Asp Asp Thr 3010 3015 3020 Ala Gly Trp Asp Thr Arg Ile Thr Glu Asp Asp Leu His Asn Glu Glu 3025 3030 3035 3040 Lys Ile Thr Gln Gln Met Asp Pro Glu His Arg Gln Leu Ala Asn Ala 3045 3050 3055 Ile Phe Lys Leu Thr Tyr Gln Asn Lys Val Val Lys Val Gln Arg Pro 3060 3065 3070 Thr Pro Lys Gly Thr Val Met Asp Ile Ile Ser Arg Lys Asp Gln Arg 3075 3080 3085 Gly Ser Gly Gln Val Gly Thr Tyr Gly Leu As n Thr Phe Thr Asn Met 3090 3095 3100 Glu Ala Gln Leu Ile Arg Gln Met Glu Gly Glu Gly Val Leu Ser Lys 3105 3110 3115 3120 Ala Asp Leu Glu Asn Pro His Pro Leu Glu Lys Lys Ile Thr Gln Trp 3125 3130 3135 Leu Glu Thr Lys Gly Val Glu Arg Leu Lys Arg Met Ala Ile Ser Gly 3140 3145 3150 Asp Asp Cys Val Val Lys Pro Ile Asp Asp Arg Phe Ala Asn Ala Leu 3155 3160 3165 Leu Ala Leu Asn Asp Met Gly Lys Val Arg Lys Asp Ile Pro Gln Trp 3170 3175 3180 Gln Pro Ser Lys Gly Trp His Asp Trp Gln Gln Val Pro Phe Cys Ser 3185 3190 3195 3200 His His Phe His Glu Leu Ile Met Lys Asp Gly Arg Lys Leu Val Val 3205 3210 3215 Pro Cys Arg Pro Gln Asp Glu Leu Ile Gly Arg Ala Arg Ile Ser Gln 3220 3225 3230 Gly Ala Gly Trp Ser Leu Arg Glu Thr Ala Cys Leu Gly Lys Ala Tyr 3235 3240 3245 Ala Gln Met Trp Ala Leu Met Tyr Phe His Arg Arg Asp Leu Arg Leu 3250 3255 3260 Ala Ser Asn Ala Ile Cys Ser Ala Val Pro Val His Trp Val Pro Thr 3265 3270 3275 3280 Ser Arg Thr Thr Trp Ser Ile His Ala His His Gln Trp Met Thr Thr 3285 3290 3295 Glu Asp Met Leu Thr Val Trp Asn Arg Val Trp Ile Glu Asp Asn Pro 3300 3305 3310 Trp Met Glu Asp Lys Thr Pro Val Thr Thr Trp Glu Asp Val Pro Tyr 3315 3320 3325 Leu Gly Lys Arg Glu Asp Gln Trp Cys Gly Ser Leu Ile Gly Leu Thr 3330 3335 3340 Ser Arg Ala Thr Trp Ala Gln Asn Ile Leu Thr Ala Ile Gln Gln Val 3345 3350 3355 3360 Arg Ser Leu Ile Gly Asn Glu Glu Phe Leu Asp Tyr Met Pro Ser Met 3365 3370 3375Lys Arg Phe Arg Lys Arg Phe Arg Glu Glu Glu Ser Glu Gly Ala Ile Trp 3380 3385 3390 <210> 15 <211> 10648 <212> DNA <213> dengue virus <400> 15 agttgttagt ctgtgtggac cgacaaggac agttccaaat cggaagcttg cttaacacag 60 ttctaacagt ttgtttgaat agagagcaga tctctggaaa aatgaaccaa cgaaaaaagg 120 tggttagacc acctttcaat atgctgaaac gcgagagaaa ccgcgtatca acccctcaag 180 ggttggtgaa gagattctca accggacttt tttctgggaa aggaccctta cggatggtgc 240 tagcattcat cacgtttttg cgagtccttt ccatcccacc aacagcaggg attctgaaaa 300 gatggggaca gttgaagaaa aataaggcca tcaggatact gattggattc aggaaggaga 360 taggccgcat gctgaacatc ttgaacggga gaaaaaggtc aacgataaca ttgctgtgct 420 tgattcccac cgtaatggcg tttcacttgt caacaagaga tggcgaaccc ctcatgatag 480 tggcaaaaca tgaaaggggg agacctctct tgtttaagac aacagagggg atcaacaaat 540 gcactctcat tgccatggac ttgggtgaaa tgtgtgagga cactgtcacg tataaatgcc 600 ccttactggt caataccgaa cctgaagaca ttgattgctg gtgcaatctc acgtctacct 660 gggtcatgta tgggacatgc acccagagcg gagaacggag acgagagaag cgctcagtag 720 ctttaacacc acattcagga atgggattgg aaacaagagc tgagacatgg atgtcatcgg 780 aaggggcttg gaagcatgct cagagagtag agagctggat actcagaaac ccaggattcg 840 cgctcttggc aggatttatg gcttatatga ttgggcaaac aggaatccag cgaactgtct 900 tctttgtcct aatgatgctg gtcgccccat cctacggaat gcgatgcgta ggagtaggaa 960 acagagactt tgtggaagga gtctcaggtg gagcatgggt cgatctggtg ctagaacatg 1020 gaggatgcgt cacaaccatg gcccagggaa aaccaacctt ggattttgaa ctgactaaga 1080 caacagccaa ggaagtggct ctgttaagaa cctattgcat tgaagcctca atatcaaaca 1140 taaccacggc aacaagatgt ccaacgcaag gagagcctta tctaaaagag gaacaagacc 1200 aacagtacat ttgccggaga gatgtggtag acagagggtg gggcaatggc tgtggcttgt 1260 ttggaaaagg aggagttgtg acatgtgcga agttttcatg ttcggggaag ataacaggca 1320 atttggtcca aattgagaac cttgaataca cagtggttgt aacagtccac aatggagaca 1380 cccatgcagt aggaaatgac acatccaatc atggagttac agccacgata actcccaggt 1440 caccatcggt ggaagtcaaa ttgccggact atggagaact aacactcgat tgtgaaccca 1500 ggtctggaat tgactttaat gagatgattc tgatgaaaat gaaaaagaaa acatggcttg 1560 tgcataagca atggtttttg gatctacctc taccatggac agcaggagca gacacatcag 1620 aggttcactg gaattacaaa gagagaatgg tgacatttaa ggttcctcat gccaagagac 1680 aggatgtgac agtgctggga tctcaggaag gagccatgca ttctgccctc gctggagcca 1740 cagaagtgga ctccggtgat ggaaatcaca tgtttgcagg acatctcaag tgcaaagtcc 1800 gtatggagaa attgagaatc aagggaatgt catacacgat gtgttcagga aagttctcaa 1860 ttgacaaaga gatggcagaa acacagcatg ggacaacagt ggtgaaagtc aagtatgaag 1920 gtgctggagc tccgtgtaaa gtccccatag agataagaga tgtgaacaag gaaaaagtgg 1980 ttgggcgtat catctcatcc acccctttgg ctgagaatac caacagtgca accaacatag 2040 agttagaacc cccctttggg gacagctaca tagtgatagg tgttggaaac agtgcattaa 2100 cactccattg gttcaggaaa gggagttcca ttggcaagat gtttgagtcc acatacagag 2160 gtgcaaaacg aatggccatt ctaggtgaaa cagcttggga ttttggttcc gttggtggac 2220 tgttcacatc attgggaaag gctgtgcacc aggtttttgg aagtgtgtat acaaccatgt 2280 ttggaggagt ctcatggatg attagaatcc taattgggtt cctagtgttg tggattggca 2340 cgaactcaag gaacacttca atggctatga cgtgcatagc tgttggagga atcactctgt 2400 ttctgggctt cacagttcaa gcagacatgg gttgtgtggt gtcatggagt gggaaagaat 2460 tgaagtgtgg aagcggaatt tttgtggttg acaacgtgca cacttggaca gaacagtaca 2520 aatttcaacc ggatcccca gcgagactag cgtctgcaat attgaatgcc cacaaagatg 2580 gggtctgtgg aattagatca accacgaggc tggaaaatgt catgtggaag caaataacca 2640 acgagctaaa ttatgttctc tgggaaggag gacatgacct cactgtagtg gctggggatg 2700 tgaagggggt gttgaccaaa ggcaagagag cactcacacc cccagtgaat gatctgaaat 2760 attcatggaa gacatgggga aaagcaaaaa tcttcacccc agaagcaaga aatagcacat 2820 ttttaataga cggaccagac acctccgaat gccccaatga acgaagagca tggaactttc 2880 ttgaggtgga agactatgga tttggcatgt tcacgaccaa catatggatg aaattccgag 2940 aaggaagttc agaagtgtgt gaccacaggt taatgtcagc ggcaattaaa gatcagaaag 3000 ctgtgcatgc tgacatgggt tattggatag agagctcaaa aaaccagacc tggcagatag 3060 agaaagcatc tcttattgaa gtgaaaacat gtctgtggcc caagacccac acatgtgga 3120 gcaatggagt gctggaaagc cagatgctca ttccaaaatc atatgcgggc cctttttcac 3180 agcacaatta ccgccagggc tatgccacgc aaaccgtggg cccatggcac ttaggcaaat 3240 tagagataga ctttggagaa tgccccggaa caacagtcgc aattcaggag gattgtgacc 3300 atagaggccc atctttgagg accaccactg catctggaaa actagtcacg caatggtgct 3360 gccgctcctg cacgatgcct cccttaaggt tcttgggaga agatgggtgc tggtatggga 3420 tggagattag gcccttgagt gaaaaagaag agaacatggt caaatcacag gtaacggccg 3480 gacagggcac atcagaaact ttttctatgg gtctgttgtg cctgaccttg tttgtggaag 3540 aatgcttgag gagaagagtc actaggaaac acatgatatt ggttgtggtg atcactcttt 3600 gtgccatcat cctaggaggc ctcacatgga tggacttact acgagccctc atcatgttgg 3660 gggacactat gtctggtaga ataggaggac agatccacct agccatcatg gcagtgttca 3720 agatgtcacc aggatacgtg ctgggtgtgt ttttaaggaa actcacttca agagagacag 3780 cactaatggt aataggaatg gccatgacaa cggtgctttc aattccacat gaccttatgg 3840 aactcattga tggaatatca ctggggctaa ttttgctaaa aatagtgaca cattttgaca 3900 acacccaagt gggaacctta gccctttcct tgaccttcat aagatcaaca atgccattgg 3960 tcatggcttg gaggaccatt atggctgtgt tgtttgtggt cacactcatt cctttgtgca 4020 ggacaagctg tcttcaaaaa cagtctcatt gggtagaaat aacagcactc atcctaggag 4080 cccaagctct gccagtgtac ctaatgactc ttatgaaagg agcctcaaga agatcttggc 4140 ctcttaacga gggcataatg gctgtgggtt tggttagtct cttaggaagc gctcttttaa 4200 agaatgatgt ccctttagct ggcccaatgg tggcaggagg cttacttctg gcggcttacg 4260 tgatgagtgg tagctcagca gatctgtcac tagagaaggc cgccaatgtg cagtgggatg 4320 aaatggcaga cataacaggc tcaagcccaa tcatagaagt gaagcaggat gaagatggct 4380 ctttctccat acgggacgtc gaggaaacca atatgataac ccttttggtg aaactggcac 4440 tgataacagt gtcaggtctc taccccttgg caattccagt cacaatgacc ttatggtaca 4500 tgtggcaagt gaaaacacaa agatcaggag ccctgtggga cgtcccctca cccgctgcca 4560 ctcaaaaagc cgcactgtct gaaggagtgt acaggatcat gcaaagaggg ttatttggga 4620 aaactcaggt tggagtaggg atacacatgg aaggtgtatt tcacacaatg tggcatgtaa 4680 caagaggatc agtgatctgc catgagactg ggagattgga gccatcttgg gctgacgtca 4740 ggaatgacat gatatcatac ggtgggggat ggagacttgg agacaaatgg gacaaagaag 4800 aagatgttca ggtcctcgcc atagaaccag gaaaaaatcc taaacatgtc caaacgaaac 4860 ccggcctttt caagacccta actggagaaa ttggagcagt aacattagat ttcaaacccg 4920 gaacgtctgg ttctcccatc atcaacagga aaggaaaagt catcggactc tatggaaatg 4980 gagtagttac caaatcaggt gattacgtca gtgccataac gcaagccgaa agaattggag 5040 agccagatta tgaagtggat gaggacattt ttcgaaagaa aagattaact ataatggact 5100 tacaccccgg agctggaaag acaaaaagaa ttcttccatc aatagtgaga gaagccttaa 5160 aaaggaggct gcgaaccttg attttggctc ccacgagagt ggtggcggcc gagatggaag 5220 aggccctacg tggactgcca atccgttatc agaccccagc tgtgaaatca gaacacacag 5280 gaagagagat tgtagacctc atgtgtcatg caaccttcac aacaagactt ttgtcatcaa 5340 ccagagttcc aaattacaac ctcatagtga tggatgaagc acatttcacc gatccttcta 5400 gtgtcgcggc tagaggatac atctcgacca gggtggaaat gggagaggca gcagccatct 5460 tcatgaccgc aacccctccc ggagcgacag atccctttcc ccagagcaac agcccaatag 5520 aagacatcga gagggaaatt ccggaaaggt catggaacac agggttcgac tggataacag 5580 actaccaagg gaaaactgtg tggtttgttc ccagcataaa agctggaaat gacattgcaa 5640 attgtttgag aaagtcggga aagaaagtta tccagttgag taggaaaacc tttgatacag 5700 agtatccaaa aacgaaactc acggactggg attttgtggt cactacagac atatctgaaa 5760 tgggggccaa ttttagagct gggagagtga tagaccctag gagatgcctc aagccagtta 5820 tcctaacaga tgggccagag agagtcattt tagcaggtcc tattccagtg actccagcaa 5880 gcgctgctca gagaagaggg cgaataggaa ggaacccagc acaagaagac gaccaatacg 5940 ttttctccgg agacccacta aaaaatgatg aagatcatgc ccactggaca gaagcaaaga 6000 tgctgcttga caatatctac accccagaag ggatcattcc aacattgttt ggtccggaaa 6060 gggaaaaaac ccaagccatt gatggagagt ttcgcctcag aggggaacaa aggaagactt 6120 ttgtggaatt aatgaggaga ggagaccttc cggtgtggct gagctataag gtagcttctg 6180 ctggcatttc ttacaaagat cgggaatggt gcttcacagg ggaaaggaat aaccaaattt 6240 tagaagaaaa catggaggtt gaaatttgga ctagagaggg agaaaagaaa aagctaaggc 6300 caagatggtt agatgcacgt gtatacgctg accccatggc tttgaaggat tttaaggagt 6360 ttgctagtgg aaggaagagc ataactctcg acatcctaac agagattgcc agtttgccaa 6420 cttacctttc ctctagggcc aagctcgccc ttgataacat agtcatgctc cacacaacag 6480 aaagaggagg gagggcctac caacacgccc tgaacgaact cccggagtca ctggaaacac 6540 ttatgcttgt agctttacta ggtgctatga cagcaggtat cttcctgttt ttcatgcaag 6600 ggaaaggaat agggaaattg tcaatgggtt tgataaccat tgcggtggct agtggcttgc 6660 tctgggtagc agaaattcaa ccccagtgga tagcggcctc aatcatacta gagttttttc 6720 tcatggtact gttgataccg gaaccagaaa aacaaaggac cccacaagac aatcaattga 6780 tctacgtcat attgaccatt ctcaccatta ttggtctcat agcagccaac gagatggggc 6840 tgattgaaaa aacaaaaacg gattttgggt tttaccaggt aaaaacagaa accaccatcc 6900 tcgatgtgga cttgagacca gcttcagcat ggacgctcta tgcagtagcc accacaattc 6960 tgactcccat gctgagacac accatagaaa acacgtcggc caacctatct ctagcagcca 7020 ttgccaacca ggcggccgtc ctaatggggc ttggaaaagg atggccgctc cacagaatgg 7080 acctcggtgt gccgctgtta gcaatgggat gctattctca agtgaaccca acaactttga 7140 cagcatcctt agtcatgctt ttagtccatt atgcaataat aggtccagga ttgcaggcaa 7200 aagccacaag agaggcccag aaaaggacag ctgctgggat catgaaaaac cccacggtgg 7260 acgggataac agtaatagat ctagaaccaa tatcctatga cccaaaattt gaaaagcaat 7320 tagggcaggt catgctactc gtcttgtgtg ctggacaact actcttgatg agaacaacat 7380 gggctttctg tgaagtcttg actttggcca caggaccaat cttgaccttg tgggagggca 7440 acccgggaag gttttggaac acgaccatag ccgtatccac cgccaacatt ttcaggggaa 7500 gttacctggc gggagctgga ctggcttttt cactcataaa gaatgtacaa acccctagga 7560 ggggaactgg gaccacagga gagacactgg gagagaagtg gaagagacag ctaaactcat 7620 tagacagaaa agagtttgaa gagtataaaa gaagtggaat actagaagtg gacaggactg 7680 aagccaagtc tgccctgaaa gatgggtcta aaatcaagca tgcagtatct agagggtcca 7740 gtaagattag atggattgtt gagagaggga tggtaaagcc aaaagggaaa gttgtagatc 7800 ttggctgtgg gagaggagga tggtcttatt acatggcgac gctcaagaac gtgactgaag 7860 tgaaagggta tacaaaagga ggtccaggac atgaagaacc gattcccatg gctacttatg 7920 gctggaattt ggtcaaactc cattcagggg ttgacgtgtt ctacaaaccc acagagcaag 7980 tggacaccct gctctgtgat attggggagt catcttctaa tccaacaata gaggaaggaa 8040 gaacattaag agttttgaag atggtggagc catggctctc ttcaaaacct gaattctgca 8100 tcaaagtcct taacccctac atgccaacag tcatagaaga gctggagaaa ctgcagagaa 8160 aacatggtgg gaaccttgtc agatgcccgc tgtccaggaa ctccacccat gagatgtatt 8220 gggtgtcagg agcgtcggga aacattgtga gctctgtgaa cacaacatca aagatgttgt 8280 tgaacaggtt cacaacaagg cataggaaac ccacttatga gaaggacgta gatcttgggg 8340 caggaacgag aagtgtctcc actgaaacag aaaaaccaga catgacaatt attgggagaa 8400 ggcttcagcg attgcaagag gagcacaaag aaacctggca ttatgatcag gaaaacccat 8460 acagaacctg ggcgtatcat ggaagctatg aagctccttc gacaggctct gcatcctcca 8520 tggtgaacgg ggtagtaaaa ctgctaacaa aaccttggga tgtggttcca atggtgaccc 8580 agttagccat gacagacaca accccttttg ggcaacaaag agtgttcaaa gagaaggtgg 8640 ataccagaac accacaacca aaacccggta cacgaatggt tatgaccacg acagccaatt 8700 ggctgtgggc cctccttggg aagaagaaaa atcccagact gtgcacaagg gaagagttca 8760 tctcaaaagt tagatcaaac gcagccatag gcgcagtctt tcaggaagaa cagggatgga 8820 catcagccag tgaagctgtg aatgacagcc ggttttggga actggttgac aaagaaaggg 8880 ccctacacca ggaagggaaa tgtgaatcgt gtgtctacaa catgatggga aaacgtgaga 8940 aaaagttagg agagtttggc agagccaagg gaagccgagc aatctggtac atgtggctgg 9000 gagcgcggtt tctggaattt gaagccctgg gttttttgaa tgaagatcac tggtttggca 9060 gagaaaattc atggagtgga gtggaagggg aaggtctgca cagattggga tatatcctgg 9120 aggagataga caagaaggat ggagacctaa tgtatgctga tgacacagca ggctgggaca 9180 caagaatcac tgaggatgac cttcaaaatg aagaactgat cacggaacag atggcccccc 9240 accacaagat cctagccaaa gccattttca aactaaccta tcaaaacaaa gtggtgaaag 9300 tcctcagacc cacaccgaga ggagcggtga tggatatcat atccaggaaa gaccaaagag 9360 gtagtggaca agttggaaca tatggtttga acacattcac caacatggaa gttcaactca 9420 tccgccaaat ggaagctgaa ggagtcatca cacaagatga catgcagaac ccaaaagggt 9480 tgaaagaaag agttgagaaa tggctgaaag agtgtggtgt cgacaggtta aagaggatgg 9540 caatcagtgg agacgattgc gtggtgaagc ccctggatga gaggtttggc acttccctcc 9600 tcttcttgaa cgacatggga aaggtgagga aagacattcc gcagtgggaa ccatctaagg 9660 gatggaaaaa ctggcaagag gttccttttt gctcccacca ctttcacaag atcttcatga 9720 aggatggccg ctcactagtt gttccatgta gaaaccagga tgaactgata gggagagcca 9780 gaatctcgca gggggctgga tggagcttaa gagaaacagc ctgcctgggc aaagcttacg 9840 cccagatgtg gtcgctcatg tacttccaca gaagggatct gcgtttagcc tccatggcca 9900 tatgctcagc agttccaacg gaatggtttc caacaagcag aacaacatgg tcaatccacg 9960 ctcatcatca gtggatgacc actgaagata tgctcaaagt gtggaacaga gtgtggatag 10020 aagacaaccc taatatgact gacaagactc cagtccattc gtgggaagat ataccttacc 10080 tagggaaaag agaggatttg tggtgtggat ccctgattgg actttcttcc agagccacct 10140 gggcgaagaa cattcacacg gccataaccc aggtcagaaa cctgatcgga aaagaggaat 10200 acgtggatta catgccagta atgaaaagat acagcgctcc ttcagagagt gaaggagttc 10260 tgtaattacc aacaacaaac accaaaggct attgaagtca ggccacttgt gccacggctt 10320 gagcaaaccg tgctgcctgt agctccgcca ataatgggag gcgtgaaatc cctagggagg 10380 ccatgcgcca cggaagctgt acgcgtggca tattggacta gcggttagag gagacccctc 10440 ccatcactga caaaacgcag caaaaggggg cccgaagcca ggaggaagct gtactcctgg 10500 tggaaggact agaggttaga ggagaccccc ccaacacaaa aacagcatat tgacgctggg 10560 aaagaccaga gatcctgctg tctctgcaac atcaatccag gcacagagcg ccgcaagatg 10620 gattggtgtt gttgatccaa caggttct 10648 <210> 16 <211> 3387 <212> PRT <213> dengue virus <400> 16 Met Asn Gln Arg Lys Lys Val Val Arg Pro Pro Phe Asn Met Leu Lys 1 5 10 15 Arg Glu Arg Asn Arg Val Ser Thr Pro Gln Gly Leu Val Lys Arg Phe 20 25 30 Ser Thr Gly Leu Phe Ser Gly Lys Gly Pro Leu Arg Met Val Leu Ala 35 40 45 Phe Ile Thr Phe Leu Arg Val Leu Ser Ile Pro Pro Thr Ala Gly Ile 50 55 60 Leu Lys Arg Trp Gly Gln Leu Lys Lys Asn Lys Ala Ile Arg Ile Leu 65 70 75 80 Ile Gly Phe Arg Lys Glu Ile Gly Arg Met Leu Asn Ile Leu Asn Gly 85 90 95 Arg Lys Arg Ser Thr Ile Thr Leu Leu Cys Leu Ile Pro Thr Val Met 100 105 110 Ala Phe His Leu Ser Thr Arg Asp Gly Glu Pro Leu Met Ile Val Ala 115 120 125 Lys His Glu Arg Gly Arg Pro Leu Leu Phe Lys Thr Thr Glu Gly Ile 130 135 140 Asn Lys Cys Thr Leu Ile Ala Met Asp Leu Gly Glu Met Cys Glu Asp 145 150 155 160 Thr Val Thr Tyr Lys Cys Pro Leu Leu Val Asn Thr Glu Pro Glu Asp 165 170 175 Ile Asp Cys Trp Cys Asn Leu Thr Ser Thr Trp Val Met Tyr Gly Thr 180 185 190 Cys Thr Gln Ser Gly Glu Arg Arg Arg Glu Lys Arg Ser Val Ala Leu 195 200 205 Thr Pro His Ser Gly Met Gly Leu Glu Thr Arg Ala Glu Thr Trp Met 210 215 220 Ser Ser Glu Gly Ala Trp Lys His Ala Gln Arg Val Glu Ser Trp Ile 225 230 235 240 Leu Arg Asn Pro Gly Phe Ala Leu Leu Ala Gly Phe Met Ala Tyr Met 245 250 255 Ile Gly Gln Thr Gly Ile Gln Arg Thr Val Phe Phe Val Leu Met Met 260 265 270 Leu Val Ala Pro Ser Tyr Gly Met Arg Cys Val Gly Val Gly Asn Arg 275 280 285 Asp Phe Val Glu Gly Val Ser Gly Gly Ala Trp Val Asp Leu Val Leu 290 295 300 Glu His Gly Gly Cys Val Thr Thr Met Ala Gln Gly Lys Pro Thr Leu 305 310 315 320 Asp Phe Glu Leu Thr Lys Thr Thr Ala Lys Glu Val Ala Leu Leu Arg 325 330 335 Thr Tyr Cys Ile Glu Ala Ser Ile Ser Asn Ile Thr Thr Ala Thr Arg 340 345 350 Cys Pro Thr Gln Gly Glu Pro Tyr Leu Lys Glu Glu Gln Asp Gln Gln 355 360 365 Tyr Ile Cys Arg Arg Asp Val Val Asp Arg Gly Trp Gly Asn Gly Cys 370 375 380 Gly Leu Phe Gly Lys Gly Gly Val Val Thr Cys Ala Lys Phe Ser Cys 385 390 395 400 Ser Gly Lys Ile Thr Gly Asn Leu Val Gln Ile Glu Asn Leu Glu Tyr 405 410 415 Thr Val Val Val Thr Val His Asn Gly Asp Thr His Ala Val Gly Asn 420 425 430 Asp Thr Ser Asn His Gly Val Thr Ala Thr Ile Thr Pro Arg Ser Pro 435 440 445 Ser Val Glu Val Lys Leu Pro Asp Tyr Gly Glu Leu Thr Leu Asp Cys 450 455 460 Glu Pro Arg Ser Gly Ile Asp Phe Asn Glu Met Ile Leu Met Lys Met 465 470 475 480 Lys Lys Lys Thr Trp Leu Val His Lys Gln Trp Phe Leu Asp Leu Pro 485 490 495 Leu Pro Trp Thr Ala Gly Ala Asp Thr Ser Glu Val His Trp Asn Tyr 500 505 510 Lys Glu Arg Met Val Thr Phe Lys Val Pro His Ala Lys Arg Gln Asp 515 520 525 Val Thr Val Leu Gly Ser Gln Glu Gly Ala Met His Ser Ala Leu Ala 530 535 540 Gly Ala Thr Glu Val Asp Ser Gly Asp Gly Asn His Met Phe Ala Gly 545 550 555 560 His Leu Lys Cys Lys Val Arg Met Glu Lys Leu Arg Ile Lys Gly Met 565 570 575 Ser Tyr Thr Met Cys Ser Gly Lys Phe Ser Ile Asp Lys Glu Met Ala 580 585 590 Glu Thr Gln His Gly Thr Thr Val Val Lys Val Lys Tyr Glu Gly Ala 595 600 605 Gly Ala Pro Cys Lys Val Pro Ile Glu Ile Arg Asp Val Asn Lys Glu 610 615 620 Lys Val Val Gly Arg Ile Ile Ser Ser Thr Pro Leu Ala Glu Asn Thr 625 630 635 640 Asn Ser Ala Thr Asn Ile Glu Leu Glu Pro Pro Phe Gly Asp Ser Tyr 645 650 655 Ile Val Ile Gly Val Gly Asn Ser Ala Leu Thr Leu His Trp Phe Arg 660 665 670 Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ser Thr Tyr Arg Gly Ala 675 680 685 Lys Arg Met Ala Ile Leu Gly Glu Thr Ala Trp Asp Phe Gly Ser Val 690 695 700 Gly Gly Leu Phe Thr Ser Leu Gly Lys Ala Val His Gln Val Phe Gly 705 710 715 720 Ser Val Tyr Thr Thr Met Phe Gly Gly Val Ser Trp Met Ile Arg Ile 725 730 735 Leu Ile Gly Phe Leu Val Leu Trp Ile Gly Thr Asn Ser Arg Asn Thr 740 745 750 Ser Met Ala Met Thr Cys Ile Ala Val Gly Gly Ile Thr Leu Phe Leu 755 760 765 Gly Phe Thr Val Gln Ala Asp Met Gly Cys Val Val Ser Trp Ser Gly 770 775 780 Lys Glu Leu Lys Cys Gly Ser Gly Ile Phe Val Val Asp Asn Val His 785 790 795 800 Thr Trp Thr Glu Gln Tyr Lys Phe Gln Pro Glu Ser Pro Ala Arg Leu 805 810 815 Ala Ser Ala Ile Leu Asn Ala His Lys Asp Gly Val Cys Gly Ile Arg 820 825 830 Ser Thr Thr Arg Leu Glu Asn Val Met Trp Lys Gln Ile Thr Asn Glu 835 840 845 Leu Asn Tyr Val Leu Trp Glu Gly Gly His Asp Leu Thr Val Val Ala 850 855 860 Gly Asp Val Lys Gly Val Leu Thr Lys Gly Lys Arg Ala Leu Thr Pro 865 870 875 880 Pro Val Asn Asp Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala Lys 885 890 895 Ile Phe Thr Pro Glu Ala Arg Asn Ser Thr Phe Leu Ile Asp Gly Pro 900 905 910 Asp Thr Ser Glu Cys Pro Asn Glu Arg Arg Ala Trp Asn Phe Leu Glu 915 920 925 Val Glu Asp Tyr Gly Phe Gly Met Phe Thr Thr Asn Ile Trp Met Lys 930 935 940 Phe Arg Glu Gly Ser Ser Glu Val Cys Asp His Arg Leu Met Ser Ala 945 950 955 960 Ala Ile Lys Asp Gln Lys Ala Val His Ala Asp Met Gly Tyr Trp Ile 965 970 975 Glu Ser Ser Lys Asn Gln Thr Trp Gln Ile Glu Lys Ala Ser Leu Ile 980 985 990 Glu Val Lys Thr Cys Leu Trp Pro Lys Thr His Thr Leu Trp Ser Asn 995 1000 1005 Gly Val Leu Glu Ser Gln Met Leu Ile Pro Lys Ser Tyr Ala Gly Pro 1010 1015 1020 Phe Ser Gln His Asn Tyr Arg Gln Gly Tyr Ala Thr Gln Thr Val Gly 1025 1030 1035 1040 Pro Trp His Leu Gly Lys Le u Glu Ile Asp Phe Gly Glu Cys Pro Gly 1045 1050 1055 Thr Thr Val Ala Ile Gln Glu Asp Cys Asp His Arg Gly Pro Ser Leu 1060 1065 1070 Arg Thr Thr Thr Ala Ser Gly Lys Leu Val Thr Gln Trp Cys Cys Arg 1075 1080 1085 Ser Cys Thr Met Pro Pro Leu Arg Phe Leu Gly Glu Asp Gly Cys Trp 1090 1095 1100 Tyr Gly Met Glu Ile Arg Pro Leu Ser Glu Lys Glu Glu Asn Met Val 1105 1110 1115 1120 Lys Ser Gln Val Thr Ala Gly Gln Gly Thr Ser Glu Thr Phe Ser Met 1125 1130 1135 Gly Leu Leu Cys Leu Thr Leu Phe Val Glu Glu Cys Leu Arg Arg Arg 1140 1145 1150 Val Thr Arg Lys His Met Ile Leu Val Val Val Ile Thr Leu Cys Ala 1155 1160 1165 Ile Ile Leu Gly Gly Leu Thr Trp Met Asp Leu Leu Arg Ala Leu Ile 1170 1175 1180 Met Leu Gly Asp Thr Met Ser Gly Arg Ile Gly Gly Gln Ile His Leu 1185 1190 1195 1200 Ala Ile Met Ala Val Phe Lys Met Ser Pro Gly Tyr Val Leu Gly Val 1205 1210 1215 Phe Leu Arg Lys Leu Thr Ser Arg Glu Thr Ala Leu Met Val Ile Gly 1220 1225 1230 Met Ala Met Thr Thr Val Leu Ser Ile Pro His Asp Leu Met Glu Leu 1235 1240 1245 Ile Asp Gly Ile Ser Leu Gly Leu Ile Leu Leu Lys Ile Val Thr His 1250 1255 1260 Phe Asp Asn Thr Gln Val Gly Thr Leu Ala Leu Ser Leu Thr Phe Ile 1265 1270 1275 1280 Arg Ser Thr Met Pro Leu Val Met Ala Trp Arg Thr Ile Met Ala Val 1285 1290 1295 Leu Phe Val Val Thr Leu Ile Pro Leu Cys Arg Thr Ser Cys Leu Gln 1300 1305 1310 Lys Gln Ser His Trp Val Glu Ile Thr Ala Leu Ile Leu Gly Ala Gln 1315 1320 1325 Ala Leu Pro Val Tyr Leu Met Thr Leu Met Lys Gly Ala Ser Arg Arg 1330 1335 1340 Ser Trp Pro Leu Asn Glu Gly Ile Met Ala Val Gly Leu Val Ser Leu 1345 1350 1355 1360 Leu Gly Ser Ala Leu Leu Lys Asn Asp Val Pro Leu Ala Gly Pro Met 1365 1370 1375 Val Ala Gly Gly Leu Leu Leu Ala Ala Tyr Val Met Ser Gly Ser Ser 1380 1385 1390 Ala Asp Leu Ser Leu Glu Lys Ala Ala Asn Val Gln Trp Asp Glu Met 1395 1400 1405 Ala Asp Ile Thr Gly Ser Ser Pro Ile Ile Glu Val Lys Gln Asp Glu 1410 1415 1420 Asp Gly Ser Phe Ser Ile Arg Asp Val Glu Glu Thr Asn Met Ile Thr 1425 1430 1435 1440 Leu Leu Val Lys Leu Ala Le u Ile Thr Val Ser Gly Leu Tyr Pro Leu 1445 1450 1455 Ala Ile Pro Val Thr Met Thr Leu Trp Tyr Met Trp Gln Val Lys Thr 1460 1465 1470 Gln Arg Ser Gly Ala Leu Trp Asp Val Pro Ser Pro Ala Ala Thr Gln 1475 1480 1485 Lys Ala Ala Leu Ser Glu Gly Val Tyr Arg Ile Met Gln Arg Gly Leu 1490 1495 1500 Phe Gly Lys Thr Gln Val Gly Val Gly Ile His Met Glu Gly Val Phe 1505 1510 1515 1520 His Thr Met Trp His Val Thr Arg Gly Ser Val Ile Cys His Glu Thr 1525 1530 1535 Gly Arg Leu Glu Pro Ser Trp Ala Asp Val Arg Asn Asp Met Ile Ser 1540 1545 1550 Tyr Gly Gly Gly Trp Arg Leu Gly Asp Lys Trp Asp Lys Glu Asp 1555 1560 1565 Val Gln Val Leu Ala Ile Glu Pro Gly Lys Asn Pro Lys His Val Gln 1570 1575 1580 Thr Lys Pro Gly Leu Phe Lys Thr Leu Thr Gly Glu Ile Gly Ala Val 1585 1590 1595 1600 Thr Leu Asp Phe Lys Pro Gly Thr Ser Gly Ser Pro Ile Ile Asn Arg 1605 1610 1615 Lys Gly Lys Val Ile Gly Leu Tyr Gly Asn Gly Val Val Thr Lys Ser 1620 1625 1630 Gly Asp Tyr Val Ser Ala Ile Thr Gln Ala Glu Arg Ile Gly Glu Pro 1635 1640 1645 Asp Tyr Glu Val Asp Glu Asp Ile Phe Arg Lys Lys Arg Leu Thr Ile 1650 1655 1660 Met Asp Leu His Pro Gly Ala Gly Lys Thr Lys Arg Ile Leu Pro Ser 1665 1670 1675 1680 Ile Val Arg Glu Ala Leu Lys Arg Arg Leu Arg Thr Leu Ile Leu Ala 1685 1690 1695 Pro Thr Arg Val Val Ala Ala Glu Met Glu Glu Ala Leu Arg Gly Leu 1700 1705 1710 Pro Ile Arg Tyr Gln Thr Pro Ala Val Lys Ser Glu His Thr Gly Arg 1715 1720 1725 Glu Ile Val Asp Leu Met Cys His Ala Thr Phe Thr Thr Arg Leu Leu 1730 1735 1740 Ser Ser Thr Arg Val Pro Asn Tyr Asn Leu Ile Val Met Asp Glu Ala 1745 1750 1755 1760 His Phe Thr Asp Pro Ser Ser Val Ala Ala Arg Gly Tyr Ile Ser Thr 1765 1770 1775 Arg Val Glu Met Gly Glu Ala Ala Ala Ile Phe Met Thr Ala Thr Pro 1780 1785 1790 Pro Gly Ala Thr Asp Pro Phe Pro Gln Ser Asn Ser Pro Ile Glu Asp 1795 1800 1805 Ile Glu Arg Glu Ile Pro Glu Arg Ser Trp Asn Thr Gly Phe Asp Trp 1810 1815 1820 Ile Thr Asp Tyr Gln Gly Lys Thr Val Trp Phe Val Pro Ser Ile Lys 1825 1830 1835 1840 Ala Gly Asn Asp Ile Ala As n Cys Leu Arg Lys Ser Gly Lys Lys Val 1845 1850 1855 Ile Gln Leu Ser Arg Lys Thr Phe Asp Thr Glu Tyr Pro Lys Thr Lys 1860 1865 1870 Leu Thr Asp Trp Asp Phe Val Val Thr Thr Asp Ile Ser Glu Met Gly 1875 1880 1885 Ala Asn Phe Arg Ala Gly Arg Val Ile Asp Pro Arg Arg Cys Leu Lys 1890 1895 1900 Pro Val Ile Leu Thr Asp Gly Pro Glu Arg Val Ile Leu Ala Gly Pro 1905 1910 1915 1920 Ile Pro Val Thr Pro Ala Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly 1925 1930 1935 Arg Asn Pro Ala Gln Glu Asp Asp Gln Tyr Val Phe Ser Gly Asp Pro 1940 1945 1950 Leu Lys Asn Asp Glu Asp His Ala His Trp Thr Glu Ala Lys Met Leu 1955 1960 1965 Leu Asp Asn Ile Tyr Thr Pro Glu Gly Ile Ile Pro Thr Leu Phe Gly 1970 1975 1980 Pro Glu Arg Glu Lys Thr Gln Ala Ile Asp Gly Glu Phe Arg Leu Arg 1985 1990 1995 2000 Gly Glu Gln Arg Lys Thr Phe Val Glu Leu Met Arg Arg Gly Asp Leu 2005 2010 2015 Pro Val Trp Leu Ser Tyr Lys Val Ala Ser Ala Gly Ile Ser Tyr Lys 2020 2025 2030 Asp Arg Glu Trp Cys Phe Thr Gly Glu Arg Asn Asn Gln Ile Leu Glu 2035 2040 2045 Glu Asn Met Glu Val Glu Ile Trp Thr Arg Glu Gly Glu Lys Lys Lys 2050 2055 2060 Leu Arg Pro Arg Trp Leu Asp Ala Arg Val Tyr Ala Asp Pro Met Ala 2065 2070 2075 2080 Leu Lys Asp Phe Lys Glu Phe Ala Ser Gly Arg Lys Ser Ile Thr Leu 2085 2090 2095 Asp Ile Leu Thr Glu Ile Ala Ser Leu Pro Thr Tyr Leu Ser Ser Arg 2100 2105 2110 Ala Lys Leu Ala Leu Asp Asn Ile Val Met Leu His Thr Thr Glu Arg 2115 2120 2125 Gly Gly Arg Ala Tyr Gln His Ala Leu Asn Glu Leu Pro Glu Ser Leu 2130 2135 2140 Glu Thr Leu Met Leu Val Ala Leu Leu Gly Ala Met Thr Ala Gly Ile 2145 2150 2155 2160 Phe Leu Phe Phe Met Gln Gly Lys Gly Ile Gly Lys Leu Ser Met Gly 2165 2170 2175 Leu Ile Thr Ile Ala Val Ala Ser Gly Leu Leu Trp Val Ala Glu Ile 2180 2185 2190 Gln Pro Gln Trp Ile Ala Ala Ser Ile Ile Leu Glu Phe Phe Leu Met 2195 2200 2205 Val Leu Leu Ile Pro Glu Pro Glu Lys Gln Arg Thr Pro Gln Asp Asn 2210 2215 2220 Gln Leu Ile Tyr Val Ile Leu Thr Ile Leu Thr Ile Ile Gly Leu Ile 2225 2230 2235 2240 Ala Ala Asn Glu Met Gly Le u Ile Glu Lys Thr Lys Thr Asp Phe Gly 2245 2250 2255 Phe Tyr Gln Val Lys Thr Glu Thr Thr Ile Leu Asp Val Asp Leu Arg 2260 2265 2270 Pro Ala Ser Ala Trp Thr Leu Tyr Ala Val Ala Thr Thr Ile Leu Thr 2275 2280 2285 Pro Met Leu Arg His Thr Ile Glu Asn Thr Ser Ala Asn Leu Ser Leu 2290 2295 2300 Ala Ala Ile Ala Asn Gln Ala Ala Val Leu Met Gly Leu Gly Lys Gly 2305 2310 2315 2320 Trp Pro Leu His Arg Met Asp Leu Gly Val Pro Leu Leu Ala Met Gly 2325 2330 2335 Cys Tyr Ser Gln Val Asn Pro Thr Thr Leu Thr Ala Ser Leu Val Met 2340 2345 2350 Leu Leu Val His Tyr Ala Ile Ile Gly Pro Gly Leu Gln Ala Lys Ala 2355 2360 2365 Thr Arg Glu Ala Gln Lys Arg Thr Ala Ala Gly Ile Met Lys Asn Pro 2370 2375 2380 Thr Val Asp Gly Ile Thr Val Ile Asp Leu Glu Pro Ile Ser Tyr Asp 2385 2390 2395 2400 Pro Lys Phe Glu Lys Gln Leu Gly Gln Val Met Leu Leu Val Leu Cys 2405 2410 2415 Ala Gly Gln Leu Leu Leu Met Arg Thr Thr Trp Ala Phe Cys Glu Val 2420 2425 2430 Leu Thr Leu Ala Thr Gly Pro Ile Leu Thr Leu Trp Glu Gly Asn Pro 2435 2440 2445 Gly Arg Phe Trp Asn Thr Thr Ile Ala Val Ser Thr Ala Asn Ile Phe 2450 2455 2460 Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu Ala Phe Ser Leu Ile Lys 2465 2470 2475 2480 Asn Val Gln Thr Pro Arg Arg Gly Thr Gly Thr Thr Gly Glu Thr Leu 2485 2490 2495 Gly Glu Lys Trp Lys Arg Gln Leu Asn Ser Leu Asp Arg Lys Glu Phe 2500 2505 2510 Glu Glu Tyr Lys Arg Ser Gly Ile Leu Glu Val Asp Arg Thr Glu Ala 2515 2520 2525 Lys Ser Ala Leu Lys Asp Gly Ser Lys Ile Lys His Ala Val Ser Arg 2530 2535 2540 Gly Ser Ser Lys Ile Arg Trp Ile Val Glu Arg Gly Met Val Lys Pro 2545 2550 2555 2560 Lys Gly Lys Val Val Asp Leu Gly Cys Gly Arg Gly Gly Trp Ser Tyr 2565 2570 2575 Tyr Met Ala Thr Leu Lys Asn Val Thr Glu Val Lys Gly Tyr Thr Lys 2580 2585 2590 Gly Gly Pro Gly His Glu Glu Pro Ile Pro Met Ala Thr Tyr Gly Trp 2595 2600 2605 Asn Leu Val Lys Leu His Ser Gly Val Asp Val Phe Tyr Lys Pro Thr 2610 2615 2620 Glu Gln Val Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser Ser Asn 2625 2630 2635 2640 Pro Thr Ile Glu Glu Gly Ar g Thr Leu Arg Val Leu Lys Met Val Glu 2645 2650 2655 Pro Trp Leu Ser Ser Lys Pro Glu Phe Cys Ile Lys Val Leu Asn Pro 2660 2665 2670 Tyr Met Pro Thr Val Ile Glu Glu Leu Glu Lys Leu Gln Arg Lys His 2675 2680 2685 Gly Gly Asn Leu Val Arg Cys Pro Leu Ser Arg Asn Ser Thr His Glu 2690 2695 2700 Met Tyr Trp Val Ser Gly Ala Ser Gly Asn Ile Val Ser Ser Val Asn 2705 2710 2715 2720 Thr Thr Ser Lys Met Leu Leu Asn Arg Phe Thr Thr Arg His Arg Lys 2725 2730 2735 Pro Thr Tyr Glu Lys Asp Val Asp Leu Gly Ala Gly Thr Arg Ser Val 2740 2745 2750 Ser Thr Glu Thr Glu Lys Pro Asp Met Thr Ile Ile Gly Arg Arg Leu 2755 2760 2765 Gln Arg Leu Gln Glu Glu His Lys Glu Thr Trp His Tyr Asp Gln Glu 2770 2775 2780 Asn Pro Tyr Arg Thr Trp Ala Tyr His Gly Ser Tyr Glu Ala Pro Ser 2785 2790 2795 2800 Thr Gly Ser Ala Ser Met Val Asn Gly Val Val Lys Leu Leu Thr 2805 2810 2815 Lys Pro Trp Asp Val Val Pro Met Val Thr Gln Leu Ala Met Thr Asp 2820 2825 2830 Thr Thr Pro Phe Gly Gln Gln Arg Val Phe Lys Glu Lys Val Asp Thr 2835 2840 2845 Arg Thr Pro Gln Pro Lys Pro Gly Thr Arg Met Val Met Thr Thr Thr 2850 2855 2860 Ala Asn Trp Leu Trp Ala Leu Leu Gly Lys Lys Lys Asn Pro Arg Leu 2865 2870 2875 2880 Cys Thr Arg Glu Glu Phe Ile Ser Lys Val Arg Ser Asn Ala Ala Ile 2885 2890 2895 Gly Ala Val Phe Gln Glu Glu Gln Gly Trp Thr Ser Ala Ser Glu Ala 2900 2905 2910 Val Asn Asp Ser Arg Phe Trp Glu Leu Val Asp Lys Glu Arg Ala Leu 2915 2920 2925 His Gln Glu Gly Lys Cys Glu Ser Cys Val Tyr Asn Met Met Gly Lys 2930 2935 2940 Arg Glu Lys Lys Leu Gly Glu Phe Gly Arg Ala Lys Gly Ser Arg Ala 2945 2950 2955 2960 Ile Trp Tyr Met Trp Leu Gly Ala Arg Phe Leu Glu Phe As Glu Ala Leu 2965 2970 2975 Gly Phe Leu Glu Asp His Trp Phe Gly Arg Glu Asn Ser Trp Ser 2980 2985 2990 Gly Val Glu Gly Glu Gly Leu His Arg Leu Gly Tyr Ile Leu Glu Glu 2995 3000 3005 Ile Asp Lys Lys Asp Gly Asp Leu Met Tyr Ala Asp Asp Thr Ala Gly 3010 3015 3020 Trp Asp Thr Arg Ile Thr Glu Asp Asp Leu Gln Asn Glu Glu Leu Ile 3025 3030 3035 3040 Thr Glu Gln Met Ala Pro His His Lys Ile Leu Ala Lys Ala Ile Phe 3045 3050 3055 Lys Leu Thr Tyr Gln Asn Lys Val Val Lys Val Leu Arg Pro Thr Pro 3060 3065 3070 Arg Gly Ala Val Met Asp Ile Ile Ser Arg Lys Asp Gln Arg Gly Ser 3075 3080 3085 Gly Gln Val Gly Thr Tyr Gly Leu Asn Thr Ph e Thr Asn Met Glu Val 3090 3095 3100 Gln Leu Ile Arg Gln Met Glu Ala Glu Gly Val Ile Thr Gln Asp Asp 3105 3110 3115 3120 Met Gln Asn Pro Lys Gly Leu Lys Glu Arg Val Glu Lys Trp Leu Lys 3125 3130 3135 Glu Cys Gly Val Asp Arg Leu Lys Arg Met Ala Ile Ser Gly Asp Asp 3140 3145 3150 Cys Val Val Lys Pro Leu Asp Glu Arg Phe Gly Thr Ser Leu Leu Phe 3155 3160 3165 Leu Asn Asp Met Gly Lys Val Arg Lys Asp Ile Pro Gln Trp Glu Pro 3170 3175 3180 Ser Lys Gly Trp Lys Asn Trp Gln Glu Val Pro Phe Cys Ser His His 3185 3190 3195 3200 Phe His Lys Ile Phe Met Lys Asp Gly Arg Ser Leu Val Val Pro Cys 3205 3210 3215 Arg Asn Gln Asp Glu Leu Ile Gly Arg Ala Arg Ile Ser Gln Gly Ala 3220 3225 3230 Gly Trp Ser Leu Arg Glu Thr Ala Cys Leu Gly Lys Ala Tyr Ala Gln 3235 3240 3245 Met Trp Ser Leu Met Tyr Phe His Arg Arg Asp Leu Arg Leu Ala Ser 3250 3255 3260 Met Ala Ile Cys Ser Ala Val Pro Thr Glu Trp Phe Pro Thr Ser Arg 3265 3270 3275 3280 Thr Thr Trp Ser Ile His Ala His His Gln Trp Met Thr Thr Glu Asp 3285 3290 3295 Met Leu Lys Val Trp Asn Arg Val Trp Ile Glu Asp Asn Pro Asn Met 3300 3305 3310 Thr Asp Lys Thr Pro Val His Ser Trp Glu Asp Ile Pro Tyr Leu Gly 3315 3320 3325 Lys Arg Glu Asp Leu Trp Cys Gly Ser Leu Ile Gly Leu Ser Ser Arg 3330 3335 3340 Ala Thr Trp Ala Lys Asn Ile His Thr Ala Ile Thr Gln Val Arg Asn 3345 3350 3355 3360 Leu Ile Gly Lys Glu Glu Tyr Val Asp Tyr Met Pro Val Met Lys Arg 3365 3370 3375Tyr Ser Ala Pro Ser Glu Ser Glu Gly Val Leu 3380 3385

Claims (37)

A unit dose of a hepatitis A vaccine and a dengue vaccine composition for use in a method of preventing hepatitis A and dengue disease in a subject or population of subjects, comprising:
The method comprises administering unit doses of the hepatitis A vaccine and the dengue vaccine composition simultaneously on the same day, the unit dose comprising a tetravalent dengue virus composition comprising four wildly attenuated dengue virus strains A unit dose of a hepatitis A vaccine and a dengue vaccine composition. The method according to claim 1,
wherein the hepatitis A vaccine is an inactivated virus vaccine. The method according to claim 1 or 2,
The dengue vaccine composition comprises, upon reconstitution with 0.5 mL of a pharmaceutically acceptable diluent, hepatitis A vaccine and a unit dose:
(i) a chimeric dengue serotype 2/1 strain at a concentration of at least 3.3 log10 pfu/0.5 mL;
(ii) a dengue serotype 2 strain at a concentration of at least 2.7 log10 pfu/0.5 mL;
(iii) a chimeric dengue serotype 2/3 strain at a concentration of at least 4.0 log10 pfu/0.5 mL, and
(iv) a chimeric dengue serotype 2/4 strain at a concentration of at least 4.5 log10 pfu/0.5 mL. 4. The method according to any one of claims 1 to 3,
wherein said subject population or subjects are seronegative for all dengue fever serotypes. 5. The method according to any one of claims 1 to 4,
The unit dose of said dengue vaccine composition is administered by subcutaneous injection, said hepatitis A vaccine is administered by intramuscular injection, said injection is preferably administered to the arm, more preferably to the deltoid region of the arm, Hepatitis A vaccine and unit dose. 6. The method of claim 5,
The unit dose of said dengue vaccine composition and said hepatitis A vaccine are administered at different anatomical sites, eg, to the contralateral arm. 7. The method according to any one of claims 1 to 6,
A hepatitis A vaccine and a unit dose, wherein the unit doses of two of said dengue vaccine compositions are administered within 12 months or more, or within 6 months, or within 3 months. 8. The method of claim 7,
In particular, hepatitis A vaccine and unit dose comprising administering two unit doses of said dengue vaccine composition and one dose of said hepatitis A vaccine according to the following schedule:
- on day 0 a first unit dose of said dengue vaccine composition and a first concurrent administration of said hepatitis A vaccine, and
- a second administration of a second unit dose of said dengue vaccine composition after said first concurrent administration, eg about 3 months later. 9. The method according to any one of claims 1 to 8,
wherein said subject population or subjects are between 2 and 60 years of age. 10. The method according to any one of claims 1 to 9,
wherein said subject population or subjects are from an area endemic to dengue fever, hepatitis A vaccine and unit dose. 10. The method according to any one of claims 1 to 9,
Hepatitis A vaccine and unit dose, wherein said subject population or subjects are from a dengue non-endemic area, preferably a dengue fever non-endemic and hepatitis A non-endemic area. 12. The method according to any one of claims 1 to 11,
wherein said hepatitis A vaccine comprises hepatitis A virus derived from hepatitis A virus strain HM-175. 13. The method according to any one of claims 1 to 12,
The hepatitis A vaccine and unit dose, wherein the hepatitis A vaccine comprises inactivated hepatitis A virus, and wherein the inactivated hepatitis A virus is derived from hepatitis A virus strain HM-175. 14. The method according to any one of claims 1 to 13,
wherein the hepatitis A vaccine comprises an inactivated hepatitis A virus, wherein the inactivated hepatitis A virus is adsorbed on aluminum. 15. The method of claim 14,
wherein the aluminum is aluminum hydroxide or aluminum hydroxyphosphate sulfate. 16. The method according to any one of claims 1 to 15,
The hepatitis A vaccine comprises an inactivated hepatitis A virus, and the hepatitis A vaccine comprises a phosphate buffered saline solution and an excipient dissolved therein in the form of amino acids and polysorbate. Volume. 17. The method of any one of claims 1 to 16,
wherein said hepatitis A vaccine comprises a hepatitis A virus expressing a viral antigen at a concentration ranging from 500 ELISA Units (EL.U.) to 2000 ELISA Units (EL.U.). 18. The method according to any one of claims 1 to 17,
The method comprises determining whether there was a prior dengue infection and/or a prior hepatitis A infection in the subject population or subject prior to administration of the hepatitis A vaccine and prior to administration of a unit dose of the dengue vaccine composition. not or
Hepatitis A vaccine and unit dose, wherein the hepatitis A serum status and/or the dengue fever serum status of the subject population or subjects are unknown prior to administration of the hepatitis A vaccine and prior to administration of the unit dose of the dengue vaccine composition. 19. The method of claim 18,
The method comprises prior dengue infection and/or prior hepatitis A in said subject population or said subject before, during, and at any time after the administering step of said hepatitis A vaccine and said unit dose of said dengue vaccine composition. does not involve the step of determining whether a hepatitis infection has been present, or
wherein the hepatitis A serum status and/or the dengue fever serum status of the subject population or of the subjects are determined prior to, during, and at any time after the administering step of the hepatitis A vaccine and a unit dose of the dengue vaccine composition. Unknown hepatitis A vaccine and unit dose. 20. The method of any one of claims 1 to 19,
The method comprises a hepatitis A vaccine and unit dose comprising a primary vaccination comprising the steps of:
(A) unit dose and A of a tetravalent dengue virus composition in need of protection against dengue infection and hepatitis A infection without determining whether there was a prior dengue infection and/or prior hepatitis A infection selecting a subject for administration of a hepatitis vaccine, and
(B) administering to said subject a first unit dose of a tetravalent dengue virus composition and hepatitis A vaccine, and optionally
(C) administering to the subject a unit dose of at least one additional tetravalent dengue virus composition within 3 to 12 months of administration of the first unit dose, and optionally
(D) administering to the subject a dose of at least one additional hepatitis A vaccine within 6 to 18 months of administration of the first unit dose. 21. The method of any one of claims 1 to 20,
The method comprises a hepatitis A vaccine and unit dose comprising a primary vaccination comprising the steps of:
(A) selecting a subject for administration of a unit dose of a tetravalent dengue virus composition and a hepatitis A vaccine in need of protection against dengue infection and hepatitis A infection, and
(B) administering to the subject a first unit dose of a tetravalent dengue virus composition and a hepatitis A vaccine, and
(C) administering to the subject a unit dose of two additional tetravalent dengue virus compositions at about 6 and about 12 months of administration of the first unit dose and at about 6 or about 12 months of administration of the first unit dose administering a hepatitis A vaccine to the subject. 22. The method of claim 21,
Hepatitis A vaccine and unit dose, wherein step (A) is performed without determining whether there has been a prior hepatitis A infection. 23. The method of any one of claims 3-22,
Upon reconstitution of the dengue vaccine composition with a pharmaceutically acceptable diluent, (i), (ii), (iii), and (iv) provide a total concentration of pfu/0.5 mL, and a total concentration of pfu/0.5 mL Based on the 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%, and the concentration of (i) in pfu/0.5 mL is at least 1 %, and the concentration of (iii) of pfu/0.5 mL is at least 6%, at least 8%, or at least 10%, or at least 12%, or at least 14%, or at least 16%, or at least 18%. Hepatitis Vaccine and Unit Dosage. 24. The method of any one of claims 1 to 23,
wherein said method provides compatibility between said dengue vaccine composition and said hepatitis A vaccine. 25. The method of any one of claims 1-24,
wherein said method provides synergy between said dengue vaccine composition and said hepatitis A vaccine. 26. The method of any one of claims 1 to 25,
The method provides for non-inferiority in a non-inferiority clinical study comprising at least 60 or at least 120 healthy subjects divided into one subject population and one control subject population, wherein the subject population is the type A Hepatitis A vaccine and unit dose, wherein unit doses of hepatitis vaccine and said dengue vaccine composition are administered simultaneously on the same day, and wherein said control subject population receives said hepatitis A vaccine and placebo administration simultaneously on the same day. 27. The method of any one of claims 1-26,
wherein the hepatitis A vaccine is administered simultaneously at unit doses of the hepatitis A vaccine and the dengue vaccine composition on the same day and is seronegative for hepatitis A at baseline and seronegative for all dengue virus serotypes at baseline A hepatitis A vaccine and unit dose that provides a hepatitis A seroprotection rate of at least 95% or at least 98% on Day 30 after administration (Day 0/1) to a subject population of at least 30 or at least 50 healthy subjects. 28. The method of any one of claims 1-27,
The method provides a difference in hepatitis A seroprotection rate with respect to hepatitis A mono-administration, wherein the difference is seronegative with respect to hepatitis A at baseline and seronegative with respect to all dengue virus serotypes at baseline. as determined in a non-inferiority clinical study involving at least 60 or at least 120 healthy subjects,
The healthy subject
e) a subject population of at least 30 or at least 50 healthy subjects concurrently administered on the same day of administration (Day 0/1) of unit doses of said hepatitis A vaccine and said dengue vaccine composition, and
f) divided into a control subject population of at least 30 or at least 50 healthy subjects receiving concurrently on the same day of administration of hepatitis A vaccine and placebo (Day 0/1);
the difference is determined between the hepatitis A seroprotection rate of the control subject population at 30 days after administration (Day 0/1) and the hepatitis A seroprotection rate of the subject population at 30 days post administration (Day 0/1);
wherein the difference has an upper bound of binding within a two-sided 95% confidence interval of less than 10%. 27. The method of any one of claims 1-26,
The hepatitis A vaccine is administered to a subject population of at least 30 or at least 50 healthy subjects who at baseline are seronegative with respect to hepatitis A, receiving unit doses of the hepatitis A vaccine and the dengue vaccine composition simultaneously on the same day. provide a hepatitis A seroprotection rate of at least 95% or at least 98% or at least 99% at 30 days post-dose (Day 0/1), wherein the healthy subject has at least one dengue virus serotype serotype at baseline. Hepatitis A vaccine and unit dose, comprising healthy subject(s) who are positive and healthy subject(s) who are seronegative for all dengue virus serotypes at baseline. 27. The method of any one of claims 1-26,
The method provides a difference in hepatitis A seroprotection rate with respect to hepatitis A mono-administration, wherein the difference comprises a ratio comprising at least 60 or at least 120 healthy subjects who are seronegative with respect to hepatitis A at baseline. -determined in an inferiority clinical study, said healthy subject being healthy subject(s) seropositive at baseline with respect to at least one dengue virus serotype and healthy subject(s) seronegative with respect to all dengue virus serotypes at baseline ), including
The healthy subject
e) a subject population of at least 30 or at least 50 healthy subjects receiving administration of unit doses of said hepatitis A vaccine and said dengue vaccine composition simultaneously on the same day (Day 0/1), wherein the subject population is at baseline a population of subjects comprising healthy subject(s) seropositive for at least one dengue virus serotype and healthy subject(s) seronegative for all dengue virus serotypes at baseline, and
f) a control subject population of at least 30 or at least 50 healthy subjects receiving concurrent administration of hepatitis A vaccine and placebo on the same day (Day 0/1), wherein the control subject population is at least one dengue virus at baseline divided into a control subject population comprising healthy subject(s) seropositive with respect to serotype and healthy subject(s) seronegative for all dengue virus serotypes at baseline;
the difference is determined between the hepatitis A seroprotection rate of the control subject population on day 30 post-dose (Day 0/1) and the hepatitis A seroprotection rate of the subject population on day 30 post-dose (Day 0/1), and ,
wherein the difference has an upper bound of binding within a two-sided 95% confidence interval of less than 10%. 31. The method of any one of claims 1-30,
wherein said subject or population of subjects is exposed to a hepatitis A virus outbreak and/or a dengue virus outbreak. 32. The method of any one of claims 1-31,
The method comprises administering at least 30 unit doses of said hepatitis A vaccine and said dengue vaccine composition simultaneously on the same day and being seronegative with respect to hepatitis A vaccine at baseline and seronegative with respect to all dengue virus serotypes at baseline. Anti-hepatitis A virus antibody geometric mean concentration of at least 70 mIU/ml or at least 80 mIU/ml or at least 90 mIU/ml on Day 30 after administration (Day 0/1) to a subject population of persons or at least 50 healthy subjects Hepatitis A vaccine and unit dose, providing (GMC). 33. The method of any one of claims 1 to 32,
Hepatitis A vaccine and unit dose, wherein it is safe to administer unit doses of said hepatitis A vaccine and said dengue vaccine composition simultaneously to said subject or population of subjects on the same day. 34. The method of any one of claims 1 to 33,
Hepatitis A vaccine and unit dose, without any serious side effects associated with the simultaneous administration on the same day. 35. The method of any one of claims 1 to 34,
The method comprises at least 30 patients who received unit doses of said hepatitis A vaccine and said dengue vaccine composition simultaneously on the same day and were seronegative for hepatitis A at baseline and seronegative for all dengue virus serotypes at baseline. or at Day 30 after administration (Day 0/1) to a subject population of at least 50 healthy subjects.
- at least 110 or at least 140 or at least 150 for dengue serotype 1;
- at least 3000 or at least 3500 or at least 3900 for dengue serotype 2;
- at least 100 or at least 120 or at least 140 for dengue serotype 3; and/or
- hepatitis A vaccine and unit dose, providing a geometric mean titer (GMT) of neutralizing antibody as measured by an MNT50 of at least 80 or at least 110 or at least 140 against dengue serotype 4. 36. The method of any one of claims 1-35,
wherein said subject or population of subjects is between 18 and 60 years of age. (a) a first container holding the hepatitis A vaccine; and
(b) a second container holding a unit dose of a dengue vaccine composition; a kit for hepatitis A and dengue disease comprising at least a box comprising:
wherein said unit dose comprises a tetravalent dengue virus composition comprising four wildly attenuated dengue virus strains.

Images