Coadministration of tetravalent dengue vaccine with HPV vaccine

Abstract

The invention relates to a HPV vaccine composition and a dengue vaccine composition for use in a method of preventing an HPV-associated disease and dengue disease in a subject or in a subject population, the method comprising concomitantly administering the HPV vaccine composition and the dengue vaccine composition, wherein the dengue vaccine composition is a tetravalent dengue composition comprising live, attenuated DENV-1, DENV-2, DENV-3 and DENV-4 strains and wherein the method provides combined vaccine efficacy against all four serotypes of the dengue virus and non-inferiority of the immune respone to the HPV vaccine composition compared to a mono-administratrion of the HPV vaccine composition.

Description

COADMINISTRATION OF TETRAVALENT DENGUE VACCINE WITH HPV VACCINEFIELD OF THE INVENTION

[0001] The present invention relates to a method for concomitantly administering a dengue vaccine composition with a Human Papillomavirus (HPV) vaccine to a subject or a subject population. The method according to this invention provides immune responses against DENV-1, DENV-2, DENV-3 and DENV-4 and against HPV.BACKGROUND OF THE INVENTION

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

[0003] Dengue disease is a mosquito-borne disease caused by infection with a dengue virus. Dengue virus infections can lead to debilitating and painful symptoms, including a sudden high fever, headaches, joint and muscle pain, nausea, vomiting and skin rashes. To date, four serotypes of dengue virus have been identified: dengue-1 (DENV-I), dengue-2 (DENV-2), dengue-3 (DENV-3) and dengue-4 (DENV-4). Dengue virus serotypes 1-4 can also cause dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). In the most severe cases, DHF and DSS can be life threatening. Dengue viruses cause 50-100 million cases of debilitating dengue fever, 500,000 cases of DHF / DSS, and more than 20,000 deaths each year, a large portion of which are children. All four dengue virus serotypes are endemic throughout the tropical regions of the world and constitute the most significant mosquito-borne viral threat to humans there. Dengue viruses are transmitted to humans primarily by Aedes ae^y / 7 mosquitoes, but also by Aedes albopictusmaap\.o&,. Infection with one dengue virus serotype results in life-long protection from re-infection by that serotype, but does not prevent secondary infection by one of the other three dengue virus serotypes. In fact, previous infection with one dengue virus serotype may lead to an increased risk of severe disease (DHF / DSS) upon secondary infection with a different serotype.

[0004] Human papillomavirus (HPV) is a common virus that is passed from one person to another through direct skin-to-skin contact during sexual activity. Most sexually active people will be infected with HPV at some time in their lives and the infection is most common in people in their late teens and early 20s. There are about 40 types of HPV that can infect the genital areas of men and women. While most HPV types cause no symptoms and are cleared by the body's immune system, some HPV types are persistent and can cause cervical cancer in women and other less common cancers - like cancers of the anus, penis, vagina, vulva and oropharynx or warts in the genital areas of men and women, called genital warts.

[0005] HPV vaccination prevents HPV-associated cervical cancers as well as HPV-associated cancers of the anus, vulva, vagina, and oropharynx. The vaccination can also prevent HPV-associated genital warts. HPV vaccination is recommended in particular for 11 and 12 year-old girls. It is also recommended for girls and women 13 through 26 years of age who have not yet been vaccinated or completed the vaccine series. HPV vaccine can also be given to girlsbeginning at age 9 years. The CDC recommends 11 to 12 year olds girls get two doses of HPV vaccine to protect against cancers caused by HPV. More recently, vaccination of boys in the same age ranges has also been recommended.

[0006] The routine HPV vaccination schedule for adolescents who start the vaccination series before the 15th birthday includes two doses of an HPV vaccine. The two doses are usually separated by 6 to 12 months. The minimum interval between doses is five calendar months. A three dose schedule is recommended for subjects who start the series on or after the 15th birthday and for subjects with certain immunocompromising conditions (such as cancer, HIV infection, or taking immunosuppressive drugs). The second dose is usually given 1 to 2 months after the first dose and the third dose 6 months after the first dose. The minimum interval between the first and second doses of vaccine is usually 4 weeks. The minimum interval between the second and third doses of vaccine is usually 12 weeks. The minimum interval between the first and third doses is usually 5 calendar months. If the vaccination series is interrupted, the series does not need to be restarted.

[0007] Available HPV vaccines include Gardasil® 9, which is a recombinant 9-valent HPV (9vHPV) vaccine for preventing HPV serotypes 6, 11, 16, 18, 31, 33, 45, 52, and 58. The HPV vaccine does not include any live or inactivated HPV, but the LI proteins of the respective HPV types.

[0008] Although incorporating a dengue vaccine into an existing HPV vaccination program could help improve dengue vaccine coverage, it has not yet been demonstrated that concomitant administration of dengue vaccine and HPV vaccine is non-inferiorto administration of an HPV vaccine alone or of a dengue vaccine alone. The dengue vaccine Dengvaxia has been evaluated in two clinical studies with bivalent and quadrivalent HPV vaccines (Human Papillomavirus Vaccine, Recombinant) (305 dengue seropositive subjects at baseline, 9 to 14 years of age and 197 dengue seropositive subjects at baseline, 9 to 13 years of age). The non-inferiority of the humoral immune response to bivalent and quadrivalent HPV vaccines / Dengvaxia at 28 days after the last injection could not be assessed because the number of evaluable subjects was limited (see Dengvaxia EU " Summary of Product Characteristics" retrieved at https: / / www.ema.europa.eu / documents / product-information / dengvaxia-epar-product-information_en.pdf).

[0009] Hence, there is a need for a safe and effective method of simultaneously preventing dengue disease and HPV-associated cancers or genital warts. In particular, there is a need for HPV and dengue vaccines which provide non-inferiority when administered concomitantly to a subject or subject population.OBJECTS AND SUMMARY

[0010] It is an object to provide a safe and effective protection against dengue disease and HPV. In this context it is beneficial that vaccination can be conducted simultaneously for more than one disease in order to avoid additional medical appointments, but also ensuring that there are no interactions between the co administered vaccines which could affect the desired immune responses.

[0011] It is an object to provide a method of administration for preventing HPV and dengue disease which is useful in typical vaccination settings wherein the subjects are unaware of their serostatus for dengue and / or HPV and a corresponding serotest is unavailable, unpractical or unreliable.

[0012] It is an object to provide a safe and effective vaccine for preventing HPV and dengue disease in a subject or subject population and a corresponding method of preventing HPV and dengue disease in a subject or a subject population from a dengue-endemic and dengue non-endemic region and for a broad range of ages, in particular for subjects between 9 to 14 years of age, and typically independent of previous exposure to any dengue virus serotypeand / or to HPV virus and independent of corresponding seropositivity or seronegativity with respect to dengue and / or HPV before vaccination.

[0013] It is an object to provide vaccines and a corresponding method of preventing HPV and dengue disease which avoids testing for individual dengue and / or HPV serostatus before individual administration of a HPV and a dengue vaccine to a subject or subject population, or analysis of seroprevalence rates of dengue and / or HPV in subjects or subject populations to be vaccinated.

[0014] It is an object to provide a dengue vaccine and an HPV vaccine which can be safely co-administered with TDV as travel vaccines before an international travel of a subject to dengue endemic countries and a method of safely administering these vaccines.

[0015] Therefore, one aspect is directed to a method of preventing dengue disease as well as HPV.

[0016] Another aspect is directed to a method of preventing HPV and dengue disease in a subject or subject population, the method comprising concomitantly administering an HPV vaccine and a dengue vaccine composition, wherein said dengue vaccine composition is a tetravalent dengue virus composition including four live, attenuated dengue virus strains.

[0017] Another aspect relates to a combination of an HPV vaccine composition and a dengue vaccine composition for use in a method of preventing an HPV-associated disease and dengue disease in a subject or in a subject population, by concomitantly administering the HPV vaccine composition and the dengue vaccine composition, wherein the dengue vaccine composition is a tetravalent dengue virus composition comprising live, attenuated DENV-1, DENV-2, DENV-3 and DENV-4 strains.

[0018] Another aspect relates to an HPV vaccine composition and a dengue vaccine composition for use in a method of preventing an HPV-associated disease and dengue disease in a subject or in a subject population, the method comprising concomitantly administering the HPV vaccine composition and the dengue vaccine composition, wherein the dengue vaccine composition is a tetravalent dengue virus composition comprising live, attenuated DENV-1, DENV-2, DENV-3 and DENV-4 strains and wherein there is no significant reduction in immunogenicity of the HPV vaccine composition compared to a mono-administration of the HPV vaccine composition and no significant reduction in immunogenicity of the dengue vaccine composition compared to a mono-administration of the dengue vaccine composition.

[0019] Another aspect relates to an HPV vaccine composition and a dengue vaccine composition for use in a method of preventing an HPV-associated disease and dengue disease in a subject or in a subject population, the method comprising concomitantly administering the HPV vaccine composition and the dengue vaccine composition, wherein the dengue vaccine composition is a tetravalent dengue virus composition comprising live, attenuated DENV-1, DENV-2, DENV-3 and DENV-4 strains and wherein there is no significant interaction between the HPV vaccine composition and the dengue vaccine composition affecting either the immunogenicity of the HPV vaccine composition or the immunogenicity of the dengue vaccine composition.

[0020] Another aspect relates to an HPV vaccine composition and a dengue vaccine composition for use in a method of preventing an HPV-associated disease and dengue disease in a subject or in a subject population, the method comprising concomitantly administering the HPV vaccine composition and the dengue vaccine composition, wherein the dengue vaccine composition is a tetravalent dengue virus composition comprising live, attenuated DENV-1, DENV-2, DENV-3 and DENV-4 strains and wherein the dengue vaccine composition provides combined vaccine efficacy againstall four serotypes of the dengue virus and wherein the immune response to the HPV vaccine composition is non inferior compared to a mono-administration of the HPV vaccine composition.

[0021] Another aspect relates to an HPV vaccine composition and a dengue vaccine composition for use in a method of preventing an HPV-associated disease and dengue disease in a subject or in a subject population, the method comprising concomitantly administering the HPV vaccine composition and the dengue vaccine composition, wherein the dengue vaccine composition is a tetravalent dengue virus composition comprising live, attenuated DENV-1, DENV-2, DENV-3 and DENV-4 strains and wherein the method provides combined vaccine efficacy against all four serotypes of the dengue virus and non-inferiority of the immune response to the HPV vaccine composition compared to a monoadministration of the HPV vaccine composition.

[0022] The HPV-associated disease may be selected from HPV-associated cancers or genital warts.

[0023] In one embodiment, the dengue vaccine composition comprises a chimeric dengue serotype 2 / 1 strain, a dengue serotype 2 strain, a chimeric dengue serotype 2 / 3 strain, and a chimeric dengue serotype 2 / 4 strain.

[0024] In another embodiment, the HPV vaccine composition is a bivalent, quadrivalent or nonavalent HPV vaccine composition.

[0025] In another embodiment, a unit dose of the dengue vaccine composition upon reconstitution with 0.5 mL of a pharmaceutically acceptable diluent comprises(i) a chimeric dengue serotype 2 / 1 strain in a concentration of at least 3.3 loglO pfu / 0.5 mL,(ii) a dengue serotype 2 strain in a concentration of at least 2.7 loglO pfu / 0.5 mL,(iii) a chimeric dengue serotype 2 / 3 strain in a concentration of at least 4.0 loglO pfu / 0.5 mL, and(iv) a chimeric dengue serotype 2 / 4 strain in a concentration of at least 4.5 loglO pfu / 0.5 mL,wherein optionally (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL and based on said total concentration of pfu / 0.5 ml 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%, and 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%, e.g. at least 10%.

[0026] The subject population or subject may be seronegative to all dengue serotypes.

[0027] In another embodiment, the dengue vaccine composition is administered by subcutaneous injection and the HPV vaccine composition is administered by intramuscular injection, and wherein optionally said injections are administered to the arm, optionally to the deltoid region of the arm.

[0028] For example, the dengue vaccine composition and the HPV vaccine composition are administered to different anatomical sites, optionally to opposite arms.

[0029] In another embodiment, two doses of the dengue vaccine composition are administered within 12 months or more, or within six months, or within three months.

[0030] In another embodiment, the method comprises the administration of two unit doses of the dengue vaccine composition and two unit doses of said HPV vaccine composition, optionally according to the following schedule: a first concomitant administration of a first unit dose of the dengue vaccine composition and a first unit dose of the HPV vaccine composition on day 0,a second administration of a second unit dose of the dengue vaccine composition after the first concomitant administration, such as about 3 months after day 0, anda third administration of a second unit dose of the HPV vaccine composition after the second administration, such as about 6 months after day 0.

[0031] In another embodiment, the subject population or subject is 9 to <15 years or 9 to 14 years of age.

[0032] In another embodiment, the subject population or subject is from a dengue endemic region, optionally from Thailand.

[0033] In another embodiment, the subject population or subject is female.

[0034] In another embodiment, the method does not include a step of determination whether there was a previous dengue infection and / or wherein the dengue serostatus of the subject population or of the subject is unknown before the concomitant administration the HPV vaccine composition and the dengue vaccine composition and / or wherein the subject or subject population has no laboratory confirmed history of dengue and / or wherein the method does not include a step of assessing a previous dengue infection through an appropriately validated serological test, e.g. by the method as disclosed herein such as the MNT50 test described in Example 2 or any serotesting with adequate performance in terms of specificity and cross reactivity based on the locale disease epidemiology.

[0035] In another embodiment, the HPV vaccine composition is a nonavalent HPV vaccine composition and the method provides non-inferiority of the immune response to the nonavalent HPV vaccine composition compared to a mono-administration of the nonavalent HPV vaccine composition.

[0036] In another embodiment, the method provides non-inferiority of the immune response to the HPV vaccine composition in a non-inferiority clinical study including at least 600 healthy subjects divided into one subject population and into one control subject population, wherein the subject population receives the HPV vaccine composition and the dengue vaccine composition concomitantly once on day 1 (month 0) followed by the dengue vaccine composition once on day 90 (month 3) and the HPV vaccine composition once on day 180 (month 6) and the control subject population receives the HPV vaccine composition once on day 1 (month 0) followed by the HPV vaccine composition once on day 180 (month 6).

[0037] In another embodiment, the method provides non-inferiority of the immune response to the dengue vaccine composition compared to a mono-administration of the dengue vaccine composition.

[0038] In another embodiment, wherein the method provides a seropositivity rate of at least 95% or at least 98% or at least 99.5% or 100% for each dengue serotype at day 120 (month 4) after a first concomitant administration of a first unit dose of the dengue vaccine composition and a first unit dose of the HPV vaccine composition at day 1, and optionally after a second administration of a second unit dose of the dengue vaccine composition at day 90 (month 3).

[0039] Another aspect of the invention relates to a kit comprising(a) a first container containing an HPV vaccine composition, and(b) a second container containing a dengue vaccine composition, wherein the dengue vaccine composition is a tetravalent dengue virus composition including four live, attenuated dengue virus strains.DEFINITIONS

[0040] In describing the present invention, the following terms are to be used as indicated below. As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise.

[0041] As used herein, the terms "unit dose of a dengue vaccine composition", "unit dose" and "unit dose of the invention as described herein" refer to the amount of a dengue vaccine which is administered to a subject in a single dose. In one embodiment, one unit dose is present in a vial and this unit dose is administered to a subject, e.g. optionally after reconstitution. In one embodiment, more than one unit dose of the dengue vaccine composition may be present in a vial so that with the content of one vial more than one subject can be vaccinated.

[0042] As used herein, the term "unit dose of an HPV vaccine composition" refers to the amount of an HPV vaccine which is administered to a subject in a single dose. In one embodiment, one unit dose is present in a vial and this unit dose is administered to a subject, e.g. optionally after reconstitution. In one embodiment, more than one unit dose of the HPV vaccine composition may be present in a vial so that with the content of one vial more than one subject can be vaccinated.

[0043] A "lyophilized unit dose" or "unit dose in lyophilized form" of the dengue vaccine composition refers to the unit dose that is obtained by subjecting a given volume of the liquid dengue vaccine composition, such as 0.5 mL, to lyophilization. Thus, the aqueous formulations of the dengue vaccine composition being produced by combining the pharmaceutically acceptable excipients and the dengue virus composition comprising the four dengue virus strains, such as TDV-1 to TDV-4, is subjected to lyophilization to obtain the lyophilized unit dose.

[0044] A "reconstituted unit dose" or "unit dose in reconstituted form" of the dengue vaccine composition is obtained from the lyophilized dose by reconstitution with a pharmaceutically acceptable diluent. The diluent does not contain dengue virus. The reconstituted unit dose is a liquid which can be administered to a subject, for example by injection, such as subcutaneous injection or intramuscular injection.

[0045] As used herein, the term "upon reconstitution with 0.5 mL" is not limiting the reconstitution to be performed using 0.5 mL of the diluent, but refers to the concentration of the dengue viruses that will be present in the reconstituted unit dose when 0.5 mL diluent are used for reconstitution. While using a different volume for reconstitution (e.g. 0.8 mL) will result in a different concentration of dengue viruses in the reconstituted unit dose, the administration of the total volume of the unit dose (e.g. 0.8 mL) will result in the same total amount of dengue virus being administered.

[0046] As used herein, a "concentration of at least X loglO pfu / 0.5 mL" refers to the concentration of a dengue serotype in 0.5 mL, but is not limiting the unit dose to be 0.5 mL. If the unit dose has a volume different than 0.5 mL, or is lyophilized from a volume different than 0.5 mL, or is reconstituted with a volume different than 0.5 mL, said concentration will differ from the "concentration of at leastX loglO pfu / 0.5 mL". However, if the unit dose has a volume of 0.5 mL, or is lyophilized from a volume of 0.5 mL, or is reconstituted with a volume of 0.5 mL, said concentration will be the "concentration of at least X loglO pfu / 0.5 mL". Thus, while the concentration may differ, the total amount of virus in the unit dose remains the same.

[0047] As used herein, the term "dengue serotype" refers to a species of dengue virus which is defined by its cell surface antigens and therefore can be distinguished by serological methods known in the art. At present, four serotypes of dengue virus are known, i.e. dengue serotype 1 (DENV-1), dengue serotype 2 (DENV-2), dengue serotype 3 (DENV-3) and dengue serotype 4 (DENV-4).

[0048] As used herein, the term "tetravalent dengue virus composition" refers to a dengue virus composition comprising four different immunogenic components from the four different dengue serotypes DENV-1, DENV-2, DENV-3 and DENV-4, e.g. comprising four different live, attenuated dengue viruses, each representing one dengue serotype, and which aims to stimulate or induce immune responses to all four dengue serotypes.

[0049] As used herein, the term "live attenuated dengue virus" refers to a viable dengue virus which is mutated to provide reduced virulence. The live attenuated dengue virus can be a dengue virus in which all components are derived from the same dengue serotype or it can be a chimeric dengue virus having parts from two or more dengue serotypes or a mixed chimeric dengue virus having parts from other flaviviruses.

[0050] A "virus strain" and in particular a "dengue virus strain" is a genetic subtype of a virus, in particular of a dengue virus, which is characterized by a specific nucleic acid sequence. A dengue serotype may comprise different strains with different nucleic acid sequences which have the same cell surface antigens. A dengue virus strain can be a dengue virus in which all components are derived from the same dengue serotype or it can be a chimeric dengue virus having parts from two or more dengue serotypes.

[0051] As used herein, " TDV-2" refers to a molecularly characterized and cloned dengue serotype 2 strain derived from the live attenuated DEN-2 PDK-53 virus strain. The PDK-53 strain is described for example in Bhamarapravati et al. (1987) Bulletin of the World Health Organization 65(2): 189-195. In one embodiment, the TDV-2 strain served as a backbone for the chimeric TDV-1, TDV-3 and TDV-4 strains into which parts from the TDV-1, TDV-3 and TDV-4 strains were introduced.

[0052] A "non-chimeric dengue virus" or "non-chimeric dengue serotype strain" or "non-chimeric dengue strain" comprises only parts from one dengue serotype. In particular, a non-chimeric dengue virus does not include parts from a different flavivirus such as yellow fever virus, Zika virus, West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, tick-borne encephalitis virus. TDV-2 is an example of a non-chimeric dengue virus.

[0053] A "chimeric dengue virus" or "chimeric dengue serotype strain" or "chimeric dengue strain" comprises parts from at least two different dengue serotypes. As used herein, the chimeric dengue virus does not include parts from a different flavivirus such as yellow fever virus, Zika virus, West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, tick-borne encephalitis virus. In particular, the chimeric dengue virus described herein does not include parts from the yellow fever virus. As used herein, a "chimeric dengue serotype 2 / 1 strain" or " DENV-2 / 1 chimera" or " TDV-1" refers to a dengue virus chimeric construct which comprises parts from both DENV-2 and DENV-1. 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, a "chimeric dengue serotype 2 / 3 strain" or " DENV-2 / 3 chimera" or " TDV-3" refers to a dengue virus chimeric construct which comprises parts from both DENV-2 and DENV- 3. 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, a "chimeric dengue serotype 2 / 4 strain" or " DENV-2 / 4 chimera" or " TDV-4" refers to a dengue virus chimeric construct which comprises parts from both DENV-2 and DENV- 4. 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. A mixed chimeric dengue virus has parts from other flaviviruses.

[0054] As used herein, " TDV" refers to a tetravalent live attenuated dengue vaccine that comprises a mixture of the four live attenuated dengue virus strains TDV-1, TDV- 2, TDV-3 and TDV-4 expressing surface antigens from the four dengue serotypes DENV-1, DENV-2, DENV-3 and DENV-4, respectively. In one embodiment (e.g. also in the examples), TDV-1 has the nucleotide sequence according to SEQ ID No. 1 and / or the amino acid sequence according to SEQ ID No. 2. In one embodiment, TDV-2 has the nucleotide sequence according to SEQ ID No. 3 and / or the amino acid sequence according to SEQ ID No. 4. In one embodiment, TDV-3 has the nucleotide sequence according to SEQ ID No. 5 and / or the amino acid sequence according to SEQ ID No. 6. In one embodiment, TDV-4 has the nucleotide sequence according to SEQ ID No. 7 and / or the amino acid sequence according to SEQ ID No. 8. In a specific embodiment, TDV is Qdenga®. Qdenga® has been approved by the EMA (Agency product number EMEA / G / C / 005155). An exemplary method for manufacturing TDV is disclosed in international patent application WO 2023 / 147337.

[0055] As used herein, the term "dengue disease" refers to the disease which is caused by infection with dengue virus. Symptoms of dengue disease include sudden high fever, headaches, joint and muscle pain, nausea, vomiting and skin rashes. 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 present with severe manifestations of plasma leakage and hemorrhage. When a subject with DHF experiences shock he or she will be categorized as having DSS. Symptoms of DSS include bleeding that may appear as tiny spots of blood on the skin and larger patches of blood under the skin. Prolonged shock is the main factor associated with complications including massive gastrointestinal hemorrhage that can lead to death. As used herein, DHF cases are defined as VCD cases meeting WHO 1997 DHF criteria. In the context of preventing dengue disease in elderly subjects, the term "preventing dengue disease" typically includes preventing DHF and / or DSS. In the context of preventing dengue disease in elderly subjects, the term "preventing dengue disease" typically includes preventing severe end-organ manifestations of dengue such as hepatomegaly and acute renal failure.

[0056] As used herein, "preventing dengue disease" refers to preventing a subject from developing one or more symptoms of dengue disease because of an infection with a dengue virus. In particular, preventing dengue disease is achieved by vaccinating or inoculating a subject with a dengue vaccine composition, such as the reconstituted unit dose described herein. As used herein, the term "prophylactically treating dengue disease" is equivalent to "preventing dengue disease". In a particular embodiment, preventing dengue disease includes preventing DHS and / or DSS.

[0057] As used herein, the terms "virologically confirmed dengue disease", " VCD case", or " VCD fever" refer to febrile illness or illness clinically suspected to be dengue disease with a positive serotype-specific reverse transcriptase polymerase chain reaction (RT-PCR). The term "virologically confirmable dengue" disease refers to a subject having febrile illness or illness clinically suspected to be dengue disease, wherein testing the subject, e.g. using RT-PCR, would confirm the presence of at least one dengue serotype. Severe forms of VCD fever will be identified as follows: Dengue Hemorrhagic Fever (DHF) was defined according to the WHO 1997 criteria. Severe dengue was defined through an assessment of an independent Dengue Case Adjudication Committee which will assess all hospitalized VCD cases (severe / non-severe) based on criteria redefined in a charter. All non-hospitalized cases are considered non-severe.

[0058] As used herein, the term "febrile illness" is defined as temperature >38°C on any 2 of 3 consecutive days.

[0059] As used herein, the terms "virologically confirmed dengue disease with hospitalization", is considered to be a surrogate for severe dengue and the "incidence of virologically confirmed dengue disease with hospitalization" is used as a safety parameter. As used herein, the "relative risk with respect to virologically confirmed dengue diseasewith hospitalization" means the number of events of virologically confirmed dengue disease with hospitalization divided by the number of subjects treated with the unit dose as disclosed herein over the number of events of virologically confirmed dengue disease with hospitalization divided by the number of subjects treated with placebo. If the "relative risk with respect to virologically confirmed dengue disease with hospitalization" is 1 or lower the vaccine provides for the same or less risk for virologically confirmed dengue disease with hospitalization as placebo and is considered "safe". In this context the risk of virologically-confirmed dengue disease with hospitalization may be also 0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, or 0.1 or less, in particular when determined from 30 days after a second administration until 12 months after a second administration, in particular when determined in age groups selected from the age group of 4 to 16 year old subjects, the age group of 4 to under 9 year old subjects, the age group of 2 to under 9 year old subjects, the age group of 4 to 5 year old subjects, the age group of 6 to 11 year old subjects, and the age group of 12 to 16 year old subjects.

[0060] As used herein, an alternative or additional criterion for considering a dengue vaccine to be "safe" is a vaccine efficacy (VE) with respect to virologically confirmed dengue disease with hospitalization of 0% or higher. This means that the vaccine provides for the same likelihood or less for virologically confirmed dengue disease with hospitalization as placebo. In particular considered "safe" is the combined vaccine efficacy against virologically-confirmed dengue with hospitalization against all four serotypes with a 2-sided 95% confidence interval, wherein the lower bound is more than 25%, in particular when measured against placebo in a subject population of at least 1,500 or at least 2,000 healthy subjects (in particular when measured in age groups selected in particular from the age group of 4 to 16 year old subjects, the age group of 4 to under 9 year old subjects, the age group of 2 to under 9 year old subjects, the age group of 4 to 5 year old subjects, the age group of 6 to 11 year old subjects, the age group of 9 to 14 year old subjects, and the age group of 12 to 16 year old subjects) being seronegative against all serotypes at baseline or being seropositive against at least one serotype at baseline, in particular when said unit dose or said placebo is administered at least twice within less than 6 months, such as within 3 months, about from first administration or from 30 days after the second or last administration of the administration schedule until at least 12 months, until 12 to 18 months, until 12 months, or until 18 months after the second or last administration of the administration schedule. In particular, the lower bound may be more than 30%, more than 40%, more than 50%, more than 60%, more than 65%, more than 66%, more than 67%, more than 68% more than 70%, or more than 75%. In particular, the 2-sided 95% confidence interval of the combined vaccine efficacy against virologically confirmed dengue with hospitalization against all four serotypes when comparing seropositive and seronegative subjects provides for lower bounds of the 2-sided confidence interval which are within 10% points or within 15% points or within 20% points. In a particular embodiment "safe" means providing a combined vaccine efficacy against virologically-confirmed dengue with hospitalization against all four serotypes with a 2-sided 95% confidence interval, wherein the lower bound is more than 65%, when measured against placebo in a subject population of at least 5,000 healthy 4 to 16 year old subjects irrespective of serostatus at baseline from first administration of the administration schedule until 12 to 18 months after the last administration of the administration schedule.

[0061] If one of the criteria as defined above for the term "safe" is fulfilled, the dengue vaccine is considered safe within the meaning of this invention. In this context, safe in particular refers to a dengue vaccine that is safe for all subjects irrespective of their serostatus at baseline. This means that the dengue vaccine can be administered without the need to determine the occurrence of a previous dengue infection in the subject before administration. For example, the dengue vaccine is safe as defined above with respect to all age groups starting from 4 years of age and typically irrespective of the serostatus, in particular from 4 years of age to 60 years of age, or 4 years of age to 16 years of age, or 9 to 14 years of age. Relevant subgroups in this context are under 9 years of age, from 2 years of age to under 9 years of age, from 4 years of age to under 9 years of age, 4 to 5 years of age, 6 to 11 years of age, 9 to 14 years ofage and 12 to 16 years of age or any age group within 4 to 16 years of age. For further definitions of VE against virologically confirmed dengue disease with hospitalization reference is made to the disclosure below with respect to certain methods of treatment.

[0062] As used herein, "vaccine efficacy" or " VE" measure the proportionate reduction in cases among vaccinated persons. Vaccine efficacy (VE) is measured by calculating the risk of disease among vaccinated and unvaccinated persons and determining the percentage reduction in risk of disease among vaccinated persons relative to unvaccinated persons. The greater the percentage reduction of illness in the vaccinated group, the greater the vaccine efficacy. For example, a VE of 90% indicates a 90% reduction in disease occurrence among the vaccinated group, or a 90% reduction from the number of cases you would expect if they have not been vaccinated. The vaccine efficiency is calculated by the formula: 100 * (1 - HR), wherein HR is the Hazard Ratio which is defined as the Hazard rate of vaccine (Av) divided by the Hazard rate of placebo (Ac), i.e. HR = Av / Ac. Av denote the hazard rate for the subjects vaccinated with a tetravalent dengue vaccine composition as disclosed herein and Ac denote the hazard rate for unvaccinated subjects, i.e. subjects receiving placebo. The hazard rate ratio HR is estimated from a Cox proportional hazard model with study vaccine as a factor, adjusted for age, and stratified by region. As used herein the ternT'combined vaccine efficacy against all four serotypes" is defined as the vaccine efficacy in relation to the risk of dengue disease irrespective of the serotype being responsible for the virologically confirmed dengue disease and the subject baseline serostatus. A vaccine is considered "effective" in case the combined vaccine efficacy is 30% or more. In this context the combined vaccine efficacy may be also 40% or more, 50% or more, 60% or more, 70% or more, 72% or more, or 80% or more, in particular when determined from 30 days after a second administration until 12 months after a second administration or 18 months after a second vaccination, in particular when determined in age groups selected from the age group of 4 to 16 year old subjects, the age group of 4 to under 9 year old subjects, the age group of 2 to under 9 year old subjects, the age group of 4 to 5 year old subjects, the age group of 6 to 11 year old subjects, the age group of 9 to 14 years, and / or the age group of 12 to 16 year old subjects. In this context, effective in particular refers to a vaccine that is effective for all subjects irrespective of their serostatus at baseline. For example, the vaccine is effective with respect to all age groups starting from 4 years of age and typically irrespective of the serostatus, in particular from 4 years of age to 60 years of age or from 4 years of age to 16 years of age or 9 to 14 years of age and irrespective of the serostatus. Relevant subgroups in this context are under 9 years of age, from 2 years of age to under 9 years of age, from 4 years of age to under 9 years of age, 4 to 5 years of age, 6 to 11 years of age, 9 to 14 years of age and 12 to 16 years of age or any age group within 4 to 16 years of age. In certain embodiments "effective" means providing a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease with a 2-sided 95% confidence interval, wherein the lower bound is more than 60%, when measured against placebo in a subject population of at least 500 or at least 600 or at least 5,000 healthy subjects irrespective of serostatus at baseline and 4 to 16 or 9 to 14 years of age, from the first administration of the administration schedule until 18 months after the last administration of the administration schedule. Further specific efficacies can be defined. As used herein, "combined vaccine efficacy against all four serotypes in seronegative subjects" refers to the efficacy measured in subjects which are seronegative at baseline. As used herein, "vaccine efficacy against a specific serotype, e.g. serotype 1" refers to the efficacy in relation to a specific serotype being responsible for the virologically confirmed dengue disease. As used herein, "combined vaccine efficacy against all four serotypes against virologically confirmed dengue with hospitalization" refers to the efficacy wherein only virologically confirmed dengue cases with hospitalization are considered. Such vaccine efficacies can be determined with respect to subjects being seronegative or seropositive at baseline and for different age groups.

[0063] As used herein, the "relative risk" of dengue disease means the number of events of virologically confirmed dengue disease divided by the number of subjects treated with the dengue vaccine composition as disclosed hereinover the number of events of virologically confirmed dengue disease divided by the number of subjects treated with placebo. As used herein the term"combined relative risk against all four serotypes" is defined as the relative risk in relation to the risk of dengue disease irrespective of the serotype being responsible for the virologically confirmed dengue disease and the subject baseline serostatus.

[0064] As used herein, "vaccinating" or "inoculating" refers to the administration of a vaccine to a subject, with the aim to prevent the subject, from developing one or more symptoms of a disease. As used herein, "vaccinating against dengue disease" or "inoculating against dengue disease" refers to the administration of a dengue vaccine composition to a subject, with the aim to prevent 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. The primary vaccination is defined as the primary administration schedule for administering the composition or unit dose as disclosed herein to establish a protective immune response and e.g. consists of two administrations e.g. within three months. Whenever an administration is mentioned within this disclosure such administration refers to the primary vaccination unless it is specified as booster vaccination. The booster vaccination refers to an administration or administration schedule which takes place after the primary vaccination e.g. at least 1 year, or 4 to 4.5 years, or even 5 or 10 years after the last administration, e.g. the second administration, of the primary vaccination schedule. The booster administration attempts at enhancing or reestablishing the immune response of the primary vaccination.

[0065] As used herein, the terms "subject" or "subjects" are limited to human subjects (e.g. infants, children or adults). The terms "elderly subject" or "elderly subjects" refer to subjects with an age of more than 60 years, such as 61 years to 100 years, 61 years to 90 years, 61 years to 80 years, 61 years to 75 years, or 61 years to 70 years.

[0066] As used herein, "subject population" refers to a group of subjects. The subject population may refer to least 40 subjects, at least 50 subjects, at least 60 subjects, at least 100 subjects or at least 1000 subjects and is defined by certain parameters. The parameters that may be used to define a subject population include, but are not limited to, the age of the subjects, whether the subjects are from a dengue endemic region or from a dengue non-endemic region and / or the serostatus of the subjects.

[0067] As used herein, "endemic region" refers to a region where a disease or infectious agent is constantly present and / or usually prevalent in a population within this region. As used herein, "non-endemic region" refers to a region from which the disease is absent or in which it is usually not prevalent. Accordingly, a "dengue endemic region" refers to geographic areas in which an infection with dengue virus is constantly maintained at a baseline level. A "dengue non-endemic region" is a geographic area in which an infection with dengue virus is not constantly maintained at a baseline level. Accordingly, subject populations or subjects "from a dengue endemic region" or "from a dengue non-endemic region" refer to subject populations or subjects living in geographic areas as defined above. Whether a geographic area or a subject population is dengue-endemic or not can be determined by different calculatory methods such as the ones described in 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 supplementary material. Overviews of dengue endemic regions and dengue epidemiology are regularly published, for example, by the WHO or CDC. Typical dengue-endemic regions are in Latin America, Southeast Asia and the Pacific islands and dengue endemic countries include, but are not limited to, Australia, Brazil, Bangladesh, Colombia, China, Dominican Republic, Indonesia, India, Mexico, Malaysia, Nicaragua, Nigeria, Pakistan, Panama, Philippines, Puerto Rico, Singapore, Sri Lanka, Thailand and Vietnam. The area's force of infection is measured by seroprevalence surveys provided as seroprevalence rate. Areas with very high force of infection are considered to have a seroprevalence rate of more than 80%. As used herein the term "region" when it concerns seroprevalence rates refers to a geographic area where the seroprevalence rate could be determined or is known, e.g. a village, a town, a city, a region, a county, a state, a province or parts of the foregoing or a whole country.

[0068] As used herein, "serostatus" refers to the amount of antibodies a subject has with respect to a certain infectious agent, in particular dengue virus. As used herein, "seronegative" or "seronaive" means that the subject does not have neutralizing antibodies against any one of dengue serotypes DENV-1, DENV-2, DENV-3 and DENV-4 in the serum. A seronegative or seronaive subject or subject population is defined by a neutralizing antibody titer of less than 10 for each one of the four dengue serotypes. A subject or subject population having a neutralizing antibody titer of equal to or more than 10 for at least one dengue serotype is defined as being "seropositive" with respect to said dengue serotype. Serostatus at baseline refers to the serostatus before the administration of a dengue vaccine composition as described herein.

[0069] As used herein, a "neutralizing antibody titer" refers to the amount of antibodies in the serum of a subject that neutralize the respective dengue serotype. The neutralizing antibody titer against DENV-1, DENV-2, DENV-3 and DENV-4 is determined in a serum sample of the subject using known methods such as the plaque reduction neutralization test (PRNT) as described in the WHO Guidelines (World Health Organization Department of Immunization Vaccines Biologicals (2007) Guidelines for plaque reduction neutralization testing of human antibodies to dengue viruses, WHO / IVB / 07.07) or a microneutralization (MNT50) assay as described herein. For example, as used herein, the "ratio of not more than 20 for the neutralizing antibody titer of dengue serotype 2 to the neutralizing antibody titer of dengue serotype 4" means that the neutralizing antibody titer of dengue serotype 2 is divided by the neutralizing antibody titer of dengue serotype 4 and that the ratio obtained hereby is no more than 20. In other words, the neutralizing antibody titer of dengue serotype 2 is not more than 20-times higher than the neutralizing antibody titer of dengue serotype 4 in the subject.

[0070] As used herein, the terms "geometric mean neutralizing antibody titer" and " GMT" refer to the geometric mean value of the titer of neutralizing antibodies against the corresponding dengue serotype in the serum of subjects in a subject population. The geometric mean value is calculated by a well-known formula. As used herein, the "ratio of not more than 20 for the GMT of dengue serotype 2 to the GMT of dengue serotype 4" means that the geometric mean neutralizing antibody titer of dengue serotype 2 (GMT DENV-2) is divided by the geometric mean neutralizing antibody titer of dengue serotype 4 (GMT DENV-4) and that the ratio obtained hereby is no more than 20. In other words, the geometric mean neutralizing antibody titer of dengue serotype 2 is not more than 20-times higher than the geometric mean neutralizing antibody titer of dengue serotype 4 in the subject population.

[0071] As used herein, an "immune response" refers to a subject's response to the administration of a vaccine, for example the dengue vaccine. In particular, the immune response to the administration of a dengue vaccine includes the formation of neutralizing antibodies to one or more dengue serotypes. It may also include the stimulation of a cell-mediated response or the formation of antibodies to non-structural proteins such as NS1. An immune response to the administration of a dengue vaccine is stimulated by the administration of a unit dose of the invention as described herein, if the titer of neutralizing antibodies against at least one dengue virus serotype and typically against all four dengue virus serotypes is increased after administration of a unit dose. An immune response to the administration of a dengue vaccine is stimulated by the administration of a unit dose as described herein, if the secretion of interferon gamma by peripheral blood mononuclear cells stimulated with peptides from dengue virus proteins is increased after said administration of said unit dose. An immune response to the administration of a dengue vaccine is stimulated by the administration of a unit dose of the invention as described herein, if the titer of antibodies to non-structural proteins such as NS1 is increased after said administration of said unit dose. In a particular embodiment, the administration of a reconstituted unit dose of the present invention as described herein stimulates the formation of neutralizing antibodies to one or more dengue serotypes, a cell-mediated response and the formation of antibodies to non-structural proteins such as NS1.

[0072] As used herein, a "balanced immune response" to the administration of a dengue vaccine means that the immune response to the four dengue serotypes is sufficient to provide protection against infection by all four dengue serotypes and typically the immune response to the four dengue serotypes has a similar strength. In particular, the neutralizing antibody titer against the four dengue serotypes at day 180 or day 365 after administration of a first reconstituted unit dose of the invention as described herein is similar, i.e. it differs by less than factor 30, by less than factor 25 or by less than factor 20.

[0073] The „total concentration in pfu / 0.5 ml" which serves as a base value for the calculation of the percentage concentration for each individual component of a tetravalent dengue vaccine is shown for one exemplary tetravalent vaccine composition comprising dengue serotype 1 in a concentration of 3.60 Iogl0pfu / 0.5 ml, a dengue serotype 2 concentration of 4.00 Iogl0pfu / 0.5 ml, a dengue serotype 3 concentration of 4.60 Iogl0pfu / 0.5 ml and a dengue serotype 4 concentration of 5.11 Iogl0pfu / 0.5 ml. Primarily, the logarithmic values of the concentrations are converted into numerical values. The results of this conversion are 4xl03pfu / 0.5 ml for serotype 1, lxlO4pfu / 0.5ml for serotype 2, 4xl04pfu / 0.5 ml for serotype 3 and 1.3xl05pfu / 0.5 ml for serotype 4. The total concentration in pfu / 0.5 ml is the sum of the preceding numerical values resulting in 1.84 xlO5pfu / 0.5 ml.

[0074] The "percentage concentration" for each of the serotypes 1, 2, 3 and 4 is obtained by dividing the numerical concentration value (expressed as pfu / 0.5 ml) of an individual serotype by the total concentration (expressed in pfu / 0.5 ml) and multiplying the result by 100 i.e.:Percentage concentration of serotype 1 = (4xl03pfu / 0.5 ml 4- 1.84 xlO5pfu / 0.5 ml) x 100 = 2%Percentage concentration of serotype 2 = (lxlO4pfu / 0.5ml 4- 1.84 xlO5pfu / 0.5 ml) x 100 = 5%Percentage concentration of serotype 3 = (4xl04pfu / 0.5 ml - 1.84 xlO5pfu / 0.5 ml) x 100 = 22%Percentage concentration of serotype 4 = (1.3xl05pfu / 0.5 ml 4- 1.84 xlO5pfu / 0.5 ml) x 100 = 71%.The percentage concentrations are rounded to whole numbers.

[0075] As used herein "simultaneous" or "concomitant" administration means an administration of at least two different vaccines such as a dengue vaccine and an HPV vaccine on the same day. " On the same day" has the ordinary meaning of within 24 hours, such as e.g. within one calendar day. The concomitant administration may be administered during the same medical appointment, e.g. by the same medical practitioner. In the context of the invention, the combined administration of two vaccines refers to combining the administration schedule of a dengue vaccine composition, such as a tetravalent dengue composition, with the administration schedule of a HPV vaccine composition, such as a 9vHPV vaccine.

[0076] As used herein "sequential" administration means an administration of at least two different vaccines, such as a dengue vaccine and an HPV vaccine on different or subsequent days, such as within 90 days, but in a combined administration schedule.

[0077] As used herein, "solicited systemic adverse events" in children under 6 years are defined as fever, irritability / fussiness, drowsiness and loss of appetite that occurred within 14 days after each vaccination, and in children of 6 years or more are defined as fever, headache, asthenia, malaise and myalgia that occurred within 14 days after each vaccination.

[0078] As used herein, "solicited local adverse events" are injection site pain, injection site erythema and injection site swelling that occurred within 7 days after each vaccination.

[0079] As used herein, "unsolicited adverse events" are any adverse events (AEs) that are not solicited local orsystemic AEs, as defined above.

[0080] As used herein, a "serious adverse event" or " SAE" is any untoward medical occurrence or effect that at any dose results in death, is life-threatening, requires inpatient hospitalization or prolongation of existing hospitalization, results in persistent or significant disability / incapacity, is a congenital anomaly I birth defect or is medically important due to other reasons than the above mentioned criteria.

[0081] The relationship of each AE, including solicited systemic AEs (solicited local AEs are considered as related) to trial vaccine(s) will be assessed using the following categories: As used herein, " IP-Related AE" or "vaccine related AE" means that there is suspicion that there is a relationship between the vaccine and the AE (without determining the extent of probability); there is a reasonable possibility that the vaccine contributed to the AE. As used herein, " Non-IP Related" or "non-vaccine related" means that there is no suspicion that there is a relationship between the vaccine and the AE; there are other more likely causes and administration of the vaccine is not suspected to have contributed to the AE.

[0082] As used herein, a subject or subject population being, e.g., "2 to 60 years of age" or "9 to 15 years of age" refers to a subject or subject population being 2 to 60 years of age or 9 to 15 years of age on the first day of the administration of the dengue vaccine composition as described herein.

[0083] As used herein "%-points" refers to the difference of two %-values in a %-value. For example two values in % which are within 5 %-points refers to e.g. one value at 1% and a second value at 6%.

[0084] As used herein, the term "determination of the previous dengue infection in the subject before administration" means that a previous dengue infection is assessed before vaccination in that there is a laboratory confirmed history of dengue or through an appropriately validated serological test, e.g. by the method as disclosed herein such as the MNT50 test described in Example 2 or any serotesting with adequate performance in terms of specificity and cross reactivity based on the locale disease epidemiology.

[0085] As used herein % w / v refers to % mg / ml wherein e.g. 150 mg / ml are 15% w / v.

[0086] As used herein, the term " Human Papillomavirus" may be abbreviated as " HPV".

[0087] As used herein, the term "placebo" may be abbreviated as " Pbo".

[0088] As used herein, "seropositivity for HPV" is defined as an anti-HPV titer greater than or equal to the prespecified serostatus cut-off for a given HPV type. The serostatus cut-off is well known for the person skilled in the art and is typically the antibody titer level above the assay's lower limit of quantification that distinguishes sera samples classified by clinical likelihood of HPV infection and positive or negative status by previous versions of cLIA or equivalent assay. An exemplary assay for determining an anti-HPV titer is disclosed in Nolan et al., " Development and Validation of Two Optimized Multiplexed Serologic Assays for the 9-Valent Human Papillomavirus Vaccine Types", American Society for Microbiology, March / April 2023, Volume 8, Issue 2.

[0089] As used herein, " HPV seronegative at baseline" or " HPV naive (at baseline)" or "seronegativity for HPV" is defined as an anti-HPV titer less than the pre-specified serostatus cut-off for a given HPV type.

[0090] As used herein, "at baseline" refers to the time point of the last measurement of a subject's serostatus prior to the first vaccination.

[0091] As used herein, the term " CCID" refers to the quantity of virus (e.g. vaccinal virus) infecting 50% of the cell culture. The CCID50 assay is a limit dilution assay with statistical titer calculation (Morrison D et al, J Infect Dis. 2010; 201(3):370-7)).

[0092] " Non-inferiority" or "non-inferior", as used herein, with respect to a concomitant or simultaneous on the same day administration of a HPV vaccine and a tetravalent dengue vaccine is in particular concluded, if the upper bound of the 95% confidence interval (CI) for the GMT ratio (Group 2-HPV alone / Group 1-HPV+dengue) is less than the non-inferiority margin of 1.5. A non-inferiority clinical study is a study designed to provide a comparison between at least two methods of treatments, in the present case between a concomitant or simultaneous administration of a dengue vaccine and an HPV vaccine and a mono-administration of either a dengue vaccine or an HPV vaccine. Such a non-inferiority clinical study can also be used to assess whether there is no significant reduction in immunogenicity of the HPV vaccine composition compared to a mono-administration of the HPV vaccine composition and / or whether there is no significant reduction in immunogenicity of the dengue vaccine composition compared to a mono-administration of the dengue vaccine composition. Likewise, such a non-inferiority clinical study can also be used to assess there is no significant interaction between the HPV vaccine composition and the dengue vaccine composition effecting either the immunogenicity of the HPV vaccine composition or the immunogenicity of the dengue vaccine composition.

[0093] As used herein, the term "control subject population" refers to a group of subjects which does not receive a simultaneous administration of a HPV vaccine and a unit dose of a dengue vaccine composition, but a single verum (such as an HPV vaccine or a unit dose of a dengue vaccine composition) on the same day in a clinical study setting as e.g. in a non-inferiority clinical study.

[0094] As used herein, the term " HPV-associated disease" refers to a disease which is caused by infection with the Human Papillomavirus. Examples of an HPV-associated disease include different types of cancer, for instance, cervical cancer, cancers of the anus, vulva and vagina, penis, head and neck, and the oropharynx. Anogenital HPV infection can also result in benign skin and mucosal tumors, including anogenital warts (also known as condylomata acuminata or venereal warts).

[0095] As used herein " HPV-associated cancers or genital warts" refers to cancers and genital wards caused by an HPV infection, respectively. The HPV serotypes 16, 18, 45, 31, 33, 52, 58, also referred to as "high-risk" HPV serotypes, are the types most common in cervical cancers, wherein the two HPV serotypes 16 and 18 cause about 70% of cervical cancers worldwide. The HPV serotypes 6 and 11, also referred to as "low-risk" HPV serotypes, cause genital warts.

[0096] As used herein, a "bivalent HPV vaccine" refers to an HPV vaccine that provides protection against the HPV serotypes 16 and 18. Examples for bivalent HPV vaccines include Cervarix®, Cecolin® and Walrinvax®.

[0097] As used herein, a "quadrivalent HPV vaccine" refers to an HPV vaccine that provides protection against HPV serotypes 6, 11, 16 and 18. Examples for quadrivalent HPV vaccines include Gardasil® and Cervavac®.

[0098] As used herein, a "nonavalent HPV vaccine" or "9vHPV vaccine" refers to an HPV vaccine that provides protection against the HPV serotypes 6, 11, 16, 18, 31, 33, 45, 52, and 58. In particular, the 9vHPV vaccine is used to prevent cervical, vulvar, vaginal, and anal cancers, precancerous or dysplastic lesions, and genital warts caused by one or more of these HPV serotypes. Examples for nonavalent HPV vaccines include Gardasil® 9.BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Figure 1: Genetic structure of the four dengue strains contained in TDV. The solid red triangles indicate the three attenuating mutations present in the 5'NCR, NS1 and NS3 proteins. The TDV-1, TDV-3 and TDV-4 strains are chimeric viruses where the prM and E genes from dengue serotype 1, 3 and 4, respectively, are inserted into the TDV-2 backbone.

[0100] Figure 2: Schematic drawing illustrating the microneutralization test (MNT) used to determine the titer of neutralizing antibodies.

[0101] Figure 3: Flow diagram of the clinical trial of Example 3.

[0102] Figure 4: Cumulative incidence of A) virologically confirmed dengue cases and B) hospitalized virologically-confirmed dengue cases overtime during Part 1 study period by baseline serostatus (safety set data; data presented truncated at Month 18). Tables show numbers of participants under follow-up at various time points to end of Part 1 study period.

[0103] Figure 5: Study design of phase III study described in example 3.

[0104] Figure 6: Scheme of the trial design of the simultaneous HPV and TDV administration study described in example 4. M, month; TDV, Takeda's Dengue Tetravalent Vaccine; 9vHPV, Recombinant 9-valent human papillomavirus vaccine.

[0105] Figure 7: GMTs of neutralizing antibodies for each HPV type at day 210 (Pre-Protocol Set). A negative logic antibody value is presented for HPV-45 and HPV-52 (day 1) as the imputed half value of the pre-specified LLOQ is 0.5 for these serotypes.

[0106] Figure 8: Figure 8A: Non-inferiority comparisons of GMTs of neutralizing antibodies for each HPV type at day 210 (Per-Protocol Set). Figure 8B: Comparisons of GMTs of neutralizing antibodies for each HPV type at day 210 (Per-Protocol Set). HPV, Human Papillomavirus; GMT, geometric mean titer; CI, confidence interval.

[0107] Figure 9: Seropositivity rates for each HPV type at day 210 (Per-Protocol Set). Seropositivity for HPV was defined as an anti-HPV titer > pre-specified serostatus cut-off for a given HPV type.

[0108] Figure 10: GMTs of neutralizing antibodies for each dengue serotype by dengue baseline seropositivity at day 120 (Per-Protocol Set).

[0109] Figure 11: Seropositivity rates for each dengue serotype by dengue baseline seropositivity at day 120 (Per-Protocol Set).

[0110] Figure 12: Comparison of GMTs by serotype in clinical trials DEN-308 and DEN-301 at day 120 in subjects seronegative at baseline (Per-Protocol Set).

[0111] Figure 13: Solicited local adverse events within 7 days after each vaccination (Safety Set).

[0112] Figure 14: Solicited systemic adverse events within 14 days after each vaccination (Safety Set).

[0113] Figure 15: Unsolicited adverse events up to 28 days after each vaccination (Safety Set).DETAILED DESCRIPTIONDengue virus strains

[0114] The dengue virus is a single stranded, positive sense RNA virus of the family fla viviridae. The taxonomy is outlined in Table 1. The family flaviviridae includes three genera, flavivirus, hepacivirus and pestivirus. The genus flavivirus contains highly pathogenic and potentially hemorrhagic fever viruses, such as yellow fever virus and dengue virus, encephalitic viruses, such as Japanese encephalitis virus, Murray Valley encephalitis virus and West Nile virus, and a number of less pathogenic viruses.Table 1. Dengue Virus Taxonomy of the GMO Parental StrainFamily FlaviviridaeGenus FlavivirusSpecies Dengue virusStrains Dengue Serotype 2 (Strain 16681), Strain DEN-2 PDK-53GMO parent TDV-2

[0115] The flavivirus genome comprises in 5' to 3' direction (see Figure 1):a 5'-noncoding region (5'-NCR),a capsid protein (C) encoding region,a pre-membrane protein (prM) encoding region,an envelope protein (E) encoding region,a region encoding nonstructural proteins (NSI, NS2A, NS2B, NS3, NS4A, NS4B, NS5) anda 3' noncoding region (3'-NCR).

[0116] The viral structural proteins are C, prM and E, and the nonstructural proteins are NSI to NS5. The structural and nonstructural proteins are translated as a single polyprotein and processed by cellular and viral proteases.

[0117] The unit dose of the invention as described herein comprises a dengue virus composition that comprises four live attenuated dengue virus strains (tetravalent dengue virus composition) representing dengue serotype 1, dengue serotype 2, dengue serotype 3 and dengue serotype 4. In specific embodiments, the composition comprises at least one non-chimeric dengue virus. For example, the composition comprises chimeric dengue viruses and optionally at least one non-chimeric dengue virus, such as a molecularly characterized and cloned dengue serotype 2 strain derived from the live attenuated DEN-2 PDK-53 virus strain (TDV-2), 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 the other dengue serotypes, resulting in the following chimeric dengue strains:- a DENV-2 / 1 chimera (TDV-1),- a DENV-2 / 3 chimera (TDV-3) and- a DENV-2 / 4 chimera (TDV-4).

[0118] The genetically modified tetravalent dengue vaccine TDV is based on a molecularly characterized and cloned dengue-2 virus strain (TDV-2). This attenuated TDV-2 strain was generated by cDNA cloning of the attenuated laboratory-derived DEN-2 PDK-53 virus strain that was originally isolated at Mahidol University, Bangkok, Thailand (Kinney et al. (1997) Virology 230(2): 300-308). DEN-2 PDK-53 was generated by 53 serial passages in primary dog kidney (PDK) cells at 32°C (Bhamarapravati et al. (1987) Bull. World Health Organ. 65(2): 189-195).

[0119] The atenuated DEN-2 PDK-53 strain (the precursor of TDV-2) was derived from the wild type virus strain DEN-216681 (SEQ ID NO 11) and differs in nine nucleotides from the wild type as follows (Kinney et al. (1997) Virology 230(2): 300-308):(i) 5'-noncoding region (NCR)-57 (nt-57 C-to-T): major atenuation 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 atenuation locus(v) NS2A-181 Leu-to-Phe (nt-4018 C-to-T)(vi) NS3-250 Glu-to-Val (nt-5270 A-to-T): major atenuation locus(vii) nt-5547 (NS3 gene) T-to-C silent mutation(viii) NS4A-75 Gly-to-Ala (nt-6599 G-to-C)* nt-8571 C-to-T (NS5 gene) silent mutation

[0120] The three nucleotide changes located in the 5' noncoding region (NCR) (nucleotide 57) (mutation (i)), the NS-1 (amino acid 828 of SEQ ID NO. 4) (mutation (iv)) and NS-3 genes (amino acid 1725 of SEQ ID NO. 4) (mutation (vi)) form the basis for the atenuation 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 the "attenuating mutations" and are comprised in TDV-1, TDV-2, TDV-3 and TDV-4.Table 2. Attenuating mutations in the common genetic backbone of all TDV strains Location of Mutation Nucleotide Change in TDV-2 Amino Acid Change in TDV-2 5' Noncoding Region (5'NCR) 57 C to T Not applicable (silent) Nonstructural Protein 1 (NS1) 2579 G to A 828 Gly to AspNonstructural Protein 3 (NS3) 5270 A to T 1725 Glu to Vai

[0121] In one embodiment, TDV-2 comprises in addition to the three atenuating mutations one or more mutations selected from:a) a mutation in the prM gene at nucleotide 524 from adenine to thymine resulting in an amino acid change at position 143 from aspartic acid to valine, and / orb) a silent mutation in the E gene at nucleotide 2055 from cytosine to thymine, and / orc) a mutation in the NS2A gene at nucleotide 4018 from cytosine to thymine resulting in an amino acid change at position 1308 from leucine to phenylalanine, and / ord) a silent mutation in the NS3 gene at nucleotide 5547 from thymine to cytosine, and / ore) a mutation in the NS4A gene at nucleotide 6599 from guanine to cytosine resulting in an amino acid change at position 2168 from glycine to alanine, and / orf) a silent mutation in the prM gene at nucleotide 900 from thymine to cytosine.The silent mutation in the NS5 gene at nucleotide 8571 from cytosine to thymine of DEN-2 PDK-53 is not present in the TDV-2 strain.

[0122] In another embodiment, TDV-2 comprises in addition to the three atenuating mutations one or more mutations selected from:g) a mutation in the prM gene at nucleotide 592 from adenine to guanine resulting in an amino acid change at position 166 from lysine to glutamic acid, and / orh) a mutation in the NS5 gene at nucleotide 8803 from adenine to guanine resulting in an amino acid change at position 2903 from isoleucine to valine.

[0123] In another embodiment, TDV-2 comprises in addition to the three attenuating mutations the mutations a) and g), such as the mutations a), g), c), e) and h), or the mutations a), g), c), e), h) and b), or the mutations a), g), c), e), h), b) and d), or the mutations a) to h). The nucleotide positions and amino acids positions of TDV-2 refer to the nucleotide sequence as shown in SEQ ID NO. 3 and amino acid sequence as shown in SEQ ID NO. 4.

[0124] The dengue virus structural envelope (E) protein and pre-membrane (prM) protein have been identified as the primary antigens that elicit a neutralizing protective antibody response (Plotkin 2001). For creation of the tetravalent dengue vaccine (TDV), TDV-2 was modified by replacing the nucleic acid sequence encoding the DENV-2 prM and E glycoproteins with the nucleic acid sequence encoding the corresponding wild type prM and E glycoproteins from the DENV-1, DENV-3, and DENV-4 wild type strains DENV-1 16007, DENV-3 16562 or DENV-4 1036 virus, respectively, (see Table 3) using standard molecular genetic engineering methods (Huang et al. (2003) J. Virol. 77(21): 11436-11447).Table 3. Viral origin of prM / E gene regions of the TDV virus strainsNucleotide Amino acid Virus Strain Origin Source Referencesequence sequence DENV-1 16007 Thailand, 1964 DHF / DSS Halstead and SEQ ID NO. 9 SEQ ID NO. 10 patient Simasthien, 1970DENV-2 16681 Thailand, 1964 DHF / DSS Halstead and SEQ ID NO. 11 SEQ ID NO. 12 patient Simasthien, 1970DENV-3 16562 Philippines, 196 DHF patient Halstead and SEQ ID NO. 13 SEQ ID NO. 14 Simasthien, 1970DENV-4 1036 Indonesia, 197( DF patient Gubleretal., 1979 SEQ ID NO. 15 SEQ ID NO. 16

[0125] A diagram of the four TDV strains comprised in the dengue vaccine composition is shown in Figure 1.

[0126] The chimeric dengue strains TDV-1, TDV-3 and TDV-4 express the surface antigens prM and E of the DENV-1, DENV-3 or DENV-4 viruses, as depicted in Table 3 respectively, and retain the genetic alterations responsible for the attenuation of TDV-2. Thus, each of the TDV-1, TDV-3 and TDV-4 strains comprises the attenuating mutations described in Table 2.

[0127] In one embodiment, TDV-1 comprises in addition to the three attenuating mutations one or more mutations selected from:c) a mutation in the NS2A gene at nucleotide 4018 from cytosine to thymine resulting in an amino acid change at position 1308 from leucine to phenylalanine, and / ord) a silent mutation in the NS3 gene at nucleotide 5547 from thymine to cytosine, and / ore) a mutation in the NS4A gene at nucleotide 6599 from guanine to cytosine resulting in an amino acid change at position 2168 from glycine to alanine, and / ori) a silent mutation in the E gene at nucleotide 1575 from thymine to cytosine, and / orj) a silent mutation in the junction site between the prM-E gene and the DEN-2 PDK-53 backbone at nucleotide 453 from adenine to guanine, and / ork) a mutation in the junction site between the prM-E gene and the DEN-2 PDK-53 backbone at nucleotides 2381 / 2382 from thymine-guanine to cytosine-cytosine resulting in an amino acid change at position 762 from valine to alanine.

[0128] In another embodiment, TDV-1 comprises in addition to the three attenuating mutations one or more mutations selected from:l) a mutation in the NS2A gene at nucleotide 3823 from adenine to cytosine resulting in an amino acid change at position 1243 from isoleucine to leucine, and / orm) a mutation in the NS2B gene at nucleotide 4407 from adenine to thymine resulting in an amino acid change at position 1437 from glutamic acid to aspartic acid, and / orn) a silent mutation in the NS4B gene at nucleotide 7311 from adenine to guanine.

[0129] In another embodiment, the TDV-1 strain comprises in addition to the three attenuating mutations the mutations I) and m), such as the mutations I), m), c) and e), or the mutations I), m), c), e), d) and n), or the mutations I), m), c), e), d), n), i), j) and k). The nucleotide positions and amino acids positions of TDV-1 refer to the nucleotide sequence as shown in SEQ ID NO. 1 and amino acid sequence as shown in SEQ ID NO. 2.

[0130] In one embodiment, TDV-3 comprises in addition to the three attenuating mutations one or more mutations selected from:c) a mutation in the NS2A gene at nucleotide 4012 from cytosine to thymine resulting in an amino acid change at position 1306 from leucine to phenylalanine, and / ord) a silent mutation in the NS3 gene at nucleotide 5541 from thymine to cytosine, and / ore) a mutation in the NS4A gene at nucleotide 6593 from guanine to cytosine resulting in an amino acid change at position 2166 from glycine to alanine, and / orj) a silent mutation in the junction site between the prM-E gene and the DEN-2 PDK-53 backbone at nucleotide 453 from adenine to guanine, and / ork) a mutation in the junction site between the prM-E gene and the DEN-2 PDK-53 backbone at nucleotides 2375 / 2376 from thymine-guanine to cytosine-cytosine resulting in an amino acid change at position 760 from valine to alanine, and / oro) a silent mutation in the prM gene at nucleotide 552 from cytosine to thymine, and / orp) a mutation in the E gene at nucleotide 1970 from adenine to thymine resulting in an amino acid change at position 625 from histidine to leucine.

[0131] In another embodiment, TDV-3 comprises in addition to the three attenuating mutations one or more mutations selected from:q) a mutation in the E gene at nucleotide 1603 from adenine to thymine resulting in an amino acid change at position 503 from threonine to serine, and / orr) a silent mutation in the NS5 gene at nucleotide 7620 from adenine to guanine.

[0132] In another embodiment, TDV-3 comprises in addition to the three attenuating mutations the mutations p) and q), such as the mutations p), q), c) and e), or the mutations p), q), c), e), d) and r), or the mutations p), q), c), e), d),r)< j), k) and o). The nucleotide positions and amino acids positions of TDV-3 refer to the nucleotide sequence as shown in SEQ ID NO. 5 and amino acid sequence as shown in SEQ ID NO. 6.

[0133] In one embodiment, TDV-4 comprises in addition to the three attenuating mutations one or more mutations selected from:c) a mutation in the NS2A gene at nucleotide 4018 from cytosine to thymine resulting in an amino acid change at position 1308 from leucine to phenylalanine, and / ord) a silent mutation in the NS3 gene at nucleotide 5547 from thymine to cytosine, and / ore) a mutation in the NS4A gene at nucleotide 6599 from guanine to cytosine resulting in an amino acid change at position 2168 from glycine to alanine, and / orj) a silent mutation in the junction site between the prM-E gene and the DEN-2 PDK-53 backbone at nucleotide 453 from adenine to guanine, and / ork) a mutation in the junction site between the prM-E gene and the DEN-2 PDK-53 backbone at nucleotides 2381 / 2382 from thymine-guanine to cytosine-cytosine resulting in an amino acid change at position 762 from valine to alanine, and / ors) a mutation in the C gene at nucleotide 396 from adenine to cytosine resulting in an amino acid change at position 100 from arginine to serine, and / ort) a silent mutation in the E gene at nucleotide 1401 from adenine to guanine, and / oru) a mutation in the E gene at nucleotide 2027 from cytosine to thymine resulting in an amino acid change at position 644 from alanine to valine, and / orv) a mutation in the E gene at nucleotide 2275 from adenine to cytosine resulting in an amino acid change at position 727 from methionine to leucine.

[0134] In another embodiment, TDV-4 comprises in addition to the three attenuating mutations one or more mutations selected from:w) a silent mutation in the C gene at nucleotide 225 from adenine to thymine, and / orx) a mutation in the NS2A gene at nucleotide 3674 from adenine to guanine resulting in an amino acid change at position 1193 from aspartic acid to glycine, and / ory) a mutation in the NS2A gene at nucleotide 3773 from adenine to an adenine / guanine mix resulting in an amino acid change at position 1226 from lysine to a lysine / arginine mix, and / orz) a silent mutation in the NS3 gene at nucleotide 5391 from cytosine to thymine, and / oraa) a mutation in the NS4A gene at nucleotide 6437 from cytosine to thymine resulting in an amino acid change at position 2114 from alanine to valine, and / orbb) a silent mutation in the NS4B gene at nucleotide 7026 from thymine to a thymine / cytosine mix, and / or cc) a silent mutation in the NS5 gene at nucleotide 9750 from adenine to cytosine.

[0135] In another embodiments, TDV-4 comprises in addition to the three attenuating mutations the mutation s), u) and v), such as the mutations s), u), v), c), e), x), y) and aa), or the mutations s), u), v), c), e), x), y), aa) and w), or the mutations s), u), v), c), e), x), y), aa), w), d), z), bb) and cc), or the mutations s), u), v), c), e), x), y), aa), w), d), z), bb), cc), j), k) and t). The nucleotide positions and amino acids positions of TDV-4 refer to the nucleotide sequence as shown in SEQ ID NO. 7 and amino acid sequence as shown in SEQ ID NO. 8.

[0136] In an exemplary embodiment, TDV-1 has the nucleotide sequence of SEQ ID NO. 1, TDV-2 has the nucleotide sequence of SEQ ID NO. 3, TDV-3 has the nucleotide sequence of SEQ ID NO. 5, and / or TDV-4 has the nucleotide sequence of SEQ ID NO. 7. In a further exemplary embodiment, TDV-1 has the amino acid sequence of SEQ ID NO. 2, TDV-2 has the amino acid sequence of SEQ ID NO. 4, TDV-3 has the amino acid sequence of SEQ ID NO. 6, and TDV-4 has the amino acid sequence of SEQ ID NO. 8. In a further exemplary embodiment, TDV-1 has a nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 2, TDV-2 has a nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 4, TDV-3 has a nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 6, and TDV-4 has a nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 8.Table 4. Sequences of the TDV virus strainsSEQ ID NO. dengue virus strain sequence typeSEQ ID NO. 1 TDV-1 nucleotide sequenceSEQ ID NO. 2 TDV-1 amino acid sequenceSEQ ID NO. 3 TDV-2 nucleotide sequenceSEQ ID NO.4 TDV-2 amino acid sequenceSEQ ID NO. 5 TDV-3 nucleotide sequenceSEQ ID NO. 6 TDV-3 amino acid sequenceSEQ ID NO. 7 TDV-4 nucleotide sequenceSEQ ID NO.8 TDV-4 amino acid sequence

[0137] Thus, in a specific embodiment, the unit dose of the invention as described herein comprises the live 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 comprise the prM and E regions of DENV-1, -3 and -4, respectively. In another specific 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, 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, TDV-3 is characterized by the nucleotide sequence according to SEQ ID No. 5 and the amino acid sequence according to SEQ ID No. 6 and TDV-4 is characterized by the nucleotide sequence according to SEQ ID No. 7 and the amino acid sequence according to SEQ ID No. 8.

[0138] The E protein of DENV-3 has two fewer amino acids than the E protein of DENV-2. Therefore, the nucleotides 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 less and 2 amino acids less than the original TDV-2 nucleotide and amino acid positions, respectively.Denoue vaccine composition

[0139] Described herein is a unit dose of a dengue vaccine composition. The dengue vaccine composition comprises a tetravalent dengue virus composition, also referred to as dengue virus composition, and pharmaceutically acceptable excipients.Dengue virus composition, virus concentrations and %-concentrations

[0140] Described is a unit dose of a dengue vaccine composition, wherein the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains:(i) a dengue serotype 1 such as in a concentration of at least 3.3 loglO pfu / 0.5 mL,(ii) a dengue serotype 2 such as in a concentration of at least 2.7 loglO pfu / 0.5 mL,(iii) a dengue serotype 3 such as in a concentration of at least 4.0 loglO pfu / 0.5 mL, and(iv) a dengue serotype 4 such as in a concentration of at least 4.5 loglO pfu / 0.5 mL.

[0141] In one embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains:(i) a dengue serotype 1 such as in a concentration of at least 3.3 loglO pfu / 0.5 mL to 3.8 loglO pfu / 0.5 mL, (ii) a dengue serotype 2 such as in a concentration of at least 2.7 loglO pfu / 0.5 mL,(iii) a dengue serotype 3 such as in a concentration of at least 4.0 loglO pfu / 0.5 mL, and(iv) a dengue serotype 4 such as in a concentration of at least 4.5 loglO pfu / 0.5 ml or 4.6 loglO pfu / 0.5 mL, optionally to 6.2 loglO pfu / 0.5 ml.

[0142] Described herein is a unit dose of a dengue vaccine composition, wherein the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains:(i) a chimeric dengue serotype 2 / 1 strain in a concentration of at least 3.3 loglO pfu / 0.5 mL,(ii) a dengue serotype 2 strain in a concentration of at least 2.7 loglO pfu / 0.5 mL,(iii) a chimeric dengue serotype 2 / 3 strain in a concentration of at least 4.0 loglO pfu / 0.5 mL, and(iv) a chimeric dengue serotype 2 / 4 strain in a concentration of at least 4.5 loglO pfu / 0.5 mL.

[0143] In one embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains:(i) a chimeric dengue serotype 2 / 1 strain in a concentration of at least 3.3 loglO pfu / 0.5 mL to 3.8 loglO pfu / 0.5 ml,(ii) a dengue serotype 2 strain in a concentration of at least 2.7 loglO pfu / 0.5 mL,(iii) a chimeric dengue serotype 2 / 3 strain in a concentration of at least 4.0 loglO pfu / 0.5 mL, and(iv) a chimeric dengue serotype 2 / 4 strain in a concentration of at least 4.5 loglO pfu / 0.5 mL or at least 4.6 loglO pfu / 0.5 mLto optionally 6.2 loglO pfu / 0.5 ml.

[0144] In a specific embodiment, the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3 and the chimeric dengue serotype 2 / 4 strain is TDV-4.

[0145] In one embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains wherein:(i) the dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 loglO pfu / 0.5 mLto 5.3 loglO pfu / 0.5 mL,(ii) the dengue serotype 2 (e.g. dengue serotype 2 strain) has a concentration of 2.7 loglO pfu / 0.5 mL to 5.0 loglO pfu / 0.5 mL,(iii) the dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) has a concentration of 4.0 loglO pfu / 0.5 mL to 6.0 loglO pfu / 0.5 mL, and(iv) the dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) has a concentration of 4.5 loglO pfu / 0.5 mLto 6.5 loglO pfu / 0.5 mL.

[0146] In one such embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains wherein:(i) the dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 loglO pfu / 0.5 mLto 5.0 loglO pfu / 0.5 mL,(ii) the dengue serotype 2 (e.g. dengue serotype 2 strain) has a concentration of 2.7 loglO pfu / 0.5 mL to 4.9 loglO pfu / 0.5 mL,(iii) the dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) has a concentration of 4.0 loglO pfu / 0.5 mLto 5.7 loglO pfu / 0.5 mL, and(iv) the dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) has a concentration of 4.5 loglO pfu / 0.5 mL to 6.2 loglO pfu / 0.5 mL.

[0147] In a further such embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains wherein:(i) a dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 loglO pfu / dose to 5.0 loglO pfu / dose,(ii) a dengue serotype 2 (e.g. dengue serotype 2 strain) has a concentration of 2.7 loglO pfu / dose to 4.9 loglO pfu / dose,(iii) a dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) has a concentration of 4.0 loglO pfu / dose to 5.7 loglO pfu / dose, and(iv) a dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) has a concentration of 4.5 loglO pfu / dose to 5.5 loglO pfu / dose.

[0148] In a further such embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains wherein:(i) a dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 loglO pfu / dose to 4.1 loglO pfu / dose,(ii) a dengue serotype 2 (e.g. dengue serotype 2 strain) has a concentration of 2.7 loglO pfu / dose to 3.6 loglO pfu / dose,(iii) a dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) has a concentration of 4.0 loglO pfu / dose to 4.7 loglO pfu / dose, and(iv) a dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) has a concentration of 4.5 loglO pfu / dose to 5.3 loglO pfu / dose.

[0149] In a further such embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains wherein:(i) a dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 loglO pfu / 0.5 mL to 3.6 loglO pfu / 0.5 mL,(ii) a dengue serotype 2 (e.g. dengue serotype 2 strain) has a concentration of 2.7 loglO pfu / 0.5 mL to 4.0 loglO pfu / 0.5 mL,(iii) a dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) has a concentration of 4.0 loglO pfu / 0.5 mL to 4.6 loglO pfu / 0.5 mL, and(iv) a dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) has a concentration of 4.5 loglO pfu / 0.5 ml or 4.6 loglO pfu / 0.5 mLto 5.1 loglO pfu / 0.5 mL.

[0150] In another embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains wherein:(i) the dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) has a concentration of 4.3 loglO pfu / 0.5 mL to 4.4 loglO pfu / 0.5 mL,(ii) the dengue serotype 2 (e.g. dengue serotype 2 strain) has a concentration of 3.7 loglO pfu / 0.5 mL to 3.8 loglO pfu / 0.5 mL,(iii) the dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) has a concentration of 4.5 loglO pfu / 0.5 mL to 5.0 loglO pfu / 0.5 mL, and(iv) the dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) has a concentration of 5.5 loglO pfu / 0.5 mLto 5.6 loglO pfu / 0.5 mL.

[0151] In a specific embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains wherein:(i) the dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) has a concentration of 4.4 loglO pfu / 0.5 mL,(ii) the dengue serotype 2 (e.g. dengue serotype 2 strain) has a concentration of 3.8 loglO pfu / 0.5 mL, (iii) the dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) has a concentration of 4.5 loglO pfu / 0.5 mL, and(iv) the dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) has a concentration of 5.6 loglO pfu / 0.5 mL.

[0152] In another specific embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains wherein:(i) the dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) has a concentration of 3.6 loglO pfu / 0.5 mL,(ii) the dengue serotype 2 (e.g. dengue serotype 2 strain) has a concentration of 4.0 loglO pfu / 0.5 mL, (iii) the dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) has a concentration of 4.6 loglO pfu / 0.5 mL, and(iv) the dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) has a concentration of 5.1 loglO pfu / 0.5 mL.

[0153] In another specific embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains wherein the arithmetic sum of all four serotypes is less than 6.7 loglO pfu / 0.5 mL, for example less than 5.5 loglO pfu / 0.5 mL. In certain such embodiments, the arithmetic sum of all four serotypes is at least 4.6 loglO pfu / 0.5 mL. In an exemplary embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains wherein the arithmetic sum of all four serotypes is in the range of 4.6 loglO pfu / 0.5 mL to 6.7 loglO pfu / 0.5 mL, e.g. in the range of 4.6 loglO pfu / 0.5 mL to 5.5 loglO pfu / 0.5 mL.

[0154] For example, in said embodiments the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3 and the chimeric dengue serotype 2 / 4 strain is TDV-4. For example, 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, 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, TDV-3 is characterized by the nucleotide sequence according to SEQ ID No. 5 and the amino acid sequence according to SEQ ID No. 6 and TDV-4 is characterized by the nucleotide sequence according to SEQ ID No. 7 and the amino acid sequence according to SEQ ID No. 8.

[0155] Described herein is a unit dose of a dengue vaccine composition, wherein the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains:(i) a dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) in a concentration of at least 3.3 loglO pfu / dose,(ii) a dengue serotype 2 (e.g. dengue serotype 2 strain) in a concentration of at least 2.7 loglO pfu / dose, (iii) a dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) in a concentration of at least 4.0 loglO pfu / dose, and(iv) a dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) in a concentration of at least 4.5 loglO pfu / dose.

[0156] In one embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains wherein:(i) the dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 loglO pfu / dose to 5.3 loglO pfu / dose,(ii) the dengue serotype 2 (e.g. dengue serotype 2 strain) has a concentration of 2.7 loglO pfu / dose to 5.0 loglO pfu / dose,(iii) the dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) has a concentration of 4.0 loglO pfu / dose to 6.0 loglO pfu / dose, and(iv) the dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) has a concentration of 4.5 loglO pfu / dose to 6.5 loglO pfu / dose.

[0157] In one such embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains wherein:(i) the dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 loglO pfu / dose to 5.0 loglO pfu / dose,(ii) the dengue serotype 2 (e.g. dengue serotype 2 strain) has a concentration of 2.7 loglO pfu / dose to 4.9 loglO pfu / dose,(iii) the dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) has a concentration of 4.0 loglO pfu / dose to 5.7 loglO pfu / dose, and(iv) the dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) has a concentration of 4.5 loglO pfu / dose to 6.2 loglO pfu / dose.

[0158] In a further such embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains wherein:(i) a dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 loglO pfu / dose to 5.0 loglO pfu / dose,(ii) a dengue serotype 2 (e.g. dengue serotype 2 strain) has a concentration of 2.7 loglO pfu / dose to 4.9 loglO pfu / dose,(iii) a dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) has a concentration of 4.0 loglO pfu / dose to 5.7 loglO pfu / dose, and(iv) a dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) has a concentration of 4.5 loglO pfu / dose to 5.5 loglO pfu / dose.

[0159] In a further such embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains wherein:(i) a dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 loglO pfu / dose to 4.1 loglO pfu / dose,(ii) a dengue serotype 2 (e.g. dengue serotype 2 strain) has a concentration of 2.7 loglO pfu / dose to 3.6 loglO pfu / dose,(iii) a dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) has a concentration of 4.0 loglO pfu / dose to 4.7 loglO pfu / dose, and(iv) a dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) has a concentration of 4.5 loglO pfu / dose to 5.3 loglO pfu / dose.

[0160] In a further such embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains wherein:(i) a dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 loglO pfu / dose to 3.6 loglO pfu / dose,(ii) a dengue serotype 2 (e.g. dengue serotype 2 strain) has a concentration of 2.7 loglO pfu / dose to 4.0 loglO pfu / dose,(Hi) a dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) has a concentration of 4.0 loglO pfu / dose to 4.6 loglO pfu / dose, and(iv) a dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) has a concentration of 4.5 loglO pfu / dose 4.6 loglO pfu / dose to 5.1 loglO pfu / dose.

[0161] In another embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains wherein:(i) the dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) has a concentration of 4.3 loglO pfu / dose to 4.4 loglO pfu / dose,(ii) the dengue serotype 2 (e.g. dengue serotype 2 strain) has a concentration of 3.7 loglO pfu / dose to 3.8 loglO pfu / dose,(iii) the dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) has a concentration of 4.5 loglO pfu / dose to 5.0 loglO pfu / dose, and(iv) the dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) has a concentration of 5.5 loglO pfu / dose to 5.6 loglO pfu / dose.

[0162] In a specific embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains wherein:(i) the dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) has a concentration of 4.4 loglO pfu / dose, (ii) the dengue serotype 2 (e.g. dengue serotype 2 strain) has a concentration of 3.8 loglO pfu / dose, (iii) the dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) has a concentration of 4.5 loglO pfu / dose, and(iv) the dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) has a concentration of 5.6 loglO pfu / dose.

[0163] In another specific embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains wherein:(i) the dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) has a concentration of 3.6 loglO pfu / dose, (ii) the dengue serotype 2 (e.g. dengue serotype 2 strain) has a concentration of 4.0 loglO pfu / dose, (iii) the dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) has a concentration of 4.6 loglO pfu / dose, and(iv) the dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) has a concentration of 5.1 loglO pfu / dose.

[0164] In another specific embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains wherein the arithmetic sum of all four serotypes is less than 6.7 loglO pfu / dose, for example less than 5.5 loglO pfu / dose. In certain such embodiments, the arithmetic sum of all four serotypes is at least 4.6 loglO pfu / dose. In an exemplary embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains wherein the arithmetic sum of all four serotypes is in the range of 4.6 loglO pfu / dose to 6.7 loglO pfu / dose, e.g. in the range of 4.6 log 10 pfu / dose to 5.5 loglO pfu / dose.

[0165] In one embodiment in the composition (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL and based on said concentration, the concentration of (iii) at least 10% of the total concentration in pfu / 0.5 mL.

[0166] In one embodiment in the composition (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL and based on said total concentration the concentration of (ii) in pfu / 0.5 mL is less than 10%, and 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)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%.

[0167] For example, the concentration in the reconstituted unit dose of (iii) in pfu / 0.5 mL is at least 10%.

[0168] In one embodiment in the composition (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL and based on said total concentration the concentration of (ii) in pfu / 0.5 mL is less than 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 6%.

[0169] For example, in said embodiments the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3 and the chimeric dengue serotype 2 / 4 strain is TDV-4. For example, 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, 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, TDV-3 is characterized by the nucleotide sequence according to SEQ ID No. 5 and the amino acid sequence according to SEQ ID No. 6 and TDV-4 is characterized by the nucleotide sequence according to SEQ ID No. 7 and the amino acid sequence according to SEQ ID No. 8.

[0170] The concentration of the different dengue viruses can be determined by an immuno-focus assay known in the art. For example, the concentration may be determined by an immuno-focus assay wherein serial dilutions of dengue virus are applied to monolayers of adherent cells, such as Vero cells. After a period of time which allows infectious viruses to bind to the cells and to be taken up by the cells, an overlay containing thickening agents, such as agarose or carboxymethylcellulose, is added to prevent diffusion of viruses so that progeny viruses can only infect cells adjacent to the original infected cells. After a period of incubation to allow viral replication, cells are fixed and stained using serotype-specific anti-dengue monoclonal antibodies and a secondary antibody such as an antibody labeled with alkaline phosphatase. The foci are stained by adding a suitable substrate for the enzyme attached to the secondary antibody, such as 5-bromo-4-chloro-3-indolyl-phosphate / nitro blue tetrazolium phosphatase substrate. The number of plaques on the plate corresponds to the plaque forming units of the virus in the solutions applied to the cells. For example, a concentration of 1,000 pfu / pl indicates that 1 pl of the solution applied to the cells contains enough viruses to produce 1,000 plaques in a cell monolayer.

[0171] In an exemplary embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains, wherein a chimeric dengue serotype 2 / 1 strain, a dengue serotype 2 strain, a chimeric dengue serotype 2 / 3 strain, and a chimeric dengue serotype 2 / 4 strain provide a total concentration in pfu / 0.5 mL. The term "total concentration in pfu / 0.5 mL" or "total concentration in pfu / dose" is the sum of the concentrations of the dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain), dengue serotype 2 (e.g. the dengue serotype 2 strain), the dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) and the dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain), for example the sum of the concentrations of TDV-1, TDV-2, TDV-3 and TDV-4, and is defined as 100% of the dengue virus concentration as determined by pfu (plaque forming units) in 0.5 mL or in a dose.

[0172] In one embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains, wherein a dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain), a dengue serotype 2 (e.g. dengue serotype 2 strain), a dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain), and a dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) provide a total concentration in pfu / 0.5 mL, wherein based on said total concentration the concentration of a dengue serotype 2 (e.g. dengue serotype 2strain) measured in pfu / 0.5 mL is less than 10% of the total concentration, or less than 8%, or less than 6% of the total concentration, and wherein the concentration of a dengue serotype 4 (e.g. 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, based on said total concentration the concentration of a dengue serotype 2 (e.g. dengue serotype 2 strain) measured in pfu / 0.5 mL is 0.3 to 10% or 0.5 to 8% of the total concentration and the concentration of a dengue serotype 4 (e.g. 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 the dengue serotype 2 (e.g. dengue serotype 2 strain) is lower than the concentration of the dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain).

[0173] In one such embodiment, the concentration of a dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) measured in pfu / 0.5 mL is at least 1% of the total concentration, and / or the concentration of a dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) measured in pfu / 0.5 mL is at least 6% of the total concentration, or at least 7% or 8%, 10%, 12%, 14%, 16% or 18% of the total concentration. In one such embodiment, the concentration of a dengue serotype 2 (e.g. chimeric dengue serotype 2 / 1 strain) measured in pfu / 0.5 mL is 1% to 7% or 2% to 6% or 2.0% to 5.0% of the total concentration, and / or the concentration of a dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) measured in pfu / 0.5 mL is 6% to 25% or 7% to 25% or 10% to 25% or 18% to 25% of the total concentration. This means that the concentration of the dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) is lower than the concentration of the dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain).

[0174] In a specific embodiment, the concentration of a dengue serotype 2 strain, such as TDV-2, measured in pfu / 0.5 mL is less than 10% of the total concentration, e.g. less than 6% or less than 2%, the concentration of a dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain), such as TDV-4, measured in pfu / 0.5 mL is at least 50% of the total concentration, e.g. at least 65%, the concentration of a dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain), such as TDV-1, measured in pfu / 0.5 mL is at least 1% of the total concentration, e.g. between 1% and 7% or 2.0% to 5.0%, and the concentration of a dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain), such as TDV-3, measured in pfu / 0.5 mL is at least 6% of the total concentration, e.g. between 6% and 25% or 10% to 25% or 18% to 25%.

[0175] In a further specific embodiment, a dengue virus composition comprising a dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain), a dengue serotype 2 (e.g. dengue serotype 2 strain), a dengue serotype 1 (e.g. chimeric dengue serotype 2 / 3 strain), and a dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain), such as TDV-1, TDV-2, TDV-3 and TDV-4, is provided, wherein the concentration of the dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) measured in pfu / 0.5 mL is at least 1% of the total concentration, e.g. between 1% and 7% or 2.0% and 5.0%, the concentration of the dengue serotype 2 (e.g. dengue serotype 2 strain) measured in pfu / 0.5 mL is less than 10% of the total concentration, e.g. less than 6% or less than 2% and the concentration of the dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) measured in pfu / 0.5 mL is at least 6% of the total concentration, e.g. between 6% and 25% or 10% to 25% or 18% to 25%. In one specific embodiment, the dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) has the highest concentration of all four dengue serotypes.

[0176] In a further specific embodiment, the dengue vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains, wherein the concentration of the dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) measured in pfu / 0.5 mL is 1% to 7% of the total concentration, the concentration of the dengue serotype 2 (e.g. dengue serotype 2 strain) measured in pfu / 0.5 mL is less than 8% of the total concentration, such as in the range of 1% to 8% of the total concentration, the concentration of the dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) measured in pfu / 0.5 mLis at least 10% of the total concentration, and the concentration of the dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) measured in pfu / 0.5 mL isat least 65% of the total concentration, such as in the range of 65% to 80%. In certain such embodiments, the arithmetic sum of all four serotypes is in the range of 4.6 loglO pfu / 0.5 mL to 6.7 loglO pfu / 0.5 mL, e.g. in the range of 4.6 loglO pfu / 0.5 mLto 5.5 loglO pfu / 0.5 mL.

[0177] In a further specific embodiment the dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) such as TDV-1 and the dengue serotype 2 (e.g. dengue serotype 2 strain) such as TDV-2 are present each in a concentration based on the total concentration in pfu / 0.5 mL which is within 5%-points of each other and / or are together less than about 10% of the total concentration in pfu / 0.5 mL. In certain such embodiments the dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) such as TDV-3 is at least about 10% of the total concentration in pfu / 0.5 mL and / or the dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) such as TDV-4 is at least about 70% of the total concentration in pfu / 0.5 mL. In certain such embodiments the dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) such as TDV-4 represents the highest concentration in the composition of all four serotypes, e.g. with at least about 70% of the total concentration in pfu / 0.5 mL, dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) such as TDV-3 represents the second highest concentration in the composition of all four serotypes, e.g. with at least about 10% of the total concentration in pfu / 0.5 mL, and dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) such as TDV-1 and dengue serotype 2 (e.g. dengue serotype 2 strain) such as TDV-2 each represent lower concentrations than the concentration of serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) such as TDV-3, and optionally together represent less than about 10% of the total concentration in pfu / 0.5 mL.

[0178] Specifically, in said embodiments the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3 and the chimeric dengue serotype 2 / 4 strain is TDV-4. For example, 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, 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, TDV-3 is characterized by the nucleotide sequence according to SEQ ID No. 5 and the amino acid sequence according to SEQ ID No. 6 and TDV-4 is characterized by the nucleotide sequence according to SEQ ID No. 7 and the amino acid sequence according to SEQ ID No. 8.

[0179] According to a further embodiment, the chimeric dengue serotype 2 / 4 strain, such as TDV-4, has the highest concentration in the dengue vaccine composition, followed by the chimeric dengue serotype 2 / 3 strain, such as TDV-3, followed by the chimeric dengue serotype 2 / 1 strain, such as TDV-1, followed by the dengue serotype 2 strain, such as TDV-2. In an exemplary embodiment, the dengue serotype 2 strain has the lowest concentration of the four strains present in the dengue vaccine composition.

[0180] Whenever reference is made to a concentration / 0.5ml, this does not limit the volume of the unit dose described herein to 0.5ml. 0.5ml is the reference volume for the determination of the concentrations of the virus strains in the composition in pfu / ml. The volume and / or amount per unit dose is described in the respective chapter.Pharmaceutically acceptable excipients

[0181] Described herein is a unit dose of a dengue vaccine composition, wherein the dengue vaccine composition comprises one or more pharmaceutically acceptable excipients.

[0182] In some embodiments, the dengue vaccine composition comprises at least one non-reducing disaccharide, at least one poloxamer, urea, at least one amino acid having a positively charged side chain at neutral pH, tromethamine, and human serum albumin.

[0183] In one embodiment, the dengue vaccine composition comprises at least one non-reducing disaccharide, wherein the non-reducing disaccharide is trehalose and / or sucrose, at least one poloxamer, wherein the poloxamer is poloxamer 407, urea, at least one amino acid having a positively charged side chain at neutral pH, wherein said amino acid is arginine, lysine and / or histidine, tromethamine in its free base form and tromethamine hydrochloride, and human serum albumin.

[0184] In one embodiment, the dengue vaccine composition comprises trehalose, poloxamer, urea, arginine hydrochloride, tromethamine, human serum albumin, chloride salts and phosphate salts. Preferably, the chloride salts comprise or consist of sodium chloride and potassium chloride. Preferably, the phosphate salts comprise or consist of potassium dihydrogen phosphate and disodium hydrogen phosphate. Most preferably, the excipients are a combination consisting of o,o-trehalose dihydrate, poloxamer 407 (Kolliphor® P407 (F-127)), urea, L-arginine hydrochloride, tromethamine, Tris-HCI, human serum albumin, potassium dihydrogen phosphate, disodium hydrogen phosphate dihydrate, potassium chloride and sodium chloride.

[0185] In one embodiment, the unit dose is a lyophilized unit dose of the dengue vaccine composition. The excipient concentrations post reconstitution of said unit dose can also be expressed in quantity per unit dose. In a preferred embodiment, a dose to be administered in 0.5 mL contains or consists of the following excipients: a,a-trehalose dihydrate in an amount of 18 to 30 mg / dose, preferably 20 to 28 mg / dose, more preferably 22 to 26 mg / dose (e.g.23.64 mg / dose), poloxamer (e.g. Kolliphor® P407 (F127)) in an amount of 0.1 to 1.5 mg / dose, preferably 0.5 to 1.0 mg / dose, more preferably 0.6 to 0.8 mg / dose (e.g. 0.71 mg / dose), humans serum albumin in an amount of 0.05 to 0.5 mg / dose, preferably 0.05 to 0.4 mg / dose, more preferably 0.1 to 0.3 mg / dose (e.g. 0.24 mg / dose), tromethamine in an amount of 0.1 to 0.7 pmoles / dose, preferably 0.2 to 0.6 pmoles / dose, more preferably 0.3 to 0.5 pmoles / dose (e.g. 0.39 pmoles / dose), Tris HCI in an amount of 0.5 to 6.0 pmoles / dose, preferably 1.0 to 5.0 pmoles / dose, more preferably 1.0 to 3.0 pmoles / dose (e.g. 1.97 pmoles / dose), L-Arginine HCI in an amount of 0.5 to 8.0 pmoles / dose, preferably 1.0 to 6.0 pmoles / dose, more preferably 2.0 to 4.0 pmoles / dose (e.g. 2.81 pmoles / dose), Urea in an amount of 5.0 to 20.0 pmoles / dose, preferably 8.0 to 16.0 pmoles / dose, more preferably 10.0 to 14.0 pmoles / dose (e.g. 11.81 pmoles / dose), potassium dihydrogen phosphate in an amount of 0.001 to 1.0 pmoles / dose, preferably 0.01 to 0.1 pmoles / dose, more preferably 0.05 to 0.09 pmoles / dose (e.g. 0.07 pmoles / dose), disodium hydrogen phosphate dihydrate in an amount of 0.01 to 1.0 pmoles / dose, preferably 0.1 to 0.6 pmoles / dose, more preferably 0.3 to 0.5 pmoles / dose (e.g. 0.38 pmoles / dose), potassium chloride in an amount of 0.001 to 1.0 pmoles / dose, preferably 0.01 to 0.5 pmoles / dose, more preferably 0.05 to 0.2 pmoles / dose (e.g. 0.10 pmoles / dose) and Sodium chloride in an amount of 15.0 to 40.0 pmoles / dose, preferably 15.0 to 35.0 pmoles / dose, more preferably 25.0 to 30.0 pmoles / dose (e.g. 26.66 pmoles / dose).

[0186] In some embodiments, the dengue vaccine composition comprises a non-reducing sugar, a surfactant, a protein and an inorganic salt. For example, the non-reducing sugar is trehalose, the surfactant is poloxamer 407, the protein is human serum albumin and the inorganic salt is sodium chloride.

[0187] In one embodiment, the unit dose of a dengue vaccine composition comprises the following pharmaceutically acceptable excipients:from about 10 % w / v to about 20 % w / v o,o-trehalose dihydrate or an equimolar amount of other forms of a,a-trehalose,from about 0.5 % w / v to about 1.5 % w / v poloxamer 407,from about 0.05 % w / v to about 2 % w / v human serum albumin, andfrom about 70 mM to 140 mM sodium chloride.

[0188] In one embodiment, the unit dose of a dengue vaccine composition comprises the following pharmaceutically acceptable excipients when measured in 0.5 ml:from about 10 % w / v to about 20 % w / v a,a-trehalose or an equimolar amount of other forms of a,a- trehalose,from about 0.5 % w / v to about 1.5 % w / v poloxamer 407,from about 0.05 % w / v to about 2 % w / v human serum albumin, andfrom about 70 mM to 140 mM sodium chloride, and optionallyhas a pH of 7 to 8.5.

[0189] In one embodiment, the unit dose of a dengue vaccine composition comprises the following pharmaceutically acceptable excipients when measured in 0.5ml:from about 143 mg / ml to about 185 mg / ml o,a-trehalose dihydrate or an equimolar amount of other forms of a,a-trehalose,from about 9.1 mg / ml to about 12.4 mg / ml poloxamer 407,from about 0.88 % mg / ml to about 1.32 mg / ml human serum albumin, andfrom about 70 mM to 140 mM sodium chloride, and optionallyhas a pH of 7 to 8.5.

[0190] In a specific embodiment, the lyophilized unit dose of the invention as described herein comprises the following pharmaceutically acceptable excipients:about 15 % w / v a,a-trehalose dihydrate,about 1 % w / v poloxamer 407,about 0.1 % w / v human serum albumin, andabout 100 mM sodium chloride.

[0191] In a specific embodiment, the lyophilized unit dose of the invention as described herein comprises the following pharmaceutically acceptable excipients when measured in 0.5ml:about 15 % w / v a,a-trehalose,about 1 % w / v poloxamer 407,about 0.1 % w / v human serum albumin, andabout 100 mM sodium chloride.

[0192] In a specific embodiment, the lyophilized unit dose of the invention as described herein comprises the following pharmaceutically acceptable excipients:about 82.9 mg o,o-trehalose dihydrate,about 5 mg poloxamer 407,about 0.5 mg human serum albumin, andabout 50 pmoles sodium chloride.

[0193] In a specific embodiment, the reconstituted unit dose of the invention as described herein comprises the following pharmaceutically acceptable excipients:about 15 % w / v a,a-trehalose dihydrate,about 1 % w / v poloxamer 407,about 0.1 % w / v human serum albumin, andabout 137 mM sodium chloride, and optionallyhas a pH of 7 to 8.5.

[0194] In a specific embodiment, the reconstituted unit dose of the invention as described herein comprises the following pharmaceutically acceptable excipients when measured in 0.5 ml:about 15 % w / v a,o-trehalose,about 1 % w / v poloxamer 407,about 0.1 % w / v human serum albumin, and optionallyabout 137 mM sodium chloride and optionallyhas a pH of 7 to 8.5.

[0195] In a specific embodiment, the reconstituted unit dose of the invention as described herein comprises the following pharmaceutically acceptable excipients:about 82.9 mg o,o-trehalose dihydrate,about 5 mg poloxamer 407,about 0.5 mg human serum albumin, and optionallyabout 68.5 pmoles sodium chloride, and optionallyhas a pH of 7 to 8.5.

[0196] The human serum albumin may be a native or recombinant human serum albumin (rHSA). The poloxamer 407 may be e.g. Pluronic F127.

[0197] In one embodiment, the unit dose further comprises a buffer. The buffer may be phosphate buffered saline (PBS). The buffer may include at least one of sodium chloride (NaCI), monosodium dihydrogen phosphate (Na^PCU), disodium hydrogen phosphate (Na2HPOi), potassium chloride (KCI), and potassium dihydrogen phosphate (KH2PO4). In a specific embodiment, the buffer may include disodium hydrogen phosphate (NazHPC^), potassium chloride (KCI), and potassium dihydrogen phosphate (KH2PO4). The buffer may have a pH in the range of 7.0 to 8.5 at 25°C.Unit dose

[0198] Described herein is a unit dose of a dengue vaccine composition comprising a tetravalent dengue virus composition as described herein and pharmaceutically acceptable excipients as described herein.

[0199] Described herein a unit dose of a dengue vaccine composition as described above e.g. of(i) a dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) with a concentration of at least 3.3 loglO pfu / 0.5 mL,(ii) a dengue serotype 2 (e.g. dengue serotype 2 strain) with a concentration of at least 2.7 loglO pfu / 0.5 mL, (iii) a dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) with a concentration of at least 4.0 loglO pfu / 0.5 mL, and(iv) a dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) with a concentration of at least 4.5 loglO pfu / 0.5 mL.

[0200] For example, the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3, and the chimeric dengue serotype 2 / 4 strain is TDV-4. In a specific example, 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, 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, TDV-3 is characterized by the nucleotide sequence according to SEQ ID No. 5 and the amino acid sequence according to SEQ ID No. 6 and TDV-4 is characterized by the nucleotide sequence according to SEQ ID No. 7 and the amino acid sequence according to SEQ ID No. 8.

[0201] In one embodiment, the unit dose is lyophilized. In one such embodiment, the lyophilized unit dose is obtained by subjecting a volume of 0.5 mL of the aqueous dengue vaccine composition produced by combining pharmaceutically acceptable excipients as described herein and the dengue vaccine composition as described herein comprising the four dengue virus strains, in particular TDV-1 to TDV-4, to lyophilization. In a specific embodiment the residual moisture content as determined by Karl Fischer Determination is equal to or less than 5.0%, e.g. equal to or less than 3%.

[0202] In another embodiment, the unit dose is reconstituted. The reconstituted unit dose is obtained by subjecting the lyophilized unit dose to reconstitution with a pharmaceutically acceptable diluent, such as before administration of the dengue vaccine. In one such embodiment, reconstitution will be accomplished by adding a pharmaceutically acceptable diluent, such as water for injection, phosphate buffered saline or an aqueous sodium chloride solution, to the lyophilized unit dose. In one embodiment, an aqueous sodium chloride solution, such as a 37 mM aqueous sodium chloride solution, is added to the lyophilized unit dose for reconstitution. In one such embodiment, the lyophilized unit dose will be reconstituted with 0.3 to 0.8 mL, or 0.4 to 0.7 mL, or 0.5 mL of diluent. In an exemplary embodiment, the lyophilized unit dose is reconstituted with 0.3 to 0.8 mL, 0.4 to 0.7 mL or 0.5 mL of 37 mM aqueous sodium chloride solution. In another exemplary, the lyophilized unit dose is reconstituted with 0.5 mL of 37 mM aqueous sodium chloride solution. The reconstituted unit dose can subsequently be administered subcutaneously.

[0203] In a specific embodiment, the unit dose in lyophilized form is the final product after manufacture of the unit dose and the storage form of the unit dose, wherein the unit dose in reconstituted form is prepared before administration of the unit dose to a subject.

[0204] Described herein is a unit dose of a dengue vaccine composition comprising a tetravalent virus composition including four live attenuated dengue virus strains, wherein the unit dose is lyophilized and upon reconstitution with 0.5 mL of a pharmaceutically acceptable diluent comprises:(i) a dengue serotype 1, such as a chimeric dengue serotype 2 / 1 strain, in a concentration of at least 3.3 loglO pfu / 0.5 ml,(ii) a dengue serotype 2, such as a dengue serotype 2 strain, in a concentration of at least 2.7 loglO pfu / 0.5 ml, (iii)a dengue serotype 3, such as a chimeric dengue serotype 2 / 3 strain, in a concentration of at least 4.0 loglO pfu / 0.5 ml, and(iv) a dengue serotype 4, such as a chimeric dengue serotype 2 / 4 strain, in a concentration of at least 4.5 loglO pfu / 0.5 ml.

[0205] In one embodiment, the reconstituted unit dose has a volume of e.g. 0.5 mL, wherein upon reconstitution with a pharmaceutically acceptable diluent (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL and based on said concentration, the concentration of (iii) at least 10% of the total concentration in pfu / 0.5 mL.

[0206] In another embodiment the reconstituted unit dose has a volume of e.g. 0.5 mL, wherein upon reconstitution with a pharmaceutically acceptable diluent (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL and based on said total concentration the concentration of (ii) in pfu / 0.5 mL is less than 10%, and 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) 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%.

[0207] For example, the concentration in the reconstituted unit dose of (iii) in pfu / 0.5 mL is at least 10%.

[0208] In one embodiment the reconstituted unit dose has a volume of e.g. 0.5 mL, wherein upon reconstitution with a pharmaceutically acceptable diluent (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL and based on said total concentration the concentration of (ii) in pfu / 0.5 mL is 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 6%.

[0209] In one embodiment, a lyophilized unit dose of a dengue vaccine composition comprises upon reconstitution with 0.5 mL of a pharmaceutically acceptable diluent a dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) with a concentration of at least 3.3 loglO pfu / 0.5 mL, a dengue serotype 2 (e.g. dengue serotype 2 strain) with a concentration of at least 2.7 loglO pfu / 0.5 mL, a dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) with a concentration of at least 4.0 loglO pfu / 0.5 mL, and a dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) with a concentration of at least 4.5 loglO pfu / 0.5 mL and pharmaceutically acceptable excipients as described herein, wherein the unit dose is e.g. formulated in 0.5 mL before lyophilization. For example, the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3 and the chimeric dengue serotype 2 / 4 strain is TDV-4. In another exemplary 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, 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, TDV-3 is characterized by the nucleotide sequence according to SEQ ID No. 5 and the amino acid sequence according to SEQ ID No. 6 and TDV-4 is characterized by the nucleotide sequence according to SEQ ID No. 7 and the amino acid sequence according to SEQ ID No. 8.

[0210] In one such embodiment, the lyophilized unit dose is obtained by lyophilizing 0.5 mL of a dengue vaccine composition comprising a dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) in a concentration of 3.3 loglO pfu / dose to 5.0 loglO pfu / 0.5 mL, a dengue serotype 2 (e.g. dengue serotype 2 strain) in a concentration of 2.7 loglO pfu / dose to 4.9 loglO pfu / 0.5 mL, a dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) in a concentration of 4.0 loglO pfu / dose to 5.7 loglO pfu / 0.5 mL, and a dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) in a concentration of 4.5 loglO pfu / dose to 5.5 loglO pfu / 0.5 mL and pharmaceutically acceptable excipients as described herein. For example, the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3 and the chimeric dengue serotype 2 / 4 strain is TDV-4.

[0211] In one such embodiment, the lyophilized unit dose is obtained by lyophilizing 0.5 mL of a dengue vaccine composition comprising a dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) in a concentration of 3.3 loglO pfu / 0.5 mL to 3.6 loglO pfu / 0.5 mL, a dengue serotype 2 (e.g. dengue serotype 2 strain) in a concentration of 2.7 loglO pfu / 0.5 mL to 4.0 loglO pfu / 0.5 mL, a dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) in a concentration of 4.0 loglO pfu / 0.5 mLto 4.6 loglO pfu / 0.5 mL, and a dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) in a concentration of 4.5 loglO pfu / 0.5 mL or 4.6 loglO pfu / 0.5 mL to 5.1 loglO pfu / 0.5 mL and pharmaceutically acceptable excipients as described herein. For example, the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3 and the chimeric dengue serotype 2 / 4 strain is TDV-4.

[0212] In certain embodiments, the lyophilized unit dose refers to 0.5 mL before lyophilization, wherein TDV-2 and TDV-4 are present in certain relative amounts, based on the total concentration of TDV-1, TDV-2, TDV-3 and TDV-4 in pfu / 0.5 mL, and the concentration of TDV-2 measured in pfu / 0.5 mL is less than 10% or less than 8% or less than6%, 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%.

[0213] In certain embodiments, the reconstituted unit dose has a volume of 0.5 mL and TDV-2 and TDV-4 are present in certain relative amounts, based on the total concentration of TDV-1, TDV-2, TDV-3 and TDV-4 in pfu / 0.5 mL, and the concentration of TDV-2 measured in pfu / 0.5 mL is less than 10% or less than 8% or less than 6%, and the concentration of TDV-4 measured in pfu / 0.5 mL is at least 50% or at least 65%. In some of these embodiments, the concentration of TDV-1 measured in pfu / 0.5 mL is at least 1% and / or the concentration of TDV-3 measured in pfu / 0.5 mL is at least 6%, 7%, 8%, 10%, 12%, 14%, 16% or at least 18%.

[0214] In a further specific embodiment, the reconstituted unit dose has a volume of 0.5 mL and comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains, wherein the concentration of the dengue serotype 1 (e.g. dengue serotype 2 / 1 strain) measured in pfu / 0.5 mL is 1% to 7% of the total concentration, the concentration of the dengue serotype 2 (e.g. dengue serotype 2 strain) measured in pfu / 0.5 mL is less than 8% of the total concentration, such as in the range of 1% to 8% of the total concentration, the concentration of the dengue serotype 3 (e.g. dengue serotype 2 / 3 strain) measured in pfu / 0.5 mL is at least 10% of the total concentration, and the concentration of the dengue serotype 4 (e.g. dengue serotype 2 / 4 strain) measured in pfu / 0.5 mL is at least 65% of the total concentration, such as in the range of 65% to 80%. In certain such embodiments, the arithmetic sum of all four serotypes is in the range of 4.6 loglO pfu / 0.5 mLto 6.7 loglO pfu / 0.5 mL, e.g. in the range of 4.6 loglO pfu / 0.5 mLto 5.5 loglO pfu / 0.5 mL.

[0215] In a further specific embodiment, the reconstituted unit dose has a volume of 0.5 mL and comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains, wherein the dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) such as TDV-1 and the dengue serotype 2 (e.g. dengue serotype 2 strain) such as TDV-2 are present each in a concentration based on the total concentration in pfu / 0.5 mL which is within 5%-points of each other and / or are together less than about 10% of the total concentration in pfu / 0.5 mL. In certain such embodiments the dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) such as TDV-3 is at least about 10% of the total concentration in pfu / 0.5 mL and / or the dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) such as TDV-4 is at least about 70% of the total concentration in pfu / 0.5 mL. In certain such embodiments the dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) such as TDV-4 represents the highest concentration in the composition of all four serotypes, e.g. with at least about 70% ofthe total concentration in pfu / 0.5 mL, dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) such as TDV-3 represents the second highest concentration in the composition of all four serotypes, e.g. with at least about 10% of the total concentration in pfu / 0.5 mL, and dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) such as TDV-1 and dengue serotype 2 (e.g. dengue serotype 2 strain) such as TDV-2 each represent lower concentrations than the concentration of serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) such as TDV-3, and optionally together represent less than about 10% of the total concentration in pfu / 0.5 mL.

[0216] The lyophilized unit dose reconstituted in 0.5 mL will provide the above concentrations for the four dengue serotypes. While the unit dose of a dengue vaccine composition as described herein refers to the concentrations ofthe dengue serotypes in 0.5 mL, the lyophilized unit dose can be reconstituted with other volumes of a pharmaceutically acceptable diluent, such as an aqueous sodium chloride solution, without changing the absolute virus amount administered or the ratios of the viruses to one another.

[0217] In certain embodiments, the lyophilized unit dose of the invention is prepared from a solution comprising a non-reducing sugar, a surfactant, a protein and an inorganic salt.

[0218] In certain embodiments, the lyophilized unit dose of the invention is prepared from a solution comprising trehalose, poloxamer 407, human serum albumin and sodium chloride.

[0219] In certain embodiments, the lyophilized unit dose of the invention is prepared from a solution comprising about 10 % w / v to about 20 % w / v a,a-trehalose dihydrate or an equimolar amount of other forms of a,a-trehalose, from about 0.5 % w / v to about 1.5 % w / v poloxamer 407, from about 0.05 % w / v to about 2 % w / v human serum albumin, and about 70 mM to about 120 mM sodium chloride.

[0220] In specific embodiments, the lyophilized unit dose of the invention as described herein is prepared from a solution comprising about 15 % w / v a,a-trehalose dihydrate, about 1 % w / v poloxamer 407, about 0.1 % w / v human serum albumin and about 100 mM sodium chloride.

[0221] In one embodiment, the solution from which the lyophilized unit dose is prepared further comprises a buffer. The buffer may be phosphate buffered saline (PBS). The buffer may include at least one of sodium chloride (NaCI), monosodium dihydrogen phosphate (Na^PCM), disodium hydrogen phosphate (NazHPCM), potassium chloride (KCI), and potassium dihydrogen phosphate (KH2PO4). In a specific embodiment, the buffer may include disodium hydrogen phosphate (NazHPC^), potassium chloride (KCI), and potassium dihydrogen phosphate (KH2PO4). The buffer may have a pH in the range of about 7.0 to about 8.5 at 25°C or a pH of about 6.8 to about 7.6 at 25°C, such as a pH of about7.2 at 25°C.

[0222] In specific embodiments, the reconstituted unit dose of the invention as described herein comprising about 15 % w / v a,a-trehalose dihydrate, about 1 % w / v poloxamer 407, about 0.1 % w / v human serum albumin and about 137 mM sodium chloride. The reconstituted unit dose may have a pH of about 7.0 to about 8.5 at 25°C, e.g. a pH of about 7.2 at 25°C.

[0223] The unit dose of the invention as described herein activates multiple arms of the immune system -neutralizing antibodies, cellular immunity and anti-NSl 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 dengue seronegative and dengue seropositive subject populations or subjects against dengue disease.

[0224] In one embodiment, one unit dose is present in a container, e.g. a vial, and said unit dose is administered to a subject after reconstitution. In one embodiment, more than one unit dose of the dengue vaccine composition may be present in a container, e.g. a vial, so that with the content of one container, e.g. a vial, more than one subject can be vaccinated. In one embodiment, the container comprising more than one unit doses of the invention as described herein is used for providing the reconstituted unit dose to be used in the methods of the invention as described herein.

[0225] In another embodiment, the vial comprises one to ten unit dose(s), such as one unit dose or multiple (e.g. five) unit doses, of the dengue vaccine formulation. In another embodiment, the dengue vaccine formulation comprises 5.0 mg / vial urea, 4.1 mg / vial L-arginine hydrochloride, 0.33 mg / vial tromethamine and 2.17 mg / vial Tris-HCI as excipients.

[0226] In certain embodiments, the container comprising the unit dose of the invention is part of a kit. Thus, the invention is directed in part to a kit for preparing a reconstituted unit dose comprising a lyophilized unit dose of the present invention as described herein, and a pharmaceutically acceptable diluent for reconstitution.

[0227] In certain embodiments, the diluent for reconstitution provided in a container, e.g. a vial, or a pre-filled syringe. In some embodiments, the diluent for reconstitution is selected from water for injection, phosphate buffered saline or an aqueous sodium chloride solution. In a specific embodiment, the diluent for reconstitution is 30 to 40 mM sodium chloride, such as 37 mM sodium chloride.

[0228] In certain embodiments, the kit may further comprise an HPV vaccine, such as GARDASIL® 9. In some embodiments, the HPV vaccine may be in a separate container, such as a vial. In another embodiment, the HPV vaccine and the unit dose of the invention may be in the same container. Thus, the invention is directed in part to a combined dengue / HPV vaccine, wherein the unit dose of the invention as described herein is combined with an HPV vaccine. Such a combined dengue / HPV vaccine comprises the unit dose of the invention as described herein and a HPV vaccine, such as GARDASIL® 9, in the same formulation. In certain embodiments, the invention is directed to a kit comprising such a combined dengue / HPV vaccine and a unit dose of the invention as described herein.HPV vaccine

[0229] GARDASIL® 9, an HPV vaccine from Merck, is a non-infectious recombinant 9-valent vaccine prepared from the purified virus-like particles (VLPs) of the major capsid (LI) protein of HPV serotypes 6, 11, 16, 18, 31, 33, 45, 52, and 58. The LI proteins can be produced by separate fermentations using recombinant Saccharomyces cerevisiae and self-assembled into VLPs. The fermentation process may involve growth of S. cerevisiae on chemically defined fermentation media which include vitamins, amino acids, mineral salts, and carbohydrates. The VLPs can be released from the yeast cells by cell disruption and purified by a series of chemical and physical methods. The purified VLPs can be adsorbed on preformed aluminum-containing adjuvant (amorphous aluminum hydroxyphosphate sulfate or AAHS). The 9-valent HPV VLP vaccine is e.g. a sterile liquid suspension that is prepared by combining the adsorbed VLPs of each HPV serotype and additional amounts of the aluminum-containing adjuvant and the final purification buffer. GARDASIL 9 is a sterile suspension for intramuscular administration. Each 0.5-mL dose contains approximately 30 pg of HPV serotype 6 LI protein, 40 pg of HPV serotype 11 LI protein, 60 pg of HPV serotype 16 LI protein, 40 pg of HPV serotype 18 LI protein, 20 pg of HPV serotype 31 LI protein, 20 pg of HPV serotype 33 LI protein, 20 pg of HPV serotype 45 LI protein, 20 pg of HPV serotype 52 LI protein, and 20 pg of HPV serotype 58 LI protein. Each 0.5-mL dose of the vaccine also contains approximately 500 pg of aluminum (provided as AAHS), 9.56 mg of sodium chloride, 0.78 mg of L-histidine, 50 pg of polysorbate 80, 35 pg of sodium borate.

[0230] Other available HPV vaccines include Cervarix®, which is a bivalent HPV vaccine for preventing HPV types 16 and 18 and Gardasil® which is a quadrivalent HPV vaccine for preventing HPV types 6, 11, 16 and 18.

[0231] Further vaccines that are currently not licensed in the European Union or the United states include the bivalent Cecolin® and Walrinvax® for preventing HPV types 16 and 18 and the quadrivalent HPV vaccine Cervavac®, for additionally preventing HPV types 6 and 11.

[0232] All vaccines can be prepared using recombinant DNA and cell-culture technology from the purified LI structural protein, which self-assembles to form HPV type-specific empty shells, termed virus-like particles (VLPs).

[0233] Typically, HPV vaccines do not contain live biological products or viral DNA and are therefore non-infectious. HPV vaccines can use different expression systems, may contain adjuvants and typically do not contain antibiotics or preservative agents.Combined Vaccine Composition

[0234] Described herein is a combined vaccine composition comprising an HPV antigen such as 9vHPV and a dengue antigen such as the tetravalent dengue vaccine, TDV, as disclosed herein or any other suitable tetravalent live attenuated dengue virus vaccine.

[0235] In certain embodiments, the combined vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains:(i) a dengue serotype 1 such as in a concentration of at least 3.3 loglO pfu / 0.5 mL,(ii) a dengue serotype 2 such as in a concentration of at least 2.7 loglO pfu / 0.5 mL,(iii) a dengue serotype 3 such as in a concentration of at least 4.0 loglO pfu / 0.5 mL, and(iv) a dengue serotype 4 such as in a concentration of at least 4.5 loglO pfu / 0.5 mL.

[0236] In certain embodiments, the combined vaccine composition comprises a tetravalent dengue virus composition including four live attenuated dengue virus strains:(i) a chimeric dengue serotype 2 / 1 strain in a concentration of at least 3.3 loglO pfu / 0.5 mL to 3.8 loglO pfu / 0.5 ml,(ii) a dengue serotype 2 strain in a concentration of at least 2.7 loglO pfu / 0.5 mL,(iii) a chimeric dengue serotype 2 / 3 strain in a concentration of at least 4.0 loglO pfu / 0.5 mL, and(iv) a chimeric dengue serotype 2 / 4 strain in a concentration of at least 4.5 loglO pfu / 0.5 mL or at least 4.6 loglO pfu / 0.5 mL to optionally 6.2 loglO pfu / 0.5 ml.

[0237] For example, in said embodiments the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3 and the chimeric dengue serotype 2 / 4 strain is TDV-4. For example, 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, 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, TDV-3 is characterized by the nucleotide sequence according to SEQ ID No. 5 and the amino acid sequence according to SEQ ID No. 6 and TDV-4 is characterized by the nucleotide sequence according to SEQ ID No. 7 and the amino acid sequence according to SEQ ID No. 8.

[0238] In certain embodiments of the combined vaccine composition, 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 based on said 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%, and 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%.

[0239] In certain embodiments of the combined vaccine composition, 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. For example, the non-reducing sugar is trehalose, the surfactant is poloxamer 407, the protein is human serum albumin and the inorganic salt is sodium chloride.

[0240] Furthermore, any vaccine excipients or combinations thereof known to the person skilled in the art, e.g. disclosed in WO 2018 / 027075 Al, can be used for the combined vaccine composition.

[0241] In one embodiment, the unit dose of a dengue vaccine composition comprises the following pharmaceutically acceptable excipients:from about 10 % w / v to about 20 % w / v o,o-trehalose dihydrate or an equimolar amount of other forms of a,a-trehalose,from about 0.5 % w / v to about 1.5 % w / v poloxamer 407,from about 0.05 % w / v to about 2 % w / v human serum albumin, andfrom about 70 mM to 140 mM sodium chloride.

[0242] In certain embodiments of the combined vaccine composition, the dengue vaccine composition comprises other dengue vaccines such as Dengvaxia®. Dengvaxia® is a tetravalent dengue vaccine with mixed chimeric dengue viruses based on a yellow fever backbone, CYD-TDV (Dengvaxia®, Sanofi Pasteur, Lyon, France), and has been licensed in several countries based on the clinical demonstration of an overall vaccine efficacy (VE) against virologically-confirmed dengue (VCD) of 56-61% in children in Asia and Latin America (Capeding MR et al. Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in Asia: a phase 3, randomised, observer-masked, placebo-controlled trial. Lancet 2014, 384:1358-65; Villar LA et al. Safety and immunogenicity of a recombinant tetravalent dengue vaccine in 9-16 year olds: a randomized, controlled, phase II trial in Latin America. Pediatr Infect Dis J 2013, 32:1102-9). The preparation of these particular strains CYD1, CYD2, CYD3 and CYD4 has been described in detail in international patent applications WO 98 / 37911, WO 03 / 101397, W007 / 021672, WO 08 / 007021, WO 08 / 047023 and WO 08 / 065315, to which reference may be made for a precise description of the processes for their preparation. The corresponding nucleotide sequences of the prM-E regions of CYD1, CYD2, CYD3 and CYD4 are provided in WO2016034629 and SEQID NOs are set out in Table 16 of this reference.

[0243] In certain embodiments of the combined vaccine composition, the quantity of a chimeric dengue virus within CYD-TDV lies within a range of about 105CCID50 to about 106CCID50. The quantity of a live attenuated chimeric dengue virus of each of serotypes 1 to 4 comprised in the CYD dosage form, e.g. Dengvaxia®, may be equal.

[0244] In such embodiments, the CYD-TDV is dissolved / dissolvable in a solution containing 0.4% NaCI.

[0245] In certain embodiments of the combined vaccine composition, the dengue vaccine composition comprises other dengue vaccines such as TV003 or TV005. In some embodiments, the other dengue vaccine comprises vaccine components rDENlA30, rDEN2 / 4A30, rDEN3A30 / 31 and rDEN4A30, preferably wherein each of these components is present at a concentration of 1 to 4 logioPFU, such as 2 to 2.5 logioPFU. TV003, developed by the U. S. National Institute of Allergy and Infectious Diseases, comprises vaccine components rDENlA30, rDEN2 / 4A30, rDEN3A30 / 31 and rDEN4A30, wherein each of these components is present at a concentration of 3 logioPFU. TV005 is similar to TV003 with the difference that the concentration of rDEN2 / 4A30 in TV005 is 4 logioPFU. The vaccines TV003 and TV005 and their vaccine components as well as their production are described in more detail in WO 2008 / 022196 A2 and S. S. Whitehead, Expert Rev Vaccines, 2016, 15(4): 509 to 517. Using recombinant DNA technology, two attenuation strategies were utilized for preparing rDENlA30, rDEN2 / 4A30, rDEN3A30 / 31 and rDEN4A30, the vaccine components, for example, of TV003 orTV005: deletions in the 3' untranslated region and structural gene chimerization. For example, the component rDEN4A30 contains all the structural and non -structural proteins of a wild type DENV-4, but is attenuated by a 30-nucleotide deletion in the 3' untranslated region (denoted " A30"). The other vaccine components are also attenuated due to the 30-nucleotide deletion in the 3' untranslated region. In addition, rDEN3A30 / 31 includes a 31 nucleotide deletion in the 3' untranslated region (shown in detail in Fig. lc and Fig. 13 of WO 2008 / 022196 A2). The rDEN2 / 4A30 component was created by substituting the prM and E genes of DENV-2 into the rDEN4A30 genome. The complete genomic sequences of dengue strains which can be used to produce TV003 or TV005 are available under the Genbank accession numbers in Table A of WO 2008 / 022196 Al.

[0246] In certain embodiments of the combined vaccine composition, the HPV vaccine is a bivalent, quadrivalent or nonavalent HPV vaccine composition.

[0247] In certain embodiments, the invention is directed to the combined vaccine composition, wherein the combined vaccine is included in a dose comprising a liquid, wherein the liquid has a volume of 0.5 ml, 1 ml, or 1.5 ml.

[0248] In certain embodiments, the combined vaccine composition is provided in one single vial in a liquid form or in a dehydrated form, such as a lyophilized form.

[0249] In certain embodiments, the combined vaccine composition is obtained from mixing a unit dose of a dengue vaccine composition and a dose of an HPV vaccine composition in a syringe.

[0250] Described herein is also a method of administering any of the above combined vaccine compositions to a subject or subject population.

[0251] In certain embodiments of said methods, the combined vaccine composition is administered subcutaneously or intramuscularly.Method of preventing dengue disease and HPV associated diseases and uses

[0252] Described herein is a method of preventing dengue disease as well as HPV-associated diseases such as HPV-associated cancers or genital warts in a subject. Thus, in certain embodiments the invention is directed to a method of preventing dengue disease in a subject, comprising administering to the subject a unit dose, e.g. a reconstituted unit dose of the invention as described herein, wherein the method further comprises preventing HPV-associated diseases such as HPV-associated cancers or genital warts in the subject by concomitant administration of a HPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, to the subject.

[0253] Described herein is a method of preventing dengue disease as well as HPV-associated diseases such as HPV-associated cancers or genital warts in a subject population. Thus, in certain embodiments a method of preventing dengue disease in a subject population comprises administering to the subject population a unit doses, e.g. a reconstituted unit dose of the invention as described herein, wherein the method further comprises preventing HPV-associated diseases such as HPV-associated cancers or genital warts in the subject population by concomitant administration of a HPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, to the subject population.

[0254] Described herein is a method for preventing dengue disease and HPV-associated diseases such as HPV-associated cancers or genital warts in a subject population comprising administering to the subject population at least a first unit dose, e.g. a reconstituted unit dose of the invention as described herein, wherein certain ratios of geometric mean neutralizing antibody titers (GMTs) at day 180 or 365 after administration of said first unit dose to the subject population are achieved, and concomitantly administering a HPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, to the subject population. According to some embodiments, the geometric mean neutralizing antibody titer for dengue serotype 2 (GMT DENV-2) and the geometric mean neutralizing antibody titer for dengue serotype 4 (GMT DENV-4) when tested in at least 40, or at least 50, or at least 60 subjects at day 180 or day 365 after at least a first administration of said reconstituted unit dose of the invention as described herein, and optionally a second administration of a reconstituted unit dose of the invention as described herein 90 days after said first administration, provide a ratio of GMT DENV-2: GMT DENV-4 of not more than 50, or not more than 40, or not more than 30, or not more than 20. In some of these embodiments, the ratio of GMT DENV-2: GMT DENV-1 is not more than 20, or not more than 18, or not more than 15 at day 180 or 365 after administration of said first reconstituted unit dose, and / orthe ratio of GMT DENV-2: GMT DENV-3 is not more than 20, or not more than 18, or not more than 15 at day 180 or 365 after administration of said first reconstituted unit dose.

[0255] Described herein is a method for preventing dengue disease and HPV-associated diseases such as HPV-associated cancers or genital warts in a subject comprising administering to the subject at least a first unit dose, e.g. a reconstituted unit dose of the invention as described herein, wherein certain ratios of neutralizing antibody titers at day 180 or 365 after administration of said first unit dose to the subject are achieved, and concomitantly administering a HPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, to the subject. According to some embodiments, the neutralizing antibody titer for dengue serotype 2 and the neutralizing antibody titer for dengue serotype 4 at day 180 or day 365 after at least a first administration of the reconstituted unit dose of the invention as described herein, and optionally a second administration of a reconstituted unit dose of the invention as described herein 90 days after said first administration, provide a ratio of neutralizing antibody titer for DENV-2: neutralizing antibody titer for DENV-4 of not more than 50, or not more than 40, or not more than 30, or not more than 20. In some of these embodiments, the ratio of the neutralizing antibody titers of DENV-2: DENV-1 is not more than 20, or not more than 18, or not more than 15 at day 180 or 365 after administration of said first reconstituted unit dose, and / or the ratio of the neutralizing antibody titers of DENV-2: DENV-3 is not more than 20, or not more than 18, or not more than 15 at day 180 or 365 after administration of said first reconstituted unit dose.

[0256] In some embodiments, the geometric mean neutralizing antibody titers (GMTs) of a subject population or the neutralizing antibody titers of a subject are determined in accordance with a microneutralization test.

[0257] In certain embodiments of the methods, the unit dose of the invention as described herein and the HPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, are administered simultaneously. In some of these embodiments the simultaneous administration is on day 0 or day 90, e.g. on day 0. In other embodiments, the administration of the unit dose of the invention as described herein and the HPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, are done sequentially. In other embodiments, the method comprises a concomitant administration and subsequent sequential administrations of unit doses of the dengue vaccine composition and the HPV vaccine composition.

[0258] In certain embodiments of the methods, the dengue vaccine composition, e.g. a reconstituted unit dose of the invention as described herein is administered by subcutaneous or intramuscular injection and the HPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, is administered by intramuscular injection. According to some embodiments the injections are administered to the arm, e.g. to the deltoid region of the arm. According to some of these embodiments the subcutaneous or intramuscular injection of the unit dose of the invention as described herein and the intramuscular injection of the HPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, are administered to different anatomical sites, such as to opposite arms, in particular when the vaccines are administered simultaneously.

[0259] In certain embodiments of said methods, 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 12 months or more, or within 6 months, or within three months, such as at day 0 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. Such a third administration may act as a booster and may be administered between 6 to 12 months after the first administration, such as 12 months after the first administration, or later than 12 months after the first administration, such as 12 months after the second administration.

[0260] In certain embodiments of said methods, two reconstituted unit doses of the invention as described herein and two doses of a HPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, are administered, in particular according to the following schedule:- a first simultaneous administration of the first unit dose, e.g. first reconstituted unit dose and the first dose of said HPV vaccine on day 0,- a second administration of the second unit dose, e.g. second reconstituted unit dose after said first simultaneous administration, such as 3 months later and e.g. on day 90, and- a third administration of the second dose of said HPV vaccine after said second administration, such as 3 months later and e.g. on day 180.

[0261] In certain embodiments, the method comprises simultaneous administration of a unit dose of a dengue vaccine composition, such as the dengue unit dose of the invention as described herein, and an HPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, wherein the primary vaccination of the HPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, consists of one administration (i.e., of one dose) of said HPV vaccine. The dengue vaccine composition, for example, may be a tetravalent dengue vaccine composition comprising a dengue serotype 1 strain, a dengue serotype 2 strain, a dengue serotype 3 strain, and a dengue serotype 4 strain. The dengue serotype 1 strain may be a chimeric dengue serotype 2 / 1 strain, the dengue serotype 2 strain may be a non-chimeric dengue serotype 2 strain, the dengue serotype 3 strain may be a chimeric dengue serotype 2 / 3 strain, and the dengue serotype 4 strain may be a chimeric dengue serotype 2 / 4 strain. The dengue serotype 1 strain may have a nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 2, the dengue serotype 2 strain may have a nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 4, the dengue serotype 3 strain may have a nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 6, and the dengue serotype 4 strain may have a nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 8.

[0262] In some embodiments, the method comprises simultaneous administration of a unit dose of a dengue vaccine composition, such as the dengue unit dose of the invention as described herein, and an HPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, wherein the primary vaccination of the HPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, consists of one administration (i.e., of one dose) of said HPV vaccine as described above, and wherein the method comprises:a) simultaneous administration of a first unit dose of the dengue vaccine composition and the HPV vaccine on Day 0, andb) administration of a second unit dose of the dengue vaccine composition after said simultaneous administration, preferably wherein the second unit dose of the dengue vaccine composition is administered on Day 90 or 3 months after said simultaneous administration, and optionallyc) administration of a third unit dose of the dengue vaccine composition after the administration of the second unit dose of the dengue vaccine composition, wherein the administration of the third unit dose of the dengue vaccine composition is a booster vaccination, wherein the booster administration is administered e.g. at least 12 months, such as 12 months to 5 years, after the administration of the first unit dose of the dengue vaccine composition.

[0263] In some embodiments, the method comprises simultaneous administration of a unit dose of a dengue vaccine composition, such as the dengue unit dose of the invention as described herein, and an HPV vaccine, inparticular a 9vHPV vaccine, such as GARDASIL® 9, wherein the primary vaccination of the HPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, consists of one administration (i.e., of one dose) of said HPV vaccine as described above, and wherein the method comprises:a) administration of a first unit dose of the dengue vaccine composition on Day 0, andb) simultaneous administration of a second unit dose of the dengue vaccine composition and the HPV vaccine after said administration of the first unit dose of the dengue vaccine composition, preferably wherein the simultaneous administration is on Day 90 or 3 months after the administration of said first unit dose, and optionallyc) administration of a third unit dose of the dengue vaccine composition after said simultaneous administration, wherein the administration of the third unit dose of the dengue vaccine composition is a booster vaccination, wherein the booster administration is administered e.g. at least 12 months, such as 12 months to 5 years, after the administration of the first unit dose of the dengue vaccine composition.

[0264] In some embodiments, the method comprises simultaneous administration of a unit dose of a dengue vaccine composition, such as the dengue unit dose of the invention as described herein, and an HPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, wherein the primary vaccination of the HPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, consists of one administration (i.e., of one dose) of said HPV vaccine as described above, and wherein the method comprises:a) administration of a first unit dose of the dengue vaccine composition on Day 0 or at month 0, and b) administration of a second unit dose of the dengue vaccine composition after said administration of said first unit dose of the dengue vaccine composition, preferably wherein the second unit dose of the dengue vaccine composition is administered on Day 90 or 3 months after the administration of the first unit dose, and c) simultaneous administration of a third unit dose of the dengue vaccine composition and the HPV vaccine after the administration of the second unit dose, wherein the administration of the third unit dose of the dengue vaccine composition is a booster vaccination, wherein the booster administration is administered e.g. at least 12 months, such as 12 months to 5 years, after the administration of the first unit dose of the dengue vaccine composition.

[0265] In certain embodiments of said methods, the subject or subject population is seronegative to all dengue serotypes. In certain embodiments of said methods, the unit dose, e.g. the reconstituted unit dose of the invention as described herein is administered subcutaneously or intramuscularly to a subject or subject population and the HPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, is administered intramuscularly to a subject or subject population, and the subject or the subject population is seronegative with respect to all dengue serotypes. In other embodiments, the subject or subject population is seropositive with respect to at least one dengue serotype.

[0266] In certain embodiments of said methods, the unit dose of the invention as described herein and the HPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, are administered to a subject or subject population from a dengue endemic region. In certain embodiments, the unit dose, e.g. the reconstituted unit dose of the invention as described herein is administered subcutaneously or intramuscularly and the HPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, is administered intramuscularly to a subject or subject population from a dengue endemic region.

[0267] In certain embodiments the invention is directed to said methods, wherein the unit dose, e.g. the reconstituted unit dose of the invention as described herein is administered subcutaneously or intramuscularly and wherein the HPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, is administered intramuscularly to a subject or subject population of 9 to 26 years of age, such as 9 to 14 years of age. In some embodiments the subject or subject population is from a dengue endemic region. According to some of these embodiments, the subject or subject population is female. According to some of these embodiments, the subject or subject population is seronegative for all four dengue serotypes.

[0268] In a specific embodiment, the method of preventing dengue disease and HPV-associated diseases such as HPV-associated cancers or genital warts in a subject comprises administering to the subject a subcutaneous or intramuscular injection of the reconstituted unit dose of the invention as described herein and an intramuscular injection of the HPV vaccine, in particular a 9vHPV vaccine, such as GARDASIL® 9, according to the above described administration schedule, wherein the subject is a female subject of 9 to 14 years of age from a dengue endemic region such as Thailand.

[0269] In another specific embodiment, the method provides for combined vaccine efficacy against all four serotypes of the dengue virus and non-inferiority of the immune response to the HPV vaccine composition compared to a mono-administration of the HPV vaccine composition.

[0270] In another specific embodiment, the HPV vaccine composition is a nonavalent HPV vaccine composition and the method provides non-inferiority of the immune response to the nonavalent HPV vaccine composition compared to a mono-administration of the nonavalent HPV vaccine composition.

[0271] In another specific embodiment, the method provides non-inferiority of the immune response to the HPV vaccine composition in a non-inferiority clinical study including at least 600 healthy subjects divided into one subject population and into one control subject population, wherein the subject population receives the HPV vaccine composition and the dengue vaccine composition concomitantly once on day 1 (month 0) followed by the dengue vaccine composition once on day 90 (month 3) and the HPV vaccine composition once on day 180 (month 6) and the control subject population receives the HPV vaccine composition once on day 1 (month 0) followed by the HPV vaccine composition once on day 180 (month 6).

[0272] In another specific embodiment, the method provides non-inferiority of the immune response to the dengue vaccine composition compared to a mono-administration of the dengue vaccine composition.

[0273] In another specific embodiment, the method provides a seropositivity rate of at least 95% or at least 98% or at least 99.5% or 100% for each of dengue serotype 1, 2, 3, and 4 after a simultaneous administration of a unit dose of a dengue vaccine composition, such as the unit dose of the dengue vaccine composition according to the invention, and one dose of an HPV vaccine. The method may provide for a tetravalent seropositivity rate (against all four dengue serotypes) of at least 95% or at least 98% or at least 99.5% or 100% after a simultaneous administration of a unit dose of a dengue vaccine composition, such as the unit dose of the dengue vaccine composition according to the invention, and one dose of an HPV vaccine. In a yet more specific embodiment, the method provides for such seropositivity rates in a subject population including at least 100 or 200 healthy subjects or between 100 and 300 healthy subjects. The method may provide for such seropositivity rates in a subject population seropositive at baseline and / or seronegative at baseline. In a yet more specific embodiment, such seropositivity rates are provided at day 120 (month 4) after a simultaneous administration of a unit dose of the dengue vaccine composition and one dose of an HPV vaccine.

[0274] In another specific embodiment, the method provides a seropositivity rate of at least 95% or at least 98% or at least 99.5% or 100% for each dengue serotype at day 120 (month 4) after a first concomitant administration of a first unit dose of the dengue vaccine composition and a first unit dose of the HPV vaccine composition. For example, the method provides a seropositivity rate of at least 95% or at least 98% or at least 99.5% or 100% for each dengue serotype at day 120 (month 4) after a first concomitant administration of a first unit dose of the dengue vaccine composition and a first unit dose of the HPV vaccine composition at day 1 and after a second administration of a second unit dose of the dengue vaccine composition at day 90 (month 3).

[0275] In some embodiments, the method provides for a geometric mean neutralizing antibody titer against dengue serotype 1 of at least about 1000, at least about 1500, or at least about 2000, or between about 1500 to about 2500 as determined in at least 100 subjects seropositive to dengue at baseline, optionally as determined on Day 120 after an administration of a first unit dose of a dengue vaccine composition (such as the unit dose of the dengue vaccine composition according to the invention) simultaneously with an HPV vaccine on Day 0 and of a second unit dose of a dengue vaccine composition (such as the unit dose of the dengue vaccine composition according to the invention) on Day 90 or 3 months after the simultaneous administration. The lower limit of the 95% confidence interval for the GMT is above 0. In some embodiments, the geometric mean neutralizing antibody titers (GMTs) are determined in accordance with a microneutralization test.

[0276] In some embodiments, the method provides for a geometric mean neutralizing antibody titer against dengue serotype 2 of at least about 2500, at least about 3000, or at least about 3500, or between about 3500 to about 4500 as determined in at least 100 subjects seropositive to dengue at baseline, optionally as determined on Day 120 after an administration of a first unit dose of a dengue vaccine composition (such as the unit dose of the dengue vaccine composition according to the invention) simultaneously with an HPV vaccine on Day 0 and of a second unit dose of a dengue vaccine composition (such as the unit dose of the dengue vaccine composition according to the invention) on Day 90 or 3 months after the simultaneous administration. The lower limit of the 95% confidence interval for the GMT is above 0. In some embodiments, the geometric mean neutralizing antibody titers (GMTs) are determined in accordance with a microneutralization test.

[0277] In some embodiments, the method provides for a geometric mean neutralizing antibody titer against dengue serotype 3 of at least about 500, at least about 800, or at least about 1000, or between about 500 to about 1500 as determined in at least 100 subjects seropositive to dengue at baseline, optionally as determined on Day 120 after an administration of a first unit dose of a dengue vaccine composition (such as the unit dose of the dengue vaccine composition according to the invention) simultaneously with an HPV vaccine on Day 0 and of a second unit dose of a dengue vaccine composition (such as the unit dose of the dengue vaccine composition according to the invention) on Day 90 or 3 months after the simultaneous administration. The lower limit of the 95% confidence interval for the GMT is above 0. In some embodiments, the geometric mean neutralizing antibody titers (GMTs) are determined in accordance with a microneutralization test.

[0278] In some embodiments, the method provides for a geometric mean neutralizing antibody titer against dengue serotype 4 of at least about 500, at least about 700, or at least about 900, or between about 800 to about 1200 as determined in at least 100 subjects seropositive to dengue at baseline, optionally as determined on Day 120 after an administration of a first unit dose of a dengue vaccine composition (such as the unit dose of the dengue vaccine composition according to the invention) simultaneously with an HPV vaccine on Day 0 and of a second unit dose of a dengue vaccine composition (such as the unit dose of the dengue vaccine composition according to the invention) on Day 90 or 3 months after the simultaneous administration. The lower limit of the 95% confidence interval for the GMTis above 0. In some embodiments, the geometric mean neutralizing antibody titers (GMTs) are determined in accordance with a microneutralization test.

[0279] In some embodiments, the method provides for a geometric mean neutralizing antibody titer against dengue serotype 1 of at least about 1000, at least about 1500, or at least about 2000, or between about 1500 to about 2500, against dengue serotype 2 of at least about 2500, at least about 3000, or at least about 3500, or between about 3500 to about 4500, against dengue serotype 3 of at least about 500, at least about 800, or at least about 1000, or between about 500 to about 1500, and against dengue serotype 4 of at least about 500, at least about 700, or at least about 900, or between about 800 to about 1200 as determined in at least 100 subjects seropositive to dengue at baseline, optionally as determined on Day 120 after an administration of a first unit dose of a dengue vaccine composition (such as the unit dose of the dengue vaccine composition according to the invention) simultaneously with an HPV vaccine on Day 0 and of a second unit dose of a dengue vaccine composition (such as the unit dose of the dengue vaccine composition according to the invention) on Day 90 or 3 months after the simultaneous administration. The lower limit of the 95% confidence interval for the GMT is above 0. In some embodiments, the geometric mean neutralizing antibody titers (GMTs) are determined in accordance with a microneutralization test.

[0280] In some embodiments, the method provides for a geometric mean neutralizing antibody titer against dengue serotype 1 of at least about 150, at least about 200, or at least about 250, or between about 250 to about 350 as determined in at least 100 subjects seronegative to dengue at baseline, optionally as determined on Day 120 after an administration of a first unit dose of a dengue vaccine composition (such as the unit dose of the dengue vaccine composition according to the invention) simultaneously with an HPV vaccine on Day 0 and of a second unit dose of a dengue vaccine composition (such as the unit dose of the dengue vaccine composition according to the invention) on Day 90 or 3 months after the simultaneous administration. The lower limit of the 95% confidence interval for the GMT is above 0. In some embodiments, the geometric mean neutralizing antibody titers (GMTs) are determined in accordance with a microneutralization test.

[0281] In some embodiments, the method provides for a geometric mean neutralizing antibody titer against dengue serotype 2 of at least about 500, at least about 600, or at least about 800, or between about 600 to about 1000 as determined in at least 100 subjects seronegative to dengue at baseline, optionally as determined on Day 120 after an administration of a first unit dose of a dengue vaccine composition (such as the unit dose of the dengue vaccine composition according to the invention) simultaneously with an HPV vaccine on Day 0 and of a second unit dose of a dengue vaccine composition (such as the unit dose of the dengue vaccine composition according to the invention) on Day 90 or 3 months after the simultaneous administration. The lower limit of the 95% confidence interval for the GMT is above 0. In some embodiments, the geometric mean neutralizing antibody titers (GMTs) are determined in accordance with a microneutralization test.

[0282] In some embodiments, the method provides for a geometric mean neutralizing antibody titer against dengue serotype 3 of at least about 150, at least about 200, or at least about 250, or between about 250 to about 350 as determined in at least 100 subjects seronegative to dengue at baseline, optionally as determined on Day 120 after an administration of a first unit dose of a dengue vaccine composition (such as the unit dose of the dengue vaccine composition according to the invention) simultaneously with an HPV vaccine on Day 0 and of a second unit dose of a dengue vaccine composition (such as the unit dose of the dengue vaccine composition according to the invention) on Day 90 or 3 months after the simultaneous administration. The lower limit of the 95% confidence interval for the GMT is above 0. In some embodiments, the geometric mean neutralizing antibody titers (GMTs) are determined in accordance with a microneutralization test.

[0283] In some embodiments, the method provides for a geometric mean neutralizing antibody titer against dengue serotype 4 of at least about 150, at least about 200, or at least about 250, or between about 250 to about 350 as determined in at least 100 subjects seronegative to dengue at baseline, optionally as determined on Day 120 after an administration of a first unit dose of a dengue vaccine composition (such as the unit dose of the dengue vaccine composition according to the invention) simultaneously with an HPV vaccine on Day 0 and of a second unit dose of a dengue vaccine composition (such as the unit dose of the dengue vaccine composition according to the invention) on Day 90 or 3 months after the simultaneous administration. The lower limit of the 95% confidence interval for the GMT is above 0. In some embodiments, the geometric mean neutralizing antibody titers (GMTs) are determined in accordance with a microneutralization test.

[0284] In some embodiments, the method provides for a geometric mean neutralizing antibody titer against dengue serotype 1 of at least about 150, at least about 200, or at least about 250, or between about 250 to about 350, against dengue serotype 2 of at least about 500, at least about 600, or at least about 800, or between about 600 to about 1000, against dengue serotype 3 of at least about 150, at least about 200, or at least about 250, or between about 250 to about 350, and against dengue serotype 4 of at least about 150, at least about 200, or at least about 250, or between about 250 to about 350 as determined in at least 100 subjects seronegative to dengue at baseline, optionally as determined on Day 120 after an administration of a first unit dose of a dengue vaccine composition (such as the unit dose of the dengue vaccine composition according to the invention) simultaneously with an HPV vaccine on Day 0 and of a second unit dose of a dengue vaccine composition (such as the unit dose of the dengue vaccine composition according to the invention) on Day 90 or 3 months after the simultaneous administration. The lower limit of the 95% confidence interval for the GMT is above 0. In some embodiments, the geometric mean neutralizing antibody titers (GMTs) are determined in accordance with a microneutralization test.

[0285] In some embodiments, the method provides for a seropositivity rate (as preferably determined in at least 150 or at least 200 healthy subjects, such as between 200 and 250 healthy subjects) of at least 95%, at least 99%, or 100% against HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58, wherein the seropositivity rate is determined after a simultaneous administration of a unit dose of a dengue vaccine composition, such as the unit dose of the dengue vaccine composition according to the invention, and the nonvalent HPV vaccine comprising antigens of HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58, such as Gardasil® 9. In more specific embodiments, the seropositivity rates against HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58 are determined at Day 210 after the simultaneous administration. In yet more specific embodiments, the seropositivity rates against HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58 are determined at Day 210 after the simultaneous administration and the vaccine schedule is as follows:a) simultaneous administration of the unit dose of the dengue vaccine composition and of a first dose of the nonvalent HPV vaccine on Day 0,b) administration of a second unit dose of the dengue vaccine composition, preferably on Day 90 or three months after the simultaneous administration, andc) administration of a second dose of the nonvalent HPV vaccine on Day 180.

[0286] In some embodiments, the method provides for geometric mean neutralizing antibody titers (as preferably determined in at least 150 or at least 200 healthy subjects, such as between 200 and 250 healthy subjects) of at least about 500 or between about 500 to about 8000 against HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-45,HPV-52, and HPV-58, wherein the geometric mean neutralizing antibody titers are determined after a simultaneous administration of a unit dose of a dengue vaccine composition, such as the unit dose of the dengue vaccine composition according to the invention, and the nonvalent HPV vaccine comprising antigens of HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58, such as Gardasil® 9. In more specific embodiments, the geometric mean neutralizing antibody titers against HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58 are determined at Day 210 after the simultaneous administration. The lower limit of the 95% confidence interval for the GMT is above 0. In some embodiments, the geometric mean neutralizing antibody titers (GMTs) are determined in accordance with a competitive Luminex immunoassay such as described in Example 4 of this application. In yet more specific embodiments, the geometric mean neutralizing antibody titers against HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58 are determined at Day 210 after the simultaneous administration and the vaccine schedule is as follows:a) simultaneous administration of the unit dose of the dengue vaccine composition and of a first dose of the nonvalent HPV vaccine on Day 0,b) administration of a second unit dose of the dengue vaccine composition, preferably on Day 90 or three months after the simultaneous administration, andc) administration of a second dose of the nonvalent HPV vaccine on Day 180.

[0287] In another specific embodiment, the subject population or subject is from a dengue endemic region. More specifically, the subject population or subject may be from Singapore and the HPV vaccination schedule may be completed between the ages 9 - 26 years, e.g. as early as possible within this age range. The number of HPV doses required may depend on the age of the subject at time of receiving the first dose of HPV vaccination. For example, if the first dose is given when the subject is younger than 15 years old, 2 doses may suffice while 3 doses of the HPV vaccine may be necessary if the subject is older than 15 years old when receiving the first dose. The vaccine can be a bivalent HPV vaccine, e.g. Cervarix® and the subject can be female.

[0288] In another exemplary embodiment, the subject population or subject may be from Indonesia and the vaccination scheme may comprise two doses of the HPV vaccine administered 6 months apart. In this embodiment, the subject can be female and / or 11 to 12 years old.

[0289] In another exemplary embodiment, the subject population or subject may be from Malaysia and the vaccination scheme may comprise two doses of the HPV vaccine administered 6 months apart. In this embodiment, the subject can be female and / or 13 years old.

[0290] In another exemplary embodiment, the subject population or subject may be from Thailand and the vaccination scheme may comprise two doses of the HPV vaccine administered 6 months apart. In this embodiment, the subject can be female and / or 11 years old.

[0291] In another exemplary embodiment, the subject population or subject may be from Mexico and the vaccination scheme may comprise two doses of the HPV vaccine administered 6 months apart. In this embodiment, the subject can be female and / or 9 to 11 years old.

[0292] In another exemplary embodiment, the subject population or subject may be from China and the vaccine is Gardasil®, Gardasil® 9, Cervarix®, Cecolin® or Walrinvax™.Method of preventing and uses, method of inoculating against dengue disease and uses

[0293] Described herein is a method of preventing dengue disease (in particular virologically confirmable dengue, VCD) in a subject. Thus, in certain embodiments the invention is directed to a method of preventing dengue disease in a subject, comprising administering to the subject, a unit dose / tetravalent dengue virus composition, in particular a reconstituted unit dose of the invention as described herein.

[0294] Described herein is also a method of preventing dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Thus, in certain embodiments the invention is directed to a method of preventing dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), comprising administering to the subject a unit dose / tetravalent dengue virus composition of the invention, in particular a reconstituted unit dose of the invention as described herein.

[0295] Described herein is a method of inoculating a subject against virologically confirmable dengue disease with a tetravalent dengue virus composition including four live attenuated dengue virus strains representing serotype 1, serotype 2, serotype 3 and serotype 4, wherein in particular the tetravalent dengue virus composition includes a chimeric dengue serotype 2 / 1 strain and a dengue serotype 2 strain and a chimeric dengue serotype 2 / 3 strain and a chimeric dengue serotype 2 / 4 strain, wherein in particular the dengue serotype 2 strain is derived from the wild type virus strain DEN-2 16681 (SEQ ID NO 11) and differs in at least three nucleotides from the wild type as follows: a) 5'-noncoding region (NCR)-57 (nt-57 C-to-T): major attenuation locusb) NS1-53 Gly-to-Asp (nt- 2579 G-to-A): major attenuation locusc) NS3-250 Glu-to-Val (nt-5270 A-to-T): major attenuation locus; andwherein the three chimeric dengue strains are derived from the serotype 2 strain by replacing the structural proteins prM and E from serotype 2 strain with the corresponding structural proteins from the other dengue serotypes, resulting in the following chimeric dengue strains:a DENV-2 / 1 chimera,a DENV-2 / 3 chimera anda DENV-2 / 4 chimera.

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

[0297] The tetravalent dengue virus composition for such a method may be in the form of a unit dose comprising: (i) a dengue serotype 1 in a concentration of at least 3.3 loglO pfu / 0.5 ml,(ii) a dengue serotype 2, in a concentration of at least 2.7 log 10 pfu / 0.5 ml,(iii) a dengue serotype 3, in a concentration of at least 4.0 loglO pfu / 0.5 ml, and(iv) a dengue serotype 4, in a concentration of at least 4.5 loglO pfu / 0.5 ml.

[0298] In one embodiment of the method, the unit dose is lyophilized and, upon reconstitution with 0.5 mL of a pharmaceutically acceptable diluent, comprises:(i) a dengue serotype 1 in a concentration of at least 3.3 loglO pfu / 0.5 ml,(ii) a dengue serotype 2, in a concentration of at least 2.7 loglO pfu / 0.5 ml,(iii) a dengue serotype 3, in a concentration of at least 4.0 loglO pfu / 0.5 ml, and(iv) a dengue serotype 4, in a concentration of at least 4.5 loglO pfu / 0.5 ml.

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

[0300] In another embodiment of the method and corresponding use, the method comprising a primary vaccination with only two administrations of the unit dose comprises the steps of:(A) administering a first unit dose of the tetravalent dengue virus composition to the subject, and(B) administering a second unit does of the tetravalent dengue virus composition to the subject within 3 months of administration of the first unit dose.According to this embodiment the administration of only two doses within 3 months is sufficient to provide effective protection against a subsequent dengue infection.

[0301] Such method may provide a combined vaccine efficacy against all four serotypes in preventing virologically confirmable dengue disease with a 2-sided 95% confidence interval, wherein the lower bound is more than 60%, when measured against placebo in a subject population of at least 5,000 healthy subjects irrespective of serostatus at baseline and 14 to 16 years of age, from the first administration of the administration schedule until 18 months after the second administration of the administration schedule.

[0302] Such method may also provide a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease with a 2-sided 95% confidence interval, wherein the lower bound is more than 45 %, when measured against placebo in a subject population of at least 1,500 or at least 2,000 healthy subjects seronegative against all serotypes at baseline and 14 to 16 years of age, from 30 days after the second administration of the administration schedule until 18 months after the second administration of the administration schedule.

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

[0304] In certain embodiments, said methods have a vaccine efficacy against serotype 1 in preventing virologically confirmable dengue disease with a 2-sided 95% confidence interval, wherein the lower bound is more than 25%, when measured against placebo in a subject population of at least 1,500, or at least 2,000, or at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) irrespective of serostatus at baseline and 4 to 16 years of age from 30 days post second administration until 12 to 18 months (e.g. at 12 or at 18 months) after the second administration. In certain such embodiments, the lower bound is more than 30%, is more than 35% is more than 40%, is more than 45%, is more than 50%, or is more than 54%. In certain such embodiments the subject population of at least 1,500 is seronegative against all serotypes at baseline and the lower bound is more than 35%. In certain such embodiments the seronegative and seropositive population each provide a vaccine efficacy against serotype 1 with a 2-sided 95% confidence interval, wherein the lower bounds are within 10%-points.

[0305] In certain embodiments, said methods have a vaccine efficacy against serotype 1 in preventing virologically confirmable dengue disease, when measured against placebo in a subject population of at least 1,500, or at least 2,000, or at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) irrespective of serostatus at baseline and 4 to 16 years of age from 30 days post second administration until 12 to 18 months (e.g. at 12 or at 18 months) after the second administration. In certain such embodiments, the vaccine efficacy is more than 40%, is more than 50%, is more than 60%, or is more than 65%. In certain such embodiments the subject population of at least 1,500 is seronegative against all serotypes at baseline. In certain such embodiments the seronegative and seropositive population each provide a vaccine efficacy against serotype 1 which are within 5 %-points.

[0306] In certain embodiments, said methods have a vaccine efficacy against serotype 2 in preventing virologically confirmable dengue disease with a 2-sided 95% confidence interval, wherein the lower bound is more than 25%, when measured against placebo in a subject population of at least 1,500, or at least 2,000, or at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) irrespective of serostatus at baseline and 4 to 16 years of age from 30 days post second administration until 12 to 18 months (e.g. at 12 or at 18 months) after the second administration. In certain such embodiments, the lower bound is more than 50%, is more than 60%, is more than 70%, is more than 80%, or is more than 85%. In certain such embodiments the subject population of at least 1,500, is seronegative against all serotypes at baseline. In certain such embodiments the seronegative and seropositive population each provide a vaccine efficacy against serotype 2 with a 2-sided 95% confidence interval, wherein the lower bounds are within 5 %-points.

[0307] In certain embodiments, said methods have a vaccine efficacy against serotype 2 in preventing virologically confirmable dengue disease, when measured against placebo in a subject population of at least 1,500, or at least 2,000, or at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) irrespective of serostatus at baseline and 4 to 16 years of age from 30 days post second administration until 12 to 18 months (e.g. at 12 or at 18 months) after the second administration. In certain such embodiments, the vaccine efficacy is more than 60%, is more than 70%, is more than 80%, or is more than 90%. In certain such embodiments the subject population of at least 1,500 is seronegative against all serotypes at baseline. In certain such embodiments the seronegative and seropositive population each provide a vaccine efficacy against serotype 2 which are within 5 %-points.

[0308] The efficacy of the method is further described in more detail below in this the section.

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

[0310] According to one embodiment such a method does not include a step of determination whether there was a previous dengue infection in the subject before administration of the unit dose or wherein the serostatus of the subject is unknown before administration of the unit dose.

[0311] According to one embodiment such a method does not include a step of determination of a previous dengue infection in the subjects at any time before, during or after the steps of administration or wherein the serostatus of the subject is unknown at any time before, during or after the steps of administration.

[0312] In one embodiment, the methods does not require the testing of the serostatus before vaccination and thus allows immediate treatment and outbreak control. According to certain embodiments the use is for a method wherein the subject is exposed to a dengue outbreak. In certain such embodiments the outbreak is due to a dengue serotype 2 and / or due to a serotype 1.

[0313] According to one embodiment of such a method the subject is from a region wherein the seroprevalence rate is unknown and / or wherein the seroprevalence rate is below 80%, or below 70%, or below 60%.

[0314] According to another embodiment the subject is from a region wherein the dengue seroprevalence rate at age 9 is above 40%, such as at least 50% or at least 60%.

[0315] According to one embodiment of such a method the subject is seronegative at baseline and is from a region or travels to a region wherein the seroprevalence rate is high with respect to serotype 1 and / or serotype 2 i.e. 80%, or 90% or above.

[0316] According to this embodiment the vaccine and corresponding method is safe for seronegative and seropositive subjects and thus does not require an analysis of the serostatus or a determination of a previous dengue infection or a high seroprevalence rate in the region. Such a method may provide a combined vaccine efficacy against virologically-confirmed dengue with hospitalization against all four serotypes with a 2-sided 95% confidence interval, wherein the lower bound is more than 65%, when measured against placebo in a subject population of at least 5,000 healthy 4 to 16 year old subjects irrespective of serostatus at baseline, such as in at least 1,500 healthy 4 to 16 year old subjects seronegative at baseline, from first administration of the administration schedule until 12 to 18 months after the second administration of the administration schedule. The 2-sided 95% confidence interval of the combined vaccine efficacy against virologically confirmed dengue with hospitalization against all four serotypes when comparing seropositive and seronegative subjects may provide for lower bounds of the 2-sided confidence interval which are within 10% points or within 15% points or within 20% points. The method may be safe with respect to serotype 1 and serotype 2 which may therefore be used in outbreak situations due to serotype 1 and / or serotype 2 or even for seronegative subjects (e.g. travelers) or subjects with unknown serostatus in regions with very high seroprevalence rates (>80%) due to serotype 1 and / or serotype 2.

[0317] The safety of the method is further described in more detail in the section "method of preventing, method of inoculating".

[0318] According to one embodiment such a method does not include the active surveillance with respect to febrile illness of the subject after the administration of the first- and second-unit dose. During active surveillance any subject with febrile illness (defined as fever >38°C on any 2 of 3 consecutive days) can be asked to return to the site for dengue fever evaluation by the Investigator. Subjects / guardians will typically be contacted at least weekly to ensure robust identification of febrile illness by reminding subjects / guardians of their obligation to return to the site in case of febrile illness. This contact will typically be implemented through appropriate methods that may differ in each trial site (e.g, phone calls, text messaging, home visits, school-based surveillance).

[0319] According to one embodiment such a method does not include vaccine immunogenicity analysis including GMTs for dengue neutralizing antibodies.

[0320] According to one embodiment such a method does not include a reactogenicity analysis. Such a reactogenicity analysis relates to solicited local AEs (injection site pain, injection site erythema, and injection site swelling) and solicited systemic AEs (child < 6 years: fever, irritabil ity / fussiness, drowsiness and loss of appetite; child > 6 years: asthenia, fever, headache, malaise and myalgia) which will e.g. be assessed for 7 days and 14 days, respectively, following each vaccination (vaccination day included) via collection of diary cards.

[0321] According to one embodiment the method does not include an active surveillance, an immunogenicity analysis and a reactogenicity analysis.

[0322] According to such embodiments the vaccine and the corresponding method of inoculation are safe and therefore do not require further steps of surveillance or analysis.

[0323] In view of the above the method according to one embodiment comprises a primary vaccination consisting of the steps of:(A) selecting a subject for administration of the unit doses of the tetravalent dengue virus composition in need for protection against dengue infection without determination of a previous dengue infection, and(B) administering a first unit dose of the tetravalent dengue virus composition to the subject, 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.

[0324] Therefore the method of inoculating can be finalized without determination of a previous dengue infection. The method further optionally comprises at least 1 years after the administration of the second unit dose a booster dose of the unit dose.

[0325] Selecting the subject may include all types of considerations but typically not the determination of a previous dengue infection. The selection may include consideration of the age, health conditions, and threat of infection. The threat of infection includes consideration of the seroprevalence rate in the region in which the subject normally lives or intends to travel, the serotype specific seroprevalence rate and an outbreak situation or serotype specific outbreak situations. The subject may be selected due to its exposure to serotype 1 and / or serotype 2 or due to the fact it requires protection against a specific dengue serotype, i.e. serotype 1 and / or serotype 2.

[0326] According to the invention the method can be applied to subjects of all kinds of ages. According to one embodiment the subject is under 9 years of age, or 4 to 5 years of age, or 6 to 11 years of age or 12 to 16 years, or 6 to 16 years of age or 4 to 16 years of age, or 2 to 17 years of age, or 9 years of age, or over 9 years of age, or 9 to 17 years of age,, or 18 to 45 years of age, or 46 to 60 years of age, or over 60 years of age.

[0327] In certain embodiments of said methods, the method is safe.

[0328] For example, such a method provides a combined vaccine efficacy against virologically-confirmed dengue with hospitalization against all four serotypes with a 2-sided 95% confidence interval, wherein the lower bound is more than 65%, when measured against placebo in a subject population of at least 5,000 healthy 4 to 16 year old subjects irrespective of serostatus at baseline from first administration of the administration schedule until 12 to 18 months after the last administration of the administration schedule.

[0329] In certain embodiments of said methods, the method is effective.

[0330] For example, such a method provides a combined vaccine efficacy against all four serotypes in preventing virologica lly confirmable dengue disease with a 2-sided 95% confidence interval, wherein the lower bound is more than 60%, when measured against placebo in a subject population of at least 5,000 healthy subjects irrespective of serostatus at baseline and 14 to 16 years of age, from the first administration of the administration schedule until 18 months after the last administration of the administration schedule.

[0331] In certain embodiments of said methods, the subject is seronegative to all dengue serotypes.

[0332] Further, described herein is a method of preventing dengue disease (in particular virologically confirmable dengue, VCD) in a subject population. Thus, in certain embodiments the method of preventing dengue disease in a subject population comprises administering to the subject population a unit dose, in particular a reconstituted unit dose of the invention as described herein.

[0333] Further, described herein is a method for preventing dengue disease (in particular virologically confirmable dengue, VCD) in a subject population comprising administering to the subject population at least a first reconstituted unit dose of the invention as described herein, wherein certain ratios of geometric mean neutralizing antibody titers (GMTs) at day 180 or 365 after administration of said first unit dose to the subject population are achieved. According to some embodiments, the geometric mean neutralizing antibody titer for dengue serotype 2 (GMT DENV-2) and the geometric mean neutralizing antibody titer for dengue serotype 4 (GMT DENV-4) when tested in at least 40, or at least 50, or at least 60 subjects at day 180 or day 365 after at least a first administration of said reconstituted unit dose of the invention as described herein, and optionally a second administration of a reconstituted unit dose of the invention as described herein 90 days after said first administration, provide a ratio of GMT DENV-2: GMT DENV-4 of not more than 50, or not more than 40, or not more than 30, or not more than 20. In some of these embodiments, the ratio of GMT DENV-2: GMT DENV-1 is not more than 20, or not more than 18, or not more than 15 at day 180 or 365 after administration of said first reconstituted unit dose, and / or the ratio of GMT DENV-2: GMT DENV-3 is not more than 20, or not more than 18, or not more than 15 at day 180 or 365 after administration of said first reconstituted unit dose.

[0334] Further, described herein is a method for preventing dengue disease (in particular virologically confirmable dengue, VCD) in a subject comprising administering to the subject at least a first reconstituted unit dose of the invention as described herein, wherein certain ratios of neutralizing antibody titers at day 180 or 365 after administration of said first unit dose to the subject are achieved. According to some embodiments, the neutralizing antibody titer for dengue serotype 2 and the neutralizing antibody titer for dengue serotype 4 at day 180 or day 365 after at least a first administration of the reconstituted unit dose of the invention as described herein, and optionally a second administration of a reconstituted unit dose of the invention as described herein 90 days after said first administration, provide a ratio of neutralizing antibody titer for DENV-2: neutralizing antibody titer for GMT DENV-4 of not more than 50, or not more than 40, or not more than 30, or not more than 20. In some of these embodiments, the ratio of the neutralizing antibody titers of DENV-2: DENV-1 is not more than 20, or not more than 18, or not more than 15 at day 180 or 365 after administration of said first reconstituted unit dose, and / or the ratio of the neutralizing antibody titers of DENV-2: DENV-3 is not more than 20, or not more than 18, or not more than 15 at day 180 or 365 after administration of said first reconstituted unit dose.

[0335] The geometric mean neutralizing antibody titers (GMTs) of a subject population or the neutralizing antibody titers of a subject are determined in accordance with the microneutralization test disclosed herein, for example according to the method described in Example 2. Without wishing to be bound to any theory, it is presently understood that a method inducing a more balanced immune response due to the administration of the reconstituted unit dose of the invention as described herein, in terms of less differences between the geometric mean neutralizing antibody titers (GMTs) against the four dengue serotypes or the neutralizing antibody titers against the four dengue serotypes, is beneficial to the subject or subject population to be vaccinated. In particular, it is understood that a much greater response to any one of the four serotypes, such as to DENV-2 in comparison to the other serotypes, is less beneficial.

[0336] Further, described herein is a method for preventing dengue disease (in particular virologically confirmable dengue, VCD) in a subject or subject population wherein the method provides a seropositivity rate in a subject population of at least 50 subjects including the administration of two unit doses subcutaneously at day 1 and at day 90, wherein the subjects of the subject population are seronegative to all dengue serotypes at baseline. In certain such embodiments, at least 80% of the subject population are seropositive for all four dengue serotypes at least one month after administration of the first unit dose, such as at day 30, and / or at least 80% of the subject population are seropositive for all four dengue serotypes before or at the time of the administration of the second unit dose, such asat day 90, and / or at least 80%, or at least 85%, or at least 90%, or at least 95% of the subject population are seropositive for all four dengue serotypes after the administration of the second unit dose, such as at day 120, and / or at least 80%, or at least 85%,or at least 90% of the subject population are seropositive for all four dengue serotypes after the administration of the second unit dose, such as at day 270.

[0337] Further, described herein is a method for preventing dengue disease (in particular virologically confirmable dengue, VCD) in a subject or subject population wherein the method provides a seropositivity rate in a subject population of at least 100 subjects including administration of two unit doses subcutaneously at day 1 and at day 90, wherein the subjects of the subject population comprises from 20% to 40% subjects who are seronegative to all dengue serotypes and from 60% to 80% subjects who are seropositive to at least one dengue serotype at base line, wherein at day 120 and / or day 270 the seropositivity rate for all four dengue serotypes in the seronegative part of the subject population and the seropositivity rate for all four dengue serotypes in the seropositive part of the subject population do not deviate more than 10%-points and / or wherein at day 120 the seropositivity rate for all four dengue serotypes in the seronegative part of the subject population and the seropositivity rate for all four dengue serotypes in the seropositive part of the subject population do not deviate more than 5%-points.

[0338] Further, described herein is a method of preventing virologically confirmable dengue disease in a subject or subject population comprising administering to the subject or subject population a reconstituted unit dose of a tetravalent dengue virus composition including four live, attenuated dengue serotypes, in particular the virus strains as described herein.

[0339] Further, described herein is a method of preventing virologically confirmable dengue disease with hospitalization in a subject or subject population comprising administering to the subject or subject population a reconstituted unit dose of a tetravalent dengue virus composition including four live, attenuated dengue serotypes, in particular the virus strains as described herein.

[0340] In certain embodiments, the method includes a reconstituted unit dose / tetravalent dengue virus composition of a dengue vaccine composition administered for preventing dengue disease in a subject or a subject population, the reconstituted unit dose comprising: a tetravalent virus composition including four live attenuated dengue virus strains, wherein a unit dose is lyophilized and upon reconstitution with 0.5 mL of a pharmaceutically acceptable diluent the reconstituted unit dose is obtained which comprises:(i) a dengue serotype 1, such as a chimeric dengue serotype 2 / 1 strain, in a concentration of at least 3.3 loglO pfu / 0.5 ml,(ii) a dengue serotype 2, such as a dengue serotype 2 strain, in a concentration of at least 2.7 loglO pfu / 0.5 ml,(iii) a dengue serotype 3, such as a chimeric dengue serotype 2 / 3 strain, in a concentration of at least 4.0 loglO pfu / 0.5 ml, and(vi) a dengue serotype 4, such as a chimeric dengue serotype 2 / 4 strain, in a concentration of at least 4.5 loglO pfu / 0.5 ml.

[0341] For example, the reconstituted unit dose / tetravalent dengue virus composition is used in the method of preventing dengue disease of the present invention, wherein upon reconstitution of the unit dose with a pharmaceutically acceptable diluent (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL and based on said total concentration the concentration of (ii) in pfu / 0.5 mL 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 6% and wherein the subject or subject population is of 18 to 60 years of age.

[0342] In another specific embodiment, the reconstituted unit dose / tetravalent dengue virus composition is used in the method of preventing dengue disease of the present invention, wherein upon reconstitution with a pharmaceutically acceptable diluent (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL and based on said total concentration the concentration of (ii) in pfu / 0.5 mL is less than 10%, and 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) in pfu / 0.5 mL is at least 8% and wherein the subject or subject population is of 2 to 17 years of age.

[0343] In certain embodiments of said methods, said unit dose comprises a tetravalent dengue virus composition including four live attenuated dengue serotypes, in particular the virus strains described herein wherein the serotypes have certain concentrations as described herein with respect to the virus composition and unit dose such as:(i) a dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) has a concentration of 3.3 loglO pfu / dose to 5.0 loglO pfu / dose, or 3.3 loglO pfu / 0.5 mLto 5.0 loglO pfu / 0.5 mL(ii) a dengue serotype 2 (e.g. dengue serotype 2 strain) has a concentration of 2.7 loglO pfu / dose to 4.9 loglO pfu / 0.5 dose, or 2.7 loglO pfu / 0.5ml to 4.9 loglO pfu / 0.5ml(iii) a dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) has a concentration of 4.0 loglO pfu / dose to 5.7 loglO pfu / 0.5 dose, or 4.0 loglO pfu / 0.5 mL to 5.7 loglO pfu / 0.5 mL and(iv) a dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) has a concentration of 4.5 loglO pfu / dose to 5.5 loglO pfu / 0.5 dose, or 4.5 loglO pfu / 0.5 mL to 5.5 loglO pfu / 0.5 mL.

[0344] In exemplary such embodiments, the subject or subject population is of 2 to 17 years of age, such as 4 to 16 years of age, e.g. less than 9 years of age. In other exemplary embodiments, the subject or subject population is 4-5 years of age, 6-11 years of age, 9 to 14 years of age or 12-16 years of age.

[0345] In certain embodiments of said methods, said unit dose upon reconstitution with 0.5 mL of a pharmaceutically acceptable diluent has a concentration of 3.3 loglO pfu / 0.5 mLto 3.6 loglO pfu / 0.5 mL for dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain), has a concentration of 2.7 loglO pfu / 0.5 mLto 4.0 loglO pfu / 0.5 mL for dengue serotype 2 (e.g. dengue serotype 2 strain), has a concentration of 4.0 loglO pfu / 0.5 mL to 4.6 loglO pfu / 0.5 mL for dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) and has a concentration of 4.5 loglO pfu / 0.5 mL or 4.6 loglO pfu / 0.5 mL to 5.1 loglO pfu / 0.5 mL for dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain). In exemplary such embodiments, the subject or subject population is of 2 to 17 years of age, such as 4 to 16 years of age, and e.g. less than 9 years of age. In other exemplary embodiments, the subject or subject population is 4-5 years of age, 6-11 years of age, 9 to 14 years of age or 12-16 years of age.

[0346] In certain embodiments of said methods, the concentration of the dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) measured in pfu / 0.5 mL is 1% to 7% of the total concentration, the concentration of the dengue serotype 2 (e.g. dengue serotype 2 strain) measured in pfu / 0.5 mL is less than 8% of the total concentration, such as in the range of 1% to 8% of the total concentration, the concentration of the dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) measured in pfu / 0.5 mL is at least 10% of the total concentration, and the concentration of the dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) measured in pfu / 0.5 mL is at least 65% of the total concentration, such as in the range of 65% to 80%. In certain such embodiments, the arithmetic sum of all four serotypes is in the range of 4.6 loglO pfu / 0.5 mL to 6.7 loglO pfu / 0.5 mL, e.g. in the range of 4.6 loglO pfu / 0.5 mL to 5.5 loglO pfu / 0.5 mL For example, in said embodiments the subject or subject population is of 2 to 17 years of age, such as 4 to 16 years of age, such as less than 9 years of age. In other exemplary embodiments, the subject or subject population is 4-5 years of age, 6-11 years of age, 9 to 14 years of age or 12-16 years of age.

[0347] In a further exemplary embodiment of said methods, the dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) such as TDV-1 and the dengue serotype 2 (e.g. dengue serotype 2 strain) such as TDV-2 are present each in a concentration based on the total concentration in pfu / 0.5 mL which is within 5%-points of each other and / or are together less than about 10% of the total concentration in pfu / 0.5 mL. In certain such embodiments the dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) such as TDV-3 is at least about 10% of the total concentration in pfu / 0.5 mL and / or the dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) such as TDV-4 is at least about 70% of the total concentration in pfu / 0.5 mL. In certain such embodiments the dengue serotype 4 (e.g. chimeric dengue serotype 2 / 4 strain) such as TDV-4 represents the highest concentration in the composition of all four serotypes, such as with at least about 70% of the total concentration in pfu / 0.5 mL, dengue serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) such as TDV-3 represents the second highest concentration in the composition of all four serotypes, e.g. with at least about 10% of the total concentration in pfu / 0.5 mL, and dengue serotype 1 (e.g. chimeric dengue serotype 2 / 1 strain) such as TDV-1 and dengue serotype 2 (e.g. dengue serotype 2 strain) such as TDV-2 each represent lower concentrations than the concentration of serotype 3 (e.g. chimeric dengue serotype 2 / 3 strain) such as TDV-3, and optionally together represent less than about 10% of the total concentration in pfu / 0.5 mL.

[0348] For example, the chimeric dengue serotype 2 / 1 strain is TDV-1, the dengue serotype 2 strain is TDV-2, the chimeric dengue serotype 2 / 3 strain is TDV-3 and the chimeric dengue serotype 2 / 4 strain is TDV-4. In a specific example, 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, 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, TDV-3 is characterized by the nucleotide sequence according to SEQ ID No. 5 and the amino acid sequence according to SEQ ID No. 6 and TDV-4 is characterized by the nucleotide sequence according to SEQ ID No. 7 and the amino acid sequence according to SEQ ID No. 8.

[0349] In certain embodiments of said methods, 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, such as to the deltoid region of the arm. In other embodiments of said methods, the reconstituted unit dose of the invention as described herein is administered by intramuscular injection.

[0350] In certain embodiments of said methods, the reconstituted unit dose is administered to a subject of unknown serostatus and / or wherein no test has been carried out to determine whether the subject is seropositive or seronegative (before) the unit dose as described herein is administered. In certain embodiments said methods do not include a step of determination of a previous dengue infection in the subject or subjects. In certain embodiments, said methods do not include the analysis of the seroprevalence rate in the region or is conducted in a region with a seroprevalence of below 80%, below 70% or below 60%. In certain embodiments of the methods the serostatus of the subject is unknown. In such embodiments the serostatus is not determined at any time before and after administration in relation to this method. In certain embodiments the method is used in an outbreak situation. In certain embodiments methods are conducted outside a clinical trial.

[0351] In certain embodiments of said methods, the subject or subject population is seronegative to all dengue serotypes.

[0352] In certain embodiments of said methods, 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 six months, or within three months, and optionally at least 4 weeks apart such as at day 0 and day 90 or at day 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. Such a third administration may act as a booster and may beadministered between 6 to 12 months after the first administration, such as 12 months after the first administration, or later than 12 months after the first administration, such as 12 months (1 year) after the second administration or even 5 years or longer after the first or second administration.

[0353] In certain embodiments, the method comprises or consists of a single unit dose of the invention being administered.

[0354] In certain embodiments of said methods, the reconstituted unit dose of the invention as described herein is administered subcutaneously to a subject or subject population that is seronegative with respect to all dengue serotypes. In other embodiments, the subject or subject population is seropositive with respect to at least one dengue serotype.

[0355] In certain embodiments of said methods, the unit dose of the invention as described herein is administered to a subject or subject population from a dengue endemic region. In some of these embodiments, the subject or subject population is from Singapore, Dominican Republic, Panama, Philippines, Colombia, Puerto Rico or Thailand, in particular from Singapore, Dominican Republic, Panama, or Philippines. In an exemplary embodiment, the subject or subject population is from Asia Pacific or from Latin America. In some other of these embodiments, the subject or subject population is from Thailand, Sri Lanka, Philippines, Panama, Nicaragua, Dominican Republic, Colombia or Brazil. In other embodiments, the subject or subject population is from a dengue non-endemic region. Such a subject population or such a subject may be vaccinated according to the present invention in the context of traveling to a dengue endemic region. In certain embodiments, the reconstituted unit dose of the invention as described herein is administered subcutaneously to a subject, or subject population that is from a dengue endemic region or a dengue non-endemic region.

[0356] In certain embodiments of said methods, the reconstituted unit dose of the invention as described herein is administered subcutaneously to a subject or subject population of 2 to 60 years of age. In some embodiments, the subjects or subject population are adults of more than 17 years, or more than 18 years, or 18 to 60 years. In further specific embodiments, the subjects or subject population are adults of more than 21 years, or 21 to 60 years, or 21 to 45 years of age.

[0357] In certain embodiments of said methods, the reconstituted unit dose of the invention as described herein is administered subcutaneously to children and adolescents of 2 to 17 years of age. In some embodiments, the subjects or subject population are less than 9 years of age, or less than 4 years of age. In some embodiments, the subjects or subject population are from 2 to 9 years of age, or from 2 to 5 years of age, or from 4 to 9 years of age or from 6 to 9 years of age. In other embodiment, the subject or subject population is 4 to 16 years of age. In some such embodiments, the subject or subject population is 4-5 years of age, 6-11 years of age or 12-16 years of age. Optionally, the subject or subject population is seronegative with respect to all dengue serotypes.

[0358] In certain embodiments of said methods, wherein the unit dose of the invention as described herein is administered to a pediatric subject or pediatric subject population of less than 2 years of age, e.g. of 2 months to 2 years or 2 months to 1.5 years or 2 months to 1 year. According to some of these embodiments, the pediatric subject or pediatric subject population is seronegative and / or from a dengue endemic region.

[0359] In certain embodiments of said methods, the reconstituted unit dose of the invention as described herein is administered to a pediatric subject or pediatric subject population of less than 2 years of age, e.g. of 2 months to 2 years or 2 months to 1.5 years or 2 months to 1 year, e.g. by subcutaneous injection. According to some of theseembodiments, the pediatric subject or pediatric subject population is seronegative and / or from a dengue endemic region.

[0360] In certain embodiments of said methods, the subject or subject population is 4-5 years of age and from Asia Pacific, 6-11 years of age and from Asia Pacific, or 12-16 years of age and from Asia Pacific. In other embodiments, the subject or subject population is 4-5 years of age and from Latin America, 6-11 years of age and from Latin America, or 12-16 years of age and from Latin America.

[0361] In certain embodiments of said methods, the subject or subject population is 4-5 years of age and seropositive for at least 1 dengue serotype, 6-11 years of age and seropositive for at least 1 dengue serotype, or 12-16 years of age and seropositive for at least 1 dengue serotype. In other embodiments, the subject or subject population is 4-5 years of age and seronegative for all dengue serotypes, 6-11 years of age and seronegative for all dengue serotypes, or 12-16 years of age and seronegative for all dengue serotypes.

[0362] In certain embodiments of said methods, the subject or subject population is from Asia Pacific or Latin America and seropositive for at least one dengue serotype at baseline. In other embodiments, the subject or subject population is from Asia Pacific or Latin America and seronegative for at all dengue serotype at baseline.

[0363] In certain embodiments of said methods, the subject or subject population is from Asia Pacific, seropositive for at least one dengue serotype at baseline and 4-5 years of age, 6-11 years of age, or 12-16 years of age. In other embodiments, the subject or subject population is from Asia Pacific, seronegative for all dengue serotypes at baseline and 4-5 years of age, 6-11 years of age, or 12-16 years of age. In yet other embodiments, the subject or subject population is from Latin America, seropositive for at least one dengue serotype at baseline and 4-5 years of age, 6-11 years of age, or 12-16 years of age. In other embodiments, the subject or subject population is from America, seronegative for all dengue serotypes at baseline and 4-5 years of age, 6-11 years of age, or 12-16 years of age.

[0364] In certain embodiments of said methods, the subject or subject population had prior vaccination against Yellow Fever. In other embodiments, the subject or subject population had prior vaccination against Japanese Encephalitis. In yet other embodiments, the subject or subject population had no prior vaccination against Yellow Fever. In other embodiments, the subject or subject population had no prior vaccination against Japanese Encephalitis. Prior vaccination indicates a vaccination prior to 30 days after a second administration, such as within 4 months after the first administration, with the reconstituted unit dose as described herein.

[0365] The present invention shows that in particular seronegative subjects show a more balanced immune response to the four dengue serotypes after administration of the reconstituted unit dose of the invention as described herein. It is therefore contemplated that the unit dose of the invention as described herein and methods of the present invention as described herein may provide a more robust immune response in a subject population including both seropositive and seronegative subjects. This balanced response and balanced efficacy and safety is required to allow inoculation without prior serostatus analysis which is a major advantage in vaccination programs and in particular in outbreak situations.

[0366] Further, described herein is a method of preventing virologically confirmable dengue disease in a subject comprising administering to the subject a tetravalent dengue virus composition including four dengue virus strains representing serotype 1, serotype 2, serotype 3 and serotype 4, wherein the virus strains are optionally live, attenuated dengue virus strains.

[0367] Further, described herein is a method of preventing virologically confirmable dengue disease in a subject consisting of administering to the subject a tetravalent dengue virus composition including four dengue virus strains representing serotype 1, serotype 2, serotype 3 and serotype 4, wherein the virus strains are optionally live, attenuated dengue virus strains.

[0368] In certain embodiments of said methods, there is no step of determining the serostatus of the subject at baseline. In other words, said methods do not comprise a determination of a previous dengue infection of the subject at baseline before the administration of the tetravalent dengue virus composition. In particular, such methods are safe and effective. Thus, in certain such embodiments, the subject has not been tested for the presence a previous dengue infection.

[0369] In certain embodiments of said methods, the vaccine administration is safe irrespective of whether there is a determination that the subject had a previous dengue infection before the administration of the tetravalent dengue virus composition. In particular, such methods are also effective.

[0370] In certain embodiments, the method is safe and / or effective.

[0371] In certain embodiments of said methods, the composition includes at least one chimeric dengue virus. In certain embodiments of said methods, the composition includes at least one non-chimeric dengue virus. In certain embodiments of said methods, the composition includes at least one non-chimeric dengue virus and at least one chimeric dengue virus, in particular a chimeric dengue serotype 2 / 1 strain and a dengue serotype 2 strain and a chimeric dengue serotype 2 / 3 strain and a chimeric dengue serotype 2 / 4 strain. The details of the composition are described above.

[0372] Therefore, in certain embodiments, said methods have a vaccine efficacy, e.g. a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease with a 2-sided 95% confidence interval, wherein the lower bound is more than 25%, when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) irrespective of serostatus at baseline and e.g. 14 to 16 years of age, wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or placebo is administered e.g. at least twice within less than 6 months, such as within 3 months, after first administration or 30 days after the second / last administration until at least 12 to 18 months (e.g. at 12 or at 18 months) after the second / last administration. In some embodiments, said methods have a vaccine efficacy, e.g. a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease with a 2-sided 95% confidence interval, wherein the lower bound is more than 25%, when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) irrespective of serostatus at baseline, wherein a reconstituted unit dose or tetravalent dengue virus composition as described herein or placebo is administered at least once, until 15 to 21 months (e.g. 15 or 21 months) after the first administration of the administration schedule. In certain such embodiments, the lower bound is more than 30%, more than 40%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70% or more than 72%. For example, said reconstituted unit dose or placebo is administered subcutaneously within about 3 months, such as on days 0 and 90.

[0373] Therefore, in certain embodiments, said methods have a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease with a 2-sided 95% confidence interval, wherein the lower bound is more than 60%, when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) irrespective of serostatus at baseline and 4 to 16 years of age, wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or placebo isadministered e.g. at least twice within less than 6 months, such as within 3 months, after the first administration until 18 months after the last administration. In these embodiments, the lower bound is e.g. more than 62%, more than 64%, more than 66%, more than 68%, or more than 69%.

[0374] In certain embodiments, said methods have a vaccine efficacy, e.g. a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease of more than 30%, when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) irrespective of serostatus at baseline and e.g. 14 to 16 years of age, wherein a reconstituted unit dose or tetravalent dengue virus composition as described herein or placebo is administered at least twice within less than 6 months, such as within 3 months, after first administration or 30 days after the second administration / last administration until at least 12 months or until 12 to 18 months (e.g. at 12 or at 18 months) after the second administration / last administration. In certain embodiments, said methods have a vaccine efficacy, e.g. a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease of more than 30%, when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) irrespective of serostatus at baseline, wherein a reconstituted unit dose or tetravalent dengue virus composition as described herein or placebo is administered at least once, until 15 months after the first administration of the administration schedule. In certain such embodiments, the vaccine efficacy is more than 40%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 78%, more than 79% or about 80%. For example, said reconstituted unit dose or placebo is administered subcutaneously within about 3 months, such as on days 0 and 90.

[0375] Therefore, in certain embodiments, said methods have a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease of more than 66%, when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) irrespective of serostatus at baseline and 14 to 16 years of age, wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or placebo is administered e.g. at least twice within less than 6 months, such as within 3 months, after the first administration until 18 months after the last administration. In these embodiments, the vaccine efficacy is e.g. more than 68%, more than 70%, more than 72%, or more than 74%.

[0376] In certain embodiments, said methods have a vaccine efficacy, e.g. a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease with hospitalization with a 2-sided 95% confidence interval, wherein the lower bound is more than 0%, when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) irrespective of serostatus at baseline, wherein a reconstituted unit dose or tetravalent dengue virus composition as described herein or placebo is administered at least twice within less than 6 months, such as within 3 months, 30 days after the second administration until at least 18 months after the second administration. In certain such embodiments, the lower bound is more than 10%, is more than 20%, is more than 30%, is more than 40%, is more than 50%, is more than 55%, is more than 60%, is more than 65%, is more than 70% or is more than 80%, or more than 90%.

[0377] In certain embodiments, said methods have a combined vaccine efficacy against all four dengue serotypes in seronegative subjects with a 2-sided 95% confidence interval, wherein the lower bound is more than 25%, when measured against placebo in a subject population of at least 1,500 or at least 2,000 healthy subjects being seronegative against all serotypes at baseline, wherein said unit dose / tetravalent dengue virus composition or said placebo is administered at least twice within less than 6 months, such as within 3 months, about 30 days after the second administration of the administration schedule until at least 12 months or until 12 to 18 months (e.g. at 12 or at 18months) after the second administration of the administration schedule. In certain such embodiments, the lower bound is more than 30%, is more than 40%, is more than 50%, or is more than 55%.

[0378] In certain embodiments, said methods have a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease with a 2-sided 95% confidence interval, wherein the lower bound is more than 25%, when measured against placebo in a subject population of at least 1,500 or at least 2,000 or at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) being seronegative against all serotypes at baseline and 4 to 16 years of age, wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or placebo is e.g. administered at least twice within less than 6 months, such as within 3 months, from 30 days post last administration until 12 to 18 months (e.g. at 12 months or at 18 months) after the last administration. In certain such embodiments, the lower bound is more than 30%, is more than 35%, is more than 40%, or is more than 45%.

[0379] In certain embodiments, said methods have a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease with a 2-sided 95% confidence interval, wherein the lower bound is more than 25%, when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) being seropositive at baseline and 4 to 16 years of age, wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or placebo is e.g. administered at least twice within less than 6 months, such as within 3 months, from 30 days post last administration until 12 to 18 months (e.g. at 12 months or at 18 months) after the last administration. In certain such embodiments, the lower bound is more than 40%, is more than 45%, is more than 50%, is more than 60%, or is more than 65%.

[0380] In certain embodiments, said methods have a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease with a 2-sided 95% confidence interval, wherein the lower bound is more than 25%, when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) being seropositive at baseline being or seronegative against all serotypes at baseline and 4 to 16 years of age, wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or placebo is e.g. administered at least twice within less than 6 months, such as within 3 months, from 30 days post last administration until 12 to 18 months (e.g. at 12 months or at 18 months) after the last administration. In certain such embodiments, the difference between the lower bound provided by the seropositive subjects at baseline and the subjects seronegative against all serotypes at baseline is no more than 15%-points.

[0381] In certain embodiments, said methods have a combined vaccine efficacy against all four dengue serotypes in seronegative subjects of more than 30%, when measured against placebo in a subject population of at least 1,500 or at least 2,000 healthy subjects being seronegative against all serotypes at baseline, wherein said unit dose / tetravalent dengue virus composition or said placebo is administered at least twice within less than 6 months, such as within 3 months, 30 days after the second administration until at least 12 months or until 12 to 18 months (e.g. at 12 or at 18 months) after the second administration. In certain such embodiments, the combined vaccine efficacy against all four dengue serotypes in seronegative subjects is more than 40%, is more than 50%, is more than 60%, is more than 65%, or is more than 70%.

[0382] In certain embodiments, said methods have a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease, when measured against placebo in a subject population of at least 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) being seronegative against all serotypes at baseline and 4 to 16 years of age, wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or placebo is e.g. administered at least twice within less than 6 months,such as within 3 months, from 30 days post last administration until 12 to 18 months (e.g. at 12 months or at 18 months) after the last administration. In certain such embodiments said vaccine efficacy is more than 30%, is more than 40%, is more than 50%, is more than 55%, is more than 60%, or is more than 65%.

[0383] In certain embodiments, said methods have a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease, when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) being seropositive at baseline and 4 to 16 years of age, wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or placebo is e.g. administered at least twice within less than 6 months, such as within 3 months, from 30 days post last administration until 12 to 18 months (e.g. at 12 months or at 18 months) after the last administration. In certain such embodiments the said vaccine efficacy is more than 40%, is more than 50%, is more than 60%, is more than 65%, is more than 70%, or is more than 75%.

[0384] In certain embodiments, said methods have a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease with a 2-sided 95% confidence interval, wherein the lower bound is more than 25%, when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) being seropositive at baseline being or seronegative against all serotypes at baseline and 4 to 16 years of age, wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or placebo is e.g. administered at least twice within less than 6 months, such as within 3 months, from 30 days post last administration until 12 to 18 months (e.g. at 12 months or at 18 months) after the last administration. In certain such embodiments, the difference between the lower bound provided by the seropositive subjects at baseline and the subjects seronegative against all serotypes at baseline is no more than 15%-points, or is no more than 10%-points.

[0385] In certain embodiments, said methods have a combined vaccine efficacy against all four dengue serotypes with a 2-sided 95% confidence interval, wherein the lower bound is more than 25%, when measured against placebo in a subject population of at least 1,000 healthy subjects 4 to 5 years of age at the time of randomization and irrespective of serostatus at baseline, wherein said unit dose / tetravalent dengue virus composition or said placebo is administered at least twice within less than 6 months, such as within 3 months, about 30 days after the second administration of the administration schedule until at least 12 months or until 12 to 18 months (e.g. at 12 or at 18 months) after the second administration of the administration schedule. In certain such embodiments, the lower bound is more than 30%, is more than 40%, is more than 45%.

[0386] In certain embodiments, said methods have a combined vaccine efficacy against all four dengue serotypes of more than 30%, when measured against placebo in a subject population of at least 1,000 healthy subjects 4 to 5 years of age at the time of randomization and irrespective of serostatus at baseline, wherein said unit dose / tetravalent dengue virus composition or said placebo is administered at least twice within less than 6 months, such as within 3 months, 30 days after the second administration until at least 12 months or until 12 to 18 months (e.g. at 12 or at 18 months) after the second administration. In certain such embodiments, the combined vaccine efficacy against all four dengue serotypes is more than 40%, is more than 50%, is more than 60%, is more than 65%, or is more than 70%.

[0387] In certain embodiments, said methods have a combined vaccine efficacy against all four dengue serotypes with a 2-sided 95% confidence interval, wherein the lower bound is more than 25%, when measured against placebo in a subject population of at least 1,000 healthy subjects 6 to 11 years of age at the time of randomization and irrespective of serostatus at baseline, wherein said unit dose / tetravalent dengue virus composition or said placebo is administered at least twice within less than 6 months, such as within 3 months, about 30 days after the secondadministration of the administration schedule until at least 12 months or until 12 to 18 months (e.g. at 12 or at 18 months) after the second administration of the administration schedule. In certain such embodiments, the lower bound is more than 30%, is more than 40%, is more than 50%, is more than 60%, or is more than 70%.

[0388] In certain embodiments, said methods have a combined vaccine efficacy against all four dengue serotypes of more than 30%, when measured against placebo in a subject population of at least 1,000 healthy subjects 6 to 11 years of age at the time of randomization and irrespective of serostatus at baseline, wherein said unit dose / tetravalent dengue virus composition or said placebo is administered at least twice within less than 6 months, such as within 3 months, 30 days after the second administration until at least 12 months or until 12 to 18 months (e.g. at 12 or at 18 months) after the second administration. In certain such embodiments, the combined vaccine efficacy against all four dengue serotypes is more than 40%, is more than 50%, is more than 60%, is more than 70%, is more than 75%, or is more than 80%.

[0389] In certain embodiments, said methods have a combined vaccine efficacy against all four dengue serotypes with a 2-sided 95% confidence interval, wherein the lower bound is more than 25%, when measured against placebo in a subject population of at least 1,000 healthy subjects 12 to 16 years of age at the time of randomization and irrespective of serostatus at baseline, wherein said unit dose / tetravalent dengue virus composition or said placebo is administered at least twice within less than 6 months, such as within 3 months, about 30 days after the second administration of the administration schedule until at least 12 months or until 12 to 18 months (e.g. at 12 or at 18 months) after the second administration of the administration schedule. In certain such embodiments, the lower bound is more than 30%, is more than 40%, is more than 50%, is more than 60%, is more than 65%, or is more than 68%.

[0390] In certain embodiments, said methods have a combined vaccine efficacy against all four dengue serotypes of more than 30%, when measured against placebo in a subject population of at least 1,000 healthy subjects 12 to 16 years of age at the time of randomization and irrespective of serostatus at baseline, wherein said unit dose / tetravalent dengue virus composition or said placebo is administered at least twice within less than 6 months, such as within 3 months, 30 days after the second administration until at least 12 months or until 12 to 18 months (e.g. at 12 or at 18 months) after the second administration. In certain such embodiments, the combined vaccine efficacy against all four dengue serotypes is more than 40%, is more than 50%, is more than 60%, is more than 70%, is more than 75%, or is more than 80%.

[0391] In certain embodiments, said methods have a vaccine efficacy against dengue serotype 1 with a 2-sided 95% confidence interval, wherein the lower bound is more than 25%, when measured against placebo in a subject population of at least 5,000 healthy subjects, or at least 10,000 healthy subjects, or at least 15,000 healthy subjects irrespective of serostatus at baseline, wherein said unit dose / tetravalent dengue virus composition or said placebo is administered at least twice within less than 6 months, such as within 3 months, about 30 days after the second administration of the administration schedule until at least 12 months or until 12 to 18 months (e.g. at 12 or at 18 months) after the second administration of the administration schedule. In certain such embodiments, the lower bound is more than 30%, is more than 40%, or is more than 50%.

[0392] In certain embodiments, said methods have a vaccine efficacy against serotype 1, in preventing virologically confirmable dengue disease with a 2-sided 95% confidence interval, wherein the lower bound is more than 25%, when measured against placebo in a subject population of at least 1,500, or at least 2,000, or at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) irrespective of serostatus at baseline and 4 to 16 years of age, wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or placebo is e.g. administered at least twice within less than 6 months, such as within 3 months, from 30 days post last administrationuntil 12 to 18 months (e.g. at 12 or at 18 months) after the last administration. In certain such embodiments, the lower bound is more than 30%, is more than 35% is more than 40%, is more than 45%, is more than 50%, or is more than 54%. In certain such embodiments the subject population of at least 1,500 is seronegative against all serotypes at base line and the lower bound is more than 35%. In certain such embodiments the seronegative and seropositive population each provide a vaccine efficacy against serotype 1 with a 2-sided 95% confidence interval, wherein the lower bounds are within 10 %-points.

[0393] In certain embodiments, said methods have a vaccine efficacy against dengue serotype 1 of more than 30%, when measured against placebo in a subject population of at least 5,000 healthy subjects, or at least 10,000 healthy subjects, or at least 15,000 healthy subjects irrespective of serostatus at baseline, wherein said unit dose / tetravalent dengue virus composition or said placebo is administered at least twice within less than 6 months, such as within 3 months, 30 days after the second administration until at least 12 months or until 12 to 18 months (e.g. at 12 or at 18 months) after the second administration. In certain such embodiments, the vaccine efficacy against dengue serotype 1 is more than 40%, is more than 50%, is more than 60%, is more than 65%, or is more than 70%.

[0394] In certain embodiments, said methods have a vaccine efficacy against serotype 1, in preventing virolog ically confirmable dengue disease, when measured against placebo in a subject population of at least 1,500, or at least 2,000, or at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) irrespective of serostatus at baseline and 4 to 16 years of age, wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or placebo is e.g. administered at least twice within less than 6 months, such as within 3 months, from 30 days post last administration until 12 to 18 months (e.g. at 12 or at 18 months) after the last administration. In certain such embodiments, the vaccine efficacy is more than 40%, is more than 50%, is more than 60%, or is more than 65%. In certain such embodiments the subject population of at least 1,500 is seronegative against all serotypes at base line. In certain such embodiments the seronegative and seropositive population each provide a vaccine efficacy against serotype 1 which are within 5 %-points.

[0395] In certain embodiments, said methods have a vaccine efficacy against dengue serotype 2 with a 2-sided 95% confidence interval, wherein the lower bound is more than 25%, when measured against placebo in a subject population of at least 5,000 healthy subjects, or at least 10,000 healthy subjects, or at least 15,000 healthy subjects irrespective of serostatus at baseline, wherein said unit dose / tetravalent dengue virus composition or said placebo is administered at least twice within less than 6 months, such as within 3 months, about 30 days after the second administration of the administration schedule until at least 12 months or until 12 to 18 months (e.g. at 12 or at 18 months) after the second administration of the administration schedule. In certain such embodiments, the lower bound is more than 30%, is more than 40%, is more than 50, is more than 60, is more than 70, is more than 80, or is more than 90%.

[0396] In certain embodiments, said methods have a vaccine efficacy against serotype 2, in preventing virolog ically confirmable dengue disease with a 2-sided 95% confidence interval, wherein the lower bound is more than 25%, when measured against placebo in a subject population of at least 1,500, or at least 2,000, or at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) irrespective of serostatus at baseline and 4 to 16 years of age, wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or placebo is e.g. administered at least twice within less than 6 months, such as within 3 months, from 30 days post last administration until 12 to 18 months (e.g. at 12 or at 18 months) after the last administration. In certain such embodiments, the lower bound is more than 50%, is more than 60%, is more than 70%, is more than 80%, or is more than 85%. In certain such embodiments the subject population of at least 1,500, is seronegative against all serotypes. In certainsuch embodiments the seronegative and seropositive population each provide a vaccine efficacy against serotype 2 with a 2-sided 95% confidence interval, wherein the lower bounds are within 5 %-points.

[0397] In certain embodiments, said methods have a vaccine efficacy against dengue serotype 2 of more than 30%, when measured against placebo in a subject population of at least 5,000 healthy subjects, or at least 10,000 healthy subjects, or at least 15,000 healthy subjects irrespective of serostatus at baseline, wherein said unit dose / tetravalent dengue virus composition or said placebo is administered at least twice within less than 6 months, such as within 3 months, 30 days after the second administration until at least 12 months or until 12 to 18 months (e.g. at 12 or at 18 months) after the second administration. In certain such embodiments, the vaccine efficacy against dengue serotype 2 is more than 40%, is more than 50%, is more than 60%, is more than 70%, is more than 80, or is more than 90%.

[0398] In certain embodiments, said methods have a vaccine efficacy against serotype 2, in preventing virolog ically confirmable dengue disease, when measured against placebo in a subject population of at least 1,500, or at least 2,000, or at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) irrespective of serostatus at baseline and 4 to 16 years of age, wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or placebo is e.g. administered at least twice within less than 6 months, such as within 3 months, from 30 days post last administration until 12 to 18 months (e.g. at 12 or at 18 months) after the last administration. In certain such embodiments, the vaccine efficacy is more than 60%, is more than 70%, is more than 80%, or is more than 90%. In certain such embodiments the subject population of at least 1,500 is seronegative against all serotypes at base line. In certain such embodiments the seronegative and seropositive population each provide a vaccine efficacy against serotype 2 which are within 5 %-points.

[0399] In certain embodiments, said methods have a vaccine efficacy against dengue serotype 3 with a 2-sided 95% confidence interval, wherein the lower bound is more than 25%, when measured against placebo in a subject population of at least 5,000 healthy subjects, or at least 10,000 healthy subjects, or at least 15,000 healthy subjects irrespective of serostatus at baseline, wherein said unit dose / tetravalent dengue virus composition or said placebo is administered at least twice within less than 6 months, such as within 3 months, about 30 days after the second administration of the administration schedule until at least 12 months after the second administration of the administration schedule. In certain such embodiments, the lower bound is more than 30%, is more than 40%.

[0400] In certain embodiments, said methods have a vaccine efficacy against dengue serotype 3 of more than 30%, when measured against placebo in a subject population of at least 5,000 healthy subjects, or at least 10,000 healthy subjects, or at least 15,000 healthy subjects irrespective of serostatus at baseline, wherein said unit dose / tetravalent dengue virus composition or said placebo is administered at least twice within less than 6 months, such as within 3 months, 30 days after the second administration until at least 12 months after the second administration. In certain such embodiments, the vaccine efficacy against dengue serotype 3 is more than 40%, is more than 50%, is more than 55%, or is more than 60%.

[0401] In certain embodiments, said methods have a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease with hospitalization with a 2-sided 95% confidence interval, wherein the lower bound is more than 25%, when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) irrespective of serostatus at baseline and 4 to 16 years of age, wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or placebo is e.g. administered at least twice within less than 6 months, such as within 3 months, from first administration until 12 to 18 months (e.g. at 12 months or at 18 months) after the last administration, or from 30 days post lastadministration until 12 to 18 months (e.g. at 12 or at 18 months) after the last administration. In certain such embodiments, the lower bound is more than 10%, is more than 20%, is more than 30%, is more than 40%, is more than 50%, is more than 55%, is more than 60%, is more than 65%, is more than 66%, is more than 67%, is more than 70%, is more than 75%, is more than 77%, or is more than 80%.

[0402] In certain embodiments, said methods have a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease with hospitalization, when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) irrespective of serostatus at baseline and 4 to 16 years of age, wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or placebo is e.g. administered at least twice within less than 6 months, such as within 3 months, from first administration until 12 to 18 months (e.g. at 12 months or at 18 months) after the last administration, or from 30 days post last administration until 12 to 18 months (e.g. at 12 or at 18 months) after the last administration. In certain such embodiments, the vaccine efficacy is more than is more than 70%, is more than 75%, is more than 80%, or is more than 82%, or is more than 85%, more than 88%.

[0403] In certain embodiments, said methods have a combined vaccine efficacy against virologically-confirmed dengue with hospitalization against all four serotypes with a 2-sided 95% confidence interval, wherein the lower bound is more than 25%, when measured against placebo in a subject population of at least 1,500 or at least 2,000 healthy subjects being seronegative against all serotypes at baseline, wherein said unit dose / tetravalent dengue virus composition or said placebo is administered at least twice within less than 6 months, such as within 3 months, about 30 days after the second administration of the administration schedule until at least 12 months or until 12 to 18 months (e.g. at 12 or at 18 months) after the second administration of the administration schedule. In certain such embodiments, the lower bound is more than 30%, is more than 40%, is more than 50%, is more than 60%, is more than 70%, or is more than 75%.

[0404] In certain embodiments, said methods have a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease with hospitalization with a 2-sided 95% confidence interval, wherein the lower bound is more than 25%, when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) being seronegative against all serotypes at baseline and 4 to 16 years of age, wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or placebo is e.g. administered at least twice within less than 6 months, such as within 3 months, from 30 days post last administration until 12 to 18 months (e.g. at 12 months or at 18 months) after the last administration. In certain such embodiments, the lower bound is more than 60%, is more than 65%, is more than 66%, is more than 67%, is more than 70%, is more than 75%, is more than 77% or is more than 80%.

[0405] In certain embodiments, said methods have a combined vaccine efficacy against virologically-confirmed dengue with hospitalization against all four serotypes of more than 30%, when measured against placebo in a subject population of at least 1,500 or at least 2,000 healthy subjects, healthy subjects being seronegative against all serotypes at baseline, wherein said unit dose / tetravalent dengue virus composition or said placebo is administered at least twice within less than 6 months, such as within 3 months, 30 days after the second administration until at least 12 months or until 12 to 18 months (e.g. at 12 or at 18 months) after the second administration. In certain such embodiments, the combined vaccine efficacy against virologically confirmed dengue with hospitalization against all four serotypes is more than 40%, is more than 50%, is more than 60%, is more than 70%, is more than 80%, or is more than 90%.

[0406] In certain embodiments, said methods have a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease with hospitalization, when measured against placebo in a subjectpopulation of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) being seronegative against all serotypes at baseline and 4 to 16 years of age, wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or placebo is e.g. administered at least twice within less than 6 months, such as within 3 months, from 30 days post last administration until 12 to 18 months (e.g. at 12 months or at 18 months) after the last administration. In certain such embodiments, the said vaccine efficacy is more than 60%, is more than 65%, is more than 66%, is more than 67%, is more than 70%, is more than 75%, is more than 77%, is more than 80, or is more than 85%.

[0407] In certain embodiments, said methods have a combined vaccine efficacy against virologically-confirmed dengue with hospitalization against all four serotypes with a 2-sided 95% confidence interval, wherein the lower bound is more than 25%, when measured against placebo in a subject population of at least 1,500 or at least 2,000 healthy subjects being seropositive at baseline, wherein said unit dose / tetravalent dengue virus composition or said placebo is administered at least twice within less than 6 months, such as within 3 months, about 30 days after the second administration of the administration schedule until at least 12 months or until 12 to 18 months (e.g. at 12 or at 18 months) after the second administration of the administration schedule. In certain such embodiments, the lower bound is more than 30%, is more than 40%, is more than 50%, is more than 60%, is more than 70%, or is more than 80%.

[0408] In certain embodiments, said methods have a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease with hospitalization with a 2-sided 95% confidence interval, wherein the lower bound is more than 25%, when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) being seropositive at baseline and 4 to 16 years of age, wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or placebo is e.g. administered at least twice within less than 6 months, such as within 3 months, from 30 days post last administration until 12 to 18 months (e.g. at 12 months or at 18 months) after the last administration. In certain such embodiments, the lower bound is more than 60%, is more than 65%, is more than 70%, is more than 75%, or is more than 80%.

[0409] In certain embodiments, said methods have a combined vaccine efficacy against virologically-confirmed dengue with hospitalization against all four serotypes of more than 30%, when measured against placebo in a subject population at least 1,500 or of at least 2,000 healthy subjects, healthy subjects being seropositive at baseline, wherein said unit dose / tetravalent dengue virus composition or said placebo is administered at least twice within less than 6 months, such as within 3 months, 30 days after the second administration until at least 12 months or until 12 to 18 months (e.g. at 12 or at 18 months) after the second administration. In certain such embodiments, the combined vaccine efficacy against virologically confirmed dengue with hospitalization against all four serotypes is more than 40%, is more than 50%, is more than 60%, is more than 70%, is more than 80%, or is more than 90%.

[0410] In certain embodiments, said methods have a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease with hospitalization, when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) being seropositive at baseline and 4 to 16 years of age, wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or placebo is e.g. administered at least twice within less than 6 months, such as within 3 months, from 30 days post last administration until 12 to 18 months (e.g. at 12 months or at 18 months) after the last administration. In certain such embodiments, the vaccine efficacy is more than 75%, is more than 70%, is more than 80%, is more than 85%, or is more than 90%.

[0411] In certain embodiments, said methods have a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease with hospitalization with a 2-sided 95% confidence interval, wherein the lower bound is more than 25%, when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) being seropositive at baseline being or seronegative against all serotypes at baseline and 4 to 16 years of age, wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or placebo is e.g. administered at least twice within less than 6 months, such as within 3 months, from 30 days post last administration until 12 to 18 months (e.g. at 12 months or at 18 months) after the last administration. In certain such embodiments, the difference between the lower bound provided by the seropositive subjects at baseline and the subjects seronegative against all serotypes at baseline is no more than 15%-points.

[0412] In certain embodiments, said methods have a combined vaccine efficacy against all four serotypes, in preventing virologically confirmable dengue disease with hospitalization, when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) being seropositive at baseline being or seronegative against all serotypes at baseline and 4 to 16 years of age, wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or placebo is e.g. administered at least twice within less than 6 months, such as within 3 months, from 30 days post last administration until 12 to 18 months (e.g. at 12 months or at 18 months) after the last administration. In certain such embodiments, the difference between the vaccine efficacy provided by the seropositive subjects at baseline and the subjects seronegative against all serotypes at baseline is no more than 10%-points or no more than 5%-points.

[0413] In certain embodiments, said methods have a relative risk, e.g. a combined relative risk against all four serotypes, with a 2-sided 95% confidence interval, wherein the upper bound is less than 0.75, when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) irrespective of serostatus at baseline, wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or placebo is administered at least twice within less than 6 months, such as within 3 months, 30 days after the second administration until at least 12 months after the second administration. In certain such embodiments, the upper bound is less than 0.70, less than 0.65, less than 0.60, less than 0.55, less than 0.50, less than 0.45, less than 0.40, less than 0.35, less than 0.30 or less than 0.28. For example, said reconstituted unit dose or placebo is administered subcutaneously within about 3 months, such as on days 0 and 90.

[0414] In certain embodiments, said methods have a relative risk, e.g. a combined relative risk against all four serotypes, of less than 0.70, when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) irrespective of serostatus at baseline, wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or placebo is administered at least twice within less than 6 months, such as within 3 months, 30 days after the second administration until at least 12 months after the second 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. For example, said reconstituted unit dose or placebo is administered subcutaneously within about 3 months, such as on days 0 and 90.

[0415] In certain embodiments of said methods, virologically confirmable dengue disease occurs in less than 2.5% of the subjects, when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) irrespective of serostatus at baseline, wherein a reconstituted unit dose / tetravalent dengue virus composition as described herein or placebo is administered at least twice within less than 6 months, such as within 3 months, 30 days after the second administration until at least 12 months or at least18 months after the second administration. In certain such embodiments, virologically confirmable dengue disease occurs in less than 2.0% of the subjects, less than 1.5% of the subjects, less than 1.0% of the subjects, less than 0.8% of the subjects, or less than 0.6% of the subjects. For example, said reconstituted unit dose or placebo is administered subcutaneously within about 3 months, such as on days 0 and 90.

[0416] In certain embodiments, said methods have a combined vaccine efficacy against all four serotypes with a 2-sided 95% confidence interval, wherein the lower bound is more than 61.0%, or more than 65.0 or more than 70.0% or more than 72.0% when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) from endemic irrespective of serostatus at baseline and being selected from the group consisting of 4 to 16 year old subjects at the time of randomization, wherein said unit dose / tetravalent dengue virus composition or said placebo is administered at least twice within 6 months or less, about 30 days after the last administration of the administration schedule until at least 12 or 13 months after the last administration of the administration schedule.

[0417] In certain embodiments, said methods have a combined vaccine efficacy against all four serotypes of more than 66 %, or of more than 70%, or of more than 75%, or of more than 77%, or of more than 80.0%, when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects) from endemic areas irrespective of serostatus at baseline and being selected from the group consisting of 4 to 16 year old subjects at the time of randomization, wherein said unit dose / tetravalent dengue virus composition or said placebo is administered at least twice within 6 months or less, about 30 days after the last administration of the administration schedule until at least 12 months or 13 month after the last administration of the administration schedule.

[0418] In certain embodiments of said methods, the combined vaccine efficacy against all four serotypes is measured about 30 days after the last administration of the administration schedule until 12 or 13 months after the last administration of the administration schedule.

[0419] In certain embodiments of said methods, said unit dose or said placebo is administered at least twice within three months, in particular at about day 1 and about day 90, and the combined vaccine efficacy against all four serotypes is measured 30 days after the second administration until 12 or 13 months after the second administration of the administration schedule.

[0420] In certain embodiments, said methods are effective and safe. In some of these embodiments, the subject or subject population is under 9 years of age, under 4 years of age, or under 2 years of age or from 2 to 9 years of age, or from 2 to 5 years of age, or from 4 to 9 years of age or from 6 to 9 years of age. Optionally the subject is seronegative with respect to all dengue serotypes.

[0421] In certain embodiments, said methods have a relative risk for virologically confirmed dengue with hospitalization of 1 or less, or 0.8 or less, or 0.6 or less, when measured against placebo in a subject population of at least 5,000 healthy subjects (or at least 10,000, or at least 15,000 healthy subjects). In some of these embodiments, the subject or subject population is under 9 years of age, under 4 years of age, or under 2 years of age or from 2 to 9 years of age, or from 2 to 5 years of age, or from 4 to 9 years of age or from 6 to 9 years of age. Optionally the subject is seronegative with respect to all dengue serotypes.

[0422] In certain embodiments of said methods, the healthy subjects of the subject population are 4 to 16 years of age. In some of such embodiments, the healthy subjects of the subject population are 4 to 5 years of age, 6 to 11 years of age, or 12 to 16 years of age.

[0423] In certain embodiments, the invention is directed to said methods, wherein the healthy subjects of the subject population are defined as being healthy in view of the exclusion criteria specified in Example 3.

[0424] In certain embodiments of said methods, the healthy subjects of the subject population are from Asia Pacific or Latin America.

[0425] In certain embodiments of said methods, the healthy subjects of the subject population are seropositive with respect to at least one serotype. In other embodiments, the healthy subjects of the subject population are seronegative with respect to all serotypes.

[0426] In certain embodiments of said methods, the healthy subjects of the subject population are 4-5 years of age and from Asia Pacific, 6-11 years of age and from Asia Pacific, or 12-16 years of age and from Asia Pacific. In other embodiments, the healthy subjects of the subject population are 4-5 years of age and from Latin America, 6-11 years of age and from Latin America, or 12-16 years of age and from Latin America.

[0427] In certain embodiments of said methods, the healthy subjects of the subject population are 4-5 years of age and seropositive for at least 1 dengue serotype, 6-11 years of age and seropositive for at least 1 dengue serotype, or 12-16 years of age and seropositive for at least 1 dengue serotype. In other embodiments, the healthy subjects of the subject population are 4-5 years of age and seronegative for all dengue serotypes, 6-11 years of age and seronegative for all dengue serotypes, or 12-16 years of age and seronegative for all dengue serotypes.

[0428] In certain embodiments of said methods, the healthy subjects of the subject population are from Asia Pacific or Latin America and seropositive for at least one dengue serotype at baseline. In other embodiments, the healthy subjects of the subject population are from Asia Pacific or Latin America and seronegative for at all dengue serotype at baseline.

[0429] In certain embodiments of said methods, the healthy subjects of the subject population are from Asia Pacific, seropositive for at least one dengue serotype at baseline and 4-5 years of age, 6-11 years of age, or 12-16 years of age. In other embodiments, the healthy subjects of the subject population are from Asia Pacific, seronegative for all dengue serotypes at baseline and 4-5 years of age, 6-11 years of age, or 12-16 years of age. In yet other embodiments, the healthy subjects of the subject population are from Latin America, seropositive for at least one dengue serotype at baseline and 4-5 years of age, 6-11 years of age, or 12-16 years of age. In other embodiments, the healthy subjects of the subject population are from America, seronegative for all dengue serotypes at baseline and 4-5 years of age, 6-11 years of age, or 12-16 years of age.

[0430] In certain embodiments of said methods, the healthy subjects of the subject population had prior vaccination against Yellow Fever. In other embodiments, the healthy subjects of the subject population had no prior vaccination against Yellow Fever. Prior vaccination indicates a vaccination prior to the first vaccination with the reconstituted unit dose as described herein.

[0431] In certain embodiments of said methods, the healthy subjects of the subject population had prior vaccination against Japanese Encephalitis. In other embodiments, the healthy subjects of the subject population had no prior vaccination against Japanese Encephalitis.

[0432] In certain embodiments of said methods, the healthy subjects of the subject population received Dengvaxia® within the administration regimen as described herein or within 4.5 years after administration of the first dose. In certain embodiments of said methods, the occurrence of vaccine related serious adverse events is less than 0.1%.

[0433] In certain embodiments of said methods, the occurrence of vaccine related unsolicited adverse events occurring within 4 weeks of administration is less than 2%.

[0434] In certain embodiments of said methods, the occurrence of vaccine related solicited adverse events occurring within 2 weeks of administration is less than 35%.

[0435] In certain embodiments of said methods, the occurrence of vaccine related solicited local reactions occurring within 1 weeks of administration is less than 40%.

[0436] In certain embodiments, the method does not increase the risk of virologically confirmed dengue with hospitalization in the individual, such as in a seronegative individual.

[0437] The above methods are also to be considered in the context of a unit dose for use in such methods or in the context of a use of such a unit dose for use in the manufacture of a medicament for such methods.

[0438] In certain embodiments, a tetravalent dengue vaccine such as Dengvaxia® is used for inoculating against dengue disease. Dengvaxia® is a tetravalent dengue vaccine based on a yellow fever backbone, CYD-TDV (Dengvaxia®, Sanofi Pasteur, Lyon, France), and has been licensed in several countries based on the clinical demonstration of an overall vaccine efficacy (VE) against virologically-confirmed dengue (VCD) of 56-61% in children in Asia and Latin America (Capeding MR et al. Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in Asia: a phase 3, randomised, observer-masked, placebo-controlled trial. Lancet 2014, 384:1358-65; Villar LA et al. Safety and immunogenicity of a recombinant tetravalent dengue vaccine in 9-16 year olds: a randomized, controlled, phase II trial in Latin America. Pediatr Infect Dis J 2013, 32:1102-9). The preparation of these particular strains CYD1, CYD2, CYD3 and CYD4 has been described in detail in international patent applications WO 98 / 37911, WO 03 / 101397, W007 / 021672, WO 08 / 007021, WO 08 / 047023 and WO 08 / 065315, to which reference may be made for a precise description of the processes for their preparation. The corresponding nucleotide sequences of the prM-E regions of CYD1, CYD2, CYD3 and CYD4 are provided in WO2016034629 and SEQID NOs are set out in Table 16 of this reference.

[0439] In certain embodiments of said methods, the unit dose disclosed herein which may comprise a chimeric dengue serotype 2 / 1 strain, a live attenuated dengue serotype 2 strain, a chimeric dengue serotype 2 / 3 strain and a chimeric dengue serotype 2 / 4 strain, and Dengvaxia® disclosed herein are administered simultaneously on the same day to the subject or to the subject population.

[0440] In certain embodiments of said methods, the unit dose disclosed herein, which may comprise a chimeric dengue serotype 2 / 1 strain, a live attenuated dengue serotype 2 strain, a chimeric dengue serotype 2 / 3 strain and a chimeric dengue serotype 2 / 4 strain, is administered to the subject or to the subject population on day 0 / 1 as a first administration and Dengvaxia® disclosed herein is subsequently administered to the subject or to the subject population within three months from the first administration, such as on day 90 from the first administration, as a second administration. Alternatively, Dengvaxia® disclosed herein is administered to the subject or to the subject population on day 0 / 1 as a first administration and the unit dose disclosed herein, which in particular comprises a chimeric dengue serotype 2 / 1 strain, a live attenuated dengue serotype 2 strain, a chimeric dengue serotype 2 / 3 strainand a chimeric dengue serotype 2 / 4 strain, is administered subsequently to the subject or the subject population within three months from the first administration, such as on day 90 from the first administration, as a second administration.

[0441] In certain embodiments of said methods, the dengue vaccine composition comprises other dengue vaccines such as TV003 or TV005. TV003, developed by the U. S. National Institute of Allergy and Infectious Diseases, comprises vaccine components rDENlA30, rDEN2 / 4A30, rDEN3A30 / 31 and rDEN4A30, wherein each of these components is present at a concentration of 3 logioPFU. TV005 is similar to TV003 with the difference that the concentration of rDEN2 / 4A30 in TV005 is 4 logioPFU. The vaccines TV003 and TV005 and their vaccine components as well as their production are described in more detail in WO 2008 / 022196 A2 and S. S. Whitehead, Expert Rev Vaccines, 2016, 15(4): 509 to 517. Using recombinant DNA technology, two attenuation strategies were utilized for the vaccine components of TV003 or TV005: deletions in the 3' untranslated region and structural gene chimerization. For example, the component rDEN4A30 contains all the structural and non -structural proteins of a wild type DENV-4, but is attenuated by a 30-nucleotide deletion in the 3' untranslated region (denoted " A30"). The other vaccine components are also attenuated due to the 30-nucleotide deletion in the 3' untranslated region. In addition, rDEN3A30 / 31 includes a 31 nucleotide deletion in the 3' untranslated region (shown in detail in Fig. lc and Fig. 13 of WO 2008 / 022196 A2). The rDEN2 / 4A30 component was created by substituting the prM and E genes of DENV-2 into the rDEN4A30 genome. The complete genomic sequences of dengue strains which can be used to produce TV003 or TV005 are available under the Genbank accession numbers in Table A of WO 2008 / 022196 Al.

[0442] In certain embodiments of said methods, the unit dose disclosed herein, which may comprise a chimeric dengue serotype 2 / 1 strain, a live attenuated dengue serotype 2 strain, a chimeric dengue serotype 2 / 3 strain and a chimeric dengue serotype 2 / 4 strain, and TV003 or TV005 disclosed herein are administered simultaneously on the same day to the subject or to the subject population.

[0443] In certain embodiments of said methods, the unit dose disclosed herein, which may comprise a chimeric dengue serotype 2 / 1 strain, a live attenuated dengue serotype 2 strain, a chimeric dengue serotype 2 / 3 strain and a chimeric dengue serotype 2 / 4 strain, is administered to the subject or the subject population on day 0 / 1 as a first administration and wherein TV003 or TV005 disclosed herein is subsequently administered to the subject or to the subject population within three months from the first administration, such as on day 90 from the first administration, as a second administration. Alternatively, TV003 or TV005 disclosed herein is administered to the subject or to subject population on day 0 / 1 as a first administration and the unit dose disclosed herein, which in particular comprises a chimeric dengue serotype 2 / 1 strain, a live attenuated dengue serotype 2 strain, a chimeric dengue serotype 2 / 3 strain and a chimeric dengue serotype 2 / 4 strain, is administered subsequently to the subject or to the subject population within three months from the first administration, such as on day 90 from the first administration, as a second administration.EXAMPLES

[0444] The following Examples are included to demonstrate certain aspects and embodiments of the invention as described in the claims. It should be appreciated by those of skill in the art, however, that the following description is illustrative only and should not be taken in any way as a restriction of the invention.Example 1: Preparation of the dengue virus strains.

[0445] Methods used to generate chimeric dengue strains TDV-1, -3 and -4 were standard molecular cloning andDNA engineering methods and are e.g. described in Huang etal. (2003) J. Virology 77(21): 11436-11447. The following well-known methods were used to construct and introduce the prM-E genes of dengue serotypes 1, 3 and 4 into the TDV-2 backbone: Reverse-transcriptase PCR (RT-PCR), PCR, restriction enzyme digestion, DNA fragment ligation, bacterial transformations by electroporation, plasmid DNA preparations, in vitro transcription by T7 RNA polymerase, and transfection of Vero cells by electroporation.

[0446] The different dengue serotypes are mixed in certain concentrations provided in Example 4. The mixture of dengue serotypes is present in a dengue vaccine composition and combined with a composition of pharmaceutically acceptable excipients resulting in a dengue vaccine composition comprising 15% w / v a, a trehalose dihydrate, 1% w / v poloxamer 407, 0.1% w / v human serum albumin and 100 mM sodium chloride. The dengue vaccine composition is lyophilized and 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 comprises 15% w / v a, a trehalose dihydrate, 1% w / v poloxamer 407, 0.1% w / v human serum albumin and 137 mM sodium chloride.Example 2: Microneutralization test

[0447] Immunogenicity was measured by a microneutralization assay to each one of the four dengue serotypes with titers defined as the dilution resulting in a 50% reduction in plaque values (MNT50). Briefly, on day 1 Vero cells were seeded on 96-well assay plates in DMEM and 10% FBS at a density of 2.5 x 105cells / ml and incubated at 37°C for 24 hours. On day 2 serial dilutions of the heat-inactivated antibody-containing test and control sera samples (dilutions range 1:10 to 1:20480) were prepared and mixed with a constant concentration of dengue viruses, in particular DENV-1 strain 16007, DENV-2 strain 16681, DENV-3 strain 16562 and DENV-4 strain 1036, (target 60-80 pfu / well) in a 96 well microtiter plate and incubated overnight at 2-8°C to enable the neutralization of the virus by the antibodies present in the sera. After the incubation the mixture of virus and antibodies was transferred onto the 96 well plates with Vero cells and the plates were incubated at 37 °C for 90-120 minutes to infect the Vero cells. A 1% methylcellulose overlay in DMEM was applied to the plate to restrict spread of progeny virus and the plate was incubated for 46-70 hours at 34°C depending on the Dengue serotype:DENV1 - 66±2 hoursDENV2 - 70±2 hoursDENV3 - 66±2 hoursDENV4 - 46±2 hours

[0448] After the incubation the cells were washed twice with PBS and fixed by adding cold methanol and incubating for 60 minutes at a temperature of < -20°C. After fixing the plates were dried and washed three times with washing buffer (lx PBS, pH 7.4 with 0.5% Tween), before 50 pl of serotype-specific anti-dengue monoclonal antibodies in blocking solution (2.5% nonfat dry milk in PBST) per well were added and incubated with the cells for 18±4 hours at 2-8°C.

[0449] The monoclonal antibodies were made as described in Gentry et al. (1982) Am. 1 Trop. Med. Hyg. 31, 548-555; Henchai et al. (1985) Am. J. Trop. Med. Hyg. 34, 162-169; and Henchai et al. (1982) Am. J. Trop. Med. Hyg.31(4):830-6). Briefly, the anti-DENV-1 HBD was made against dengue 1 strain Hawaii, Envelope, the anti-DENV-2 was made against dengue 2 strain New Guinea C, Envelope, isotype 1, the anti-DENV-3 HBD was made against dengue 3 strain H87, Envelope, isotype 2A, and the anti-DENV-4 HBD was made against dengue 4 strain H241, Envelope, isotype 2A.

[0450] After incubation, the plates were washed three times with washing buffer and 50 pl of a secondary peroxidase labelled goat anti-mouse IgG (H + L) (KPL Cat#074-1806) in blocking solution was added and incubated for 90 to 120 minutes at 37°C. Then the plates were washed three times with washing buffer and 50 pl of precipitant substrate (2-amino-9-ethyl carbazole (AEC) tablet in 2.5 ml DMSO, 47.5 ml 50mM acetate buffer and 250 pl hydrogen peroxide) were added and the mixture was incubated for 20 minutes at room temperature. Finally, the substrate was removed, the plates were rinsed with dH2O and dried.

[0451] Sample titers are calculated using the linear regression method and reported as MNT50 titers for each sample. Clinical data are reported as a geometric mean titer for all the individual MNT50 titers in each treatment group. Briefly, the number of infectious foci in each well was counted and the titer of neutralizing antibodies was determined by comparing the percent reduction of infectious foci centers in wells containing antibody (test samples) in comparison to wells containing virus alone. The MNT50 was calculated using the following linear regression equation:MNT50 = 10A[(50-c) / m]) where c = y intercept of regression line and m = slope of regression line

[0452] Each test sample was tested in triplicates and the titer was calculated from the average of the triplicates. A schematic drawing of the steps performed in this test is provided in Figure 2.Example 3: Phase III clinical trial in children

[0453] A Phase III, double-blind, randomized, and placebo-controlled trial in 20100 subjects aged 4 to 16 years living in Thailand, Sri Lanka, Philippines, Panama, Nicaragua, Dominican Republic, Colombia or Brazil was performed evaluating the efficacy, safety and immunogenicity of a tetravalent dengue vaccine referred to hereinafter as TDV (TDV-1, TDV-2, TDV-3 and TDV-4 as described herein). The trial includes 3 parts. Part 1 evaluates vaccine efficacy (VE) and lasts until both of the following 2 criteria are fulfilled: (i) 120 cases of dengue fever are confirmed and (ii) minimum duration of subject follow-up of 12 months post-second vaccination. Part 2 is for an additional 6 months to evaluate VE and for secondary efficacy analyses. Part 3 will evaluate long-term safety by following participants for side effects and will last an additional 3 years.

[0454] Part 1: Active surveillance for the primary assessment of efficacy in all subjects. During this time subjects were contacted at least weekly to ensure identification of febrile illness that could potentially be due to dengue. This part commenced on the day of vaccination and finished once both of the following 2 criteria were fulfilled: (i) 120 cases of dengue fever are confirmed and (ii) minimum duration of subject follow-up of 12 months post-second vaccination. The end of Part 1 was defined for each subject so that the duration of follow up after the second vaccination was approximately the same for all subjects. Virologically confirmed cases in Part 1 count towards the primary efficacy objective if occurring at least 30 days post-second vaccination. Part 1 was finished 12 months post-second vaccination.

[0455] Part 2: Active surveillance for an additional 6 months for each subject following the completion of Part 1, I, i.e. 18 month post second vaccination. During this time subjects were contacted at least weekly to ensure identification of febrile illness that could potentially be due to dengue. Virologically confirmed cases in Parts 1 and 2 contribute towards the secondary efficacy objectives.

[0456] Part 3: Modified active surveillance for the assessment of safety in all subjects following the completion of Part 2 and lasting 3 years for each subject. The modified surveillance during Part 3 will maintain at least weekly contacts through Part 3 of the trial, but the intensity of investigation will be modified based on the need for hospitalization. Surveillance will identify febrile illness of any severity that could potentially be due to dengue.

[0457] Criteria for Inclusion include:• The subject was aged 4 to 16 years inclusive, at the time of randomization.• Individuals who were in good health at the time of entry into the trial as determined by medical history, physical examination (including vital signs) and clinical judgment of the Investigator.• The subject and / or the subject's parent / guardian signed and dated an assent / written informed consent form where applicable, and any required privacy authorization prior to the initiation of any trial procedures, after the nature of the trial has been explained according to local regulatory requirements.• Individuals who can comply with trial procedures and are available for the duration of follow-up.

[0458] Exclusion criteria include:1. Febrile illness (temperature >38°C) or moderate or severe acute illness or infection at the time of randomization.2. History or any illness that, in the opinion of the Investigator, might interfere with the results of the trial or pose an additional risk to the subject due to participation in the trial, including but not limited to:a. Known hypersensitivity or allergy to any of the vaccine components.b. Female subjects (post-menarche) who are pregnant or breastfeeding.c. Individuals with any serious chronic or progressive disease according to judgment of the Investigator (e.g., neoplasm, insulin-dependent diabetes, cardiac, renal or hepatic disease, neurologic or seizure disorder or Guillain-Barre syndrome).d. Known or suspected impairment / alteration of immune function, including:i. Chronic use of oral steroids (equivalent to 20 mg / day prednisone >12 weeks / >2 mg / kg body weight / day prednisone >2 weeks) within 60 days prior to Day 1 (Month 0) (use of inhaled, intranasal, or topical corticosteroids is allowed).ii. Receipt of parenteral steroids (equivalent to 20 mg / day prednisone > 12 weeks / >2 mg / kg body weight / day prednisone >2 weeks) within 60 days prior to Day 1 (Month 0).Hi. Administration of immunoglobulins and / or any blood products within the 3 months prior to Day 1 (Month 0) or planned administration during the trial.iv. Receipt of immunostimulants within 60 days prior to Day 1 (Month 0).v. Immunosuppressive therapy such as anti-cancer chemotherapy or radiation therapy within 6 months prior to Day 1 (Month 0).vi. Human Immunodeficiency Virus (HIV) infection or HIV-related disease. vii. Genetic immunodeficiency.3. Receipt of any other vaccine within 14 days (for inactivated vaccines) or 28 days (for live vaccines) prior to Day 1 (Month 0) or planning to receive any vaccine within 28 days after Day 1 (Month 0).4. Participation in any clinical trial with another investigational product 30 days prior to Day 1 (Month 0) or intent to participate in another clinical trial at any time during the conduct of this trial.5. Previous participation in any clinical trial of a dengue candidate vaccine, or previous receipt of a dengue vaccine.6. First degree relatives of individuals involved in trial conduct.7. Females of childbearing potential who are sexually active, and who have not used any of the acceptable contraceptive method for at least 2 months prior to Day 1 (Month 0).8. Females of childbearing potential who are sexually active, and who refuse to use an acceptable contraceptive method up to 6 weeks post-second vaccination.9. Deprived of freedom by administrative or court order, or in an emergency setting, or hospitalized involuntarily.10. Current alcohol abuse or drug addiction that may interfere with the subject's ability to comply with trialprocedures.11. Identified as an employee of the Investigator or trial center, with direct involvement in the proposed trial or other trials under the direction of that Investigator or trial center.

[0459] Eligible subjects were randomized (2:1) into two treatment groups: groups 1 received one subcutaneous (SC) dose of TDV in the upper arm on Day 1 and on Day 90, and group 2 received one subcutaneous dose of placebo in the upper arm on Day 1 and on Day 90. Randomization was stratified by region (Asia Pacific and Latin America) and age range (children aged 4-5 years, 6-11 years, and 12-16 years) to ensure each age range has the appropriate ratio of TDV to placebo in each region. After randomization dropouts were not replaced. Study Day 1 is defined to be the date of the first dose administration of TDV or placebo. The TDV was prepared as described in Example 1. Each subcutaneous dose of TDV was 0.5 mL and the concentration of the four dengue serotypes in the TDV vaccine in each dose was 3.6 logio PFU / dose, 4.0 logio PFU / dose, 4.6 logio PFU / dose and 5.1 logic PFU / dose of TDV-1, TDV-2, TDV-3 and TDV-4, respectively.

[0460] The „total concentration in pfu / 0.5 ml" which serves as a base value for the calculation of the percentage concentration for each individual component o...

Claims

CLAIMS1. An HPV vaccine composition and a dengue vaccine composition for use in a method of preventing an HPV-associated disease and dengue disease in a subject or in a subject population, the method comprising concomitantly administering the HPV vaccine composition and the dengue vaccine composition, wherein the dengue vaccine composition is a tetravalent dengue virus composition comprising live, attenuated DENV-1, DENV-2, DENV-3 and DENV-4 strains and wherein the dengue vaccine composition provides combined vaccine efficacy against all four serotypes of the dengue virus and wherein the immune response to the HPV vaccine composition is non-inferior compared to a mono-administration of the HPV vaccine composition.

2. The HPV vaccine composition and the dengue vaccine composition for use according to claim 1, wherein the HPV-associated disease is selected from HPV-associated cancers or genital warts.

3. The HPV vaccine composition and the dengue vaccine composition for use according to claim 1 or 2, wherein the dengue vaccine composition comprises a chimeric dengue serotype 2 / 1 strain, a dengue serotype 2 strain, a chimeric dengue serotype 2 / 3 strain, and a chimeric dengue serotype 2 / 4 strain.

4. The HPV vaccine composition and the dengue vaccine composition for use according to any of the preceding claims, wherein the HPV vaccine composition is a bivalent, quadrivalent or nonavalent HPV vaccine composition.

5. The HPV vaccine composition and the dengue vaccine composition for use according to any of the preceding claims, wherein a unit dose of the dengue vaccine composition upon reconstitution with 0.5 mLof a pharmaceutically acceptable diluent comprises(i) a chimeric dengue serotype 2 / 1 strain in a concentration of at least 3.3 loglO pfu / 0.5 mL,(ii) a dengue serotype 2 strain in a concentration of at least 2.7 log 10 pfu / 0.5 mL,(iii) a chimeric dengue serotype 2 / 3 strain in a concentration of at least 4.0 loglO pfu / 0.5 mL, and(iv) a chimeric dengue serotype 2 / 4 strain in a concentration of at least 4.5 loglO pfu / 0.5 mL,wherein optionally (i), (ii), (iii), and (iv) provide a total concentration of pfu / 0.5 mL and based on said total concentration of pfu / 0.5 ml 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%, and 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 10%.

6. The HPV vaccine composition and the dengue vaccine composition for use according to any of the preceding claims, wherein the subject population or subject is seronegative to all dengue serotypes.

7. The HPV vaccine composition and the dengue vaccine composition for use according to any of the preceding claims, wherein the dengue vaccine composition is administered by subcutaneous injection and the HPV vaccine composition is administered by intramuscular injection, and wherein optionally said injections are administered to the arm, optionally to the deltoid region of the arm.

8. The HPV vaccine composition and the dengue vaccine composition for use according to any of the preceding claims, wherein the dengue vaccine composition and the HPV vaccine composition are administered to different anatomical sites, optionally to opposite arms.

9. The HPV vaccine composition and the dengue vaccine composition for use according to any of the preceding claims, wherein two doses of the dengue vaccine composition are administered within 12 months or more, or within six months, or within three months.

10. The HPV vaccine composition and the dengue vaccine composition for use according to any of the preceding claims, comprising the administration of two unit doses of the dengue vaccine composition and two unit doses of said HPV vaccine composition, optionally according to the following schedule:a first concomitant administration of a first unit dose of the dengue vaccine composition and a first unit dose of the HPV vaccine composition on day 0,a second administration of a second unit dose of the dengue vaccine composition after the first concomitant administration, such as about 3 months after day 0, anda third administration of a second unit dose of the HPV vaccine composition after the second administration, such as about 6 months after day 0.

11. The HPV vaccine composition and the dengue vaccine composition for use according to any of the preceding claims, wherein the subject population or subject is 9 to 14 years of age.

12. The HPV vaccine composition and the dengue vaccine composition for use according to any of the preceding claims, wherein the subject population or subject is from a dengue endemic region, optionally from Thailand.

13. The HPV vaccine composition and the dengue vaccine composition for use according to any of the preceding claims, wherein the subject population or subject is female.

14. The HPV vaccine composition and the dengue vaccine composition for use according to any of the preceding claims, wherein the method does not include a step of determination whether there was a previous dengue infection and / or wherein the dengue serostatus of the subject population or of the subject is unknown before the concomitant administration the HPV vaccine composition and the dengue vaccine composition and / or wherein the subject or subject population has no laboratory confirmed history of dengue and / or wherein the method does not include a step of assessing a previous dengue infection through an appropriately validated serological test.

15. The HPV vaccine composition and the dengue vaccine composition for use according to any of the preceding claims, wherein the HPV vaccine composition is a nonavalent HPV vaccine composition and the method provides noninferiority of the immune response to the nonavalent HPV vaccine composition compared to a mono-administration of the nonavalent HPV vaccine composition.

16. The HPV vaccine composition and the dengue vaccine composition for use according to any of the preceding claims, wherein the method provides non-inferiority of the immune response to the HPV vaccine composition in a noninferiority clinical study including at least 600 healthy subjects divided into one subject population and into one control subject population, wherein the subject population receives the HPV vaccine composition and the dengue vaccine composition concomitantly once on day 1 (month 0) followed by the dengue vaccine composition once on day 90 (month 3) and the HPV vaccine composition once on day 180 (month 6) and the control subject population receives the HPV vaccine composition once on day 1 (month 0) followed by the HPV vaccine composition once on day 180 (month 6).

17. The HPV vaccine composition and the dengue vaccine composition for use according to any of the preceding claims, wherein the method provides a seropositivity rate of at least 95% or at least 98% or at least 99.5% or 100% for each dengue serotype at day 120 (month 4) after a first concomitant administration of a first unit dose of the dengue vaccine composition and a first unit dose of the HPV vaccine composition.

18. A kit comprising(a) a first container containing an HPV vaccine composition, and(b) a second container containing a dengue vaccine composition, wherein the dengue vaccine composition is a tetravalent dengue virus composition including four live, attenuated dengue virus strains.

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