WO2014083194A1

WO2014083194A1 - Methods for inducing antibodies

Info

Publication number: WO2014083194A1
Application number: PCT/EP2013/075183
Authority: WO
Inventors: Alain Bouckenooghe; Remi Forrat; Denis Crevat; Jean Lang; Bruno Guy; Yves Girerd-Chambaz; Nathalie MANTEL; Isabelle Legastelois; Jiansheng Yao; Veronique Barban
Original assignee: Sanofi Pasteur
Priority date: 2012-11-30
Filing date: 2013-11-29
Publication date: 2014-06-05
Also published as: BR112015012515B1; BR112015012515A2

Abstract

The present invention provides agents for use in methods of inducing neutralising antibodies against the four serotypes of dengue virus, wherein said agents are administered in conjunction with a measles vaccine, a mumps vaccine and a rubella vaccine.

Description

METHODS FOR INDUCING ANTIBODIES

BACKGROUND OF THE INVENTION

Dengue is the second most important infectious tropical disease after malaria with approximately one half of the world's population living in areas where there is a risk of epidemic transmission. There are estimated to be 50-100 million cases of dengue disease every year resulting in 500,000 patients being hospitalized for hemorrhagic dengue fever (DHF) and resulting in approximately 25,000 deaths. Dengue disease infections are endemic in more than 100 tropical countries and hemorrhagic dengue fever has been documented in 60 of these countries (Gubler, 2002, TRENDS in Microbiology, 10: 100-103).

Dengue disease is caused by four antigenically distinct, but closely related dengue virus serotypes of the flavivirus genus (Gubler ef al., 1988, in: Epidemiology of arthropod-borne viral disease. Monath TPM, editor, Boca Raton (FL): CRC Press: 223-60; Kautner ef al., 1997, J. of Pediatrics, 131 : 516-524; Rigau-Perez et al., 1998, Lancet, 352: 971 -977; Vaughn et al., 1997, J. Infect. Dis., 176: 322-30).

Dengue disease is usually transmitted by injection of the dengue virus during the blood meal of an Aedes aegypti mosquito infected by the virus. After an incubation period of 4-10 days, the illness begins abruptly and is followed by three phases: febrile (2 to 7 days), critical (24-48 hours - during which severe complications may occur) and recovery (48-72 hours). During the critical phase, life threatening complications such as hemorrhages, shock and acute organ impairment may occur. A proper management of these unpredictable outcomes can reduce the case fatality rate. Cure of dengue fever is complete after 7 to 10 days, but prolonged asthenia is normal. Reduced leukocyte and platelet numbers are frequently observed.

Dengue haemorrhagic fever (DHF) is a potentially deadly complication of dengue virus infection. DHF is characterized by a high fever and symptoms of dengue fever, but with extreme lethargy and drowsiness. Increased vascular permeability and abnormal homeostasis can lead to a decrease in blood volume, hypotension, and in severe cases, hypovolemic shock and internal bleeding. Two factors appear to play a major role in the occurrence of DHF - rapid viral replication with a high level of viremia (the severity of the disease being associated with the level of viremia; Vaughn et al., 2000, J. Inf. Dis., 181 : 2-9) and a major inflammatory response with the release of high levels of inflammatory mediators (Rothman and Ennis, 1999, Virology, 257: 1 -6). The mortality rate for hemorrhagic dengue fever can reach 10% without treatment, but is < 1 % in most centres with access to treatment (WHO Technical Guide, 1986. Dengue hemorrhagic fever: diagnosis, treatment and control, p. 1 -2. World Health Organization, Geneva, Switzerland).

Dengue shock syndrome (DSS) is a common progression of DHF and is frequently fatal. DSS results from generalized vasculitis leading to plasma leakage into the extravascular space. DSS is characterized by rapid and poor volume pulse, hypotension, cold extremities, and restlessness.

In Asia, DHF and DSS are observed primarily in children, with approximately 90% of those with DHF being less than 15 years of age (Malavige et al., 2004, Postgrad Med. J., 80: 588-601 ; Meulen et al., 2000, Trap. Med. Int. Health, 5:325-9). In contrast, outbreaks in the Caribbean and Central America have predominantly affected adults (Malavige et al., 2004, Postgrad Med. J., 80: 588-601 ).

The four serotypes of dengue virus possess approximately 60-80% sequence homology. Infection with one dengue serotype provides durable homologous immunity but limited heterologous immunity. (Sabin, 1952, Am. J. Trap. Med. Hyg., 1 : 30-50). Accordingly, an individual that has been infected with one serotype of dengue may subsequently become infected with a different serotype. In the past, it has been considered that a second infection arising from a different dengue virus serotype is theoretically a risk factor for the development of DHF, since the majority of patients that exhibit DHF have been previously exposed to at least one of the other four serotypes of dengue viruses.

Current treatment for dengue fever is symptomatic, with bed rest, control of the fever and pain through antipyretics and analgesics, and adequate drinking. The treatment of hemorrhagic dengue fever requires balancing of liquid losses, replacement of coagulation factors and the infusion of heparin.

Measles is a disease caused by a Paramyxovirus of the genus Morbillivirus. Measles infections are most serious in children in developing countries, where the mortality rates can be as high as 2% to 15%. Pneumonia is the most common severe complication from measles and is associated with the greatest number of measles-associated deaths. The rash is intense and often haemorrhagic; it resolves after marked desquamation. Inflammation of the mucosa leads to stomatitis and diarrhoea. There are other severe complications when the disease affects the brain.

Mumps is a disease caused by a Paramyxovirus of the genus Rubulavirus. A classic symptom of mumps is parotidis (inflammation of the salivary glands), which develops within 16 to 18 days after exposure to the virus. Subjects may also present with fever, headache and myalgia. Complications of mumps infection include orchitis in males (more often when the virus infects adults than infants) and sterility, as well as mastitis in females.

Rubella is a disease caused by a Togavirus of the genus Rubivirus. Usually, a rash on the face and neck develops within 2 weeks after exposure to the virus. The volume of glands increases and subjects experience fever, malaise, and conjunctivitis. Complications of Rubella infection include brain damage.

Measles, mumps and rubella are diseases that may be prevented by a single administration of a measles, mumps and rubella combination vaccine. Such a vaccine is generally referred to as an MM R vaccine.

The existing childhood vaccination programme contains a number of vaccines against a range of disease agents. There has been some success in minimizing the frequency of vaccination by combining multiple vaccinations into a single dosage form. However, there is the potential for incompatibility among the different agents in a single dosage form. Additionally, the administration of multiple vaccines at a single time also creates issues for effective vaccination. Whenever a multivalent vaccine is administered (or multiple monovalent vaccines are co-administered), each individual antigen induces an immunological response. It is therefore possible to inhibit the immune system's ability to adequately respond to all of the antigens administered and to not provide a durable protective response to one or more of the antigens.

The present invention addresses the foregoing needs by providing agents for use in methods of inducing neutralising antibodies against the four serotypes of dengue virus, wherein said agents are administered in conjunction with a measles vaccine, a mumps vaccine and a rubella vaccine. SUMMARY OF THE INVENTION

The present invention provides an agent which comprises a dengue antigen of serotype 1 or a nucleic acid construct or viral vector which is able to express a dengue virus-like particle (VLP) of serotype 1 ; an agent which comprises a dengue antigen of serotype 2 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 2; an agent which comprises a dengue antigen of serotype 3 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 3; and an agent which comprises a dengue antigen of serotype 4 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 4; wherein each dengue antigen is independently selected from the group consisting of:

(a) a live attenuated dengue virus;

(b) an inactivated dengue virus;

(c) a live attenuated or inactivated chimeric dengue virus and

(d) a dengue virus-like particle (VLP); for use in a method of inducing neutralising antibodies against four serotypes of dengue virus, wherein said method comprises the administration of said agents to said mammal in conjunction with a measles vaccine.

The present invention further provides an agent for use in a method of inducing neutralising antibodies against at least one serotype of dengue virus in a mammal, wherein said agent comprises:

(i) at least one dengue antigen selected from the group consisting of:

(a) a live attenuated dengue virus;

(b) an inactivated dengue virus;

(c) a live attenuated or inactivated chimeric dengue virus and

(d) a dengue virus-like particle (VLP); or

(ii) at least one nucleic acid construct or viral vector which is able to express in a human cell at least one dengue antigen which is a dengue VLP;

and wherein said method comprises the administration of said agent to said mammal in conjunction with a measles vaccine.

The invention additionally provides a measles vaccine for use in a method of inducing antibodies against measles virus in a mammal, wherein said method comprises the administration of said measles vaccine to said mammal in conjunction with an agent comprising a dengue antigen of serotype 1 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 1 , an agent comprising a dengue antigen of serotype 2 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 2, an agent comprising a dengue antigen of serotype 3 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 3 and an agent comprising a dengue antigen of serotype 4 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 4; wherein each dengue antigen is independently selected from the group consisting of:

(a) a live attenuated dengue virus;

(b) an inactivated dengue virus;

(c) a live attenuated or inactivated chimeric dengue virus; and

(d) a dengue virus-like particle (VLP).

In addition, the invention provides a method of inducing antibodies against four serotypes of dengue virus, measles virus in a mammal, comprising the administration to said mammal of an agent comprising a dengue antigen of serotype 1 or a nucleic acid construct or viral vector which is able to express a dengue virus-like particle (VLP) of serotype 1 ; an agent comprising a dengue antigen of serotype 2 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 2; an agent comprising a dengue antigen of serotype 3 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 3 and an agent comprising a dengue antigen of serotype 4 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 4 in conjunction with a measles vaccine, wherein said agents are as defined herein.

Further, the present invention relates to the use of an agent which comprises a dengue antigen of serotype 1 or a nucleic acid construct or viral vector which is able to express a dengue virus-like particle (VLP) of serotype 1 ; an agent which comprises a dengue antigen of serotype 2 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 2; an agent which comprises a dengue antigen of serotype 3 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 3; and an agent which comprises a dengue antigen of serotype 4 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 4; wherein each dengue antigen is independently selected from the group consisting of:

(a) a live attenuated dengue virus;

(b) an inactivated dengue virus;

(c) a live attenuated or inactivated chimeric dengue virus; and

(d) a dengue virus-like particle (VLP);

for the manufacture of a medicament for the induction of neutralising antibodies against four serotypes of dengue virus, wherein said induction comprises the administration of said agents to said mammal in conjunction with a measles vaccine.

The invention additionally provides the use of a measles vaccine in the manufacture of a medicament for the induction of antibodies against measles virus in a mammal, wherein said induction comprises the administration of said measles vaccine to said mammal in conjunction with an agent comprising a dengue antigen of serotype 1 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 1 , an agent comprising a dengue antigen of serotype 2 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 2, an agent comprising a dengue antigen of serotype 3 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 3 and an agent comprising a dengue antigen of serotype 4 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 4; wherein each dengue antigen is independently selected from the group consisting of: (a) a live attenuated dengue virus;

(b) an inactivated dengue virus;

(c) a live attenuated or inactivated chimeric dengue virus; and

(d) a dengue virus-like particle (VLP).

DESCRIPTION OF THE FIGURES

Figure 1 illustrates geometric means of titres (GMTs) for antibodies against measles, mumps and rubella following vaccination with an MMR vaccine composition when said MMR vaccine composition has been administered sequentially or in conjunction with a tetravalent dengue composition .

Figure 2 illustrates GMTs of antibodies against each serotype of dengue virus prior to administration of the tetravalent dengue composition and after each dose of said composition (PRNT assay).

Figure 3 illustrates the construction of the YF-VAX cDNA by RT-PCR and cloning.

DEFINITIONS

The term "Dengue disease", as used herein, refers to the clinical symptoms exhibited by an individual following infection by any one of the four Dengue virus serotypes. Since 1970, clinical dengue has been classified according to World Health Organization guidelines as (i) dengue fever or (ii) dengue hemorrhagic fever (World Health Organization. Dengue hemorrhagic fever: Diagnosis, treatment, prevention and control 2^nd Ed. Geneva: WHO, 1997; ISBN 92 4 154500 3). In 2009, the WHO issued new guidelines that classify clinical dengue as (i) dengue with or without warning signs or (ii) severe dengue. Both classifications are shown in Figures 1 & 2 of Srikiatkachorn et al., Clin. Infect. Dis. (201 1 ) 53(6): 563. According to the earlier classification, dengue fever is characterized by at least two symptoms selected from headache, arthralgia, retro- orbital pain, rash, myalgia, hemorrhagic manifestations, and leucopenia, together with supportive serology or occurrence at the same location and time as other confirmed dengue cases. Progression to Dengue hemorrhagic fever is confirmed when fever, hemorrhagic manifestations, thrombocytopenia and evidence of plasma leakage are all observed. According to the more recent classification, diagnosis of dengue requires the presence of fever and at least two clinical symptoms selected from nausea, vomiting, rash, aches and pains, a positive tourniquet test, or any warning signs selected from abdominal pain and tenderness, persistent vomiting, clinical fluid accumulation, mucosal bleed, lethargy or restlessness, liver enlargement greater than 2 cm or an increase in hematocrit concurrent with a rapid decrease in platelet count. Severe dengue is diagnosed when any of the following events are observed: severe plasma leakage leading to shock or respiratory distress, severe bleeding as evaluated by clinicians or severe organ involvement.

The term "Dengue hemorrhagic fever or DHF", as used herein, refers to dengue disease wherein fever, hemorrhagic manifestations, thrombocytopenia and evidence of plasma leakage are all observed. DHF, as used herein, may be further defined on the basis of its severity. For instance, DHF may be defined as being of Grade I, Grade II, Grade III or Grade IV (World Health Organization. Dengue hemorrhagic fever: Diagnosis, treatment, prevention and control 2^nd Ed. Geneva: WHO, 1997; ISBN 92 4 154500 3). Grade I is defined as fever accompanied by nonspecific constitutional symptoms; the only haemorrhagic manifestation is a positive tourniguet test and/or easy bruising. Grade II is defined as spontaneous bleeding in addition to the manifestations of Grade I patients, usually in the form of skin or other haemorrhages. Grade III is defined as circulatory failure manifested by a rapid, weak pulse and narrowing of pulse pressure or hypotension, with the presence of cold clammy skin and restlessness. Grade IV is defined as profound shock with undetectable blood pressure or pulse.

The term "virologicallv-confirmed dengue", as used herein, refers to an acute febrile episode which is confirmed to be induced by a dengue virus, e.g. by reverse transcriptase polymerase chain reaction (RT-PCR) or by a dengue non-structural 1 (NS1 ) protein enzyme-linked immunosorbent assay (ELISA). In the RT-PCR method, serum samples are tested according to the method of Callahan et al, J. Clin. Microbiol. (2001 ) 39: 4119. Briefly, RNA is extracted from the serum to discard potential Tag polymerase inhibitors or interfering factors, using a commercial kit. Then an RT-PCR reaction is carried out with serotype specific primers from the dengue NS5 gene seguence. Results are expressed as a concentration of logioGEQ (genome eguivalent)/ml_, by comparison with standards containing known concentrations of viral genomic serotype-specific nucleic acid seguences integrated into plasmids. In the ELISA method, 50 μΙ_ of patient serum, a positive control, a negative control, or a cut-off control are diluted 1 :2 in sample diluent and combined with 100 μΙ_ of diluted horseradish peroxidase (HRP)-labelled anti-NS1 monoclonal Ab (MAb). The diluted serum and conjugate are added to capture anti-NS1 MAb-coated microwells, and plates are incubated for 90 minutes at 37°C. Capture MAb/NS1/HRP-labelled-MAb complexes are formed when NS1 is present in the serum. Complexes are detected via a colorimetric reaction in positive wells which is induced by adding 160 μΙ_ of 3,3',5,5'-tetramethylbenzidine (TMB) substrate and incubating for 30 minutes at room temperature in the dark. The reaction is stopped with the addition of 100 μΙ_ of stop solution (1 N H₂S04) and the plate is read. A sample ratio is determined for each sample by dividing the average optical density (OD) of the test sample by the average OD of the cut-off control (tested in guadruplicate). Sample ratios of <0.5, 0.5-<1 .0, and >1 are indicative of negative, eguivocal, and positive results, respectively. The term "severe viroloqicallv-confirmed dengue", as used herein, refers to dengue haemorrhagic fever (DHF) as defined by the 1997 WHO classification and further characterized by the following additional list of symptoms: haemorrhage requiring blood transfusion, objective evidence of capillary permeability, signs of circulatory failure or visceral manifestations.

The term "dengue shock syndrome", as used herein, refers to the most severe complications of DHF as defined above. According to the 1997 WHO classification, DSS corresponds to DHF of Grades III and IV.

The term "dengue fever virus", "dengue virus" and "DEN" are used interchangeably. They refer to positive single-strand RNA viruses belonging to the Flavivirus genus of the family of Flaviviridae. There are four different serotypes of dengue virus (serotypes 1 , 2, 3 and 4), which possess approximately 60-80% seguence homology. The organization of the genome comprises the following elements: a 5' non-coding region (NCR), a region encoding structural proteins (capsid (C), pre-membrane (prM) and envelope (E)) and a region encoding non-structural proteins (NS1 - NS2A-NS2B-NS3-NS4A-NS4B-NS5) and a 3' NCR. The dengue viral genome encodes an uninterrupted coding region which is translated into a single polyprotein which undergoes post- translational processing. The prM-E protein seguence as translated from the nucleic acid coding region may be numbered in various ways: (i) the total prM-E protein seguence is numbered from position 1 to position 661 , with the preM protein seguence designated as position 1 to position 90/91 , the M protein seguence designated as position 91 /92 to position 166 and the E protein seguence designated as position 167 to position 661 ; (ii) the prM and M protein seguences are numbered together, i.e. from position 1 to position 166 of the total seguence and E is numbered separately from position 1 to position 495; (iii) the prM, M and E seguences are numbered separately, i.e. prM is numbered from position 1 to 90/91 , M is numbered from 1 to 75/76 and E from position 1 to position 495. In the present disclosure the E protein is always numbered from position 1 to position 495. For example, a residue designated herein as E-154 refers to position

154 of the E protein.

The term "monovalent", as used herein, refers to a agent or vaccine which comprises at least one antigen of a single serotype of a pathogen (e.g. a dengue antigen of serotype 1 ) or at least one antigen derived from a single pathogen (e.g. a measles virus) and which will elicit an antibody response against only that single serotype of the pathogen or that single pathogen when administered to an immunocompetent mammal. A composition according to the present invention is termed "multivalent" when it contains antigens from multiple serotypes of a single pathogen (e.g. an agent comprising dengue antigens of at least two different serotypes) and/or antigens from multiple different pathogens (e.g. the measles, mumps and rubella (MMR) vaccine), which, when administered to an immunocompetent mammal, will elicit an antibody response against all serotypes of the pathogen and/or all the pathogens represented in the composition. The nomenclature used for such compositions is consistent with conventional nomenclature. For example, a composition is considered "bivalent", "trivalent", "tetravalent", "pentavalent", "hexavalent", "heptavalent" or "octavalent" when it contains antigens from two, three, four, five, six, seven or eight serotypes of the same pathogen and/or antigens from two, three, four, five, six, seven or eight different pathogens. Examples of multivalent compositions according to the present invention include a bivalent, trivalent or tetravalent dengue composition, a trivalent MMR vaccine composition, a heptavalent composition comprising four different serotypes of dengue antigen, measles, mumps and rubella vaccines in combination or an octavalent composition comprising four different serotypes of dengue antigen, measles, mumps, rubella and varicella zoster virus (VZV) vaccines in combination. Multivalent compositions may be prepared by simple mixing of monovalent compositions. Such multivalent compositions may be prepared in advance at the point of manufacture or may be combined at the time of administration to the subject. The administration of dengue antigens of all four serotypes (i.e. serotypes 1 to 4) may be achieved by the administration of four separate monovalent compositions, two separate bivalent compositions, a trivalent and a monovalent composition or a tetravalent composition.

The term "dengue antigen" refers to a substance which is capable of inducing antibodies against a dengue virus by the administration of such dengue antigen to an immunocompetent mammal. Examples of dengue antigens include inactivated dengue viruses, live attenuated dengue viruses, live attenuated or inactivated chimeric dengue viruses and dengue virus-like particles (VLPs). A dengue antigen may be classified as being of serotype 1 , 2, 3 or 4 depending on the serotype of the virus or VLP which constitutes said dengue antigen.

The term "inactivated virus", as used herein, refers to a virus that is incapable of replication to any significant degree in cells permissive for replication of the corresponding wild type virus. Viruses may be inactivated by a number of means well known to those skilled in the art. Examples of methods for inactivating a virus include chemical treatments, or radiation treatments (including heat or electromagnetic radiation typically in the forms of X-ray or ultraviolet radiation).

The term "inactivated dengue virus", as used herein refers to an inactivated wild type virus containing all the dengue structural proteins (envelope, premembrane/membrane and capsid proteins) and inactivated viral RNA. An inactivated dengue virus may also refer to an inactivated chimeric dengue virus. Inactivated dengue viruses are for instance described in United States Patent No. 6,254,873.

The term "live attenuated virus" or "LAV", as used herein, refers to a virus which is not able to induce a disease state characterised by the same sets of symptoms associated with the corresponding wild-type virus. Examples of live attenuated viruses are well known in the art. A live attenuated virus may be prepared from a wild type virus, for example, by recombinant DNA technology, site directed mutagenesis, genetic manipulation, serial passages on replication- competent cells, chemical mutagenesis treatment or electromagnetic radiation.

The term "live attenuated dengue virus", as used herein, refers to a live dengue virus derived from a virulent wild type dengue virus by genetic modification resulting in attenuation of virulence and an inability to induce a disease state characterised by the same sets of symptoms associated with the corresponding wild type dengue virus. Examples of live attenuated dengue viruses useful in the practice of the present invention include VDV-1 , VDV-2, and the strains described for example in applications WO 02/66621 , WO 00/57904, WO 00/57908, WO 00/57909, WO 00/57910, WO 02/0950075 and WO 02/102828. Live attenuated dengue viruses of serotype 1 which may be used in the method of the invention include VDV-1 . Live attenuated dengue viruses of serotype 2 which may be used in the method of the invention include VDV-2 and LAV-2.

"VDV" and "Vero dengue vaccine" are used interchangeably herein and designate a live attenuated dengue virus capable of replication in Vero cells and capable of inducing a specific humoral response, including the induction of neutralizing antibodies, in a human.

The DEN-1 16007/PDK13 strain, also called "LAV1 ", is derived from wild-type DEN-1

(dengue virus serotype 1 ) 16007 strain which has undergone 1 1 passages through primary dog kidney (PDK) cells (DEN-1 16007/PDK1 1 ). LAV1 has been described in patent application EP1 159968 in the name of Mahidol University and has been filed with the National Microorganisms Cultures Collection (CNCM) under number I-2480. "VDV-1 " is a virus derived from LAV1 by subseguent adaptation to Vero cells; in this regard, the RNA from LAV1 has been extracted and purified before being transfected into Vero cells. The VDV-1 strain has subseguently been obtained by plate purification and amplification in Vero cells. The VDV-1 strain has 14 additional mutations in comparison with the DEN-1 16007/PDK13 strain (13 passes through PDK cells). A process for preparing and characterizing the VDV-1 strain has been described in the international patent application published under number WO 06/134433 in the names of Sanofi Pasteur and the Center for Disease Control and Prevention.

The DEN-2 16681/PDK53 strain, also known as "LAV2", has been obtained from wild-type strain DEN-2 (dengue virus serotype 2) 16681 which has undergone 50 passes through PDK cells (DEN-2 16681/PDK50). LAV2 has been described in in patent application EP1159968 in the name of Mahidol University and has been filed with the National Microorganisms Cultures Collection (CNCM) under number 1 -2481. "VDV-2" is a strain derived from LAV2 by subseguent adaptation to Vero cells; in this regard, the RNA from LAV2 has been extracted and purified before being transfected in Vero cells. The VDV-2 strain has subseguently been obtained by plate purification and amplification in Vero cells. The VDV-2 strain has 10 additional mutations in comparison with the DEN-2 16681/PDK53 strain (53 passes through PDK cells), including 4 silent mutations. A process for preparing and characterizing the VDV-2 strain has been described in the international patent application published under number WO 06/134443 in the names of Sanofi Pasteur and the Center for Disease Control and Prevention. The complete nucleic acid sequence (RNA) of the VDV-2 strain is as shown in SEQ ID NO: 24. The sequence of the E protein of the VDV-2 strain is as shown in SEQ ID NO: 26 and the sequence of the M protein of the VDV-2 strain is as shown in SEQ ID NO: 27. The nucleic acid sequence (RNA) of the prM-E region of the VDV-2 strain is as shown in SEQ ID NO: 25.

The VDV 1 and 2 strains are prepared by amplification in Vera cells. The viruses produced are harvested and clarified from cell debris by filtration. The DNA is digested by treatment with enzymes. Impurities are eliminated by ultrafiltration. Infectious titers may be increased by a concentration method. After adding a stabilizer, the strains are stored in lyophilized or frozen form before use and then reconstituted when needed.

In the context of the present invention, a "dengue chimera" or a "chimeric dengue virus" means a recipient flavivirus in which the genetic backbone has been modified by exchanging the genomic seguences encoding the prM and E proteins of the recipient flavivirus by the corresponding seguences of a dengue virus. Typically, the recipient flavivirus may be attenuated. The recipient flavivirus may be a yellow fever (YF) virus such as the attenuated YF17D, YF17DD and YF17D204 (YF-VAX®) viruses; in that case, such chimeras are referred to as YF/dengue chimeras. The recipient flavivirus may also be a dengue virus and in that case, it is referred to as a dengue/dengue chimera, the dengue virus serotype characteristic of the prM and E proteins being identical or different from the recipient dengue virus serotype characteristic of the genetic backbone. When the serotypes are identical, the recipient dengue virus and the dengue virus from which the prM and E protein encoding seguences originate, are two different virus strains of the same serotype. For use in the present invention, chimeric dengue viruses are typically YF/dengue chimeras. Chimeric dengue viruses are preferably inactivated or live attenuated chimeric dengue viruses. Advantageously, the recipient flavivirus of a live attenuated chimeric dengue virus of the present invention is YF17D or YF17D204 (YF-VAX®). According to one embodiment a dengue chimera is an inactivated virus. According to an alternative embodiment a dengue chimera is a live attenuated virus. Chimeric dengue viruses that can be used in the agents of the present invention include Chimerivax™ Dengue Serotype 1 (referred to herein as CYD-1 ), Chimerivax™ Dengue Serotype 2 (referred to herein as CYD-2), Chimerivax™ Dengue Serotype 3 (referred to herein as CYD-3) and Chimerivax™ Dengue Serotype 4 (referred to herein as CYD-4).

Further examples of chimeric dengue viruses useful in the practice of the present invention include the dengue/YF chimeric viruses described in patent application WO 98/3791 1 and the dengue/dengue chimeras such as those described in patent applications WO 96/40933 and WO 01/60847. In one embodiment, a chimeric YF/dengue virus according to the present invention comprises the genomic backbone of the attenuated yellow fever virus strain YF17D (Theiler M. and Smith H.H., 1937, J. Exp. Med., 65. 767-786), e.g. viruses YF17D/DEN-1 , YF17D/DEN-2, YF17D/DEN-3 and YF17D/DEN-4. Examples of YF17D strains which may be used include YF17D204 (YF-VAX(R), Sanofi Pasteur, Swiftwater, PA, USA; Stamaril(R), Sanofi Pasteur, Marcy I'Etoile, France; ARILVAX(TM), Chiron, Speke, Liverpool, UK; FLAVIMUN(R), Berna Biotech, Bern, Switzerland; YF17D-204 France (X15067, X15062); YF17D-204.234 US (Rice et al., 1985, Science, 229: 726-733), or the related strains YF17DD (Genbank access number U17066), YF17D-213 (Genbank access number U17067) and the strains YF17DD described by Galler et al. (1998, Vaccines, 16(9/10): 1024-1028). In another embodiment, the chimeric YF/dengue virus comprises the genomic backbone of the attenuated yellow fever virus strain YF17D204 (YF- VAX®).

One example of a chimeric dengue virus particularly suitable for use in the practice of the present invention is a Chimerivax dengue virus. As used herein, a "Chimerivax dengue virus", is a live attenuated chimeric YF/dengue virus which comprises the genomic backbone of a YF17D or YF17D204 (YF-VAX®) virus in which the nucleic acid sequences encoding the pre-membrane (prM) and envelope (E) proteins have been replaced by nucleic acid sequences encoding the corresponding structural proteins of a dengue virus. A preferred chimeric dengue virus for use in the present invention is a live attenuated chimeric YF/dengue virus which comprises the genomic backbone of a YF17D virus in which the nucleic acid sequences encoding the pre-membrane (prM) and envelope (E) proteins have been replaced by nucleic acid sequences encoding the corresponding structural proteins of a dengue virus. A preferred chimeric dengue virus for use in the present invention is a live attenuated chimeric YF/dengue virus which comprises the genomic backbone of a YF17D204 (YF-VAX®) virus in which the nucleic acid sequences encoding the pre- membrane (prM) and envelope (E) proteins have been replaced by nucleic acid sequences encoding the corresponding structural proteins of a dengue virus. Construction of such Chimerivax viruses may be achieved in accordance with, or in substantial accordance with, the teaching of Chambers, et al. (1999, J. Virology 73(4):3095-3101 ). The particular Chimerivax (CYD) dengue viruses described in the examples have been generated by using prM and E sequences from strains DEN 1 PU0359 (TYP1 140), DEN2 PU0218, DEN3 PaH881/88 and DEN 4 1228 (TVP 980) and the genomic backbone of YF17D virus. Those particular strains are referred to herein as "CYD- 1", "CYD-2", "CYD-3" and "CYD-4" respectively. The preparation of these particular CYD-1 , CYD-2, CYD-3 and CYD-4 strains has been described in detail in international patent applications WO 98/3791 1 , WO 03/101397, WO 07/021672, 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. Alternatively, other dengue fever virus strains as described herein may be used as a source of nucleic acids to facilitate construction of chimeric viruses useful in the practice of the present invention, for example in the construction of other Chimerivax dengue serotype 1 (CYD-1 ), Chimerivax dengue serotype 2 (CYD-2), Chimerivax dengue serotype 3 (CYD-3) and Chimerivax dengue serotype 4 (CYD-4) strains.

In an alternative embodiment a chimeric dengue virus usable in the present invention is a recipient flavivirus in which the genetic backbone has been modified by exchanging (i) the nucleic sequence encoding the E protein of the recipient flavivirus with a corresponding nucleic acid sequence from a dengue virus and (ii) the nucleic acid sequence encoding the prM protein of the recipient flavivirus with a corresponding sequence of a non-dengue flavivirus, e.g. a Japanese Encephalitis virus (JEV). Typically, such a chimeric dengue virus may be a live attenuated virus or an inactivated virus. Examples of such chimeric dengue viruses are described in WO 201 1/138586.

The term "virus-like particles" or "VLPs", as used herein, refers to virus particles that do not contain replicative genetic material but present at their surface a dengue E protein in a repetitive ordered array similar to the virion structure. Typically, dengue VLPs also contain dengue prM and/or M, and E proteins. VLPs may be produced in vitro (Zhang et al, J. Virol. (201 1 ) 30 (8):333). VLPs may also be produced in vivo. To that end, at least one nucleic acid construct (e.g. DNA or RNA) encoding prM and E dengue proteins may be introduced into a cell of a mammal, e.g. a human, via methods known in the art, e.g. via use of a viral vector. Any viral vector may be used provided it is able to contain and express both prM and E dengue virus sequences. Non-limiting examples of viral vectors that may be used in the method of the present invention include the poxviruses (e.g. the attenuated pox Ankara virus) and the measles virus. For use in the present invention, a particular category of viral vector expressing VLPs in vivo includes replication-deficient pseudoinfectious (PIV) viruses, e.g. according to the Replivax™ technology. (Rumyantsev AA, et al. Vaccine. 201 1 Jul 18;29(32):5184-94).

The term "replication-defective pseudo-infectious virus", as used herein, refers to a virion particle that is replication-defective in vivo, owing to the absence in their genome of an essential sequence of the replicative cycle, for example the sequence encoding a capsid protein. However, the virion particles can propagate in a culture of helper cells that provide for the essential sequence(s) in trans. Replication-deficient pseudoinfectious viruses for use in the present invention include any virus according to the above definition which is capable of expressing the prM and E proteins of a dengue virus of any serotype. Examples include replication defective flavivirus / dengue chimeras such as replication defective West Nile virus / dengue, Japanese Encephalitis virus / dengue and YF / dengue chimeras.

The ability of the agents of the present invention to provoke an immune response in a mammal (e.g. induce the production of neutralizing antibodies) can be assessed, for example, by measuring the neutralizing antibody titre raised against each of the dengue antigen(s) administered to said mammal. The neutralizing antibody titre may be measured by the Plaque Reduction Neutralization Test (PRNT₅₀) test. Briefly, neutralizing antibody titre is measured in sera collected from subjects at least 28 days following administration of the agents of the present invention. Serial, two-fold dilutions of sera (previously heat-inactivated) are mixed with a constant challenge- dose of each dengue virus of serotype 1 , 2, 3 or 4 as appropriate (expressed as PFU/mL). The mixtures are inoculated into wells of a microplate with confluent Vero cell monolayers. After adsorption, cell monolayers are incubated for a few days. The presence of dengue virus infected cells is indicated by the formation of infected foci and a reduction in virus infectivity due to the presence of neutralising antibodies in the serum samples can thus be detected. The reported value (end point neutralization titre) represents the highest dilution of serum at which > 50 % of dengue challenge virus (in foci counts) is neutralized when compared to the mean viral focus count in the negative control wells (which represents the 100% virus load). The end point neutralization titres are presented as discontinuous values. The lower limit of quantification (LLOQ) of the assay is 10 (1/dil). It is commonly considered that seroconversion occurs when the titer is superior or equal to 10 (1/dil). As PRNT tests may slightly vary from a laboratory to another the LLOQ may also slightly vary. Accordingly, in a general manner, it is considered that seroconversion occurs when the titre is superior or equal to the LLOQ of the test. Neutralising antibody titres were considered in the following references (Guirakhoo et al, J. Virol. (2004) 78 (9): 4761 ; Libraty et al, PLoS Medicine (2009) 6 (10); Gunther et al, Vaccine (2011 ) 29: 3895) and Endy et al, J. Infect. Dis. (2004), 189(6): 990-1000).

The term "CCID50", as used herein, refers to the quantity of 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).

As used herein, the expression "flavivirus-naive" refers to a mammal, e.g. a human, who has not been infected by a flavivirus nor previously immunized with a flavivirus vaccine. A serum sample taken from said mammal will produce a negative result in a flavivirus ELISA or PRNT assay.

As used herein, the expression "dengue-naive" refers to a mammal, e.g. a human, who has not been infected by a dengue virus nor previously immunized with a dengue vaccine. A serum sample taken from said subject will produce a negative result in a dengue ELISA or PRNT assay.

As used herein, the expression "flavivirus-immune" refers to a mammal, e.g. a human, who has been infected or immunized by a flavivirus before administration of the agents of the present invention. A serum sample taken from said mammal will produce a positive result in a flavivirus ELISA or PRNT assay.

As used herein, the expression "dengue-immune" refers to a mammal who has been infected by a dengue virus or immunized by a dengue vaccine before administration of the agents of the present invention. A serum sample taken from said subject will produce a positive result in a dengue ELISA or PRNT assay.

As used herein, the expression "in conjunction with" refers to the mode of administration of the agent(s) of the present invention and the vaccine(s) of the present invention. The agent(s) and the vaccine(s) of the present invention are administered in conjunction with each other when the agent(s) and the vaccine(s) of the present invention have been administered to a mammal in a manner that results in the dengue antigen(s) and the antigen(s) contained in the vaccine(s) of the invention (e.g. dengue antigen(s) and the measles virus or the dengue antigen(s) and the measles, mumps and rubella viruses) being presented to the immune system of the mammal concurrently. As a non-limiting example, the agent(s) and the vaccine(s) of the present invention are administered in conjunction when the agent(s) and vaccine(s) are administered to the mammal (e.g. by sub-cutaneous injection) directly one after the other. If the agents of the present invention are combined into a multivalent (e.g. tetravalent) composition and the measles, mumps and rubella vaccines are combined into a trivalent MMR vaccine composition, then, as a non-limiting example, the multivalent dengue composition and the trivalent MMR vaccine composition are administered in conjunction with one another if the multivalent dengue composition and the trivalent MMR vaccine composition are administered to the mammal directly one after the other. The agent(s) and vaccine(s) of the present invention may be considered to be administered in conjunction with one another if they are all administered to a mammal, e.g. a human, within a time period of less than one week, preferably less than one day, preferably less than three hours, preferably less than one hour or preferably simultaneously. The agent(s) and the vaccine(s) of the present invention may be considered to be administered in conjunction with one another if they are administered to a mammal, e.g. a human, during a single visit to a physician. The agent(s) and vaccine(s) of the present invention may be considered to be administered in conjunction with one another if they are mixed together to form a single (e.g. heptavalent) composition (e.g. at the point of manufacture or at the point of administration to the mammal) which is then administered to the mammal (e.g. via sub-cutaneous injection using a single needle).

The term "mammal", as used herein, refers to individuals of the mammalian family, including, for example, primates such as humans. It has been demonstrated that dengue viruses are capable of infecting mammals in addition to humans, see, e.g. Dengue infection in neotropical forest mammals, de Thoissy, et al. (2009) Vector Borne Zoonotic Disease 9(2):157-70. A mammal according to the present invention includes both mammals who have never been exposed to dengue, measles, mumps, rubella and/or VZV virus(es) (i.e. immunologically naive) or those who have been previously exposed to one or more dengue virus serotypes and/or measles, mumps, rubella and/or VZV including mammals who have exhibited the symptoms of one or more of the disease states associated with dengue, mumps, measles, VZV or rubella viral infections (i.e. not naive). An immunocompetent mammal is a mammal possessing a functional immune system capable of eliciting the production of neutralizing antibodies when said mammal is exposed to an antigen as described herein.

In accordance with the present invention, a "method of protecting", as used herein, results in a reduction in the severity or in the likelihood of developing dengue disease in a human exposed to a dengue virus or a reduction in the severity or in the likelihood of developing measles, mumps or rubella in a human exposed to a measles, mumps or rubella virus. Advantageously, said reduction is statistically significant. For example, a method of protecting, according to the present invention, may result in a reduction in at least one symptom of dengue disease as defined herein or a reduction in a combination of any two or more of those symptoms. The protection may result in any one or more of the following:

(i) a statistically significant reduction in the incidence or likelihood of, e.g. the prevention of, symptomatic virologically-confirmed dengue disease caused by dengue virus of any serotype;

(ii) a statistically significant reduction in the incidence or likelihood of, e.g. the prevention of, symptomatic virologically-confirmed dengue disease caused by dengue virus of any one of serotypes 1 , 3 or 4;

(iii) a statistically significant reduction in the incidence or likelihood of, e.g. the prevention of, symptomatic dengue disease caused by dengue virus of any serotype;

(iv) a statistically significant reduction in the incidence or likelihood of, e.g. the prevention of, symptomatic dengue disease caused by dengue virus of any one of serotypes 1 , 3 or 4;

(v) a statistically significant reduction in the incidence or likelihood of, e.g. the prevention of, severe virologically-confirmed dengue caused by dengue virus of any serotype;

(vi) a statistically significant reduction in the incidence or likelihood of, e.g. the prevention of, severe dengue disease caused by dengue virus of any serotype;

(vii) a statistically significant reduction in the incidence or likelihood of, e.g. the prevention of, dengue hemorrhagic fever cases of Grades I to IV caused by dengue virus of any serotype;

(viii) a statistically significant reduction in the incidence or likelihood of, e.g. the prevention of, DHF cases of Grade I caused by dengue virus of any serotype;

(ix) a statistically significant reduction in the incidence or likelihood of, e.g. the prevention of, DHF cases of Grade II caused by dengue virus of any serotype; (x) a statistically significant reduction in the incidence or likelihood of, e.g. the prevention of, DHF cases of Grade III caused by dengue virus of any serotype;

(xi) a statistically significant reduction in the incidence or likelihood of, e.g. the prevention of, DHF cases of Grade IV caused by dengue virus of any serotype;

(xii) a statistically significant reduction in the incidence or likelihood of, e.g. the prevention of, fever or a reduction in the mean duration and/or intensity of fever;

(xiii) a statistically significant reduction in the incidence or likelihood of, e.g. the prevention of, plasma leakage as defined by a change in haematocrit or a reduction in the mean value for plasma leakage as defined by a change in haematocrit;

(xiv) a statistically significant reduction in the incidence or likelihood of, e.g. the prevention of, thrombocytopenia or a reduction in the mean value for thrombocytopenia;

(xv) a statistically significant reduction in the incidence or likelihood of, e.g. the prevention of, increases in the level of liver enzymes including alanine aminotransferase (ALT) and aspartate aminotransferase (AST);

(xvi) a statistically significant reduction in the incidence or likelihood of, e.g. the prevention of, hospitalization due to virologically-confirmed dengue disease caused by dengue virus of any serotype;

(xvii) a statistically significant reduction in the incidence or likelihood of, e.g. the prevention of, hospitalization due to dengue disease caused by dengue virus of any serotype;

(xviii) a statistically significant reduction in the length of hospital stay due to virologically- confirmed dengue disease.

(xix) a statistically significant reduction in the length of hospital stay due to dengue disease.

The duration and intensity of fever are monitored and recorded according to standard hospital procedures. In a human subject, a fever (i.e. a febrile episode) is defined as the observance of two temperature readings of at least 37.5°C measured twice over an interval of at least 4 hours. Measurements of haematocrit, thrombocytopenia and hepatic enzyme levels are standard tests well-known to the person of skill in the art, for example as described in the pharmacopeia.

Protection against dengue disease, for example as defined in points (i) to (xix) above, may be demonstrated in respect of dengue disease caused by a particular dengue virus serotype. For example, protection against dengue disease, as defined herein, may be demonstrated in respect of dengue disease caused by a dengue virus of serotype 1 , a dengue virus of serotype 2, a dengue virus of serotype 3 or a dengue virus of serotype 4. Advantageously, protection against dengue disease, as defined herein, may be demonstrated in respect of dengue disease caused by, for example, dengue virus of serotype 1 or serotype 3, dengue virus of serotype 1 or serotype 4, dengue virus of serotype 3 or serotype 4, dengue virus of serotype 1 or serotype 2, dengue virus of serotype 2 or serotype 3, dengue virus of serotype 2 or serotype 4, dengue virus of serotype 1 , 2 or 3, dengue virus of serotype 1 , 3 or 4, dengue virus of serotype 2, 3 or 4 or dengue virus of serotype 1 , 2, 3 or 4. Advantageously, protection against dengue disease, as defined herein, may be demonstrated in respect of dengue disease caused by dengue virus of serotype 1 , 3 or 4.

Mumps, Measles and Rubella Vaccines

In the practice of the present invention, vaccination of a mammal against measles, mumps and rubella may be achieved by the administration of a monovalent mumps vaccine in conjunction with a monovalent measles vaccine and a monovalent rubella vaccine. Alternatively, vaccination of a mammal against measles, mumps and rubella may be achieved by the administration of a trivalent (i.e. combined) vaccine composition comprising a measles vaccine, a mumps vaccine and a rubella vaccine. Such trivalent vaccine compositions are commonly referred to as "MMR" vaccines. In a particular embodiment of the present invention, measles, mumps and rubella vaccines according to the present invention may additionally include a varicella zoster virus (VZV) vaccine (e.g. an inactivated or attenuated varicella zoster virus). Such a VZV vaccine may be administered as a separate monovalent vaccine, or it may be included in the trivalent MMR composition to form a tetravalent composition. Such tetravalent compositions are commonly referred to as MMRV vaccines. References in the present specification to the administration of separate measles, mumps and rubella vaccines or the administration of a trivalent MMR vaccine composition may also be understood to refer to the administration of separate measles, mumps rubella and VZV vaccines and the administration of a tetravalent MMRV vaccine composition respectively.

The preparation of vaccines against measles, mumps, rubella and/or VZV is well known to those of skill in the art. Examples of measles virus strains useful in the preparation of a measles vaccine include the Enders-Edmonston, Edmonston-Zagreb and Schwarz measles strains. Examples of mumps virus strains useful on the preparation of a mumps vaccine include the Jeryl Lynn, Urabe AM 9, RIT 4385 and Rubini strains. Examples of rubella virus strains useful in the preparation of rubella vaccines include the Wistar RA 27/3 and Wistar RA 27/3M strains. Monovalent mumps, measles and rubella vaccines have been approved for use in humans and are commercially available. Examples of VZV strains useful in the preparation of VZV vaccines include the Oka/Merck and Oka strains. An example of a commercial monovalent mumps vaccine useful in the practice of the present invention is the Mumpsvax® vaccine (Merck & Co, Whitehouse Station, NJ, USA). An example of a commercially available monovalent measles vaccine useful in the practice of the present invention is the Attenuvax® vaccine (Merck & Co, Whitehouse Station, NJ, USA). An example of a commercially available monovalent rubella vaccine useful in the practice of the present invention is the Meruvax® II vaccine (Merck & Co, Whitehouse Station, NJ, USA). Examples of commercially available monovalent attenuated VZV vaccines include the Varivax® and Zostavax® vaccines (Merck & Co, Whitehouse Station, NJ, USA) and Okavax (Sanofi Pasteur SA, Lyon FR).

Trivalent MMR vaccines may be prepared using strains of mumps, measles and rubella viruses as described above. Trivalent MMR vaccines for vaccination against mumps, measles and rubella have been approved by regulatory authorities for human use and are commercially available. Examples of commercially available trivalent MMR vaccines include the M-M-R® II vaccine (commercially available from Merck & Co, Whitehouse Station, NJ USA), the Triviraten Berna® (also referred to as the Berna-MMR) vaccine (commercially available from Berna Biotech, Basel, Switzerland), the Priorix™ vaccine (commercially available from Glaxo SmithKline Biologies, Rixensart, Belgium), and the Trimovax® vaccine (commercially available Sanofi Pasteur SA, Lyon, France).

Tetravalent MMRV vaccines may be prepared using strains of measles, mumps, rubella and VZV as described above. Tetravalent MMRV compositions for vaccination against measles, mumps, rubella and VZV have been approved for human use and are commercially available. Examples of tetravalent MMRV compositions include ProQuad (Merck and Company, Whitehouse Station NJ USA) and Priorix Tetra® (commercially available from Glaxo SmithKline Biologies, Rixensart, Belgium).

OVERVIEW OF SEVERAL EMBODIMENTS

Preferably, the agents for use in a method of inducing neutralizing antibodies against four serotypes of dengue virus according to the present invention are administered to said mammal in conjunction with a measles vaccine, a mumps vaccine and a rubella vaccine.

Preferably, the agent for use in a method of inducing neutralizing antibodies against at least one serotype of dengue virus according to the present invention is administered to said mammal in conjunction with a measles vaccine, a mumps vaccine and a rubella vaccine.

Advantageously, the present invention is directed to an agent which is a dengue antigen of serotype 1 or a nucleic acid construct or viral vector which is able to express a dengue virus-like particle (VLP) of serotype 1 ; an agent which is a dengue antigen of serotype 2 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 2; an agent which is a dengue antigen of serotype 3 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 3; and an agent which is a dengue antigen of serotype 4 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 4; wherein each dengue antigen is independently selected from the group consisting of:

(a) a live attenuated dengue virus;

(b) an inactivated dengue virus;

(c) a live attenuated or inactivated chimeric dengue virus; and

(d) a dengue virus-like particle (VLP);

for use in a method of inducing neutralising antibodies against four serotypes of dengue virus, wherein said method comprises the administration of said agents to said mammal in conjunction with a measles vaccine, a mumps vaccine and a rubella vaccine.

Advantageously, an agent according to the present invention as described herein is an immunostimulatory agent. More advantageously, an agent according to the present invention as described herein is a prophylactic agent. Most advantageously, an agent according to the present invention as described herein is a vaccine. An immunostimulatory agent, as defined herein, refers to an agent that directly or indirectly stimulates the immune system of an immunocompetent mammal. An example of an indirect immunostimulatory agent is a nucleic acid construct (e.g. as described herein) which is able to express a polypeptide wherein said polypeptide is able to stimulate the immune system of an immunocompetent mammal.

Preferably, a mammal according to the present invention is a human. Preferably a human according to the present invention is less than 18 years of age or less than 12 years of age. For example, a human according to the present invention may be 0-17 years of age, 0-1 1 years of age, 4-17 years of age, 4-1 1 years of age, 4-6 years of age, 6-8 years of age, 8-10 years of age, 2-8 years of age, 2-1 1 years of age, 2-14 years of age, 9-16 years of age, 12-17 years of age or 18-45 years of age. Preferably, a human according to the present invention is 4-11 years of age, 2-14 years of age or 9-16 years of age. A human according to the present invention may be at least 9 months old or less than 9 months old. For instance a human according to the present invention may be about 9 months to 16 years of age, about 9 months to 14 years of age, about 9 months to 1 1 years of age, about 9 months to 8 years of age, about 9 months to 5 years of age, about 9 months to 3 years of age, about 9 months to 2 years of age or about 9 months to 18 months old. A human according to the present invention may preferably be about 12 to about 24 months old or preferably about 12 to about 15 months old. A human according to the present invention may be at least 9 months old, with no history of severe allergy to any component of the agents or vaccines as defined herein, no congenital or acquired immune deficiency, no symptomatic H IV infection and said human should not be pregnant or breast feeding. A mammal, e.g. a human as defined herein, according to the present invention may advantageously reside in particular countries, areas or regions of the world. For instance, a mammal, e.g. a human, may advantageously reside in a dengue endemic area. A dengue endemic area according to the present invention may comprise those American countries or parts thereof which fall within the tropics and sub-tropics. A dengue endemic area according to the present invention may thus comprise any one or more of the following: Brazil, Venezuela, Colombia, Ecuador, Peru, Bolivia, Paraguay, Panama, Costa Rica, Nicaragua, Honduras, El Salvador, Guatemala, Belize, Mexico, the USA and the islands of the Caribbean. In a particular embodiment, a dengue endemb area of the present invention may consist of the following: Brazil, Colombia, Honduras, Mexico and Puerto Rico. A dengue endemic area according to the present invention may also include south Asian and Oceania countries within the tropics and sub-tropics. A dengue endemic area according to the present invention may thus comprise any one or more of the following: India, Myanmar (Burma), Thailand, Laos, Vietnam, Cambodia, Indonesia, Malaysia, Singapore, the Philippines, Taiwan, Papua New Guinea and Australia. In a dengue endemic area according to the present invention, a particular serotype, strain or genotype of wild type dengue virus may be the dominant circulating strain. For example, a dengue virus of serotype 2 may be characterised as having an Asian I or an Asian/American genotype. Asian/American genotype strains are characterised by a genomb sequence that encodes at least one of, at least two of, at least three of, at least four of, at least five of or all six of the following residues Arg, Asn, Asp, Thr, Gly and His at positions prM-16, E-83, E-203, E-226, E-228 and E-346 respectively (wherein prM- 16 designates position 16 of the prM protein and E-83 etc. designates position 83 of the E protein). Asian I genotype strains are characterised by a genomic sequence that encodes at least one of, at least two of, at least three of, at least four of, at least five of or all six of the following residues He, Lys, Asn, Arg, Glu and Tyr at positions prM-16, E-83, E-203, E-226, E-228 and E-346 respectively (see Table 1 of Hang et al., PLoS NTD, 4(7): e757). A preferred dengue endemic area according to the present invention is one in which a dengue virus having an Asian/American genotype is the dominant circulating strain, i.e. at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the cases of dengue disease in said dengue endemic area are caused by dengue virus having an Asian/American genotype. A preferred dengue endemic area according to the present invention is one in which a dengue virus of any one or more of serotypes 1 , 3 or 4 is/are the dominant circulating serotype(s), i.e. at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the cases of dengue disease are caused by dengue virus of serotypes 1 , 3 or 4.

Advantageously, the agents for use in a method according to the present invention are present in a single tetravalent composition. Alternatively, the agents for use in a method according to the present invention are present in four separate monovalent compositions. Alternatively, the agents for use in a method according to the present invention are present in two separate bivalent compositions. Alternatively, the agents for use in a method according to the present invention are present in a bivalent composition and two monovalent compositions. Alternatively, the agents for use in a method according to the present invention are present in a trivalent composition and a monovalent composition. When the agents for use in a method according to the present invention are administered to said mammal (at least partly) in a multivalent composition (e.g. two bivalent compositions, a bivalent composition and two monovalent compositions, a trivalent and a monovalent composition or a tetravalent composition), those agents may have been provided in (a) multivalent composition(s) and then administered to said mammal in (a) multivalent composition(s). Alternatively, when the agents for use in a method according to the present invention are administered to said mammal (at least partly) in a multivalent composition (e.g. two bivalent compositions, a bivalent composition and two monovalent compositions, a trivalent and a monovalent composition or a tetravalent composition), those agents may have been provided separately (i.e. in monovalent form) or provided in lower order multivalent form (e.g. two bivalent compositions, a bivalent composition and two monovalent compositions or a trivalent composition and a monovalent composition) and then combined into a (higher order) multivalent composition prior to administration to a mammal in a method according to the present invention.

According to one aspect of the invention, the agents and the measles, mumps and rubella vaccines for use in a method of the present invention may be present in a single heptavalent composition. In another aspect of the invention, the agents and the measles, mumps, rubella and VZV vaccines for use in a method of the present invention may be present in a single octavalent composition. In another aspect of the invention, the agents and the measles vaccine for use in a method of the present invention may be present in a single pentavalent composition.

Preferably, the agents for use in a method according to the present invention each comprise a dengue antigen as described herein. Preferably, the agents for use in a method according to the present invention each comprise a dengue antigen selected from the group consisting of (a) a live attenuated virus; (b) an inactivated dengue virus and (c) a live attenuated or inactivated chimeric dengue virus. Preferably, the agents for use in a method according to the present invention each comprise a dengue antigen independently selected from the group consisting of (a) a live attenuated virus and (b) a live attenuated chimeric dengue virus. Preferably, the agents for use in a method according to the present invention each comprise a dengue antigen independently selected from the group consisting of (a) a live attenuated virus; (b) an inactivated dengue virus and (c) a live attenuated or inactivated chimeric dengue virus. Preferably, the agents for use in a method according to the present invention each comprise a dengue antigen which is a live attenuated chimeric dengue virus. Preferably, the agents for use in a method of the present invention each comprise a dengue antigen which is a dengue virus, e.g. a live attenuated dengue virus, an inactivated dengue virus, a live attenuated chimeric dengue virus or an inactivated chimeric dengue virus. Preferably, the agents for use in the present invention each independently comprise a dengue antigen which is a live attenuated dengue virus or a live attenuated chimeric dengue virus. Preferably, the agents for use in a method of the present invention each comprise a live attenuated chimeric dengue virus.

Preferably a dengue antigen of serotype 1 for use in a method according to the present invention is selected from the group consisting of LAV1 , VDV1 , CYD-1 or a DEN-1 chimeric virus, e.g. a YF/DEN-1 chimeric virus, comprising a nucleotide sequence encoding the prM and E proteins of the VDV1 strain. Preferably a dengue antigen of serotype 1 for use in a method according to the present invention is CYD-1 .

Preferably a dengue antigen of serotype 2 for use in a method according to the present invention is selected from the group consisting of LAV2, VDV2, CYD-2, CYD-LAV, CYD-BID, CYD- PR, CYD-MD or a DEN-2 chimeric virus, e.g. a YF/DEN-2 chimeric virus, comprising a nucleotide sequence encoding prM and E proteins, wherein said nucleotide sequence has at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO; 6, SEQ ID NO: 7 or SEQ ID NO: 1. Preferably a dengue antigen of serotype 2 for use in a method according to the present invention is CYD-2. Preferably a dengue antigen of serotype 2 for use in a method according to the present invention is VDV2.

Preferably a dengue antigen of serotype 2 for use in a method according to the present invention is selected from the group consisting of CYD-LAV, CYD-BID, CYD-PR, CYD-MD or a DEN-2 chimeric virus, e.g. a YF/DEN-2 chimeric virus, comprising a nucleotide sequence encoding prM and E proteins, wherein said nucleotide sequence has at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to a nucleotide sequence selected from the group consisting of

SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 1 .

Preferably, a dengue antigen of serotype 2, e.g. a dengue virus of serotype 2, for use in a method according to the present invention comprises a polypeptide having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% identity to SEQ ID NO: 12. Preferably said polypeptide comprises a valine residue at the position within the polypeptide that corresponds to position 251 of SEQ ID NO: 12. Preferably, said polypeptide comprises a threonine residue at the position within the polypeptide that corresponds to position 226 of SEQ ID NO: 12, a glycine residue at the position within the polypeptide that corresponds to position 228 of SEQ ID NO: 12 and a valine residue at the position within the polypeptide that corresponds to position 251 of SEQ ID NO: 12. Preferably, a dengue antigen of serotype 2 which is a dengue virus for use in a method according to the present invention comprises a nucleotide sequence having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% identity to a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 1 . Preferably the prM and E protein sequence encoded by said nucleotide sequence comprises a leucine residue at position 24 of prM, a threonine residue at position 125 of prM and a valine residue at position 251 of E (wherein the prM and M sequences are numbered together, i.e. from position 1 to position 166 and the E sequence is numbered separately from position 1 to position 495).

Preferably, a dengue antigen of serotype 2, e.g. a dengue virus of serotype 2, for use in a method according to the present invention comprises a polypeptide having at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99% identity or 100% identity to a sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17. Preferably said polypeptide comprises a valine residue at the position within the polypeptide that corresponds to position 251 of SEQ ID NO: 12. Preferably, said polypeptide comprises a threonine residue at the position within the polypeptide that corresponds to position 226 of SEQ ID NO: 12, a glycine residue at the position within the polypeptide that corresponds to position 228 of SEQ ID NO: 12 and a valine residue at the position within the polypeptide that corresponds to position 251 of SEQ ID NO: 12.

Preferably, a dengue antigen of serotype 2, e.g. a dengue virus of serotype 2, for use in a method according to the present invention comprises a polypeptide having at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99% identity or 100% identity to a sequence selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 16. Preferably said polypeptide comprises a valine residue at the position within the polypeptide that corresponds to position 251 of SEQ ID NO: 12. Preferably, said polypeptide comprises a threonine residue at the position within the polypeptide that corresponds to position 226 of SEQ ID NO: 12, a glycine residue at the position within the polypeptide that corresponds to position 228 of SEQ ID NO: 12 and a valine residue at the position within the polypeptide that corresponds to position 251 of SEQ ID NO: 12.

Preferably a dengue antigen of serotype 2, e.g. a live attenuated dengue virus of serotype

2 or a chimeric dengue virus of serotype 2, for use in a method according to the present invention comprises a nucleotide sequence encoding prM and E proteins, wherein said nucleotide sequence has at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or 100% identity to the RNA equivalent of a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO; 6, SEQ ID NO: 7 and SEQ ID NO: 1 . Preferably the prM and E protein sequence encoded by said nucleotide sequence comprises a leucine residue at position 24 of prM, a threonine residue at position 125 of prM and a valine residue at position 251 of E (wherein the prM and M sequences are numbered together, i.e. from position 1 to position 166 and the E sequence is numbered separately from position 1 to position 495). Preferably the prM and E protein sequence encoded by said nucleotide sequence comprises a leucine residue at position 24 of prM, a threonine residue at position 125 of prM, a threonine residue at position 226 of E, a glycine residue at position 228 of E and a valine residue at position 251 of E (wherein the prM and M sequences are numbered together, i.e. from position 1 to position 166 and the E sequence is numbered separately from position 1 to position 495).

Preferably a dengue antigen of serotype 2, e.g. a live attenuated dengue virus of serotype 2 or a chimeric dengue virus of serotype 2, for use in a method according to the present invention comprises a nucleotide sequence encoding prM and E proteins, wherein said nucleotide sequence has at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to the RNA equivalent of a nucleotide sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 7. Preferably the prM and E protein sequence encoded by said nucleotide sequence comprises a leucine residue at position 24 of prM, a threonine residue at position 125 of prM and a valine residue at position 251 of E (wherein the prM and M sequences are numbered together, i.e. from position 1 to position 166 and the E sequence is numbered separately from position 1 to position 495). Preferably the prM and E protein sequence encoded by said nucleotide sequence comprises a leucine residue at position 24 of prM, a threonine residue at position 125 of prM, a threonine residue at position 226 of E, a glycine residue at position 228 of E and a valine residue at position 251 of E (wherein the prM and M sequences are numbered together, i.e. from position 1 to position 166 and the E sequence is numbered separately from position 1 to position 495).

Preferably, a dengue antigen of serotype 2, e.g. a dengue virus of serotype 2, for use in a method according to the present invention comprises a first polypeptide having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% identity to SEQ ID NO: 12 and a second polypeptide having at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% identity to SEQ ID NO: 23. Preferably said first polypeptide comprises a valine residue at the position within the polypeptide that corresponds to position 251 of SEQ ID NO: 12 and said second polypeptide comprises a threonine residue at the position with the polypeptide that corresponds to position 34 of SEQ ID NO: 23. Preferably said first polypeptide comprises a threonine residue at the position within the polypeptide that corresponds to position 226 of SEQ ID NO: 12, a glycine residue at the position within the polypeptide that corresponds to position 228 of SEQ ID NO: 12 and a valine residue at the position within the polypeptide that corresponds to position 251 of SEQ ID NO: 12 and said second polypeptide comprises a threonine residue at the position with the polypeptide that corresponds to position 34 of SEQ ID NO: 23. Preferably when an agent for use in the present invention comprises a polypeptide having at least 90% identity to SEQ ID NO: 23, said polypeptide comprises a threonine residue at the position with the polypeptide that corresponds to position 34 of SEQ ID NO: 23.

It is one aim of the present invention to provide an optimized tetravalent dengue composition (i.e. a composition comprising an agent which comprises a dengue antigen of serotype 1 , an agent which comprises a dengue antigen of serotype 2, an agent which comprises a dengue antigen of serotype 3 and an agent which comprises an antigen of serotype 4 as defined herein) which provides an improved neutralising antibody response against dengue virus of serotype 2 (when compared with the neutralising antibody response generated by CYD-1 , CYD-2, CYD-3 and CYD-4 as defined in Example 1 ) and which may be used in a method of the present invention.

Accordingly, in one aspect, the agents of the present invention each comprise a dengue antigen, wherein the dengue antigens of serotypes 1 , 3 and 4 are each a live attenuated chimeric dengue virus and the dengue antigen of serotype 2 is a live attenuated dengue virus which comprises a nucleic acid sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 24. Preferably said nucleic acid sequence has at least 92%, at least 94%, at least 96%, at least 98%, at least 99% or 100% sequence identity to the sequence as set forth in SEQ ID NO: 24. Preferably the nucleotides at the positions within said nucleic acid sequences (that have at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% sequence identity to the sequence as set forth in SEQ ID NO: 24) which correspond to positions 736, 1619, 4723, 5062, 9191 , 10063, 10507, 57, 524, 2055, 2579, 4018, 5547, 6599 and 8571 of SEQ ID NO: 24 are not mutated, i.e. retain the nucleotide appearing at those positions in SEQ ID NO: 24.

Accordingly, in another aspect, the agents for use in the method of the present invention each comprise a dengue antigen, wherein:

i) the dengue antigen of serotype 1 is a YF/dengue chimeric dengue virus (i.e. a recipient yellow fever virus in which the genetic backbone of the YF virus has been modified by exchanging the sequences encoding the prM and E proteins of the YF virus by the corresponding sequences of a dengue serotype 1 virus);

ii) the dengue antigen of serotype 2 is a live attenuated dengue virus of serotype 2 which comprises a nucleic acid sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 24;

iii) the dengue antigen of serotype 3 is a YF/dengue chimeric dengue virus (i.e. a recipient yellow fever virus in which the genetic backbone of the YF virus has been modified by exchanging the sequences encoding the prM and E proteins of the YF virus by the corresponding sequences of a dengue serotype 3 virus) and

iv) the dengue antigen of serotype 4 is a YF/dengue chimeric dengue virus (i.e. a recipient yellow fever virus in which the genetic backbone of the YF virus has been modified by exchanging the sequences encoding the prM and E proteins of the YF virus by the corresponding sequences of a dengue serotype 4 virus).

Preferably, said recipient YF virus (which forms the genetic backbone of the YF/dengue chimeric viruses of serotypes 1 , 3 and 4) is an attenuated YF virus. For example, said recipient YF virus may be an attenuated YF virus selected from the group consisting of YF 17D, YF 17DD and YF 17D204. Preferably, the YF/dengue chimeric viruses of serotypes 1 , 3 and 4 are respectively a Chimerivax dengue serotype 1 (i.e. a CYD-1 ), a Chimerivax dengue serotype 3 (i.e. a CYD-3) and a Chimerivax dengue serotype 4 (i.e. a CYD-4).

A reference herein to a nucleic acid sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 24 may preferably be read as a nucleic acid sequence having at least 92%, at least 94%, at least 96%, at least 98%, at least 99% or 100% sequence identity to the sequence as set forth in SEQ ID NO: 24. Preferably the nucleotides at the positions within said nucleic acid sequences (that have at least 90% at least 92%, at least 94%, at least 96%, at least 98% or at least 99% sequence identity to the sequence as set forth in SEQ ID NO: 24) which correspond to positions 736, 1619, 4723, 5062, 9191 , 10063, 10507, 57, 524, 2055, 2579, 4018, 5547, 6599 and 8571 of SEQ ID NO: 24 are not mutated, i.e. retain the nucleotide appearing at those positions in SEQ ID NO: 24.

Advantageously, when an agent for use in a method according to the present invention comprises a dengue antigen of serotype 2 (for example for use in combination with a dengue antigen of each of serotypes 1 , 3 and 4 as described above and elsewhere herein (e.g. dengue antigens of serotypes 1 , 3 and 4 which are live attenuated chimeric dengue viruses, e.g. YF/dengue chimeric dengue viruses)), said dengue antigen of serotype 2 is a live attenuated dengue virus which comprises a nucleic acid sequence having 100% sequence identity to the sequence as set forth in SEQ ID NO: 24 or a live attenuated dengue virus which comprises a nucleic acid sequence having at least one and no more than 20 nucleotide substitutions when compared with the sequence as set forth in SEQ ID NO: 24. Preferably said live attenuated dengue virus comprises a nucleic acid having at least one and no more than 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3 or 2 nucleotide substitutions when compared with the sequence as set forth in SEQ ID NO: 24. Preferably the nucleotides at the positions within said nucleic acid sequences which correspond to positions 736, 1619, 4723, 5062, 9191 , 10063, 10507, 57, 524, 2055, 2579, 4018, 5547, 6599 and 8571 of SEQ ID NO: 24 are not mutated, i.e. retain the nucleotide appearing at those positions in SEQ ID NO: 24.

Advantageously, when an agent for use in a method according to the present invention comprises a dengue antigen of serotype 2 (for example for use in combination with a dengue antigen of each of serotypes 1 , 3 and 4 as described above and elsewhere herein (e.g. dengue antigens of serotypes 1 , 3 and 4 which are live attenuated chimeric dengue viruses, e.g. YF/dengue chimeric dengue viruses)), said dengue antigen of serotype 2 is a live attenuated dengue virus which comprises a nucleic acid sequence having 100% sequence identity to the sequence as set forth in SEQ ID NO: 24 or a live attenuated dengue virus which comprises a nucleic acid sequence having at least one and no more than 20 base mutations, deletions or insertions when compared with the sequence as set forth in SEQ ID NO: 24. Preferably, said live attenuated dengue virus of serotype 2 comprises a nucleic acid sequence that has at least one and no more than 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3 or 2 base mutations, deletions or insertions when compared with the sequence as set forth in SEQ ID NO: 24. Preferably the nucleotides at the positions within said nucleic acid sequence that correspond to positions 736, 1619, 4723, 5062, 9191 , 10063, 10507, 57, 524, 2055, 2579, 4018, 5547, 6599 and 8571 of SEQ ID NO: 24 are not mutated, i.e. retain the nucleotide appearing at those positions in SEQ ID NO: 24.

It is also preferred that when an agent for use in a method according to the present invention comprises a dengue antigen of serotype 2 (e.g. a dengue antigen which is a live attenuated dengue virus or a live attenuated chimeric dengue virus of serotype 2) said dengue antigen is capable of inducing neutralizing antibodies in humans and is capable of inducing a balanced immune response when used in the context of a tetravalent dengue vaccine composition. It is also preferred that said dengue antigen of serotype 2 results in low or absent viremia in humans. It is also preferred that said dengue antigen of serotype 2 (when used in the method of the present invention in the context of an agent comprising a dengue antigen of serotype 1 , an agent comprising a dengue antigen of serotype 3 and an agent comprising a dengue antigen of serotype 4) provides an improved neutralising antibody response against dengue virus of serotype 2 when compared with the neutralising antibody response generated by CYD-1 , CYD-2, CYD-3 and CYD-4 as defined in Example 1 .

Advantageously, the agents for use in the method of the present invention each comprise a dengue antigen, wherein: (i) the dengue antigen of serotype 1 is a live attenuated chimeric dengue virus other than CYD-1 or said dengue antigen of serotype 1 is CYD-1 ; (ii) the dengue antigen of serotype 2 is a live attenuated dengue virus, other than VDV-2, which comprises a nucleic acid sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 24 or said dengue antigen of serotype 2 is VDV-2; (iii) the dengue antigen of serotype 3 is a live attenuated chimeric dengue virus other than CYD-3 or said dengue antigen of serotype 3 is CYD-3 and (iv) the dengue antigen of serotype 4 is a live attenuated chimeric dengue virus other than CYD-4 or said dengue antigen of serotype 4 is CYD-4. In this context, the VDV-2 strain is the strain derived from the DEN-2 16681/PDK53 strain (LAV2) by subsequent adaptation to Vera cells, wherein said VDV-2 strain has 10 additional mutations in comparison with the DEN-2 16681/PDK53 strain including four silent mutations. Advantageously, the agents for use in the method of the present invention each comprise a dengue antigen, wherein: (i) the dengue antigen of serotype 1 is a live attenuated chimeric dengue virus other than CYD-1 or said dengue antigen of serotype 1 is CYD-1 ; (ii) the dengue antigen of serotype 2 is a live attenuated dengue virus, other than VDV-2, which comprises a nucleic acid sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 24 or said dengue antigen of serotype 2 is VDV-2; (iii) the dengue antigen of serotype 3 is a live attenuated chimeric dengue virus other than CYD-3 or said dengue antigen of serotype 3 is CYD-3 and (iv) the dengue antigen of serotype 4 is a live attenuated chimeric dengue virus other than CYD-4 or said dengue antigen of serotype 4 is CYD-4. In this context, the VDV-2 strain is the strain comprising the nucleic acid sequence as set forth in SEQ ID NO: 24.

Advantageously, the agents for use in the method of the present invention each comprise a dengue antigen, wherein the dengue antigens of serotypes 1 , 3 and 4 are each a live attenuated chimeric dengue virus and said dengue antigen of serotype 2 is a live attenuated dengue virus which comprises a nucleic acid sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 24 and wherein said dengue antigens of serotypes 1 , 2, 3 and 4 are not CYD-1 , VDV-2, CYD-3 and CYD-4 respectively. Preferably said nucleic acid sequence has at least 92%, at least 94%, at least 96%, at least 98% or at least 99% sequence identity to the sequence as set forth in SEQ ID NO: 24. Preferably the nucleotides at the positions within said nucleic acid sequences (that have at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% sequence identity to the sequence as set forth in SEQ ID NO: 24) which correspond to positions 736, 1619, 4723, 5062, 9191 , 10063, 10507, 57, 524, 2055, 2579, 4018, 5547, 6599 and 8571 of SEQ ID NO: 24 are not mutated, i.e. retain the nucleotide appearing at those positions in SEQ ID NO: 24.

Advantageously, in another aspect, the agents for use in the method of the present invention each comprise a dengue antigen, wherein:

i) the dengue antigen of serotype 1 is a YF/dengue chimeric dengue virus other than a CYD-1 or the dengue antigen of serotype 1 is a CYD-1 ;

ii) the dengue antigen of serotype 2 is a live attenuated dengue virus of serotype 2 which comprises a nucleic acid sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 24, wherein said dengue antigen of serotype 2 is not a live attenuated dengue virus of serotype 2 which comprises a nucleic acid sequence having 100% sequence identity to the sequence as set forth in SEQ ID NO: 24 or the dengue antigen of serotype 2 is a live attenuated dengue virus of serotype 2 which comprises a nucleic acid sequence having 100% sequence identity to the sequence as set forth in SEQ ID NO: 24; iii) the dengue antigen of serotype 3 is a YF/dengue chimeric dengue virus other than a CYD-3 or the dengue antigen of serotype 3 is a CYD-3; and

iv) the dengue antigen of serotype 4 is a YF/dengue chimeric dengue virus other than a CYD-4 or the dengue antigen of serotype 4 is a CYD-4.

In this aspect of the invention, said nucleic acid sequence having at least 90% sequence identity to the sequence as set forth in SEQ ID NO: 24 preferably has at least 92%, at least 94%, at least 96%, at least 98% or at least 99% sequence identity to the sequence as set forth in SEQ ID NO: 24. Preferably the nucleotides at the positions within said nucleic acid sequences (that have at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% sequence identity to the sequence as set forth in SEQ ID NO: 24) which correspond to positions 736, 1619, 4723, 5062, 9191 , 10063, 10507, 57, 524, 2055, 2579, 4018, 5547, 6599 and 8571 of SEQ ID NO: 24 are not mutated, i.e. retain the nucleotide appearing at those positions in SEQ ID NO: 24.

An agent comprising a dengue antigen of serotype 2 which is advantageous for use in the method of the present invention (for example for use in combination with an agent comprising a dengue antigen of serotype 1 , an agent comprising a dengue antigen of serotype 3 and an agent comprising a dengue antigen of serotype 4 as described above and elsewhere herein (e.g. dengue antigens of serotypes 1 , 3 and 4 which are YF/dengue chimeric dengue viruses)) is an agent comprising a dengue antigen of serotype 2 wherein said dengue antigen is a chimeric dengue virus comprising a nucleic acid sequence having at least 90% identity, at least 92%, at least 94%, at least 96%, at least 98%, at least 99% or 100% sequence identity to the sequence as set forth in SEQ ID NO: 25. Preferably the nucleotides at the positions within said nucleic acid sequence which correspond to positions 524, 736, 1619 and 2055 of SEQ ID NO: 24 are not mutated (i.e. maintain the nucleotide appearing in SEQ ID NO: 24 at those positions).

An agent comprising a dengue antigen of serotype 2 which is advantageous for use in the method of the present invention (for example for use in combination with an agent comprising a dengue antigen of serotype 1 , an agent comprising a dengue antigen of serotype 3 and an agent comprising a dengue antigen of serotype 4 as described above and elsewhere herein (e.g. dengue antigens of serotypes 1 , 3 and 4 which are YF/dengue chimeric dengue viruses)) is an agent comprising a dengue antigen of serotype 2 wherein said dengue antigen is a chimeric dengue virus comprising a nucleic acid sequence having at least 90% identity, at least 92%, at least 94%, at least 96%, at least 98%, at least 99% or 100% sequence identity to the prM-E nucleotide sequence from the LAV-2 strain (i.e. the RNA equivalent of SEQ ID NO: 4). Preferably the nucleotides at the positions within said prM-E nucleotide sequence which correspond to positions 524, 736, 1619 and 2055 of SEQ ID NO: 24 are not mutated (i.e. maintain the nucleotide appearing in the RNA equivalent of SEQ ID NO: 4 at those positions). Preferably said dengue antigen is not CYD-LAV. An agent comprising a dengue antigen of serotype 2 which is advantageous for use in the method of the present invention (for example for use in combination with an agent comprising a dengue antigen of serotype 1 , an agent comprising a dengue antigen of serotype 3 and an agent comprising a dengue antigen of serotype 4 as described above and elsewhere herein (e.g. dengue antigens of serotypes 1 , 3 and 4 which are YF/dengue chimeric dengue viruses)) is an agent comprising a dengue antigen of serotype 2 wherein said dengue antigen is a live attenuated dengue virus or a chimeric dengue virus comprising a nucleic acid sequence having at least 90% identity, at least 92%, at least 94%, at least 96%, at least 98%, at least 99% or 100% sequence identity to the prM-E nucleotide sequence from the MD-1280 strain (i.e. the RNA equivalent of SEQ ID NO: 7). Preferably said dengue antigen is not CYD-MD.

An agent comprising a dengue antigen of serotype 2 which is advantageous for use in the method of the present invention (for example for use in combination with an agent comprising a dengue antigen of serotype 1 , an agent comprising a dengue antigen of serotype 3 and an agent comprising a dengue antigen of serotype 4 as described above and elsewhere herein (e.g. dengue antigens of serotypes 1 , 3 and 4 which are YF/dengue chimeric dengue viruses)) is an agent comprising a dengue antigen of serotype 2 wherein said dengue antigen is live attenuated dengue virus or a chimeric dengue virus comprising a nucleic acid sequence having at least 90% identity, at least 92%, at least 94%, at least 96%, at least 98%, at least 99% or 100% sequence identity to the prM-E nucleotide sequence of the clinical trial circulating strain (i.e. the RNA equivalent of SEQ ID NO: 1 ).

Preferably an agent which comprises a dengue antigen of serotype 2 for use in the method of the present invention comprises a polypeptide comprising: (i) a sequence having at least one and no more than five amino acid substitutions with respect to the sequence as set forth in SEQ ID NO: 13; (ii) a sequence having at least one and no more than five amino acid substitutions with respect to the sequence as set forth in SEQ ID NO: 14; (iii) a sequence having at least one and no more than five amino acid substitutions with respect to the sequence as set forth in SEQ ID NO: 15; (iv) a sequence having at least one and no more than five amino acid substitutions with respect to the sequence as set forth in SEQ ID NO: 16 or (v) a sequence having at least one and no more than five amino acid substitutions with respect to the sequence as set forth in SEQ ID NO: 17. Preferably said sequences (i) to (v) have at least one and no more than four amino acid substitutions, at least one and no more than three amino acid substitutions, at least one and no more than two amino acid substitutions or only one amino acid substitution. Preferably said sequences comprise a valine residue at the position within the polypeptide that corresponds to position 251 of SEQ ID NO: 12. Preferably, said sequences comprise a threonine residue at the position within the polypeptide that corresponds to position 226 of SEQ ID NO: 12, a glycine residue at the position within the polypeptide that corresponds to position 228 of SEQ ID NO: 12 and a valine residue at the position within the polypeptide that corresponds to position 251 of SEQ ID NO: 12.

Preferably, an agent comprising a dengue antigen of serotype 2, e.g. a dengue virus of serotype 2 such as a live attenuated dengue virus or a live attenuated chimeric dengue virus, for use in a method according to the present invention comprises a polypeptide having the sequence as set forth in SEQ ID NO: 28. In a preferred embodiment, the amino acids at positions X₂, X₃, Xg and Xii of SEQ ID NO: 28 are Asn, Thr, Gly and His respectively. In another preferred embodiment, the amino acids at positions Xi and X₃ of SEQ ID NO: 28 are lie and Thr respectively. In another preferred embodiment, the amino acids at positions X-i, X₂, X3, Xs and Xn of SEQ ID NO: 28 are Met, Asn, Arg, Thr and His respectively. In another preferred embodiment, the amino acids at positions Xi, X₂, X₃, X₈, Xg, X11 , X12 and X13 of SEQ ID NO: 28 are He, Asn, Thr, Thr, Gly, His, Ser and lie respectively. In another preferred embodiment, the amino acids at positions X-i , X₂, X₃, X₈, X₉ and Xn of SEQ ID NO: 28 are lie, Arg, Thr, Lys, Glu and Tyr respectively.

Preferably, the agents comprising a dengue antigen of serotype 2 as described herein comprise an E protein that comprises a valine residue at the position corresponding to position 251 of SEQ ID NO: 12 an M protein that comprises a threonine residue at the position corresponding to position 34 of SEQ ID NO: 23. Preferably, the agents comprising a dengue antigen of serotype 2 as described herein comprise a nucleotide sequence encoding a prM and E protein, wherein the prM and E protein sequences encoded by said nucleotide sequence comprise a leucine residue at position 24 of prM, a threonine residue at position 125 of prM and a valine residue at position 251 of E (wherein the prM and M sequences are numbered together, i.e. from position 1 to position 166 and the E sequence is numbered separately from position 1 to position 495).

Preferably, a dengue antigen of serotype 2 for use in a method as described herein is not LAV2. It is also preferred that a dengue antigen of serotype 2 for use in a method as described herein is not VDV2. It is also preferred that a dengue antigen of serotype 2 for use in a method as described herein is not CYD-2. Preferably, a dengue antigen of serotype 2 for use in a method as described herein is not CYD-LAV. Preferably, a dengue antigen of serotype 2 for use in a method as described herein is not CYD-BID. Preferably, a dengue antigen of serotype 2 for use in a method as described herein is not CYD-PR. Preferably, a dengue antigen of serotype 2 for use in a method as described herein is not CYD-MD.

Preferably a dengue antigen of serotype 3 for use in a method according to the present invention is selected from the group consisting of CYD-3 or an alternative YF/DEN-3 chimeric virus. Preferably a dengue antigen of serotype 3 for use in a method according to the present invention is CYD-3.

Preferably a dengue antigen of serotype 4 for use in a method according to the present invention is selected from the group consisting of CYD-4 or an alternative YF/DEN-4 chimeric virus. Preferably a dengue antigen of serotype 4 for use in a method according to the present invention is CYD-4.

The dengue antigens of serotypes 1 , 2, 3 and 4 as described herein may be combined together in any combination possible for use as agents according to the present invention. In other words, the agents according to the present invention may be formed by independently selecting a dengue antigen of serotype 1 as described herein, a dengue antigen of serotype 2 as described herein, a dengue antigen of serotype 3 as described herein and a dengue antigen of serotype 4 as described herein. For example, the dengue antigens of serotypes 1 , 2, 3 and 4 according to the present invention may respectively be: (i) CYD-1 , CYD-2, CYD-3 and CYD-4; (ii) CYD-1 , CYD-LAV, CYD-3 and CYD-4; iii) CYD-1 , CYD-BID, CYD-3 and CYD-4; (iv) CYD-1 , CYD-PR, CYD-3 and CYD-4 or (v) CYD-1 , CYD-MD, CYD-3 and CYD-4; (vi) CYD-1 , LAV-2, CYD-3 and CYD-4; (vii) CYD-1 , VDV-2, CYD-3 and CYD-4.

When a dengue antigen of serotype 1 according to the present invention is CYD-1 , a dengue antigen of serotype 3 according to the present invention is CYD-3 and a dengue antigen of serotype 4 according to the present invention is CYD-4, a dengue antigen of serotype 2 according to the present invention may be any of the dengue antigens of serotype 2 as described herein. For example, such a dengue antigen of serotype 2 may be a DEN-2 chimeric virus, e.g. a YF/DEN-2 chimeric virus, comprising a polypeptide having at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NO: 13 (i.e. LAV-2 E protein sequence), SEQ ID NO: 14 (i.e. BID-V585 E protein sequence), PR/DB023 SEQ ID NO: 15 (i.e. PR/DB023 protein sequence), SEQ ID NO: 16 (i.e. MD1280 E sequence) or SEQ ID NO: 17.

When a dengue antigen of serotype 1 according to the present invention is CYD-1 , a dengue antigen of serotype 3 according to the present invention is CYD-3 and a dengue antigen of serotype 4 according to the present invention is CYD-4, a dengue antigen of serotype 2 according to the present invention may be a DEN-2 chimeric virus, e.g. a YF/DEN-2 chimeric virus, comprising a nucleotide sequence encoding prM and E proteins, wherein said nucleotide sequence has at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to the nucleotide sequence encoding prM and E proteins from the serotype 2 strains LAV-2 (i.e. SEQ ID NO: 4), BID-V585 (i.e. SEQ ID NO: 5), PR/DB023 (i.e. SEQ ID NO: 6) or MD1280 (SEQ ID NO: 7) or wherein said nucleotide sequence has at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 1 .

In further embodiments, the dengue antigens of serotypes 1 , 3 and 4 for use in a method according to the present invention may respectively be: a dengue antigen, e.g. a dengue virus, comprising the E protein sequence of CYD-1 , a dengue antigen, e.g. a dengue virus, comprising the E protein sequence of CYD-3 and a dengue antigen, e.g. a dengue virus, comprising the E protein sequence of CYD-4. In such embodiments, the dengue antigen of serotype 2, e.g. a dengue virus, may comprise a polypeptide having at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NO: 13 (i.e. LAV-2 E protein sequence), SEQ ID NO: 14 (i.e. BID-V585 E protein sequence), PR/DB023 SEQ ID NO: 15 (i.e. PR/DB023 protein sequence), SEQ ID NO: 16 (i.e. MD1280 E sequence) or SEQ ID NO: 17.

Preferably, an inactivated or a live attenuated chimeric dengue virus according to the present invention comprises an envelope (E) protein from a dengue virus of a first serotype and one or more proteins other than an envelope protein from: (i) a second dengue virus serotype, said first and said second serotype being different from each other, or (ii) a flavivirus other than a dengue virus. Preferably, an inactivated or a live attenuated chimeric dengue virus according to the present invention comprises an envelope (E) protein and a membrane (M) protein from a dengue virus of a first serotype and one or more proteins other than an envelope protein or a membrane protein from: (i) a second dengue virus serotype, said first and said second serotype being different from each other, or (ii) a flavivirus other than a dengue virus. Preferably said flavivirus other than a dengue virus is a yellow fever virus. Preferably said flavivirus other than a dengue virus is an attenuated yellow fever virus. Preferably the flavivirus other than a dengue virus is an attenuated yellow fever virus selected from the group consisting of the attenuated virus strain YF17D, an attenuated YF17D derivative strain including YF17DD and YF17D204 (YF-VAX®) and any other attenuated yellow fever virus strain as described in Monath, Expert Rev. Vaccines 4(4), 553-574 (2005).

The exact quantity of a dengue virus to be administered to a mammal according to the invention may vary, for example, according to the age and the weight of the mammal or the frequency of administration. Generally, where a dengue antigen according to the present invention is a live attenuated dengue virus or a live attenuated chimeric dengue virus, the amount of said virus comprised in a agent of the present invention lies within a range of from about 10³ to about 10⁶ CCID₅₀, for example within a range of from about 5 x 10³ to about 5 x 10⁵ CCID₅₀, for example within a range of from about 1 x 10⁴ to about 1 x 10⁵ CCID₅₀, for example about 10⁵ CCID₅₀. Generally, where a dengue antigen according to the present invention is an inactivated dengue virus, the quantity of said inactivated dengue virus comprised in a agent of the present invention lies within a range of from about 10⁴ to about 10⁸ CCID₅₀ equivalent, preferably within a range of from about 5 x 10⁴ to about 5 x 10⁷ CCID₅₀ equivalent, preferably within a range of from about 1 x 10⁴ to about 1 x 10⁶ CCID₅₀ equivalent, preferably about 10⁵ CCID₅₀ equivalent. Generally, where a dengue antigen according to the present invention is a VLP, the quantity of a VLP comprised in a agent of the present invention lies within a range of from about 100 ng to about 100 ig of said VLP, preferably within a range of from about 100 ng to about 50 [ig, preferably within a range of from about 100 ng to about 20 [ig, preferably about 1 ig to 10 ig. The amount of VLP can be determined by ELISA. Advantageously, a agent according to the present invention comprises an effective amount of a dengue antigen as defined herein.

The agent(s) and the vaccine(s) for use in a method according to the present invention may further comprise a pharmaceutically acceptable carrier or excipient. A pharmaceutically acceptable carrier or excipient according to the present invention means any solvent or dispersing medium etc., commonly used in the formulation of pharmaceuticals and vaccines to enhance stability, sterility and deliverability of the active agent and which does not produce any secondary reaction, for example an allergic reaction, in mammals, e.g. in humans. The excipient is selected on the basis of the pharmaceutical form chosen, the method and the route of administration. Appropriate excipients, and requirements in relation to pharmaceutical formulation, are described in "Remington's Pharmaceutical Sciences" (19th Edition, A.R. Gennaro, Ed., Mack Publishing Co., Easton, PA (1995)). Particular examples of pharmaceutically acceptable excipients include water, phosphate-buffered saline (PBS) solutions and a 0.3% glycine solution. A agent according to the present invention may advantageously comprise 0.4% saline and 2.5% human serum albumin (HSA).

The agent(s) and the vaccine(s) for use in a method according to the present invention may optionally contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, human serum albumin, essential amino acids, nonessential amino acids, L-arginine hydrochlorate, saccharose, D-trehalose dehydrate, sorbitol, tris(hydroxymethyl)aminomethane and/or urea. In addition, the agents for use in a method may optionally comprise pharmaceutically acceptable additives including, for example, diluents, binders, stabilizers, and preservatives. Preferably a composition comprising a live attenuated dengue virus or a live attenuated chimeric dengue virus according to the present invention further comprises a stabilizer as described in WO 2010/003,670. For example, said stabilizer comprises (in an aqueous solution without proteins of animal origin and without added salts having divalent cations), a buffer, 2.5% to 6.5% of sorbitol, 2.5% to 13% of sucrose, 0 to 7.5% of trehalose and/or 0 to 7.5% of any other disaccharide or trisaccharide, 0.2% to

0.5% of urea and 0.8% to 2.5% of an amino acid mixture comprising arginine (Arg), cystine (Cys- Cys), histidine (His), isoleucine (lie), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), valine (Val), alanine (Ala), asparagine (Asn), aspartic acid (Asp), glutamb acid (Glu), glycine (Gly), proline (Pro) and serine (Ser). Preferably, said stabilizer comprises one or more buffers chosen from the group comprising TRIS (tris(hydroxymethyl)aminomethane), HEPES (2-(4-(2-hydroxyethyl)-1 -piperazinyl)ethane-sulfonic acid), potassium phosphate and sodium phosphate. When said stabilizer comprises TRIS, the TRIS is present at a concentration of from about 5 to about 10 mM. When said stabilizer comprises HEPES, the HEPES is present at a concentration of from about 7.5 to about 20 mM. Preferably, the stabilizer comprises 3.8% (w/v) of sorbitol, 7.5% (w/v) of sucrose, 5.5% (w/v) of trehalose, 0.25% (w/v) of urea and 1 .5% (w/v) of the above-described amino acid mixture.

The agent(s) according to the present invention may be administered in multiple doses. Doses of such agents may be administered in an initial dosage regimen followed by booster administrations. For example, such agents may be administered in an initial dosage regimen comprising one, two or three doses or more than three doses, e.g. four doses. Preferably, the first dose and the third dose of the initial dosage regimen are to be administered approximately twelve months apart. For example, an initial dosage regimen according to the present invention comprises three doses, wherein the first and third doses of said dosage regimen are to be administered approximately twelve months apart. Advantageously, the agents which comprise a dengue antigen or which comprise a nucleic acid construct or viral vector which is able to express a dengue VLP according to the present invention are to be administered in a first dose, a second dose and a third dose. In such an embodiment, said first dose and said third dose may be administered approximately twelve months apart. For instance, said agents may be administered in a first dose, a second dose and a third dose, wherein said second dose is to be administered about six months after said first dose and wherein said third dose is to be administered about twelve months after said first dose. Alternatively, said second dose is to be administered at about three to four months (e.g. at about three-and-a-half months) after said first dose and said third dose is to be administered about twelve months after said first dose. Advantageously, said first dose is administered to a human subject who is about 12 months of age.

The measles, mumps and rubella vaccines may be administered in conjunction with any of the doses of the agents which comprise a dengue antigen or which comprise a nucleic acid construct or viral vector which is able to express a dengue VLP according to the present invention. Preferably, the measles, mumps and rubella vaccines are administered in conjunction with either the first or the second dose of the agents which comprise a dengue antigen or which comprise a nucleic acid construct or viral vector which is able to express a dengue VLP according to the present invention. Preferably, the measles, mumps and rubella vaccines are administered in conjunction with the first dose of the agents which comprise a dengue antigen or which comprise a nucleic acid construct or viral vector which is able to express a dengue VLP according to the present invention.

Advantageously, when the agents according to the present invention are administered in conjunction with a measles vaccine, said agents and said measles vaccine are administered in conjunction to a mammal, e.g. a human, who is about 12 months of age. An agent according to the present invention may be administered in two doses. Preferably, the first dose and the second dose are to be administered approximately about six to twelve months after the first dose. Preferably, the second dose is to be administered at eight months after the first dose. Preferably the second dose is administered at about eight-and-a-half to nine months after the first dose. In this embodiment of the invention, the measles, mumps and rubella vaccines are administered in conjunction with either of the doses of the agent which comprises a dengue antigen or which comprise a nucleic acid construct or viral vector which is able to express a dengue VLP according to the present invention

An agent according to the present invention may be administered in a single dose. In this embodiment of the invention, said agent is administered in conjunction with the measles, mumps and rubella vaccines.

Preferably when the agents and the vaccines of the present invention are co-administered to a mammal, e.g. a human, the agent(s) and the vaccine(s) of the present invention are administered to said mammal, e.g. a human, at distinct anatomical sites (i.e. the agent(s) at one anatomical site and the vaccine(s) at a distinct anatomical site. By distinct anatombal sites, it is meant anatomical sites that are drained by different lymphatic nodes. For example the right arm and the left arm are considered to be distinct sites, Further non-limiting examples include right arm/right thigh, left arm/left thigh, right arm/left thigh, left arm/right thigh etc. The vaccine(s) and the agent(s) are advantageously administered in the upper arm and in the thigh, respectively. However, they may also be respectively administered in the left and right upper arms or in the left and right thighs or vice versa. When administered in the upper arm, the deltoid region is preferred.

The agents of the present invention may be administered to a mammal, e.g. a human, in accordance with the following procedure:

(1 ) At time TO_, a human being is injected subcutaneously in one arm with a tetravalent composition comprising 10^5± CCID₅₀ of each of CYD-1 to 4 and subcutaneously in the other arm with an MMR vaccine. Both injections are performed within a period of 3 hours. In one embodiment, the human being is less than 36 months of age at the time T₀. In one embodiment, the MMR vaccine is Trimovax® (Sanofi Pasteur Lyon FR).

(2) Approximately six months after TO (at time T1 ), the same human being who received the initial immunization described above, receives a second subcutaneous administration of a tetravalent composition comprising 10^5± CCID₅₀ of each of CYD-1 to 4. No MMR vaccine is required to be administered at this time.

(3) Approximately six months after T1 (at time T2), the same human being who received the initial immunization described above, receives a third subcutaneous administration of a tetravalent composition comprising 10^5± CCID₅₀ of each of CYD-1 to 4. No MMR vaccine is required to be administered at this time.

Preferably, the measles, mumps and rubella vaccines for use in a method according to the present invention are present in a single trivalent measles, mumps and rubella (MMR) vaccine composition. Alternatively, the measles, mumps and rubella vaccines for administration in conjunction with the agents according to the present invention are present as three separate monovalent vaccines. Alternatively, the measles, mumps and rubella vaccines for administration in conjunction with the agents according to the present invention are present in a bivalent vaccine composition and a separate monovalent vaccine composition. In such an embodiment, any two of the measles, mumps and rubella vaccines may be present together in the bivalent vaccine composition. Where the measles mumps and rubella vaccines are present in a multivalent composition, said vaccines may be provided and administered as a multivalent vaccine composition or said vaccines may be provided as separate vaccines and then combined into a multivalent vaccine composition prior to administration.

Advantageously, the present invention provides an agent which comprises a dengue antigen of serotype 1 or a nucleic acid construct or viral vector which is able to express a dengue virus-like particle (VLP) of serotype 1 ; an agent which comprises a dengue antigen of serotype 2 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 2; an agent which comprises a dengue antigen of serotype 3 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 3; and an agent which comprises a dengue antigen of serotype 4 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 4; wherein each dengue antigen is independently selected from the group consisting of:

(a) a live attenuated dengue virus;

(b) an inactivated dengue virus;

(c) a live attenuated or inactivated chimeric dengue virus; and

(d) a dengue virus-like particle (VLP);

for use in a method of protecting a mammal against dengue disease, wherein said method comprises the administration of said agents to said mammal in conjunction with a measles vaccine.

(a) a live attenuated dengue virus;

(b) an inactivated dengue virus;

(c) a live attenuated or inactivated chimeric dengue virus; and

(d) a dengue virus-like particle (VLP);

for use in a method of protecting a mammal against dengue disease, wherein said method comprises the administration of said agents to said mammal in conjunction with a measles vaccine, a mumps vaccine and a rubella vaccine.

Advantageously, the antibodies induced by the measles vaccine or the measles, mumps and rubella vaccines according to the present invention are neutralising antibodies.

Advantageously, the present invention provides a measles vaccine for use in a method of protecting a mammal against measles, wherein said method comprises the administration of said measles vaccine to said mammal in conjunction with an agent comprising a dengue antigen of serotype 1 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 1 , an agent comprising a dengue antigen of serotype 2 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 2, an agent comprising a dengue antigen of serotype 3 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 3 and an agent comprising a dengue antigen of serotype 4 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 4; wherein each dengue antigen is independently selected from the group consisting of:

(a) a live attenuated dengue virus;

(b) an inactivated dengue virus;

(c) a live attenuated or inactivated chimeric dengue virus; and

(d) a dengue virus-like particle (VLP).

Advantageously, the present invention provides a measles vaccine, a mumps vaccine and a rubella vaccine for use in a method of protecting a mammal against measles, mumps and rubella, wherein said method comprises the administration of said measles, mumps and rubella vaccines to said mammal in conjunction with an agent comprising a dengue antigen of serotype 1 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 1 , an agent comprising a dengue antigen of serotype 2 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 2, an agent comprising a dengue antigen of serotype 3 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 3 and an agent comprising a dengue antigen of serotype 4 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 4; wherein each dengue antigen is independently selected from the group consisting of:

(a) a live attenuated dengue virus;

(b) an inactivated dengue virus;

(c) a live attenuated or inactivated chimeric dengue virus; and

(d) a dengue virus-like particle (VLP).

A mammal, e.g. a human, according to the present invention may be flavivirus immune, for example a dengue-immune human. A mammal, e.g. a human, according to the present invention may be flavivirus-naive. Advantageously, a mammal, e.g. a human, according to the present invention is flavivirus-immune, for example a dengue-immune human.

Preferably, the agents according to the present invention reduce the likelihood or severity of DHF. A reduction in the likelihood of DHF (i.e. a reduction in the probability of contracting DHF) may be measured by comparing the number of cases of DHF in a group of human subjects, who have received the agents according to the present invention and the number of cases of DHF in a control group of human subjects, who have not received the agents according to the present invention. A reduction in the severity of DHF may be determined by calculating the number of human subjects displaying DHF of each of Grades I, II, III or IV in a group of subjects who have received the agents according to the present invention and comparing those numbers to the equivalent numbers from a control group of human subjects who have not received the agents according to the present invention. For instance, the agents according to the present invention preferably reduce the number of cases of Grade I DHF, the number of cases of Grade II DHF, the number of cases of Grade III DHF and/or the number of cases of Grade IV DHF in those human subjects receiving the agents, when compared to the equivalent number of cases Grade I DHF, Grade II DHF, Grade III DHF and Grade IV DHF occurring in a control group of human subjects who have not received the agents according to the present invention.

Dengue disease, as defined herein, may be caused by any one of two serotypes of a dengue virus. For example, dengue disease is preferably caused by a dengue virus of serotype 1 or serotype 3, a dengue virus of serotype 1 or serotype 4, a dengue virus of serotype 3 or serotype 4, a dengue virus of serotype 1 or serotype 2, a dengue virus of serotype 2 or serotype 3, a dengue virus of serotype 2 or serotype 4. Dengue disease, as defined herein, is preferably caused by any one of three serotypes of a dengue virus. For example, dengue disease is preferably caused by a dengue virus of serotype 1 , 2 or 3, a dengue virus of serotype 1 , 3 or 4, a dengue virus of serotype 1 , 2 or 4, a dengue virus of serotype 2, 3 or 4. In another embodiment, dengue disease is caused by a dengue virus of serotype 1 , a dengue virus of serotype 2, a dengue virus of serotype 3 or a dengue virus of serotype 4.

The agents of the present invention may comprise one or more adjuvants to enhance the immunogenicity of the dengue antigen. Those skilled in the art will be able to select an adjuvant which is appropriate in the context of this invention. An adjuvant is preferably used in an agent of the invention comprising an inactivated virus or a VLP. An adjuvant may be used in an agent of the invention comprising a live attenuated virus, as long as said adjuvant does not impact replication.

Suitable adjuvants include an aluminum salt such as aluminum hydroxide gel, aluminum phosphate or alum, but may also be a salt of calcium, magnesium, iron or zinc. Further suitable adjuvants include an insoluble suspension of acylated tyrosine or acylated sugars, cationically or anionically derivatized saccharides, or polyphosphazenes. Alternatively, the adjuvant may be an oil-in-water emulsion adjuvant (EP 0399843), as well as combinations of oil-in-water emulsions and other active agents (WO 95/17210; WO 98/56414; WO 99/12565 and WO 99/1 1241 ). Other oil emulsion adjuvants have been described, such as water-in-oil emulsions (US 5,422,109; EP 0480982) and water-in-oil-in-water emulsions (US 5,424,067; EP 0480981 ). Examples of such adjuvants include MF59, AF03 (WO 2007/006939), AF04 (WO 2007/080308), AF05, AF06 and derivatives thereof. The adjuvant may also be a saponin, lipid A or a derivative thereof, an immunostimulatory oligonucleotide, an alkyl glucosamide phosphate, an oil in water emulsion or combinations thereof. Examples of saponins include Quil A and purified fragments thereof such as QS7 and QS21 .

As appreciated by skilled artisans, the agents of the present invention are suitably formulated to be compatible with the intended route of administration. Examples of suitable routes of administration include for instance intramuscular, transcutaneous, subcutaneous, intranasal, oral or intradermal. Advantageously, the route of administration is subcutaneous or intramuscular.

An agent or a vaccine of the present invention may be administered using a conventional hypodermic syringe or a safety syringe such as those commercially available from Becton Dickinson Corporation (Franklin Lakes, NJ , USA) or jet injectors. For intradermal administration, conventional hypodermic syringes may be employed using the Mantoux technique or specialized intradermal delivery devices such as the BD Soluvia(TM) microinjection system (Becton Dickinson Corporation, Franklin Lakes, NJ, USA), may be used. The volume of an agent of the present invention to be administered will depend on the method of administration. In the case of subcutaneous injections, the volume is generally between 0.1 and 1 .0 ml, preferably approximately 0.5 ml.

According to one embodiment, the invention also provides a kit comprising at least one of the agents and/or at least one of the vaccines of the present invention and instructions for the use of said at least one agent and/or at least one vaccine in a method according to the present invention. The kit can comprise at least one dose (typically in a syringe) of any of the agents or vaccines contemplated herein or a combination thereof. Advantageously, a kit according to the present invention comprises: a container comprising an agent which comprises a dengue antigen of serotype 1 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 1 as described herein; a container comprising an agent which comprises a dengue antigen of serotype 2 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 2 as described herein; a container comprising an agent which comprises a dengue antigen of serotype 3 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 3 as described herein and a container comprising an agent which comprises a dengue antigen of serotype 4 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 4 as described herein along with instructions for the use of said agents in a method according to the present invention. Advantageously, a kit according to the present invention comprises a container comprising a tetravalent composition comprising a dengue antigen of serotype 1 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 1 as described herein; a dengue antigen of serotype 2 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 2 as described herein; a dengue antigen of serotype 3 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 3 as described herein and a dengue antigen of serotype 4 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 4 as described herein, along with instructions for the use of said tetravalent composition in a method according to the present invention. Advantageously, a kit according to the present invention comprises a container comprising a measles vaccine as described herein; a container comprising a mumps vaccine as described herein and a container comprising a rubella vaccine as described herein, along with instructions for the use of said agents in a method according to the present invention. Advantageously, a kit according to the present invention comprises a container comprising a trivalent MMR vaccine composition as described herein, along with instructions for the use of said vaccine composition in a method according to the present invention. Advantageously, a kit according to the present invention comprises a first container comprising a tetravalent composition comprising a dengue antigen of serotype 1 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 1 as described herein; a dengue antigen of serotype 2 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 2 as described herein; a dengue antigen of serotype 3 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 3 as described herein and a dengue antigen of serotype 4 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 4 as described herein and a second container comprising a trivalent MMR vaccine composition as described herein. Advantageously, a kit according to the present invention comprises a first container comprising a tetravalent composition comprising a dengue antigen of serotype 1 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 1 as described herein; a dengue antigen of serotype 2 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 2 as described herein; a dengue antigen of serotype 3 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 3 as described herein and a dengue antigen of serotype 4 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 4 as described herein and a second container comprising a measles vaccine as described herein. Advantageously, a kit according to the present invention comprises a container comprising a heptavalent composition comprising a dengue antigen of serotype 1 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 1 as described herein; a dengue antigen of serotype 2 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 2 as described herein; a dengue antigen of serotype 3 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 3 as described herein and a dengue antigen of serotype 4 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 4 as described herein; a measles vaccine as described herein, a mumps vaccine as described herein and a rubella vaccine as described herein.

If the agents and vaccines in the kits as described herein are provided in lyophilized form, the kit will advantageously comprise at least one additional container holding a solution which can be used to reconstitute the lyophilisate. Pharmaceutically acceptable diluents and carriers as described herein may be used for reconstitution.

Optionally, the container(s) in the kits as described herein can be associated with administration means and/or instructions for use. Examples of administration means may include syringes for parenteral administration or delivery systems to facilitate intradermal administration.

The efficacy of the agents of the present invention in reducing the likelihood or severity of dengue disease may be measured in a number of ways. For instance the efficacy of the agents of the present invention in reducing the likelihood or severity of symptomatic virologically-confirmed dengue disease may be calculated by measuring after the administration of at least one dose of said agents (e.g. after administration of one, two or three doses of said agents): (i) the percentage of symptomatic virologically-confirmed dengue cases caused by dengue virus of any serotype;

(ii) the percentage of severe virologically-confirmed dengue cases caused by dengue virus of any serotype;

(iii) the percentage of dengue hemorrhagic fever cases of Grades I to IV caused by dengue virus of any serotype;

(iv) the percentage of DHF cases of Grade I caused by dengue virus of any serotype;

(v) the percentage of DHF cases of Grade II caused by dengue virus of any serotype;

(vi) the percentage of DHF cases of Grade III caused by dengue virus of any serotype; (vii) the percentage of DHF cases of Grade IV caused by dengue virus of any serotype;

(viii) the annual incidence rate of hospitalized virologically-confirmed dengue caused by dengue virus of any serotype; and/or

(ix) the length of hospital stay for symptomatic virologically-confirmed dengue cases caused by dengue virus of any serotype;

in a group of subjects that has received said agents and comparing those measurements with the equivalent measurements from a control group of subjects that have not received said agents, wherein the subjects in both said groups are resident in a Dengue endemic region. A statistically significant reduction in any one or more of (i) to (ix) in the group of subjects receiving the agents of the present invention when compared with the control group of subjects is indicative of the efficacy of the agents according to the present invention. In a preferred embodiment, the efficacy of the agents according to the present invention is demonstrated by a statistically significant reduction of one or more of the measures as described above, wherein the DHF cases or dengue cases are caused by dengue virus of serotypes 1 , 3 or 4.

The efficacy of the agents according to the present invention in reducing the severity or likelihood of dengue disease may also be calculated by measuring after the administration of at least one dose of said agents (e.g. after administration of one, two or three doses of said agents):

(i) the mean duration and/or intensity of fever;

(iii) the mean value for plasma leakage as defined by a change in haematocrit;

(iii) the mean value for thrombocytopenia (platelet count); and/or

(iv) the mean value of the level of liver enzymes including alanine aminotransferase (ALT) and aspartate aminotransferase (AST);

in a group of subjects that have received said agents and who have developed virologically- confirmed dengue disease and comparing those measurements with the equivalent measurements from a control group of subjects that have not received said agents and who have developed virologically-confirmed dengue disease. A statistically significant reduction in any one or more of (i) to (v) in the group of subjects who have received said agents and who have developed virologically-confirmed dengue disease when compared with the control group of subjects who have developed virologically-confirmed dengue disease is indicative of the efficacy of the agents according to the present invention in reducing the severity or likelihood of dengue disease.

Typically the efficacy of the method of protection of the invention against dengue disease, as measured e.g. by the method described in example 1 (E=100*(1 -ID_CYD/IDcontroi), where ID is the incidence density (i.e., the number of human subjects with virologically-confirmed dengue divided by the number of person-years at risk) in each group), is at least 50%, preferably at least 60%, wherein said dengue disease is caused by serotype 1 , 3 or 4. The efficacy of the method of protection being advantageously at least 70%, preferably 80% against a dengue disease caused by serotype 3 or 4. The efficacy of the method of protection being advantageously at least 90% against dengue disease caused by serotype 4.

Percent identity between two amino acid sequences or two nucleotide sequences is determined by standard alignment algorithms such as, for example, Basic Local Alignment Tool (BLAST) described in Altschul et al. (1990) J. Mol. Biol., 215: 403-410, the algorithm of Needleman et al. (1970) J. Mol. Biol., 48: 444-453; the algorithm of Meyers et al. (1988) Comput. Appl. Biosci., 4: 11 -17; or Tatusova et al. (1999) FEMS Microbiol. Lett., 174: 247-250, etc. Such algorithms are incorporated into the BLASTN, BLASTP and "BLAST 2 Sequences" programs (see www.ncbi.nlm.nih.gov/BLAST). When utilizing such programs, the default parameters can be used. For example, for nucleotide sequences the following settings can be used for "BLAST 2 Sequences": program BLASTN, reward for match 2, penalty for mismatch-2, open gap and extension gap penalties 5 and 2 respectively, gap x~dropoff 50, expect 10, word size 1 1 , filter ON. For amino acid sequences the following settings can be used for "BLAST 2 Sequences": program BLASTP, matrix BLOSUM62, open gap and extension gap penalties 1 1 and 1 respectively, gap x~dropoff 50, expect 10, word size 3, filter ON.

It is understood that the various features and preferred aspects of the present invention as disclosed herein may be combined together.

Throughout this application, various references are cited. The disclosures of these references are hereby incorporated by reference into the present disclosure.

The present invention will be further illustrated by the following examples. It should be understood however that the invention is defined by the claims, and that these examples are given only by way of illustration of the invention and do not constitute in any way a limitation thereof.

EXAMPLES

The following Examples are illustrative of the practice of the present invention and not intended to be limiting on the scope of the practice of the present invention as provided above.

Example 1 : One year follow-up in Thailand of patients receiving a tetravalent dengue composition (TDC) comprising Chimerivax™ CYD-1, CYD-2, CYD-3 and CYD-4

Methods

Study design and participants

An observer-blind, randomised, controlled, monocentre, Phase lib trial of the efficacy of the tetravalent Chimerivax™ composition (i.e. TDC comprising the particular CYD-1 strain generated from the prM and E sequences of DEN1 PU0359 (TYP1 140), the particular CYD-2 strain generated from the prM and E sequences of DEN2 PU0218, the particular CYD-3 strain generated from the prM and E sequences of DEN3 PaH881/88 and the particular CYD-4 strain generated from the prM and E sequences of DEN4 1228 (TVP 980), see WO 03/101397 and Guy et al., Vaccine (2011 ), 29(42): 7229-41 ) against virologically-confirmed dengue disease is conducted. 4002 schoolchildren aged 4-1 1 years who are in good health based on medbal history and physical examination are enrolled into the trial. The study is conducted at Ratchaburi Regional Hospital (RRH), Ratchaburi province, Muang district, Thailand. Children with acute febrile illness at enrolment, those with congenital or acquired immunodeficiency, and those receiving immunosuppressive therapy or other prohibited treatments or vaccines are excluded. Participants are randomly assigned 2:1 to receive three doses of TDC or a control product at Months 0, 6 and 12.

Products

Each of the chimeric viruses are produced and cultured on Vera cells as described in WO 03/101397, Guy et al, Vaccine (201 1 ) 29: 7229-7241 ; Guy et al, Hum. Vaccines (2010) 6 (9): 696; Guy et al, Vaccine (2010) 28: 632; Guirakhoo et al, J. Virol. (2000) 74 : 5477 ; Guirakhoo et al, J. Virol. (2001 ) 75 (16) : 7290 ; Guirakhoo et al, Virol. (June 20, 2002) 298: 146; and Guirakhoo et al, J. Virol. (2004) 78 (9): 4761 .

The TDC is presented as a lyophilized powder (previously stored at temperature of between 2°C and 8°C), which is reconstituted with 0.5 imL of solvent for injection (0.4% NaCI containing 2.5% human serum albumin). As reconstituted, each 0.5 imL dose of contains 5 ± 1 log-|₀ CCID₅₀ of each chimeric dengue serotype (1 , 2, 3 and 4) and excipients (essential amino acids, non-essential amino acids, L-arginine chlorhydrate, saccharose, D-trehalose dehydrate, sorbitol, tris(hydroxymethyl)aminoethane and urea). The control product is inactivated rabies vaccine (Verorab^®, Sanofi Pasteur, Lyon France) for the first injection of the first 50 children randomised to the control group, and 0-9% NaCI saline placebo for all other injections. All products are injected subcutaneously into the deltoid region of the upper arm.

Assessments

All children are actively followed to detect acute febrile illness based on daily surveillance of school registers during school terms for absenteeism (followed by phone calls or home visits to absentees), and twice-weekly home visits, phone calls or mobile phone text-messages throughout school holidays. In any case of febrile illness (defined as illness with two temperature readings of >37.5°C at least 4 hours apart) parents are asked to take their child to RRH for diagnosis and treatment. The surveillance system also captures spontaneous consultations at RRH. Consecutive febrile episodes separated by a symptom-free interval of >14 days are considered as separate episodes. Paired serum samples are collected at presentation (i.e., acute sample, collected no later than 7 days after fever onset) and 7-14 days later (convalescent sample) and sent to Sanofi Pasteur's Global Clinical Immunology (GCI) laboratory (Swiftwater, PA, USA) and to the Centre for Vaccine Development (CVD, Mahidol University, Thailand). Acute samples are screened for the presence of flavivirus using an initial RT-PCR assay which detects the presence of any flavivirus (using primers composed of highly conserved flavivirus sequences). Positive samples are tested for wild-type dengue virus with a serotype-specific quantitative RT-PCR, as described herein. In parallel, all acute samples are tested for the presence of dengue NS1 antigen using a commercial ELISA kit (Platelia™, Bio-Rad Laboratories, Marnes-La-Coquette, France). A virologically- confirmed episode of dengue disease is defined as a positive result in either the serotype-specific RT-PCR, or the NS1 antigen ELISA.

Active surveillance is maintained until each participant has been followed for at least 13 months after the third vaccination and until the Independent Data Monitoring Committee (IDMC) confirms that >27 cases have occurred in the per-protocol (PP) population.

All serious adverse events (SAE) are documented until the sixth month after the last vaccination, and thereafter any fatal SAE or vaccine-related SAE.

Dengue immune responses are assessed in the first 300 enrolled children at RRH in sera collected at enrolment and 28 days after each injection. Sera are also prospectively collected from all participants on Day 28 after the third injection to assess immune responses in children with virologically-confirmed dengue occurring from this timepoint. Sera are sent to GCI for measurement of serotype-specific neutralizing antibody titres against the CYD parental dengue viruses using the plaque-reduction neutralization test (PRNT₅₀) as described herein. The assay's quantitation limit is 10 (1/dil). Samples below this value are assigned the titre 5 and considered seronegative. Statistical analysis

The primary objective is to determine efficacy (E) against cases of symptomatic, virologically-confirmed dengue occurring more than 28 days after the third dose of TDC among children who are enrolled and who receive TDC as planned, according to the equation: E =100*(1 - IDc_Yo IDc_on_tr_oi), where ID is the incidence density (i.e., the number of children with virologically- confirmed dengue divided by the number of person-years at risk) in each group. With an assumed disease incidence of 1 -3%, a true efficacy of 70%, a minimum follow-up period of 1 year after the third dose of TDC, and a per protocol (PP) subject attrition rate of 7-5%/year, 4002 subjects assigned with a 2:1 ratio to TDC or control are needed to demonstrate, with more than 82% power, and 95% confidence, that efficacy is not nul. Analyses are based on the two-sided 95% confidence interval (CI) of efficacy, calculated using the Exact method (Breslow NE, Day NE. Statistical Methods in Cancer Research, Volume II - The Design and Analysis of Cohort Studies. International Agency for Research on Cancer (IARC scientific publication No. 82), Lyon, France). The primary analysis is performed on the PP population, i.e. those who satisfy the enrolment criteria, who correctly receive all three doses of the TDC at Months 0, 6 (±15 days), and 12 (±30 days), and for whom group allocation is not unmasked. This analysis is repeated on the full analysis set for efficacy, in those who receive three injections. As a secondary objective, efficacy against dengue is determined before completion of the 3-dose vaccination regimen. In an analysis defined after unblinding, efficacy against each serotype individually is investigated. Analyses for safety and immunogenicity endpoints are descriptive, using 95%CI.

Results

Of the 4002 children enrolled, 95.9% complete the three doses of TDC and 91 .8% are included in the per protocol (PP) analysis set for efficacy. TDC and control groups are comparable for age and gender. More than 90% of those sampled at baseline are positive for antibodies against dengue or JEV.

Efficacy

During the study, 131 dengue cases (131 children had 136 episodes) are virologically- confirmed. Of these, 77 occur more than 28 days after the third injection in the PP population and are included in the primary analysis: 45 cases occurred during 2522 person-years at risk in the vaccine group, while 32 cases occurred during 1251 person-years at risk in the control group. The corresponding efficacy is 30-2% (95%CI: -13-4-56-6). This finding is confirmed in the full analysis set (see Table 1 below). Efficacy after at least one injection is 33-4% (95%CI: 4- 1-53-5) and after at least two injections is 35-3% (95%CI: 3-3-56-5). Table 1 : Serotype-specific and overall efficacy of CYD TDC against virologically-confirmed dengue disease

TDC Control Efficacy

Person- Cases or Person- Cases or % (95% CI) years at Episodes* years at Episodes*

risk risk

>28 days after 3 injections

(per-protocol analysis)

Cases 2522 45 1251 32 30-2 (-13 -4-56-6)

Serotype 1 episodes 2536 9 1251 10 55-6 (-21 -6-84-0)

Serotype 2 episodes 2510 31 1250 17 9 -2 (-75 -3-51 -3)

Serotype 3 episodes 2541 1 1257 2 75-3 (-375 0-99 -6)

Serotype 4 episodes 2542 0 1263 4 100 (24-8-100)

NS1 Antigen positive 2542 4 1265 0 ND ND only episodes

>28 days after 3 injections

(Full analysis set)

Cases 2620 46 1307 34 32-5 (-8-5-57-6)

Serotype 1 episodes 2633 9 1308 10 55-3 (-22 -5-83 -9)

Serotype 2 episodes 2608 32 1307 19 15-6 (-57-6-53 -6)

Serotype 3 episodes 2638 1 1312 2 75- 1 (-378-99-6)

Serotype 4 episodes 2641 0 1320 4 100 (-24-3-100)

NS1 Antigen positive 2640 4 1322 0 ND ND only episodes

>28 days after at least 1 injection

(Full analysis set)

Cases 5089 75 2532 56 33 -4 (4 - 1-53 -5)

Serotype 1 episodes 5139 14 2564 18 61 -2 (17-4-82 - 1)

Serotype 2 episodes 5107 51 2560 26 1 -7 (-64-3-39- 8)

Serotype 3 episodes 5144 4 2565 10 80 1 (30-9-95 -4)

Serotype 4 episodes 5149 1 2577 5 90 0 (10-5-99-8)

NS1 Antigen positive 5147 5 2579 1 -150· (-1 1750-72 -0) only episodes 5

Data are number except where indicated. ND: not determined. *A 'case' was defined as a first episode of dengue fever virologically-confirmed by either serotype-specific PCRs, or NS1 antigen ELISA. Serotype-specific efficacy was calculated including all episodes of that serotype; 5 children with two virologically confirmed dengue episodes during the study were therefore included twice in the serotype-specific analysis.

Post-hoc analyses reveal differing efficacy by serotype (see Table 1 ). Efficacy against DENV1 , DENV3, and DENV4 after at least one injection is in the range 61 -2%-90 0%, compared with 1 -7% against DENV2. Efficacy against DENV1 , DENV3, and DENV4 after three injections is in the range 55.3%-100%, compared with 15.6% against DENV2. In those subjects that acquired virologically-confirmed dengue, a statistically significant reduction in the annual incidence rate of hospitalization was observed in the group receiving the TDC when compared with the control group. The relative risk (RR) after three doses was 0.523 (see Table 2). Table 2: Incidence of hospitalized virologically-confirmed dengue during the trial

Year 1 = DO to injection 3 ; Year 2 = Injection 3 to the end of Active Phase

Table 3: Rate of hospitalisation by serotype

Immunogenicity

Geometric mean titres (GMT) of neutralising antibodies against dengue serotypes λ-Α on Day 28 after the third injection in the per-protocol analysis set are, respectively, 146 (95%CI: 98-5- 217), 310 (224-431 ), 405 (307-534), and 155 (123-196) in the TDC group. In the control group these values are 23-9 (14-0-40-9), 52-2 (26-8-102), 48-9 (25-5-93-9), and 19-4 (11 -6-32-2). Post one year GMTs are respectively 76.5; 122; 94 and 153 for serotypes 1 , 2, 3 and 4.

Safety

There are 584 SAEs during this phase of the study: 366 are reported by 1 1 .8% (315/2666) of participants in the TCD group, and 218 are reported by 13.2% (176/1331 ) of participants in the control group. There are no TDC-related SAEs in the dengue group and there is one in the control group. SAEs observed are medical conditions consistent with the age group and showed no clustering within the 7- or 28-day post-administration periods.

Virologically-confirmed dengue cases occurring as a breakthrough in those subjects receiving the TDC were not more serious than those cases occurring in the control group.

Sequence of the prM-E region of circulating wild type serotype 2 strain in the trial

The nucleotide and amino acid sequence of the prM-E region of the wild type serotype 2 strain that causes the DEN-2 cases in the trial is determined. These are set out below as SEQ ID NO: 1 and SEQ ID NO: 2 respectively. The E and the M amino acid sequences of the serotype 2 strain that causes the DEN-2 cases in the trial are set out below in SEQ ID NOs: 17 and 22 respectively. >prM-E region nucleotide sequence of the clinical trial circulating strain (SEQ ID NO: 1) ttccatctaaccacacgcaacggagaaccacacatgatcgtcggtatacaggagaaaggg aaaagtcttctgttcaaaacagaggatggtgtgaacatgtgcaccctcatggctatggac cttggtgaattgtgtgaagacacaatcacgtacaagtgtcctcttctcaggcagaatgag ccagaagacatagactgttggtgcaactccacgtccacgtgggtaacctatgggacctgt accactacgggagaacataggagagaaaaaagatcagtggcactcgttccacatgtggga atgggactggagacgcgaaccgaaacatggatgtcatcagaaggggcttggaaacatgcc cagagaattgaaacttggatcctgagacatccaggcttcaccataatggcagcaatcctg gcatacaccataggaacgacacatttccagagagtcctgattttcatcctactgacagct gtcgctccttcaatgacaatgcgttgcataggaatatcaaatagagactttgtagaaggg gtttcaggaggaagttgggttgacatagtcttagaacatggaagctgtgtgacgacgatg gcaaaaaacaaaccaacattggatttcgaactgataaaaacggaagccaaacagcctgcc accctaaggaagtactgcatagaagcaaaactaaccaacacaacaacagaatcccgttgc ccaacacaaggggaacccagcctaaaagaagagcaggacaagaggttcgtctgcaaacac tccatggtagacagaggatggggaaatggatgtggattatttggaaagggaggcattgtg acctgtgctatgttcacatgcaaaaagaacatggaagggaaaatcgtgcaaccagaaaac ttggaatacaccattgtggtaacacctcactcaggggaagagcatgcggtcggaaatgac acaggaaaacacggcaaggaaatcaaagtaacaecacagagttccatcacagaagcagaa ctgacaggttatggcaccgtcacgatggagtgctccccgagaacaggcctcgacttcaat gagatggtgttgctgcagatggaaaataaagcttggctggtgcataggcaatggtttcta gacctgccattaccatggctgcccggagcggataaacaagaatcaaattggatacagaaa gaaacattggtcactttcaaaaatccccatgcgaagaaacaggatgttgttgttttagga tcccaagaaggggccatgcatacagcactcacaggagccacagaaatccaaatgtcgtca ggaaacttgctcttcactggacatctcaagtgcaggctgagaatggacaagctacagctt aaaggaatgtcatactctatgtgcacaggaaagtttaaagttgtgaaggaaatagcagaa acacaacatggaacgatagttatcagagtgcaatatgaaggggacggctctccatgtaaa attccttttgagataatggatttggaaaaaagatatgtcttaggccgcctgatcacagtc aacccaattgtaacagaaaaagacagcccagtcaacatagaagcagaacctccattcgga gacagttacatcatcataggagtagagccgggacaactgaagctcaactggttcaagaaa ggaagttctatcggccaaatgtttgagacaacgatgagaggggcgaagagaatggccatt ttgggtgacacagcctgggacttcggatccctgggaggagtgtttacatctataggaaaa gctctccaccaagtctttggagcgatctatggggctgccttcagtggggtttcatggacc atgaaaatcctcataggagtcattatcacatggataggaatgaactcacgcagcacctca ctgtctgtgtcactggtactggtgggaattgtgacactgtatttaggagtcatggtgcag gcc

> prM-E region amino acid sequence of the clinical trial circulating strain (SEQ ID NO: 2)

FHLTTRNGEPHMIVGIQEKGKSLLFKTEDGVNMCTLMAMDLGELCEDTITYKCPLLRQNE PEDIDCWCNSTSTWVTYGTCTTTGEHRREKRSVALVPHVGMGLETRTETWMSSEGAWKHA QRIETWILRHPGFTIMAAILAYTIGTTHFQRVLIFILLTAVAPSMTMRCIGISNRDFVEG VSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYCIEAKLTNTTTESRC PTQGEPSLKEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFTCKKNMEGKIVQPEN LEY IVV PHSGEEHAVGNDTGKHGKE IKV PQSSI EAELTGYGTVTMECSPRTGLDFN EMVLLQMENKAWLVHRQWFLDLPLPWLPGADKQESNWIQKETLVTFKNPHAKKQDVVVLG SQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKVVKEIAE TQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRYVLGRLITVNPIVTEKDSPVNIEAEPPFG DSYI I IGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGK ALHQVFGAIYGAAFSGVSWTMKILIGVI ITWIGMNSRSTSLSVSLVLVGIVTLYLGVMVQ

A

> E protein amino acid sequence of the clinical trial circulating strain (SEQ ID NO: 17)

MRCIGISNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYC IEAKLTNTTTESRCPTQGEPSLKEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFT CKKNMEGKIVQPENLEY IVV PHSGEEHAVGNDTGKHGKE IKVTPQSSI EAELTGYGT VTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPWLPGADKQESNWIQKETLVTF KNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYS MCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRYVLGRLITVNPIVTE KDSPVNIEAEPPFGDSYI I IGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMAILGDTAW DFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVI ITWIGMNSRSTSLSVSLV LVGIVTLYLGVMVQA

> M protein amino acid sequence of the clinical trial circulating strain (SEQ ID NO: 22)

SVALVPHVGMGLETRTETWMSSEGAWKHAQRIETWILRHPGFTIMAAILAYTIGTTHFQR VLIFILLTAVAPSMT Discussion

The main finding from this study is that a composition based on the chimeric CYD viruses is safe and efficacious. Estimated efficacy against DENV1 , 3 and 4 is in a range consistent with the 70% hypothesis and is statistically significant after at least one dose. Efficacy in a range consistent with the 70% hypothesis is not observed against DENV2. Since DENV2 is the prevalent serotype in this study, overall efficacy is diminished in this setting.

The safety and reactogenicity profile of the TDC is good, and no TDC-related SAEs and no safety signals are identified during the review of AEs and SAEs collected from over two years of active follow-up of more than 2600 subjects receiving the TDC. Theoretical safety concerns associated with the potential enhancement of the rate or severity of dengue disease by an incomplete immune response against the four serotypes of dengue have previously hampered vaccine development. In this trial, the absence of disease enhancement in the presence of an incomplete immune response against the circulating DENV2 viruses is an important and reassuring finding. For instance, cases in subjects receiving the TDC do not differ from cases in controls in terms of factors such as the duration of fever or in terms of the classical clinical signs of dengue such as bleeding, plasma leakage or thrombocytopenia. Furthermore, severe dengue was not more frequent among subjects receiving the TDC than controls at any point during the trial.

It was also demonstrated that, in those subjects that acquired virologically-confirmed dengue, a statistically significant reduction in the annual incidence rate of hospitalization was observed in the group receiving the TDC when compared with the control group. This reduction was seen in those subjects that acquired virologically-confirmed dengue of serotype 2 (see Table 3).

The results observed in respect of DENV2 may be explained by a number of contributing factors. For instance, there is a possible antigenic mismatch between the CYD2 vaccine virus and the DENV2 virus that causes disease in the trial. In the 1990s, the Asian 1 genotype of DENV2 emerged in South-East Asia, replacing the previously dominant Asian/ American lineage of viruses. Several mutations identified in Domain 2 of the E protein (E83, and in particular E226 and E228) are suggestive of changing viral fitness and antigenicity. The donor wild type virus for the CYD2 vaccine (and the challenge strain used in the PRNT₅₀) was a clinical isolate from Bangkok in 1980 (Guirakhoo F et al., J Virol 2000, 74: 5477-85). While this virus is also classified as belonging to the Asian I genotype, the above-mentioned key amino acid residues in this virus (and thus in CYD2) correspond to those of the Asian/American genotype (Hang et al PLoS Negl Trap Dis. 2010 Jul 20;4(7):e757).

Additionally, there are two extremely rare mutations in the prM-E sequence of the CYD2 vaccine that may also contribute to a mismatched immune response. These mutations are at positions prM24 and E251 (Guirakhoo et al, J. Virol. (2004) 78 (9): 4761 ). The results observed against DENV2 are not associated with an absence of immunogenicity in the PRNT₅₀ assay. Neutralising antibody responses against DENV2 after dosing of the TDC are higher than those against DENV1 and DENV3. Example 2: Immunogenicity of CYD dengue compositions and MMR vaccine in healthy toddlers aged 12 to 15 months in the Philippines

Method

Trial & Participants

A randomized, controlled, multi-centre, Phase II trial involving 210 healthy toddlers was carried out in the Philippines. The trial included four different treatment groups receiving combinations of treatments as described in Table 4:

Table 4: Treatments received by each treatment group

One objective of the trial was to describe the immunogenicity of the CYD1 -4 tetravalent composition after each dose, wherein the first dose of the composition is given alone (Group 4) or is co-administered with MMR vaccine (Group 3). A second objective of the trial was to describe the immunogenicity of the MMR vaccine, when given alone (Group 4) or co-administered with the CYD1 -4 tetravalent composition (Group 3). The "combo" Diphtheria, Tetanus, Pertussis, Poliomyelitis and Hib vaccine was administered at M9 purely to comply with the required vaccination schedule in the Philippines. No data was collected in respect of the "combo" vaccine. Products

The tetravalent dengue composition administered to the toddlers was the same particular CYD-1 , CYD-2, CYD-3 and CYD-4 Chimerivax™ tetravalent composition as described and administered in Example 1 . Briefly, each CYD dengue monovalent biological substance (i.e., serotypes 1 , 2, 3, and 4) was produced by Sanofi Pasteur in compliance with current Good Manufacturing Practice (cGMP). No raw materials of human or animal origin were used in the production. Biological substances were prepared by amplification of each serotype on serum-free Vera cells. The viral harvest, clarified from cellular debris by filtration, was then concentrated and purified by ultrafiltration and chromatography to remove host cell DNA. After stabilization, biological substances were stored at ≤ -70°C before formulation and filling. The biological product, manufactured by Sanofi Pasteur in compliance with cGMP, consists of a tetravalent lyophilized form combination that includes CYD-1 , CYD-2, CYD-3 and CYD-4. The bulk product of each serotype is thawed, diluted to the targeted dose in stabilizer solution, and pooled. After sterile filtration, the tetravalent mixture is filled into 3 mL glass vials, lyophilized, and stored at 5°C. The lyophilized powder was reconstituted with 0.5 mL of solvent for injection (0.4% NaCI containing 2.5% human serum albumin). As reconstituted, each 0.5 mL dose contained 5 ± 1 logi₀ CCID₅₀ of each chimeric dengue virus serotype (1 , 2, 3 and 4) and excipients (essential amino acids, nonessential amino acids, L-arginine chlorhydrate, saccharose, D-trehalose dihydrate, D-sorbitol, tris(hydroxymethyl)aminoethane and urea).

The first control vaccine (Control Vaccine 1 ) was Okavax® (Aventis Pasteur). The vaccine was presented as a lyophilized powder (previously stored at temperature of between 2°C and 8°C), which was reconstituted with 0.7 mL of water for injection. As reconstituted, each 0.7 mL dose of vaccine contained attenuated live varicella zoster virus (Oka strain, not less than 1000 PFU).

The second control vaccine (Control Vaccine 2) was Avaxim® 80U (Sanofi Pasteur MSD).

The vaccine was presented as a suspension for injection, which had been previously stored between 2°C and 8°C. Each 0.5 mL dose of vaccine contained 80U of inactivated Hepatitis A virus (GBM strain).

The placebo was a 0.9% solution of NaCI.

The MMR vaccine was TRIMOVAX® (Sanofi Pasteur). The vaccine was presented as a powder and diluent for suspension for injection (previously stored between 2°C and 8°C). Each 0.5 mL dose of reconstituted vaccine contained at least 1000 CCID₅₀ measles virus (Schwarz strain) cultivated on a primary culture of chicken embryo cells; at least 5000 CCID₅₀ mumps virus (Urabe AM-9 strain) cultivated in embryonated hen eggs and at least 1000 CCID₅₀ rubella virus (Wistar RA 27/3M strain) cultivated on human diploid cells. The excipient was human albumin. The combination diphtheria, tetanus, pertussis, poliomyelitis and Hib vaccine (also referred to herein as the "Combo" vaccine) was Pentaxim® (Sanofi Pasteur). The vaccine was presented as a lyophilized powder (previously stored at between 2°C and 8°C), which was reconstituted with 0.5 mL of suspension for injection. Each 0.5 mL dose of reconstituted vaccine contained >30 IU of diphtheria toxoid; >40 IU of tetanus toxoid; Bordetella pertussis antigens (25 ig toxoid, 25 [ig filamentous haemagglutinin); 40 D antigen units of Type 1 poliomyelitis virus (inactivated), 8 DU of Type 2 poliomyelitis virus (inactivated) and 32 DU of Type 3 poliomyelitis virus (inactivated) and 10ig of polysaccharide of Haemophilus influenza type b conjugated to the tetanus protein. As reconstituted, each dose contained the following excipients: saccharose, trometamol, aluminium hydroxide, Hank's medium without phenol red, acetic acid and/or sodium hydroxide for pH adjustment, formaldehyde, phenoxyethanol and water for injection.

The tetravalent dengue composition, Okavax®, placebo and Trimovax® were administered via sub-cutaneous injection to the deltoid region of the upper arm. Avaxim® 80U was administered intramuscularly in the muscle of the upper arm and Pentaxim® was administered by intramuscular injection in the anterolateral aspect of the thigh.

Inclusion and Exclusion Criteria

Eligible subjects for this trial were toddlers aged 12 to 15 months on the day of inclusion who were in good health without history of infection or previous vaccination to varicella, measles, mumps, rubella, flavivirus or hepatitis A.

Where one of the conditions listed below occurred, the physician postponed administration until the condition was resolved:

Febrile illness (temperature >38°C) or moderate or severe acute illness/infection on the day of attendance at the clinic, according to the physician's judgment; receipt of oral or injected antibiotic therapy within 72 hours prior to attendance at the clinic; any vaccination received in the 4 weeks preceding attendance at the clinic.

Statistical Methods

All main analyses were descriptive. For the main parameters, 95% confidence intervals (Cls) of point estimates were calculated using normal approximation for quantitative data and exact binomial distribution (Clopper-Pearson method) for proportions.

Immunogenicity Assessment Methods

The neutralising antibody titres produced against each of the four parental dengue virus strains that were used to generate CYD1 -4 were measured using a plaque reduction neutralisation test (PRNT₅₀) as described herein. Neutralising antibody titres were measured in sera collected at baseline (for all toddlers), 28 days post dose 2 and post dose 3 (for all toddlers from Groups 1 and 2) and 28 days post dose 1 , 2 and 3 for toddlers from Groups 3 and 4. Antibody levels against measles, mumps and rubella were measured by enzyme-linked immunosorbent assay (ELISA) in sera collected from all toddlers from Groups 3 and 4 at baseline and at 28 days post vaccination with MMR. MMR antibody measurements were performed at Pharmaceutical Product Development (PPD), Wayne, Pennsylvania, USA. The ELISAs for measles, mumps and rubella antibody levels follow the same principle with the coating antigen dependent upon the assay: either measles virus, mumps virus or rubella virus. Inactivated viral antigen was adsorbed to wells of a solid phase microtiter plate. Specific antibodies in the reference standard, serum quality controls and test samples were bound to the immobilized antigen, unbound antibodies were washed from the wells and enzyme-conjugate anti-human immunoglobulin (Ig) was added. The enzyme conjugate bound to the antigen-antibody complex. Excess conjugate was washed away and a specific colorimetric substrate was added. Bound enzyme catalyzed a hydrolytic reaction that caused colour development. After a specific time, the reaction was stopped via the addition of a stopping solution. The intensity of the colour was proportional to the amount of specific antibody bound to the wells. The results were read on a spectrophotometer (ELISA plate reader).

In the measles ELISA, a WHO International Reference and controls were included in each assay. The activity of the virus specific IgG antibodies contained in the sample is quantified by the standard curve generated using the reference standard and a four parameter logistic regression function. Results are reported in mlU/mL and the lower limit of quantitation (LLOQ) of the assays is 0.782 mlU/mL. In the mumps ELISA, controls were included in each assay. The activity of the virus specific IgG antibodies contained in the sample was quantified by the standard curve generated using the reference standard and a four parameter logistic regression function. Results are reported in antibody units and the lower limit of quantitation (LLOQ) of the assays is 4.04 Elisa units per mL (EU/mL). In the rubella ELISA, a WHO International Reference and controls were included in each assay. The activity of the virus specific IgG antibodies contained in the sample is quantified by the standard curve generated using the reference standard and a four parameter logistic regression function. Results are reported in lU/mL and the lower limit of quantitation (LLOQ) of the assays is 0.086 lU/mL.

For the rubella ELISA, the Captia Rubella IgG kit from Trinity Biotech was used. The conjugate was peroxidase-conjugated AffiniPure F(ab.)2 goat anti-human IgG Fc Fragment (Jackson ImmunoResearch Laboratories, Inc.) and the substrate was TMB peroxidase (KPL TMB Microwell Peroxidase Substrate System, AvP US NA0195). The reference serum was an internal reference from pooled human sera (healthy adult) standardised against British 2nd International standard NIBSC Code 67/182 (80 lU/mL). For the measles ELISA, the Captia Measles IgG kit from Trinity Biotech was used. The conjugate was peroxidase-conjugated AffiniPure F(ab.)2 goat anti- human IgG Fc Fragment (Jackson ImmunoResearch Laboratories, Inc.) and the substrate was TMB peroxidase (KPL TMB Microwell Peroxidase Substrate System, AvP US NA0195). The reference serum was an internal reference from pooled human sera (healthy adult) standardised against an interim reference preparation of anti-measles serum (2870 mlU/ml) For the measles ELISA, the Captia Mumps IgG kit from Trinity Biotech was used. The conjugate was peroxidase- conjugated AffiniPure F(ab.)2 goat anti-human IgG Fc Fragment (Jackson ImmunoResearch Laboratories, Inc.) and the substrate was TMB peroxidase (KPL TMB Microwell Peroxidase Substrate System, AvP US NA0195). The reference serum was an internal reference from pooled human sera (healthy adult).

Results

The results of the trial are shown in Figures 1 and 2. Figure 1 shows the Geometric Mean of Titres (GMTs) for serum antibodies against measles virus, mumps virus and rubella virus both pre and post MMR dosing. As can be seen from the figure, there was no significant difference in the GMTs (of antibodies against measles virus, mumps virus or rubella virus) between those patients receiving the MMR vaccine in conjunction with the tetravalent dengue composition (Group 3) and those patients receiving the MMR vaccine and tetravalent dengue composition sequentially (Group 4). Accordingly, it was demonstrated that administration of the MMR vaccine in conjunction with a tetravalent dengue composition did not significantly impact the immunogenicity of the MMR vaccine.

Figure 2 shows the GMTs for serum antibodies against dengue virus of each of serotypes 1 , 2, 3 and 4 (as determined by PRNT assay), prior to dosing with the tetravalent dengue composition (TDC) and post doses 1 , 2 and 3 of the TDC/control vaccine. Data is compared between a control group who received three doses of control vaccines but did not receive the TDC (Group 2); a group who received three doses of the TDC, none of which were administered in conjunction with an MMR vaccine (Groups 1 and 4 combined) and a group who received three doses of the TDC, the first dose of which was administered in conjunction with an MMR vaccine (Group 3). As can be seen from the Figure, there were no significant differences for any of the serotypes between the GMTs from Groups 1 and 4 and the GMTs from Group 3 following completion of the administration programme.

Example 3: Identification of optimized dengue strains of serotype 2

The objective of the present example is to identify dengue virus strains of serotype 2 which provide the basis for generating optimized agents according to the present invention which comprise a dengue antigen of serotype 2, wherein said optimized agents provide improved efficacy in comparison to Chimerivax™ CYD-2 when used in a method according to the present invention.

Criteria determining the selection of optimized strains include: (i) recently circulating strain; (ii) balanced selection between Asian and American strains; (iii) an optimized strain should have a prM-E sequence that is as similar as possible to a calculated global consensus sequence generated by aligning the available prM-E sequences of dengue viruses of serotype 2; (iv) amino acid variations that are predicted to impact antibody recognition should be avoided; (v) rare amino acids at particular positions in the prM and E sequences should be avoided, especially in the E protein ectodomain (a rare amino acid at a particular position is defined as a amino acid that appears at that position in less than 15% of the aligned sequences); (vi) optimized strains for which some previous laboratory experience exists are preferred and (vii) a dengue antigen that leads to a balanced immune response when used in the context of a tetravalent composition

Criteria determining the selection of optimized strains for a local dengue 2 antigen (i.e. that is especially effective against a wild type dengue virus circulating in a particular area) are criteria (i) and (vii).

Methods

Databases

Sequences are retrieved from the National Center for Biotechnology Information (NCBI) Dengue virus variation database (www.ncbi.nlm.nih.gov/genomesA/irusVariation/Database/nph- select.cgi?tax_id=12637).

Sequence analyses

Sequence alignments are performed using the MUSCLE algorithm (Edgar, R. C. (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res, 32(5):1792-1797).

Sequence alignment outputs are generated in Vector NTi version 9, module AlignX (Invitrogen). Sequence similarity searches are carried out using the BLAST algorithm (Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) Basic local alignment search tool. J Mol Biol, 215(3):403-410).

Sequence numbering for prM-E sequences

The sub-sequences included in the prM-E sequences may be numbered in various ways: (i) the total prM-E protein sequence is numbered from position 1 to position 661 , with the preM protein sequence designated as position 1 to position 90/91 , the M protein sequence designated as position 91/92 to position 166 and the E protein sequence designated as position 167 to position 661 ; (ii) the prM and M protein sequences are numbered together, i.e. from position 1 to position 166 of the total sequence and E is numbered separately from position 1 to position 495; (iii) the prM, M and E sequences are numbered separately, i.e. prM is numbered from position 1 to 90/91 , M is numbered from 1 to 75/76 and E from position 1 to position 495.

Results

Public sequences retrieval

All available dengue virus serotype 2 full length prM and E protein sequences are downloaded from the NCBI Dengue database. Download of sequences took place on two separate occasions - on 4 October 2010 and in 201 1. On the first occasion 669 sequences were downloaded and on the second occasion approximately 3200 sequences were downloaded.

Global consensus sequence generation

On each occasion, all retrieved protein sequences are aligned to generate a global consensus sequence for the prM and E proteins of dengue virus of serotype 2. By definition, the global consensus sequence is an artificial sequence containing the most frequently encountered amino acid at each position. The global consensus sequences for the 2010 alignment and the 2011 alignment only differ by two amino acids. In the 2010 alignment, the global consensus sequence contains isoleucine and valine at positions 129 and 308 respectively of the E protein (by reference to the 1 -495 E sequence numbering) and, by contrast, in the 201 1 alignment, the global consensus sequence contains valine and isoleucine at positions 129 and 308 respectively of the E protein (by reference to the 1 -495 E sequence numbering). The differences between the 2010 and 201 1 global consensus sequences are explained by the fact that the respective percentage of strains containing valine or isoleucine at those positions is close to 50%. The global consensus sequence for the prM-E amino acid sequence is therefore represented as follows:

fhittrngephmivgrqekgks llfktedgvnmctlmaidlgelcedtitykcpllrqne pedidcwcnststwvtygtctttgehrrekrsvalvphvgmgletrtetwmssegawkhv qrietwilrhpgftimaailaytigtthfqralifilltavapsmtMRCIGISNRDFVEG VSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYCIEAKLTNTTTESRC PTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFTCKKNMEGKXVQPEN LEY IVI PHSGEEHAVGNDTGKHGKE IKI PQSSI EAELTGYGTVTMECSPRTGLDFN EMVLLQMEDKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLG SQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKZVKE IAE TQHG IVIRVQYEGDGSPCKIPFE IMDLEKRHVLGRLI VNPIVTEKDSPV IEAEPPFG DSYI I IGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGK ALHQVFGAIYGAAFSGVSWTMKILIGVI ITWIGMNSRSTSLSVSLVLVGVVTLYLGVMVQ A (SEQ ID NO: 3)

The global consensus sequence for the E sequence is represented as follows:

MRCIGISNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYC IEAKLTNTTTESRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFT CKKNMEGKXVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGT VTMECSPRTGLDFNEMVLLQMEDKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTF KNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYS MCTGKFKZVKE IAETQHG IVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLI VNPIVTE KDSPVNIEAEPPFGDSYI I IGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMAILGDTAW DFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVI ITWIGMNSRSTSLSVSLV LVGVVTLYLGVMVQA (SEQ ID NO: 12) In the prM-E global consensus sequence, the global consensus prM sequence is shown in lower case letters and the global consensus E sequence is shown in upper case letters. The amino acid positions denoted as X (position 129 of the E sequence) and Z (position 308 of the E sequence) are each independently Val or He, i.e. the proportion of aligned amino acid sequences including Val or lie at those positions is close to 50%. Determination of minor amino acid residues and analysis of the Chimerivax™ CYD2 sequence

A list of variable amino acid positions is established from the global alignment containing all amino acid positions varying in at least 5% of the aligned sequences. In addition, any amino acid from the sequence of the prM and E proteins of Chimerivax™ CYD2 that do not match the global consensus sequence are also identified. The results are shown in Table 5 (N.B., in the table, the prM and M protein sequences are numbered together, i.e. from position 1 to position 166 of the total sequence and E is numbered separately from position 1 to position 495).

Table 5: Dengue virus serotype 2 variable residues and CYD2 comparison

A total of 41 amino acid positions are identified in the prM and E sequences which either vary from the global consensus sequence in at least 5% of the aligned sequences and/or differ from the sequence of the prM and E proteins in CYD2. Ten amino acid positions in the sequence of the prM and E proteins in CYD2 differ from the global consensus sequence (5 positions in E, 2 positions in M and 3 in its precursor part, see Table 5 above). Five out of the ten differing residues present a variation distribution close to 50:50, suggesting a naturally variable position. Only three positions in the CYD2 prM-E sequence appear as very minor variants (pr-24 Val, M-125 lie (i.e. M-34 He when the M protein is numbered separately) and E-251 Phe). Impact analysis of variations in the E and M proteins

To gain further insight into the variable positions, changes in the E protein ectodomain (amino acids 1 -395), the most important domain for the seroneutralisation by the immune system are further analysed.

Using information available from a published 3D structure of the soluble ectodomain of the

E protein of a dengue virus of serotype 2 (Modis, Y., et al. (2003) Proc Natl Acad Sci U S A, 100(12):6986-6991 ), a 3D model of the Dengue virus particle surface is reconstructed. This allows a fine tuned assessment of the accessibility of each amino acid from the E ectodomain, which in turn is used in association with the variability level and the nature of the amino acid change to assess a potential impact of CYD2 variations on antibody recognition.

The analysis demonstrates that two variations in the Chimerivax™ CYD2 sequence from the global consensus sequence (Val 141 and Val 164 of the E protein) are completely buried in the 3D structure and so cannot directly interact with an antibody at the surface of the virion. Position 129 of the E protein is a 50:50 variable amino acid position between Val (Chimerivax™ CYD2) and He (global consensus sequence) and the substitution is also a fully conservative change. The potential impact of these variations is therefore considered as very limited.

The variation at position 203 of the E protein (Asn in Chimerivax™ CYD2 and Asp in the global consensus sequence) could potentially have an impact (well-exposed residue, change of charge) but the distribution of the variation among strains is close to 50:50, suggesting a naturally variable position.

The variation at position 251 of the E protein of Chimerivax™ CYD2 (Phe in Chimerivax™ CYD2 and Val in the global consensus sequence) is extremely rare among retrieved strains. Such a variation could have some impact on recognition by an antibody, as it is rare, rather well exposed at the surface of the virion (29%) and corresponds to a non-conservative amino acid change.

The modeling analysis described above identifies two other position variations in the E protein that could have a potential impact on antibody recognition (positions 226 and 228), although Chimerivax™ CYD2 does not vary from the global consensus sequence at those positions. Therefore in identifying optimised serotype 2 strains, variations from the global consensus sequence at those positions (i.e. Thr at position 226 and glycine at position 228) are preferably avoided for a universal dengue 2 vaccine.

Without being bound by theory, the present inventors consider that the impact of amino acid variations in dengue virus sequences can also be assessed using a scoring method which takes into account a number of relevant factors. In particular this method takes into account the genome location of the variation (G), the nature of the amino acid change (B), 3D mapping (M) and known variants at the position in question (DB), wherein the score is calculated as G x B x M x DB. A score of 0 would be classified as no expected impact, a score of >0 to 10 would be classified as a low expected impact, a score of >10 to 25 would be classified as a median expected impact and a score of >25 would be classified as a high expected impact.

The genome location (G) score is 0 if the amino acid is located in the M part of the prM/M protein (i.e. position 92 to 166 of the prM/M sequence) or in position 396 to 495 of the E protein. The genome location score is 1 if the amino acid is located in prM part of the prM/M protein (i.e. position 1 to 91 of the prM/M sequence) or in position 1 to 395 of the E protein.

The score related to the nature of the amino acid change (B) is calculated as B = 100 - [(Blosum95 score + 6) x 10], wherein the Blosum95 score for different amino acid substitutions is as shown in Table 6 below.

Table 6

R N D E H K M W B Z X *

-3 -1 -1 -6

-2 -1 -2 -6

4 -1 -2 -6

4 0 -2 -6

-4 -5 -3 -6

-1 4 -1 -6

0 4 -2 -6

-2 -3 -3 -6

-1 0 -2 -6

-5 -4 -2 -6

-1 0 -1 -6

-4 -2 -2 -6

-5 -4 -2 -6

-3 -2 -3 -6

-1 -1 -1 -6

-1 -2 -1 -6

-6 -4 -4 -6

-4 -4 -2 -6

-5 -3 -2 -6

B = Asx, Z = Glx, X = Any and * = Stop

The M value depends on whether the amino acid is or is not located at the prM/E interface. For example, for CYD2 as used in Example 1 , the amino acids that are located at the interface are prM residues 6, 7, 39, 40, 46-54, 56, 59-65, 67, 74 and 77 and E residues 64-72, 82-84, 101 -104, 106-108 and 244-247. Where an amino acid is located at the interface, M equals 1 . Where an amino acid is not located at the interface, M = Y x SAS %. Y is 1 if the amino acid is located in an "up" position (i.e. directed towards the external environment); Y is 0.5 if the amino acid is located on the "side" of the molecule (i.e. the amino acid is neither directed towards the external environment nor towards the capsid) and Y is 0 if the amino acid is located in a "down" position (i.e. directed towards the capsid). The solvent accessibility surface % (SAS %) value is generated using the Discovery Studio 3D modeling software (Accelrys, Inc., CA, USA).

The DB value is 0 when the amino acid substitution results in an amino acid at the substitution position which is the most common amino acid at that position in the dengue sequences present in the GenBank database (http://www.ncbi.nlm.nih.gov). The DB value is 0.25 when the amino acid substitution results in an amino acid at the substitution position which is found in more than 5% of the dengue sequences present in the database (but is not the most common amino acid at that position). The DB value is 0.50 when the amino acid substitution results in an amino acid at the substitution position which is found in less than 5% of the dengue sequences present in the database (except unique substitutions). The DB value is 1 when the substitution amino acid is unique.

During replication, viruses may acquire a mutation leading to an amino acid substitution. The above-mentioned method provides a means to determine the effect of such mutations on the progeny of the mutated viruses.

Preferred sequences (i.e. sequences that are considered to be satisfactorily close to the identified consensus sequence) may have: (i) at most two, preferably one or no high-impact amino acid substitutions; (ii) at most three, preferably two or one, or no median impact amino acid substitutions; and/or (iii) at most five, four, three, two or one low impact amino acid substitutions. Identification of optimized serotype 2 strains

Optimised serotype 2 strains are identified on the basis of the selection criteria described above. A BLAST search is conducted to identify the strain having the closest sequence to the prM- E global consensus sequence in all of the available sequences. No sequence that is 100% identical to the prM-E global consensus sequence is found, but the best hit is a sequence from strain BID- V585 (NCBI Protein ID no. ACA58343; Genome ID no. EU529706; isolated from Puerto Rico in 2006) which shows only one variation from the global consensus sequence, at position 91 (Val in the global consensus sequence and lie in BID-V585). The BID-V585 prM-E sequence contains 13 variations from the Chimerivax™ CYD-2 prM-E sequence.

A further strain selection is made so as to provide geographical balance in strain origin. Therefore a recently isolated Asian strain showing a good score in the BLAST analysis (strain MD- 1280; NCBI Protein ID no. CAR65175; Genome ID no. FM21043; isolated from Viet Nam in 2004) is selected. Despite showing 6 variations with the global consensus sequence across prM-E, 3 of the 6 variations are identified as versatile positions naturally varying in more than 30% of the strains. The MD-1280 prM-E sequence contains 15 variations from the Chimerivax™ CYD-2 prM-E sequence.

A further strain selection is made on the basis of a large amount of previously accumulated experience with the strain. It is the PDK53-16681 strain, also known as the LAV-2 strain, a live- attenuated virus derived from Dengue serotype 2 16681 strain from Mahidol University (NCBI Protein ID no. AAA73186; Genome ID no. M84728; isolated from Thailand in 1964; Blok, J., et al. (1992); Virology 187 (2), 573-590). The LAV-2 prM-E sequence contains 10 variations from the global consensus sequence and 13 variations from the Chimerivax™ CYD-2 prM-E sequence.

A further strain selected on the basis of the above-mentioned criteria is strain PR/DB023 (NCBI Protein ID no. AEN71248; Genome ID no. JF804036; isolated from Puerto Rico in 2007). The PR DB023 prM-E sequence contains 3 variations from the global consensus sequence and 13 variations from the Chimerivax™ CYD-2 prM-E sequence.

None of the selected strains contain the rare amino acids present in the Chimerivax™ CYD-2 prM-E sequence, i.e. Val at pr-24, lie at M-125 and Phe at E-251 .

PrM to E nucleotide sequences of the four selected strains > LAV-2 prME nucleotide sequence (SEQ ID NO: 4)

ttccatttaaccacacgtaacggagaaccacacatgatcgtcagcagacaagagaaaggg aaaagtcttctgtttaaaacagaggttggcgtgaacatgtgtaccctcatggccatggac cttggtgaattgtgtgaagacacaatcacgtacaagtgtccccttctcaggcagaatgag ccagaagacatagactgttggtgcaactctacgtccacgtgggtaacttatgggacgtgt accaccatgggagaacatagaagagaaaaaagatcagtggcactcgttccacatgtggga atgggactggagacacgaactgaaacatggatgtcatcagaaggggcctggaaacatgtc cagagaattgaaacttggatcttgagacatccaggcttcaccatgatggcagcaatcctg gcatacaccataggaacgacacatttccaaagagccctgattttcatcttactgacagct gtcactccttcaatgacaATGCGTTGCATAGGAATGTCAAATAGAGACTTTGTGGAAGGG GTTTCAGGAGGAAGCTGGGTTGACATAGTCTTAGAACATGGAAGCTGTGTGACGACGATG GCAAAAAACAAACCAACATTGGATTTTGAACTGATAAAAACAGAAGCCAAACAGCCTGCC ACCCTAAGGAAGTACTGTATAGAGGCAAAGCTAACCAACACAACAACAGAATCTCGCTGC CCAACACAAGGGGAACCCAGCCTAAATGAAGAGCAGGACAAAAGGTTCGTCTGCAAACAC TCCATGGTAGACAGAGGATGGGGAAATGGATGTGGACTATTTGGAAAGGGAGGCATTGTG ACCTGTGCTATGTTCAGATGCAAAAAGAACATGGAAGGAAAAGTTGTGCAACCAGAAAAC TTGGAATACACCATTGTGATAACACCTCACTCAGGGGAAGAGCATGCAGTCGGAAATGAC ACAGGAAAACATGGCAAGGAAATCAAAATAACACCACAGAGTTCCATCACAGAAGCAGAA TTGACAGGTTATGGCACTGTCACAATGGAGTGCTCTCCAAGAACGGGCCTCGACTTCAAT GAGATGGTGTTGCTGCAGATGGAAAATAAAGCTTGGCTGGTGCACAGGCAATGGTTCCTA GACCTGCCGTTACCATGGTTGCCCGGAGCGGACACACAAGGGTCAAATTGGATACAGAAA GAGACATTGGTCACTTTCAAAAATCCCCATGCGAAGAAACAGGATGTTGTTGTTTTAGGA TCCCAAGAAGGGGCCATGCACACAGCACTTACAGGGGCCACAGAAATCCAAATGTCATCA GGAAACTTACTCTTCACAGGACATCTCAAGTGCAGGCTGAGAATGGACAAGCTACAGCTC AAAGGAATGTCATACTCTATGTGCACAGGAAAGTTTAAAGTTGTGAAGGAAATAGCAGAA ACACAACATGGAACAATAGTTATCAGAGTGCAATATGAAGGGGACGGCTCTCCATGCAAG ATCCCTTTTGAGATAATGGATTTGGAAAAAAGACATGTCTTAGGTCGCCTGATTACAGTC AACCCAATTGTGACAGAAAAAGATAGCCCAGTCAACATAGAAGCAGAACCTCCATTTGGA GACAGCTACATCATCATAGGAGTAGAGCCGGGACAACTGAAGCTCAACTGGTTTAAGAAA GGAAGTTCTATCGGCCAAATGTTTGAGACAACAATGAGGGGGGCGAAGAGAATGGCCATT TTAGGTGACACAGCCTGGGATTTTGGATCCTTGGGAGGAGTGTTTACATCTATAGGAAAG GCTCTCCACCAAGTCTTTGGAGCAATCTATGGAGCTGCCTTCAGTGGGGTTTCATGGACT ATGAAAATCCTCATAGGAGTCATTATCACATGGATAGGAATGAATTCACGCAGCACCTCA CTGTCTGTGACACTAGTATTGGTGGGAATTGTGACACTGTATTTGGGAGTCATGGTGCAG GCC

UPPERCASE: E coding sequence; lowercase: prM coding sequence

> BID V585 - prME nucleotide sequence (SEQ ID NO: 5)

ttccatttaaccacacgtaatggagaaccacacatgatcgttggtaggcaagagaaaggg aaaagtcttctgtttaaaacagaggatggtgttaacatgtgcaccctcatggccatagac cttggtgaattgtgtgaagatacaatcacgtacaagtgccccctcctcaggcaaaatgaa ccagaagacatagattgttggtgcaactctacgtccacatgggtaacttatgggacatgt accaccacaggagaacacagaagagaaaaaagatcagtggcactcgttccacatgtgggc atgggactggagacacgaactgaaacatggatgtcatcagaaggggcctggaaacatgtt cagagaattgaaacctggatcttgagacatccaggctttaccataatggcagcaatcctg gcatataccataggaacgacacatttccaaagggctctgatcttcattttactgacagcc gttgctccttcaatgacaATGCGTTGCATAGGAATATCAAATAGAGACTTCGTAGAAGGG GTTTCAGGAGGAAGTTGGGTTGACATAGTCTTAGAACATGGAAGTTGTGTGACGACGATG GCAAAAAATAAACCAACATTGGATTTTGAACTGATAAAAACAGAAGCCAAACAACCTGCC ACTCTAAGGAAGTACTGTATAGAAGCAAAGCTGACCAATACAACAACAGAATCTCGTTGC CCAACACAAGGGGAACCCAGTCTAAATGAAGAGCAGGACAAAAGGTTCATCTGCAAACAC TCCATGGTAGACAGAGGATGGGGAAATGGATGTGGATTATTTGGAAAGGGAGGCATTGTG ACCTGTGCTATGTTCACATGCAAAAAGAACATGGAAGGAAAAGTCGTGCAGCCAGAAAAT CTGGAATACACCATCGTGATAACACCTCACTCAGGAGAAGAGCACGCTGTAGGTAATGAC ACAGGAAAGCATGGCAAGGAAATCAAAATAACACCACAGAGCTCCATCACAGAAGCAGAA CTGACAGGCTATGGCACTGTCACGATGGAGTGCTCTCCGAGAACGGGCCTCGACTTCAAT GAGATGGTACTGCTGCAGATGGAAGACAAAGCTTGGCTGGTGCACAGGCAATGGTTCCTA GACCTGCCGTTACCATGGCTACCCGGAGCGGACACACAAGGATCAAATTGGATACAGAAA GAGACGTTGGTCACTTTCAAAAATCCCCACGCGAAGAAACAGGACGTCGTTGTTTTAGGA TCTCAAGAAGGGGCCATGCACACGGCACTTACAGGGGCCACAGAAATCCAGATGTCATCA GGAAACTTACTGTTCACAGGACATCTCAAGTGTAGGCTGAGAATGGACAAATTACAGCTT AAAGGAATGTCATACTCTATGTGTACAGGAAAGTTTAAAATTGTGAAGGAAATAGCAGAA ACACAACATGGAACAATAGTTATCAGAGTACAATATGAAGGGGACGGCTCTCCATGTAAG ATTCCTTTTGAGATAATGGATTTGGAAAAAAGACACGTCCTAGGTCGCCTGATTACAGTG AACCCAATCGTAACAGAAAAAGATAGCCCAGTCAACATAGAAGCAGAACCTCCATTCGGA GACAGCTACATCATCATAGGAGTAGAGCCGGGACAATTGAAACTCAATTGGTTCAAGAAG GGAAGTTCCATTGGCCAAATGTTTGAGACAACAATGAGAGGAGCGAAGAGAATGGCCATT TTAGGTGACACAGCCTGGGATTTTGGATCCCTGGGAGGAGTGTTTACATCTATAGGAAAG GCTCTCCACCAAGTTTTCGGAGCAATCTATGGGGCTGCTTTTAGTGGGGTCTCATGGACT ATGAAAATCCTCATAGGAGTTATTATCACATGGATAGGAATGAATTCACGTAGCACCTCA CTGTCTGTGTCACTAGTATTGGTGGGAGTCGTGACACTGTACTTGGGGGTTATGGTGCAG GCT

>PR/DB023 prME nucleotide sequence (SEQ ID NO: 6)

ttccatttaaccacacgtaatggagaaccacacatgatcgttggtaggcaagagaaaggg aaaagtcttctgttcaaaacagaggatggtgttaacatgtgtaccctcatggccatagac cttggtgaattgtgtgaagatacaatcacgtacaagtgccccctcctcaggcaaaatgaa ccagaagacatagattgttggtgcaactctacgtccacatgggtaacttatgggacatgt accaccacaggagaacacagaagagaaaaaagatcagtggcactcgttccacatgtgggc atgggactggagacacgaactgaaacatggatgtcatcagaaggggcctggaaacatgtt cagagaattgaaacctggatattgagacatccaggctttaccataatggcagcaatcctg gcatataccataggaacgacacatttccaaagggctctgatcttcattttactgacagcc gtcgctccttcaatgacaATGCGTTGCATAGGAATATCAAATAGAGACTTCGTAGAAGGG GTTTCAGGAGGAAGTTGGGTTGACATAGTCTTAGAACATGGAAGTTGTGTGACGACGATG GCAAAAAATAAACCAACATTGGATTTTGAACTGATAAAAACAGAAGCCAAACAACCTGCC ACTCTAAGGAAGTACTGTATAGAAGCAAAGCTGACCAATACAACAACAGAATCTCGTTGC CCAACACAAGGGGAACCCAGTCTAAATGAAGAGCAGGACAAAAGGTTCATCTGCAAACAC TCCATGGTAGACAGAGGATGGGGAAATGGATGTGGATTATTTGGAAAAGGAGGCATTGTA ACCTGTGCTATGTTCACATGCAAAAAGAACATGGAAGGAAAAGTTGTGCTGCCAGAAAAT CTGGAATACACCATCGTGATAACACCTCACTCAGGAGAAGAGCACGCTGTAGGTAATGAC ACAGGAAAACATGGCAAGGAAATTAAAATAACACCACAGAGTTCCATCACAGAAGCAGAA CTGACAGGCTATGGCACTGTCACGATGGAGTGCTCTCCGAGAACGGGCCTCGACTTCAAT GAGATGGTGCTGCTGCAGATGGAAGACAAAGCCTGGCTGGTGCACAGGCAATGGTTCCTA GATCTGCCGTTACCATGGCTACCCGGAGCGGACACACAAGGATCAAATTGGATACAGAAA GAGACGTTGGTCACTTTCAAAAATCCCCACGCGAAGAAACAGGACGTCGTTGTTTTAGGA TCTCAAGAAGGGGCCATGCACACGGCACTTACAGGGGCCACAGAAATCCAGATGTCATCA GGAAACTTACTGTTCACAGGACATCTCAAGTGTAGGCTGAGAATGGACAAATTACAGCTT AAAGGAATGTCATACTCTATGTGTACAGGAAAGTTTAAAATTGTGAAGGAAATAGCAGAA ACACAACATGGAACAATAGTTATCAGAGTACAATATGAAGGGGACGGCTCTCCATGTAAG ATTCCTTTTGAGATAATGGATTTAGAAAAAAGACACGTCCTAGGTCGCCTGATTACAGTG AACCCAATCGTAACAGAAAAAGATAGCCCAGTCAACATAGAAGCAGAACCTCCATTCGGA GACAGCTACATCATCATAGGAGTAGAGCCGGGACAATTGAAACTCAATTGGTTCAAGAAG GGAAGTTCCATTGGCCAAATGTTTGAGACAACAATGAGAGGAGCGAAGAGAATGGCCATT TTAGGTGACACAGCCTGGGATTTTGGATCCCTGGGAGGAGTGTTTACATCTATAGGAAAG GCTCTCCACCAAGTTTTCGGAGCAATCTATGGGGCTGCTTTTAGTGGGGTCTCATGGACT ATGAAAATCCTCATAGGAGTTATCATCACATGGATAGGAATGAATTCACGTAGCACCTCA CTGTCTGTGTCACTAGTATTGGTGGGAGTCGTGACACTGTACTTGGGGGTTATGGTGCAG GCT

>MD1280 prME nucleotide sequence (SEQ ID NO: 7)

ttccatttaaccacacgaaatggagaaccacacatgatcgttggcagacaagagaaaggga aaagccttctgtttaaaacagaggatggtgtgaacatgtgtaccctcatggccattgatc ttggtgaattgtgtgaagatacaatcacgtacaagtgccccctcctcaggcagaatgaac cagaagatatagattgttggtgcaactccacgtccacatgggtaacttatgggacgtgta ccaccacaggagaacacagaagagaaaaaagatcagtggcactcgttccacatgtgggta tgggactggagacacgaactgaaacatggatgtcgtcagaaggggcctggaaacacgctc agagaattgaaacttggatcttgagacatccaggctttaccataatggcagcaatcctgg catataccgtaggaacgacacatttccaaagggccctgattttcatcttactggcagctg tcgctccttcaatgacaATGCGTTGCATAGGAATATCAAATAGAGACTTTGTAGAAGGGG TTTCAGGAGGAAGCTGGGTTGACATAGTCTTAGAACATGGAAGTTGTGTGACGACAATGG CAAAAAATAAACCAACACTGGATTTTGAACTGATAAAAACAGAAGCCAAACAACCTGCCA CTCTAAGGAAGTACTGTATAGAGGCAAAGCTGACCAATACAACAACAGAATCTCGTTGCC CAACACAAGGGGAACCCAGTCTAAATGAAGAGCAGGACAAAAGGTTCGTCTGCAAACACT CCATGGTAGACAGAGGATGGGGAAATGGATGTGGATTATTTGGAAAGGGAGGCATTGTGA CCTGTGCTATGTTCACATGCAAAAAGAACATGGAAGGAAAAATCGTGCAACCAGAAAATT TGGAATACACCATCGTGATAACACCTCACTCAGGAGAAGAGCACGCTGTAGGTAATGACA CAGGAAAACATGGTAAGGAAATTAAAATAACACCACAGAGTTCCATCACAGAAGCAGAAC TGACAGGCTATGGCACAGTCACGATGGAGTGCTCTCCGAGAACGGGCCTTGACTTCAATG AGATGGTGCTGCTGCAGATGGAAGATAAAGCTTGGCTGGTGCACAGGCAATGGTTCCTAG ACCTGCCGTTACCATGGCTACCCGGAGCGGACACACAAGGATCAAATTGGATACAGAAAG AGACATTGGTCACTTTCAAAAATCCCCACGCGAAGAAGCAGGATGTCGTTGTTTTAGGAT CTCAAGAAGGAGCCATGCACACGGCACTCACAGGGGCCACAGAAATCCAGATGTCATCAG GAAACTTACTATTCACAGGACATCTCAAATGCAGGCTGAGAATGGACAAACTACAGCTCA AAGGAATGTCATACTCTATGTGTACAGGAAAGTTTAAAATTGTGAAGGAAATAGCAGAAA CACAACATGGAACAATAGTTATCAGAGTACAATATGAAGGAGACGGCTCTCCATGTAAGA TCCCTTTTGAAATAATGGATTTGGAAAAAAGACATGTCTTAGGTCGCCTGATTACAGTTA ATCCGATCGTAACAGAAAAAGATAGCCCAGTCAACATAGAAGCAGAACCTCCATTCGGAG ACAGCTACATCATTATAGGAGTAGAGCCGGGACAATTGAAACTCAACTGGTTCAAGAAAG GAAGTTCCATCGGCCAAATGTTTGAGACGACAATGAGAGGAGCAAAGAGAATGGCCATTT TAGGTGACACAGCCTGGGATTTTGGATCTCTGGGAGGAGTGTTTACATCTATAGGAAAGG CTCTCCACCAAGTTTTCGGAGCAATCTATGGGGCTGCCTTTAGTGGGGTTTCATGGACTA TGAAAATCCTCATAGGAGTCATCATCACATGGATAGGAATGAATTCACGTAGCACCTCAC TGTCTGTGTCACTAGTATTGGTGGGAATCATAACACTGTACTTGGGAGCTATGGTGCAGG CT

Corresponding protein prM to E sequences of the four selected strains

>LAV2 prME protein sequence (SEQ ID NO: 8)

fhittrngephmivsrqekgks llfktevgvnmctlmamdlgelcedtitykcpllrqne pedidcwcnststwvtygtcttmgehrrekrsvalvphvgmgletrtetwmssegawkhv qrietwilrhpgftmmaailaytigtthfqralifilltavtpsmtMRCIGMSNRDFVEG VSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYCIEAKLTNTTTESRC PTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFRCKKNMEGKVVQPEN LEY IVI PHSGEEHAVGNDTGKHGKE IKI PQSSI EAELTGYGTVTMECSPRTGLDFN EMVLLQMENKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLG SQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKVVKEIAE TQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFG DSYI I IGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGK ALHQVFGAIYGAAFSGVSWTMKILIGVI ITWIGMNSRSTSLSVTLVLVGIVTLYLGVMVQ

A

>LAV2 E protein sequence (SEQ ID NO: 13)

MRCIGMSNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYC IEAKLTNTTTESRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFR CKKNMEGKVVQPENLEY IVI PHSGEEHAVGNDTGKHGKE IKI PQSSI EAELTGYGT VTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTF KNPHAKKQDVVVLGSQEGAMHTALTGATE IQMSSGNLLFTGHLKCRLRMDKLQLKGMSYS MCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTE KDSPVNIEAEPPFGDSYI I IGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMAILGDTAW DFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVI ITWIGMNSRSTSLSVTLV LVGIVTLYLGVMVQA

>LAV2 M protein sequence (SEQ ID NO: 18)

svalvphvgmgletrtetwmssegawkhvqrietwilrhpgftmmaaila tigtthfqr alifilltavtpsmt

>BID/V585 prME protein sequence (SEQ ID NO: 9)

fhittrngephmivgrqekgks llfktedgvnmctlmaidlgelcedtitykcpllrqne pedidcwcnststwvtygtctttgehrrekrsvalvphvgmgletrtetwmssegawkhv qrietwilrhpgftimaailaytigtthfqralifilltavapsmtMRCIGISNRDFVEG VSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYCIEAKLTNTTTESRC PTQGEPSLNEEQDKRFICKHSMVDRGWGNGCGLFGKGGIVTCAMFTCKKNMEGKVVQPEN LEYTIVITPHSGEEHAVGNDTGKHGKE IKITPQSSITEAELTGYGTVTMECSPRTGLDFN EMVLLQMEDKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLG SQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKIVKEIAE TQHGTIVIRVQYEGDGSPCKIPFE IMDLEKRHVLGRLITVNPIVTEKDSPV IEAEPPFG DSYI I IGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGK ALHQVFGAIYGAAFSGVSWTMKILIGVI ITWIGMNSRSTSLSVSLVLVGVVTLYLGVMVQ A

>BID/V585 E protein sequence (SEQ ID NO: 14)

MRCIGISNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYC IEAKLTNTTTESRCPTQGEPSLNEEQDKRFICKHSMVDRGWGNGCGLFGKGGIVTCAMFT CKKNMEGKVVQPENLEY IVI PHSGEEHAVGNDTGKHGKE IKI PQSSI EAELTGYGT VTMECSPRTGLDFNEMVLLQMEDKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTF KNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYS MCTGKFKIVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTE KDSPVNIEAEPPFGDSYI I IGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMAILGDTAW DFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVI ITWIGMNSRSTSLSVSLV LVGVVTLYLGVMVQA

>BID/V585 M protein sequence (SEQ ID NO: 19)

svalvphvgmgletrtetwmssegawkhvqrietwilrhpgftimaailaytigtthfqr alifilltavapsmt

>PR/DB023 prME protein sequence (SEQ ID NO: 10)

fhittrngephmivgrqekgks llfktedgvnmctlmaidlgelcedtitykcpllrqne pedidcwcnststwvtygtctttgehrrekrsvalvphvgmgletrtetwmssegawkhv qrietwilrhpgftimaailaytigtthfqralifilltavapsmtMRCIGISNRDFVEG VSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYCIEAKLTNTTTESRC PTQGEPSLNEEQDKRFICKHSMVDRGWGNGCGLFGKGGIVTCAMFTCKKNMEGKVVLPEN LEY IVI PHSGEEHAVGNDTGKHGKE IKI PQSSI EAELTGYGTVTMECSPRTGLDFN EMVLLQMEDKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLG SQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKIVKEIAE TQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFG DSYI I IGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGK ALHQVFGAIYGAAFSGVSWTMKILIGVI ITWIGMNSRSTSLSVSLVLVGVVTLYLGVMVQ

A

>PR/DB023 E protein sequence (SEQ ID NO: 15)

MRCIGISNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYC IEAKLTNTTTESRCPTQGEPSLNEEQDKRFICKHSMVDRGWGNGCGLFGKGGIVTCAMFT CKKNMEGKVVLPENLEYTIVITPHSGEEHAVGNDTGKHGKE IKITPQSSITEAELTGYGT VTMECSPRTGLDFNEMVLLQMEDKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTF KNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYS MCTGKFKIVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTE KDSPVNIEAEPPFGDSYI I IGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMAILGDTAW DFGSLGGVFTS IGKALHQVFGAIYGAAFSGVSWTMKILIGVI ITWIGMNSRSTSLSVSLV LVGVVTLYLGVMVQA

>PR/DB023 M protein sequence (SEQ ID NO: 20) svalvphvgmgletrtetwmssegawkhvqrietwilrhpgftimaailaytigtthfqr alifilltavapsmt

>MD1280 prME protein sequence (SEQ ID NO: 11)

fhittrngephmivgrqekgks llfktedgvnmctlmaidlgelcedtitykcpllrqne pedidcwcnststwvtygtctttgehrrekrsvalvphvgmgletrtetwmssegawkha qrietwilrhpgftimaaila tvgtthfqralifillaavapsmtMRCIGISNRDFVEG VSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYCIEAKLTNTTTESRC PTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFTCKKNMEGKIVQPEN LEY IVI PHSGEEHAVGNDTGKHGKE IKI PQSSI EAELTGYGTVTMECSPRTGLDFN EMVLLQMEDKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLG SQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKIVKEIAE TQHG IVIRVQYEGDGSPCKIPFE IMDLEKRHVLGRLI VNPIVTEKDSPV IEAEPPFG DSYI I IGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGK ALHQVFGAIYGAAFSGVSWTMKILIGVI ITWIGMNSRSTSLSVSLVLVGI ITLYLGAMVQ

A

>MD1280 E protein sequence (SEQ ID NO: 16)

MRCIGISNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYC IEAKLTNTTTESRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFT CKKNMEGKIVQPENLEY IVI PHSGEEHAVGNDTGKHGKE IKI PQSSI EAELTGYGT VTMECSPRTGLDFNEMVLLQMEDKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTF KNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYS MCTGKFKIVKE IAETQHG IVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLI VNPIVTE KDSPVNIEAEPPFGDSYI I IGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMAILGDTAW DFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVI ITWIGMNSRSTSLSVSLV LVGI ITLYLGAMVQA

>MD1280 M protein sequence (SEQ ID NO: 21)

s alvphvgmgletrtetwmssegawkhaqrietwilrhpgftimaailaytvgtthfqr alifillaavapsmt

> Consensus M sequence (SEQ ID NO: 23)

S alvphvgmgletrtetwmssegawkhvqrietwilrhpgftimaailaytigtthfqr alifilltavapsmt

> Consensus E protein sequence covering SEQ ID NOs: 13, 16 & 17 (SEQ ID NO: 28)

MRCIGXiSNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYC IEAKLTNTTTESRCPTQGEPSLX₂EEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMF X₃CKKNMEGKX₄VQPENLEY IVX₅TPHSGEEHAVGNDTGKHGKE IKX₆TPQSSI EAELTG YGTVTMECSPRTGLDFNEMVLLQMEX₇KAWLVHRQWFLDLPLPWLPGADX₈QX₉SNWIQKE TLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLK GMSYSMCTGKFKX₁₀VKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRX₁₁VLGRLIT VNPIVTEKDSPVNIEAEPPFGDSYI I IGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMA ILGDTAWDFGSLGGVFTS IGKALHQVFGAIYGAAFSGVSWTMKIL I GVI I TWIGMNSRST SLSVXi 2LVLVG IX13TLYLGX14MVQA ; wherein X, is M or I, X₂ is N or K, X₃ is or T, X,, is V or i, X₅ is V or I, X₆ is V or I, X₇ is N or D, X₈ is T or K, X₉ is G or E, X₁₀ is V or i, Xn is H or Y, X₁₂ is T or S, Xv, is V or I, X₁₄ is V or A.

Example 4: Construction of the cDNA clones corresponding to the optimized serotype 2 chimeric viruses and production of the encoded viruses

Construction of chimeric dengue viruses corresponding to the optimized serotype 2 strains is achieved using the Chimerivax™ technology substantially in accordance with the teaching of Chambers, et al. (1999, J. Virology 73(4):3095-3101 ). Reference may also be made to international patent applications WO 98/37911 , WO 03/101397, WO 07/021672, WO 08/007021 , WO 08/047023 and WO 08/065315, which detail the analogous processes used to construct the particular CYD-1 , CYD2, CYD-3 and CYD-4 used in Example 1 . Briefly, however, chimeric dengue viruses corresponding to the optimized serotype 2 strains are constructed as follows (N.B. the optimized chimeric dengue viruses are constructed using the genomic backbone of YF strain YF17D204 (YF- VAX(R), Sanofi-Pasteur, Swiftwater, PA, USA).

Construction of plasmid pSP1101

Construction of the YF-VAX cDNA clone - pJSY2284.1 (pACYC YF-Vax 5-3)

A full-length infectious cDNA clone of YF-VAX is constructed. The full-length infectious cDNA clone is based on the sequence of YF-VAX. A low copy number plasmid pACYC177 (New England Biolabs, Inc., Ipswich, MA, USA) is used to assemble the full-length cDNA clone.

A DNA sequence named as SP6 YF-Vax 5-3 is synthesized by GeneArt®. The sequence of SP6 YF-Vax 5-3 is designed in a way to facilitate an easy assembly of a full-length YF-Vax cDNA clone. The sequence is 2897 bp long and comprises the Xma I-SP6 promoter, the YF-Vax 5'UTR, the capsid, prM, M, part of E which extends to the Apa I site followed by unique sites Mlu I-

Sap l-Ngo Ml-Aat ll-CIa I for assembly, part of NS5 and further extended to 3' UTR followed by an Nru I site, which is used for run-off. This synthesized DNA sequence is flanked by EcoR V and Xho I sites. After digestion with EcoR V/Xho I, this DNA fragment is then cloned into the Aat ll/Xho I sites of low copy number plasmid pACYC177 to replace the 1615bp Aat ll/Xho I fragment. The resulting plasmid pJSY2284.1 (pACYC YF-Vax 5-3) is confirmed by sequence analysis. RT-PCR and cloning of the YF-Vax cDNA fragments spanning from the sites Apa I, Mlu I, Sap I, Ngo Ml, Aat II and Cla I and assembly of a full-length infectious cDNA clone of YF-vax (pJSY2374.5)

The yellow fever vaccine YF-VAX is grown in Vera cells, and the virus particles are concentrated. The viral RNA of YF-VAX is extracted from the concentrated virus and the cDNA copy is made by reverse transcriptase. Five cDNA fragments as shown herein are PCR amplified, TOPO cloned, sequenced and compared to the sequence of YF-VAX 2003 (see Figure 3). The PCR errors found in each fragment are corrected by either site-directed mutagenesis or fragment switching. There are too many sequence differences found in Ngo Ml-Aat II fragment after TOPO cloning, and therefore, this fragment is synthesized by GeneArt®. After final sequence confirmation, the five DNA fragments; Apa l-Mlu I, Mlu l-Sap I, Sap l-Ngo M1 , Ngo Ml-Aat II, and Aat ll-CIa I are isolated and stepwise cloned into the unique sites Apa I, Mlu I, Sap I, Ngo Ml, Aat II and Cla I in the plasmid pJSY2284.1 to obtain plasmid pJSY2374.5, which is confirmed to contain the correct sequence of YF-VAX full-length cDNA. Construction of cDNA for optimized chimeric dengue virus derived from the LA V2 strain (pSP1101) The strategy is to replace the prM and E genes of the YF-VAX® vaccine strain in the pJSY2374.5 plasmid containing the YF-VAX genome with those of the LAV2 strain, as done previously to build the CYD-1 , CYD-2, CYD-3 and CYD-4 dengue vaccines, using the Chimerivax™ technology. The resulting plasmid is pSP1101 .

In pJSY2374, restriction sites used for cloning are Xma I and Mlu I. These sites are located upstream and downstream of a 3000 bp fragment which contains: the SP6 promoter, YF17D 5'UTR, YF17D-capsid, YF17D-prM, YF17D-E and the N terminus of YF17D-NS1 . A sequence corresponding to this fragment but instead containing the prM and E genes of LAV2 flanked by Xma I and Mlu I sites is synthesized by GeneArt® and cloned into plasmid pMK-RQ (GeneArt®, Life Technologies Ltd, Paisley, U.K.) to create plasmid pMK-RQ-Seq1 . Plasmid pJSY2374.5 and pMK-RQ-Seq1 are digested by Xma I and Mlu I. The Xma l-Mlu I fragment from pMK-RQ-Seq1 is then inserted into plasmid pJSY2374.5 to form plasmid pSP1101 . XL-10 Gold Ultracompetent bacteria (Agilent Technologies, CA, USA) are used for transformation, as they are suitable for large plasmids. In a second step, positive clones are transferred into One Shot® TOP10 E. coli (Life Technologies Ltd, Paisley, U.K.), which allows the amplification of large size plasmids in significant amounts.

Plasmid pSP1101 thus allows the expression of LAV2 strain prM and E proteins with a YF- VAX replication engine. The resulting chimeric virus is designated CYD-LAV. Sequencing analysis shows no mutation as compared to the original sequences. Construction of corresponding plasmids for strains BID-V585, PR/DB023 and MD1280

An analogous strategy to that described above is used to build the plasmids corresponding to the serotype 2 strains BID-V585, PR/DB023 and MD1280. These plasmids are designated pSP1 102 (BID-V585), pSP1 103 (PR/DB023) and pSP1 104 (MD1280). The resulting chimeric viruses generated from those plasmids are designated CYD-BID, CYD-PR and CYD-MD.

Sequence analysis of the generated plasmids shows no mutations compared to the original sequences.

Generation of chimeric viruses from plasmids pSP1101, pSP1102, pSP1103 and pSP1104

In vitro transcription of RNA and generation of viruses is carried out as previously described (Guirakhoo F et al. J. Virol. 2001 ; 75:7290-304).

Example 5: Evaluation of the immunogenicity and viremia of the optimized serotype 2 chimeric viruses in a monkey model

Evaluation of immunogenicity and viremia in monkeys

Design of the study

Four groups each containing four Cynomolgus monkeys are defined. The four groups receive the following tetravalent dengue compositions (containing 5 log-|₀ CCID₅₀ of each CYD dengue serotype):

1 . Control tetravalent composition comprising CYD-1 , CYD-2, CYD-3 and CYD-4.

2. CYD-LAV tetravalent composition comprising CYD-1 , CYD-3, CYD-4 and CYD-LAV.

3. CYD-MD tetravalent composition comprising CYD-1 , CYD-3, CYD-4 and CYD-MD.

4. CYD-PR tetravalent composition comprising CYD-1 , CYD-3, CYD-4 and CYD-PR.

Monkeys receive two doses two months apart, as previously described (Guy B et al., Am J Trap Med Hyg. 2009; 80(2):302-1 1 ).

Results

Immunogenicity (SN₅₀ neutralizing response) and viremia are determined as described in the Materials and Methods section of Guy B., et al., Am. J. Trap. Med. Hyg. 2009; 80(2): 302-11 .

Table 7: SN50 neutralizing responses in monkeys immunized with optimized chimeric dengue serotype 2 viruses

DEN1 DEN2 DEN3 DEN4 DEN1 DEN2 DEN3 DEN4 control CYD TV responders 4/4 2/4 1/4 4/4 4/4 1/4 4/4 4/4

GMT 27 5 7 636 71 8 35 425

PD: Post-dose; TV: tetravalent formulation No serotype 2 viremia is observed, regardless of the serotype 2 chimeric virus administered. In respect of immunogenicity responses against DEN2, the tetravalent formulations comprising CYD-LAV, CYD-MD and CYD-PR demonstrate a higher response (both GMTs and number of responding animals) than the control formulation (see Table 7). Example 6: Assessment of tetravalent dengue vaccine formulations in flavivirus-naive adults in Mexico.

The objective of the present study was to compare the immunogenicity and viremia of a blended tetravalent dengue vaccine comprising CYD-1 (i.e. the particular Chimerivax dengue serotype 1 (CYD-1 ) strain generated from the prM and E sequences of DEN1 PU0359 (TYP 1 140)), VDV2, CYD-3 (i.e. the particular Chimerivax dengue serotype 3 (CYD-3) strain generated from the prM and E sequences of DEN3 PaH881/88) and CYD-4 (i.e. the particular Chimerivax dengue serotype 4 (CYD-4) strain generated from the prM and E sequences of DEN4 1228 (TVP 980)) with the immunogenicity and viremia of a tetravalent dengue vaccine comprising CYD-1 , CYD-2 (i.e. the particular Chimerivax dengue serotype 2 (CYD-2) strain generated from the prM and E sequences of DEN2 PU0218), CYD-3 and CYD-4. See Example 1 for more detail concerning the particular CYD-1 , CYD-2, CYD-3 and CYD-4 used in this study.

The relevant nucleotide and protein sequences of the VDV2 strain are as follows: >VDV2 nucleotide sequence (SEQ ID NO: 24)

AGUUGUUAGUCUACGUGGACCGACAAAGACAGAUUCUUUGAGGGAGCUAAGCUCAAUGUA GUUCUAACAGUUUUUUAAUUAGAGAGCAGAUCUCUGAUGAAUAACCAACGGAAAAAGGCG AAAAACACGCCUUUCAAUAUGCUGAAACGCGAGAGAAACCGCGUGUCGACUGUGCAACAG CUGACAAAGAGAUUCUCACUUGGAAUGCUGCAGGGACGAGGACCAUUAAAACUGUUCAUG GCCCUGGUGGCGUUCCUUCGUUUCCUAACAAUCCCACCAACAGCAGGGAUAUUGAAGAGA UGGGGAACAAUUAAAAAAUCAAAAGCUAUUAAUGUUUUGAGAGGGUUCAGGAAAGAGAUU GGAAGGAUGCUGAACAUCUUGAAUAGGAGACGCAGAUCUGCAGGCAUGAUCAUUAUGCUG AUUCCAACAGUGAUGGCGUUCCAUUUAACCACACGUAACGGAGAACCACACAUGAUCGUC AGCAGACAAGAGAAAGGGAAAAGUCUUCUGUUUAAAACAGAGGUUGGCGUGAACAUGUGU ACCCUCAUGGCCAUGGACCUUGGUGAAUUGUGUGAAGACACAAUCACGUACAAGUGUCCC CUUCUCAGGCAGAAUGAGCCAGAAGACAUAGACUGUUGGUGCAACUCUACGUCCACGUGG GUAACUUAUGGGACGUGUACCACCAUGGGAGAACAUAGAAGAGAAAAAAGAUCAGUGGCA CUCGUUCCACAUGUGCGAAUGGGACUGGAGACACGAACUGAAACAUGGAUGUCAUCAGAA GGGGCCUGGAAACAUGUCCAGAGAAUUGAAACUUGGAUCUUGAGACAUCCAGGCUUCACC AUGAUGGCAGCAAUCCUGGCAUACACCAUAGGAACGACACAUUUCCAAAGAGCCCUGAUU UUCAUCUUACUGACAGCUGUCACUCCUUCAAUGACAAUGCGUUGCAUAGGAAUGUCAAAU AGAGACUUUGUGGAAGGGGUUUCAGGAGGAAGCUGGGUUGACAUAGUCUUAGAACAUGGA AGCUGUGUGACGACGAUGGCAAAAAACAAACCAACAUUGGAUUUUGAACUGAUAAAAACA GAAGCCAAACAGCCUGCCACCCUAAGGAAGUACUGUAUAGAGGCAAAGCUAACCAACACA ACAACAGAAUCUCGCUGCCCAACACAAGGGGAACCCAGCCUAAAUGAAGAGCAGGACAAA AGGUUCGUCUGCAAACACUCCAUGGUAGACAGAGGAUGGGGAAAUGGAUGUGGACUAUUU GGAAAGGGAGGCAUUGUGACCUGUGCUAUGUUCAGAUGCAAAAAGAACAUGGAAGGAAAA GUUGUGCAACCAGAAAACUUGGAAUACACCAUUGUGAUAACACCUCACUCAGGGGAAGAG CAUGCAGUCGGAAAUGACACAGGAAAACAUGGCAAGGAAAUCAAAAUAACACCACAGAGU UCCAUCACAGAAGCAGAAUUGACAGGUUAUGGCACUGUCACAAUGGAGUGCUCUCCAAGA ACGGGCCUCGACUUCAAUGAGAUGGUGUUGCUGCAGAUGGAAAAUAAAGCUUGGCUGGUG CACAGGCAAUGGUUCCUAGACCUGCCGUUACCAUGGUUGCCCGGAGCGGACACACAAGAG UCAAAUUGGAUACAGAAGGAGACAUUGGUCACUUUCAAAAAUCCCCAUGCGAAGAAACAG GAUGUUGUUGUUUUAGGAUCCCAAGAAGGGGCCAUGCACACAGCACUUACAGGGGCCACA GAAAUCCAAAUGUCAUCAGGAAACUUACUCUUCACAGGACAUCUCAAGUGCAGGCUGAGA AUGGACAAGCUACAGCUCAAAGGAAUGUCAUACUCUAUGUGCACAGGAAAGUUUAAAGUU GUGAAGGAAAUAGCAGAAACACAACAUGGAACAAUAGUUAUCAGAGUGCAAUAUGAAGGG GACGGCUCUCCAUGCAAGAUCCCUUUUGAGAUAAUGGAUUUGGAAAAAAGACAUGUCUUA GGUCGCCUGAUUACAGUCAACCCAAUUGUGACAGAAAAAGAUAGCCCAGUCAACAUAGAA GCAGAACCUCCAUUUGGAGACAGCUACAUCAUCAUAGGAGUAGAGCCGGGACAACUGAAG CUCAACUGGUUUAAGAAAGGAAGUUCUAUCGGCCAAAUGUUUGAGACAACAAUGAGGGGG GCGAAGAGAAUGGCCAUUUUAGGUGACACAGCCUGGGAUUUUGGAUCCUUGGGAGGAGUG UUUACAUCUAUAGGAAAGGCUCUCCACCAAGUCUUUGGAGCAAUCUAUGGAGCUGCCUUC AGUGGGGUUUCAUGGACUAUGAAAAUCCUCAUAGGAGUCAUUAUCACAUGGAUAGGAAUG AAUUCACGCAGCACCUCACUGUCUGUGACACUAGUAUUGGUGGGAAUUGUGACACUGUAU UUGGGAGUCAUGGUGCAGGCCGAUAGUGGUUGCGUUGUGAGCUGGAAAAACAAAGAACUG AAAUGUGGCAGUGGGAUUUUCAUCACAGACAACGUGCACACAUGGACAGAACAAUACAAA UUCCAACCAGAAUCCCCUUCAAAACUAGCUUCAGCUAUCCAGAAAGCCCAUGAAGAGGAC AUUUGUGGAAUCCGCUCAGUAACAAGACUGGAGAAUCUGAUGUGGAAACAAAUAACACCA GAAUUGAAUCACAUUCUAUCAGAAAAUGAGGUGAAGUUAACUAUUAUGACAGGAGACAUC AAAGGAAUCAUGCAGGCAGGAAAACGAUCUCUGCGGCCUCAGCCCACUGAGCUGAAGUAU UCAUGGAAAACAUGGGGCAAAGCAAAAAUGCUCUCUACAGAGUCUCAUAACCAGACCUUU CUCAUUGAUGGCCCCGAAACAGCAGAAUGCCCCAACACAAAUAGAGCUUGGAAUUCGUUG GAAGUUGAAGACUAUGGCUUUGGAGUAUUCACCACCAAUAUAUGGCUAAAAUUGAAAGAA AAACAGGAUGUAUUCUGCGACUCAAAACUCAUGUCAGCGGCCAUAAAAGACAACAGAGCC GUCCAUGCCGAUAUGGGUUAUUGGAUAGAAAGUGCACUCAAUGACACAUGGAAGAUAGAG AAAGCCUCUUUCAUUGAAGUUAAAAACUGCCACUGGCCAAAAUCACACACCCUCUGGAGC AAUGGAGUGCUAGAAAGUGAGAUGAUAAUUCCAAAGAAUCUCGCUGGACCAGUGUCUCAA CACAACUAUAGACCAGGCUACCAUACACAAAUAACAGGACCAUGGCAUCUAGGUAAGCUU GAGAUGGACUUUGAUUUCUGUGAUGGAACAACAGUGGUAGUGACUGAGGACUGCGGAAAU AGAGGACCCUCUUUGAGAACAACCACUGCCUCUGGAAAACUCAUAACAGAAUGGUGCUGC CGAUCUUGCACAUUACCACCGCUAAGAUACAGAGGUGAGGAUGGGUGCUGGUACGGGAUG GAAAUCAGACCAUUGAAGGAGAAAGAAGAGAAUUUGGUCAACUCCUUGGUCACAGCUGGA CAUGGGCAGGUCGACAACUUUUCACUAGGAGUCUUGGGAAUGGCAUUGUUCCUGGAGGAA AUGCUUAGGACCCGAGUAGGAACGAAACAUGCAAUACUACUAGUUGCAGUUUCUUUUGUG ACAUUGAUCACAGGGAACAUGUCCUUUAGAGACCUGGGAAGAGUGAUGGUUAUGGUAGGC GCCACUAUGACGGAUGACAUAGGUAUGGGCGUGACUUAUCUUGCCCUACUAGCAGCCUUC AAAGUCAGACCAACUUUUGCAGCUGGACUACUCUUGAGAAAGCUGACCUCCAAGGAAUUG AUGAUGACUACUAUAGGAAUUGUACUCCUCUCCCAGAGCACCAUACCAGAGACCAUUCUU GAGUUGACUGAUGCGUUAGCCUUAGGCAUGAUGGUCCUCAAAAUGGUGAGAAAUAUGGAA AAGUAUCAAUUGGCAGUGACUAUCAUGGCUAUCUUGUGCGUCCCAAACGCAGUGAUAUUA CAAAACGCAUGGAAAGUGAGUUGCACAAUAUUGGCAGUGGUGUCCGUUUCCCCACUGUUC UUAACAUCCUCACAGCAAAAAACAGAUUGGAUACCAUUAGCAUUGACGAUCAAAGGUCUC AAUCCAACAGCUAUUUUUCUAACAACCCUCUCAAGAACCAGCAAGAAAAGGAGCUGGCCA UUAAAUGAGGCUAUCAUGGCAGUCGGGAUGGUGAGCAUUUUAGCCAGUUCUCUCCUAAAA AAUGAUAUUCCCAUGACAGGACCAUUAGUGGCUGGAGGGCUCCUCACUGUGUGCUACGUG CUCACUGGACGAUCGGCCGAUUUGGAACUGGAGAGAGCAGCCGAUGUCAAAUGGGAAGAC CAGGCAGAGAUAUCAGGAAGCAGUCCAAUCCUGUCAAUAACAAUAUCAGAAGAUGGUAGC AUGUCGAUAAAAAAUGAAGAGGAAGAACAAACACUGACCAUACUCAUUAGAACAGGAUUG CUGGUGAUCUCAGGACUUUUUCCUGUAUCAAUACCAAUCACGGCAGCAGCAUGGUACCUG UGGGAAGUGAAGAAACAACGGGCCGGAGUAUUGUGGGAUGUUCCUUCACCCCCACCCAUG GGAAAGGCUGAACUGGAAGAUGGAGCCUAUAGAAUUAAGCAAAAAGGGAUUCUUGGAUAU UCCCAGAUCGGAGCCGGAGUUUACAAAGAAGGAACAUUCCAUACAAUGUGGCAUGUCACA CGUGGCGCUGUUCUAAUGCAUAAAGGAAAGAGGAUUGAACCAACAUGGGCGGACGUCAAG AAAGACCUAAUAUCAUAUGGAGGAGGCUGGAAGUUAGAAGGAGAAUGGAAGGAAGGAGAA GAAGUCCAGGUAUUGGCACUGGAGCCUGGAAAAAAUCCAAGAGCCGUCCAAACGAAACCU GGUCUUUUCAAAACCAACGCCGGAACAAUAGGUGCUGUAUCUCUGGACUUUUCUCCUGGA ACGUCAGGAUCUCCAAUUAUCGACAAAAAAGGAAAAGUUGUGGGUCUUUAUGGUAAUGGU GUUGUUACAAGGAGUGGAGCAUAUGUGAGUGCUAUAGCCCAGACUGAAAAAAGCAUUGAA GACAACCCAGAGAUCGAAGAUCACAUUUUCCGAAAGAGAAGACUGACCAUCAUGGACCUC CACCCAGGAGCGGGAAAGACGAAGAGAUACCUUCCGGCCAUAGUCAGAGAAGCUAUAAAA CGGGGUUUGAGAACAUUAAUCUUGGCCCCCACUAGAGUUGUGGCAGCUGAAAUGGAGGAA GCCCUUAGAGGACUUCCAAUAAGAUACCAGACCCCAGCCAUCAGAGCUGAGCACACCGGG CGGGAGAUUGUGGACCUAAUGUGUCAUGCCACAUUUACCAUGAGGCUGCUAUCACCAGUU AGAGUGCCAAACUACAACCUGAUUAUCAUGGACGAAGCCCAUUUCACAGACCCAGCAAGU AUAGCAGCUAGAGGAUACAUCUCAACUCGAGUGGAGAUGGGUGAGGCAGCUGGGAUUUUU AUGACAGCCACUCCCCCGGGAAGCAGAGACCCAUUUCCUCAGAGCAAUGCACCAAUCAUA GAUGAAGAAAGAGAAAUCCCUGAACGCUCGUGGAAUUCCGGACAUGAAUGGGUCACGGAU UUUAAAGGGAAGACUGUUUGGUUCGUUCCAAGUAUAAAAGCAGGAAAUGAUAUAGCAGCU UGCCUGAGGAAAAAUGGAAAGAAAGUGAUACAACUCAGUAGGAAGACCUUUGAUUCUGAG UAUGUCAAGACUAGAACCAAUGAUUGGGACUUCGUGGUUACAACUGACAUUUCAGAAAUG GGUGCCAAUUUCAAGGCUGAGAGGGUUAUAGACCCCAGACGCUGCAUGAAACCAGUCAUA CUAACAGAUGGUGAAGAGCGGGUGAUUCUGGCAGGACCUAUGCCAGUGACCCACUCUAGU GCAGCACAAAGAAGAGGGAGAAUAGGAAGAAAUCCAAAAAAUGAGAAUGACCAGUACAUA UACAUGGGGGAACCUCUGGAAAAUGAUGAAGACUGUGCACACUGGAAAGAAGCUAAAAUG CUCCUAGAUAACAUCAACACGCCAGAAGGAAUCAUUCCUAGCAUGUUCGAACCAGAGCGU GAAAAGGUGGAUGCCAUUGAUGGCGAAUACCGCUUGAGAGGAGAAGCAAGGAAAACCUUU GUAGACUUAAUGAGAAGAGGAGACCUACCAGUCUGGUUGGCCUACAGAGUGGCAGCUGAA GGCAUCAACUACGCAGACAGAAGGUGGUGUUUUGAUGGAGUCAAGAACAACCAAAUCCUA GAAGAAAACGUGGAAGUUGAAAUCUGGACAAAAGAAGGGGAAAGGAAGAAAUUGAAACCC AGAUGGUUGGAUGCUAGGAUCUAUUCUGACCCACUGGCGCUAAAAGAAUUUAAGGAAUUU GCAGCCGGAAGAAAGUCUCUGACCCUGAACCUAAUCACAGAAAUGGGUAGGCUCCCAACC UUCAUGACUCAGAAGGCAAGAGACGCACUGGACAACUUAGCAGUGCUGCACACGGCUGAG GCAGGUGGAAGGGCGUACAACCAUGCUCUCAGUGAACUGCCGGAGACCCUGGAGACAUUG CUUUUACUGACACUUCUGGCUACAGUCACGGGAGGGAUCUUUUUAUUCUUGAUGAGCGCA AGGGGCAUAGGGAAGAUGACCCUGGGAAUGUGCUGCAUAAUCACGGCUAGCAUCCUCCUA UGGUACGCACAAAUACAGCCACACUGGAUAGCAGCUUCAAUAAUACUGGAGUUUUUUCUC AUAGUUUUGCUUAUUCCAGAACCUGAAAAACAGAGAACACCCCAAGACAACCAACUGACC UACGUUGUCAUAGCCAUCCUCACAGUGGUGGCCGCAACCAUGGCAAACGAGAUGGGUUUC CUAGAAAAAACGAAGAAAGAUCUCGGAUUGGGAAGCAUUGCAACCCAGCAACCCGAGAGC AACAUCCUGGACAUAGAUCUACGUCCUGCAUCAGCAUGGACGCUGUAUGCCGUGGCCACA ACAUUUGUUACACCAAUGUUGAGACAUAGCAUUGAAAAUUCCUCAGUGAAUGUGUCCCUA ACAGCUAUAGCCAACCAAGCCACAGUGUUAAUGGGUCUCGGGAAAGGAUGGCCAUUGUCA AAGAUGGACAUCGGAGUUCCCCUUCUCGCCAUUGGAUGCUACUCACAAGUCAACCCCAUA ACUCUCACAGCAGCUCUUUUCUUAUUGGUAGCACAUUAUGCCAUCAUAGGGCCAGGACUC CAAGCAAAAGCAACCAGAGAAGCUCAGAAAAGAGCAGCGGCGGGCAUCAUGAAAAACCCA ACUGUCGAUGGAAUAACAGUGAUUGACCUAGAUCCAAUACCUUAUGAUCCAAAGUUUGAA AAGCAGUUGGGACAAGUAAUGCUCCUAGUCCUCUGCGUGACUCAAGUAUUGAUGAUGAGG ACUACAUGGGCUCUGUGUGAGGCUUUAACCUUAGCUACCGGGCCCAUCUCCACAUUGUGG GAAGGAAAUCCAGGGAGGUUUUGGAACACUACCAUUGCGGUGUCAAUGGCUAACAUUUUU AGAGGGAGUUACUUGGCCGGAGCUGGACUUCUCUUUUCUAUUAUGAAGAACACAACCAAC ACAAGAAGGGGAACUGGCAACAUAGGAGAGACGCUUGGAGAGAAAUGGAAAAGCCGAUUG AACGCAUUGGGAAAAAGUGAAUUCCAGAUCUACAAGAAAAGUGGAAUCCAGGAAGUGGAU AGAACCUUAGCAAAAGAAGGCAUUAAAAGAGGAGAAACGGACCAUCACGCUGUGUCGCGA GGCUCAGCAAAACUGAGAUGGUUCGUUGAGAGAAACAUGGUCACACCAGAAGGGAAAGUA GUGGACCUCGGUUGUGGCAGAGGAGGCUGGUCAUACUAUUGUGGAGGACUAAAGAAUGUA AGAGAAGUCAAAGGCCUAACAAAAGGAGGACCAGGACACGAAGAACCCAUCCCCAUGUCA ACAUAUGGGUGGAAUCUAGUGCGUCUUCAAAGUGGAGUUGACGUUUUCUUCAUCCCGCCA GAAAAGUGUGACACAUUAUUGUGUGACAUAGGGGAGUCAUCACCAAAUCCCACAGUGGAA GCAGGACGAACACUCAGAGUCCUUAACUUAGUAGAAAAUUGGUUGAACAACAACACUCAA UUUUGCAUAAAGGUUCUCAACCCAUAUAUGCCCUCAGUCAUAGAAAAAAUGGAAGCACUA CAAAGGAAAUAUGGAGGAGCCUUAGUGAGGAAUCCACUCUCACGAAACUCCACACAUGAG AUGUACUGGGUAUCCAAUGCUUCCGGGAACAUAGUGUCAUCAGUGAACAUGAUUUCAAGG AUGUUGAUCAACAGAUUUACAAUGAGAUACAAGAAAGCCACUUACGAGCCGGAUGUUGAC CUCGGAAGCGGAACCCGUAACAUCGGGAUUGAAAGUGAGAUACCAAACCUAGAUAUAAUU GGGAAAAGAAUAGAAAAAAUAAAGCAAGAGCAUGAAACAUCAUGGCACUAUGACCAAGAC CACCCAUACAAAACGUGGGCAUACCAUGGUAGCUAUGAAACAAAACAGACUGGAUCAGCA UCAUCCAUGGUCAACGGAGUGGUCAGGCUGCUGACAAAACCUUGGGACGUUGUCCCCAUG GUGACACAGAUGGCAAUGACAGACACGACUCCAUUUGGACAACAGCGCGUUUUUAAAGAG AAAGUGGACACGAGAACCCAAGAACCGAAAGAAGGCACGAAGAAACUAAUGAAAAUAACA GCAGAGUGGCUUUGGAAAGAAUUAGGGAAGAAAAAGACACCCAGGAUGUGCACCAGAGAA GAAUUCACAAGAAAGGUGAGAAGCAAUGCAGCCUUGGGGGCCAUAUUCACUGAUGAGAAC AAGUGGAAGUCGGCACGUGAGGCUGUUGAAGAUAGUAGGUUUUGGGAGCUGGUUGACAAG GAAAGGAAUCUCCAUCUUGAAGGAAAGUGUGAAACAUGUGUGUACAACAUGAUGGGAAAA AGAGAGAAGAAGCUAGGGGAAUUCGGCAAGGCAAAAGGCAGCAGAGCCAUAUGGUACAUG UGGCUUGGAGCACGCUUCUUAGAGUUUGAAGCCCUAGGAUUCUUAAAUGAAGAUCACUGG UUCUCCAGAGAGAACUCCCUGAGUGGAGUGGAAGGAGAAGGGCUGCACAAGCUAGGUUAC AUUCUAAGAGACGUGAGCAAGAAAGAGGGAGGAGCAAUGUAUGCCGAUGACACCGCAGGA UGGGAUACAAAAAUCACACUAGAAGACCUAAAAAAUGAAGAGAUGGUAACAAACCACAUG GAAGGAGAACACAAGAAACUAGCCGAGGCCAUUUUCAAACUAACGUACCAAAACAAGGUG GUGCGUGUGCAAAGACCAACACCAAGAGGCACAGUAAUGGACAUCAUAUCGAGAAGAGAC CAAAGAGGUAGUGGACAAGUUGGCACCUAUGGACUCAAUACUUUCACCAAUAUGGAAGCC CAACUAAUCAGACAGAUGGAGGGAGAAGGAGUCUUUAAAAGCAUUCAGCACCUAACAAUC ACAGAAGAAAUCGCUGUGCAAAACUGGUUAGCAAGAGUGGGGCGCGAAAGGUUAUCAAGA AUGGCCAUCAGUGGAGAUGAUUGUGUUGUGAAACCUUUAGAUGACAGGUUCGCAAGCGCU UUAACAGCUCUAAAUGACAUGGGAAAGAUUAGGAAAGACAUACAACAAUGGGAACCUUCA AGAGGAUGGAAUGAUUGGACACAAGUGCCCUUCUGUUCACACCAUUUCCAUGAGUUAAUC AUGAAAGACGGUCGCGUACUCGUUGUUCCAUGUAGAAACCAAGAUGAACUGAUUGGCAGA GCCCGAAUCUCCCAAGGAGCAGGGUGGUCUUUGCGGGAGACGGCCUGUUUGGGGAAGUCU UACGCCCAAAUGUGGAGCUUGAUGUACUUCCACAGACGCGACCUCAGGCUGGCGGCAAAU GCUAUUUGCUCGGCAGUACCAUCACAUUGGGUUCCAACAAGUCGAACAACCUGGUCCAUA CAUGCUAAACAUGAAUGGAUGACAACGGAAGACAUGCUGACAGUCUGGAACAGGGUGUGG AUUCAAGAAAACCCAUGGAUGGAAGACAAAACUCCAGUGGAAACAUGGGAGGAAAUCCCA UACUUGGGGAAAAGAGAAGACCAAUGGUGCGGCUCAUUGAUUGGGUUAACAAGCAGGGCC ACCUGGGCAAAGAACAUCCAAGCAGCAAUAAAUCAAGUUAGAUCCCUUAUAGGCAAUGAA GAAUACACAGAUUACAUGCCAUCCAUGAAAAGAUUCAGAAGAGAAGAGGAAGAAGCAGGA GUUCUGUGGUAGAAAGCAAAACUAACAUGAAACAAGGCUAGAAGUCAGGUCGGAUUAAGC CAUAGUACGGAAAAAACUAUGCUACCUGUGAGCCCCGUCCAAGGACGUUAAAAGAAGUCA GGCCAUCAUAAAUGCCAUAGCUUGAGUAAACUAUGCAGCCUGUAGCUCCACCUGAGAAGG UGUAAAAAAUCCGGGAGGCCACAAACCAUGGAAGCUGUACGCAUGGCGUAGUGGACUAGC GGUUAGGGGAGACCCCUCCCUUACAAAUCGCAGCAACAAUGGGGGCCCAAGGCGAGAUGA AGCUGUAGUCUCGCUGGAAGGACUAGAGGUUAGAGGAGACCCCCCCGAAACAAAAAACAG CAUAUUGACGCUGGGAAAGACCAGAGAUCCUGCUGUCUCCUCAGCAUCAUUCCAGGCACA GAACGCCAGAAAAUGGAAUGGUGCUGUUGAAUCAACAGGUUCU

>VDV2 prME nucleotide sequence (SEQ ID NO: 25)

UUCCAUUUAACCACACGUAACGGAGAACCACACAUGAUCGUCAGCAGACAAGAGAAAGGG AAAAGUCUUCUGUUUAAAACAGAGGUUGGCGUGAACAUGUGUACCCUCAUGGCCAUGGAC CUUGGUGAAUUGUGUGAAGACACAAUCACGUACAAGUGUCCCCUUCUCAGGCAGAAUGAG CCAGAAGACAUAGACUGUUGGUGCAACUCUACGUCCACGUGGGUAACUUAUGGGACGUGU ACCACCAUGGGAGAACAUAGAAGAGAAAAAAGAUCAGUGGCACUCGUUCCACAUGUGCGA AUGGGACUGGAGACACGAACUGAAACAUGGAUGUCAUCAGAAGGGGCCUGGAAACAUGUC CAGAGAAUUGAAACUUGGAUCUUGAGACAUCCAGGCUUCACCAUGAUGGCAGCAAUCCUG GCAUACACCAUAGGAACGACACAUUUCCAAAGAGCCCUGAUUUUCAUCUUACUGACAGCU GUCACUCCUUCAAUGACAAUGCGUUGCAUAGGAAUGUCAAAUAGAGACUUUGUGGAAGGG GUUUCAGGAGGAAGCUGGGUUGACAUAGUCUUAGAACAUGGAAGCUGUGUGACGACGAUG GCAAAAAACAAACCAACAUUGGAUUUUGAACUGAUAAAAACAGAAGCCAAACAGCCUGCC ACCCUAAGGAAGUACUGUAUAGAGGCAAAGCUAACCAACACAACAACAGAAUCUCGCUGC CCAACACAAGGGGAACCCAGCCUAAAUGAAGAGCAGGACAAAAGGUUCGUCUGCAAACAC UCCAUGGUAGACAGAGGAUGGGGAAAUGGAUGUGGACUAUUUGGAAAGGGAGGCAUUGUG ACCUGUGCUAUGUUCAGAUGCAAAAAGAACAUGGAAGGAAAAGUUGUGCAACCAGAAAAC UUGGAAUACACCAUUGUGAUAACACCUCACUCAGGGGAAGAGCAUGCAGUCGGAAAUGAC ACAGGAAAACAUGGCAAGGAAAUCAAAAUAACACCACAGAGUUCCAUCACAGAAGCAGAA UUGACAGGUUAUGGCACUGUCACAAUGGAGUGCUCUCCAAGAACGGGCCUCGACUUCAAU GAGAUGGUGUUGCUGCAGAUGGAAAAUAAAGCUUGGCUGGUGCACAGGCAAUGGUUCCUA GACCUGCCGUUACCAUGGUUGCCCGGAGCGGACACACAAGAGUCAAAUUGGAUACAGAAG GAGACAUUGGUCACUUUCAAAAAUCCCCAUGCGAAGAAACAGGAUGUUGUUGUUUUAGGA UCCCAAGAAGGGGCCAUGCACACAGCACUUACAGGGGCCACAGAAAUCCAAAUGUCAUCA GGAAACUUACUCUUCACAGGACAUCUCAAGUGCAGGCUGAGAAUGGACAAGCUACAGCUC AAAGGAAUGUCAUACUCUAUGUGCACAGGAAAGUUUAAAGUUGUGAAGGAAAUAGCAGAA ACACAACAUGGAACAAUAGUUAUCAGAGUGCAAUAUGAAGGGGACGGCUCUCCAUGCAAG AUCCCUUUUGAGAUAAUGGAUUUGGAAAAAAGACAUGUCUUAGGUCGCCUGAUUACAGUC AACCCAAUUGUGACAGAAAAAGAUAGCCCAGUCAACAUAGAAGCAGAACCUCCAUUUGGA GACAGCUACAUCAUCAUAGGAGUAGAGCCGGGACAACUGAAGCUCAACUGGUUUAAGAAA GGAAGUUCUAUCGGCCAAAUGUUUGAGACAACAAUGAGGGGGGCGAAGAGAAUGGCCAUU UUAGGUGACACAGCCUGGGAUUUUGGAUCCUUGGGAGGAGUGUUUACAUCUAUAGGAAAG GCUCUCCACCAAGUCUUUGGAGCAAUCUAUGGAGCUGCCUUCAGUGGGGUUUCAUGGACU AUGAAAAUCCUCAUAGGAGUCAUUAUCACAUGGAUAGGAAUGAAUUCACGCAGCACCUCA CUGUCUGUGACACUAGUAUUGGUGGGAAUUGUGACACUGUAUUUGGGAGUCAUGGUGCAG GCC >VDV2 E protein sequence (SEQ ID NO: 26)

MRCIGMSNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYC IEAKLTNTTTESRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFR CKKNMEGKVVQPENLEY IVI PHSGEEHAVGNDTGKHGKE IKI PQSSI EAELTGYGT VTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPWLPGADTQESNWIQKETLVTF KNPHAKKQDVVVLGSQEGAMHTALTGATE IQMSSGNLLFTGHLKCRLRMDKLQLKGMSYS MCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTE KDSPVNIEAEPPFGDSYI I IGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMAILGDTAW DFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVI ITWIGMNSRSTSLSVTLV LVGIVTLYLGVMVQA

>VDV2 M protein sequence (SEQ ID NO: 27)

SVALVPHVRMGLETRTETWMSSEGAWKHVQRIETWILRHPGFTMMAAILAYTIGTTHFQR ALIFILLTAVTPSMT Study Design

In an open, randomised, controlled, phase I la trial, 150 healthy adults aged 18-45 years were enrolled at two centres in Mexico City, which is a dengue non-endemic area. Main exclusion criteria were: pregnancy or breast-feeding, human immunodeficiency virus, hepatitis B or C seropositivity, immunodeficiency or any other chronic illness that could interfere with the results, previous residence in or travel of >2 weeks to areas with high dengue endemicity, a history of flavivirus infection or previous vaccination against flavivirus disease. Women who were capable of conceiving were required to use an effective method of contraception or abstinence for at least 4 weeks before the first injection until at least four weeks after the last injection.

Participants were randomised into two groups and vaccinations were performed on Day 0 and Day 105 (±15 days). The groups received the following formulations:

Group 1 : Blended CYD VDV2 tetravalent formulation, i.e. a formulation comprising CYD-

1 , CYD-3, CYD4 and VDV2.

Group 2: Control tetravalent formulation (CYD-TDV), i.e. CYD-1 , CYD-2, CYD-3 and

CYD-4.

The formulations contained 10⁵ CCID₅₀ of each serotype of the CYD viruses and the formulation administered to Group 1 contained 10⁴ CCID₅₀ of the VDV-2 virus. Viremia

To evaluate the safety of the vaccines, the presence of CYD-1-4 or VDV-2 was assessed in serum collected 7, 14 and 21 days after each injection. Analyses were performed by the Global Clinical Immunology laboratory (Sanofi Pasteur, Swiftwater, PA, USA).

Analyses for CYD-1 -4 viremia were performed in two steps, as previously described in Poo ef a/., Pediatr Infect Dis J (201 1 ) 30: e9. Briefly, a first, non-serotype-specific, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the presence of any of the four CYD viruses. Samples that were positive in this first test were then analysed using four CYD serotype-specific quantitative RT-PCRs. In the non-serotype-specific RT-PCR, RNA was extracted from the serum using a commercial kit and an RT-PCR was carried out with primers from the yellow fever core gene sequence. In the serotype-specific RT-PCRs, RNA was again extracted from the serum using a commercial kit and an RT-PCR was carried out with serotype-specific primers from the envelope non-structural protein 1 junction gene sequence for each serotype. A dengue RT-PCR for serotype 2 was performed in group 1 since the tetravalent blending formulation administered to this group contained the VDV-2 virus.

Immunogenic ity

Antibody levels to each of the four dengue virus serotypes were determined by 50% plaque reduction neutralisation test on serum collected 28 days after each injection as well as on day 365 after the first injection. Briefly, serial 2-fold dilutions of heat-inactivated serum were mixed with a constant challenge dose of each dengue serotype DEN-1 , -2, -3, or -4 (expressed as plaque forming unit [PFU]/mL). The mixtures were inoculated into wells of a 24-well plate of confluent VERO cell monolayers. After incubation for several days, dengue virus infection is indicated by formation of plaques. The neutralising antibody titre is calculated as the highest reciprocal dilution (1/dil) of serum at which >50% reduction in viral plaque count is observed (PRNT50). The lower limit of quantitation of the dengue PRNT50 is 10; samples with titres >10 were considered seropositive.

Results

Formulations were administered to participants in Groups 1 and 2 on day 0 and day 105 of the study. There were no marked differences between the two groups with regard to the injection site or systemic reactogenicity after either the first or the second vaccination. Viremia was assessed in serum collected 7, 14 and 21 days after each injection (Table 8). The neutralising antibody titres were measured 28 days after each injection and on day 365 after the first injection (Table 8).

Table 8. Vaccine virus viremia 7, 14, or 21 days after first and second injections (n (%) with detectable and quantifiable viremia)

First injection Second injection

Group 1 Group 2 Group 1 Group 2

Blended Tetravalent Blended Tetravalent

CYD/VDV CYD-TDV CYD/VDV CYD-TDV Ϊ 4 -

Non-serotype specific

N 29 31 28 29

Detectable viremia 27 (93%) 25 (81%) 1 (4%) 1 (3%)

Quantifiable viremia 1 (3%) 2 (6%) 0 0

DENV-1

Detectable viremia 1 (3%) 4 (13%) 0 0

Quantifiable viremia 0 2 (7%) 0 0

DENV-2

Detectable viremia 0 2 (6%) 0 0

Quantifiable viremia 0 0 0 0

DENV-3

Detectable viremia 8 (28%) 7 (23%) 1 (4%) 0

Quantifiable viremia 0 0 0 0

DENV-4

Detectable viremia 24 (83%) 21 (68%) 0 0

Quantifiable viremia 0 3 (1 %) 0 0

After the first injection, detectable viremia, as determined by the non-serotype specific RT- PCR test, was observed in a similar proportion of participants in both groups (see Table 8). In the majority of cases, viremia was below the lower limit of quantitation. Analysis with the serotype- specific assays showed that CYD-4 was the most commonly detected serotype, followed by CYD- 3. After the second injection of the blended CYD VDV vaccine in Group 1 or the CYD-TDV vaccine in Group 2, viremia was only detected in one participant per group by the non-serotype-specific assay.

Accordingly, there was no significant difference between the levels of viremia induced by the blended CYD/VDV and CYD-TDV.

Table 9. Geometric mean titres (95% confidence interval) of dengue antibodies 28 days after the first and second injections and 365 days after the first injection

Group 1 Group 2

CYD/VDV blended CYD-TDV

First injection

Serotype 1 15 (9;28) 17 (10;31)

Serotype 2 17 (8;33) 32 (16;65)

Serotype 3 64 (31;133) 23 (13;39) Ϊ 5 -

Serotype 4 552 (299;1019) 468 (226;968)

Second injection

Serotype 1 54 (30;96) 28 (15;50)

Serotype 2 152 (79;293) 43 (23;79)

Serotype 3 127 (71;229) 46 (29;73)

Serotype 4 246 (159;382) 173 (97;307)

365 days post-dose 1

Serotype 1 14 (9;22) 18 (10;30)

Serotype 2 55 (32;94) 16 (9;29)

Serotype 3 36 (20;64) 11 (7;16)

Serotype 4 103 (69; 155) 72 (44; 117)

It can be seen from Table 9 that the second injection of the blended CYD VDV vaccine (Group 1 ) induced higher GMTs against serotype 2 of dengue virus than the CYD-TDV vaccine (Group 2). An improved response to serotype 2 in the blended CYD/VDV group was also observed 365 days after the first dose.

Furthermore, the second injection of the blended CYD/VDV vaccine (Group 1 ) resulted in an improved neutralising antibody response against all serotypes of dengue virus when compared with the group receiving the CYD-TDV vaccine (Group 2). Importantly, the blended CYD/VDV formulation group demonstrated a more persistent neutralising antibody response against dengue virus than the CYD-TDV group on day 365 after the first injection.

The example therefore shows that, overall, the blended CYD-1 , 3, 4/VDV2 vaccine formulation induces stronger and longer lasting immune responses against the dengue virus serotypes than the CYD-TDV vaccine while showing a similar safety profile, as determined by the levels of viremia.

Sequence Listing

SEQ ID NO. Sequence

1 Clinical trial circulating strain prM+E nucleotide sequence

2 Clinical trial circulating strain prM+E protein sequence

3 Consensus serotype 2 prM+E protein sequence

4 LAV2 prM+E nucleotide sequence

5 BID/V585 prM+E nucleotide sequence

6 PR/DB023 prM+E nucleotide sequence MD1280 prM+E nucleotide sequence

LAV2 prM+E protein sequence

BID V585 prM+E protein sequence

PR/DB023 prM+E protein sequence

MD1280 prM+E protein sequence

Consensus serotype 2 E protein sequence

LAV2 E protein sequence

BID V585 E protein sequence

PR/DB023 E protein sequence

MD1280 E protein sequence

Clinical trial circulating strain E protein sequence

LAV2 M protein sequence

BID/V585 M protein sequence

PR DB023 M protein sequence

MD1280 M protein sequence

Clinical trial circulating strain M protein sequence

Consensus serotype 2 M protein sequence

Entire nucleotide sequence of VDV2 (RNA equivalent) prM+E VDV2 nucleotide sequence (RNA equivalent)

E VDV2 protein sequence

M VDV2 protein sequence

Consensus E protein sequence covering SEQ ID NOs: 13, 16 & 17

Claims

1 . An agent which comprises a dengue antigen of serotype 1 or a nucleic acid construct or viral vector which is able to express a dengue virus-like particle (VLP) of serotype 1 ; an agent which comprises a dengue antigen of serotype 2 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 2; an agent which comprises a dengue antigen of serotype 3 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 3; and an agent which comprises a dengue antigen of serotype 4 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 4; wherein each dengue antigen is independently selected from the group consisting of:

(a) a live attenuated dengue virus;

(b) an inactivated dengue virus;

(c) a live attenuated or inactivated chimeric dengue virus; and

(d) a dengue virus-like particle (VLP);

for use in a method of inducing neutralising antibodies against four serotypes of dengue virus in a mammal, wherein said method comprises the administration of said agents to said mammal in conjunction with a measles vaccine.

2. The agents for use in a method according to claim 1 , wherein said method comprises the administration of said agents to said mammal in conjunction with a measles vaccine, a mumps vaccine and a rubella vaccine.

3. The agents for use in a method according to claim 1 or claim 2, wherein said mammal is a human.

4. The agents for use in a method according to any preceding claim, wherein said agents are present within a single tetravalent composition.

5. The agents for use in a method according to any preceding claim, wherein each of said agents comprises a dengue antigen.

6. The agents for use in a method according to claim 5, wherein each of said dengue antigens is independently selected from the group consisting of a live attenuated dengue virus and a live attenuated chimeric dengue virus.

7. The agents for use in a method according to claim 5, wherein each of said agents comprises a live attenuated chimeric dengue virus.

8. The agents for use in a method according to claim 6 or claim 7, wherein said live attenuated chimeric dengue virus comprises an envelope (E) protein from a dengue virus of a first serotype and one or more proteins other than an envelope protein from: (i) a second dengue virus serotype, said first and said second serotype being different from each other, or (ii) a flavivirus other than a dengue virus.

9. The agents for use in a method according to claim 6 or claim 7, wherein said live attenuated chimeric dengue virus comprises an envelope (E) protein and a membrane (M) protein from a dengue virus of a first serotype and one or more proteins other than an envelope protein or a membrane protein from: (i) a second dengue virus serotype, said first and said second serotype being different from each other, or (ii) a flavivirus other than a dengue virus.

10. The agents for use in a method according to claim 8 or claim 9, wherein the flavivirus other than a dengue virus is a yellow fever virus.

1 1 . The agents for use in a method according to any preceding claim, wherein the dengue antigen of serotype 1 is CYD-1 , the dengue antigen of serotype 2 is CYD-2, the dengue antigen of serotype 3 is CYD-3 and the dengue antigen of serotype 4 is CYD-4.

12. The agents for use in a method according to claim 5, wherein said dengue antigens of serotypes 1 , 3 and 4 are each a live attenuated chimeric dengue virus and the dengue antigen of serotype 2 is a live attenuated dengue virus of serotype 2, which comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 24.

13. The agents for use in a method according to claim 12, wherein said live attenuated chimeric dengue viruses are as defined in any one of claims 8 to 10.

14. The agents for use in a method according to any one of claims 1 to 10, wherein the dengue antigen of serotype 2 comprises a polypeptide having at least 90% identity to SEQ ID NO: 12.

15. The agents for use in a method according to claim 14, wherein said polypeptide comprises a valine residue at the position within the polypeptide that corresponds to position 251 of SEQ ID NO: 12.

16. The agents for use in a method according to claim 14 or claim 15, wherein said dengue antigen of serotype 2 further comprises a second polypeptide having at least 90% identity to SEQ ID NO: 23.

17. The agents for use in a method according to claim 16, wherein said second polypeptide comprises a threonine residue at the position within the polypeptide that corresponds to position 34 of SEQ ID NO: 23.

18. The agents for use in a method according to any one of claims 14 to 17, wherein 1 9 - the dengue antigen of serotype 1 is CYD-1 , the dengue antigen of serotype 3 is CYD-3 and the dengue antigen of serotype 4 is CYD-4.

19. The agents for use in a method according to any one of claims 7 to 11 and 14 to 18, wherein each of said agents comprises an amount of live attenuated chimeric dengue virus of from about 10³ to about 10⁶ CCID₅₀.

20. The agents for use in a method according to claim 19, wherein each agent comprises an amount of live attenuated chimeric dengue virus of about 10⁵ CCID₅₀.

21 . The agents for use in a method according to any preceding claim, wherein said agents further comprise a pharmaceutically acceptable carrier or excipient.

22. The agents for use in a method according to any one of claims 2 to 21 , wherein said method comprises administering a first dose, a second dose and a third dose of said agents to said mammal and wherein said second dose is to be administered about six months after said first dose and wherein said third dose is to be administered about twelve months after said first dose and wherein said measles vaccine, said mumps vaccine and said rubella vaccine are only administered in conjunction with either one of said first dose or said second dose.

23. The agents for use in a method according to claim 22, wherein said first dose is administered at about twelve months of age.

24. The agents for use in a method according to any one of claims 2 to 23, wherein said measles vaccine, said mumps vaccine and said rubella vaccine are present within a single trivalent measles, mumps and rubella (MMR) vaccine composition.

25. An agent for use in a method of inducing neutralising antibodies against at least one serotype of dengue virus in a mammal, wherein said agent comprises:

(i) at least one dengue antigen selected from the group consisting of:

(a) a live attenuated dengue virus;

(b) an inactivated dengue virus;

(c) a live attenuated or inactivated chimeric dengue virus; and

(d) a dengue virus-like particle (VLP); or

(iii) at least one nucleic acid construct or viral vector which is able to express in a human cell at least one dengue antigen which is a dengue VLP; and wherein said method comprises the administration of said agent to said mammal in conjunction with a measles vaccine.

26. The agent for use in a method according to claim 25, wherein said method comprises the administration of said agent to said mammal in conjunction with a measles vaccine, a mumps vaccine and a rubella vaccine.

27. The agent for use in a method according to claim 26 wherein said method comprises administering a first dose, a second dose and a third dose of said agent to said mammal and wherein said second dose is to be administered about six months after said first dose and wherein said third dose is to be administered about twelve months after said first dose and wherein said measles vaccine, said mumps vaccine and said rubella vaccine are only administered in conjunction with either one of said first dose or said second dose.

28. An agent for use in a method according to claim 26 or claim 27, wherein said measles vaccine, said mumps vaccine and said rubella vaccine are present within a single trivalent measles, mumps and rubella (MMR) vaccine composition.

29. A measles vaccine for use in a method of inducing antibodies against measles virus in a mammal, wherein said method comprises the administration of said measles vaccine to said mammal in conjunction with an agent comprising a dengue antigen of serotype 1 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 1 , an agent comprising a dengue antigen of serotype 2 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 2, an agent comprising a dengue antigen of serotype 3 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 3 and an agent comprising a dengue antigen of serotype 4 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 4; wherein each dengue antigen is independently selected from the group consisting of:

(a) a live attenuated dengue virus;

(b) an inactivated dengue virus;

(c) a live attenuated or inactivated chimeric dengue virus; and

(d) a dengue virus-like particle (VLP).

30. The measles vaccine for use in a method according to claim 29, wherein said measles vaccine is administered to said mammal in conjunction with a mumps vaccine and a rubella vaccine and said method further comprises the induction of antibodies against mumps virus and rubella virus.

31 . The measles, mumps and rubella vaccines for use in a method according to claim 30, wherein said measles vaccine, said mumps vaccine and said rubella vaccine are present within a single trivalent measles, mumps and rubella (MMR) vaccine composition.

32 The measles vaccine for use in a method according to claim 29, wherein said agent comprising a dengue antigen of serotype 1 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 1 , said agent comprising a dengue antigen of serotype 2 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 2, said agent comprising a dengue antigen of serotype 3 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 3 and said agent comprising a dengue antigen of serotype 4 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 4 are as defined in any one of claims 3 to 23.

33. The measles, mumps and rubella vaccines for use in a method according to claims 30 or claim 31 , wherein said agent comprising a dengue antigen of serotype 1 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 1 , said agent comprising a dengue antigen of serotype 2 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 2, said agent comprising a dengue antigen of serotype 3 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 3 and said agent comprising a dengue antigen of serotype 4 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 4 are as defined in any one of claims 2 to 24.

34. A method of inducing antibodies against four serotypes of dengue virus and measles virus in a mammal, comprising the administration to said mammal of an agent comprising a dengue antigen of serotype 1 or a nucleic acid construct or viral vector which is able to express a dengue virus-like particle (VLP) of serotype 1 ; an agent comprising a dengue antigen of serotype 2 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 2; an agent comprising a dengue antigen of serotype 3 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 3 and an agent comprising a dengue antigen of serotype 4 or a nucleic acid construct or viral vector which is able to express a dengue VLP of serotype 4 in conjunction with a measles vaccine, wherein said agents are as defined in any one of claims 1 to 24.

35. A method as claimed in claim 34, wherein said method comprises administering said agents and said measles vaccine to said mammal in conjunction with a mumps vaccine and a rubella vaccine and said method further comprises inducing antibodies against mumps virus and rubella virus.

36. A method as claimed in claim 35, wherein said measles vaccine, said mumps vaccine and said rubella vaccine are present within a single trivalent measles, mumps and rubella (MMR) vaccine composition.