Method of immunization against the four serotypes of dengue
The invention relates to a method for inducing a homologous protection against the 4 dengue serotypes in a patient, comprising the sequential administration, to sard patient, (i) of a dose of a vaccinal dengue virus of a first serotype and of a dose of a vaccinal dengue virus of a second serotype, and (ii) of a dose of a vaccinal dengue virus of a third serotype and of a dose of a vaccinal dengue virus of a fourth serotype, in which the vaccinal dengue viruses (Ii) are administered at least 30 days and at most 1 year after administration of the vaccinal dengue viruses (i).
Dengue diseases are caused by four viruses of the flavivlrus genus, of the serological type, which are similar to but distinct from an antigenic point of view (GiJbler et al., 1988 In: Epidemiology of arthropod-borne viral disease. Monath TPM, editor, Boca Raton (FL): CRC Press: 223-60; Kautner et ai., 1997, J. of Pediatrics, 131:516-624; Rigau-Perez et al., 1998, Lancet; 352: 971-977; Vaughn et al., 1997, J Infect Dis; 176: 322-30). Infection with a dengue serotype can produce a clinical disease spectrum ranging from a nonspecific viral syndrome to a severe hemorrhagic disease which is fatal. The incubation period of dengue fever after a mosquito bite is approximately 4 days (ranging from 3 to 14 days). Dengue fever is characterized by a biphasic fever, headaches, pain in various parts of the body, prostration, eruptions, lymphadenopathy and leukopenia (Kautner et al., 1997, J. of Pediatrics, 131:516-524; Rigau-P^rez et al.. 1998, Lancet; 352: 971-977). The viremic period Is the same as for febrile diseases (Vaughn et al., 1997, J. Infect. Dis.; 176: 322-30). Recovery from dengue fever occurs after 7 to 10 days, but there is usually a pnDlonged asthenia. Decreases in leukocyte and platelet count are common.
Hemorrhagic dengue Is a severe febrile disease characterized by anomalies in homeostasis and an increase in vascular pemieability which can result in hypovolemia and in hypotension (dengue with shock syndrome) often complicated by severe intema! hemorrhaging. The mortality rate of hemon-hagic dengue can be up to 10% >Aflthout treatment, but is 1% In most centers with experience in treatment (WHO technical Guide, 1986. Dengue

haemorrhagic fever: diagnosis, treatment and control, p1-2. Worid Health Organization, Geneva, Switzerland).
The routine laboratory diagnosis of dengue is based on isolation of the • virus and/or detection of antibodies specific to the dengue virus.
Dengue is the second most common tropical infectious disease after malaria, more than half the world's population (2.5 billion) living In regions where there is a risk of epidemic transmission. Each year, cases of dengue are estimated at 50-100 miillon, cases of patients hospitalized for hemorrhagic dengue at 500 000, and the number of deaths at 25 000. Dengue is endemic in Asia, in the Pacific region, in Africa, in Latin America and in the Caribbean. More than 100 tropical countries are endemic for dengue virus infections and hemon'hagic dengue has been documented in 60 of these countries (Gubier, 2002, TREIMDS in Microbiology. 10:100-103; Monath, 1994. Proc. Natl. Acad. Set.; 91; 2395-2400). A certain number of well-described factors appear to be involved in dengue: population growth; unplanned and uncontrolled urbanization, in particular in combination with poverty; an increase in air travel; the lack of effective control of mosquitoes and the deterioration of hygiene infrastructures and of public health (Gubier, 2002, TRENDS in Microbiology. 10:100-103). Individuals who travel and expatriates are increasingly warned about dengue (Shirtcliffe et al., 1998, J. Roy. Coil. Phys. Lond.; 32: 235-237). Dengue has constituted one of the main causes of febrile diseases in American troops during deployments in tropica) zones endemic for dengue (DeFraites et al., 1994, MMWR 1994; 43: 845-848).
The viruses are maintained fn a cycle which involves humans and Aedes Begypth a domestic mosquito which bites during the day, and which prefers to feed off humans. The infection in humans is initiated by injection of the virus while an infected Aedes aegypti mosquito feeds on the blood. The virus in the saliva is deposited mainly in the extravascular tissues. The first category of cells Infected after inoculation are dendritic cells, which then migrate to the lymph nodes (Wu et al., 2000, Nature Med.; 7:816-820). After an initial replication in the skin and in the lymph nodes, the virus appears in the blood during the acute febrile phase, generally for 3 to 5 days.

hAor)Ocy\es and macrophages are, with dendritic cells, among the first
targets of the dengue virus. Protection against a homotypic reinfection is
complete and probably lasts for a lifetime, but crossprotection between the
various dengue types lasts less than a few weeks to a few months (Sabin,
1952, Am. J. Trop. Med. Hyg.; 1: 30-50). Consequently, an individual can
experience an infection with a different serotype. A second infection with
dengue is in theory a risk factor for developing a severe dengue disease.
However, hemorrhagic dengue is multifactorial: these factors include the strain
of the virus Involved, and also the age, the immune status and the genetic
predisposition of the patient. Two factors play a major role In the occunrence of
hemorrhagic dengue: rapid viral replication with a high 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 substantial inflammatory response with the release of
high levels of inflammatory mediators (Rothman and Ennis, 1999, Virology;
257: 1-6). There is no specific treatment against dengue. The treatment for
dengue fever is symptomatic with confinement to bed, control of the fever and
of the pain with antipyretics and analgesics, and adequate fluid intake. The
treatment for hemoniiagic dengue requires equilibration of fluid losses,
replacement of clotting factors and heparin infusion.
Preventive measures are currently based on controlling the vector and taking personal protection steps which are difficult to implement and expensive. No vaccine against dengue has been approved at this time. Given that the four dengue serotypes are in circulation in the worid and since they have been reported as being involved in cases of dengue hemorrhagic fever, vaccination should ideally confer protection against the four serotypes of the dengue virus.
Sequential immunization strategies have previously been implemented with the aim of inducing a heterologous pnatection among the various dengue serotypes.
Thus, Price (1968, Am. J. Epid., 88 :392-397) has described a method of sequential immunization against dengue comprising a series of two infections with dengue serotype 1 and then with dengue serotype 2, which conferred pnstection in a challenge test with dengue serotype 3 or 4.

Whitehead et al. (1970, Am. J. Trop. Med. Hyg., 19:94-102) sought to determine the influence of a sequential monovalent infection with two or three of the four dengue serotypes, on the conferred heterologous immunity. Gibbons were thus initially Infected with a dengue virus serotype 1, 2, 3 or 4. Following a second infection with a heterologous serotype, a variable viremla was detected which was dependent on the sequence of infection and in particular on the serotype used for the first infection. More specifically, a second viremia appeared in gibbons initially infected with serotype 2, 3 or 4 and then challenged with serotype 1, 2 or 4.
Scherer et al. (1972, Am. J. Epid., 96:67-79) described a sequential monovalent infection comprising a first infection with one of the four dengue serotypes, followed by a second infection, or even a third infection, with a homologous or heterologous serotype. The proposed schemes did not make it possible to obtain a satisfactory protection against a challenge with a heterologous serotype,
Halstead et al. (1973, Am. J. Trop. Med. Hyg., 22 :365-374) evaluated, in monl^eys, a method of sequential immunization against dengue comprising a series of two, three or four monovalent infections with heterologous dengue serotypes 1 to 4. The authors concluded that a protection against a subsequent infection could be obtained with the immunization sequence consisting of serotypes 1, 2 then 4, followed by a challenge with serotype 3. Bivalent immunization is neither described nor suggested, Furlhemiore, the authors advise against sequential immunizations due to their laborious nature and to the random nature of the results generated.
Halstead et al. (1973, Am. J, Trop. Med. Hyg., 22 :375-381) also found that a bivalent immunization with two heterologous dengue serotypes did not protect, or only partially protected, against an infection with a third dengue serotype.
In the context of the present invention, the objective is to Induce a homologous pnDtection against the 4 dengue serotypes. The inventors demonstrated that it is possible to generate an immune response comprising antibodies which neutralize the 4 serotypes when the latter are administered sequentially in pairs.

The inventors have in particular shown that a DEN-1.2 bivalent immunization followed two months later by a DEN-3,4 bivalent immunization induces high responses against the four serotypes in all the monkeys immunized. The immune response thus generated is quantitatively and qualitatively greater (covers all the serotypes).
According to a first subject, the present invention therefore relates to vaccinai compositions comprising (i) a dose of a vaccinal dengue virus of a first serotype and a dose of a vaccinal dengue virus of a second serotype, and (ii) a dose of a vaccinal dengue virus of a third serotype and a dose of a vaccinal dengue virus of a fourth serotype, as a combination vaccinal composition against dengue for sequential administration, in which the vaccinal dengue viruses (ii) are administered at least 30 days and at most 1 year after the administration of the vaccinal dengue viruses (i).
According to one embodiment of the vaccinai compositions according to the invention, the vaccinai viruses (ii) are administered 30 days to 3 months after the administration of the vaccinai viruses (i).
According to another specific embodiment of the vaccinal compositions according to the invention, the vaccinai viruses (ii) are administered 30 days after the administration of the vaccinal viruses (i).
According to another embodiment of the vaccinal compositions according to the invention, the vaccinal dengue viruses (i) are administered in the form of a bivalent vaccinai composition-According to another embodiment of the vaccinal compositions according to the invention, the vaccinal dengue viruses (ii) are administered in the form of a bivalent vaccinal composition.
According to one specific embodiment of the vaccinal compositions according io the invention, said vaccinal dengue virus serotype 1 is selected from the group consisting of the VDV1 strain and of a ChimeriVax™ DEN-1.
According to another specific embodiment of the vaccinal compositions according to the invention, said vaccinai dengue virus serotype 2 is selected from the group consisting of the VDV2 strain and of a ChimeriVax™ DEM-2.

According to another specific embodiment of the vaccinal compositions according to the invention, said vaccinal dengue virus serotype 1 is the VDV1 strain and said vaccinal dengue virus serotype 2 Is the VDV2 strain.
According to another specific embodiment of the vaccinal compositions according to the invention, said vaccina! dengue vims serotype 1 is a ChimeriVax™ DEN-1 and said vaccinal dengue virus serotype 2 is a ChimeriVax'TM DEN-2.
According to another specific embodiment of the vaccinal compositions according to the invention, said vaccinal dengue virus serotype 3 is a ChimeriVax™ DEN-3.
According to another specific embodiment of the vaccinal compositions according to the invention, said vaccina) dengue virus serotype 4 is a ChimeriVax™ DEN-4.
According to another specific embodiment of the vaccinal compositions according to the invention, the first and second serotypes are, respectively, CYD DEN1 and CYD DEN2 and the third and fourth serotypes are. respectively, CYD DEN3 and CYD DEN4.
According to another specific embodiment of the vaccinal compositions according to the invention, the doses of vaccinal dengue viruses serotypes 1, 2, 3 and 4 are each within a range of from 10^ to 10^ CCID50.
A subject of the invention is also the use of a vaccinal dengue virus of a third serotype and of a vaccinal dengue virus of a fourth serotype, for the manufacture of a dengue vaccine intended to be administered to a patient who has received, at least 30 days and at most 1 year beforehand, a dose of a vaccinal dengue virus of a first serotype and a dose of a vaccinal dengue vinjs of a second serotype.
According to another specjfic embodiment of the use according to the invention, the third and fourth serotypes are administered in the form of a bivalent vaccinal composition.
According to another specific embodiment of the use according to the invention, the first and second serotypes are administered in the form of a bivalent vaccirial composition.

According to another specific embodiment of the use according to the invention, said vaccinal dengue vims serotype 1 is selected from the group consisting of the VDV1 strain and a ChimeriVax™ DEN-1.
According to another specific embodiment of the use according to the invention, said vaccinal dengue vims serotype 2 is selected from the group consisting of the VDV2 strain and a ChimeriVax™ DEN-2.
According to another specific embodiment of the use according to the invention, said vaccinal dengue virus serotype 1 is the VDV1 strain and said vaccinal dengue vims serotype 2 is the VDV2 strain.
According to another specific embodiment of the use according to the Invention, said vaccinai dengue vims serotype 1 is a ChimeriVax"^** DEN-1 and said vaccinai dengue vims serotype 2 is a ChimeriVax™ DEN-2,
According to another specific embodiment of the use according to the invention, said vaccinal dengue vims serotype 3 is a ChimeriVax™ DEN-3.
According to another specific embodiment of the use according to the invention, said vaccinal dengue virus serotype 4 is a ChimeriVax™ DEN-4.
According to another specific embodiment of the use according to the invention, the first and second serotypes are, respectively, CYD DEN1 and CYD DEN2 and the third and fourth serotypes are, respectively, CYD DEN3 and CYD DEN4.
According to another specific embodiment of the use according to the invention, the third and fourth senDtypes are administered 30 days to 3 months after the administration of the first and second serotypes.
According to another specific embodiment of the use according to the invention, the third and fourth serotypes are administered 30 days after the administration of the first and second serotypes.
According to another specific embodiment of the use according to the invention, the doses of vaccinai dengue viruses serotypes 1, 2, 3 and 4 are each within a range of from 10^ to 10^ CCID50,
The invention will be described in further detail in the description which follows.
Definitions

"Dengue viruses" or "DENs" are positive, single-stranded RNA vimses belonging to the Flavivirus genus of the flaviviridae family. The genomic RNA contains a type I cap at the 5* end but lacks a poly-A tail at the 3' end. The genomic organization consists of the following elements: 6' noncoding region (NCR), structural proteins (capsid (C), premembrane/membrane (prM/M). envelope (E)) and nonstmctural proteins (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5), and 3' NCR. The genomic viral RNA is associated with the capsid proteins so as to form a nucleocapsid. As for the other flaviviruses, the DEN viral genome encodes an unintenojpted coding region which is translated into a single polyprotein.
"VDV" or "Vero dengue vaccine" denotes a live attenuated dengue viral strain adapted on Vero cells and capable of inducing a specific humoral response, including the induction of neutralizing antibodies, in primates and in particular in humans.
'VDV-1" is a strain obtained from a wild-type strain DEN-1 16007 which was subjected to 11 passages on PDK cells (DEN-1 16007/PDK11), which was then amplified on Vero cells at 32''C, and the RNA of which was purified and transfected into Vero cells. The VDV-1 strain has 14 additional mutations compared to the vaccinal strain DEN-1 16007/PDK13 (13 passages on PDK -Primary Dog Kidney - cells). The DEN-1 16007/PDK13 strain, also called "LAV1", was described In patent application EP1159968 in the name of Mahidol University and was deposited with the Collection Nationale de Cultures de Microorganismes (CNCM) [National Collection of Microorganism Cultures] under the number 1-2480, The complete sequence of the VDV-1 strain is given in the sequence SEQ ID N0;1. Said strain can be readily repnaduced irom said sequence. A method of preparation and the characterization of the VDV-I strain have been described in the international patent application filed in the names of Sanofi Pasteur and of the Center for Disease Control and Prevention under the number PCT/IB 2006/001313.
"VDV-2'' Is a strain obtained fn^m a wild-type strain DEN-2 16681 which was subjected to 50 passages on PDK cells (DEN-2 16681/PDK50), and plaque-purified, and ihe RNA of which was extracted and purified before being transfected Into Vero cells. The VDV-2 strain was then obtained by plaque-

purification and amplification on Vero cells. The VDV-2 strain has 10 additional mutations compared with the vaccinal strain DEIM-2 16681/PDK53 (53 passages on PDK cells), 4 mutations of which are silent. The DEN-2 16681/PDK53 strain, also called "LAV2". was described in patent application EP1159968 in the name of Mahidol University and was deposited with the Collection Nationaie de Cultures de Micnoorganismes (CNCM) under the number 1-2481. The complete sequence of the VDV-2 strain is shown in the sequence SEQ ID N0:2. The VDV-2 strain can be readily reproduced from said sequence. A method of preparation and the characterization of the VDV-2 strain has been described in the international patent application filed in the names of Sanofi Pasteur and of the Center for Disease Control and Prevention under the number PCT/IB 2006/001513.
The term "ChimeriVax™ dengue" or "CYD" denotes a chimeric yellow fever (YF) virus which comprises the backbone of a YF virus in which the sequences encoding the premembrane and envelope proteins have been replaced with those of a DEN virus. The term "CYD-1 or CYD DEM" Is thus used to describe a chimeric YF virus containing the prM and E sequences of a dengue serotype 1 strain (DEN-1). The term **CYD-2 or CYD DEN2" is used to describe a chimeric YF virus containing the prM and E sequences of a DEN-2 strain. The term "CYD-3 or CYD DEN3*' is used to describe a chimeric YF virus containing the prM and E sequences of a DEN-3 strain. The term "CYD-4 or CYD DEN4" is used to describe a chimeric YF virus containing the prU and E sequences of a DEN-4 strain. The preparation of these ChimeriVax™ dengues has been described in detail in International patent applications WO 98/37911 and WO 03/101397, to which reference may be made for a precise description of the method for preparing them. The chimeras described in the examples were generated using the prM and E sequences derived from the DEN1 PU0359, DEN2 PR 159, DEN3 PaH881 and DEN4 TVP 980 Strains. Any strain of the dengue virus could be used in the context of the present invention for the construction of the chimeras.
Preferably, the chimeric YF virus comprises the backbone of an anenuSited yellow fever strain YF17D (Theiler M, and Smith HH (1937) J Exp. Med 65. P767-786.) (YF17P/DEN-1, YF17P/DEN-2. YF17D/DEN-3,

YF17D/DEN-4 virus). Examples of YF17D strains which can be used include YF17D204 (YF-Vax®, Sanofi Pasteur, Swtfwater, PA. USA; Stamaril®, Sanofi Pasteur, IVIarcy I'Etoiie, France; ARILVAX™, Chiron, Speke, Liverpool, UK; FLAVIIVIUN®, Bema Biotech, Bern, Switzerland); YF17D-204 France (XI5067X15062); YF170-204,234 US (Rice et al., 1985, Science, 229:726-733), or else related strains YF17DD (Genbank accession number U17066), YF17D-213 (Genbank accession number U17067) and the YF17DD strains described by Galier et al. (1998, Vaccines 16(9/10):1024-1028). Any other yellow fever virus strain sufficiently attenuated for use in humans can be used.
A "monovalent"* vaccine contains a single dengue virus serotype. A "bivalent" vaccine contains two different dengue virus serotypes, A Irivalent" vaccine contains three different dengue virus serotypes. A "tetravalent" vaccine contains four different dengue virus serotypes.
The term "patient" denotes an individual (child or adult) who may be infected with dengue, in particular an individual at risk of infection, such as, for example, an individual who travels in regions where dengue is present or an inhabitant of these regions.
Sequential immunization
The inventors have shown that the administration of the 4 serotypes in the form of two sequential bivalent administrations makes it possible to obtain an effective homologous protection against the 4 serotypes. The method according to the present invention is therefore most particularly valuable in the context of an immunization strategy against dengue.
The inventors therefore propose a method for inducing a neutralizing antibody response against the 4 dengue serotypes in a patient, comprising the sequential administration, to said patient, (i) of a dose of a vaccinal dengue y\rus of a first serotype and of a dose of a vaccinal dengue vinjs of a second senDtype. and (ii) of a dose of a vaccinal dengue virus of a third serotype and of a dose of a vaccinal dengue virus of a fourth serotype, in which the vaccinal dengue viruses (ii) are administered at least 30 days ar\6 at most 3 months after administration of the vaccinal dengue viruses (i).

in the context of the present invention, the tenm "vaccinal dengue virus" is Intended to mean any viral form of the dengue virus which is capable of inducing a specific homologous response. The vaccinal dengue virus can preferably be used in the context of an immunization program in humans against an Infection with a dengue virus.
The term "vaccinal dengue virus" is therefore intended to mean an inactivated virus, an attenuated virus, and also recombinant proteins such as the envelope protein of the dengue virus. A vaccinal virus is "inactivated" if it can no longer replicate on permissive cells. A vaccinal virus is "attenuated" if, after growth at 37*^0 or sgX on Huh-7. VERO and/or C6/C36 cells, such a virus has a titer which is at least 10 times lower than the maximum titer of the wild-type, as determined under the same culture conditions and using the same method of titration.
A vaccinal virus which exhibits reduced growth on at least one of these three cell types identified above Is considered to be attenuated in the context of the present invention.
A vaccinal vims that can be used in humans has a positive benefit/risk ratio which meets the regulatory requirements for being placed on the market A vaccinal dengue virus used in the context of the present invention is preferably attenuated such that it does not induce the disease in humans. A vaccinal virus of this type advantageously results only in side effects which are at most of moderate intensity (i.e. medium to low. or even zero) in the majority of individuals vaccinated, while at the same time conserving its ability to induce a homologous response comprising neutralizing antibodies.
By way of nonlimitlng examples of vaccinal dengue virus that can be used in the context of the present invention, mention may be made of inactivated dengue viruses, attenuated dengue viruses, such as the attenuated strains VDV-1 and VDV-2, the strains described, for example, in applications WO 02/66621, WO 00/57904, WO 00/57908. WO 00/507909, WO 00/67910 and WO 02/0950075, and also the chimeras. The chimeric viruses exhibit the characteristics of the attenuated viruses as defined above.
Any chimeric virus which expresses an envelope protein of a dengue virus and which induces an immune response comprising antibodies that

neutralize the serotype from which the protein is derived may be used in the context of the present invention. By way of nonlimiting examples, mention may be made of the ChimeriVax^^ dengues as described, for example, in WO 98/37911, and also the dengue/dengue chimeras as described, for example, in patent applications WO 96/40933 and WO 01/60847.
The vaccinal dengue virus serotype 1 can, for example, be the vaccinal strain VDV1 or a ChimeriVax™ DEN-1, in particular a YF17D/DEN-1 virus, or else a DEN-1 16007/PDK13 strain. The vaccinal dengue virus serotype 2 can, for example, be the vaccinal strain yDV2 or a ChimeriVax™ DEN-2, in particular a YF17D/DEN-2 virus, or else a DEN-2 16681/PDK53 \nrus. The vaccinal dengue virus serotype 3 can be a ChimeriVax™ DEN-3, in particular a YF17D/DEN-3 virus. The vaccinal dengue virus serotype 4 can be a ChimeriVax™ DEN-4. in particular a YF17D/DEN-4 vinjs. It can also be a "LAV4" or "DEN-4 1036/PDK48" strain, i.e. a DEN-4 1036 strain attenuated by 48 passages on PDK cells. This strain was described in patent application EP1159968 in the name of IVlahidol University and was deposited with the Collection Nationale de Cultures de Microorganismes (CNCM) under the number 1-2483.
Each ChimeriVax"^" monovalent vaccinal dengue virus (serotypes 1, 2, 3 and 4) was prepared by amplification of each serotype on Vero cells. More specifically, the four viruses are produced separately on adherent Vero cefis in serum-free medium. The viral harvest, clarified to remove the cell debris by filtration, is then concentrated and purified by ultrafiltration and chromatography in order to remove the DNA of the host cells. After the addition of a stabilizer, the vaccinal strains are stored in frozen or lyophilized form before use, and then reconstituted extemporaneously. The same method is applied for the four chimeras.
The VDV 1 and 2 strains are prepared by amplification on Vero ceils. The viruses produced are harvested and clarified to remove the celldebris by filtration. The DNA is digested by enzymatic treatment. The impurities are eliminated by uitrafiltration. The infectious titers can be increased by means of a method of concentration. After the addition of a stabilizer, the strains, are

stored in a lyophilized or frozen form before use, and then reconstituted extemporaneously.
The multivalent compositions are obtained by simple mixing of the monovalent composilions.
According to the present Invention, the 4 dengue serotypes can be administered in any order provided that they are administered in pairs sequentially, within a period of 30 days to 1 year, such as 30 days, 45 days, 60 days, 3 months, 6 months, 9 months and 1 year, being observed, advantageously a period of 30 days to 3 months, in particular a period of 1 to 2 months, being observed between the two series of administrations. The method according to the present invention can therefore be implemented with the embodiments described below;
-(i) serotypes 1 and 2; (ii) serotypes 3 and 4; or -(i) serotypes 1 and 3; (ii) serotypes 2 and 4; or -(i) serotypes 1 and 4; (ii) serotypes 2 and 3; or -(i) serotypes 2 and 3; (ii) serotypes 1 and 4; or -(i) serotypes 2 and 4; (ii) serotypes 1 and 3; or -(i) serotypes 3 and 4; (ii) serotypes 1 and 2.
According to specific embodiments, the present invention therefore covers the following schemes:
-(i) CYD DEN-1 and CYD DEN-2; (ii) CYD DEN-3 and CYD DEN-4 -(i) CYD DEN-1 and CYD DEN-3; (ii) CYD DEN-2 and CYD DEN-4 -(i) CYD DEN-1 and CYD DEN-4; (ii) CYD DEN-2 and CYD DEN-3 -(i) CYD DEN-2 and CYD DEN-3; (ii) CYD DEN-1 and CYD DEN-4 -(i) CYD DEN-2 and CYD DEN-4; (ii) CYD DEN-1 and CYD DEN-3 -(1) CYD DEN-3 and CYD DEN-4; (ii) CYD DEN-1 and CYD DEN-2 -(i) VDV-1 and CYD DEN-2; (if) CYD DEN-3 and CYD DEN-4 -(i) VDV-1 and CYD DEN-3; (ii) CYD DEN-2 and CYD DEN-4 -(i) VDV-1 and CYD DEN-4; (ii) CYD DEN-2 and CYD DEN-3 -(i) CYD DEN-2 and CYD DEN-3; (ii) VDV-1 and CYD DEN-4 -(I) CYD DEN-2 and CYD DEN-4: (ii) VDV-1 and CYD PEN-3 -(i) CYD DEN-3 and CYD DEN-4; (ii) VDV-1 and CYD DEN-2 -(i) CYD DEN-1 and VDV-2 ; (ii) CYD DEN-3 and CYD PEN-4

-(i) CYD DEN-1 and CYD DEN-3: (ii) VDV-2 and CYD DEN-4 -(i) CYD DEN-1 and CYD DEN-4; (if) VDV'2 and CYD DEN-3 -(i) VDV-2 and CYD DEN-3; (ii) CYD DEN-1 and CYD DEN-4 -(i) VDV-2 and CYD DEN-4; (ii) CYD DEN-1 and CYD DEN-3 -(i) CYD DEN-3 and CYD DEN-4; (ii) CYD DEN-1 and VDV-2 -(i) VDV-1 and VDV-2; (ii) CYD DEN-3 and CYD DEN-4 -(i) VDV-1 and CYD DEN-3; (ii) VDV-2 and CYD DEN-4 -(I) VDV-1 and CYD DEN-4; (ii) VDV-2 and CYD DEN-3 -(i) VDV-2 and CYD DEN-3; (ii) VDV-1 and CYD DEN-4 -(i) VDV-2 and CYD DEN-4; (ii) VDV-1 and CYD DEN-2 and -(i) CYD DEN-3 and CYD DEN-4; (Ii) VDV-1 and VDV-2.
In the context of the present invention, the term "dose of vaccinal virus" is Intended to mean a composition comprising an "immunoeffective amount" of the vaccinal virus, i.e. an amount of virus sufficient to induce a homologous neutralizing antibody response, which can be demonstrated, for example, by means of the seroneutralization test as described below in example 1. A serum is considered to be positive for the presence of neutralizing antibodies when the neutralizing antibody titer thus determined Is greater than or equal to 1:10.
Vaccinal strain amounts are commonly expressed In terms of viral plaque-forming units (PFU) or of 50% tissue culture infectious dose (TCID50), or else of 50% cell culture infectious dose {CCID50). For example, the compositions according to the invention can contain from 10 to 10^ CCID50, in particular from 10^ to 10^ CCID50 of vaccinal dengue virus serotype 1. 2, 3 or 4 for a monovalent or bivalent composition. Thus, in the compositions or use according to the invention, the doses of vaccinal dengue viruses serotypes 1, 2, 3 and 4 are preferably each within a range of from 10 to 10® CCID50, such as 10,10\ 10^ 10^ 10^ 10® or 10^ CCID50. in particular in a range from 10^ to 10^ CCID50. The vaccinal viruses can be used at identical or different doses, which can be adjusted according to the nature of the vaccinal virus used and to the strength of the immune response obtained.

Preferably, the homologous neutralizing antibody response is long-iasting, i.e. it can be detected in the serum at least 6 months after administration of the dengue serotypes (ii).
In the sequential administration according to the invention, the vaccinal dengue viruses of the third and fourth serotypes are administered at least 30 days and at most 12 months after the administration of the vaccinal dengue viruses of the first and second serotypes.
!n the context of the present invention, the vaccinal dengue viruses of the third and fourth serotypes can, for example, be administered 30 days to 1 year, for example 30 days, 45 days, 60 days, 3 months, 6 months, 9 months or 1 year, advantageously 30 days to 3 months, in particular 1 to 2 months, after the administration of the vaccinal dengue viruses of the first and second serotypes.
The dose of a vaccinal dengue virus of a first serotype and the dose of a vaccinal dengue virus of a second serotype are administered simultaneously In the form of two monovalent compositions, or in the form of a single bivalent composition.
Similarly, the dose of a vaccinal dengue virus of a third serotype and the dose of a vaccinal dengue vinjs of a fourth serotype are administered simultaneously. For example, the third and fourth serotypes can be administered simultaneously in the fomn of two monovalent vaccinal compositions, or In the form of a single bivalent vaccinal composition.
The vaccinal viruses are administered in the form of vaccinal compositions which can be prepared according to any method known to those skilled in the art. Usually, the viruses, generally in lyophilized form, are mixed with a pharmaceutically acceptable excipient, such as water or a phosphate-buffered saline solution, wetting agents or stabilizers. The term "pharmaceuticalfy acceptable excipient" is intended to mean any solvent, dispersing medium, filler, etc., which does not produce a side reaction, for example an allergic reaction, in humans or animals. The excipient is selected according to the pharmaceutical form chosen, and to the method and route of administration. Appropriate excipients and also the requirements in terms of

pharmaceutical formulation are described in "Remington; The Science 8t Practice of Pharmac/, \A^ich represents a reference work in the field.
Preferably, the vaccinal compositions are prepared in an injectable form, and can correspond to liquid solutions, suspensions or emulsions. The compositions can in particular include an aqueous solution buffered so as to maintain a pH of between approximately 6 and 9 (as detenriined with a pH meter at ambient temperature).
Although it is not necessary to add an adjuvant, the compositions can nevertheless include such a compound, i.e. a substance which increases, stimulates or strengthens the cellular or humoral immune response induced by the vaccina! strain administered simultaneously. Those skilled in the art are in a position to select, from the adjuvants conventionally used in the field of vaccines, an adjuvant which may be suitable in the context of the present Invention.
The vaccinal compositions according to the invention can be administered according to any route normally used In immunization, for example parenterally (in particular Intradermally, subcutaneously or intramuscularly). Preferably, the vaccinal compositions are injectable compositions administered subcutaneously in the deltoid region.
The volume of composition administered depends on the route of administration. For subcutaneous injections, the volume is generally between 0.1 and 1.0 ml, preferably approximately 0.5 ml.
The optimal period for the administration of the first and second serotypes, or preferably of all the serotypes 1 to 4. is approximately 1 to 3 months before exposure to the dengue virus. The vaccines can be administered as a prophylactic treatment for infection with a dengue virus In adults and children. Target populations therefore include individuals who may be naive (i.e. not previously immunized) or non-naive with respect to the dengue virus.
Vaccinial dengue virus serotypes 1 to 4 booster administrations can also be carried out, for example, between 6 months and 10 years, for example 6 months, 1 year, 3 years, 5 years or 10 years, after administration of the third and fourth serotypes.

The present invention also provides an immunization kit comprising 4 vaccinal dengue viruses of 4 different serotypes, in which each serotype is present in the form of a vaccine dose, or in which at least 2, or even 4 serotypes are present in the form of a bivalent composition.
For a description of the vaccine doses or monovalent or bivalent compositions and of the embodiments that are prefen^ed, reference may be made to the passages in the description relating to the method of immunization according to the present invention.
The invention is illustrated by means of the following examples.
EXAMPLES
Example 1: Sequential immunization in monkeys
The viremia and the immunogenicity were therefore tested in a monkey model. The viremia, in particular, was identified as one of the factors associated with the virulence and the severity of the disease in humans and therefore constitutes an important parameter to be taken into consideration. The immunogenicity is, for its part, a key parameter in the context of the evaluation of the protection conferred.
1.1 Materials and methods:
The experiments in monkeys were cam'ed out according to the European Directives relating to animal experimentation. The immunizations were earned out in cynomolgus monkeys {Macaca fascicularls) originating from Mauritania. The monkeys were placed in quarantine for six weeks before immunization.
The monkeys were immunized subcutaneously in the arm(s) with 0.5 ml of vaccinal composition. After a light anesthesia with ketamine (Imalgene, Merial), blood was collected by puncture from the inguinal or saphenous veins. At days 0 and 28, 5 ml of blood were sampled in order to evaluate the antibody responses, while, between days 2 and 10, 1 ml of blood was sampled in order to evaluate the viremia. The blood was collected on ice and stored on ice until

serum separation. To do this, the blood was centrifuged for 20 minutes at 4^C and the serum collected was stored at -80*C until the time of the tests.
Measurement of viremla
The post-vacclnal viremias were monitored by quantitative real-time RT-PCR (qRT-PCR). Two sets of primers and of probes located In the NS5 gene of the DEN1 and DEN2 strains were used to quantify the VDV-1 RNA and VDV-2 RNA, respectively. A third set of primers and of probes located in the NS5 gene of the YF virus was used to quantify the CYD RNA. Finally, 4 sets of primers and of probes specific for the various serotypes, located at the Junction of the E (DEN)/NS1 (YF) genes were used to Identify the serotype in the samples positive for the YF NS5 RNA (see also table I), 7 plasmids containing, under the control of the T7 promoter, the region targeted by each PCR were transcribed in vitro so as to generate a series of synthetic RNAs which were included as an internal reference in each RT-PCR assay. These synthetic RNAs were assayed by spectrophotometry, and the amount of RNA obtained was converted to number of RNA copies and expressed as GEQ (genomic equivalents).
0.140 mi of monlcey serum was extracted using the Macherey Nagel "Nucleospin 96 vinjs™" RNA extraction kit, according to the manufacturer's Instructions, and then the purified RNA was eiuted with 0.140 ml (0.090 ml, then 0.05 ml) of RNase-free water. In order to avoid repeated freezing/thawing cycles, a first quantification was carried out immediately after the extraction, on 5 \x\ of said RNA preparation. The remaining volume was frozen at 70°C.
The reaction mixtures contained, in addition to the components of the "Qiagen Qauntitect™ probes" RT-PCR quantification kit (QIagen), 10 picomoi of each primer, 4 picomoi of each pnDbe and 5 \x\ of RNA, in a total volume of 25 jil. in the case of the RNAs to be tested, 5 iil of the purified preparation were directly introduced into the reaction mixture, without any prior dilution step. The synthetic RNAs were diluted to 1/10 In RNase-free water, and 7 dilutions containing approximately 10 to 10® GEQ in 5 ^1 were quantified in parallel in order to generate the standard cun/e.

The quantification reactions were carried out on tiie Applied Biosystem ABIPrism 700™ device, using the following program: SO'^C/SO min, 95**C/15 min, then 40 cycles of 95X/15 sec-60X/60 sea The limit of quantification of the viral RNA in this test is from 2.9 to 3.3 logioGEQ/ml (800 to 2000 GEQ/ml; 4 to 10 GEQ/reaction), according to the PCR targets (standard deviation: +/-0.3 logic).
The correlation between the Infectious titer and the viral RNA quantification v^as established in parallel to the assays, by analysis of 0.140 m! of negative monkey serum samples (DO) to which a known amount of infectious particles of the viruses which were used for the immunization (CYD or VDV) were added. Said control sera were prepared at two dilutions containing approximately 1 PFU and approximately 100 PFU in 5 jxl (2.3 and 4.3 logioPFU/mi, respectively).
The primers and probes used are given in table 1 below, in which are listed, in onder, for each assay, the sense and antisense primers and the probe. Table 1


Conventionally, the dengue antibody measurement is established using the PRNT50 (50% PFU number reduction neutralization test) test. Since this test is laborious and uses up a lot of material, we developed the SN50 test, based on 50% reduction in the number of units measured in a CCID50 test. In a 96-well plate, 0.120 ml of each decomplemented serum is added to 0.480 ml of diluent (ISCOVE 4% FCS) per well. 6-fold serial dilutions are prepared by transfer of 0.150 ml of serum into 0.450 ml of diluent. 450 |il of viral dilution at 2.7 logio CCIDgo/ml are added to each well so as to obtain 25 CClDso/weil. The plate is incubated at 37*C for 1 hour. 100 \i\ of each dilution are then distributed into 6 wells of a 96-well plate into which VERO cells had been seeded 3 days before the beginning of the experiment at a density of 8000 cells/well, in 100 ^l of ISCOVE medium containing 4% FCS. After incubation at S/^C for 6 days, in the presence of 5% CO2, the cells are fixed with an ethanol/acetone (70/30) mixture at 4''C for 15 minutes, and then washed 3 times in PBS and incubated for 1 h at 37^*0 in the presence of 50 \i\ of a 1/2000 dilution of an anti-flavivirus monoclonal antibody (mAb 4G2). The plates are then washed twice and incubated for 1 h at 37^0 in the presence of 50 ^1 of a 1/1000 dilution of an alkaline phosphatase-conjugated anti-mouse IgG. The lysis piaques are visualized by adding 50 lal of a colored substrate: BCIP/NBT. The neutralizing antibody titers are calculated using the Karber formula as defined below:
logioSN50 = d + f/N (X + N/2),
in which:
d represents the dilution resulting in 100% neutralization (i.e. 6 negative
replicates, i.e. replicates exhibiting no sign of infection)
f represents the dilution factor in Iog10 (e.g. dilution factor of 1:4. f = 0.6)
N represents the number of repiicates/dilution (N-6)
X total number of wells exhibiting no sign of infection, with the exception of the
dilution d
The limit of viral detection is 10 SN50 (i.e. 1.0 logioSNSO).

The viral strains which were used for the neutralization are the DEN1 16007,
DEN2 16681. DENS 16562 orDEN4 1036 strains.
For the controls, the initial viral dilutions were re-titrated.
The con-elation between the neutralizing titer measured in the SN50 test and
the neutralizing titer measured conventionally in the PRNT50 test is:
logioPRNT50 = logioSN50 + 0.2
The mean titer (GMT) is established by calculating the geometric mean of the
titers expressed as a linear value; the samples of which the titer is less than the
detection threshold are, by convention, assigned a value equal to half this
threshold.





Briefly, the results can be summarized as follows;
- The administration scheme according to the present invention mal