Patent application title: Method of Immunization Against the 4 Serotypes of Dengue Fever
Bruno Guy (Lyon, FR)
Bruno Guy (Lyon, FR)
Véronique Barban (Craponne, FR)
Véronique Barban (Craponne, FR)
Rémi Forrat (Serezin Du Rhone, FR)
Rémi Forrat (Serezin Du Rhone, FR)
Jean Lang (Mions, FR)
Jean Lang (Mions, FR)
SANOFI PASTEUR SA
IPC8 Class: AA61K3912FI
Class name: Antigen, epitope, or other immunospecific immunoeffector (e.g., immunospecific vaccine, immunospecific stimulator of cell-mediated immunity, immunospecific tolerogen, immunospecific immunosuppressor, etc.) virus or component thereof togaviridae or flaviviridae, except hepatitis c virus (e.g., yellow fever virus, bovine viral diarrhea virus, dengue virus, equine viral arteritis virus, equine encephalitis virus, japanese b encephalitis virus, sindbis virus, flavivirus, etc.)
Publication date: 2010-09-23
Patent application number: 20100239612
The invention relates to a method for inducing protection against the 4
serotypes of dengue fever in a patient, comprising: (a) the
administration of a monovalent vaccine comprising a vaccinal virus of a
first serotype of dengue fever, and (b) the administration of a
tetravalent vaccine comprising vaccinal viruses of the four serotypes of
dengue fever,in which administration (b) is made between at least 30 days
and not more than 12 months following the first administration (a).
8. An immunization kit against dengue fever virus comprising container (a) a first container containing a monovalent vaccine comprising a vaccinal virus of a first serotype of dengue fever, (b) a second container containing a tetravalent vaccine comprising vaccinal viruses for the 4 serotypes of dengue fever.
9. The immunization kit as claimed in claim 8, comprising at least:(a) a first container containing a monovalent vaccine comprising a VDV1 or VDV2 vaccinal virus,(b) a second container containing a tetravalent vaccine comprising Chimerivax® DEN-1,2,3 and 4.
10. The immunization kit as claimed in claim 9, wherein the monovalent vaccine comprises 10.sup.4 DICC50 of VDV1 or VDV2 and the tetravalent vaccine comprises about 10.sup.5 DICC50 of Chimerivax® DEN-1,2,3 and about 10.sup.3 DICC50 of Chimerivax® DEN-4.
11. An immunization kit against dengue fever virus comprising container (a) a first container containing a monovalent vaccine comprising a VDV1 or VDV2 vaccinal virus, (b) a second container containing a tetravalent vaccine comprising vaccinal viruses for the 4 serotypes of dengue fever.
12. The immunization kit as claimed in claim 11, comprising at least:(a) a first container containing a monovalent vaccine comprising a VDV1 or VDV2 vaccinal virus,(b) a second container containing a tetravalent vaccine comprising Chimerivax® DEN-1,2,3 and 4.
13. The immunization kit as claimed in claim 12, wherein the monovalent vaccine comprises 10.sup.4 DICC50 of VDV1 or VDV2 and the tetravalent vaccine comprises about 10.sup.5 DICC50 of Chimerivax® DEN-1,2,3 and about 10.sup.3 DICC50 of Chimerivax® DEN-4.
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to provisional application Ser. No. 60/885,077, which was filed on Jan. 16, 2007, and is hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to a method for inducing protection against the 4 serotypes of dengue fever in a patient, comprising: (a) a first administration of a monovalent vaccine comprising a vaccinal virus of a first serotype of dengue fever, (b) a second administration of a tetravalent vaccine comprising vaccinal viruses of the four serotypes of dengue fever, and
in which the second administration (b) is made between at least 30 days and not more than 12 months after the first administration (a).
2. Summary of the Related Art
Dengue fevers are caused by four viruses of the flavivirus genus which are of similar serological type but differ from the antigen point of view (Gubler et al., 1988, in: Epidemiology of arthropod-borne viral disease. Monath T P M, editor, Boca Raton (Fla.): CRC Press: 223-60; Kautner et 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). Infection with a serotype of dengue fever may produce a spectrum of clinical disease from non-specific viral syndrome to severe fatal hemorrhagic disease. The incubation period for dengue fever after a mosquito bite is approximately 4 days (from 3 to 14 days). Dengue fever is characterized by a two-phase fever, headaches, pains in various parts of the body, prostration, eruptions and lymphadenopathy (Kautner et al., 1997, J. of Pediatrics, 131: 516-524; Rigau-Perez et al., 1998, Lancet, 352: 971-977). The viremic period is of the same as the febrile period (Vaughn et al., 1997, J. Infect. Dis., 176: 322-30). Cure of dengue fever is complete after 7 to 10 days, but prolonged asthenia is normal. Reduced leukocyte and platelet numbers frequently occur.
Hemorrhagic dengue fever is a severe febrile disease characterized by homeostasis abnormalities and an increase in vascular permeability which can lead to hypovolemia and hypotension (dengue fever with shock syndrome), often complicated by severe internal bleeding. The mortality rate for hemorrhagic dengue fever can reach 10% without treatment, but is 1% in most centers with experience of treatment (WHO Technical Guide, 1986. Dengue haemorrhagic fever: diagnosis, treatment and control, p. 1-2. World Health Organization, Geneva, Switzerland).
Routine laboratory diagnosis of dengue fever is based on isolation of the virus and/or the detection of antibodies specific to dengue fever virus.
Dengue is the second most important infectious tropical disease after malaria, more than 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 fever every year, 500,000 patients hospitalized for hemorrhagic dengue fever, and 25,000 deaths. Dengue fever is endemic in Asia, the Pacific, Africa, Latin America and the Caribbean. Dengue fever virus 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; Monath, 1994, Proc. Natl. Acad. Sci., 91: 2395-2400). A number of well-described factors would appear to be implicated in dengue fever--population growth, unplanned and uncontrolled urbanization, in particular associated with poverty, an increase in air travel, lack of effective mosquito control and deterioration of sanitary and public health infrastructure (Gubler, 2002, TRENDS in Microbiology, 10: 100-103). Travellers and expatriates are increasingly being warned about dengue fever (Shirtcliffe et al., 1998, J. Roy. Coll. Phys. Lond., 32: 235-237). Dengue fever has been one of the main causes of febrile diseases among American troops during deployments in tropical areas where dengue fever is endemic (DeFraites et al., 1994, MMWR, 1994, 43: 845-848).
The viruses are maintained within a cycle involving humans and Aedes aegypti, a domestic mosquito which bites during the daytime, and prefers to feed on man. Infection in man is initiated by injection of the virus during the blood meal of an infected Aedes aegypti mosquito. The salivary virus is mainly deposited in the extravascular tissues. The first category of cells to be infected after inoculation are the dentritic cells, which then migrate to the lymphatic ganglia (Wu et al., 2000, Nature Med., 7: 816-820). After initial replication in the skin and lymphatic ganglia, the virus appears in the blood in the course of the acute febrile stage, generally for 3 to 5 days.
Along with the dentritic cells, monocytes and macrophages are among the first targets of dengue fever virus. Protection against homotypic reinfection is complete and probably lasts a lifetime, but cross-protection between the different types of dengue lasts from less than a few weeks to a few months (Sabin, 1952, Am. J. Trop. Med. Hyg., 1: 30-50). As a consequence, an individual may become infected with a different serotype. A second infection due to dengue fever is in theory a risk factor for the development of severe dengue fever. However, hemorrhagic dengue fever is multifactorial--factors include the strain of virus involved and the age, immune status and genetic predisposition of the patient. Two factors play a major role in the occurrence of hemorrhagic dengue fever--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). There is no specific treatment against dengue fever. 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.
Preventive measures are currently based on control of the vector and personal protection measures which are difficult to apply and are costly. No vaccine against dengue fever has at present been approved. Given that the four serotypes of dengue fever are in circulation in the world and that they have been reported as being involved in cases of hemorrhagic dengue fever, vaccination should ideally confer protection against the four serotypes of dengue fever virus.
When immunizing with a tetravalent vaccine, it may happen that the response is induced predominantly against only one or at most 3 serotypes. There is therefore a need for a method which makes it possible to reduce interference between the different serotypes and makes it possible to induce neutralizing antibodies against the 4 serotypes of dengue fever.
SUMMARY OF THE INVENTION
The inventors have found that it is possible to generate an immune response comprising antibodies neutralizing the 4 serotypes when the vaccinal formulation which is intended to induce a response against the 4 serotypes is administered after preliminary immunization with an attenuated living vaccine of only one serotype, the second immunization being made 30 days to 12 months after the first administration.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The inventors have in particular shown that tetravalent DEN-1,2,3,4 immunization after monovalent DEN-2 immunization induces responses against the four serotypes in all the monkeys immunized. Conversely, tetravalent immunization alone only induced a satisfactory response against two out of 4 serotypes, even after a booster.
The immune response generated by the method according to the invention is therefore both quantitatively and qualitatively greater (covers all serotypes).
In accordance with a first object, this invention therefore relates to a method making it possible to induce a neutralizing antibody response against the 4 serotypes of dengue fever in a patient and comprises: (a) a first administration of a monovalent vaccine comprising a vaccinal virus of a first serotype of dengue fever, (b) a second administration of a tetravalent vaccine comprising vaccinal viruses of the 4 serotypes of dengue fever, and
in which the second administration (b) is made at least 30 days and not more than 12 months after the first administration (a).
According to a particular embodiment of the method of immunization according to the invention, the vaccinal virus used in the first administration (a) is selected from the group comprising vaccinal viruses of dengue fever of serotype 1 or 2.
According to another particular embodiment of the method of immunization according to the invention, the said vaccinal virus used in the first administration (a) is selected from the group comprising strains VDV1 and VDV2.
According to another particular embodiment of the method according to the invention, the said vaccinal viruses used in the tetravalent vaccine are Chimerivax® DEN-1,2,3 and 4.
According to another particular embodiment of the method according to the invention the quantity of vaccinal viruses of dengue fever of serotypes 1, 2, 3 and 4 lies within a range from 103 to 106 DICC50.
According to another particular embodiment of the method according to the invention, the monovalent vaccine comprises about 104 DICC50 of VDV1 or VDV2 and the tetravalent vaccine comprises about 105 DICC50 of Chimerivax® DEN-1,2,3 and 103 DICC50 of Chimerivax® DEN-4.
According to another embodiment of the method according to the invention, the second administration (b) is made 30 to 60 days after the first administration (a).
Another object of the present invention is an immunization kit against dengue fever virus comprising a container containing at least (a) a first container containing a monovalent composition or vaccine comprising a vaccinal virus of a first serotype of dengue fever, (b) a second container containing a tetravalent composition or vaccine comprising vaccinal viruses for the 4 serotypes of dengue fever.
According to one embodiment, the kit according to the invention comprises at least: (a) a first container containing a monovalent vaccine comprising a VDV1 or VDV2 vaccinal virus, (b) a second container containing a tetravalent vaccine comprising the 4 Chimerivax® DEN-1,2,3 and 4.
According to a particular embodiment, the kit according to the invention comprises a monovalent vaccine comprising about 104 DICC50 of VDV1 or VDV2 and a tetravalent vaccine comprising about 105 DICC50 of Chimerivax® DEN-1,2,3 and about 103 DICC50 of Chimerivax® DEN-4.
This invention therefore also relates to use of dengue fever vaccinal viruses for the manufacture of a monovalent vaccine and a tetravalent vaccine for immunization against dengue fever virus in which the monovalent vaccine comprises a vaccinal virus of a first serotype of dengue fever, the tetravalent vaccine comprises vaccinal viruses of the 4 serotypes of dengue fever and in which the tetravalent vaccine is administered at least 30 days and not more than 12 months after administration of the monovalent vaccine.
The invention will now be described in more detail in the description which follows.
"Dengue fever viruses" or "DEN" are positive single-strand RNA viruses belonging to the Flavivirus genus of the family of flaviviridae. The genome in RNA contains a type I end member at the 5' extremity but has no poly-A tail at the 3' extremity. The organization of the genome comprises the following elements: non-coding region (NCR) 5', structural proteins (capsid (C), pre-membrane/membrane (prM/M), envelope (E)) and non-structural proteins (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5) and NCR 3'. The viral genome RNA is associated with the capsid proteins to form a nucleocapsid. As in the case of flaviviruses, the DEN viral genome codes an uninterrupted coding region which is translated into a single polyprotein.
In the context of this invention, by "vaccinal dengue fever virus" is meant any viral form of dengue fever virus that is capable of inducing a specific immune response comprising neutralizing antibodies, which preferably includes all viral forms of dengue fever virus which can be used in the context of an immunization program in man against infection by a dengue fever virus. By vaccinal dengue fever viruses are therefore meant inactivated viruses, attenuated viruses, and recombinant proteins such as the envelope protein of dengue fever virus. Numerous examples of these are known in the art.
A vaccinal virus is regarded as being "inactivated" if it no longer replicates in permissive cells.
A vaccinal virus is regarded as being "attenuated" if after growth at 37° C. or 39° C. in Huh-7, VERO and/or C6/36 liver cells the said vaccinal virus has a maximum titer which is at least 10 times less than maximum titer obtained with the wild parent strain under the same culture conditions and as measured using the same method for determining titer. A vaccinal virus which has diminished growth in at least one of the three cell types identified above is therefore regarded as being "attenuated" in the context of this invention.
A vaccinal virus which can be used in man has a positive benefit/risk ratio, the said ratio generally satisfying statutory and regulatory requirements for obtaining a marketing authorization. A vaccinal dengue fever virus used in the context of this invention is preferably a virus which has been attenuated in such a way that it does not induce the disease in man. Advantageously, the said vaccinal virus only results in side effects of at most moderate intensity (i.e., medium to slight, or zero) in the majority of vaccinated individuals, while retaining its ability to induce a neutralizing antibody response.
Dengue fever vaccinal viruses which can be used in the context of this invention may be cited by way of non-limiting examples: inactivated vaccinal viruses, attenuated vaccinal viruses such as the attenuated strains VDV-1, VDV-2, the strains described for example in applications WO02/66621, WO0057904, WO0057908, WO0057909, WO0057910, WO02/0950075 and WO02/102828, or chimeras. Chimeric viruses have the special feature that they have the characteristics of attenuated viruses as defined above. All chimeric viruses expressing the envelope protein of a dengue fever virus and inducing an immune response comprising antibodies neutralizing the serotype from which the envelope protein originates may therefore be used in the context of this invention. Mention may be made by way of non-limiting examples of: the dengue fever Chimerivax® such as described for example in patent application WO 98/37911, dengue/dengue fever chimeras such as described for example in patent applications WO9640933 and WO0160847. The vaccinal virus of serotype 1 dengue fever may for example be the vaccinal strain VDV1 or a Chimerivax® DEN-1, in particular a YF17D/DEN-1 virus, or again a DEN-1 16007/PDK13 strain. The vaccinal virus for serotype 2 of dengue fever may for example be the vaccinal strain VDV2 or a Chimerivax® DEN-2, in particular a YF17D/DEN-2 virus, or again a DEN-2 16681/PDK53 strain. The vaccinal virus of serotype 3 of dengue fever may be a Chimerivax® DEN-3, in particular a YF17D/DEN-3 virus. The vaccinal virus of serotype 4 of dengue fever may be a Chimerivax® DEN-4, in particular a YF17D/DEN-4 virus. Reference may be made to the applications identified here for precise description of the strains mentioned and the processes for obtaining them.
"VDV" or "Vero dengue vaccine" designates an attenuated living dengue fever viral strain adapted to Vero cells (i.e. able to reproducibly replicate at significant level in Vero cells) and capable of inducing a specific humoral response, including the induction of neutralizing antibodies, in primates and particularly in man.
"VDV-1" is a strain obtained from a wild DEN-1 16007 strain which has undergone 11 passes through PDK cells (DEN-1 16007/PDK11) and which has subsequently been amplified in Vero cells at 32° C., the RNA of which has been purified and transfected in Vero cells. The VDV-1 strain has 14 additional mutations in comparison with the DEN-1 16007/PDK13 vaccinal strain (13 passes through PDK--Primary Dog Kidney--cells). The DEN-1 16007/PDK13 strain, also called "LAV1", has been described in patent application EP1159968 in the name of Mahidol University and has been filed with the National Microorganisms Cultures Collection (CNCM) under number I-2480. The complete sequence of the VDV-1 strain is given in sequence SEQ ID NO:1. This strain can easily be reproduced from that sequence. A process for preparing and characterizing the VDV-1 strain has been described in the international patent application filed under number WO/2006/134433 in the names of Sanofi-Pasteur and the Center for Disease Control and Prevention.
"VDV-2" is a strain which has been obtained from wild strain DEN-2 16681 which has undergone 50 passes through PDK cells (DEN-2 16681/PDK50), plate purified, the RNA from which has been extracted and purified before being transfected in Vero cells. The VDV-2 strain has subsequently 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 vaccinal strain (53 passes through PDK cells), including 4 silent mutations. The DEN-2 16681/PDK53 strain, also known as "LAV2", has been described 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. The complete sequence of the VDV-2 strain is given in sequence SEQ ID NO:2. The VDV-2 strain can easily be reproduced from that sequence. A process for preparing and characterizing the VDV-2 strain has been described in the international patent application filed under number WO/2006/134443 in the names of Sanofi-Pasteur and the Center for Disease Control and Prevention.
The VDV 1 and 2 strains are prepared by amplification in Vero 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.
By "ChimeriVax® dengue" or "CYD" is meant a chimeric yellow fever (YF) virus which comprises the skeleton of a YF virus in which the sequences coding for the pre-membrane and envelope proteins have been replaced by those of a DEN virus. Thus, a chimeric YF virus containing the prM and E sequences of a serotype 1 dengue fever strain (DEN-1) is called "CYD-1 or CYD DEN1". A chimeric YF containing the prM and E sequences of a DEN-2 strain is referred to as "CYD-2 or CYD DEN2". A chimeric YF virus containing the prM and E sequences of a DEN-3 strain is referred to as "CYD-3 or CYD DEN3". A chimeric YF virus containing the prM and E sequences of a DEN-4 strain is referred to as "CYD-4 or CYD DEN4". The preparation of these dengue ChimeriVax® 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 processes for their preparation. The chimeras described in the examples have been generated by using prM and E sequences from strains DEN 1 PUO359 (TYP1140), DEN2 PUO218, DEN3 PaH881/88 and DEN 4 1228 (TVP 980). Any dengue fever virus strain may be used to construct chimeras in the context of this invention.
Preferably, the chimeric YF virus comprises the skeleton of an attenuated yellow fever strain YF17D (Theiler M. and Smith H. H. (1937) J. Exp. Med., 65, p. 767-786) (viruses YF17D/DEN-1, YF17D/DEN-2, YF17D/DEN-3, YF17D/DEN-4). Examples of YF17D strains which may be used include YF17D204 (YF-Vax®, Sanofi-Pasteur, Swifwater, Pa., USA; Stamaril®, Sanofi-Pasteur, Marcy l'Etoile, France; ARILVAX®, Chiron, Speke, Liverpool, UK; FLAVIMUN®, Berna Biotech, Bern, Switzerland; YF17D-204 France (X15067, X15062); YF17D-204,234 US (Rice et al., 1985, Science, 229: 726-733), or again 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). Any other attenuated yellow fever virus strain which may be used in man may be used to construct chimeras in the context of this invention.
When the term "about" is used in conjunction with an amount it means plus or minus 10% of that amount, e.g., "about 104" means 104±103.
According to a particular embodiment, for each serotype used in the various administrations the vaccinal viruses are present in the vaccine in a quantity from 103 to 105 DICC50.
According to a particular embodiment, vaccinal viruses VDV1 or VDV2 are present in the monovalent vaccine at a level of about 104 DICC50.
According to a particular embodiment, Chimerivax® DEN-1,2,3 are present in the tetravalent vaccine at a level of about 105 DICC50 and Chimerivax® DEN-4 is present in the tetravalent vaccine at a level of about 103 DICC50.
Each monovalent ChimeriVax® dengue fever vaccinal virus (serotypes 1, 2, 3 and 4) has been prepared by amplifying each serotype in Vero cells. More specifically, the four viruses are produced separately in adhering Vero cells in a serum-free medium. The viral harvest, clarified from cell debris by filtration, is then concentrated and purified by ultrafiltration and chromatography to remove the DNA from the host cells. After adding a stabilizing agent, the vaccinal strains are stored in a frozen or lyophilized form before use and then reconstituted as needed. The same process is applied to the four chimeras.
A dose, composition or vaccine is "monovalent" when in addition to a pharmaceutically acceptable excipient it contains a vaccinal virus of a single dengue fever serotype. A dose, composition or vaccine is "tetravalent" when it contains vaccinal viruses of the four serotypes of dengue fever. Multivalent compositions are obtained by simple mixing of monovalent compositions.
By "patient" is meant a person (child or adult) who is likely to be infected by dengue fever, in particular a person at risk of infection, such as for example a person traveling in regions where dengue fever is present, or an inhabitant of those regions. The term therefore includes persons who are naive for dengue fever virus and those who are not naive.
Tetravalent Immunization Following Initial Monovalent Immunization
In a first aspect, this invention therefore relates to a method of immunization against dengue fever virus.
The inventors have in fact shown in particular that the administration of 4 serotypes 30 days to 12 months after the first administration of a monovalent vaccine makes it possible to obtain effective protection against the 4 serotypes. The method according to this invention is therefore of very particular interest in the context of an immunization strategy against dengue fever.
According to this invention, the first immunization may be performed using a monovalent composition or vaccine comprising a vaccinal virus of any of the 4 serotypes of dengue fever, the second administration being performed with all 4 vaccinal serotypes. According to a particular embodiment, a serotype 1 or 2 dengue fever vaccinal virus, preferably serotype 2, is used for the first administration. Preferably, the dengue fever vaccinal virus used in the first administration is an attenuated dengue virus and is not a chimeric virus. According to a particular embodiment, strain VDV1 or VDV2, preferably strain VDV2, is used as the vaccinal virus in the first administration.
Attenuated living vaccinal viruses are used in the second administration, preferably chimeric viruses expressing antigens for the four serotypes of dengue fever virus, in particular Chimerivax® DENT, 2, 3 and 4.
According to particular embodiments, this invention therefore includes the following systems:
(a) VDV1 (b) CYD DEN-1,2,3 and 4
(b) VDV2 (b) CYD DEN-1,2,3 and 4.
In the context of this invention, by "vaccinal composition" is meant a composition comprising an "immunoeffective quantity" of dengue fever vaccinal virus, that is to say a sufficient quantity of dengue fever vaccinal virus to induce a specific immune response comprising neutralizing antibodies, which may be revealed for example by the seroneutralization test as described in Example 1 below. A serum is regarded as being positive for the presence of neutralizing antibodies when the titer of neutralizing antibodies so determined is not less than 1:10 (unity: 1/dilution).
The quantities of vaccinal strain are commonly expressed in terms of viral plaque forming units (PFU) or doses infecting 50% of the tissue culture or again doses infecting 50% of the cell culture (DICC50). For example, compositions according to the invention may contain 10 to 106 DICC50, in particular 103 to 105 DICC50 of dengue fever vaccinal virus of serotypes 1, 2, 3 or 4 for a monovalent or tetravalent composition. Thus, in the compositions or utilizations according to the invention the doses of dengue vaccinal viruses of serotypes 1, 2, 3 and 4 preferably each lie within a range from 10 to 106 DICC50, such as 10, 102, 103, 104, 105 or 106 DICC50, in particular within a range from 103 to 105 DICC50. Vaccinal virus may be used at the same or different doses, which can be adjusted in relation to the nature of the vaccinal virus used and the intensity of the immune response obtained.
According to a particular embodiment of a method according to this invention, the quantities of attenuated live vaccinal virus in monovalent and tetravalent compositions or vaccines are 103 to 105 DICC50. According to a particular embodiment, the monovalent vaccine comprises about 104 DICC50 of VDV1 or VDV2, preferably VDV2. According to a particular embodiment, the tetravalent vaccine comprises 105 DICC50 of Chimerivax® DEN-1,2,3 and 4. According to one advantageous embodiment, the tetravalent vaccine comprises about 105 DICC50 of Chimerivax® DEN-1, 2 and 3 and about 103 DICC50 of Chimerivax® DEN-4.
In the context of this invention, the second administration (b) is performed 30 days and not more than 12 months after administration (a). According to an advantageous embodiment, the second administration is performed 30 days to 60 days after the first administration (a).
The neutralizing antibody response is advantageously durable, that is to say it can be detected in serum up to at least 6 months after the second administration.
Vaccinal viruses are administered in the form of compositions or vaccines which can be prepared by any method known to those skilled in the art. Normally, viruses, generally in lyophilized form, are mixed with a pharmaceutically acceptable excipient such as water or a phosphate-buffered saline solution, wetting agents or stabilizing agents. By "pharmaceutically acceptable excipient" is meant any solvent, dispersing medium, charge, etc., which does not produce any secondary reaction, for example an allergic reaction, in humans or animals. 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: The Science & Practice of Pharmacy", which represents a reference work in the field.
Preferably, vaccinal compositions are prepared in injectable form, and may take the form of liquid solutions, suspensions or emulsions. The compositions may in particular comprise an aqueous solution buffered in such a way as to maintain a pH between about 6 and 9 (as determined using a pH meter at ambient temperature).
Although it is not necessary to add an adjuvant, the compositions may nevertheless include such a compound, that is to say a substance which increases, stimulates or reinforces the cell or humoral immune response induced by the vaccinal virus administered simultaneously. Those skilled in the art will be able to select an adjuvant which might be appropriate in the context of this invention from the adjuvants conventionally used in the field of vaccines.
The compositions or vaccines according to the invention may be administered by any means conventionally used in vaccination, for example parenterally (in particular intradermally, subcutaneously or intramuscularly), advantageously subcutaneously. Preferably, the compositions or vaccines are injectable compositions administered subcutaneously, advantageously in the region of the left deltoid or right deltoid.
The volume of vaccine composition 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 about 0.5 ml.
The optimum period for administering all serotypes 1 to 4 is about 1 to 3 months before exposure to dengue fever virus. Vaccinations may be administered as a prophylactic treatment against infection by dengue fever virus in adults and children. Target populations therefore include persons who may be naive (i.e., not previously immunized) or non-naive with regard to dengue fever virus.
Booster administrations of dengue fever vaccinal viruses of serotypes 1 to 4 may also be used 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 second administration (b) according to the invention. Booster administrations will advantageously be performed using the same compositions or vaccines (i.e., the same vaccinal viruses) and preferably under the same conditions of administration (anatomical sites and methods of administration) as used for the 2nd administration (b).
Interference phenomena may be explained by the dominance of one or more serotypes in relation to others and are therefore independent of the technology used for preparation of the candidate vaccine (from VDV or Chimerivax®). The method according to this invention can therefore be applied in general to all dengue fever vaccinal viruses.
This invention is therefore also intended to cover use of dengue fever vaccinal viruses for the manufacture of a monovalent vaccine and a tetravalent vaccine for immunization against dengue fever virus in which the monovalent vaccine comprises the vaccinal virus of a first serotype of dengue fever, the tetravalent vaccine comprises vaccinal viruses for 4 serotypes of dengue fever, in which the tetravalent vaccine is administered at least 30 days and not later than 12 months after administration of the monovalent vaccine.
For a description of the vaccines and conditions of use in the context of use according to this invention, reference may be made to the description provided in relation to the method of immunization according to the invention.
According to another aspect, this invention has as its object an immunization kit against the four serotypes of dengue fever virus. The kit according to this invention comprises compositions or vaccines as defined above in relation to the method of immunization proposed. The kit according to the invention therefore comprises a container containing various containers containing the compositions or vaccines and advantageously, and optionally, an explanatory brochure including useful information for administration of the said compositions or vaccines.
According to one embodiment, this invention therefore relates to a kit for immunization against dengue fever virus, a container containing at least (a) a first container containing a monovalent vaccine comprising a vaccinal virus of a first serotype of dengue fever, and (b) a second container containing a tetravalent vaccine comprising vaccinal viruses for the 4 serotypes of dengue fever.
For a description of the vaccines, compositions or dengue fever vaccinal viruses which may be used in the kit according to the invention, reference may be made to the description provided above in relation to the method of immunization according to the invention.
According to a particular embodiment the kit according to the invention comprises at least: (a) a first container containing a monovalent vaccine comprising a VDV1 or VDV2 vaccinal virus, and (b) a second container containing a tetravalent vaccine comprising the 4 Chimerivax® DEN-1,2,3 and 4.
According to a particular embodiment, the kit according to the invention comprises at least one monovalent vaccine comprising about 104 DICC50 of VDV1 or VDV2 and a tetravalent vaccine comprising about 105 DICC50 of Chimerivax® DEN-1,2,3 and about 103 DICC50 of Chimerivax® DEN-4.
The kits according to the invention may contain a single example or several examples of the containers as described above.
If the vaccines used are in lyophilized form, the kit will advantageously comprise at least one additional container containing the diluent which can be used to reconstitute an injectable dose of vaccine. Any pharmaceutically acceptable diluent may be used for this purpose, conventionally water or a phosphate-buffered aqueous solution.
The invention is illustrated by the following example.
Immunization Against the 4 Serotypes of Dengue Fever Virus by Successive Injection of a Monovalent Composition Followed by a Tetravalent Composition in Monkeys
Viremia and immunogenicity were tested in a monkey model. Viremia in particular has been identified as being one of the factors associated with the virulence and severity of the disease in man, and therefore constitutes an important parameter which must be taken into consideration. As for immunogenicity, this is a key parameter in the context of evaluating the protection imparted.
1.1 Materials and methods
Experiments on monkeys were carried out in accordance with European Directives relating to animal experiments. The immunizations were performed on cynomolgus monkeys (Macaca fascicularis) originating from Mauritania. The monkeys were placed in quarantine for six weeks prior to immunization.
The monkeys were immunized subcutaneously with 0.5 ml of vaccine composition in the arm. After mild anesthesia with ketamine (Imalgene, Merial), blood was collected by puncture of the inguinal or saphenal veins. On days 0 and 28 following each immunization, 5 ml of blood were sampled in order to evaluate antibody responses, while between days 2 and 10 1 ml of blood was sampled in order to evaluate viremia. The blood was collected on ice and preserved on ice until the serum was separated off. In order 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 Viremia
Post-vaccination viremia was monitored by quantitative real time RT-PCT (qRT-PCR). Two sets of primers and probes located in the NS5 gene of the DENT and DEN2 strains were used to quantify the RNA of VDV-1 and VDV-2 respectively. A third set of 2 primers and 1 probe located in the NS5 gene of the YF virus was used to quantify the RNA of CYD. Finally, 4 sets of primers and specific probes for the different CYD serotypes located at the junction of the E (DEN)/NS1 (YF) genes were used to identify the serotype in the samples positive for NS5 YF RNA (see also Table 1). 7 plasmids containing the region targeted by each PCR, under the control of promoter T7, were transcribed in vitro to generate a series of synthetic RNA which were included in each RT-PCT test as an internal reference. The synthetic RNA were determined by spectrophotometry, the quantity of RNA obtained was converted into the number of RNA copies and expressed as GEQ (genome equivalents).
0.140 ml of monkey serum were extracted using the "Nucleospin 96 Virus®" RNA extraction kit from Macherey Nagel according to the manufacturer's instructions, and then the purified RNA was eluted with 0.140 ml (0.090 ml, then 0.05 ml) of RNase-free water. In order to avoid repeated freeze/thaw cycles, a first quantification was performed immediately after extraction on 5 μl of the said RNA preparation. The remaining volume was frozen at 70° C.
In addition to the components of the "Qiagen Qauntitect® probes" RT-PCR quantification kit (Qiagen), the reaction mixtures contained 10 picomoles of each primer, 4 picomoles of each probe and 5 μl of RNA in a total volume of 25 μl. In the case of the RNA under test, 5 μl of the purified preparation were added directly to the reaction mixture without a prior dilution stage. The synthetic RNAs were diluted 1/10 in RNAse-free water, and 7 dilutions containing approximately 10 to 106 GEQ in 5 μl were quantified in parallel in order to generate a calibration curve.
The quantification reactions were carried out using the ABIPrism 700® equipment from Applied Biosystem, using the following program: 50° C./30 min, 95° C./15 min, followed by 40 cycles of 95° C./15 sec-60° C./60 sec.
The quantification limit for viral RNA in this test is 2.9 to 3.3 log10GEQ/ml (800 to 2000 GEQ/ml; 4 to 10 GEQ/reaction), according to PCR targets (standard deviation: +/-0.3 log10).
The correlation between infectious titer and the quantification of viral RNA was established in parallel with the tests by analyzing 0.140 ml of samples of negative monkey serums (DO) to which a known quantity of infectious particles of the viruses used for immunization (CYD or VDV) had been added. The said control serums were prepared in two dilutions containing approximately 1 PFU and approximately 100 PFU in 5 μl (2.3 and 4.3 log10PFU/ml, respectively).
In the tests used in the examples, the correlation between GEQ and PFU is as follows: GEQ/PFU ratio 2.7 log10 (i.e. 1 PFU=500 GEQ) for sera positive for YF or CYDs; GEQ/PFU ratio 2.5 log10 (i.e. 1 PFU=320 GEQ) for sera positive for VDV1 or VDV2.
The quantification limits are <3.3 log10GEQ/ml (i.e. <4 PFU/ml) for YF and CYDs qRT-PCR, and <2.9 log10GEQ/ml (i.e. <2.5 PFU/ml) for VDV1 and VDV2 qRT-PCR.
The primers and probes used are shown in Table 1 below, in which the sense and anti-sense primers and the probe are listed in order for each test.
TABLE-US-00001 TABLE 1 YF YF-NS5 sense 5' GCACGGATGTAACAGACTGAAGA (23 bases) SEQ ID NO: 3 YF-NS5 antisense 5' CCAGGCCGAACCTGTCAT (18 bases) SEQ ID NO: 4 YF-NS5 5' Fam-CGACTGTGTGGTCCGGCCCATC-Tamra (22 bases) SEQ ID NO: 5 CYD1 spe CYD1 sense 5' CAT TGC AGT TGG CCT GGT AA (20 b) SEQ ID NO: 6 CYD1 antisense 5' CTT TGG CAA GAG AGA GCT CAA GT (23 b) SEQ ID NO: 7 CYD1 5' Fam-CCG ATC AAG GAT GCG CCA TCA-Tamra (21 b) SEQ ID NO: 8 CYD2 spe CYD2 sense 5' GTG GGA GTC GTG ACG CTG TA (20 b) SEQ ID NO: 9 CYD2 antisense 5' GTT GAT GGC GCA TCC TTG ATC (21 b) SEQ ID NO: 10 CYD2 5' Fam-TGG GAG TTA TGG TGG GCG CCG-Tamra (21 b) SEQ ID NO: 11 CYD3 spe CYD3 sense 5' AAA ACA CTT CCA TGT CAT TTT CAT G (25 b) SEQ ID NO: 12 CYD3 antisense 5' GTT GAT GGC GCA ACC TTG ATC (21 b) SEQ ID NO: 13 CYD3 5' Fam-TGCGATAGGAATTATCACACTCTATCTGGGAGC-Tamra (33 b) SEQ ID NO: 14 CYD4 spe CYD4 sense 5' CTT AGT ATT GTG GAT TGG CAC GAA (24 b) SEQ ID NO: 15 CYD4 antisense 5' GCG CCA ACT GTG AAA CCT AGA (21 b) SEQ ID NO: 16 CYD4 5' Fam-AGAAACACTTCAATGGCAATACGTGCAT-Tamra (39 b) SEQ ID NO: 17 VDV1 spe VDV1-NS5 sense 5' TCG CAA CAG CCT TAA CAG C (19b) SEQ ID NO: 18 VDV1-NS5 antisense 5' ACT ATC TCC CTC CCA TCC TTC (21 b) SEQ ID NO: 19 VDV1-NS5 5' Fam-TTC ACA CCA CTT CCA C-M GB/NFQ (16 b) SEQ ID NO: 20 VDV2 spec VDV2-NS5 sense 5' AAT GAC AGA CAC GAC TCC (18 b) SEQ ID NO: 21 VDV2-NS5 antisense 5' CCC AAA ACC TAC TAT CTT CAA C (22 b) SEQ ID NO: 22 VDV2-NS5 5' Fam-TGG AAG TCG GCA CGT GA-MGB/NFQ (17 b) SEQ ID NO: 23
Measurement of Neutralizing Antibodies (Seroneutralization Test) (SN50)
Conventionally, dengue fever antibodies are measured using the PRNT50 test (test of neutralization by reducing the number of PFU to 50%). As this test is cumbersome and consumes much material, we have developed the SN50 test based on a 50% reduction in the number of units measured in the DICC50 test.
In a 96 well plate, 0.120 ml of each decomplemented serum is added to 0.480 ml of diluent (ISCOVE 4% SVF) in each well. Serial dilutions of a factor 6 are performed by transferring 0.150 ml of serum into 0.450 ml of diluent. 450 μl of viral dilution containing 2.7 log10 DICC50/ml are added to each well so as to obtain 25 DICC50/well. The plate is incubated at 37° C. for 1 hour. 0.1 ml of each dilution is then distributed into 6 wells of a 96 well plate in which VERO cells have been seeded 3 days before the start of the experiment at a density of 8000 cells/well in 0.1 ml of ISCOVE 4% SVF medium. After 6 days incubation at 37° C. in the presence of 5% CO2, the cells are fixed using an ethanol/acetone (70/30) mixture at 4° C. for 15 minutes, and then washed 3 times in PBS and incubated for 1 hour at 37° C. in the presence of 0.05 ml of a 1/2000 dilution of an anti-flavivirus monoclonal antibody (mAb 4G2 obtained from an ATCC H-B112 hybridoma). The plates are then washed twice and incubated for 1 hour at 37° C. in the presence of 0.05 ml of a 1/1000 dilution of an anti-mouse IgG conjugated with alkaline phosphatase. The lysis plaques are revealed by adding 0.05 ml of a stained substrate: BCIP/NBT. The neutralizing antibody titers are calculated using the Karber formula as defined below:
d: represents the dilution providing 100% neutralization (that is 6 negative replicates, i.e. presenting no signs of infection)
f: represents the dilution factor as log10 (e.g. dilution factor of 1:4, f=0.6)
N: represents the number of replicates/dilution (N=6)
X: total number of wells having no sign of infection, with the exception of dilution d.
The limit for viral detection is 10 SN50 (i.e. 1.0 log10SN50).
The viral strains used for neutralization were the strains DENT 1 6007, DEN2 16681, DEN3 16562 or DEN4 1036.
In the case of the controls, the initial viral dilutions were re-titrated.
The correlation between the neutralizing titer measured in the SN50 test and the neutralizing titer measured conventionally in the PRNT50 test is: log10PRNT50=log10SN50+0.2.
1.2 Evaluation of Simultaneous Immunizations
2 groups of 4 monkeys of equivalent age and weight were immunized (see Table 2).
Immunization was performed subcutaneously in the arm using a 23G1 needle, with a quantity of 105 DICC50 for each CYD DEN 1 to 4 serotype for the tetravalent vaccine and a quantity of 104 DICC50 for the monovalent VDV-2.
TABLE-US-00002 TABLE 2 Composition of the groups and immunization protocol Monkeys Immunizations Group D0 D56 Group 1 Monovalent Tetravalent VDV 2 Dengue 1234 ChimeriVax Group 2 Tetravalent Tetravalent Dengue Dengue 1234 1234 ChimeriVax ChimeriVax
The immunogenicity results obtained after one immunization (D0+28) and two immunizations (D56+28) are shown in Table 3.
The viremia results are provided in Table 4.
TABLE-US-00003 TABLE 3 SN50 neutralizing titer (units 1/dil) Monkeys Immunizations D 0 + 28 D 56 + 28 ID D 0 D 56 DEN-1 DEN-2 DEN-3 DEN-4 DEN-1 DEN-2 DEN-3 DEN-4 AM762 VDV 2 CYD 10 501 -- 10 63 1005 63 200 AM839 1234 -- 802 -- -- 80 1271 63 504 AM905 20 158 -- -- 318 1010 252 506 AN011 13 1005 -- -- 252 1271 319 1010 Geometric 11 583 <10 <10 142 1131 134 477 mean AM496 CYD CYD 50 -- 16 32 100 40 80 252 AM645 1234 1234 -- -- 13 31 16 -- -- 63 AM766 -- -- -- 32 20 -- -- 80 AM813 25 -- -- 13 63 13 20 63 Geometric 13 <10 <10 25 38 11 14 95 mean --: titer < 10
TABLE-US-00004 TABLE 4 viremia analyses (units: log 10 GEQ/mL) First immunization Group Monkey D 2 D 3 D 4 D 5 D 6 D 7 D 8 D 9 D 10 1 AM762 <2.7 <2.7 <2.7 <2.7 4.77 4.89 4.84 4.47 <2.7 initial VDV 2 AM839 5.11 4.51 4.19 4.19 4.56 3.69 <2.7 <2.7 <2.7 booster CYD 1, 2, 3, 4 AM905 <2.7 <2.7 <2.7 <2.7 4.16 4.31 4.16 3.50 4.14 AN011 <2.7 <2.7 3.75 4.29 4.35 4.22 3.51 <2.7 3.28 2 AM496 4.22 3.36 3.71 4.15 3.14 <3.1 3.58 <3.1 <3.1 initial CYD 1, 2, 3, 4 AM645 4.21 3.61 2.82 3.51 3.65 3.24 <3.1 3.47 3.44 booster CYD 1, 2, 3, 4 AM766 3.97 3.06 3.38 4.19 3.80 3.73 <3.1 <3.1 <3.1 AM813 4.81 4.60 3.17 <3.1 <3.1 <3.1 <3.1 <3.1 <3.1 Booster Group Monkey D 58 D 59 D 60 D 61 D 62 D 63 D 64 D 65 D 66 1 AM762 <3.2 <3.2 <3.2 3.11 <3.2 <3.2 3.65 3.59 <3.2 initial VDV 2 AM839 4.98 4.86 4.43 3.79 <3.2 <3.2 <3.2 <3.2 <3.2 booster CYD 1, 2, 3, 4 AM905 <3.2 <3.2 3.09 3.42 3.39 <3.2 <3.2 4.08 4.22 AN011 3.55 3.42 <3.2 <3.2 3.42 3.37 4.67 5.09 4.97 2 AM496 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 initial CYD 1, 2, 3, 4 AM645 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 booster CYD 1, 2, 3, 4 AM766 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 AM813 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 <3.2 CYD1 CYD4 VDV2
In brief, the results can be summarized as follows: The method of administration according to this invention brings about a qualitative and quantitative increase in the neutralizing antibody response obtained with a system comprising two identical immunizations with tetravalent vaccine. One CYD-1,2,3,4 immunization performed after an initial monovalent VDV2 immunization induces high level responses against the four serotypes in all the monkeys, unlike a system comprising 2 immunizations of tetravalent vaccine. As expected, the initial immunization performed with VDV2 induces a response which is almost exclusively directed against serotype 2, with a low level of cross-reactivity against serotypes 1 and 4 in some animals. Viremia is observed with VDV2 after initial immunization, and is predominantly caused by CYD-4 after the second administration (b) (group 1). No noteworthy differences were observed in the viremia induced after a first immunization with tetravalent vaccine in naive animals (group 2). It can therefore be concluded that the system proposed by the invention does not encourage the emergence of viremia of serotypes 1, 3 and 4 after the second administration.
The examples therefore show that the method of immunization according to this invention improves the immunogenicity of the dengue fever vaccinal viruses without adversely affecting the latter's safety.
All publications recited herein are hereby incorporated by reference in their entirety. To the extent of conflict between a publication and the present specification, the present specification controls.
23110735DNADengue virus 1agttgttagt ctacgtggac cgacaagaac agtttcgaat cggaagcttg cttaacgtag 60ttctaacagt tttttattag agagcagatc tctgatgatc aaccaacgaa aaaagacggg 120tcgaccgtct ttcaatatgc tgaaacgcgc gagaaaccgc gtgtcaactg tttcacagtt 180ggcgaagaga ttctcaaaag gattgctctc aggccaagga cccatgaaat tggtgatggc 240tttcatagca ttcttaagat ttctagccat acccccaaca gcaggaattt tggctagatg 300gggctcattc aagaagaatg gagcgattaa agtgttacgg ggtttcaaga gagaaatctc 360aaacatgcta aacataatga acaggaggaa aagatccgtg accatgctcc ttatgctgct 420gcccacagcc ctggcgttcc atctgacgac acgaggggga gagccgcata tgatagttag 480caagcaggaa agaggaaagt cacttttgtt caagacctct gcaggtgtca acatgtgcac 540cctcattgcg atggatttgg gagagttgtg tgaggacacg atgacctaca aatgcccccg 600gatcactgag gcggaaccag atgacgttga ctgttggtgc aatgccacgg acacatgggt 660gacctatgga acgtgctctc aaactggcga acaccgacga gacaaacgtt ccgtcgcatt 720ggccccacac gtggggcttg gcctagaaac aagagccgaa acgtggatgt cctctgaagg 780tgcttggaaa cagatacaaa aagtagagac ttgggctctg agacatccag gattcacggt 840gatagccctt tttctagcac atgccatagg aacatccatc acccagaaag ggatcatttt 900cattttgctg atgctggtaa caccatctat ggccatgcga tgcgtgggaa taggcaacag 960agacttcgtg gaaggactgt caggagcaac atgggtggat gtggtactgg agcatggaag 1020ttgcgtcacc accatggcaa aaaacaaacc aacactggac attgaactct tgaagacgga 1080ggtcacaaac cctgcagttc tgcgtaaatt gtgcattgaa gctaaaatat caaacaccac 1140caccgattcg agatgtccaa cacaaggaga agccacactg gtggaagaac aagacgcgaa 1200ctttgtgtgc cgacgaacgt tcgtggacag aggctggggc aatggctgtg ggctattcgg 1260aaaaggtagt ctaataacgt gtgccaagtt taagtgtgtg acaaaactag aaggaaagat 1320agctcaatat gaaaacctaa aatattcagt gatagtcacc gtccacactg gagatcagca 1380ccaggtggga aatgagacta cagaacatgg aacaactgca accataacac ctcaagctcc 1440tacgtcggaa atacagctga ccgactacgg aacccttaca ttagattgtt cacctaggac 1500agggctagat tttaacgaga tggtgttgct gacaatgaaa aagaaatcat ggcttgtcca 1560caaacagtgg tttctagact taccactgcc ttggacctct ggggctttaa catcccaaga 1620gacttggaac agacaagatt tactggtcac atttaagaca gctcatgcaa agaagcagga 1680agtagtcgta ctaggatcac aagaaggagc aatgcacact gcgctgactg gagcgacaga 1740aatccaaacg tcaggaacga caacaatttt cgcaggacac ctaaaatgca gactaaaaat 1800ggacaaacta actttaaaag ggatgtcata tgtgatgtgc acaggctcat tcaagttaga 1860gaaagaagtg gctgagaccc agcatggaac tgttctggtg caggttaaat atgaaggaac 1920agacgcacca tgcaagattc ccttttcgac ccaagatgag aaaggagcaa cccagaatgg 1980gagattaata acagccaacc ccatagtcac tgacaaagaa aaaccagtca atattgaggc 2040agaaccaccc tttggtgaga gctacatcgt ggtaggagca ggtgaaaaag ctttgaaact 2100aagctggttc aagaaaggaa gcagcatagg gaaaatgttt gaagcaactg cccgaggagc 2160acgaaggatg gccattctgg gagacaccgc atgggacttc ggttctatag gaggagtgtt 2220cacgtctatg ggaaaactgg tacaccaggt ttttggaact gcatatggag ttttgtttag 2280cggagtttct tggaccatga aaataggaat agggattctg ctgacatggc taggattaaa 2340ttcaaggaac acgtcccttt cggtgatgtg catcgcagtt ggcatggtca cactgtacct 2400aggagtcatg gttcaggcag attcgggatg tgtaatcaac tggaaaggca gagaacttaa 2460atgtggaagc ggcatttttg tcactaatga agttcacact tggacagagc aatacaaatt 2520ccaggctgac tcccccaaga gactatcagc agccattggg aaggcatggg aggagggtgt 2580gtgtggaatc cgatcagcca ctcgtctcga gaacatcatg tggaaacaaa tatcaaatga 2640attgaaccac atcctacttg aaaatgacat gaaatttaca gtggtcgtgg gagacgttag 2700tggaatcttg gcccaaggaa aaaaaatgat taggccacaa cccatggaac acaaatactc 2760gtggaaaagc tggggaaaag ctaaaatcat aggagcggat gtacagaaca ccaccttcat 2820catcgacggc ccaaacaccc cagaatgccc tgacaatcaa agagcatgga atatttggga 2880agtagaggac tatggatttg ggattttcac gacaaacata tggttgaaat tgcgtgactc 2940ctacacccaa gtatgtgacc accggctgat gtcagctgcc attaaggaca gcaaggcagt 3000ccatgctgac atggggtact ggatagaaag tgaaaagaac gagacatgga agttggcgag 3060agcctccttt atagaagtta agacatgcat ctggccaaaa tcccacactc tatggagcaa 3120tggagttctg gaaagtgaaa tgataattcc aaagatatat ggaggaccaa tatctcagca 3180caactacaga ccaggatatt tcacacaaac agcagggccg tggcacctag gcaagttgga 3240actagatttc gatttttgtg aaggtaccac agttgttgtg gatgaacatt gtggaaatcg 3300aggaccatct ctcagaacca caacagtcac aggaaagata atccatgaat ggtgctgcag 3360atcttgtacg ctaccccccc tacgtttcaa aggggaagac gggtgttggt acggcatgga 3420aatcagacca gtgaaggaca aggaagagaa cctggtcaag tcaatggtct ctgcagggtc 3480aggagaagtg gacagctttt cactaggact gctatgcata tcaataatga ttgaagaagt 3540gatgagatcc agatggagca aaaaaatgct gatgactgga acactggctg tgttcctcct 3600tcttataatg ggacaattga catggagtga tctgatcagg ttatgtatta tggttggagc 3660caacgcttca gacaagatgg ggatgggaac aacgtaccta gctttaatgg ccactttcaa 3720aatgagacca atgttcgccg tcgggctatt atttcgcaga ctaacatcta gagaagttct 3780tcttcttaca attggcttga gcctggtggc atccgtggag ctaccaagtt ccctagagga 3840gctgggggat ggacttgcaa taggcatcat gatgttgaaa ttattgactg attttcagtc 3900acaccagcta tgggctactc tgctatcctt gacatttatt aaaacaactt tttcattgca 3960ctatgcatgg aagacaatgg ctatggtact gtcaattgta tctctcttcc ctttatgcct 4020gtccacgacc tctcaaaaaa caacatggct tccggtgctg ttgggatctc ttggatgcaa 4080accactaccc atgtttctta taacagaaaa caaaatctgg ggaaggaaga gttggcccct 4140caatgaagga attatggctg ttggaatagt tagtattcta ctaagttcac ttttaaaaaa 4200tgatgtgccg ctagccggcc cattaatagc tggaggcatg ctaatagcat gttatgtcat 4260atccggaagc tcagctgatt tatcactgga gaaagcggct gaggtctcct gggaggaaga 4320agcagaacac tcaggcgcct cacacaacat actagtagag gttcaagatg atggaaccat 4380gaagataaaa gatgaagaga gagatgacac gctcaccatt ctccttaaag caactctgct 4440ggcagtctca ggggtgtacc caatgtcaat accagcgacc ctttttgtgt ggtatttttg 4500gcagaaaaag aaacagagat caggagtgct atgggacaca cccagccccc cagaagtgga 4560aagagcagtt cttgatgatg gcatctatag aattttgcaa agaggactgt tgggcaggtc 4620ccaagtagga gtaggagttt tccaagaagg cgtgttccac acaatgtggc acgtcactag 4680gggagctgtc ctcatgtatc aaggaaaaag gctggaacca agctgggcca gtgtcaaaaa 4740agacttgatc tcatatggag gaggttggag gtttcaagga tcctggaaca cgggagaaga 4800agtacaggtg attgctgttg aaccgggaaa aaaccccaaa aatgtacaaa caacgccggg 4860taccttcaag acccctgaag gcgaagttgg agccatagcc ttagacttta aacctggcac 4920atctggatct cccatcgtaa acagagaggg aaaaatagta ggtctttatg gaaatggagt 4980ggtgacaaca agcggaactt acgttagtgc catagctcaa gctaaggcat cacaagaagg 5040gcctctacca gagattgagg acaaggtgtt taggaaaaga aacttaacaa taatggacct 5100acatccagga tcgggaaaaa caagaagata ccttccagcc atagtccgtg aggccataaa 5160aaggaagctg cgcacgctaa tcctagctcc cacaagagtt gtcgcttctg aaatggcaga 5220ggcactcaag ggagtgccaa taaggtatca gacaacagca gtgaagagtg aacacacagg 5280aaaggagata gttgacctta tgtgccacgc cactttcacc atgcgcctcc tgtctcccgt 5340gagagttccc aattataaca tgattatcat ggatgaagca cacttcaccg atccagccag 5400catagcagcc agagggtaca tctcaacccg agtgggtatg ggtgaagcag ctgcgatctt 5460tatgacagcc actcccccag gatcggtgga ggcctttcca cagagcaatg caattatcca 5520agatgaggaa agagacattc ctgagagatc atggaactca ggctatgact ggatcactga 5580ttttccaggt aaaacagtct ggtttgttcc aagcatcaaa tcaggaaatg acattgccaa 5640ctgtttaaga aaaaacggga aacgggtgat ccaattgagc agaaaaacct ttgacactga 5700gtaccagaaa acaaaaaaca acgactggga ctatgtcgtc acaacagaca tttccgaaat 5760gggagcaaat ttccgggccg acagggtaat agacccaagg cggtgtctga aaccggtaat 5820actaaaagat ggtccagagc gcgtcattct agccggaccg atgccagtga ctgtggccag 5880tgccgcccag aggagaggaa gaattggaag gaaccaaaac aaggaaggtg atcagtatat 5940ttacatggga cagcctttaa aaaatgatga ggaccacgct cattggacag aagcaaagat 6000gctccttgac aatataaaca caccagaagg gattatccca gccctctttg agccggagag 6060agaaaagagt gcagctatag acggggaata cagactgcgg ggtgaagcaa ggaaaacgtt 6120cgtggagctc atgagaagag gggatctacc agtctggcta tcctacaaag ttgcctcaga 6180aggcttccag tactccgaca gaaggtggtg cttcgatggg gaaaggaaca accaggtgtt 6240ggaggagaac atggacgtgg agatctggac aaaagaagga gaaagaaaga aactacgacc 6300tcgctggttg gacgccagaa catactctga cccactggct ctgcgcgagt ttaaagagtt 6360tgcagcagga agaagaagcg tctcaggtga cctaatatta gaaataggga aacttccaca 6420acatttgacg caaagggccc agaatgcttt ggacaacttg gtcatgttgc acaattccga 6480acaaggagga aaagcctata gacatgctat ggaagaactg ccagacacaa tagaaacgtt 6540gatgctccta gccttgatag ctgtgttgac tggtggagtg acgctgttct tcctatcagg 6600aagaggtcta ggaaaaacat ctatcggctt actctgcgtg atggcctcaa gcgcactgtt 6660atggatggcc agtgtggagc cccattggat agcggcctcc atcatactgg agttctttct 6720gatggtactg cttattccag agccagacag acagcgcact ccacaggaca accagctagc 6780atatgtggtg ataggtctgt tattcgtgat attgacagtg gcagccaatg agatgggatt 6840attggaaacc acaaagaaag acctggggat tggccatgta gctgctgaaa accaccacca 6900tgctacaatg ctggacgtag acctacatcc agcttcagcc tggaccctct atgcagtggc 6960cacaacaatc atcactccta tgatgagaca cacaattgaa aacacaacgg caaatatttc 7020cctgacagcc atcgcaaacc aagcagctat attgatggga cttgacaagg gatggccaat 7080atcgaagatg gacataggag ttccacttct cgccttgggg tgctattccc aagtgaatcc 7140gctgacactg atagcggcag tattgatgct agtagctcat tacgccataa ttggacctgg 7200actgcaagca aaagctacta gagaagctca aaaaagaaca gcggctggaa taatgaaaaa 7260tccaactgtc gacgggattg ttgcaataga cttagatccc gtggtttacg atgcaaaatt 7320tgaaaaacag ctaggccaaa taatgttgtt gatactttgc acatcacaga ttcttttgat 7380gcggactaca tgggccttgt gtgaatccat cacattggct actggacctc tgaccactct 7440ttgggaggga tctccaggaa aattctggaa caccacaata gcggtatcca tggcaaacat 7500tttcaggggg agttatctag caggagcagg tctggccttc tcattaatga aatctctagg 7560aggaggtagg agaggcacgg gagcccaagg ggaaacactg ggagaaaaat ggaaaagaca 7620actaaaccaa ctgagcaagt cagaattcaa tacttacaag aggagtggga ttatggaggt 7680ggatagatcc gaagccaaag agggactgaa aagaggagaa acaaccaaac acgcagtatc 7740gagaggaacg gccaaactga ggtggttcgt ggagaggaac cttgtgaaac cagaagggaa 7800agtcatagac ctcggttgtg gaagaggtgg ctggtcatat tattgcgctg ggctgaagaa 7860agtcacagaa gtgaaaggat acacaaaagg aggacctgga catgaggaac caatcccaat 7920ggcgacctat ggatggaacc tagtaaggct gcactccgga aaagatgtat tttttatacc 7980acctgagaaa tgtgacaccc ttttgtgtga tattggtgag tcctctccga acccaactat 8040agaggaagga agaacgttac gtgttctgaa aatggtggaa ccatggctca gaggaaacca 8100attttgcata aaaattctaa atccctatat gccgagcgtg gtagaaactc tggaacaaat 8160gcaaagaaaa catggaggaa tgctagtgcg aaacccactc tcaagaaatt ccacccatga 8220aatgtactgg gtttcatgtg gaacaggaaa cattgtgtca gcagtaaaca tgacatctag 8280aatgttgcta aatcggttca caatggctca caggaagcca acatatgaaa gagacgtgga 8340cttaggcgct ggaacaagac atgtggcagt agaaccagag gtagccaacc tagatatcat 8400tggccagagg atagagaata taaaaaatga acataagtca acatggcatt atgatgagga 8460caatccatac aaaacatggg cctatcatgg atcatatgag gttaagccat caggatcggc 8520ctcatccatg gtcaatggcg tggtgagatt gctcaccaaa ccatgggatg ttatccccat 8580ggtcacacaa atagccatga ctgataccac accctttgga caacagaggg tgtttaaaga 8640gaaagttgac acgcgcacac caaaagcaaa acgtggcaca gcacaaatta tggaagtgac 8700agccaggtgg ttatggggtt tcctttctag aaacaaaaaa cccagaattt gcacaagaga 8760ggagtttaca agaaaagtta ggtcaaacgc agctattgga gcagtgttcg ttgatgaaaa 8820tcaatggaac tcggcaaaag aagcagtgga agacgaacgg ttctgggaac ttgtccacag 8880agagagggag cttcataaac aggggaaatg tgccacgtgt gtctacaata tgatggggaa 8940gagagagaaa aaattaggag agttcggaaa ggcaaaagga agtcgtgcaa tatggtacat 9000gtggttggga gcacgcttcc tagagtttga agcccttggt ttcatgaatg aagatcactg 9060gttcagtaga gagaattcac tcagtggagt ggaaggagaa ggactccaca aacttggata 9120catactcaga gacatatcaa ggattccagg ggggaacatg tatgcagatg acacagccgg 9180atgggacaca agaataacag aggatgatct ccagaatgag gctaaaatca ctgacatcat 9240ggagcccgaa catgccctgc tggctacgtc aatctttaag ctgacctacc aaaataaggt 9300ggtaagggtg cagagaccag caaaaaatgg aaccgtgatg gatgttatat ccagacgtga 9360ccagagaggc agtggacagg ttggaactta tggcttaaac actttcacca acatggaggc 9420ccaactgata agacaaatgg agtctgaggg aatcttttta cccagcgaat tggaaacccc 9480aaatctagcc ggaagagttc tcgactggtt ggaaaaatat ggtgtcgaaa ggctgaaaag 9540aatggcaatc agcggagatg actgtgtggt gaaaccaatt gatgacaggt tcgcaacagc 9600cttaacagct ttgaatgaca tgggaaaagt aagaaaagac ataccacaat gggaaccttc 9660aaaaggatgg aatgattggc aacaagtgcc tttctgttca caccacttcc accagctaat 9720tatgaaggat gggagggaga tagtggtgcc atgccgcaac caagatgaac ttgtggggag 9780ggccagagta tcacaaggcg ccggatggag cctgagagaa accgcatgcc taggcaagtc 9840atatgcacaa atgtggcagc tgatgtattt ccacaggaga gacctgagac tggcggctaa 9900cgctatttgt tcagccgttc cagttgattg ggtcccaacc agccgcacca cctggtcgat 9960ccatgcccat caccaatgga tgacaacaga agacatgtta tcagtatgga atagggtctg 10020gatagaggaa aacccatgga tggaggataa gactcatgtg tccagttggg aagaagttcc 10080atacctagga aagagggaag atcagtggtg tggatccctg ataggcttaa cagcaagggc 10140cacctgggcc actaatatac aagtggccat aaaccaagtg agaaggctca ttgggaatga 10200gaattatcta gattacatga catcaatgaa gagattcaag aatgagagtg atcccgaagg 10260ggcactctgg taagtcaaca cattcacaaa ataaaggaaa ataaaaaatc aaatgaggca 10320agaagtcagg ccagattaag ccatagtacg gtaagagcta tgctgcctgt gagccccgtc 10380caaggacgta aaatgaagtc aggccgaaag ccacggtttg agcaagccgt gctgcctgtg 10440gctccatcgt ggggatgtaa aaacccggga ggctgcaacc catggaagct gtacgcatgg 10500ggtagcagac tagtggttag aggagacccc tcccaagaca caacgcagca gcggggccca 10560acaccagggg aagctgtacc ctggtggtaa ggactagagg ttagaggaga ccccccgcgt 10620aacaataaac agcatattga cgctgggaga gaccagagat cctgctgtct ctacagcatc 10680attccaggca cagaacgcca gaaaatggaa tggtgctgtt gaatcaacag gttct 10735210723DNADengue virus 2agttgttagt ctacgtggac cgacaaagac agattctttg agggagctaa gctcaatgta 60gttctaacag ttttttaatt agagagcaga tctctgatga ataaccaacg gaaaaaggcg 120aaaaacacgc ctttcaatat gctgaaacgc gagagaaacc gcgtgtcgac tgtgcaacag 180ctgacaaaga gattctcact tggaatgctg cagggacgag gaccattaaa actgttcatg 240gccctggtgg cgttccttcg tttcctaaca atcccaccaa cagcagggat attgaagaga 300tggggaacaa ttaaaaaatc aaaagctatt aatgttttga gagggttcag gaaagagatt 360ggaaggatgc tgaacatctt gaataggaga cgcagatctg caggcatgat cattatgctg 420attccaacag tgatggcgtt ccatttaacc acacgtaacg gagaaccaca catgatcgtc 480agcagacaag agaaagggaa aagtcttctg tttaaaacag aggttggcgt gaacatgtgt 540accctcatgg ccatggacct tggtgaattg tgtgaagaca caatcacgta caagtgtccc 600cttctcaggc agaatgagcc agaagacata gactgttggt gcaactctac gtccacgtgg 660gtaacttatg ggacgtgtac caccatggga gaacatagaa gagaaaaaag atcagtggca 720ctcgttccac atgtgcgaat gggactggag acacgaactg aaacatggat gtcatcagaa 780ggggcctgga aacatgtcca gagaattgaa acttggatct tgagacatcc aggcttcacc 840atgatggcag caatcctggc atacaccata ggaacgacac atttccaaag agccctgatt 900ttcatcttac tgacagctgt cactccttca atgacaatgc gttgcatagg aatgtcaaat 960agagactttg tggaaggggt ttcaggagga agctgggttg acatagtctt agaacatgga 1020agctgtgtga cgacgatggc aaaaaacaaa ccaacattgg attttgaact gataaaaaca 1080gaagccaaac agcctgccac cctaaggaag tactgtatag aggcaaagct aaccaacaca 1140acaacagaat ctcgctgccc aacacaaggg gaacccagcc taaatgaaga gcaggacaaa 1200aggttcgtct gcaaacactc catggtagac agaggatggg gaaatggatg tggactattt 1260ggaaagggag gcattgtgac ctgtgctatg ttcagatgca aaaagaacat ggaaggaaaa 1320gttgtgcaac cagaaaactt ggaatacacc attgtgataa cacctcactc aggggaagag 1380catgcagtcg gaaatgacac aggaaaacat ggcaaggaaa tcaaaataac accacagagt 1440tccatcacag aagcagaatt gacaggttat ggcactgtca caatggagtg ctctccaaga 1500acgggcctcg acttcaatga gatggtgttg ctgcagatgg aaaataaagc ttggctggtg 1560cacaggcaat ggttcctaga cctgccgtta ccatggttgc ccggagcgga cacacaagag 1620tcaaattgga tacagaagga gacattggtc actttcaaaa atccccatgc gaagaaacag 1680gatgttgttg ttttaggatc ccaagaaggg gccatgcaca cagcacttac aggggccaca 1740gaaatccaaa tgtcatcagg aaacttactc ttcacaggac atctcaagtg caggctgaga 1800atggacaagc tacagctcaa aggaatgtca tactctatgt gcacaggaaa gtttaaagtt 1860gtgaaggaaa tagcagaaac acaacatgga acaatagtta tcagagtgca atatgaaggg 1920gacggctctc catgcaagat cccttttgag ataatggatt tggaaaaaag acatgtctta 1980ggtcgcctga ttacagtcaa cccaattgtg acagaaaaag atagcccagt caacatagaa 2040gcagaacctc catttggaga cagctacatc atcataggag tagagccggg acaactgaag 2100ctcaactggt ttaagaaagg aagttctatc ggccaaatgt ttgagacaac aatgaggggg 2160gcgaagagaa tggccatttt aggtgacaca gcctgggatt ttggatcctt gggaggagtg 2220tttacatcta taggaaaggc tctccaccaa gtctttggag caatctatgg agctgccttc 2280agtggggttt catggactat gaaaatcctc ataggagtca ttatcacatg gataggaatg 2340aattcacgca gcacctcact gtctgtgaca ctagtattgg tgggaattgt gacactgtat 2400ttgggagtca tggtgcaggc cgatagtggt tgcgttgtga gctggaaaaa caaagaactg 2460aaatgtggca gtgggatttt catcacagac aacgtgcaca catggacaga acaatacaaa 2520ttccaaccag aatccccttc aaaactagct tcagctatcc agaaagccca tgaagaggac 2580atttgtggaa tccgctcagt aacaagactg gagaatctga tgtggaaaca aataacacca 2640gaattgaatc acattctatc agaaaatgag gtgaagttaa ctattatgac aggagacatc 2700aaaggaatca tgcaggcagg aaaacgatct ctgcggcctc agcccactga gctgaagtat 2760tcatggaaaa catggggcaa agcaaaaatg ctctctacag agtctcataa ccagaccttt 2820ctcattgatg gccccgaaac agcagaatgc cccaacacaa atagagcttg gaattcgttg 2880gaagttgaag actatggctt tggagtattc accaccaata tatggctaaa attgaaagaa 2940aaacaggatg tattctgcga ctcaaaactc atgtcagcgg ccataaaaga caacagagcc 3000gtccatgccg atatgggtta ttggatagaa agtgcactca atgacacatg gaagatagag 3060aaagcctctt tcattgaagt taaaaactgc cactggccaa aatcacacac cctctggagc 3120aatggagtgc tagaaagtga gatgataatt ccaaagaatc tcgctggacc agtgtctcaa 3180cacaactata gaccaggcta ccatacacaa ataacaggac catggcatct aggtaagctt 3240gagatggact ttgatttctg tgatggaaca acagtggtag tgactgagga ctgcggaaat 3300agaggaccct ctttgagaac aaccactgcc tctggaaaac tcataacaga atggtgctgc 3360cgatcttgca cattaccacc gctaagatac agaggtgagg atgggtgctg gtacgggatg 3420gaaatcagac cattgaagga gaaagaagag aatttggtca actccttggt cacagctgga 3480catgggcagg tcgacaactt ttcactagga gtcttgggaa tggcattgtt cctggaggaa 3540atgcttagga cccgagtagg aacgaaacat gcaatactac tagttgcagt ttcttttgtg 3600acattgatca cagggaacat gtcctttaga gacctgggaa gagtgatggt tatggtaggc 3660gccactatga cggatgacat aggtatgggc gtgacttatc ttgccctact agcagccttc 3720aaagtcagac caacttttgc agctggacta ctcttgagaa agctgacctc caaggaattg 3780atgatgacta ctataggaat tgtactcctc tcccagagca ccataccaga gaccattctt 3840gagttgactg atgcgttagc cttaggcatg atggtcctca aaatggtgag aaatatggaa 3900aagtatcaat tggcagtgac tatcatggct atcttgtgcg tcccaaacgc agtgatatta 3960caaaacgcat ggaaagtgag ttgcacaata ttggcagtgg tgtccgtttc cccactgttc 4020ttaacatcct cacagcaaaa aacagattgg ataccattag cattgacgat caaaggtctc 4080aatccaacag ctatttttct aacaaccctc tcaagaacca gcaagaaaag gagctggcca 4140ttaaatgagg ctatcatggc agtcgggatg gtgagcattt tagccagttc tctcctaaaa 4200aatgatattc ccatgacagg accattagtg gctggagggc tcctcactgt gtgctacgtg 4260ctcactggac gatcggccga
tttggaactg gagagagcag ccgatgtcaa atgggaagac 4320caggcagaga tatcaggaag cagtccaatc ctgtcaataa caatatcaga agatggtagc 4380atgtcgataa aaaatgaaga ggaagaacaa acactgacca tactcattag aacaggattg 4440ctggtgatct caggactttt tcctgtatca ataccaatca cggcagcagc atggtacctg 4500tgggaagtga agaaacaacg ggccggagta ttgtgggatg ttccttcacc cccacccatg 4560ggaaaggctg aactggaaga tggagcctat agaattaagc aaaaagggat tcttggatat 4620tcccagatcg gagccggagt ttacaaagaa ggaacattcc atacaatgtg gcatgtcaca 4680cgtggcgctg ttctaatgca taaaggaaag aggattgaac caacatgggc ggacgtcaag 4740aaagacctaa tatcatatgg aggaggctgg aagttagaag gagaatggaa ggaaggagaa 4800gaagtccagg tattggcact ggagcctgga aaaaatccaa gagccgtcca aacgaaacct 4860ggtcttttca aaaccaacgc cggaacaata ggtgctgtat ctctggactt ttctcctgga 4920acgtcaggat ctccaattat cgacaaaaaa ggaaaagttg tgggtcttta tggtaatggt 4980gttgttacaa ggagtggagc atatgtgagt gctatagccc agactgaaaa aagcattgaa 5040gacaacccag agatcgaaga tcacattttc cgaaagagaa gactgaccat catggacctc 5100cacccaggag cgggaaagac gaagagatac cttccggcca tagtcagaga agctataaaa 5160cggggtttga gaacattaat cttggccccc actagagttg tggcagctga aatggaggaa 5220gcccttagag gacttccaat aagataccag accccagcca tcagagctga gcacaccggg 5280cgggagattg tggacctaat gtgtcatgcc acatttacca tgaggctgct atcaccagtt 5340agagtgccaa actacaacct gattatcatg gacgaagccc atttcacaga cccagcaagt 5400atagcagcta gaggatacat ctcaactcga gtggagatgg gtgaggcagc tgggattttt 5460atgacagcca ctcccccggg aagcagagac ccatttcctc agagcaatgc accaatcata 5520gatgaagaaa gagaaatccc tgaacgctcg tggaattccg gacatgaatg ggtcacggat 5580tttaaaggga agactgtttg gttcgttcca agtataaaag caggaaatga tatagcagct 5640tgcctgagga aaaatggaaa gaaagtgata caactcagta ggaagacctt tgattctgag 5700tatgtcaaga ctagaaccaa tgattgggac ttcgtggtta caactgacat ttcagaaatg 5760ggtgccaatt tcaaggctga gagggttata gaccccagac gctgcatgaa accagtcata 5820ctaacagatg gtgaagagcg ggtgattctg gcaggaccta tgccagtgac ccactctagt 5880gcagcacaaa gaagagggag aataggaaga aatccaaaaa atgagaatga ccagtacata 5940tacatggggg aacctctgga aaatgatgaa gactgtgcac actggaaaga agctaaaatg 6000ctcctagata acatcaacac gccagaagga atcattccta gcatgttcga accagagcgt 6060gaaaaggtgg atgccattga tggcgaatac cgcttgagag gagaagcaag gaaaaccttt 6120gtagacttaa tgagaagagg agacctacca gtctggttgg cctacagagt ggcagctgaa 6180ggcatcaact acgcagacag aaggtggtgt tttgatggag tcaagaacaa ccaaatccta 6240gaagaaaacg tggaagttga aatctggaca aaagaagggg aaaggaagaa attgaaaccc 6300agatggttgg atgctaggat ctattctgac ccactggcgc taaaagaatt taaggaattt 6360gcagccggaa gaaagtctct gaccctgaac ctaatcacag aaatgggtag gctcccaacc 6420ttcatgactc agaaggcaag agacgcactg gacaacttag cagtgctgca cacggctgag 6480gcaggtggaa gggcgtacaa ccatgctctc agtgaactgc cggagaccct ggagacattg 6540cttttactga cacttctggc tacagtcacg ggagggatct ttttattctt gatgagcgca 6600aggggcatag ggaagatgac cctgggaatg tgctgcataa tcacggctag catcctccta 6660tggtacgcac aaatacagcc acactggata gcagcttcaa taatactgga gttttttctc 6720atagttttgc ttattccaga acctgaaaaa cagagaacac cccaagacaa ccaactgacc 6780tacgttgtca tagccatcct cacagtggtg gccgcaacca tggcaaacga gatgggtttc 6840ctagaaaaaa cgaagaaaga tctcggattg ggaagcattg caacccagca acccgagagc 6900aacatcctgg acatagatct acgtcctgca tcagcatgga cgctgtatgc cgtggccaca 6960acatttgtta caccaatgtt gagacatagc attgaaaatt cctcagtgaa tgtgtcccta 7020acagctatag ccaaccaagc cacagtgtta atgggtctcg ggaaaggatg gccattgtca 7080aagatggaca tcggagttcc ccttctcgcc attggatgct actcacaagt caaccccata 7140actctcacag cagctctttt cttattggta gcacattatg ccatcatagg gccaggactc 7200caagcaaaag caaccagaga agctcagaaa agagcagcgg cgggcatcat gaaaaaccca 7260actgtcgatg gaataacagt gattgaccta gatccaatac cttatgatcc aaagtttgaa 7320aagcagttgg gacaagtaat gctcctagtc ctctgcgtga ctcaagtatt gatgatgagg 7380actacatggg ctctgtgtga ggctttaacc ttagctaccg ggcccatctc cacattgtgg 7440gaaggaaatc cagggaggtt ttggaacact accattgcgg tgtcaatggc taacattttt 7500agagggagtt acttggccgg agctggactt ctcttttcta ttatgaagaa cacaaccaac 7560acaagaaggg gaactggcaa cataggagag acgcttggag agaaatggaa aagccgattg 7620aacgcattgg gaaaaagtga attccagatc tacaagaaaa gtggaatcca ggaagtggat 7680agaaccttag caaaagaagg cattaaaaga ggagaaacgg accatcacgc tgtgtcgcga 7740ggctcagcaa aactgagatg gttcgttgag agaaacatgg tcacaccaga agggaaagta 7800gtggacctcg gttgtggcag aggaggctgg tcatactatt gtggaggact aaagaatgta 7860agagaagtca aaggcctaac aaaaggagga ccaggacacg aagaacccat ccccatgtca 7920acatatgggt ggaatctagt gcgtcttcaa agtggagttg acgttttctt catcccgcca 7980gaaaagtgtg acacattatt gtgtgacata ggggagtcat caccaaatcc cacagtggaa 8040gcaggacgaa cactcagagt ccttaactta gtagaaaatt ggttgaacaa caacactcaa 8100ttttgcataa aggttctcaa cccatatatg ccctcagtca tagaaaaaat ggaagcacta 8160caaaggaaat atggaggagc cttagtgagg aatccactct cacgaaactc cacacatgag 8220atgtactggg tatccaatgc ttccgggaac atagtgtcat cagtgaacat gatttcaagg 8280atgttgatca acagatttac aatgagatac aagaaagcca cttacgagcc ggatgttgac 8340ctcggaagcg gaacccgtaa catcgggatt gaaagtgaga taccaaacct agatataatt 8400gggaaaagaa tagaaaaaat aaagcaagag catgaaacat catggcacta tgaccaagac 8460cacccataca aaacgtgggc ataccatggt agctatgaaa caaaacagac tggatcagca 8520tcatccatgg tcaacggagt ggtcaggctg ctgacaaaac cttgggacgt tgtccccatg 8580gtgacacaga tggcaatgac agacacgact ccatttggac aacagcgcgt ttttaaagag 8640aaagtggaca cgagaaccca agaaccgaaa gaaggcacga agaaactaat gaaaataaca 8700gcagagtggc tttggaaaga attagggaag aaaaagacac ccaggatgtg caccagagaa 8760gaattcacaa gaaaggtgag aagcaatgca gccttggggg ccatattcac tgatgagaac 8820aagtggaagt cggcacgtga ggctgttgaa gatagtaggt tttgggagct ggttgacaag 8880gaaaggaatc tccatcttga aggaaagtgt gaaacatgtg tgtacaacat gatgggaaaa 8940agagagaaga agctagggga attcggcaag gcaaaaggca gcagagccat atggtacatg 9000tggcttggag cacgcttctt agagtttgaa gccctaggat tcttaaatga agatcactgg 9060ttctccagag agaactccct gagtggagtg gaaggagaag ggctgcacaa gctaggttac 9120attctaagag acgtgagcaa gaaagaggga ggagcaatgt atgccgatga caccgcagga 9180tgggatacaa aaatcacact agaagaccta aaaaatgaag agatggtaac aaaccacatg 9240gaaggagaac acaagaaact agccgaggcc attttcaaac taacgtacca aaacaaggtg 9300gtgcgtgtgc aaagaccaac accaagaggc acagtaatgg acatcatatc gagaagagac 9360caaagaggta gtggacaagt tggcacctat ggactcaata ctttcaccaa tatggaagcc 9420caactaatca gacagatgga gggagaagga gtctttaaaa gcattcagca cctaacaatc 9480acagaagaaa tcgctgtgca aaactggtta gcaagagtgg ggcgcgaaag gttatcaaga 9540atggccatca gtggagatga ttgtgttgtg aaacctttag atgacaggtt cgcaagcgct 9600ttaacagctc taaatgacat gggaaagatt aggaaagaca tacaacaatg ggaaccttca 9660agaggatgga atgattggac acaagtgccc ttctgttcac accatttcca tgagttaatc 9720atgaaagacg gtcgcgtact cgttgttcca tgtagaaacc aagatgaact gattggcaga 9780gcccgaatct cccaaggagc agggtggtct ttgcgggaga cggcctgttt ggggaagtct 9840tacgcccaaa tgtggagctt gatgtacttc cacagacgcg acctcaggct ggcggcaaat 9900gctatttgct cggcagtacc atcacattgg gttccaacaa gtcgaacaac ctggtccata 9960catgctaaac atgaatggat gacaacggaa gacatgctga cagtctggaa cagggtgtgg 10020attcaagaaa acccatggat ggaagacaaa actccagtgg aaacatggga ggaaatccca 10080tacttgggga aaagagaaga ccaatggtgc ggctcattga ttgggttaac aagcagggcc 10140acctgggcaa agaacatcca agcagcaata aatcaagtta gatcccttat aggcaatgaa 10200gaatacacag attacatgcc atccatgaaa agattcagaa gagaagagga agaagcagga 10260gttctgtggt agaaagcaaa actaacatga aacaaggcta gaagtcaggt cggattaagc 10320catagtacgg aaaaaactat gctacctgtg agccccgtcc aaggacgtta aaagaagtca 10380ggccatcata aatgccatag cttgagtaaa ctatgcagcc tgtagctcca cctgagaagg 10440tgtaaaaaat ccgggaggcc acaaaccatg gaagctgtac gcatggcgta gtggactagc 10500ggttagggga gacccctccc ttacaaatcg cagcaacaat gggggcccaa ggcgagatga 10560agctgtagtc tcgctggaag gactagaggt tagaggagac ccccccgaaa caaaaaacag 10620catattgacg ctgggaaaga ccagagatcc tgctgtctcc tcagcatcat tccaggcaca 10680gaacgccaga aaatggaatg gtgctgttga atcaacaggt tct 10723323DNAArtificialSynthetic 3gcacggatgt aacagactga aga 23418DNAArtificialSynthetic 4ccaggccgaa cctgtcat 18522DNAArtificialSynthetic 5cgactgtgtg gtccggccca tc 22620DNAArtificialSynthetic 6cattgcagtt ggcctggtaa 20723DNAArtificialSynthetic 7ctttggcaag agagagctca agt 23821DNAArtificialSynthetic 8ccgatcaagg atgcgccatc a 21920DNAArtificialSynthetic 9gtcggagtcg tgacgctgta 201021DNAArtificialSynthetic 10gttgatggcg catccttgat c 211121DNAArtificialSynthetic 11tgggagttat ggtgggcgcc g 211225DNAArtificialSynthetic 12aaaacacttc catgtcattt tcatg 251321DNAArtificialSynthetic 13gttgatggcg catccttgat c 211433DNAArtificialSynthetic 14tgcgatagga attatcacac tctatctggg agc 331524DNAArtificialSynthetic 15cttagtattg tggattggca cgaa 241621DNAArtificialSynthetic 16gcgccaactg tgaaacctag a 211729DNAArtificialSynthetic 17agaaacactt caatggcaat gacgtgcat 291819DNAArtificialSynthetic 18tcgcaacagc cttaacagc 191921DNAArtificialSynthetic 19actatctccc tcccatcctt c 212016DNAArtificialSynthetic 20ttcacaccac ttccac 162118DNAArtificialSynthetic 21aatgacagac acgactcc 182222DNAArtificialSynthetic 22cccaaaacct actatcttca ac 222317DNAArtificialSynthetic 23tggaagtcgg cacgtga 17
Patent applications by Bruno Guy, Lyon FR
Patent applications by Jean Lang, Mions FR
Patent applications by Rémi Forrat, Serezin Du Rhone FR
Patent applications by Véronique Barban, Craponne FR
Patent applications by SANOFI PASTEUR SA
Patent applications in class Togaviridae or Flaviviridae, except hepatitis C virus (e.g., yellow fever virus, bovine viral diarrhea virus, dengue virus, equine viral arteritis virus, equine encephalitis virus, Japanese B encephalitis virus, Sindbis virus, flavivirus, etc.)
Patent applications in all subclasses Togaviridae or Flaviviridae, except hepatitis C virus (e.g., yellow fever virus, bovine viral diarrhea virus, dengue virus, equine viral arteritis virus, equine encephalitis virus, Japanese B encephalitis virus, Sindbis virus, flavivirus, etc.)