TW201402143A - Compositions and methods for administration of vaccines against dengue virus - Google Patents

Abstract

Embodiments of the present invention report compositions and methods for vaccinating a subject against all dengue virus serotypes. In some embodiments, multiple vaccine compositions may be administered to a subject in different anatomical locations in order to induce a rapid response to all dengue virus serotypes. In certain embodiments, administration of two or more vaccine compositions to a subject against all dengue virus serotypes may include two or more routes of administration.

Description

Composition of anti-dengue virus vaccine and administration method priority

This application claims priority to a portion of the continuation application of U.S. Patent Application Serial No. 13/492,884, filed on June 10, 2012, which is filed on May 28, 2010. The benefit of the non-provisional patent application Serial No. 12/790, 511, the priority of which is hereby incorporated by reference in its entirety in its entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire entire all The entire disclosure of the prior application is hereby incorporated by reference in its entirety in its entirety in its entirety in its entirety herein in its entirety herein

Field of invention

A specific example of the present invention reports a vaccine composition against all dengue virus strains and a method of administering the vaccine to a single body. In certain embodiments, the vaccine composition can be administered by subcutaneous, intradermal, intramuscular or other injection or introduction methods. In certain embodiments, injecting an anti-dengue virus type vaccine in one body comprises multiple anatomical locations on day 0. Other specific examples include subsequent injections from the first treatment to the maximum of 12 months after the initial injection. In other specific examples, no additional injection is required other than treatment on Day 0. In some specific examples, on day 0, anti-subcutaneous, intradermal, intramuscular or other modes are used. The vaccine composition of dengue virus is introduced into a body to provide protection against three or more dengue serotypes DEN-1, DEN-2, DEN-3 and DEN-4 after administration.

Background of the invention

Vaccines have been used effectively to protect against viral infections to reduce the incidence of human disease. One of the most successful viral vaccine technologies is the use of attenuated or attenuated strains ("live attenuated viruses") to immunize animals or humans. This attenuated strain does not cause disease due to limited replication after immunization. However, this restricted viral replication is sufficient to exhibit the full integrity of the viral antigen and to produce an effective and long lasting immune response to the virus. Thus, after subsequent exposure to the pathogen of the virus, the immunized system is protected from the disease. These live attenuated virus vaccines are the most successful vaccines used in public health.

Summary of invention

Specific examples of the invention are generally directed to methods and compositions for inducing protection against multiple dengue viruses in a single body by, for example, administering a multivalent dengue vaccine to the individual. Some specific examples can include introducing a vaccine composition into a body via intradermal (ID) injection. According to these specific examples, the vaccine composition can introduce a body intradermally, for example, to elicit a neutralizing antibody against three or more dengue virus serotypes. In certain embodiments, the vaccine composition can include, but is not limited to, a single dose of a multivalent dengue serotype vaccine formulation that has a predetermined ratio when administered to a single body. In other examples, the vaccine composition may include but is not limited to the initial dose of dengue vaccine formulation (e.g. tetravalent formulations, such as DENVax ^TM), may then be a dosing body is added one or more times the same formulation or different Formulation.

In this context, other aspects may be directed to eliciting a monolithic or cellular immune response in a body by introducing a vaccine composition into a body, for example, via an intradermal route, wherein the vaccine composition includes, but is not limited to, a dengue fever Viral vaccine. According to these specific examples, the disclosed compositions can be administered intradermally to a subject to modulate the production of neutralizing antibodies against three or more dengue virus serotypes in the individual. Certain aspects relate to a plurality of dengue virus serotypes or fragments thereof or attenuated compositions thereof having a predetermined composition ratio in a single vaccine composition (eg, 1:1:1, 10:11:2:2, 1: 10;10:1,3:4:3:3, 1:4:1, 5:5:4:5; or consider any ratio of three or more serotypes, so that when administering to a body In the case of a single vaccine composition, the cross-protection and extent of neutralizing antibodies against at least three dengue virus serotypes are increased in the individual.

In certain embodiments, certain advantages of using an intradermally introduced anti-dengue virus vaccine can include, but are not limited to, multiple protection (cross protection) against one or all dengue virus serotypes in one body; The vaccine dose is reduced in cost, compared to subcutaneous injection; the antibody produced against some or all of the dengue virus serotypes is regulated in one body; and the composition of the drug is not reduced in the administration of the primary anti-dengue virus vaccine composition. The pain at the place.

In some embodiments, the single dose is resistant to dengue virus Seedlings may include one or more dengue virus serotypes. Moreover, certain embodiments are directed to treating a subject with at least one additional injection of a vaccine comprising multiple dengue viruses, wherein the additional injection is administered at a separate location from the first injection, eg, on the individual proximate to the initial In the injection or in a distant anatomical position. In addition, the at least one additional intradermal injection can be performed less than 30 days after the first administration to the individual; while the other is performed 30 days and up to 12 months after the first administration of the vaccine.

Other specific examples disclosed herein relate to methods and compositions for inducing protection against all dengue virus serotypes in a body by, for example, continuously for a short period of time, in one or more than one In the anatomical position, a vaccine of the primary antibody against all dengue virus serotypes is administered to one body using two or more agents. Some specific examples may be included in one site, via intradermal (ID), subcutaneous (SC) or intramuscular (IM) injection; and continuously in another anatomical location, by ID, SC, IM; or At the second different anatomical location, a vaccine composition is introduced into one body by other methods of introduction. Other specific examples include the introduction of a dengue virus vaccine of all dengue virus serotypes into a single body using any combination of modes of administration, wherein the administration of the vaccine occurs at two or more anatomical locations on the same day, or by two or more A variety of routes are administered continuously to the individual.

Some specific examples include the continuous treatment of individuals in need of dengue virus tetravalent vaccination at two or more anatomical locations. In certain embodiments, the individual may need to be administered twice in a single day to elicit a neutralizing antibody that is resistant to dengue infection to an appropriate extent. In other embodiments, a body can be administered continuously at two or more anatomical locations The fever virus is multivalent vaccinated, and then the subject can be administered at least one third vaccine having a composition comprising a dengue virus serotype within 30 days, such as about 7, about 14, about 21 or about 28 days. Where the composition may or may not have all serotypes. In other embodiments, one body may be continually administered a dengue virus tetravalent vaccination at two or more anatomical locations on day 0, and then, for example, within about 30 days, such as about 7, about 14, about 21 Or about 28 days later, the individual is administered at least one third vaccine comprising a composition comprising a dengue virus serotype, wherein the composition may or may not have all serotypes. The vaccine compositions disclosed herein and in other specific examples may comprise two or more dengue virus serotypes at a predetermined ratio for subsequent administration in addition to the initial double vaccination. These subsequent vaccinations may be based on the individualized price of the antibody after the double injection or other criteria such as the results of the test population. In certain embodiments, subsequent vaccination may comprise only a single dengue serotype (eg, DEN-4).

In certain embodiments, the composition of the introduced individual comprises an anti-denture virus serotype vaccine, such as a tetravalent DENVax ^(TM) or another similar formulation. DENVax ^TM comprises a predetermined ratio of tetravalent dengue vaccine, wherein the vaccine based on the attenuated constructed by the DEN-2 backbone of body composition (see, e.g., 16 February 2001, submitted PCT Application No. PCT / US01 / 05142 The entire disclosure is incorporated herein by reference for all purposes. In other compositions, all dengue vaccine serotypes are in equal proportions in the composition. In still other compositions, each dengue vaccine virus serotype can be at a particular ratio to each other such that introduction of the composition elicits a sufficient level of neutralizing antibody that will provide the individual with sufficient resistance to three or more dengue fevers Virus (eg DEN-1, DEN-2, DEN-3 and/or DEN-4) is protected by infection. For example, if a body receives two or more compositions continuously at two or more anatomical locations and the individual has lower protection against one or more particular dengue virus serotypes, then the individual is reinforced with the injection. Multiple (more than two) vaccine components or a single vaccine component can be included to improve the immune response to all four dengue viruses in an individual. According to these specific examples, samples from individuals can be analyzed for resistance to dengue infection using standard methods known in the art.

In certain embodiments, the vaccine composition can be introduced into a body in multiple anatomical locations by any route to, for example, protect against three or more dengue serotypes after continuous administration. In certain instances, the vaccine composition may include, but serotypes formulation (e.g., DENVax ^TM) is not limited to the single dose comprises all dengue virus, will to a physical provide protection against at least three dengue virus serotypes its administration protection. In other embodiments, the vaccine composition can include an attenuated dengue virus serotype and other anti-pathogenic compositions (eg, Japanese encephalitis, yellow fever, West Nile fever, influenza, Chikungunya or Other) combination. Compositions contemplated herein can be administered by any method known in the art including, but not limited to, intradermal, subcutaneous, intramuscular, intranasal, inhalation, vaginal, intravenous, ingestion, and any other method. Introduction in two or more anatomical locations may include any combination of administrations, including by two or more anatomical locations by the same pattern; or by two or more different modes, including two or more separate Anatomical position. According to these specific examples, the two or more anatomical locations can include different feet. In other embodiments, the vaccination can be delivered to an individual using any device known in the art including, but not limited to, needles and syringes, jet injection, microneedle injection, patch delivery (eg, skin), intradermal delivery devices. Inhalation devices, intranasal devices, slow release microparticles, and any other acceptable vaccine delivery device.

In certain embodiments, a dual-administered vaccine composition for a dengue virus vaccine can include a composition comprising more than one chimeric dengue virus in a single composition. In certain compositions, the chimeric architecture used in this composition consists of a dengue-dengue serotype, such as dengue-1, dengue-3, and/or dengue-4 on a dengue-2 backbone. According to these specific examples, the single vaccine composition can include a live attenuated dengue virus in which an immune response is elicited against at least three and up to all four dengue virus serotypes in the individual receiving the composition. Constructs contemplated herein include live attenuated dengue viruses, including one or more live attenuated dengue viruses and one or more dengue-dengue chimeric viruses, including the attenuated dengue in a single construct. The shell and non-structural proteins of the virus and the protomembrane proteins and envelope proteins of at least the second dengue virus. In certain embodiments, the capsid and non-structural protein are derived from attenuated dengue-1, dengue-2, dengue-3, or dengue-4 virus. In other embodiments, the at least a second type of dengue virus protomembrane protein and envelope protein are dengue-2, dengue-3 or dengue-4, when the attenuated dengue virus is dengue-1; or dengue- 1. Dengue-3 or dengue-4 when the attenuated dengue virus is dengue-2; or dengue-1, dengue-2 or dengue-4 when the attenuated dengue virus is dengue-3; or Dengue-1, dengue-2 or dengue-3, when the attenuated The fever virus is dengue fever-4. Furthermore, the dengue-dengue chimeric chimeric virus may comprise a shell and a non-structural protein of an attenuated dengue-2 virus, and the protomembrane protein and envelope protein are dengue-1, dengue-3 or dengue-4.

Other specific examples include the live attenuated virus of the framework of the live attenuated virus dengue-2. Furthermore, the dengue-2 can include any dengue-2 strain. In some live attenuated dengue-2 viruses, the dengue-2 comprises the PDK-53 strain. In another embodiment, the chimeric system-nucleic acid chimera comprises a first nucleotide sequence from which a non-structural protein from an attenuated dengue-2 virus is translated, and a second yellow fever virus is transduced The second nucleotide sequence of the structural protein. In another embodiment, the structural protein can be a C, prM or E protein of a yellow fever virus. Examples of yellow fever viruses from which structural proteins can be selected include, but are not limited to, dengue-1 virus, dengue-2 virus, dengue-3 virus, dengue-4 virus, West Nile fever virus, Japanese encephalitis virus, St. Louis ( St.Louis) Encephalitis virus, yellow fever virus and sputum encephalitis virus. In further embodiments, the structural protein can be selected from non-yellow fever virus species, such as hepatitis C virus, that are closely related to the yellow fever virus.

In certain embodiments, an amino acid substitution mutation in the non-structural protein and a nucleotide substitution mutation in the 5' non-coding region can be present. This nucleotide substitution mutation occurs in the stem of the stem-loop structure preserved in all four dengue serotypes. In particular, a single mutation at NS1-53, a double mutation at NS1-53 with at 5'NC-57, a double mutation at NS1-53 with at NS3-250, and at NS1-53 The triple mutation at 5'NC-57 and at NS3-250 provides the attenuated disclosure disclosed herein. DEN-2 virus.

The genome of any dengue-2 virus comprising non-conservative amino acid substitutions at these loci can be used as a backbone in the non-toxic chimeras described herein. Furthermore, other yellow fever virus genomes containing similar mutations at the same locus after amino acid sequence or nucleotide sequence alignment and stem structure analysis can also be used as the framework structure, and are defined herein. It is equal to the attenuating mutation of the dengue-2 PDK-53 genome. The backbone, the region of the chimera comprising the 5' and 3' non-coding regions, and the region from which the non-structural protein is translated may also include further mutations to maintain stability of the non-toxic phenotype and to reduce the avirulence virus or chimera Can return to the possibility of highly toxic wild-type virus. For example, a second mutation in the stem of the stem/loop structure in the 5' non-coding region provides additional stability if necessary.

In other embodiments, in addition to those specifically described herein, the chimeric virus can include nucleotide and amino acid substitutions, deletions or insertions in its structural and non-structural proteins. The structural and non-structural proteins disclosed herein are understood to include any protein, including any of the complete protein or the sequence from which the complete protein is translated, the epitope of the protein, or comprise, for example, two or more amino acids thereof. Any fragment of the residue. Specific examples disclosed herein provide a method for making a chimeric virus described in the specific examples herein, which employs recombinant techniques to insert the desired substitutions into the appropriate framework genome.

In other embodiments, the composition can include a pharmaceutically acceptable carrier and an attenuated chimeric virus, including other dengue viruses. Amino acid sequence of serotype, other yellow fever virus species or other closely related species such as hepatitis C virus. Proteins or polypeptides containing amino acid sequences from other dengue virus serotypes, other yellow fever virus species or other closely related species can act as immunogens and, therefore, are used to elicit other dengue virus serotypes, other yellow fever An immune response to a viral species or other closely related species.

In a specific example, the use of a nucleic acid chimera comprising a nucleotide sequence from an attenuated dengue-2 virus and a nucleotide sequence from a second dengue virus (or other yellow fever virus) has been considered. Dual administration at 0 days, wherein the nucleotide sequence from the second yellow fever virus directs the synthesis of the yellow fever virus antigen. In another aspect of the invention, a composition of a vaccine comprising three or more dengue virus serotypes has been considered.

In another aspect, the method for making an immunogenic or vaccine composition uses recombinant techniques that insert the desired substitution into the appropriate yellow fever virus genome. Another object of the present invention is to provide a composition and method for conferring immunity against three or more dengue virus serotypes, while using dual administration in different anatomical regions to elicit other lymph nodes in an individual receiving the regimen .

Another object of the invention is to provide nucleic acid probes and primers for use in some rapid genetic tests, wherein the test is for the diagnosis of each vaccine virus of the invention. This object of the invention may be embodied in a polymerase chain reaction assay, a hybridization assay, or other nucleic acid sequence detection techniques known in the art. A specific example includes a nucleic acid detection system using an automated PCR substrate.

In other embodiments, a plurality of mutations can be introduced into the chimeric dengue virus to further attenuate the chimeric virus or to improve immunogenicity. In certain embodiments, the composition can include a chimeric dengue virus capable of eliciting an immune response against all four dengue virus serotypes, wherein a single composition is introduced in two anatomical locations of one body. Some specific examples are for the short-term tour of the target population of dengue-endemic countries, such as tourists.

The following figures form part of this patent specification and are included to further clarify certain specific examples. Some specific examples may be better understood by reference to one or more of these figures, or in combination with the detailed description of the specific embodiments presented.

Figure 1 shows an embodiment of a currently available intradermal injection device.

Figure 2 shows an example of an injection site in a non-human primate individual in which the individual has introduced an anti-dengue virus vaccine intradermally.

Figure 3 shows a long bar graph comparing the neutralization of dengue virus serotypes against different ratios after one (primary) administration of an anti-dengue virus vaccine via subcutaneous (SC) intradermal (ID) route. Antibody price.

Figure 4 shows a long bar graph comparing the neutralizing antibody titers against different dengue virus serotypes after a second additional dose of subcutaneous (ID) intradermal (ID) injection of anti-dengue virus vaccine. .

Figure 5 shows a bar graph of neutralizing antibody titers after subcutaneous and intradermal immunization with anti-dengue virus vaccine serotype-4 in mice.

Figures 6A and 6B show the inoculation of dengue vaccine followed by wild Schematic description of surviving mice after challenge with dengue virus. Mice are vaccinated with a dengue vaccine (eg, DENVax-4) or a buffer/placebo (eg, TFA) by the SC or ID route.

Figure 7 shows twice on day 0; or once on day 0 and once on day 42 (for example, DENVaxTM; 4:3:4:5 ratio), on days 28 and 56, Neutralizing antibody titers against DEN-1, DEN-2, DEN-3, and DEN-4.

Figure 8 shows twice on day 0; or once on day 0 and once on day 42 (eg DENVaxTM; 3:3:3:3, using approximately equal amounts), on day 28 and 56 At the time of the day, the antibody titers of DEN-1, DEN-2, DEN-3 and DEN-4 were neutralized.

Figures 9A-9D show a graph comparing neutralizing antibody titers in non-human primates after immunization with a tetravalent dengue virus vaccine SC. The two groups were vaccinated via a subcutaneous route with a needle-free device, which was used twice (0, 0) on the same day, or once on day 0 and once again on day 60 (0, 60).

Figures 10A-10B show data obtained from human clinical trials. For seropositive humans (at the beginning of the trial, humans stated that there are some antibodies to the dengue virus serotype that are somewhat devoid of antibodies), two doses (days 0 and 90) of four-valent dengue vaccine serotype formulations are provided subcutaneously or intradermally. . The extent of antibodies against each dengue serotype was analyzed on days 0, 30, 60, 90 and 120.

Figures 11A-11D show a graph comparing neutralizing antibody titers in non-human primates after subcutaneous immunization with a tetravalent serotype dengue vaccine. The second group is vaccinated twice (0,0) on the same day, or Once on day 0 and again on day 60 (0,60). Analysis of the presence of antibodies in serum at days 0, 28, 58, 73 and 90, and detection of antibodies against all four dengue serotypes (DEN-1, DEN-2, DEN-3, DEN-4) .

Figure 12 shows a single pair of dual-dose dengue virus vaccine analysis. This data shows changes in the degree of transcription of multiple genes in individuals exposed to this composition using certain protocols disclosed herein.

definition

As used herein, "a" or "an" can mean one or more than one.

As used herein, a container can include, but is not limited to, a test tube, a mini or micro-fuge tube, a tube, a vial, a microtiter plate, or a container.

As used in this patent specification, "individual" may include, but is not limited to, mammals such as humans or domesticated or wild mammals, such as dogs, cats, other domestic pets (eg, hamsters, guinea pigs, mice, rats), Snow caps, rabbits, pigs, horses, cattle, prairie dogs or zoo animals.

As used herein, "about" or "about" may mean plus or minus ten percent.

As used herein, "attenuated virus" may mean a virus that clarifies clinical signs of reduction or absence of disease when administered to an individual, such as a mammal (eg, a human or an animal).

As used herein, "continuously" may mean a short-lived approach, usually a single patient visit and within 24 hours.

As used herein, "administering" may mean delivering a vaccine or treatment to a respective animal or human by any of a number of methods, such as intradermal, subcutaneous, intramuscular, intranasal, inhalation, vaginal, Intravenous, orally, buccally, by inhalation, intranasally, or any other known in the art.

Detailed description of the preferred embodiment

In the following sections, various typical compositions and methods are described to illustrate a number of specific examples. It will be apparent to those skilled in the art that the practice of the plurality of specific examples does not require the use of all or some of the details outlined herein, but the concentration, time, and other details may be modified by routine experimentation. In some instances, well-known methods or components have not been included in the description.

Certain aspects of the invention include, but are not limited to, vaccine compositions that are administered against dengue virus.

Specific examples of the invention generally relate to methods and compositions for eliciting protective neutralizing antibodies against three or more dengue virus serotypes in an individual. Other specific examples can include introducing a vaccine composition into a body via any method known in the art including, but not limited to, intradermal, subcutaneous, intramuscular, intranasal, inhalation, oral, intranasal, vaginal, intravenous, Ingestion and any other method wherein the vaccine composition so introduced elicits neutralizing antibodies against three or more dengue virus serotypes. In certain embodiments, the vaccine composition comprises a vaccine administered to a single body against three or more dengue virus serotypes. In other embodiments, the vaccine composition comprises an initial dose of all four dengue serotypes, and then one or more other vaccine compositions are administered to a single body.

Other aspects of the invention include modulating intradermal administration to a The immune response of the body against dengue virus vaccine is compared with subcutaneous. The anti-dengue virus vaccine may comprise a composition comprising a live attenuated dengue vaccine virus, a recombinant dengue vaccine virus, a chimeric virus or a mutant thereof, all of which are in a predetermined ratio. The ratio of multiple dengue serotypes may be equal or nearly equal in performance; or the concentration of certain serotypes may appear to be higher than others, depending on the need or ability to initiate a balanced neutralizing antibody response in the individual. According to these specific examples, different dengue vaccine ratios can vary from 2 to 100,000 times (eg, spot forming units) between any two serotypes. This may depend, for example, on the number of serotypes exhibited in the formulation, the intended response, and the desired effect. Any dengue vaccine serotype formulation can be used to produce a vaccine (eg, an attenuated virus, etc.) that is administered continuously to the individual in need thereof, including but not limited to three or more A dengue virus serotype.

In other embodiments, the composition of the dengue virus vaccine formulation can be introduced to the individual before, during, or after exposure of the body to the dengue virus. According to these specific examples, one body may receive more than one continuous administration, or more than one administration may include a dengue virus formulation, selectivity, followed by one or more additional administrations at a later time. Intradermal, subcutaneous, intramuscular, intranasal, inhalation, vaginal, intravenous, oral, and any other method of application described herein can be combined with any other antiviral treatment. In certain embodiments, the intradermal, subcutaneous, intramuscular introduction of a formulation contemplated herein can be administered to any suitable region of the subject's body (eg, arms, shoulders, hips, nose, etc.). In addition, parenteral administration of the vaccine formulation can be continued in combination with other modes of administration, such as nasal Internal, pulmonary, oral, buccal or vaginal. In certain embodiments, it is contemplated that after continuous administration as described herein, the primary or booster administration can occur continuously on the same day, consecutive days, weekly, monthly, bimonthly, or other suitable treatment regimen.

Dengue fever is in Asia; Central and South America, including Colombia, Caribbean; Pacific Island; and parts of Africa and Australia. An estimated 3.6 billion people (55% of the world's population) live in areas where dengue virus infection (DVI) is at risk. Infection with dengue virus can produce a range of symptoms, from symptoms of insensitivity to debilitating but transient dengue to life-stressed hemorrhagic dengue fever (DHF) or dengue shock syndrome (DSS). There is currently no therapeutic or prophylactic vaccine for dengue fever. Given the impact of dengue on people in endemic countries and on travellers to those areas, there is a need to prevent dengue fever.

Dengue fever is a mosquito-borne viral disease that is mainly transmitted by mosquitoes and Aedes aegypti. The dengue virus (DEN) includes a single-strand, sense RNA genome of approximately 11 kb. The genome consists of three structural proteins, a shell (C), a promembrane protein (prM) and an envelope (E); and seven non-structural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. There are four different dengue virus serotypes, DEN-1, DEN-2, DEN-3 and DEN-4. The major infection of the provided serotypes triggers lifetime serotype specific immunity. However, there is no long-term cross-protective immunity against the other three dengue virus serotypes, and subsequent infection with another serotype leads to an increased chance of a more serious disease such as DHF or DSS.

Due to the increased disease associated with secondary DENV infection, need to be stimulated A multivalent (eg, tetravalent) vaccine that is resistant to immunity to more than one and up to all four serotypes of DENV. Several DENV vaccine candidates that have been attenuated by standard serial passages in cell culture have proven to be unsafe or have poor immunity. Chimeric, live, attenuated recombinant DENV vaccine candidates are known in the art, including viruses based on the attenuated gene background of the yellow fever 17D (YF-17D) vaccine virus, DENV-2 PDK-53 vaccine virus Or DENV-4 containing a 30-nucleotide 3' non-coding region (NCR) deletion.

In the development of an effective live attenuated dengue virus (DENV) vaccine, the challenge is the interference between the four dengue vaccine viruses when administered as a tetravalent formulation. Interference is revealed when one or more components of the multivalent mixture will elicit a lower immune response than those elicited by each of the individual monovalent vaccines. Vaccines with multiple pathogenic serotypes for disease, such as polio, dengue or other disturbances have been observed. To some extent, due to this interference, it has previously been found that a three-dose oral polio vaccine is required to elicit a sufficient immune response against the three key serotypes. Historical studies of live attenuated tetravalent dengue vaccines have shown that when administered in the context of multivalent formulations containing other serotypes, the DENV serotype that elicits the strongest neutralizing antibody response when administered alone tends to dominate immune response. As for the examples, a tetravalent mixture of four different live attenuated dengue vaccines showed a dominating response to the DEN-3 component and a reduced immune response to DEN-1, -2 and -4 (see, for example, Sabchareon et al. , 2002; Kitchener et al., 2006). Due to this dominance, the clinical development of the tetravalent mixture is suspended. Similarly, interference with recombinant, live attenuated viruses has been seen. Interference in the tetravalent mixture of dengue/yellow fever chimeras has been documented (Guy et al., 2009. Evaluation of Interferences between Dengue Vaccine Serotypes in a Monkey Model. Am. J. Trop Med. Hyg. 80:3012-311). In these studies, it has been found that two serotypes dominate the reaction in the tetravalent formulation of the ChimeriVax vaccine strain. The interference can be overcome by administering the two bivalent vaccine formulations sequentially in separate anatomical locations or in time, or by administering a tetravalent formulation a third time after one year. Similarly, an improved multivalent reaction of a tetravalent recombinant vaccine strain has been elucidated (in this case, a formulation comprising DENV or chimeric DENV and a deletion in the 3' non-coding region) is only possible in the first and the first The second administration was extended for a period of four months. (Blaney et al., 2005. Recombinant, live attenuated tetravalent dengue virus vaccine formulations that elicit a balanced, broad and protective neutralizing antibody response against each of the four serotypes in rhesus monkeys (Recombinant, Live- Attenuated Tetravalent Dengue Virus Vaccine Formulations Induce a Balanced, Broad, and Protective Neutralizing Antibody Response against Each of the Four Serotypes in Rhesus Monkeys). J. Virology 79: 5516-5528).

Successful vaccination often requires vaccine delivery to closely mimic natural infections. To date, all clinical trials of dengue vaccine candidates have used the SC route, using needles and syringes. The natural pathway of dengue infection is transmitted through the dermis via mosquitoes. The skin system has been thought to act as an immunologically active organ that infects the immune barrier. Highly dense reticulated specific antigen presenting cells (APCs, such as Langerhan's cells and dendritic cells) are present in the epidermis and provide protection through efficient absorption and presentation of antigen to local lymph nodes. Stay Main against infectious pathogens. These APCs subgroups, together with both resident macrophages, have shown a natural target for dengue virus infection. In view of the fact that the epidermis is rich in immunocompetent cells, it has been considered herein that the use of the intradermal route for dengue virus vaccine delivery will favor a more potent and balanced immune response to all four dengue virus serotypes. In particular, an increase in the number of native host cells that exhibit viral replication in the skin can reduce interference and permit less dominant viral replication in tetravalent formulations. In some embodiments, intradermal immunization with a multivalent, live attenuated dengue vaccine can elicit a more balanced immune response to dengue virus exposure in one body.

Some specific examples disclosed herein regarding DENVax ^TM. DENVax ^TM dengue vaccine based one kind, which is (DEN-1, DEN-2 , DEN-3 and DEN-4) produced a mixture of four strains of recombinant dengue virus immunoreactive composition of the four dengue serotypes designed. It is not intended to be limited by any particular restriction tetravalent formulations, DENVax ^TM, dengue serotype 2 vaccine components (DENVax-2) with an attenuated DEN-2 PDK-53 strains appropriate. This construct has been investigated in many clinical studies. Other dengue vaccine strains (DENVax-1, DENVax-3, and DENVax-4) are selected from the DEN-1, DEN-3, or DEN-4 structural membranes of the DEN-2 PDK-53 non-structural gene backbone. A chimera composed of a protein (prM) and an envelope (E) protein gene. These recombinant viruses exhibit surface antigens of DEN-1, DEN-3 or DEN-4, and retain the genetic alterations that are responsible for the attenuation of the DEN-2 PDK-53 strain. In certain embodiments, DENVax ^(TM) can be used as an example of a multivalent live attenuated dengue vaccine having all four dengue virus serotypes exhibited in a variety of ratios in a vaccine composition. Other specific examples relate to optimizing tetravalent vaccine administration. More specific examples of other immunization method based on DENVax ^TM.

During the exploratory process of intradermal delivery of multivalent dengue vaccines, it has been found that more than one dose of multivalent vaccine in at least two separate anatomical locations triggers a neutralizing antibody response that is approximately equal or superior over time. Separate to dose multiple doses. Furthermore, it has been found that the benefits of multiple site administration are independent of the immune pathway.

The results of this study were unexpected. Information relating to multiple subcutaneous administration of depleted, attenuated and/or recombinant virus-based tetravalent vaccines has been previously disclosed. It has been reported that the second administration of the quaternary administration 30 days after the first administration failed to achieve an increase in the neutralizing antibody titer. In comparison, the second administration of the first 120 days after the first administration improved the neutralizing antibody titer for all four dengue serotypes. Similar information is reported in clinical trials of the yellow fever/dengue recombinant vaccine (Poo et al., 2011 Ped. Inf. Dis J. 30: 1-9). It is also recommended to produce a secondary antibody response against multiple dengue viruses during the three-month period between doses. (Capeding et al, 2011 Vaccine 29: 3863-3872). These reports suggesting that two clinical studies require longer intervals, such as 6-9 months, to produce a better multivalent immune response. Recently, in earlier human immunostimulatory studies, wild-type dengue viruses have been reported to elicit a wide range of cross-reactive antibodies that last up to 6 months after the initial infection. These data support the notion that short immunization regimens are suboptimal for live attenuated vaccines: previously observed transient cross-reactive antibodies will effectively neutralize any live attenuated vaccine components in multivalent formulations. Until the present disclosure, use a multivalent live attenuated vaccine at more than one anatomical location in a short interval The immunization protocol has not been considered as a viable treatment option for individuals who require this treatment. In this regard, multi-site administration permits an immune response to less-dominant components of a multivalent live attenuated vaccine by accessing a larger amount of antigen presenting cells and/or more than one draining lymph node, and effectively reducing Vaccine interference.

In certain embodiments, the composition of the individual is introduced comprising a vaccine against all dengue virus serotypes (DEN-1, DEN-2, DEN-3, DEN-4). In other embodiments, the compositions contemplated herein may include DENVax ^(TM) or other similar formulations. In certain compositions, vaccine viruses against all dengue serotypes are in equal proportions in the composition. In still other compositions, each dengue vaccine virus serotype can be in a particular ratio to each other, such introduction of the composition provides that the individual is sufficiently resistant to all dengue viruses (eg, DEN-1, DEN-2, DEN-3, The degree of neutralizing antibody of DEN-4).

In certain embodiments, a vaccine composition for dual administration of a dengue virus vaccine can include a composition comprising more than one chimeric dengue virus in a single composition. In certain compositions, the chimeric constructs used in this composition consist of dengue-dengue serotypes, such as dengue-1, dengue-3, and/or dengue-4 on the dengue-2 backbone. According to these specific examples, a single vaccine composition can include a live attenuated dengue virus that elicits an immune response in at least three and up to all four dengue virus serotypes in an individual receiving the composition. Constructs contemplated herein include live attenuated dengue viruses, including one or more live attenuated dengue viruses and one or more dengue-dengue chimeric viruses, and more A shell and non-structural protein of a dengue virus attenuated in a single construct and an original membrane protein and an envelope protein of at least a second dengue virus. In certain embodiments, the capsid and non-structural protein are from attenuated dengue-1, dengue-2, dengue-3 or dengue-4 virus. In another specific example, when the attenuated dengue virus is dengue-1, the at least a second dengue virus has a membrane protein and an envelope protein of dengue-2, dengue-3 or dengue-4; or When the attenuated dengue virus is dengue-2, it is dengue-1, dengue-3 or dengue-4; or when the attenuated dengue virus is dengue-3, it is dengue-1, dengue- 2 or dengue-4; or when the attenuated dengue virus is dengue-4, it is dengue-1, dengue-2 or dengue-3. Furthermore, the dengue-dengue chimeric chimeric virus may comprise a shell and a non-structural protein of an attenuated dengue-2 virus, and the protomembrane protein and envelope protein are dengue-1, dengue-3 or dengue-4.

Other specific examples include live attenuated viruses in which the skeleton of the live attenuated virus is dengue-2. Furthermore, the dengue-2 can include any dengue-2 strain. In some live attenuated dengue-2 viruses, the dengue-2 comprises the PDK-53 strain. In another embodiment, the chimeric system is a nucleic acid chimera comprising a first nucleotide sequence that is translated from a non-structural protein of an attenuated dengue-2 virus, and a second yellow fever virus The second nucleotide sequence of the structural protein. In another embodiment, the structural protein can be a C, prM or E protein of a yellow fever virus. Examples of yellow fever viruses from which structural proteins can be selected include, but are not limited to, dengue-1 virus, dengue-2 virus, dengue-3 virus, dengue-4 virus, West Nile fever virus, Japanese encephalitis virus, St. Louis encephalitis Virus, yellow fever virus and tick-borne encephalitis virus. In further embodiments, the structural protein can be selected from non-yellow fever virus species, such as hepatitis C virus, that are closely related to the yellow fever virus.

In certain embodiments, an amino acid substitution mutation in the non-structural protein and a nucleotide substitution mutation in the 5' non-coding region can be present. This nucleotide substitution mutation occurs in the stem of the stem-loop structure preserved in all four dengue serotypes. In particular, a single mutation at NS1-53, a double mutation at NS1-53 and at 5'NC-57, a double mutation at NS1-53 with at NS3-250, and at NS1-53 The triple mutation at 5'NC-57 and at NS3-250 provides the attenuated DEN-2 virus disclosed herein.

The genome of any dengue-2 virus containing non-conservative amino acid substitutions at these loci can be used as a backbone in the non-toxic chimeras described herein. Furthermore, after the amino acid sequence or nucleotide sequence alignment and stem structure analysis, other yellow fever virus genomes containing similar mutations at the same locus can also be used as the framework structure, and are defined herein as It is equal to the attenuated mutation of the dengue-2 PDK-53 genome. The backbone, the region of the chimera comprising the 5' and 3' non-coding regions and the region from which the non-structural protein is translated may also include further mutations to maintain the stability of the non-toxic phenotype and to reduce the avirulence virus or chimera. The possibility of returning to a deadly wild-type virus. For example, a second mutation in the 5' non-coding region in the stem of the stem/loop structure can provide additional stability if necessary.

In other specific examples, except those specifically described herein In addition, the chimeric virus may include nucleotide and amino acid substitutions, deletions or insertions in its structural and non-structural proteins. The structural and non-structural proteins disclosed herein are understood to include any protein, including the complete protein or any gene whose sequence is translated, the epitope of the protein, or comprise, for example, two or more amino acid residues thereof. Any fragmentation. Specific examples disclosed herein provide a method for making a chimeric virus described in the specific examples herein by using recombinant techniques to insert the desired substitutions into the appropriate framework genome.

In other embodiments, the composition can include a pharmaceutically acceptable carrier and an attenuated chimeric virus, including from other dengue virus serotypes, other yellow fever virus species, or other closely related species, such as hepatitis C virus. Amino acid sequence. Proteins or polypeptides containing amino acid sequences from other dengue virus serotypes, other yellow fever virus species or other closely related species can act as immunogens and, therefore, are used to elicit other dengue virus serotypes, other yellow fever An immune response to a viral species or other closely related species.

In one embodiment, in view of the use of a nucleic acid chimera comprising a nucleotide sequence from an attenuated dengue-2 virus and a nucleotide sequence from a second dengue virus (or other yellow fever virus), at 0th Double administration in days, wherein the nucleotide sequence from the second yellow fever virus directs the synthesis of the yellow fever virus antigen. In another aspect of the invention, a vaccine composition comprising three or more dengue virus serotypes is contemplated.

In another aspect, a method of using recombinant techniques to make an immunogenic or vaccine composition by inserting the desired substitution When in the yellow fever virus genome. Another object of the present invention is to provide a composition and method for conferring immunity against three or more dengue virus serotypes, which simultaneously use dual administration in different anatomical regions to elicit an individual receiving the regimen Other lymph nodes.

Another object of the present invention is to provide nucleic acid probes and primers for use in some rapid genetic tests, wherein the test is used in the diagnosis of each of the vaccine viruses of the present invention. This object of the invention may be embodied in a polymerase chain reaction assay, a hybridization assay, or other nucleic acid sequence detection techniques known in the art. A specific example includes a nucleic acid detection system using an automated PCR substrate.

In other embodiments, a plurality of mutations can be introduced into the chimeric dengue virus to further attenuate the chimeric virus or to improve immunogenicity. In certain embodiments, the composition can include a chimeric dengue virus that elicits an immune response to all four dengue virus serotypes, wherein a single composition is introduced in two anatomical locations of one body. Some specific examples are about short-term visits to the target population of dengue-endemic countries, such as tourists.

Certain specific examples disclosed herein relate to a method and composition for rapidly eliciting protection against all dengue virus serotypes in an individual by administering the vaccine continuously in more than one anatomical location, for example, on the same day. To one body against all dengue virus serotypes. Some specific examples may include introducing a vaccine composition into a body at an anatomical location via intradermal (ID) or subcutaneous (SC) injection or other mode of administration; then, by ID, SC or other mode of administration, At least another second vaccine composition is introduced at another anatomical location. Some specific examples include the use of any mode of administration a combination of dengue virus vaccines that incorporate all dengue virus serotypes into a body, wherein the administration of the vaccine to the system occurs at two or more anatomical locations on day 0, or by two or more different way. Some specific examples include the use of the same mode of administration but at different anatomical locations.

Certain dengue virus vaccine compositions described herein have a dosage range of 10 ² to 5 x 10 ⁶ PFU per serotype in the composition. Other compositions contemplated herein (e.g., subsequent vaccination) include compositions having a dose less than or greater than the range after primary immunization, based on the immune response in the individual. In some embodiments, the ratio of multiple dengue vaccine virus serotypes can vary depending on individual needs and immune response.

In certain embodiments, the compositions contemplated herein at the time of the first vaccination or in any subsequent vaccination can include a tetravalent dengue virus composition. According to these specific examples, the composition can include DENVax ^(TM) or other similar tetravalent formulations at equal or equivalent ratios or at predetermined serotype ratios. Other specific examples may include different formulations (e.g., serotype ratios) for each vaccine composition administered upon primary vaccination or any subsequent vaccination (e.g., less than 30 days later).

Some specific examples herein include treating an individual in need of such a vaccine at two or more anatomical locations on day 0, such as after about 7, about 14, about 21, or about 28 days within 30 days. At least a second vaccine is administered with a composition comprising a dengue virus serotype that may or may not have all serotypes. In some embodiments, all dengue virus serotypes are present in the vaccine formulation for each inoculation. For subsequent administration, the vaccine composition disclosed for subsequent administration herein may comprise a predetermined ratio of two or more A variety of dengue virus serotypes.

In some embodiments, the composition of the individual introduced comprises all dengue virus serotypes. In certain embodiments, the vaccine composition comprises a plurality of formulations of DENVax ^(TM) or other similar formulations. In certain vaccine compositions, the ratio of DEN-1:DEN-2:DEN-3:DEN-4 can be 3:3:3:3, 4:3:4:5, 5:4:5:5 , 5:4:5:5, 5:5:5:5, 5:5:5; 10, 10:1:10:100 or other ratios, in a single composition, between the two serotypes ratio may be from about 2 to about 100,000-fold difference _{(e.g., DENVax 4: 3: 4:} 5 TM , etc.). In certain embodiments, a dengue serotype ratio can be DEN-1 at 2x10 ⁴ : DEN-2 at 5x10 ⁴ : DEN-3 at 1x10 ⁵ : DEN-4 at 3x10 ⁵ PFUs; or DEN- 1 at 8x10 ³ : DEN-2 at 5x10 ³ : DEN-3 at 1x10 ⁴ : DEN-4 at 2x10 ⁵ PFUs. In some compositions, all dengue vaccine serotypes are in equal proportions in the composition. In still other compositions, each dengue vaccine virus serotype may be in a particular ratio to another serotype, such that introduction of the composition provides the individual with sufficient or more than sufficient neutralizing antibody level to confer protection against all dengue fever Viruses (eg, dengue 1, 2, 3, and 4). For example, if on day 0, after two or more consecutive vaccinations at two or more anatomical locations, the individual has lower protection against one or more particular dengue virus serotypes, then The booster injection of the individual can include increasing the concentration of the one or more dengue vaccine virus serotypes (which clarify lower neutralizing antibodies) to provide better protection against all dengue virus patterns. According to these specific examples, immunological responses to dengue serotype infections (e.g., dengue-1, -2, -3, -4) can be analyzed for samples from one body using standard methods known in the art.

In certain embodiments, the vaccine composition can be introduced into a body simultaneously or continuously in multiple anatomical locations to, for example, protect against all dengue serotypes (eg, cross-protection). In certain instances, the vaccine composition may include, but are not limited to all serotypes of vaccine virus Dengue single formulation (e.g., DENVax ^TM), which is administered to a regeneration to provide maximum protection against all dengue serotype virus . In other embodiments, a vaccine composition can include a combination of an attenuated dengue virus serotype and other anti-pathogenic components (eg, Japanese encephalitis, West Nile fever, influenza, etc.). Compositions contemplated herein can be administered by any method known in the art including, but not limited to, intradermal, subcutaneous, intramuscular, intranasal, inhalation, vaginal, intravenous, ingested, and any other method. Introduction in two or more anatomical locations can include any combination of administrations, including by the same pattern in two or more anatomical locations; or by two different modes, including two separate anatomical locations. According to these specific examples, the two or more anatomical locations can include different feet.

For example, if a subject receives two or more consecutive vaccinations at two or more anatomical locations on day 0, and that the individual does not elicit differential neutralizing antibodies against one or more particular dengue virus serotypes To the extent that the individual is vaccinated, the concentration of the one or more dengue vaccine virus serotypes (which clarify a lower degree of neutralizing antibody) may be increased to provide complete protection against infection by all dengue virus patterns. According to these specific examples, the resistance of a sample from one body to dengue infection can be analyzed using standard methods known in the art.

In certain embodiments, the dose of the vaccine composition can be introduced continuously into a single body at multiple anatomical locations on day 0, for example to protect against all dengue serotypes (eg, cross-protection). In certain instances, the vaccine compositions can include, but is not limited to three or four dengue single composition (e.g., DENVax ^TM) virus serotype, which was administered to a neutralizing antibody to initiate regeneration will provide maximum protection To the extent of infection with all dengue virus serotypes. Thus, a particular individual may need to receive only a clinical visit once and receive adequate protection to tour or remain in the area with the dengue virus for a predetermined period of time (eg, 30 days). In other embodiments, the vaccine composition can include an attenuated dengue virus serotype and against other pathogens (eg, yellow fever virus, such as Japanese encephalitis, West Nile fever; or other viruses, such as influenza, etc.) A combination of vaccine compositions. Compositions contemplated herein can be administered by any method known in the art including, but not limited to, intradermal, subcutaneous, intramuscular, intranasal, inhalation, vaginal, intravenous, ingested, and any other method. Introduction in two or more anatomical locations may include any combination of administrations, including in two or more anatomical locations by the same pattern; or by two or more different modes, including two or more separate Anatomical location. According to these specific examples, the two or more anatomical locations can include different feet, different tissues, intranasal, such as drops (eg, for the eyes), intramuscularly in two or more locations.

In certain embodiments, a vaccine composition disclosed herein can be a chimeric construct, which can include a mixture of constructs that comprise at least three dengue serotypes in a vaccine composition for administration to a subject. . In other embodiments, the dengue virus vaccine can include attenuated yellow A construct of a fever virus skeleton having a plurality of dengue serotype substitutions showing each of four serotypes, wherein the constructs can be mixed in a composition for administration as a vaccine.

Chimeras contemplated and described herein can be substituted for a corresponding structural gene and replaced with a desired structural gene by using recombinant engineering techniques well known to those skilled in the art, and one or more yellow fevers The structural protein gene of the virus is spliced into a dengue virus genomic backbone (e.g., PDK-53) or an equivalent thereof as described above, wherein immunity against the yellow fever virus is desired. Furthermore, using the sequences provided in the sequence listing, known nucleic acid synthesis techniques can be used to synthesize the nucleic acid molecules from which the yellow fever virus protein is translated and into the appropriate vector. Thus, non-toxic, immunological viruses are produced using recombinant engineering techniques known to those skilled in the art.

As mentioned above, the gene to be embedded in the backbone translates a yellow fever virus (eg, other dengue virus serotype) structural proteins. Preferably, the yellow fever virus gene line to be inserted is a gene for the C protein, the PrM protein and/or the E protein. The sequence embedded in the dengue-2 backbone can be used to decipher both PrM and E structural proteins. The C, prM and E structural proteins can be deduced from the sequence embedded in the dengue-2 backbone. The dengue virus skeleton is the PDK-53 dengue-2 virus genome, and the gene (DEN-2/1) including the junction of the C, prM and/or E structural proteins from which dengue-1 is translated, and the dengue fever is translated. a gene for the binding of -3 PrM and/or E structural protein (DEN-2/3), or a gene for the binding of the PrM and/or E structural protein of dengue-4 (DEN-2/4) . In a specific example, the translation of the dengue The conjugated gene of the structural protein of the heat-3 virus directs the synthesis of the E protein comprising leucine at the amino acid position 345.

In another embodiment, the chimera of the C structural protein from which the dengue-2 virus is translated directs the synthesis of a C protein comprising a serine at position 100 of the amino acid, and comprises a translation of the dengue - A gene for the binding of a structural protein of 4, which directs the synthesis of an E protein comprising leucine at the amino acid position 447.

In still other embodiments, a chimera can interpret the C structural protein of the dengue-2 virus and direct the synthesis of a C protein comprising a serine at the amino acid position 100, and include a dengue A gene for the binding of a structural protein of -4, which directs the synthesis of lysine comprising an amino acid at position 447 of amino acid and E protein comprising valerine at amino acid position 364. The structural proteins described herein can be present in the viral chimeras of the invention, such as any combination of only yellow fever virus structural proteins or yellow fever virus structural proteins.

This chimera can be manipulated by recombination of whole genome length cDNA colonies from both DEN-2 16681 wild type virus and either PDK-53 dengue-2 virus variant. The unselected PDK-53 vaccine includes a mixture of two genotype variants, designated PDK53-E and PDK53-V herein. The PDK53-V variant includes all nine PDK-53 vaccine-specific nucleotide mutations, including the Glu to Val mutation at the amino acid position NS3-250. The PDK53-E variant includes eight of the nine mutations of the PDK-53 vaccine and the NS3-250-Glu of the parental 16681 virus. The infectious cDNA colony constructs the variants of the two, and the virus lines from the two colonies are reduced in mice. poison. The attenuated phenotype markers of the DEN-2 PDK-53 virus include small spot size, temperature sensitivity (especially in LLC-MK.sub.2 cells), limited replication (especially in C6/36 cells), Attenuation of neonatal mice (especially, loss of neurotoxicity in unweaned mice) and reduction in the incidence of viremia in monkeys. A chimeric system useful as a candidate vaccine is constructed in the genetic background of two DEN-2 PDK-53 variants, all including mutations in the non-structural regions of the genome, including 5 in the 5' non-coding region. 'NC-57 C to T (16681-to-PDK-53); and mutations in the amino acid sequence of the non-structural protein, such as, for example, NS1-53 Gly-to-Asp and NS3-250 Glu to Val .

Can be assessed by screening of the aforementioned attenuated phenotypic markers for non-toxicity and by immunogenicity screening, including the translation of nucleotide sequences of structural proteins of other yellow fever virus or dengue virus serotypes. A suitable chimeric virus or nucleic acid chimera is useful as a vaccine. Antigenicity and immunogenicity can be assessed using in vitro or in vivo reactivity with yellow fever virus antibodies or immunoreactive serum using routine screening procedures known to those skilled in the art.

Yellow fever virus vaccine

In certain embodiments, chimeric viruses and nucleic acid chimeras provide live attenuated viruses useful as immunogens or vaccines. These chimeras are highly immunogenic while producing a non-hazardous pathogen or lethal effect.

Effective vaccination against all dengue virus strain vaccines has been difficult. In order to prevent DHF/DSS from occurring in individuals vaccinated against only one dengue virus serotype vaccine, trivalent or tetravalent dengue virus is required. The vaccine is rapidly immunized to provide immunity to all four serotypes simultaneously. A tetravalent vaccine is produced by combining dengue-2 PDK-53 with the above-described dengue-2/1, dengue-2/3 and dengue-2/4 chimeras in a pharmaceutical carrier suitable for administration as a multivalent vaccine. .

The chimeric virus or nucleic acid chimera can comprise a structural gene of a wild-type or attenuated virus in a lethal or attenuated DEN-2 viral backbone. For example, the chimera can display a structural protein gene of wild-type DEN-1 16007 virus or its candidate PDK-13 vaccine derivative in any of the DEN-2 PDK-53 backgrounds.

The tetravalent formulation, such as DENVax ^(TM), can be prepared by mixing a predetermined amount of each monovalent vaccine component. Depending on the input valency of each vaccine component, the defined monovalent vaccine volume can be added to the final volume of the vaccine formulation to 0.1 milliliters (e.g., for intradermal use) or 0.5 milliliters (e.g., for subcutaneous use). The remaining volume of the tetravalent vaccine may comprise DENVax ^TM Rhaponticum sugar (15%) in saline buffer, F127 (1%) and human serum albumin (0.1%) of diluent to reduce the stability of the live Toxic vaccine formulation. In certain embodiments, at least three dengue virus serotypes can be presented in a single ratio at a predetermined ratio. For example, dengue-1 to dengue-4 constructs can be presented in a single composition, with one live attenuated virus serotype presenting more, compared to other constructs. For example, the PFU of dengue-4 can be several times higher than other dengue viruses because it clarifies a reduced response.

method Nucleic acid amplification

Nucleic acids can be used in any formulation or used to produce Any formulation considered in the text. The nucleic acid sequence used as a template for amplification may be a virus isolated according to standard methods (for example, dengue virus). The nucleic acid sequence can be genomic DNA or partial or whole cell RNA. If RNA is used, it may be desirable to convert the RNA into a complementary cDNA. In certain embodiments, the RNA is whole cell RNA and used directly as a template for amplification. Any method known in the art for amplifying nucleic acid molecules (e.g., PCR, LCR, Qbeta replicase, etc.) has been contemplated.

Performance protein or peptide

The gene can be expressed in any number of different recombinant DNA expression systems to produce a large number of multi-peptide products which can then be purified and used in the methods and compositions reported herein. Any method known in the art for producing and using constructs has been considered. In some embodiments, a gene or gene fragment from which one or more multi-peptides are translated can be embedded in a performance vector by standard selection or sub-selection techniques known in the art.

Proteins, peptides and/or antibodies or fragments thereof can be detected or analyzed by any method known in the art. In some embodiments, methods such as gel electrophoresis or column chromatography can be used to separate and analyze molecules.

Electrophoresis

Electrophoresis can be used to separate molecules (eg, macromolecules such as proteins or nucleic acids) based on their size and charge. Many variations in electrophoresis are known in the art. The solution through which the molecules move can be random, usually in a capillary or it can be embedded in a matrix or other material known in the art. Common matrices can include, but are not limited to, polyacrylamide gels, agarose gels, mass spectrometers, dots, and filter paper.

Some specific examples use a gene or gene fragment from which a multi-peptide is translated, which can be inserted into a performance vector by standard sub-selection techniques. A performance vector can be used which produces the recombinant multi-peptide, such as a fusion protein, allowing rapid affinity purification of a peptide or protein. Examples of such fusion protein expression systems are the glutathione S-converting enzyme system (Pharmacia, Piscataway, NJ), the maltose binding protein system (NEB, Beverley, MA), the FLAG system (IBI, New Haven, CT), and 6xHis system (Qiagen, Chatsworth, CA).

Medical formulation

Any pharmaceutical formulation known in the art for use in vaccines is contemplated herein. In certain embodiments, the formulation can include one or more dengue virus serotypes in a plurality of ratios in a single vaccine. It is contemplated that the formulation may include other agents for inoculation of a subject, including but not limited to other active or inactive ingredients or compositions known to those skilled in the art.

All vaccine viruses contemplated herein can be administered as a vaccine composition, wherein the composition can be prepared by any method known to those skilled in the art. In certain embodiments, the viral composition is lyophilized and mixed with a pharmaceutically acceptable excipient (eg, water, phosphate buffered saline (PBS), wetting agent, and the like). In other embodiments, the vaccine composition can include a stabilizer that is known to reduce the deterioration of the formulation and to extend the idle life of the composition.

In other embodiments, an adjuvant may be added to the composition to administer, increase, stimulate or reinforce a cellular or humoral immune response to a vaccination as described herein. Has been considered in the art known and disclosed in this article The composition is compatible with any adjuvant.

Some specific examples herein relate to the amount or dose or volume of administration of a tetravalent dengue virus composition, and the amount or dosage may be based on the route of administration and other specifications of the individual (eg, age, state of health, weight) from which the vaccine was obtained. Depending on).

It is contemplated that the compositions described herein can be administered to an individual living in a region having a dengue virus, to an individual traveling to a region having a dengue virus, or to other individuals such as any human that may have symptoms of dengue or other dengue virus or animal. In certain embodiments, an individual traveling to a region having a dengue virus may be recommended to administer one or more vaccine compositions for about 1 to about 3 months prior to dengue virus exposure (eg, two or more on day 0) Kind) The vaccines herein can be administered as a prophylactic treatment to prevent infection in adults and children. With regard to exposure to the dengue virus and vaccine protocols disclosed herein, the individual can be a primary or non-initial individual.

Set

Other specific examples pertain to the use of the methods described herein (e.g., application or administration of vaccines) and kits of compositions. Some specific examples relate to the use of kits with vaccine compositions to prevent or treat individuals who have been exposed or suspected of being exposed to one or more dengue viruses. In certain instances, the kit may comprise one or more than one form of dengue virus serotype formulation a predetermined ratio (e.g., attenuated vaccine, trivalent or tetravalent formulations, DENVax ^TM). The kit can be portable, for example, capable of being transported and used in remote areas of dengue-endemic areas, such as military installations or remote villages. Other kits can be used in health equipment to treat individuals who have been exposed to one or more dengue viruses or who are suspected of being exposed to the risk of dengue virus.

The kit may also include suitable receptacles such as containers, vials, cartridges, mini or microcentrifuge tubes, test tubes, flasks, bottles, syringes or other containers. If additional components or medicaments are provided, the kit can include one or more additional receptacles in which the medicament or component can be placed. The kits herein will also typically include a tool that is closely constrained for commercial sale to contain the medicament (e.g., container), composition, and any other medicament container. The container may include an injection or blow molded plastic container in which the desired vial is retained. The compositions described may optionally require one or more additional agents, such as immunological agents or other antiviral agents, anti-fungal or antibacterial agents, for example, the composition is used as a vaccine against one or more additional microorganisms.

In other embodiments, the kit can include a device for administering one or more vaccines to a subject, such as an ID, SQ, IM, an inhaler, an intranasal applicator, or a vaccine composition for administration as disclosed herein. Other devices.

The following examples are included to illustrate certain specific examples presented herein. It will be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent the techniques found to function well in the practice disclosed herein. However, it will be apparent to those skilled in the art that <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt;

Example Example 1

Previous studies have revealed that natural infection of each DENV (dengue virus) serotype results in persistence of dengue fever caused by homologous serotypes. In some embodiments, an effective dengue vaccine administration closely mimics a natural infection and can provide a mode of administration as an anti-dengue virus vaccine. Specific examples reported herein may relate to the natural route of infection of dengue virus (DENV) infection, similar to intradermal delivery by a delivery host, mosquito bites. In some embodiments, an intradermal injection can be used to deposit the vaccine virus into the same tissue. The skin is a highly accessible organ and exhibits an effective immune barrier, primarily due to the presence of Langerhans cells (LCs) resident in the epidermis. Skin immunity elicits a broad range of immune responses, including body fluids, cells, and mucous membranes; and has the potential to bypass the effects of pre-existing immunity in the immunogenicity of vaccines administered.

Some specific examples of intradermal (ID) administration of tetravalent dengue vaccines have been reported in individuals in need of such treatment. A typical method of administration intradermally lines were on four cynomolgus monkey (Cynomolgus macaques), which is administered by intradermal be administered a ^{DENVax TM ((DENVax-1:} 1x10 5 PFU, DENVax-2: 1x10 5 PFU, DENVax3: 1x10 ⁵ PFU, DENVax 4: 1x10 ⁵ PFU) Dengue virus vaccine). To achieve an equivalent viral dose, use a needleless syringe to register 0.15 ml of vaccine in three closely spaced positions (see Figures 1 and 2 below). Figure 1 shows an intradermal injection device (e.g., PharmaJet® or other intradermal device) for intradermal inoculation.

Figure 2 illustrates the vaccination sites after vaccination with PharmaJet devices on cynomolgus monkeys. After 60 days, the animals were treated with the same formulation Add the same way. Serum samples were collected at predetermined intervals on days 15, 30, 58, 74 and 91, and the presence of neutralizing antibodies directed against the four dengue serotypes was tested. PRNT (spot reduction neutralization assay, known in the art for quantifying the extent of anti-DEN neutralizing antibodies) is performed on serum samples.

It has been elucidated that after neutral administration or after secondary administration, the neutralizing antibody titer is significantly higher after ID administration, as compared with SC administration (p<0.05 for DENV-1 and DENV-2) (see Figure 3); (p < 0.05 for all DENV serotypes) (see Figure 4). Since the position of the closely spaced lines in the same area, and each dose of the composition of all four viruses, this vaccine delivery pattern closely similar to the single administration DENVax ^TM. Figure 3 illustrates the geometric mean force price of 50% PRNT (spot reduction neutralization power price) at day 58 (58 days after the main administration). Figure 4 illustrates the 50% PRNT geometric mean force price at day 74 (14 days after the second dose on day 60). As can be seen in the graph, for all four dengue virus neutralizing antibody power prices, after intradermal administration is higher, compared to subcutaneous administration. In addition, the number of animals elucidating the neutralizing antibody response ("serum antibody conversion" defined as PRNT > 10) was greater after the first dose of vaccine (see Table 1, which shows serum antibody conversion after primary and secondary immunization). Percentage of animals in each of the four dengue serotypes).

The immunized animals were tested for protection against immunity by challenge with wild-type dengue virus. In cynomolgus macaques, wild-type dengue virus infection leads to viral replication and viremia, but there are no clinical signs. On day 91, two monkeys were challenged with DENV-1 (dengue virus serotype 1) and two monkeys were challenged with DEN-2 (dengue virus serotype 2). Serum samples were collected daily for 11 days after challenge initiation. The extent of dengue virus RNA in the sample was measured by quantitative real-time polymerase chain reaction reaction (q-rtPCR), and viable viral power was measured by virus isolation and spot formation on Vero cells. The results are shown in Tables 2 and 3. On day 91, anti-DEN-1 neutralizing antibodies were post-immunized ("post") for 14 days only prior to challenge initiation ("pre-excitation") and day 105. Viremia is provided as the number of days ("cycle") from which the live DEN-1 virus can be isolated from the blood sample and the log 10 of the peak force value isolated from each animal. Viral RNA is provided as the number of days ("cycles") in which viral RNA can be detected in serum samples and the extent of peak viral RNA in each monkey, expressed as log10 of the number of viral RNA genomes detected.

After challenge with immunization, the SC and ID immunized animals were completely protected from viremia induced by DEN-1 or DEN-2 (compared to control animals that elucidated a significant long-term viremia). In all ID-immunized animals, but not all SC-immunized animals, there was also a lack of viral RNA replication and lack of antibody potency after challenge immunization (compare ID animals with SC injected CY0181, CY0172 or control animals). These data suggest that the protection is "sterile" and prevents any viral replication after priming.

Example 2

In another embodiment, optimized DENVax ^(TM) formulations delivered at different locations and at different times will be tested in non-human primates. The group of eight cynomolgus macaques will each contain 1x10 ⁵ spot forming units (PFU), 1x10 ⁴ PFU, 1x10 ⁵ PFU and 1x10 ⁵ PFU of DENVax ^TM -1, DENVax ^TM -2, DENVax ^TM -3 and DENVax ^TM -4 (abbreviated as 5: 4: 5: 5) DENVax ^TM formulation immunization. The second dose will be administered in 0.1 ml ID. The groups will be one dose per arm on day 0; one dose in one arm on day 0, and one dose on the other arm on day 7; or one on day 0 One dose in the arm, and one of the other arms in the other arm is immunized on day 60. These groups will be in the same dose (5:4:5:5) at three locations on day 0 and three locations at the same time on the other day as the other arms; and Group comparison of the same dose in a single 0.5 ml SC immunization in one arm and at day 60 in the other arms at day 0. The control group will only be immunized with vaccine excipients (no vaccine virus). After immunization, blood samples were collected at 0, 7 (for peak viremia), 15, 30, 60, and 90 days to test for neutralizing antibodies against the four dengue virus serotypes by PRNT50. PBMCs collected on days 30, 60, and 90 will also monitor IFN-[gamma] secretion by the ELISPOT assay. On day 90, two animals from each group will be challenged with wild-type DEN-1, DEN-2, DEN-3 or DEN-4 virus. Clinical signs and temperatures (twice daily), changes in food consumption (once a day), and body weight (weekly) will be monitored for the animals that are stimulated. In addition, 11 days after the challenge, all animals will be bled daily to monitor viremia and hematology parameters. Again, the rate and duration of PRNT response and protection against all four DEN viruses after 90 days of challenge will be assessed. Intradermal administration in multiple locations and in distinguishable anatomical locations is more effective than subcutaneous administration with a single large bolus. Multiple locations provide vaccine exposure to more antigen presenting cells. A distinguishable anatomical location permits the vaccine to enter multiple lymph nodes. In addition, the booster immunization of dengue vaccines was only administered after 30 days or longer of antibody development in mice, primates, and human clinical trials. At this time, the neutralizing antibody inhibits the response to the live virus vaccine. It has previously been shown that primates are less effective at one month after primary immunization than at four months after primary immunization. It has been postulated that a high degree of homologous and heterologous antibodies circulating after the initial immunization can inhibit viral replication in the second dose. Although prolonged (two months or longer) immunization can circumvent this inhibition, it has not been tested whether the shorter immunization interval accelerates the immunization protocol before the development of a potent neutralizing antibody response can be excellent. This shortened protocol can be advantageous in popular countries or for travelers, where exposure to dengue virus at the risk of such immunity can put it at risk.

Example 3

In another embodiment, human clinical trials have been initiated to study safety and immunogenicity with 0.1 ml of two DENVax ^(TM) formulations administered by ID or SC injection. 12 individuals in the groups at day 0 and 90, for example a low dose formulation DENVax ^TM (individual is ^{8x10 3 PFU, 5x10 3 PFU,} 1x10 4 PFU and 2x10 ⁵ PFU of DENVax ^TM -1, -2, -3 and -4) or high doses (2 x 10 ⁴ PFU, 5 x ^{10 4} PFU, 1 x 10 ⁵ PFU and 3 x 10 ⁵ PFU are each DENVax ^TM -1, -2, -3 and -4) DENVax ^TM ID or SC immunization. Two control groups injected SC or ID with phosphate buffered saline. Any side effects of the patient and any significant changes in hematology or blood chemistry parameters will be monitored. Serum samples will be collected to measure vaccine viral replication and neutralizing antibody responses during the inter-cycle period.

Example 4

In AG129 mice, DENVax ^TM transdermal administration of the immunogenicity and efficacy. In another embodiment, two studies were performed to compare the effects of the route of administration on the immunogenicity and efficacy of DENVax ^(TM) in AG129 mice. In one embodiment, by use of a needle and syringe after SC injection at the dorsal skin or ID injection into the foot pad, and measuring antibody responses compared monovalent DENVax ^TM -4 (e.g., against one dengue virus serotype Vaccine) is immunogenic in AG129 mice. AG129 mice Group 8 to 50 microliters and the respective final volume of 100 microliters ID or SC injection of 10 ⁵ PFU / dose of the chimeric vaccine DENVax ^TM -4. Six weeks after the initial screening, animals from each treatment group were supplemented with 10 ⁵ PFU DENVax ^TM -4 or TFA via the corresponding ID or SC route. Mice were bled on days 31 and 58 and the collected serum was counted to measure the neutralizing antibody response.

DENVax ^TM -4 immunized via the ID route the lead-out after adding 5 times higher neutralizing antibody response to DEN-4, the comparison of the response elicited by the SC route (see, e.g., FIG. 4). The anti-DEN-4 response caused by either of the immunological pathways has significant anti-DEN-3 cross-neutralizing activity, but is not resistant to the DEN-1 or DEN-2 serotype. Figure 4 shows a rat at AG129 DENVax ^TM -4 fitted after the second immunization and in primary antibody response. Mice were bled on days 31 and 58 and the collected serum was stored to measure the neutralizing antibody response using the spot reduction test (PRNT50).

Two weeks after the addition, the animals from each group were divided into two groups and immunized with 10 ⁶ PFU of DEN-1 (Mochizuki strain) or DEN-2 (New Guinea C strain) virus. The clinical signs of the immunized animals were monitored and the survival rate recorded within 5 weeks was recorded. Mice immunized via the ID route showed no signs of disease after DEN-1 challenge (Fig. 5A). In the SC immunization cohort, only one mouse died of infection, while the remaining animals did not show any significant signs of infection (Fig. 5B). In comparison, on day 13 after DEN-1 challenge immunization, all control animals died of infection (Fig. 5A). After DEN-2 stimulate the immune, day 25, all only DENVax ^TM -4 immunized animals are killed via the SC route of infection, the mean survival time (MST) 19.5 days as compared to control mice (the FTA), its All died on the 17th day after the immunization (MST = 12.5 days) (Fig. 5B). Comparison fifty percent survival mice from infection by the immune ID DENVax ^TM -4, five weeks until the end of the monitoring period (Figure 5B). 5A and 5B show DENVax ^TM -4 immunized mice survived the AG129 to DEN-1 (a) or DEN-2 (b) viral challenge immunized. The clinical signs of the immunized animals were monitored and the survival rate recorded within 5 weeks was recorded.

In a second study, the immunogenicity of a tetravalent DENVaxTM vaccine administered in SC or ID in mice (eg, AG129) was tested. AG129 mice each group of six individual 100 [mu] l or 50 [mu] l (final volume) of DENVax ^TM SC or ID injection. In the rat system was 5: 4: 5: 5 ( 10 5 PFU DENVax TM -1, -3 , and -4 and 10 ⁴ PFU DENVax ^TM -2) fitting the dose level of compound of the DENVax ^TM vaccine immunization. Primary prevention 42 days after inoculation, all immunized animals receiving 5: 4: 5: 5 DENVax TM (10 5 PFU DENVax TM -1, -3 , and -4 and 10 ⁴ PFU DENVax ^TM -2) reinforcing injection. Blood samples were collected at 42 and 56 days to measure the neutralizing antibody response to each DEN virus serotype.

As shown in Table 4, both primary and secondary neutralizing antibody responses have been elicited for all four DEN serotypes. After the addition, the neutralizing anti-DEN-1, DEN-3, and DEN-4 antibody titers were increased by 2, 5, and 2 times, respectively, in the ID-injected mouse cohort, as compared with SC immunized animals. In the two groups, the neutralization reaction to the DEN-2 virus was comparable. Generation of anti-DEN-1>DEN-2>DEN-3>DEN-4 dominant neutralizing antibody response by immunization via the SC pathway Line, where the neutralization price is 5120, 1280, 640 and 80 respectively. The level of neutralizing antibody response after ID administration has shifted as follows: DEN-1>DEN-3>DEN-2>DEN-4, wherein the neutralizing antibody titers are 10240, 3840, 1280, and 160, respectively.

Materials and methods

Mice: AG129 mice have a "complete" immune system; lack of interferon (IFN)-α/β and -γ receptors. This model has been used to describe dengue infection. Other studies: Pathogenesis, cell tropism, and ADE have also been tested. This model permits immunization with DEN-1 and DEN-2.

Non-human primates: Cynomolgus, a rhesus monkey carrying a virus (viremia), but no disease is revealed.

Rapid drug administration study Example 5

In a typical study, comparisons were made with conventional needle injections. Vaccine delivery without needle administration to assess the immune response to different medicinal routes and dosing regimens in a non-human primate model for a tetravalent dengue vaccine. The quantifiable endpoints for this non-human primate study are: i) the maximum geometric mean neutralizing antibody titer for each of the four dengue serotypes in non-human primates; and ii) Excitation immunity of dengue serotypes.

In this study, two dosing schedules were evaluated: two doses (at different anatomical locations) on day 0, compared to two doses (0.60) provided separately for 60 days. In this study, the use of tetravalent formulations (e.g. DENVax ^TM) is a high dose formulation immunized. This vaccine lot is the same material used for the two period 1 studies to be performed. The high dose of the tetravalent vaccine formulation ^{2x10 4 PFU DEN-1,5x10 4 PFU} DEN-2,1x10 5 PFU and ^{3x10 5 PFU DEN-3 DEN-} 4 composition. This research design for non-human primate studies is shown in Table 5.

On days 0, 3, 5, 7, 10, 12, 14, 53, 64, 67, 88, 91, 93, 95, 97, 99, 101, 102, and 104, each inoculation and wild type dengue A serum sample is collected after the virus is stimulated to analyze the dengue viremia of the sample. Serum samples were also collected at days 0, 30, 53, 75, 88, and 104 to measure the extent of neutralizing antibodies elicited by the tetravalent formulation by administration via a needle/syringe or ID syringe.

Serum samples were collected during the specified intervals during the study procedure. Serum (pre-added) collected on days 0, 30, and 88 has been tested for neutralizing antibodies against dengue-1, dengue-2, dengue-3, and dengue-4. The GMT antibody titer is shown in Table 6 below.

In this study, all 42 animals were seronegative at the start of the study and showed no neutralizing antibody titers for any of the four dengue serotypes on day 0. After the animal to play DENVax ^TM First, the results of day 30 at day 0 showed, by ID or SC route of administration and the animals receiving DENVax ^TM is two (one in each arm) dengue -1, Dengue-2 and Dengue-4 (Groups 1 and 4) show high school and antibody prices. The serum antibody conversion ratio on day 30 was 100% for both groups, as compared to groups 2 and 3. The two groups maintained high and medium antibody response levels up to the 88th day before the virus elicited immunity.

For live attenuated vaccines, post-immunization vaccine virus replication is an important measure of vaccine absorption and vaccine safety. After at a live attenuated tetravalent vaccine formulation (DENVax ^TM) first and second immunization, the vaccine evaluation in non-human primates of viral replication. After the first immunization, the qRT-PCR assay was used to test the presence of viral RNA from the vaccine strain on serum samples collected on days 0, 3, 5, 7, 10, 12, and 14 (see Table 7). ).

Viral RNA was not detected on day 0 (pre-vaccination) and day 3 (post-immunization). For all cohorts, viral RNA was detected only on the dengue-2 serotype from day 5 to day 14 after inoculation after the first immunization. For groups 1, 3, 5 and 6, no end point price was observed 14 days after immunization. Watch peaks for groups 1 and 4 on day 10 and groups 5 and 6 on days 7 and 10 (table 7). Viral RNA was not detected in any of the groups evaluated on days 64 and 67 (4 and 7 days after 2 days of dosing) after the second immunization.

On day 90, three animals from each cohort were challenged with wild-type dengue-2 or dengue-4 to elucidate the efficacy following immunization with the tetravalent formulation. Protected animals should show a lack of wild-type dengue virus infection and replication. After immunization with 10 ⁶ PFU of wild-type dengue 2 (New Guinea C strain) and dengue 4 (814669 strain) virus, all populations were at 91, 93, 95, 97, 99, 101, 102 and 104 days. group analysis of wild-type virus to provoke an immune (dengue fever and dengue -2 -4) replication (table 8) dengue vaccine (eg DENVax ^TM): stimulate the immune viremia.

Viral RNA of wild-type elicited immune virus was detected only in cohort 7 that had received PBS. For dengue-2, viral RNA was detected in 3 of 3 animals on days 93-97. For dengue-4, viral RNA was detected in only 1 of 3 animals on day 95. An important observation in the cohort of immunization with tetravalent formulations was that no viral RNA was detected for dengue-2 or dengue-4 challenged immune virus. These results suggest that immunization against both dengue-2 and dengue-4 wild-type viruses is conferred by immunization with a tetravalent formulation of any dosing schedule tested.

Overall, this non-human primate study clearly shows a novel dosing schedule for the administration of two doses of a tetravalent formulation at two distinct locations (eg, different arms) on day 0. The degree of neutralizing antibody elicited is equal to or higher than those observed for the more traditional initial and additional immunization schedules for 2 to 3 months delivery. For the group receiving two doses on day 0, the onset of the immune response was faster and longer lasting. A needle-free ID or SC syringe was applied to increase the immune response, thus observing a higher price.

Example 6 Rapid immunization study of AG129 mice

In another typical study, a novel dosing schedule was designed that explores the administration of two doses of vaccine at two distinct locations on a single occasion or a shorter dosing interval between two doses of vaccine. The vaccinated individual has the capacity elasticity to return for the second immunization. Previously developed standard dengue vaccines typically require three doses over the course of a year to achieve a robust multivalent dengue immune response. With regard to the vaccination schedule presented herein, an immunological response to the maximum dose of intradermal administration at each of the two locations of at least two anatomy, and in some specific instances, is assessed (see Table 9). Performing this method, to some extent, activates immune cells and antigen-presenting cells in two different lymph nodes on day 0 to elicit a higher degree and a more robust dengue-specific immune response, with separate 7, 14 or 42 days of intradermal administration of two doses of comparison. In one study, the two routes of administration, SC and ID pathways were compared using conventional 42-day intervals between inoculations. Tetravalent formulations (DENVax ^TM; each serotype 3: 3: 3: 3 ratio) of the low dose formulation of immunized mice, wherein the formulation in a 0.05 ml volume of each dengue-1, -2, -3 and -4 (e.g., DENVax ^TM -1, -2, -3, and -4) 10 ³ PFU, whose line via the intradermal route (in the footpad) is provided. The field portion of this study was performed prior to the initiation of this infection. The research design department is shown in Table 9 below.

Appearing in the collected mouse serum, the neutralizing antibody titer against dengue 1-4 was measured by a microneutralization test. Throughout the study, serum was collected at specific time points and borrowed The lifespan of this immune response was studied by maintaining the study group until day 160 (longer than 5 months after the start of the study). The results obtained from the sera collected on days 28 and 56 after immunization are set forth in Table 10.

In the previous study, a conventional initial animal dosing schedule was used on day 0 and then a booster vaccination was performed on day 42 to assess the immune response to the tetravalent dengue vaccine. Both initial and additional vaccination are administered by the subcutaneous (SC) route. This dosing schedule is included in the study for comparison with a novel dosing schedule. Initially, one study (shown in Table 10) used a conventional dosing interval that provided two doses separately for 42 days to compare SC and ID administration routes. The results indicate that there is no significant difference in the neutralized antibodies elicited between the SC and ID pathways in this mouse model. This study further explored whether a two-dose administration (one of each of the two foot pads) at two anatomical locations on day 0 could trigger a standard dosing schedule similar to the above (two doses separated for 42 days). And the extent of the antibody. The results show that at day 0, at each location at two, via the ID route to DENVax ^TM tetravalent immune maximum dose formulation, for all four dengue serotypes induced neutralizing antibody levels equal to conventional administration The size of the schedule. The effect of a single vaccine dose administered by the ID route was also investigated (Group A). On Day 0, administration of a single dose of DENVax ^(TM) produced a slightly lower antibody response compared to the two doses on Day 0 (Comparative Groups A and B). Increasing the period between 7 and 42 days between the two doses did increase the antibody response beyond the observed level. Assessment of the lifespan of the dengue immune response revealed that the neutralizing antibody titers for all four dengue serotypes remained high on day 160 post-immunization, regardless of the route of administration and dosing schedule (data not shown).

Overall, the results suggest that the level of neutralizing antibodies elicited by the intradermal route of administration is equivalent to those observed for the subcutaneous route. Furthermore, on day 0, administration of two doses at two different locations by the ID route elicited a robust neutralizing antibody response equivalent to the conventional dosing schedule. The antibody response elicited has a long persistence and only a slight decrease. Animals did not show an increase in rickets and mortality. This study demonstrates that the administration of two doses of vaccine at two distinguishable locations is a viable option for immunization because the antibody valence and period produced by the immune response are equal in size to the two doses provided in separate 42 days. Those. These dosing regimens will benefit travelers who are in the epidemic area of dengue and other people who need rapid protection due to exposure to dengue virus.

Example 7 Another rapid immunization study of AG129 mice

The goal of this study was to determine whether the two doses administered at the two location IDs on day 0 would trigger a higher degree and a more robust dengue-specific immune response compared to the separate 42-day ID administration. Hypothesis to be tested for the maximum dose of vaccine administered within two leather whether the position of each activated transported to the lymph nodes of two different immune cells and antigen presenting cells, thus reducing interference in Four DENVax ^TM component of the vaccine. The design of this AG129 mouse study is shown in Table 11 below.

In this typical method, two different vaccine dose levels (low and medium doses) were used for immunization, using a novel dosing schedule to administer two doses on day 0, compared to two doses separated for 42 days. Mouse-based low-dose formulations DENVax ^TM (3: 3: 3: 3), to 0.05 ml volume, provided via the intradermal route (in the footpad) administration, wherein the formulation by the DENVax ^TM -1, - 2, -3 and 4 10 ³ PFU of each composition; or DENVax ^TM dose formulation (4: 3: 4: 5) administered in 0.05 ml volume, wherein the formulation comprises 10 ⁴ PFU DENVax ^TM -1, 10 ³ PFU DENVax ^TM -2, 10 ⁴ PFU DENVax ^TM -3 and 10 ⁵ PFU DENVax ^TM -4. On day 0, all mice were immunized and groups 2 and 4 were added on day 42. Serum for antibody analysis was collected on days 14, 41 and 56 after primary vaccination and analyzed using spot reduction micro-neutralization assay to measure the extent of neutralizing antibodies to all four dengue serotypes. The immunogenic results obtained from stocked rat serum samples are shown in Table 12.

In this embodiment, a low dose or tetravalent vaccine (e.g. DENVax ^TM) formulation was checked after immunization early inoculation 14 days, elicit neutralizing antibodies against all four dengue serotypes, and at day 0 When one or two doses are administered, it has nothing to do. On day 0 in the group receiving two and three, the formulation dose DENVax ^TM on day 28, and especially for DEN-1 DEN-3 induced a slightly higher neutralizing antibody titer, and in On day 0, only a single dose group (groups 2 and 4) was compared. The antibody titer obtained from the serum collected on day 56 indicates that the neutralizing antibody response continues and does not decline, regardless of whether the animal is supplemented on day 42 or only on day 0. The results obtained in this study further support the use of a novel dosing schedule for administration of two doses at two distinct locations (e.g., immunologically) on day 0.

The ELISPOT dengue virus neutralizing power price was calculated using a 50% NMS cutoff at a starting dilution of 1:20. Serum from individual animals in a group was stocked and tested in triplicate.

Example 8

Figures 9A-9D show a comparison of the neutralizing antibody titers achieved in non-human primates after immunization with tetravalent DENVax, including DENVax-1 (1x10 ⁵ PFU); DENVax- 2 (1x10 ⁴ PFU); DENVax-3 (1x10 ⁵ PFU); DENVax-4 (1x10 ⁶ PFU). The two groups were vaccinated via the subcutaneous route with a needle-free PharmaJet device twice on the same day (0, 0), or once on day 0 and again on day 60 (0, 60). The presence of antibodies to serum was analyzed at 0, 30, 53, 75, and 88 days, and detection of antibodies against four dengue serotypes (DEN-1, DEN-2, DEN-3, DEN-4) was analyzed.

In another embodiment, a serum-negative human subject is immunized with a two-dose tetravalent formulation, DENVax, comprising DENVax-1 (1x10 ⁴ PFU); DENVax-22 (1x10 ³ PFU); DENVax3 (1x10) ⁴ PFU); DENVax-4 (1x10 ⁵ PFU). The immunization route is subcutaneous or intradermal, and the vaccination is provided separately for 90 days. The extent of antibodies against each dengue serotype was analyzed at 0, 30, 60, 90 and 120 days. The vaccine elicits neutralizing antibodies against all four serotypes. However, when comparing the immune pathways, the extent of serum antibody conversion is different. Overall, in this study, the resulting intradermal route of immunization showed a more "balanced" immune response and the antibody levels were more equal, as compared to the subcutaneous route.

Figure 10 shows data obtained from human clinical trials in Colombia. Two doses of the tetravalent formulation DENVax are provided subcutaneously or intradermally to seropositive humans, wherein the DENVax comprises DENVax-1 (1x10 ⁴ PFU); DENVax-2 (1x10 ³ PFU); DENVax-3 (1x10 ⁴ PFU) ;DENVax-4 (1x10 ⁵ PFU). The extent of antibodies against each dengue serotype was analyzed at 0, 30, 60, 90 and 120 days.

In this exemplary method, at day 0 simultaneously in two of the tetravalent vaccine (e.g. ^{DENVax TM DENVax-1: 2x10 4} PFU, DENVax-2: 5x10 4 PFU, DENVax-3: 1x10 5 PFU, DENVax -4:3x10 ⁶ PFU) Immunize non-human primates, or two doses at 0 and 60 days, respectively. The vaccine elicits neutralizing antibodies against all four dengue serotypes. At 90 days post inoculation, the neutralizing antibody titers of the two cohorts were relatively equal (Figure 11). However, the kinetics of the immune response in the group receiving the second immunization on day 0 was faster. The results obtained in this study further support the use of a novel dosing schedule for administering two doses at two immunologically distinguishable locations on day 0.

Figure 11 shows a comparison of the neutralization antibody titers in non-human primates after subcutaneous immunization with tetravalent DENVax, including DENVax-1 (1x10 ⁵ PFU); DENVax-2 ( 1x10 ⁴ PFU); DENVax-3 (1x10 ⁵ PFU); DENVax-4 (1x10 ⁶ PFU). The two groups were vaccinated twice (0,0) on the same day, or once on day 0 and again on day 60 (0,60). The presence of serum antibodies was analyzed at 0, 28, 58, 73, and 90 days, and detection of antibodies against four dengue serotypes (DEN-1, DEN-2, DEN-3, DEN-4) was analyzed.

Example 9

Figure 12 shows a single pair of dual-dose dengue virus vaccine analysis. This data shows a change in the degree of transcription of multiple genes in an individual after 0,0 or a single injection, but the amount of change in double dose is greater. Therefore, in an individual who has dual administration of the composition disclosed herein on the same day, the initiation of innate immunity can be large.

The four dengue virus serotypes (DENV-1-4) are the most popular mosquito-borne virus disease in humans worldwide. Tetravalent vaccines are under development, but until this application, multiple immunizations over a period of six months to one year are required. The Rapid Immune Response (RIS) elicits an immune response to all four DENV serotypes and requires fewer visits to health providers, thus increasing vaccine capacity elasticity and triggering rapid serum antibody conversion, which will increase safety and protection for people in endemic countries. Travellers and military personnel are not affected by dengue fever. Study RIS using a variety of tetravalent formulations with a predetermined ratio, which is vaccinated at the beginning At the time of the visit (Day 0), the two largest vaccine doses were administered at two different anatomical locations. This vaccination strategy produces an efficient initial response to all four dengue virus serotypes and a long-term (3 months) potency of neutralizing antibody responses, such as with traditional first-time and second weeks after weeks or months Agent (additional) comparison. In addition, analysis of the innate immune response following primary immunization supports the notion that the initial efficiency provided by RIS is the result of a higher amount of immunological markers stimulated by the immunological method, as compared to single dose immunization.

Compositions disclosed herein include chimeric dengue virus compositions in which one skeleton of the dengue virus is tunable and one or more other dengue virus components.

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in accordance with the present disclosure. Although the compositions and methods have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes may be made to the compositions and methods, and to the steps of the methods described herein or in the order of the steps. Without leaving the concept, spirit and scope of this article. More particularly, for the reagents described herein, certain chemically and physiologically related agents may be substituted while achieving the same or similar results. All such similar substitutes and modifications which are apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the scope of the appended claims.

Claims (22)

A single vaccine composition comprising at least three live attenuated dengue virus serotypes or fragments thereof for use in rapidly eliciting an immune response against at least three dengue virus serotypes in a body, wherein at least two doses of the single vaccine composition Two or more anatomical locations are administered on the same day to an individual in need of the composition, in which neutralizing antibodies are raised to combat at least three dengue virus serotypes. A single vaccine composition according to claim 1, wherein at least one additional booster administration of the active attenuated dengue vaccine formulation is administered from 1 to 180 days after administration as claimed in claim 1. A single vaccine composition according to claim 1, wherein the single vaccine composition comprises a plurality of monovalent vaccines of the three or more dengue virus serotypes in a predetermined ratio in the single vaccine composition. A single vaccine composition according to claim 1, wherein the single vaccine composition comprises a plurality of monovalent vaccines of the three or more dengue virus serotypes in an equivalent ratio in the single vaccine composition. A single vaccine composition according to claim 2, wherein the attenuated dengue vaccine formulation to be used in the at least one additional booster administration is the same as the single vaccine composition administered at the same time as claim 1. The single vaccine composition of claim 2, wherein the attenuated dengue vaccine formulation to be used in the at least one additional booster administration is different from the single vaccine composition used in the simultaneous administration of claim 1, and comprises One or more of the serotypes of the dengue viruses at a predetermined concentration Valuation vaccine. A single vaccine composition according to claim 6, wherein the concentration of the dengue virus serotype comprises a concentration of one or more dengue virus serotypes that are higher than the concentration of the formulation used for administration on the same day as claim 1. A single vaccine composition according to claim 7, wherein the high concentration is 2 to 100,000 times greater than the concentration used in the single formulation of claim 1. A single vaccine composition according to claim 1, wherein the two or more anatomical locations comprise different anatomical locations using the same mode of administration. A single vaccine composition as claimed in claim 1, wherein the two or more anatomical locations comprise different anatomical locations using different modes of administration. A single vaccine composition according to claim 1, wherein the mode of administration of the vaccine comprises subcutaneous (SC), intradermal (ID) or intramuscular (IM). The single vaccine composition of claim 1, the composition further comprising at least one immunogenic agent for the individual. A single vaccine composition according to claim 1, wherein the composition comprises a tetravalent single vaccine composition representing all four dengue virus serotypes. A single vaccine composition according to claim 1, wherein the composition comprises all four dengue virus serotypes in a predetermined ratio. The single vaccine composition of claim 1, wherein the live attenuated dengue virus comprises one or more live attenuated dengue viruses and one or more dengue-dengue chimeric viruses, and further comprises the attenuated dengue virus shell And non-structural proteins and at least one second dengue virus Pre-membrane protein and envelope protein. A single vaccine composition according to claim 15 wherein the shell and non-structural protein are derived from an attenuated dengue-1, dengue-2, dengue-3 or dengue-4 virus. The single vaccine composition of claim 15, wherein when the attenuated dengue virus is dengue-1, the protomembrane protein and envelope protein of the at least one second dengue virus are dengue-2, dengue-3 or Dengue-4; or, when the attenuated dengue virus is dengue-2, it is dengue-1, dengue-3 or dengue-4; or, when the attenuated dengue virus is dengue-3, It is dengue-1, dengue-2 or dengue-4; or when the attenuated dengue virus is dengue-4, it is dengue-1, dengue-2 or dengue-3. The single vaccine composition of claim 15, wherein the dengue-dengue chimeric virus comprises a shell and non-structural protein of the attenuated dengue-2 virus, and dengue-1, dengue-3 or dengue-4 Protomembrane protein and envelope protein. A vaccine kit comprising: a single vaccine composition according to any one of claims 1 or 14; and at least one device, wherein the device can be used to administer the composition to two different anatomical locations of a body . The kit of claim 19, wherein the single vaccine composition comprises the predetermined ratio of the three or more dengue virus serotypes. A kit of claim 19, comprising two or more vaccine compositions, wherein at least one vaccine composition comprises approximately four of the average distribution A dengue virus serotype, and at least a second vaccine composition comprising a predetermined ratio of all four dengue virus serotypes. The kit of claim 19, further comprising two or more devices selected from the group consisting of an intradermal injection device, a hypodermic injection device, and an intramuscular device.