# Patent

## Title
Compositions, methods and uses for dengue virus serotype-4 constructs

## Abstract

Embodiments herein report compositions, methods and uses for dengue-4 (DENV-4) virus constructs. Some embodiments concern a composition that includes, but is not limited to, DENV-4 virus constructs alone or in combination with other constructs, can be used in a vaccine composition to induce an immune response in a subject. In certain embodiments, compositions can include constructs of more than one serotypes of dengue virus, such as dengue-1 virus, dengue-2 virus, or dengue-3 virus in combination with DENV-4 virus constructs disclosed herein. In other embodiments, DENV-4 constructs disclosed herein can be combined in a composition with other flavivirus constructs to generate a vaccine against more than one flavivirus. Other embodiments provide methods and uses for DENV-4 virus constructs in vaccine compositions that when administered to a subject induce an immune response in the subject against DENV-4 that is improved by modified constructs compared to other vaccine compositions.

## Background
PRIORITY CLAIM
This application claims priority to U.S. Provisional Application No. 61/724,190 filed Nov. 8, 2012 and U.S. Provisional Application No. 61/788,536 filed Mar. 15, 2013. All prior applications are incorporated herein in their entirety by reference for all purposes.


FEDERALLY FUNDED RESEARCH
This invention was made with Government support under R43 AI084291 awarded by the National Institutes of Health. The Government has certain rights in this invention.


FIELD
Embodiments herein report compositions, methods and uses for dengue-virus 4 (DENV-4) constructs. Some embodiments concern a composition that includes, but is not limited to, DENV-4 virus constructs that alone or in combination with other agents can be used in a vaccine composition. In certain embodiments, compositions can include chimera constructs of more than one serotypes of dengue virus, such as dengue-1 (DEN-1) virus, dengue-2 (DEN-2) virus, or dengue-3 (DEN-3) virus in combination with DENV-4 virus chimera constructs in di-, tri or tetravalent formulations. In other embodiments, DENV-4 chimera constructs (dengue-dengue chimeras) disclosed herein can be combined with other flavivirus constructs. Certain embodiments include DENV-4 chimeric constructs having components of other dengue serotypes, such as structural elements. Other embodiments provide methods and uses for DENV-4 virus chimera constructs in vaccine compositions that when administered to a subject induces an immune response in the subject against DENV-4 that is improved compared to other constructs.
BACKGROUND
Infection with dengue virus can lead to a painful fever of varying severity. To date, five serotypes of dengue virus have been identified: dengue-1 (DEN-1), dengue-2 (DEN-2), or dengue-3 (DEN-3), dengue-4 (DENV-4) and dengue-5 (DEN-5). Dengue fever is caused by infection of a dengue virus. Dengue virus serotypes 1-4 can also cause dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). The most severe consequences of infection, DHF and DSS, can be life threatening. Dengue viruses cause 50-100 million cases of debilitating dengue fever, 500,000 cases of DHF/DSS, and more than 20,000 deaths each year. To date, there is no effective vaccine to protect against dengue fever and no drug treatment for the disease. Mosquito control efforts have been ineffective in preventing dengue outbreaks in endemic areas or in preventing further geographic spread of the disease. It is estimated that 3.5 billion people are threatened by infection with dengue virus. In addition, dengue virus is a leading cause of fever in travelers to endemic areas, such as Asia, Central and South America, and the Caribbean.
All four dengue virus serotypes are endemic throughout the tropical and subtropical regions of the world and constitute the most significant mosquito-borne viral threat to humans worldwide. Dengue viruses are transmitted to humans primarily by Aedes aegypti mosquitoes. Infection with one dengue virus serotype results in life-long protection from re-infection by that serotype, but does not prevent secondary infection by one of the other three dengue virus serotypes. In fact, previous infection with one dengue virus serotype can lead to an increased risk of severe disease (DHF/DSS) upon secondary infection with a different serotype. The development of an effective vaccine represents an important approach to the prevention and control of this global emerging disease.
SUMMARY
Embodiments herein concern compositions, methods and uses of DENV chimera constructs, for example DENV-4. In some embodiments, a composition can include DENV-4 virus chimera constructs alone or in combination with other dengue virus serotype constructs or live, attenuated dengue viruses of the same or other serotypes or other flavivirus constructs capable of inducing an immune response to a target virus (e.g. dengue virus). Other embodiments can include a composition of a live, attenuated virus construct against DENV-4 and optionally, one or more live, attenuated viral constructs against DEN-1, DEN-2 and DEN-3. In other embodiments, an immunogenic composition is provided that includes a DENV-4 live, attenuated chimeric virus construct with strong immunogenicity when introduced to a subject. In accordance with these embodiments, these live, attenuated viral constructs can be used alone or in combination with one or more other DEN-1, DEN-2 and DEN-3 constructs, and a pharmaceutically acceptable excipient to generate a vaccine formulation against dengue virus serotypes. In certain embodiments, monovalent, bivalent, trivalent or tetravalent pharmaceutically effective formulations against one or more dengue viruses are generated. In certain embodiments, an immunogenic composition can include one or more of DEN-1, DEN-2, DEN-3 dengue-dengue chimeric constructs in combination with a chimeric DENV-4 construct disclosed herein.
In certain embodiments, an immunogenic composition including a DENV-4 construct of the present invention in combination with one or more of DEN-1, DEN-2 and DEN-3 can be used to confer simultaneous protection against two or more dengue virus serotypes in a single vaccine administration. In other embodiments, an immunogenic composition including DEN-1, DEN-2, DEN-3 and modified or mutated DENV-4 constructs of embodiments disclosed herein can be administered to a subject to induce improved immunogenic responses against each dengue virus serotype and where immune response interference to DENV-4 is reduced.
In certain embodiments, DENV-4 constructs can include a dengue-dengue chimeric construct having adaptive mutations in the structural or non-structural regions of DENV-4. In other embodiments, DENV-4 constructs can include a backbone of another dengue virus serotype, DEN-1, DEN-2 or DEN-3. In yet other embodiments, a chimeric construct can include a DEN-2 backbone where DENV-4 structural or non-structural regions of DENV-4 are substituted for DEN-2 structural or non-structural regions. In accordance with these embodiments, a DEN-2 backbone can include any live attenuated DEN-2 virus. In other embodiments, a DEN-2 backbone can include live attenuated DEN-2 PDK-53 virus as a backbone where the live attenuated DEN-2 PDK virus further includes structural proteins of one or more of prM (premembrane) and E (envelope) structural proteins of DENV-4. In addition, a DEN-2 PDK-53 backbone can include additional mutations or reversions of mutations of DEN-2 PDK-53 generating a novel construct in order to enhance in vitro growth, or in vivo the immune response to DENV-4 in a subject upon administration.
In some embodiments, a current dengue chimeric construct denoted as DENVax-4 strain (SEQ ID NO:21) was modified to contain a capsid/PrM junction of the DEN-2 backbone to be more genetically similar to that of DENV-4 instead of DEN-2 in order to improve replication efficiency of the virus both in vitro for production and in vivo as a construct of use for inducing an immune response to DENV-4. The current strain of DEN-4, DENVax-4, has a capsid/PrM sequence that is identical to DEN-2 instead of DEN-4, possibly creating an inefficient transcription and translation from the genomic RNA, which is different than that of wild type DENV-4.
In some embodiments, structural protein genes can include prM and E genes of DENV-4 on another dengue virus backbone (e.g. dengue-2, DEN-2 PDK-53), making a dengue-dengue chimera. For example, a DEN-4 construct, in certain embodiments can include those construct termed DENVax-4e (Capsid 107 Cysteine to Tyrosine; DenVax-4b backbone, modifications at Capsid/prM junction), DENVax-4f (where the PDK-53 backbone NS2A and NS4A mutations are reverted to that of 16681) or DENVax-4h (Envelope 417 Glu to Lys) (see for example FIG. 4) where for certain constructs the DEN-2 PDK-53 backbone has one or more reversions to wild-type DEN-2 (e.g. in the non-coding region (NCR) or a non-structural region (NS2 etc.)) and one or more mutations in the DENV-4 structural region (e.g. prM or E), while encoding one or more structural proteins of DENV-4 (e.g. strain 1036). A modified DENV-4 construct disclosed herein can include a modified attenuated DEN-2 PDK-53 backbone, having one or more modified structural proteins of DENV-4 strain 1036. In some embodiments, one or more mutations present in live, attenuated DEN-2 PDK-53 virus can be reverted back to a wildtype nucleic acid (which may be a silent mutation) or another nucleic acid to produce constructs herein that generate a modified DEN-2/DENV-4 construct having increased replication ability and immunogenicity without affecting its attenuation or safety but may affect growth and/or replication of the DEN-4 virus. In certain embodiment, the reversions may lead to increased growth and/or replication.
In other embodiments, a modified DENV-4 construct can incorporate mutations introduced to one or more structural regions and/or non-structural regions of DENV-4 in order to generate constructs inducing an improved immunological response while maintaining safety and viral attenuation. For example, a modified or mutated dengue-dengue chimera of DEN-2/DENV-4 may contain mutations at one or more non-structural regions of a DEN-2 PDK-53 backbone, such as NS2A, and NS4A, and/or mutations at 5′ non-coding region (5′NCR). In another embodiment, a modified DENV-4 chimera construct can include NS2A and NS4A of DEN-2 16681 by reverting mutations at NS2A and NS4A of PDK-53 (e.g. an M-L substitution at NS4A). Some embodiments include a modified DENV-4 chimera construct having 5′NCR, NS2A and NS4A of DEN-2 16681 by reverting corresponding mutations in the DEN-2 PDK-53 backbone of a target construct. Other embodiments can include a modified DENV-4 chimera construct having 5′NCR of DEN-2 16681 by reverting corresponding mutations in the DEN-2 PDK-53 backbone. A modified DEN-4 chimera construct can also include DEN-2 PDK-53 backbone, and encode one or more structural proteins of DEN-4 strain H241. It is contemplated that, to induce an immune response, any DEN-4 structural protein can be substituted for structural regions of a chimeric virus containing a dengue-2 serotype backbone (e.g. PDK-53 or modified PDK-53). In some embodiments, a modified DEN-4 construct contains live attenuated DEN-2 PDK-53 as a backbone, and DEN-4 structural proteins where mutations can be introduced to modify structural regions of a DEN-4 (e.g. strain 1036).
In other embodiments, mutations can be introduced to capsid/prM junction amino acid sequences of a DENV virus in order to increase immunogenicity of a construct containing such a mutation. For example, a mutation in DEN-4 can be a Cys-Tyr mutation at capsid position 107 of the DEN-4. In other embodiments, it is contemplated that the cysteine in position 107 can be mutated to any other aromatic amino acid with a hydrophobic side chain (see for example DEN-4e). Other DEN-2 PDK-53 reversion of a chimeric construct can be found in NS2A or NS4A. Yet other embodiments include a DEN-4 construct where a DEN-2 backbone comprises PDK-53 (MVS, SEQ ID NO:21) where amino acid positions 102-107 of the capsid region of PDK-53 are converted to a homologous DEN-4 counterpart amino acid to generate DENV-4b (see for example, FIG. 4). These backbone constructs can then further comprise a cysteine in the capsid region to aromatic amino acid in position (e.g. tyrosine, tryptophan etc). In certain embodiments, this construct is represented by SEQ ID NO:22 or SEQ ID NO:23.
Other DENV-4 constructs disclosed herein can include an amino acid substitution at Envelope position 417. For example, DEN-4 strain 1036 strain sequence or equivalent strain position thereof where a PDK-53 (MVS DEN2/4, SEQ ID NO:21) backbone of Dengue-2 with DEN-4 structural proteins is provided. Embodiments include further mutating Envelope position 417 from a negative to a positively charged side-chain amino acid (e.g. lysine). It is contemplated that any charged side chain will provide increased immunogenicity of the DEN-4 construct without affecting its safety or attenuation. In certain embodiments, this construct is represented by SEQ ID NO:24 or SEQ ID NO:25.
In certain embodiments, DEN-2 PDK-53 reversions of a chimeric DENV construct have the 5′ NC, NS1 and NS3 mutations found in DEN-2 PDK-53 MVS while having other reversions or mutations. It has been demonstrated that these three mutations can be important for attenuation (e.g. small plaque size, reduced growth rate, lower titer, increased temperature sensitivity and decreased neurovirulence compared to a control).
In other embodiments, DEN-2 PDK-53 genome backbones can be used to generate chimeric constructs of DEN-1 and DEN-3, where one or more structural protein genes of DEN-2 PDK-53 genome can be replaced by one or more structural protein genes of DEN-1 and DEN-3. These constructs can include a combination of both DEN-1 and DEN-3 in a single chimera having a DEN-2 PDK-53 backbone. In some embodiments, a structural protein can be the C, prM or E protein of DEN-1 and/or DEN-3. In certain embodiments, structural protein genes include the prM and E genes of DEN-1 or DEN-3. These hybrid/chimeric viruses express the surface antigens of DEN-1, DEN-3 or DENV-4 while retaining the attenuation phenotypes of the parent DEN-2. In certain embodiments, these constructs can be represented by SEQ ID NO:15, DEN-2/DEN-1 and SEQ ID NO: 19, DEN-2/DEN-3 where these constructs can be used in di-, tri or tetravalent compositions disclosed herein.
In some embodiment, constructs disclosed herein can include chimeric constructs of DENV-4, DEN-2, DEN-1, and DEN-3 expressing surface antigens of DEN-1, DEN-3 and DENV-4 using attenuated DEN-2 PDK-53 virus as a backbone.
Some embodiments disclose methods for making modified or mutated DENV-4 constructs of use in any vaccine composition including, but not limited to, a single vaccine composition having only DENV-4 constructs, a mixture single vaccine composition capable of inducing an immune response against two or more dengue virus serotypes, a mixture single vaccine composition having chimeric (and non-chimeric) constructs disclosed herein in combination with other flavivirus constructs capable in inducing an immune response to a flavivirus as well as one or more dengue virus serotypes that include DENV-4.



BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain embodiments. Some embodiments may be better understood by reference to one or more of these drawings alone or in combination with the detailed description of specific embodiments presented.
FIGS. 1A-1B illustrates (A) a representative schematic diagram of structural and non-structural genes of DEN-2 PDK-53, and (B) exemplary results of certain chimeric dengue virus constructs of some embodiments disclosed herein expressing structural proteins of different dengue virus serotypes respectively in the DEN-2 PDK-53 backbone.
FIG. 2 is a table that illustrates an exemplary experimental design for analyzing certain vaccines including DENV-4 chimeric constructs of some embodiments disclosed herein in non-human primates.
FIG. 3 represents an exemplary graph of an experiment related to production of DENV-4 neutralizing antibody titers produced by an animal model immunized with a vaccine composition having DENV-4 chimeric viral constructs of certain embodiments disclosed herein.
FIG. 4 represents a schematic of certain DENV-4 chimera constructs disclosed in some embodiments herein and used in vaccine formulations alone or in combination with other live, attenuated virus constructs.
FIG. 5 is a table illustrating some exemplary data of analysis of plaque phenotypes, Vero cell titers and mosquito growth of some DENV-4 virus constructs of certain embodiments disclosed herein.
FIGS. 6A-6F are photographs providing exemplary phenotypes of ImmunoFoci (plaques) results of an exemplary experiment using certain DENV-4 chimera constructs including DENV-4 wild type (A), DENVax-4 (B), DENVax-4e (C), DENVax-4h (D), DENVax-4i (E), and DENVax-4j (F).
FIG. 7 is a graphic representation of viral titers over time of various DENV-4 chimeric constructs after growth in mammalian cells, illustrated as “Days Post Infection.”
FIG. 8 is a graphic representation of viral titers over time of various DENV-4 chimeric constructs after growth in mosquito cells illustrated as “Days Post Infection.”
FIG. 9 is a flow chart representing an acceptable animal model used to test various DENV-4 chimeric constructs to induce an immune response to DENV-4 in the animal model.
FIG. 10 illustrates an alignment of various DENV-4 chimeric constructs, illustrating common sequences and exemplary mutations and/or reversions.
FIG. 11 illustrates an alignment of various DENV-4 chimeric constructs, illustrating common sequences and exemplary mutations and/or reversions.
FIG. 12 illustrates a schematic representation of a live, attenuated virus construct of DEN-2 virus.
FIG. 13 illustrates an exemplary schematic of cloning of various DENV-4 constructs disclosed herein.
FIG. 14 represents a graph comparing various DENV-4 constructs growth in mosquito cells.
FIG. 15 represents an exemplary histogram plot comparing immunogenicity in mice of various DENV-4 constructs disclosed herein.
FIG. 16 represents an exemplary graph illustrating efficacy using various DEN-4 constructs in mice to protect immunized mice against challenge by wt-DENV, represented as survival.
FIG. 17 represents an alignment of the flavivirus envelope protein.
FIG. 18 represents a schematic of a DENV virus including open reading frames for proteins of DENV.
FIG. 19 represents a graphic illustration of titer achieved after growth of various DEN-4 constructs in a mammalian cell line contemplated of use herein.
FIG. 20 represents various levels of neutralizing antibodies after vaccination with various dengue constructs in non-human primates post vaccination, represented as days post vaccination.
FIG. 21 represents various levels of neutralizing antibodies after vaccination with various dengue constructs in non-human primates post vaccination, represented as days post vaccination.
FIG. 22 represents various levels of dengue constructs in a mouse post vaccination, represented as days post vaccination.
FIG. 23 represents various levels of dengue constructs in a mouse post vaccination, represented as days post vaccination.



DEFINITIONS
As used herein, “a” or “an” may mean one or more than one of an item.
As used herein the specification, “subject” or “subjects” may include, but are not limited to, mammals such as humans (e.g. adults and juveniles) or mammals, domesticated or wild, for example dogs, cats, other household pets (e.g. hamster, guinea pig, mouse, rat), ferrets, rabbits, pigs, horses, cattle, prairie dogs, wild rodents, or zoo animals.
As used herein, the terms “chimeric construct,” “virus chimera,” “chimeric virus,” “flavivirus chimera” and “chimeric flavivirus” can mean a construct comprising a portion of the nucleotide sequence of a dengue-2 virus and further nucleotide sequence that is not from dengue-2 virus or is from a different dengue virus serotype or a different flavivirus. A “dengue chimera” comprises at least two different dengue virus serotypes. Examples of other dengue viruses or flaviviruses include, but are not limited to, sequences from dengue-1 virus, dengue-3 virus, dengue-4 virus, West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, tick-borne encephalitis virus, yellow fever virus and any combination thereof.
As used herein, “nucleic acid chimera” can mean a construct disclosed herein including nucleic acid sequences comprising a portion of the nucleotide sequence of a dengue-2 virus and further one or more nucleotide sequences are not of the same origin as the nucleotide sequence of the dengue-2 virus. Correspondingly, any chimeric flavivirus, any dengue chimera or flavivirus chimera disclosed herein can be recognized as an example of a nucleic acid chimera.
DESCRIPTION
In the following sections, various exemplary compositions and methods are described in order to detail various embodiments. It will be obvious to one skilled in the art that practicing the various embodiments does not require the employment of all or even some of the specific details outlined herein, but rather that concentrations, times and other specific details may be modified through routine experimentation. In some cases, well-known methods or components have not been included in the description.
In accordance with embodiments of the present invention, there may be employed conventional molecular biology, protein chemistry, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Animal Cell Culture, R. I. Freshney, ed., 1986).
Embodiments herein concern compositions, methods and uses for inducing improved immune responses against DEN-4 alone or in combination with one or more agents for inducing immune responses against other dengue virus serotypes or flaviviruses in a subject. In accordance with these embodiments, live, attenuated dengue viruses and nucleic acid chimeras of DEN-4 are generated and used in immunogenic compositions disclosed herein. Some embodiments concern modified or mutated DEN-4 constructs. Some embodiments concern introducing mutations and/or reversions into DEN-4 chimeric constructs to modify the amino acid sequence of the chimeric construct. Some embodiments concern introducing mutations and/or reversions into the DEN-2 PDK-53 backbone constructs to modify the amino acid sequence or RNA sequence of the chimeric construct. In certain embodiments, mutations and/or reversions into DEN-4 chimeric constructs to modify the amino acid sequence of the chimeric construct can include mutations upstream of the C/prM cleavage site of a known chimeric construct referred to as DENVax-4 (SEQ ID NO: 21) by techniques including, but not limited to mutagenesis.
Embodiments herein concern compositions, methods and uses of DENV-4 virus chimera constructs. In some embodiments, a composition can include DENV-4 virus chimera constructs alone or in combination with other dengue virus serotype constructs or live, attenuated dengue viruses of the same or other serotypes or other flavivirus constructs capable of inducing an immune response to a target virus (e.g. dengue virus or other flavivirus). Other embodiments can include a composition of a live, attenuated virus construct against DENV-4 and optionally, one or more live, attenuated viral constructs against DEN-1, DEN-2 and DEN-3. In yet other embodiments, an immunogenic composition is provided that includes a DENV-4 live, attenuated chimeric virus constructs with increased immunogenicity compared to other known constructs when introduced to a subject. In accordance with these embodiments, these live, attenuated viral chimera constructs can be used alone or in combination with one or more other DEN-1, DEN-2 and DEN-3 constructs (e.g. live, attenuated viruses or chimeras), and a pharmaceutically acceptable excipient to generate a vaccine formulation against one or more dengue virus serotypes. In certain embodiments, monovalent, bivalent, trivalent or tetravalent pharmaceutically effective formulations against one or more dengue viruses are generated. In certain embodiments, an immunogenic composition can include one or more of DEN-1, DEN-2, DEN-3 dengue-dengue chimeric constructs or live, attenuated dengue virus in combination with a chimeric DENV-4 construct disclosed herein.
In certain embodiments, an immunogenic composition including a DENV-4 construct of the present invention in combination with one or more of DEN-1, DEN-2 and DEN-3 can be used to confer simultaneous protection against two or more dengue virus serotypes in a single vaccine administration. In other embodiments, an immunogenic composition including DEN-1, DEN-2, DEN-3 and modified or mutated DENV-4 constructs of embodiments disclosed herein can be administered to a subject to induce improved immunogenic responses against each dengue virus serotype and where immune response interference to DENV-4 is reduced.
In certain embodiments, DENV constructs can include a dengue-dengue chimeric construct having adaptive mutations in the structural or non-structural regions of the backbone (PDK-53) or structural regions of DEN-4. In other embodiments, DENV-4 constructs can include a backbone of another dengue virus serotype, DEN-1, DEN-2 or DEN-3. In yet other embodiments, a chimeric construct can include a DEN-2 backbone where DENV-4 structural or non-structural regions of DENV-4 are substituted for DEN-2 structural and/or non-structural regions. In accordance with these embodiments, a DEN-2 backbone can include any live, attenuated DEN-2 virus having safety and efficacy while inducing an immune response to DEN-2. In other embodiments, a DEN-2 backbone can include live, attenuated DEN-2 PDK-53 (53 passages in primary dog kidney cells (PDK)) or derived from DEN-16681 strain virus as a backbone where the live, attenuated DEN-2 PDK-53 virus further includes structural proteins of one or more of prM (premembrane) and E (envelope) structural proteins of DENV-4. In addition, a DEN-2 PDK-53 backbone can include additional mutations or reversions of mutations of DEN-2 PDK-53 in order to enhance the immune response to DENV-4 in a subject upon administration (see for example FIG. 4).
In some embodiments, structural protein genes can include prM and E genes of DENV-4 on another dengue virus backbone, making a dengue-dengue chimera. For example, a DENV-4 construct, in certain embodiments can include those construct termed DENVax-4e, DENVax-4f, or DENVax-4h (see for example FIG. 4) where the DEN-2 PDK-53 backbone has one or more reversions to wild-type DEN-2 amino acids (e.g. in the non-coding region (NCR) or a non-structural region (NS1 etc.)) and one or more mutations in the DENV-4 structural region (e.g. prM or E), while encoding one or more structural proteins of DENV-4 (e.g. strain 1036). A modified DENV-4 construct disclosed herein can include a modified attenuated DEN-2 PDK-53 backbone, having one or more modified structural regions of DENV-4 strain 1036. In some embodiments, one or more mutations present in live, attenuated DEN-2 PDK-53 virus can be reverted back to a control amino acid or another amino acid to produce constructs herein that generate a modified DEN-2/DENV-4 chimeric construct (when compared to MVS sequence SEQ ID. NO: 21) having increased immunogenicity without affecting its safety or attenuation but may affect in vitro growth and/or replication of the DEN-4 virus. In certain embodiment, the reversions may lead to increased growth and/or replication.
In other embodiments, a modified DENV-4 construct can incorporate mutations introduced to one or more structural regions and/or non-structural regions of DENV-4 in order to generate constructs inducing an improved immunological response while maintaining safety and viral attenuation. For example, a modified or mutated dengue-dengue chimera of DEN-2/DENV-4 may contain mutations at one or more non-structural regions of a DEN-2 PDK-53 backbone, such as NS2A, and NS4A, and/or mutations at 5′ non-coding region (5′NCR). In another embodiment, a modified DENV-4 chimera construct can include NS2A and NS4A of DEN-2 16681 by reverting mutations at NS2A and NS4A of PDK-53 (e.g. an M-L substitution at NS4A). Some embodiments include a modified DENV-4 chimera construct having 5′NCR, NS2A and NS4A of DEN-2 16681 by reverting corresponding mutations in the DEN-2 PDK-53 backbone of a target construct. Other embodiments can include a modified DENV-4 chimera construct having 5′NCR of DEN-2 16681 by reverting corresponding mutations in the DEN-2 PDK-53 backbone. A modified DENV-4 chimera construct can also include DEN-2 PDK-53 backbone, and encode one or more structural proteins of DENV-4 strain H241.
It is contemplated that DENV-4 structural proteins can substitute for structural or non-structural regions of a dengue-2 serotype backbone (e.g. PDK-53 or modified PDK-53 identified herein) In some embodiments, a modified DENV-4 construct contains live attenuated modified DEN-2 PDK-53 as a backbone, and DENV-4 structural proteins where mutations can be introduced to modify structural regions of a DENV-4 (e.g. strain 1036). In some embodiments, mutations can be introduced to capsid/prM junction amino acid sequences of DENV-4 in order to increase replication and/or immunogenicity of a construct containing such a mutation. For example, a mutation in DENV-4 can be a C-Y mutation at capsid position 107 of the DENV-4 (see for example, DENVax-4e). In accordance with these embodiments, a cysteine can be mutated to an aromatic amino acid (e.g. tyrosine or other) on a modified PDK-53 backbone (DENV-4b). Other mutations can include an amino acid substitution at Envelope position 417 (glutamic acid, E) in a DENV-4 1036 strain sequence (see for example, DENVax-4h) or equivalent position thereof in another DENV-4, where a negative amino acid is replaced by a positive amino acid with a charged side chain (e.g. lysine, arginine, histidine etc.). Other DEN-2 PDK-53 reversion of a chimeric construct can be found in the NS2A or NS4A regions.
In other embodiments, DEN-2 PDK-53 genome backbones can be used to generate chimeric dengue virus constructs of DEN-1 and DEN-3, where one or more structural or non-structural protein genes of DEN-2 PDK-53 genome can be replaced by one or more structural protein or non-structural genes of DEN-1 and DEN-3. These constructs can include a combination of both DEN-1 and DEN-3 structural or non-structural genes in a single chimera having a DEN-2 PDK-53 backbone. In some embodiments, a structural protein can be the C, prM or E protein of DEN-1 and/or DEN-3. In certain embodiments, structural protein genes include the prM and E genes of DEN-1 or DEN-3 or a combination thereof. These hybrid/chimeric viruses can express surface antigens of DEN-1, DEN-3 or DENV-4 while retaining the attenuation phenotypes of the parent DEN-2.
In some embodiment, constructs disclosed herein can include chimeric constructs of DENV-4, DEN-2, DEN-1, and DEN-3 expressing surface antigens of DEN-1, DEN-3 and DENV-4 using attenuated DEN-2 PDK-53 or live, attenuated DEN-2 16681 virus (or a dengue-2 virus with one or more reversion of any of the mutations found in dengue-2 serotype PDK-53 back to its wildtype 16681 virus) as a backbone. In addition, constructs that are part of a pharmaceutical composition can include other agents such as other live, attenuated viruses (e.g. DEN-2, other flaviviruses). Further, other agents of use in these compositions can include other pharmaceutically acceptable anti-viral agents, adjuvants or stabilizing agents to reduce degradation of the live, attenuated viruses.
Some embodiments herein disclose methods for making modified or mutated DENV-4 constructs of use in any vaccine composition against DENV-4 including, but not limited to, a single vaccine composition having only DENV-4 constructs, a mixture of dengue virus constructs of a single vaccine composition capable of inducing an immune response against two or more dengue virus serotypes, a mixture in a single vaccine composition having chimeric (and non-chimeric) constructs disclosed herein in combination with other flavivirus constructs capable in inducing an immune response to a different flavivirus (e.g. yellow fever, West Nile, Japanese encephalitis etc.) as well as one or more dengue virus serotypes that include DENV-4.
In other embodiments, other combinations are contemplated of use with DENV-4 constructs disclosed herein. For example, a dengue virus serotype 1 wild-type virus passaged in PDK cells 13 times is designated as DEN-1 PDK-13 virus. Other vaccine candidates are DEN-2 PDK-53, DEN-3 PGMK-30/FRhL-3 (e.g. thirty passages in primary green monkey kidney cells, followed by three passages in fetal rhesus lung cells) and DENV-4 PDK-48. These four candidate vaccine viruses were derived by tissue culture passage of wild-type parental DEN-1 16007, DEN-2 16681, DEN-3 16562 and DENV-4 1036 viruses, respectively. Any of these existing live, attenuated dengue viruses are contemplated of use in combination with the DENV-4 chimeric virus constructs disclosed herein.
Previous human clinical trials with these attenuated viruses have indicated that DEN-2 PDK-53 has the lowest infectious dose (50% minimal infectious dose of 5 plaque forming units or PFU) in humans, is strongly immunogenic, and produces no apparent safety concerns. The DEN-1 PDK-13, DEN-3 PGMK-30/FRhL-3 and DENV-4 PDK-48 vaccine virus candidates have higher 50% minimal infectious doses of 10,000, 3500, and 150 PFU, respectively, in humans.
DEN-2 PDK-53 virus vaccine candidate, henceforth abbreviated PDK-53, has several measurable biological markers associated with attenuation, including temperature sensitivity, small plaque size, decreased replication in mosquito C6136 cell culture, decreased replication in intact mosquitoes, loss of neurovirulence for suckling mice and decreased incidence of viremia in monkeys. Clinical trials of the candidate PDK-53 vaccine have demonstrated its safety and immunogenicity in humans. Furthermore, the PDK-53 vaccine induces dengue virus-specific T-cell memory responses in human vaccine recipients.
In certain embodiments, a nucleic acid molecule can include a chimeric flavivirus construct having a nucleic acid sequences encoding nonstructural proteins and at least one or more structural proteins from a live, attenuated dengue-2 virus and at least encoding one or more structural proteins from a second flavivirus, wherein the chimeric construct further comprises one or more mutations comprising a mutation in the envelope (E) protein at a position synonymous to amino acid 417, a mutation in the capsid protein at a position synonymous to position 107, and a mutation in NS4A at a position synonymous to amino acid position 17. In other embodiments, a nucleic acid can further include a mutation in the envelope (E) protein at a position synonymous to amino acid 417 that changes the wild type glutamic acid to a lysine. Yet other nucleic acid molecules disclosed herein can further include a mutation in the capsid (C) protein at a position synonymous to amino acid 107 that changes a cysteine to a tyrosine. In other nucleic acid molecules, the mutation in the NS4A protein at a position synonymous to amino acid 17 changes methionine (e.g. wild type sequence) to a leucine. It is contemplated that the second flavivirus can be a DENV-1, DENV-3 or DENV-4. In certain embodiments, the nucleic acid molecule can include a second flavivirus that is DENV-4. In other embodiments, the nucleic acid molecules having a live attenuated dengue-2 backbone contains a mutation at position 57 in the 5′NCR, at position 53 of ns1 and position 250 of ns3. According to these embodiments, a live, attenuated dengue-2 virus contains a mutation at position 53 of ns1 and position 250 of ns1 plus a dengue-1, 3 or 4 substitution of one or more structural proteins. In certain embodiments, a nucleic acid construct can be DENV-4e (SEQ ID NO:22), DENV-4h (SEQ ID NO:24) or DENV-4i (SEQ ID NO:9) capable of inducing an immune response to dengue-4 virus in a subject. It is contemplated herein that the structural sequences of dengue virus serotype 4 can be substituted using dengue-virus 1 or 3 and further contain the above referenced mutations for increasing an immune response to the construct.
Some embodiments concern a nucleic acid molecule having a chimeric flavivirus construct including a nucleic acid sequences encoding nonstructural proteins and at least one or more structural proteins from a live, attenuated dengue-2 virus and at least encoding one or more structural proteins from a second flavivirus, wherein the attenuated dengue-2 contains a mutation at position 53 of ns1 and position 250 of ns3 but does not contain mutations in NS2A or NS4A. In certain embodiments, a nucleic acid construct can be DENV-4f (SEQ ID NO:30). In other embodiments, nucleotide position 674 can be mutated to C from its wild-type nucleotide of G of DENV-4f. In yet other embodiments, a mixture of DENV-2/DENV-4 constructs of the instant application can be combined in a pharmaceutically acceptable composition of use as an immunogenic agent against dengue virus infection.
In certain embodiments, an attenuated dengue-2 virus backbone of DENV-2/DENV-4 constructs can further include one or more mutations/substitutions at positions 102-107 to a wild-type dengue 4 sequence (e.g. 1086) in a DENV-2/DENV-4 construct. For example, one or more of TITLLC at respective positions 102-107 from dengue-4 can replace wild type dengue-2 virus one or more of AGMIIM, at synonymous positions 102-107, respectively. In accordance with these embodiments, a DEN-2/DEN-4 construct having a substitution in this region can further include a mutation of cysteine to an aromatic amino acid (e.g. tyrosine, tryptophan etc.).
In other embodiments, a DENV-2/DENV-4 construct of any immunogenic compositions disclosed herein can be DENV-4g (SEQ ID NO:28) or DENV-4j (SEQ ID NO:32).
Immunogenic flavivirus chimeras having a dengue-2 virus backbone and at least one structural protein of dengue-4 virus can be used for preparing the dengue virus chimeras and methods for producing the dengue virus or flavivirus chimeras are described. The immunogenic flavivirus chimeras are provided, alone or in combination, in a pharmaceutically acceptable carrier as immunogenic compositions to minimize, inhibit, or immunize individuals against infection by one or more dengue virus or flaviviral strains, such as dengue virus serotypes DENV-4, alone or in combination with DEN-2, DEN-3 and DEN-1. When combined, the immunogenic flavivirus chimeras may be used as multivalent vaccines to confer simultaneous protection against infection by more than one species or strain of flavivirus. In certain embodiments, the flavivirus chimeras are combined in an immunogenic composition useful as a bivalent, trivalent or tetravalent vaccine against the known dengue virus serotypes or confer immunity to other pathogenic flaviviruses by including nucleic acids encoding one or more proteins from a different flavivirus. The nucleic acid sequence for each of the DEN-1, DEN-2, DEN-3 and DENV-4 viruses can be used to generate a probe for use in detecting dengue virus in a biological sample in order, for example, to assess efficacy of the vaccine and/or level of a dengue virus infection.
In some embodiments, avirulent, immunogenic flavivirus chimeras provided herein contain the nonstructural protein genes of the attenuated dengue-2 virus (e.g. PDK-53), or the equivalent thereof, and one or more of the structural protein genes or immunogenic portions thereof of the flavivirus against which immunogenicity is to be induced in a subject. For example, some embodiments concern a chimera having attenuated dengue-2 virus PDK-53 genome as the viral backbone, and one or more structural protein genes encoding capsid, premembrane/membrane, or envelope of the PDK-53 genome, or combinations thereof, replaced with one or more corresponding structural protein genes from DENV-4 virus or other flavivirus to be protected against, such as a different flavivirus or a different dengue virus serotype. In accordance with these embodiments, the PDK-53 backbone is further mutated or reverted to increase immunogenicity of the construct. Further, a nucleic acid chimera disclosed herein can have functional properties of the attenuated dengue-2 virus and is avirulent, but expresses antigenic epitopes of the structural gene products of DENV-4 in addition to other flaviviruses and is immunogenic (e.g. induces an immune response to the gene products in a subject). The mutations and/or reversions do not affect the attenuation and/or safety of the chimeric construct.
In another embodiment, a nucleic acid chimera can be a nucleic acid chimera having, but not limited to, a first nucleotide sequence encoding nonstructural proteins from an attenuated dengue-2 virus, and a second nucleotide sequence encoding a structural protein from dengue-4 virus alone or in combination with another flavivirus. In other embodiments, the attenuated dengue-2 virus can be vaccine strain PDK-53 or 16681. Some embodiments include structural proteins of one or more of C, prM or E protein of a dengue or other flavivirus. Examples of flaviviruses from which the structural protein may be selected include, but are not limited to, DEN-1, DEN-2, DEN-3, West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, yellow fever virus and tick-borne encephalitis virus in combination with the DENV-4 constructs disclosed herein. In other embodiments, the structural protein may be selected from non-flavivirus species that are closely related to the flaviviruses, such as hepatitis C virus.
Other aspects disclosed herein include that chimeric viruses can include nucleotide and amino acid substitutions, deletions or insertions for example, in the DEN-2 PDK-53; these changes can reduce interference with immunogenicity responses to DENV-4 virus. These modifications can be made in structural and nonstructural proteins alone or in combination with the example modifications disclosed herein.
Embodiments herein include structural and nonstructural proteins of a flavivirus that can be any protein including or any gene encoding the sequence of the complete protein, an epitope of the protein, or any fragment comprising, for example, five or more amino acid residues thereof.
Certain embodiments disclosed herein provide for method for making the chimeric viruses of this invention using recombinant techniques, by inserting the required substitutions into the appropriate backbone genome.
Flavivirus Chimeras
Dengue virus types 1-4 (DEN-1 to DENV-4) are mosquito-borne flavivirus pathogens. The flavivirus genome contains a 5′-noncoding region (5′-NC), followed by a capsid protein (C) encoding region, followed by a premembrane/membrane protein (prM) encoding region, followed by an envelope protein (E) encoding region, followed by the region encoding the nonstructural proteins (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5) and finally a 3′ noncoding region (3′NC) (See for example FIG. 1A). The viral structural proteins are C, prM and E, and the nonstructural proteins are NS1-NS5. The structural and nonstructural proteins are translated as a single polyprotein and processed by cellular and viral proteases.
Structure of Dengue Virus Genome




Flavivirus chimeras can be constructs formed by fusing non-structural protein genes from one type, or serotype, of dengue virus or virus species of the flaviviridae, with protein genes, for example, structural protein genes, from a different type, or serotype, of dengue virus or virus species of the flaviviridae. Alternatively, a flavivirus chimera disclosed herein is a construct formed by fusing non-structural protein genes from one type, or serotype, of dengue virus or virus species of the flaviviridae, with further nucleotide sequences that direct the synthesis of polypeptides or proteins selected from other dengue virus serotypes or other viruses of the flaviviridae.
In other embodiments, avirulent, immunogenic flavivirus chimeras provided herein contain the nonstructural protein genes of the attenuated dengue-2 virus, or the equivalent thereof, and one or more of the structural protein genes, or antigenic portions thereof, of the flavivirus against which immunogenicity is to be conferred.
Other suitable flaviviruses for use in constructing the flavivirus chimeras can be wild-type, virulent DEN-1 16007, DEN-2 16681, DEN-3 16562 and DENV-4 1036 and attenuated, vaccine-strain DEN-1 PDK-13, DEN-2 PDK-53, DEN-3 PMK-30/FRhL-3 and DENV-4 PDK-48. Genetic differences between the DEN-1, DEN-2, DEN-3 and DENV-4 wild type/attenuated virus pairs are contemplated along with changes in the amino acid sequences encoded by the viral genomes. Any DENV-4 strain of use herein would contain synonymous mutations to the constructs contemplated and/or disclosed herein.
Sequence listings for DEN-2 PDK-53 correspond to the DEN-2 PDK-53-V variant, wherein genome nucleotide position 5270 is mutated from an A to a T and amino acid position 1725 of the polyprotein or amino acid position 250 of the NS3 protein contains a valine residue. The DEN-2 PDK-53 variant without this nucleotide mutation, DEN-2 PDK-53-E, differs from PDK-53-V only in this one position. DEN-2 PDK-53-E has an A at nucleotide position 5270 and a glutamate at polyprotein amino acid position 1725, NS3 protein amino acid position 250. It is understood that embodiments herein can include modified DEN-2 PDK-53 that include one or more reversions/mutations of these positions to the native derived sequence.
Sequence listings for DEN-3 16562 correspond to the variant wherein genome nucleotide position 1521 is a T and amino acid position 476 of the polyprotein or amino acid position 196 of the E protein contain a leucine. A second variant, present in DEN-3 16562 cultures has a T at nucleotide position 1521 and amino acid position 476 of the polyprotein or amino acid position 196 of the E protein contain a serine.
Sequence listings for DENV-4 PDK-48 correspond to the variant wherein genome nucleotide positions: 6957 is a T and amino acid position 2286 of the polyprotein and amino acid position 44 of NS4B protein is a phenylalanine, 7546 is a T and amino acid position 2366 of the polyprotein and amino acid position 240 of NS4B protein is a valine, and 7623 is a T and amino acid position 2508 of the polyprotein and amino acid position 21 of NS5 protein is a tyrosine.
In certain embodiments, designations of the chimeras are based on the DEN-2 virus-specific infectious clone backbones and the structural genes (prM-E or C-prM-E) insert of other flaviviruses. DEN-2 for the dengue-2 backbone, followed by the strain from which the structural genes are inserted. The particular backbone variant is reflected in next. The particular DEN-2 backbone variant from which the chimera was constructed is indicated by the following letter placed after a hyphen, parent 16681 (P), PDK-53-E (E), or PDK-53-V (V); the last letter indicates the C-prM-E structural genes from the parental (P) strain or its vaccine derivative (V) or the prM-E structural genes from the parental (P) or its vaccine derivative (V1). For example; DEN-2/1-VP denotes the chimera comprising the attenuated DEN-2 PDK-53V backbone comprising a valine at NS3-250 and the C-prM-E genes from wild-type DEN-1 16007; DEN-2/1-VV denotes the DEN-2 PDK-53V backbone with the vaccine strain of dengue-1, DEN-1 PDK-13; DEN-2/1-VP1 denotes the DEN-2 PDK-53V backbone and the prM-E genes from wild-type DEN-1 16007; DEN-2/3-VP1 denotes the DEN-2 PDK-53V backbone and the prM-E genes from wild-type DEN-3 16562; DEN-2/4VP1 denotes the DEN-2 PDK-53V backbone and the prM-E genes from wild-type DENV-4 1036; and DEN-2/WN-PP1 denotes the DEN-2 16681 backbone and the prM-E genes from West Nile NY99. Other chimeras disclosed herein are indicated by the same manner.
In one embodiment, chimeras disclosed herein contain attenuated DEN-2 virus PDK-53 genome as the viral backbone, in which the structural protein genes encoding C, prM and E proteins of the PDK-53 genome, or combinations thereof, are replaced with the corresponding structural protein genes from DENV-4 virus and optionally, another flavivirus to be protected against, such as a different flavivirus or a different dengue virus strain. Newly discovered flaviviruses or flavivirus pathogens can also be incorporated into the DEN-2 backbone.
In the nonstructural protein regions, a Gly-to-Asp (wild type-to-PDK-53) mutation was discovered at nonstructural protein NS1-53 (genome nucleotide position 2579); a Leu-to-Phe (wild type-to-PDK-53) mutation was discovered at nonstructural protein NS2A-181 (genome nucleotide position 4018); a Glu-to-Val (wild type-to-PDK-53) mutation was discovered at nonstructural protein NS3-250 (genome nucleotide position 5270); and a Gly-to-Ala mutation (wild type-to-PDK-53) was discovered at nonstructural protein NS4A-75 (genome nucleotide position 6599).
Attenuated PDK-53 virus strain has a mixed genotype at genome nt 5270. A significant portion (approximately 29%) of the virus population encodes the non-mutated NS3-250-Glu that is present in the wild type DEN-2 16681 virus rather than the NS3-250-Val mutation. As both genetic variants are avirulent, this mutation may not be necessary in an avirulent chimera.
Previously, it was discovered that avirulence of the attenuated PDK-53 virus strain can be attributed to mutations in the nucleotide sequence encoding nonstructural proteins and in the 5′ noncoding region. For example, a single mutation at NS1-53, a double mutation at NS1-53 and at 5′NC-57, a double mutation at NS1-53 and at NS3-250 and a triple mutation at NS1-53, at 5′NC-57 and at NS3-250, result in attenuation of the DEN-2 virus. Therefore, the genome of any dengue-2 virus containing such non-conservative amino acid substitutions or nucleotide substitutions at these loci can be used as a base sequence for deriving the modified PDK-53 viruses disclosed herein. Another mutation in the stem of the stem/loop structure in the 5′ noncoding region will provide additional avirulent phenotype stability, if desired. Mutations to this region disrupt potential secondary structures important for viral replication. A single mutation in this short (only 6 nucleotide residues in length) stem structure in both DEN and Venezuelan equine encephalitis viruses disrupts the formation of the hairpin structure. Further mutations in this stem structure decrease the possibility of reversion at this locus, while maintaining virus viability.
Mutations disclosed herein can be achieved by any method known in the art including, but not limited to, site-directed mutagenesis, direct synthesis, deletion, or other method using techniques known to those skilled in the art. It is understood by those skilled in the art that the virulence screening assays, as described herein and as are well known in the art, can be used to distinguish between virulent and avirulent backbone structures.
Construction of Flavivirus Chimeras
Flavivirus chimeras described herein can be produced by splicing one or more of the structural protein genes of the flavivirus against which immunity is desired into a PDK-53 dengue virus genome backbone, or other methods known in the art, using recombinant engineering to remove the corresponding PDK-53 gene and replace it with a dengue-4 virus gene or other gene known in the art.
Alternatively, nucleic acid sequences of any construct disclosed herein, nucleic acid molecules encoding the flavivirus proteins, may be synthesized using any known nucleic acid synthesis techniques and inserted into an appropriate vector. Avirulent, immunogenic viruses of embodiments herein can therefore be produced using recombinant engineering techniques known to those skilled in the art.
A target gene can be inserted into the backbone that encodes a flavivirus structural protein of interest for DENV-4, alone or in combination with another flavivirus. A flavivirus (e.g. dengue virus) gene to be inserted can be a gene encoding a C protein, a PrM protein and/or an E protein. For example, a sequence inserted into the dengue-2 backbone can encode both PrM and E structural proteins, or just a single structural protein. A sequence inserted into the dengue-2 backbone can encode all or one of C, prM and E structural proteins.
Suitable chimeric viruses or nucleic acid chimeras containing nucleotide sequences encoding structural proteins of other flaviviruses or dengue virus serotypes can be evaluated for usefulness as vaccines by screening them for phenotypic markers of attenuation that indicate avirulence and by screening them for immunogenicity. Antigenicity and immunogenicity can be evaluated using in vitro and/or in vivo reactivity with flavivirus antibodies or immunoreactive serum using routine screening procedures known to those skilled in the art.
Dengue Virus Vaccines
In certain embodiments, chimeric viruses and nucleic acid chimeras can provide live, attenuated viruses useful as immunogens or vaccines. Some embodiments include chimeras that exhibit high immunogenicity to dengue-4 virus while producing no dangerous pathogenic or lethal effects.
To reduce occurrence of DHF/DSS in subjects vaccinated against only one serotype of dengue virus, a di-, tri or tetravalent vaccine is needed to provide simultaneous immunity for two to all four serotypes of the virus. A tetravalent vaccine can be produced by combining live, attenuated dengue-2 (e.g. dengue-2 PDK-53) with dengue-2/1, dengue-2/3, and dengue-2/4 novel chimeras described herein in a suitable pharmaceutical carrier for administration as a multivalent vaccine against all four dengue virus serotypes. Other formulations can include divalent or trivalent formulations of the above where the formulation includes one or more novel DENV-4 chimeric construct.
Chimeric viruses or nucleic acid chimeras disclosed in certain embodiments herein can include structural genes of either wild-type or attenuated viruses in a virulent or an attenuated DEN-2 virus backbone. For example, the chimera may express the structural protein genes of wild-type DENV-4 1036 virus, and its candidate vaccine derivative in either DEN-2 PDK-53 backgrounds. In certain embodiments, pharmaceutical or experimental compositions disclosed herein can include one or more constructs having the designation of DENVax-4e, DENVax-4g, and/or DENVax-4h alone, or in combination with other flavivirus constructs. In certain examples, these constructs can be used in combination with one or more master virus seed (MVS) constructs disclosed herein (e.g. DEN-1/DEN-2). Other embodiments can include DENV-4 constructs disclosed herein in combination with other flavivirus chimeras such as those made on a Yellow Fever backbone or West Nile backbone or other flavivirus backbone where these flavivirus chimeras are capable of forming a chimeric construct with a dengue virus serotype that when introduced to a subject induces an immune response to the virus in the subject.
Viruses used in the chimeras described herein can be grown using techniques known in the art. Virus plaque titrations are then performed and plaques counted in order to assess the viability and phenotypic characteristics of the growing cultures. Wild type viruses are passaged through cultured cell lines to derive attenuated candidate starting materials.
Chimeric infectious clones can be constructed from the various dengue serotype clones available. The cloning of virus-specific cDNA fragments can also be accomplished, if desired. The cDNA fragments containing the structural protein or nonstructural protein genes can be amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) from dengue virus RNA with various primers. Amplified fragments can be cloned into the cleavage sites of other intermediate clones. Intermediate, chimeric dengue virus clones can then be sequenced to verify the accuracy of the inserted dengue virus-specific cDNA.
In certain embodiments, full genome-length chimeric plasmids constructed by inserting the structural protein or nonstructural protein gene region of dengue serotype viruses into vectors are obtainable using recombinant techniques well known to those skilled in the art.
Nucleotide and Amino Acid Analysis
In certain embodiments, PDK-53, contains no amino acid mutations in the E protein relative to wild type dengue-2 virus; DEN-1, DEN-3 and DENV-4 attenuated viruses can have amino acid mutations in the E protein. Wild-type DEN-3 16562 has been demonstrated to comprise traces of a variant comprising a T at nucleotide position 1521 which directs incorporation of a leucine at polyprotein position 476, amino acid residue position 476 of the E protein. Each of the latter three viruses can possess a Glu-to-Lys (parent-to-vaccine) mutation in the E protein, although the mutation is located at a different amino acid residue in the E protein. This substitution causes a shift from a negatively charged amino acid to a positively charged one. The Glu-to-Lys substitution in the E protein of DENV-4 vaccine virus was the only mutation present in the E protein, while the E proteins of DEN-1 and DEN-3 vaccine viruses had five and three amino acid mutations, respectively.
In certain embodiments, an NS1-53 mutation occurs in the DEN-2 PDK-53 virus and is significant for the attenuated phenotype of this virus, because the NS1-53-Gly of the DEN-2 16681 virus is conserved in nearly all flaviviruses, including the tick-borne viruses, sequenced to date. DENV-4 virus constructs disclosed herein can contain an amino acid mutation in the NS1 protein at position 253. This locus, which is a Gln-to-His mutation in DENV-4 PDK-48 virus is Gln in all four wild serotypes of dengue virus. This Gln residue is unique to the dengue viruses within the flavivirus genus. The NS1 protein is a glycoprotein that is secreted from flavivirus-infected cells. It is present on the surface of the infected cell and NS1-specific antibodies are present in the serum of virus-infected individuals. Protection of animals immunized with NS1 protein or passively with NS1-specific antibody has been reported.
Certain mutations are found in NS2A, NS2B, NS4A, and NS4B proteins of the DEN-1, -2, -3 and -4 attenuated strains that are conservative in nature. The NS4A-75 and NS4A-95 mutations of DEN-2 and DENV-4 vaccine viruses, respectively, occurred at sites of amino acid conservation among dengue viruses, but not among flaviviruses in general.
Flaviviral NS3 protein possesses at least two recognized functions: the viral proteinase and RNA helicase/NTPase. The 698-aa long (DEN-2 virus) NS3 protein contains an amino-terminal serine protease domain (NS3-51-His, -75-Asp, -135-Ser catalytic triad) that is followed by sequence motifs for RNA helicase/NTPase functions (NS3-196-GAGKT), -284-DEAH, -459-GRIGR (SEQ ID NO:26), previously presented). None of the mutations in the NS3 proteins of DEN-1, DEN-2, or DEN-3 virus occur within a recognized motif. NS3-510 Tyr-to-Phe mutation in DEN-1 PDK-13 virus is a conservative mutation. Since the wild-type DEN-2, -3 and -4 viruses contain Phe at this position, it is unlikely that the Tyr-to-Phe mutation plays a role in the attenuation of DEN-1 virus. The NS3-182 Glu-to-Lys mutation in DEN-1 PDK-13 virus occurred at a position that is conserved as Asp or Glu in most mosquito-borne flaviviruses and it may play some role in attenuation. This mutation was located 15 amino acid residues upstream of the GAGKT (SEQ ID NO:27) helicase motif. In certain dengue-2 viruses, the NS3-250-Glu in DEN-2 16681 virus is conserved in all mosquito-borne flaviviruses except for yellow fever virus.
Nucleic acid probes of use in certain embodiments herein selectively hybridize with nucleic acid molecules encoding the DEN-1, DEN-3 and DENV-4 viruses or complementary sequences thereof. By “selective” or “selectively” is meant a sequence which does not hybridize with other nucleic acids to prevent adequate detection of the dengue virus. Therefore, in the design of hybridizing nucleic acids, selectivity will depend upon the other components present in a sample. Hybridizing nucleic acids should have at least 70% complementarity with the segment of the nucleic acid to which it hybridizes. As used herein to describe nucleic acids, the term “selectively hybridizes” excludes the occasional randomly hybridizing nucleic acids, and thus, has the same meaning as “specifically hybridizing.” The selectively hybridizing nucleic acids disclosed herein can have at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, and 99% complementarity with the segment of the sequence to which it hybridizes, preferably 85% or more.
Sequences, probes and primers which selectively hybridize to the encoding nucleic acid or the complementary, or opposite, strand of the nucleic acid are contemplated. Specific hybridization with nucleic acid can occur with minor modifications or substitutions in the nucleic acid, so long as functional species-specific hybridization capability is maintained. By “probe” is meant nucleic acid sequences that can be used as probes or primers for selective hybridization with complementary nucleic acid sequences for their detection or amplification, which probes can vary in length from about 5 to 100 nucleotides, or preferably from about 10 to 50 nucleotides, or most preferably about 18-24 nucleotides.
If used as primers, the composition preferably includes at least two nucleic acid molecules which hybridize to different regions of the target molecule so as to amplify a desired region. Depending on the length of the probe or primer, the target region can range between 70% complementary bases and full complementarity and still hybridize under stringent conditions. For example, for the purpose of detecting the presence of the dengue virus, the degree of complementarity between the hybridizing nucleic acid (probe or primer) and the sequence to which it hybridizes is at least enough to distinguish hybridization with a nucleic acid from other organisms.
Nucleic acid sequences encoding the DENV-4, DEN-3 or DEN-1 virus can be inserted into a vector, such as a plasmid, and recombinantly expressed in a living organism to produce recombinant dengue virus peptides and/or polypeptides.
Nucleic Acid Detection Methods
A rapid genetic test that is diagnostic for each of the vaccine viruses described herein is provided by the current invention. This embodiment of the invention enhances analyses of viruses isolated from the serum of vaccinated humans who developed a viremia, as well as enhancing characterization of viremia in nonhuman primates immunized with the candidate vaccine viruses.
These sequences include a diagnostic TaqMan probe that serves to report the detection of the cDNA amplicon amplified from the viral genomic RNA template by using a reverse-transciptase/polymerase chain reaction (RT/PCR), as well as the forward and reverse amplimers that are designed to amplify the cDNA amplicon, as described below. In certain instances, one of the amplimers has been designed to contain a vaccine virus-specific mutation at the 3′-terminal end of the amplimer, which effectively makes the test even more specific for the vaccine strain because extension of the primer at the target site, and consequently amplification, will occur only if the viral RNA template contains that specific mutation.
Automated PCR-based nucleic acid sequence detection system can be used, which is becoming widely used in diagnostic laboratories. The TaqMan assay is a highly specific and sensitive assay that permits automated, real time visualization and quantitation of PCR-generated amplicons from a sample nucleic acid template. TaqMan can determine the presence or absence of a specific sequence. In this assay, a forward and a reverse primer are designed to anneal upstream and downstream of the target mutation site, respectively. A specific detector probe, which is designed to have a melting temperature of about 10° C., higher than either of the amplimers and containing the vaccine virus-specific nucleotide mutation or its complement (depending on the strand of RT/PCR amplicon that is being detected), constitutes the third primer component of this assay. A probe designed to specifically detect a mutated locus in one of the chimeric constructs can contain a specific nucleotide change for detecting any mutation.
One strategy for diagnostic genetic testing makes use of molecular beacons. The molecular beacon strategy also utilizes primers for RT/PCR amplification of amplicons, and detection of a specific sequence within the amplicon by a probe containing reporter and quencher dyes at the probe termini. In this assay, the probe forms a stem-loop structure. The 5′- and 3′-terminal reporter dye and quencher dye, respectively, are located at the termini of the short stem structure, which brings the quencher dye in close juxtaposition with the reporter dye. The stem-structure is melted during the denaturation step of the RT/PCR assay. If the target viral RNA contains the target sequence and is amplified by the forward and reverse amplimers, the opened loop of the probed hybridizes to the target sequence during the annealing step of the cycle. When the probe is annealed to either strand of the amplicon template, the quencher and reporter dyes are separated, and the fluorescence of the reporter dye is detected. This is a real-time identification and quantitation assay that is very similar to the TaqMan assay. The molecular beacons assay employs quencher and reporter dyes that differ from those used in the TaqMan assay.
Pharmaceutical Formulations
Any pharmaceutical formulation known in the art for a vaccine is contemplated herein. In certain embodiments, a formulation can contain, DENV-4 constructs alone or one or more additional DEN serotype (or other flavivirus compositions) in various ratios in combination with DENV-4 constructs disclosed herein, depending on predetermined exposure to or existence of dengue virus subtype prevalence in a region. It is contemplated that formulations can contain other agents of use in vaccination of a subject including, but not limited to other active or inactive ingredients or compositions known to one skilled in the art. In certain embodiments, an adjuvant may be included in a formulation disclosed herein.
Other aspects of the present invention can include modulating an immune response to a vaccine against dengue virus to a subject. Vaccines against dengue virus may include a composition comprising ratios of serotypes of dengue virus, live attenuated dengue virus, or fragments thereof such as proteins or nucleic acids derived or obtained from dengue virus serotypes. Ratios of various serotypes may be equal or certain serotypes may be represented more than others depending on need or exposure or potential exposure to the virus. In accordance with these embodiments, a ratio may be a 1:2, 1:3, 1:4, 1:10, 1:20; 1:1:1, 1:2:2, 1:2:1, 1:1:1:1, 1:2:1:2; 1:3:1:3, 2:3:3:3, 5:4:5:5, 4:4:4:5, 1:2:2, 4:4:5:5, 4:4:5:6 or any ratio for any of serotypes 1, 2, 3 in combination with the DENV-4 constructs disclosed herein, depending on for example, number of serotypes represented in the formulation, predetermined response and effect desired. The last number represents the amount of DENV-4 construct in a formulation. Each number represents the power of ten (6=106 PFU). It is contemplated that any dengue virus serotype formulation may be used to generate a vaccine (e.g. attenuated virus etc.) of use in administration to a subject in need thereof.
Embodiments herein provide for administration of compositions to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo. By “biologically compatible form suitable for administration in vivo” is meant a form of the active agent (e.g. pharmaceutical protein, peptide, or gene etc. of the embodiments) to be administered in which any toxic effects are outweighed by the therapeutic effects of the active agent. Administration of a therapeutically active amount of the therapeutic compositions is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example, a therapeutically active amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of antibody to elicit a desired response in the individual. Dosage regima may be adjusted to provide the optimum therapeutic response.
In one embodiment, the compound (e.g. pharmaceutical protein, peptide etc. of the embodiments) may be administered in a convenient manner such as subcutaneous, intravenous, intradermal, by oral administration, inhalation, transdermal application, intradermal, intravaginal application, topical application, intranasal or rectal administration. Depending on the route of administration, the active compound may be coated in a material to protect the compound from degradation by enzymes, acids and other natural conditions that may inactivate the compound. In one embodiment, the compound may be administered intranasally, such as inhalation.
A compound may be administered to a subject in an appropriate carrier or diluent, co-administered with enzyme inhibitors or in an appropriate carrier such as liposomes. The term “pharmaceutically acceptable carrier” as used herein is intended to include diluents such as saline and aqueous buffer solutions. It may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. The active agent may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
Pharmaceutical compositions suitable for injectable use may be administered by means known in the art. For example, sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion may be used. In all cases, the composition can be sterile and can be fluid to the extent that easy syringability exists. It might be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms such as bacteria and fungi. The pharmaceutically acceptable carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of microorganisms can be achieved by heating, exposing the agent to detergent, irradiation or adding various antibacterial or antifungal agents.
Sterile injectable solutions can be prepared by incorporating active compound in the required amount with one or a combination of ingredients enumerated above, as required.
Aqueous compositions can include an effective amount of a therapeutic compound, peptide, epitopic core region, stimulator, inhibitor, and the like, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Compounds and biological materials disclosed herein can be purified by means known in the art.
Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above. It is contemplated that slow release capsules, timed-release microparticles, and the like can also be employed. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
The active therapeutic agents may be formulated within a mixture to comprise about 102 to about 5×106 PFU of each construct contemplated herein. Single dose or multiple doses can also be administered on an appropriate schedule for a predetermined condition. In certain embodiments, a dual dose on day 0 may be administered in single anatomical or multiple anatomical locations in order to induce an immune response with reduced interference or different lymph nodes. In certain embodiments, a mono-, bi-, tri- or tetravalent formulation of dengue virus constructs may be administered to a subject. Any of these formulations can be provided to a subject as a single or in multiple doses. In certain embodiments, one dose can be administered then a boost some time later may be provided.
In another embodiment, nasal solutions or sprays, aerosols or inhalants may be used to deliver the compound of interest. Additional formulations that are suitable for other modes of administration include suppositories and pessaries. A rectal pessary or suppository may also be used. In general, for suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% 2%.
The pharmaceutical compositions containing the α1-antitrypsin, analog thereof, or inhibitor of serine protease activity or a functional derivative thereof may be administered to individuals, particularly humans, for example by subcutaneously, intramuscularly, intranasally, orally, topically, transdermally, parenterally, gastrointestinally, transbronchially and transalveolarly.
In certain embodiments of the methods of the present invention, the subject may be a mammal such as a human or a veterinary and/or a domesticated animal.
In one embodiment of the present invention, methods provide for vaccinating a subject preparing to travel to a country with dengue virus. In other embodiments, a subject may be a resident in an endemic area. It is contemplated that a subject may be administered a single injection or dual injections on day 0, optionally followed by a boost less than 30 days, 2 months, 3 months, 6 months or as much as one year later.
Kits
Other embodiments concern kits of use with the methods (e.g. methods of application or administration of a vaccine) and compositions described herein. Some embodiments concern kits having vaccine compositions of use to prevent or treat subjects having, exposed or suspected of being exposed to one or more dengue viruses. In certain embodiments, a kit may contain one or more than one formulation of dengue virus serotype(s) (e.g. attenuated vaccines) at predetermined ratios. Kits can be portable, for example, able to be transported and used in remote areas such as military installations or remote villages. Other kits may be of use in a health facility to treat a subject having been exposed to one or more dengue viruses or suspected of being at risk of exposure to dengue virus.
Kits can also include a suitable container, for example, vials, tubes, mini- or microfuge tubes, test tube, flask, bottle, syringe or other container. Where an additional component or agent is provided, the kit can contain one or more additional containers into which this agent or component may be placed. Kits herein will also typically include a means for containing the agent, composition and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. Optionally, one or more additional agents such as immunogenic agents or other anti-viral agents, anti-fungal or antibacterial agents may be needed for compositions described, for example, for compositions of use as a vaccine against one or more additional microorganisms.
Embodiments of the present invention are further illustrated by the following non-limiting examples, which are not to be construed in any way as imposing limitations upon the scope thereof. It should be appreciated by those of skill in the art that the techniques disclosed in the Examples which follow represent techniques discovered to function well in the practices disclosed herein, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope herein.
EXAMPLES
Example 1
In certain exemplary methods, DENV-4 chimera constructs are generated of use in pharmaceutically acceptable compositions.
FIG. 1A illustrates an exemplary viral backbone of some embodiments disclosed herein referred to as DEN-2 PDK-53 genome (previously disclosed in PCT/US01/05142, U.S. application Ser. No. 10/204,252, each incorporated herein by reference in their entirety for all purposes) which can be used to generate modified DENV-4 constructs (also indicated as a second generation). As shown in FIG. 1A, specific amino acid substitution mutations in the nonstructural regions, such as NS1-G53D, NS3-E250V and a nucleotide substitution mutation in the 5′ non-coding region, C57T in the DEN-2 PDK-53 genome have been identified. Original DENV-1, 3, and 4 constructs (also indicated as first generation) were generated by replacing the coding sequence of prM and E in the DENV-2 backbone with structural coding sequences of respective serotypes (FIG. 1B). To generate modified DENVax-4 constructs that can boost replication efficiency in vitro and in vivo, and improve immunogenicity in the host, point mutations were introduced to the non-coding region, structural protein coding sequences and/or non-structural regions of the original DENVax-4 construct. For example, modifications can be made to the amino acid sequence upstream of the current DENVax-4 C/prM cleavage site to mimic wild-type dengue-4 as opposed to dengue-2 (which DENVax-4 currently contains).
Some additional modifications in certain exemplary DENV-4 constructs are provided in Table 1. Illustrated below are sequences included in certain modified DENVax-4 constructs (DENVax-4b, DENVax-4c, and DENVax-4d) aligned to the wild type sequence of DENV-2, DENV-1 and original DENVax-4. The selected changes in these sequences are in bold and underlined:












       C-100      Capsid
---
 prM













DENV-2:
NILNRRRRSAGMIIMLIPTVMA

FHLTTRN
SEQ ID NO: 1



 


DENV-4 WT:
NILNGRKRSTITLLCLIPTVMA

FHLSTRD
SEQ ID NO: 2


 


DENVax-4ori:
NILNRRRSSAGMIIMLIPTVMA

FHLTTRD
SEQ ID NO: 3


 


DENVax-4b:
NILNRRRSSTITLLCLIPTVMA

FHLSTRD
SEQ ID NO: 4


 


DENVax-4c:
NILNGRKRSTITLLCLIPTVMA

FHLSTRD
SEQ ID NO: 5


 


DENVax-4d:
*ILNGRKRSTITLLCLIPTVMA

FHLSTRD
SEQ ID NO: 6






FIG. 4 presents a schematic map of the original and modified DENV-4 constructs. DENVax-4e is generated using DENVax-4b sequence as a backbone, and further incorporates a C to Y mutation at amino acid 107 of Capsid protein. DENVax4f, DENVax4g and DENVax4j were built on the DENVax-4a backbone, but contain wild type reversions of the attenuating mutations of the PDK-53 sequence as indicated in FIG. 4: DENVax-4f contains wild type (DEN-2 16681) NS2A and NS4A; DENVax-4g contains wild type 5′NCR, NS2A and NS4A; and DENVax-4j contains wild type 5′NCR. DENVax4h has an E to K substitution at Envelope 417 in the DENV4 1036 sequence. DENVax4i has a methionine to leucine amino acid substitution at NS4A position 17 (synonymous position 6424 A to T of the genomic nucleic acid or amino acid position 2110 of the construct). DENVax4k has the PrM/E genes from DENV-4 H241 strain instead of 1036 strain (Table 1). Mutations constructed in DENVax4e, h, and i were based on sequence data from DENVax4 and DENVax-4b serial passages in Vero cells.
DENVax-4b contains 7 total amino acid changes, DENVax-4c contains 9 total amino acid changes, and DENVax-4d contains an amino acid deletion resulting in a frame shift of the sequence. RNA was transcribed and electroporated into Vero cells for amplification and virus rescue. DENVax-4b and DENVax-4c were tested for growth efficiency in Vero and C6/36 cells in a growth kinetics experiment, with DENVax4-P2, DENVax4-P8 and DENVax2-P2 used as controls. DENVax-4-P2 and DENVax-2-P2 are virus samples that have not been genotypically selected by viral plaque purification, a process in which individual viral plaques are picked from a DENVax-infected Vero cell monolayer, and then over-layed with agarose gel containing neutral red to visualize single plaques. DENVax-4-P8 had been selected by plaque purification to obtain a virus stock with a clonal DENVax-4 genotype. This procedure is done to generate a master seed virus (MVS) with no reverted attenuating mutations. DENVax-4c did not reach an adequate peak titer after growth in Vero cells, and had a slower initial growth rate than either DENVax-4 or DENVax-4b. There was no significant difference between the peak titers of DENVax-4 and DENVax-4b in the Vero growth curve, and both had similar initial growth rates. In the analysis C6/36 mosquito cells, growth of DENVax-4b and DENVax-4c both reached peak titers that were significantly less than wild type DENV-4, confirming their attenuation.







TABLE 1







Exemplary DENV-4 chimeric constructs: Differences in the constructs


compared to DENV-4/DEN-2 PDK-53 previously disclosed








Exemplary



DENV-4



Constructs
Modifications





DENVax-4e
Capsid 107 C to Y mutation at position 416 with



G mutated to A of DENVax-4b backbone


DENVax-4f
Attenuating mutations in NS2 and NS4 reverted to



corresponding sequences of Dengue virus strain



16681


DENVax-4g
Attenuating mutations in 5′ NCR, NS2 and NS4



reverted to corresponding sequences of Dengue



virus serotype 2 strain 16681


DENVax-4h
Envelope 417 E to K mutation at position 2185 with



G mutated to A of DENVax-4b backbone


DENVax-4i
NS4A 17 M to L mutation at position 6424 with A



mutated to T of DENVax-4b backbone


DENVax-4j
Attenuating mutations in 5′ NCR reverted to



corresponding sequences of Dengue virus serotype 2



strain 16681


DENVax-4k
Coding sequence of prM and E of DENV-2 backbone



replaced with structural coding sequences of Dengue



virus serotype 4 strain H241









Example 2
Generation of Full-Length Infectious cDNA Clones
To generate full-length infectious cDNA clones containing modified DENVax-4 construct sequences, a multi-step digestion/ligation scheme can be used. It can have the following steps: 1) insert the AgeI/MluI synthetic fragment including a modified nucleic acid sequence or a modified structural protein coding sequence (for example, b, c or d, or any of the above constructs) into pD2/3-PP1-5′ to generate pD2/4i-b, c or d; 2) digest the pDENVax-4 full length cDNA clone to extract the MluI/NgoMIV fragment and insert it into the corresponding position of pD2/4i-b, c or d to obtain pD2/4i-b, c or d; 3) digest the pDENVax-4 full length cDNA clone to extract NgoMIV/XbaI fragment and insert it into the corresponding position of pD2/4i-b, c or d to generate the full length infectious clones containing modified sequences. The sequences of the final full-length infectious clones were confirmed by sequence analysis.
Virus Generation
The cDNA clones for each of the modified construct was transcribed into genomic viral RNA. The RNA was transformed into Vero cells by electroporation. The viruses were grown for 12 days while monitoring CPE, and harvested. This first harvest after electroporation was termed P1 (Passage 1). The subsequent amplifications and passages were called P2, P3, etc. There was limited CPE for the original DENVax-4, and DENVax-4b, and c, whereas DENVax-4d did not generate any noticeable CPE. Unlike other viruses when grown in vitro, the dengue viruses do not produce much CPE when/grown in Vero cells. Although the DENVax-4d virus did not generate any CPE in vitro, it was amplified in parallel with the other strains. Amplified P1 viruses (for example, DENVax-4b, c, d P1) produced high enough titers to perform sequence analysis and growth curve experiments. DENVax-4d had no titer and therefore, no virus was made after electroporation.
The DENVax-4b and -4c viruses were fully sequenced. The DENVax-4b-P2 virus had two mutations. Nucleotide 416 was at the position of a modification engineered in the capsid (near the C/prM junction) of the DENVax-4b virus. Since this was a mixed population, the nt 416 was reverting back to the “A” nucleotide instead of the engineered “G” nucleotide, causing the expected amino acid arginine to instead be a lysine. The second mutation found in the DENVax-4b-P2 virus was at nucleotide 8769. This caused a change in the amino acid from the expected glutamine to a proline. This mutation was in the NS5 gene region of the infectious clone. The DENVax-4c-P2 virus had four mutations. They were all complete conversions, unlike the DENVax-4b virus that had mixed populations. The mutation at nucleotide 400 in the capsid region of the genome affected an engineered modification at the C/prM junction. This caused the expected amino acid at that position to be a proline instead of threonine. The other three mutations were in the nonstructural genes, two of which caused amino acid changes, and one that was a silent mutation.
Phenotypic and Genetic Characterization of the Viruses
Phenotypic characterization of the viruses was performed in Vero cells and C6/36 (Aedes albopictus) cells. FIGS. 5-6 represent exemplary phenotypic character of the virus variants generated from the modified DENVax-4 constructs. Plaque size is an important attenuation marker, and was analyzed for each of the virus variants. As shown in FIG. 5, the plaque size of the DENVax-4b viruses was roughly 0.3 cm in diameter. The plaque size of the DENVax-4c viruses was about 0.1 cm in diameter. The plaque size of the DENVax-4h viruses was about 0.3 cm in diameter. The plaque size of the DENVax-4i viruses was about 0.1 cm in diameter. The plaque size of the DENVax-4c viruses was about 0.2 cm in diameter. Each of the sizes represents an average of 5-10 plaques. The plaques in the b and c candidate viruses, especially DENVax-4c were not homogenous. There was a mixed population of viruses with some small and some large plaques. The sequence variability of these small and large plaques can be characterized for possible nucleotide changes which would contribute to attenuation and fitness in vitro. If needed, these mutations could be engineered into further DENVax-4 variants. FIGS. 6A-6F present exemplary results of ImmunoFoci of the wild type DENV-4 virus (A) and each of the DENVax-4b-f virus variants (B-F).
In some exemplary methods, growth curves of DENV-4 constructs generated viruses were determined. Monolayers of Vero cells were infected at a MOI of 0.001 with various DENV-4 construct compositions (for example, DENVax-4b-j, and DENVax-4P1). In some exemplary experiments, samples were taken every other day through day 13, and aliquots were titrated. The DENVax-2-P2 virus reached peak titers more rapidly. The DENVax-4b virus was similar in peak titer and growth rate to the DENVax-4-P2 and P8 viruses. The DENVax-4c virus initially had a slower growth rate, but it finally reached a similar peak titer. The efficiency and peak titers of the DENVax-4b and DENVax-4c viruses were comparable to the original DENVax-4. In other exemplary experiments, samples were obtained every other day from day 2 through day 12. Harvested media was retained and stabilized at days 7, 9 and 11 for further study. Samples from day 2 to 12 were titered by IFA. The DENVax-4e-4h viruses showed similar peak titer to control DENVax-4 (FIG. 7).
To demonstrate attenuation, the replication efficiency of each vaccine virus should be decreased in C6/36 mosquito cells as compared to the wild-type virus. This phenotype is an essential safety feature of DENVax vaccine viruses, to ensure that there is no potential for transmission of the attenuated chimeric viruses in nature. In some exemplary methods, growth in C6/36 mosquito cells was conducted to compare the growth characteristics of the DENVax4b and DENVax-4c viruses to the wild-type dengue 4 virus (strain 1036). In other exemplary experiments, comparison was done between DENVax4e-4j and original DENVax4. Duplicate flasks of C6/36 cells were infected at a MOI of 0.001 with each of the P2 viruses (-b, -c, and wild-type) and grown for 14 days. The dengue 4 wild-type virus (WT D4 1036) replicated most efficiently and to the highest titer. The DENVax-4b virus replicated reasonably well in the C6/36 cells, reaching a peak titer of 2.7×106 pfu/mL by day 14. DENVax4c virus was very slow growing until after day 6 when growth was accelerated until day 14, and reaching a peak titer of 2.2×104 pfu/mL. At day 6, both DENVax-4b and DENVax4c were similarly attenuated for growth as compared to the wild-type in C6/36 cells, and had similar titers to the original DENVax-4. In other exemplary experiments, chimeric dengue viruses of certain embodiments herein were grown in C6/36 mosquito cells. Growth in the mosquito cells DENVax4e-4j was compared to a control DENVax4. Duplicate flasks of C6/36 cells were infected at a MOI of 0.001 with each of the P2 viruses and grown for 12 days. Samples were harvested on day 2 through day 12. Growth in the mosquito cells were compared to growth in Vero cell. Culture media was obtained and stabilized at days 7, 9 and 11. Samples from day 2 to 12 were titered by IFA and analyzed for virus production. Virus titer and growth of the constructs were compared to control DENVax4 (see FIG. 8).
FIGS. 7-8 represent exemplary graphs illustrating growth curves of viruses of various DENV4 constructs in Vero cells (FIG. 7) and in C6/36 mosquito cells (FIG. 8). Several novel DENVax4 constructs of certain embodiments herein produced higher titers in Vero cells (FIG. 7) than the control DENVax4 and lower titers in mosquito cells (FIG. 8) than control DENVax4, as desired for further assessment in animal models (see Table 2 below).
Safety and Efficacy of the Virus Variants in Mice
Studies were performed to analyze the immunogenicity of DENVax-4b, c or other variant viruses. Table 2 presents an exemplary study design. Groups of 10 AG129 mice were vaccinated intradermally (in the footpad) with 104 PFU each of the original DENVax-4, and the separate variants. A control group was injected with excipient solution only. These mice received a booster dose after 42 days. Serum samples for serology were taken on days 42 and 56 post primary inoculation, and seroconversion was analyzed by PRNT. There was no significant improvement in the immunogenicity of DENVax-4 when using the second generation DENVax-4b and c variants as immunogens.







TABLE 2







Summary of Mouse Study Design












Number of
Number of












Group
Dose
immunizations
animals














1
DENVax-4
104 PFU
2
10


2
DENVax-4b
104 PFU
2
10


3
DENVax-4c
104 PFU
2
10


4
TFA
NA
2
10









To further modify and amplify the immunogenicity of DENVax-4b, this virus was adapted in vitro to Vero cell growth. This was performed by 10 blind passages in Vero cells, with the hypothesis that adaptation in mammalian cell culture would boost the replicative capacity of the virus and thus its immunogenicity. This “new” second generation DENVax-4 was termed DENVax-4b-P10 (Passage-10).
Another study was performed to evaluate different tetravalent formulations of dengue virus vaccine including three separate DEN4 viruses; first generation, second generation (4b construct) and the homologous wildtype DEN4 1036. Groups of 6 mice were injected intradermally with a tetravalent vaccine formulation containing 104 PFU DENVax-1, 103 PFU DENVax-2, 104 PFU DENVax-3 and 105 PFU DENVax 4 (4:3:4:5) in a 50 μL volume. Control mice were injected by the same route with 105 PFU of 1st generation, 2nd generation or wildtype 1036 monovalent DENV4. A group of mice injected with diluent only was included as a control. All mice received a booster injection on day 42 with the corresponding vaccine formulation, and mice were bled on days 0, 21, 41 and 56 post primary inoculation to assess the presence of neutralizing antibodies against all four DENV serotypes. On day 56, mice from groups 4, 5 and 6 were challenged with DENV-2 (NGC strain) to assess for survival (there is currently no mouse-adapted lethal DENV4 strain to use for efficacy analysis). As illustrated in Table 3, the first and second generation DENVax-4 vaccines have comparable immunogenic profile when administered alone. However, when they are given in the context of tetravalent dengue virus vaccine, immune responses to DENV-4 are diminished due to interference affecting only the second generation DENVax-4. Wild type DENV4 was highly immunogenic and in the context of tetravalent dengue virus vaccine it interferes and suppresses the neutralizing antibody responses elicited by the other three dengue virus vaccines. Wild type DENV4, the first generation and the second generation DENVax-4 provided partial protection against heterologous DENV-2 challenge.
Safety and Efficacy of Second Generation DENVax in NHP







TABLE 3







Mouse study #2 study design and PRNT results














Group
Dose
DENV1
DENV2
DENV3
DENV4
















1
DENVax (1ST 
4:3:4:5
1280
640
>1280
640



generation DENVax-4)

(320)
(320)
(320)
(160)


2
DENVax (DENVax-4 
4:3:4:5
160
160
320
640



was replaced with wild 

(320)
(160)
(160)
(640)



type DENV-4)







3
DENVax (DENVax-4 
4:3:4:5
>1280
640
>1280
80



was replaced with 2nd 

(640)
(320)
(640)
 (80)



generation DENVax-4)







4
DENVax-4 (2nd 
10{circumflex over ( )}5
X
X
X
320



generation)




 (80)


5
Wild type DENV-4
10{circumflex over ( )}5
X
X
X
640








(320)


6
DENVax-4 (1st 
10{circumflex over ( )}5
X
X
X
320



generation)




 (80)









The immunogenicity of DENVax in groups of four Cynomologus macaques was evaluated after subcutaneous injection of each tetravalent DENVax formulation (Table 4 and FIG. 2) in 0.5 mL injection volume. The goal of this study was to evaluate the lead second generation DENVax-4 construct, as compared with the first generation DENVax-4. Additionally, different methods were explored to improve the neutralizing antibody responses to DENV-4 elicited by DENVax. These included testing a formulation which contained 10× the dose of DENVax-4 as used previously (termed “new formulation”), and also lowering the DENVax-2 component of the tetravalent mixture, as this vaccine is the most immunogenic of the four vaccine virus strains. Different dosing days were tested, and priming and boosting on the same day were compared (Day 0) to priming on Day 0 and boosting on Day 60. Samples for serology were taken on days 0, 28, 58, 73, and 90. The kinetics of neutralizing antibody titers against DENV-4 variant virus and the original was shown in FIG. 3 as an example, where comparable levels were observed.







TABLE 4







NHP Study Design:










Groups
Vaccine Source
Formulation
Treatment





1
DENVax Formulation
2e4, 5e4, 1e5, 3e5
2 dose (day 0)



(Clinical Stock)




2
DENVax Formulation
2e4, 5e4, 1e5, 3e5(4b)
2 dose (day 0)



(4b-P10)




3
DENVax Formulation
2e4, 5e4, 1e5, 3e6
2 dose (day 0)



(Clinical Stock)





w/10x DENVax-4




4
DENVax Formulation
2e4, 5e4, 1e5, 3e6
2 dose (0, 60)



(Clinical Stock)





w/10x DENVax-4




5
DENVax New
2e4, 1e4, 1e5, 3e6
2 dose (0, 60)



formulation









Vaccine formulations for the NHP studies were prepared in bulk. Group 1 received vaccine which was cGMP manufactured and is identical to the vaccine which is currently being tested in human clinical trials. Vaccines were given to the NHPs and subsequently back-titrated to determine the actual dose. These results are presented in Table 5 below.







TABLE 5







Vaccine Stocks-back titration results:
















desired



desired




iFFU/dose
dose

desired dose
back
dose
back


DENVax
GP1
GP2
back titration
GP3 and 4
titration
GP5
titration





D1
2.00E+04
2.00E+04
1.90E+04
2.00E+04
1.80E+04
2.00E+04
1.80E+04


D2
5.00E+04
5.00E+04
2.60E+04
5.00E+04
2.40E+04
1.00E+04
3.60E+03


D3
1.00E+05
1.00E+05
1.80E+05
1.00E+05
1.40E+05
1.00E+05
1.70E+05


D4
3.00E+05
300000
2.10E+05
3.00E+06
2.90E+06
3.00E+06
3.00E+06




(4b)









Samples for viremia were taken after the primary dose on days −11 (baseline), 3, 5, 7, 9, 11, 13, 15, 17, 21, 28, 58, 62 and 66. RNA was extracted from the serum sample and virus titer was determined by tetraplex qRT-PCR assay. The only virus which gave any detectable viremia was DENVax-2, and this resolved by day 21 after primary vaccination. No virus was detected after the booster dose of vaccine was administered.
Serology was evaluated using a high throughput PRNT assay in a 96 well plate. Comparison of the first and second generation DENVax-4 (Group 1 compared to group 2) showed no significant difference in immunogenicity of these two viruses (Table 6).







TABLE 6







Geometric Mean Titers of NHP study.

















Group
DEN
d0
d28
d58
d73
d90
d128
d149
d181
d210




















1
1
5
226.3
134.5
160.0
95.1
67.3
95.1
226.3
226.3


5455a
2
5
1810.2
1280.0
905.1
905.1
761.1
761.1
761.1
380.5


0.0
3
5
226.3
190.3
320.0
226.3
95.1
134.5
226.3
160.0



4
5
28.3
20.0
134.5
56.6
23.8
33.6
113.1
67.3


2
1
5
33.6
16.8
47.6
40.0
23.8
23.8
20.0
67.3


5455b
2
5
1076.3
1280.0
905.1
1076.3
1076.3
1280.0
640.0
640.0


0.0
3
5
67.3
33.6
226.3
80.0
23.8
28.3
28.3
33.6



4
5
23.8
11.9
80.0
47.6
23.8
33.6
23.8
20.0


3
1
5
80.0
47.6
56.6
67.3
40.0
33.6
134.5
134.5


5456
2
5
380.5
320.0
538.2
538.2
190.3
508.0
269.1
269.1


0.0
3
5
95.1
80.0
134.5
95.1
40.0
40.0
160.0
113.1



4
5
95.1
134.5
160.0
160.0
134.5
134.5
320.0
226.3


4
1
5
16.8
16.8
80.0
56.6
23.8
23.8
16.8
20.0


5456
2
5
134.5
134.5
269.1
320.0
134.5
134.5
95.1
80.0


0.60
3
5
23.8
23.8
134.5
95.1
23.8
20.0
28.3
28.3



4
5
23.8
20.0
134.5
113.1
56.6
80.0
28.3
28.3


5
1
5
20.0
11.9
134.5
134.5
33.6
20.0
28.3
14.1


5356
2
5
28.3
8.4
95.1
80.0
10.0
5.9
5.0
5.0


0.60
3
5
23.8
11.9
190.3
190.3
23.8
28.3
33.6
28.3



4
5
33.6
33.6
269.1
134.5
113.1
56.6
80.0
67.3









Evaluating a higher dose of DENVax-4 (Group 3) revealed that a significant difference in the kinetics of DENV-4 neutralizing antibody responses could be obtained. The peak titers remained roughly equivalent (within a 2-fold dilution range) when comparing the two different vaccine formulations, but the rate in which the peak titer to DENV-4 was obtained was much earlier when the immunization was performed with a greater amount of DENVax-4. Further, when the amount of DENVax-2 was lowered in the formulation (Group 5), this had no marked difference in the kinetics or peak titer of DENV-4 in serum responses in this tetravalent DENVax vaccine.
FIG. 9 represents a flow chart of one exemplary procedure for testing safety and efficacy of the virus construct disclosed herein in an animal model. For example, composition(s) that include one or more chimeric Dengue viruses of use as a vaccine, such as compositions of DENVax4e, DENVax4f or DENVax4h, or other chimeric constructs or a control composition can be tested in animal models, e.g. AG129. Animals can be primed on day 0, and subsequently boosted with the same or different Dengue virus vaccine compositions on or before day 30 or day 42 or other appropriate time. Then, the animals can be challenged with exposure to one or more Dengue virus serotypes to assess induction of an immune response to the challenge. Blood samples can be collected, for example, on days 0, 30, 41, 48, 52 or 84 or other appropriate day to test for neutralizing antibodies, and on other days such as 5 and 47 to test for viremia (or as deemed appropriate for the composition and administration protocol chosen).
Virus Cloning and Rescue to P1
Plasmid mutagenesis was used to create the new DENV-4 chimeric clones of use in vaccine compositions disclosed herein. Primers coding for point mutations were synthesized and used to amplify an entire new infectious clone. The template was digested by DpnI and the subsequent plasmid was sequenced. RNA was transcribed and electroporated into Vero cells to create a virus at passage level 0 (P0) and then amplified by a single passage in Vero cells (P1).
Modifications at the Capsid/PrM junction in the DENVax-4b clone did not appear to cause an increase in DENV-4 immunogenicity in NHP studies. Using the sequencing data from the blind serial passages of DENV-4 and DENVax-4b, 3 point mutations that may increase Vero cell adaptation were identified. In addition some attenuating mutations were reverted back to the wild-type sequence to increase immunogenicity in mice. As presented in FIG. 6, the larger IFU size and lytic phenotypes of DENVax-4e and DENVax-4h illustrates the potential that either of these clones may show an increase in immunogenicity in mice. Experiments to analyze the growth kinetics of each of the new DENVax-4 clones were conducted to determine whether the inserted modifications increase Vero cell adaptation. Test in A129 mice to analyze immunogenicity are also conducted.
Growth Kinetics
Serial passaging of Dengue vaccine strains in Vero cells is a classic method for selecting strains which are better fit to grow in vitro. In these exemplary growth experiments, DENVax-2 (FIG. 1) was included as a control and displayed the highest initial titer at day 2, followed by DENVax-4b-P10, DENVax-4, and DENVax-4b-P1. At the end of the growth period (day 12) DENVax-2 had the highest peak titer, followed by DENVax-4b-P10, DENVax-4b-P1, and DENVax-4.
Amino Acid Changes in Sequences
DENVax4-P10 genomic sequencing demonstrated mutations which corresponded to amino acids E-417 E-K (Glu-Lys) and NS4A-17 M-L. DENVax4b-P10 sequencing showed a mutation which corresponded to amino acid C-107 C-Y. The E-417 E-K mutation changes the amino acid residue so that an amine group (NH2) is substituted for a carbonyl hydroxyl group. However, the R group is still charged and remains hydrophilic. The NS4A-17 M-L mutation results in removal of a sulfate from the R group, but maintains non-polarity resulting in a hydrophobic amino acid. The C-107 C-Y mutation results in drastic change in the R group. Cysteine has an SH group that is capable of forming disulfide bonds, while tyrosine has a carbon benzene ring with a hydroxyl group. This causes the amino acid residue to become hydrophilic instead of hydrophobic, affecting its interaction with the other amino acid R groups.
FIG. 19 is a graphic representation of titers of DENVax-4 constructs during growth kinetics experiment. The day each sample was taken is plotted on the x-axis and the titer is plotted on the y-axis.
Neutralizing Antibodies in NHP Vaccinated with DENVax
In certain methods, evaluation of the DENV-4 chimeric construct viruses took place with a larger study testing immunization regimens. The immunogenicity of DENVax-4b compared to that of DENVax-4a was tested. Groups 1 and 2 were vaccinated on Day 0 with 2 doses and given no booster vaccination. Equivalent titers of either DENVax-4 (diamonds) or DENVax-4b (squares) were used in all doses. No significant differences in geometric mean titers (GMT) of neutralizing antibodies were found between any of the serotypes including DENV-4. This suggests that using DENVax-4b in tetravalent DENVax does affect or increase the neutralizing antibody response against DENV-4. FIG. 20: GMT values comparing Groups 1 (DENVax-4, diamonds) and 2 (DENVax-4b, squares). GMT values are measurements of neutralizing antibody values from Plaque Reduction Neutralization Technique.
Neutralizing antibody responses in Groups 1 and 3 were compared to determine whether increasing the dose of DENVax-4 in tetravalent DENVax increased immunogenicity against DENV-4. Results demonstrated that primates immunized with a higher dose of DENVax-4 showed an increased in GMT of primary neutralizing antibodies detected in the first 60 days compared to those immunized with traditional tetravalent DENVax. There was no significant difference in GMT between the other DENV serotypes (FIG. 21). This suggests that a higher dose of DENVax-4 improves the neutralizing antibody response against DENV-4.
Neutralizing antibody responses were compared in Groups 1 (diamonds) and 4 (squares) to test the effect of the immunization schedule on immunogenicity (FIG. 22). FIG. 22 illustrates GMT values demonstrating effect of vaccine schedule on neutralizing antibody response. This indicates that 2 doses on Day 0 and no boost has no adverse effects compared to 1 dose on Day 0 and 1 dose on Day 60. Finally, data from Groups 4 (diamonds) and 5 (squares) were compared to see if decreasing the dose of DENVax-2 had an effect on immunogenicity of DENVax-4. Results show no significant difference in GMT against DENV-4, but GMT against DENV-2 is significantly reduced in Group 5. This indicates that the antibody response elicited by the DENVax-2 dose does not have an adverse effect on the antibody response elicited by DENVax-4 (FIG. 23). FIG. 23 represents GMT values for Groups 4 (diamonds) and 5 (squares) for all DENV serotypes. Results were determined using Plaque Reduction Neutralization Technique.
The results of these experiments support that constructs disclosed herein improve DENV-4 neutralizing antibody responses. Increased DENVax-4 in the dose formulation does show a significant increase in neutralizing antibody production. The DENVax-2 dose also does not appear to have an impact on neutralizing antibody responses of DENVax-4. There may be little to no difference in antibody production between the prime 2 doses vaccination method and prime and boost vaccination method. Sufficient neutralizing antibody titers were produced against DENV-4 1036, the strain that is currently used in DENVax-4. A successful DENV-4 vaccine should be able to adequately neutralize multiple strains of wild type DENV-4 including newly evolved strains with genome modifications, different genotypes and different phenotypes.
During sequencing there were three point mutations identified between passage 1 and passage 10 in both constructs. These mutations were located in the capsid region of DENVax-4b and in the prM and envelope genes of DENVax-4 as previously discussed. Incorporating these mutations into the constructs may provide increased growth in Vero cells by decreasing the attenuation of the virus, which could improve immunogenicity. As disclosed herein DENVax-4h had a 2-fold increase in neutralizing antibody titers while DENVax-4e had a 1.5-fold increase in neutralizing antibody titers. In other methods, increased DENV-4 immunogenicity is tested and compared to other bivalent, trivalent and tetravalent construct compositions.
Materials and Methods
Cell Culture
Vero cells are mammalian cells derived from African Green Monkey kidney. The Vero cell line used in the in vitro experiments. Vero cells were grown at 37° C. in Dulbecco's Modification of Eagle's Medium (DMEM, Mediatech Inc., Manassas Va.) supplemented with 10% Fetal Bovine serum (FBS, Hyclone, Logan Utah), 2% L-glutamine (Hyclone), and 1% Penicillin-Streptomycin (Pen-Strep, Hyclone). To passage the cells Tryple Express solution (Life Technologies, Grand Island N.Y.) was used to remove the cells from the flask surface.
Viral Infection of Vero Monolayers
Vero cells were seeded on T-75 cm2 flasks approximately 48 hours before infection. DMEM supplemented with 10% FBS, 2% L-glutamine, and 1% Penicillin-Streptomycin was used as cell growth medium. Upon cell confluency, 1 flask was trypsinized using 4 mL of a 0.25% trypsin solution diluted 1:5 in PBS. Cells were counted to establish an MOI. Two of the remaining flasks were infected at a predetermined MO1 in 1 mL of either DENVax-4-P2 (1st generation DENVax-4) or DENVax-4b-P3 or other construct and diluted in BA-1 diluent (Bovine serum albumin, 1×M199, 0.05M Tris-HCL, 1× L-glutamine, 7.5% Sodium bicarbonate, 1× Pen-strep, 1× Fungizone). Viruses were adsorbed onto Vero cells for 90 minutes with rocking every 10 minutes to prevent drying of cell monolayers. After adsorption 20 mL DMEM supplemented with 5% FBS was added to each flask. Flasks were incubated for 7 days at 37° C.
Viral Harvests and Subsequent Infections: Blind Passage
After a pre-determined period, the CPE was observed on each flask and viral supernatant was harvested and stabilized in 20% FBS for storage at −80° C. Previously seeded confluent T-75 cm2 Vero flasks were infected with 1 mL of the viral supernatant from the preceding flask. Virus was adsorbed for 90 minutes with rocking every 10 minutes. After viral adsorption, 20 mL DMEM 5% FBS was added to each flask. New non-infected control flasks were plated every 7 days. This process was repeated every 7 days for 10 subsequent weeks, yielding 10 passages per virus denoted either DENVax-4-P2-P1 through P10 or DENVax-4b-P3-P1 through P10 or other indicated denotations for the various constructs.
Plaque Titration of Viruses
Samples from DENVax-4-P2 and DENVax-4b-P3 and other chimeric constructs were taken weeks 1, 5 and 10 were plaque titrated to measure titer. Virus samples were serially diluted 1×10−1 to 1×10−6 in BA-1 diluent. Samples were plaque titrated in triplicate, and 100 uL of each dilution was adsorbed to a pre-seeded 6-well plate of Vero cells for 90 minutes with rocking every 8 minutes. After adsorption, wells were overlayed with 4 mL of BSS/Agar (NaCl, KCl, NaH2PO4—H2O, glucose, CaCl2-2H2O, Mg504-7H2O) solution and incubated for 4 days at 37° C. On day 4 wells were overlayed with 2 mL BSS/Agar/Neutral Red solution and incubated overnight at 37° C. Plaques were counted on Days 5, 6 and 7.
Growth Curve Analysis
Growth kinetics of the adapted strains were analyzed by performing a growth curve on Vero cells. Vero flasks were seeded as previously described. On day 0 a confluent flask of Vero cells was counted to calculate the virus PFU needed to infect the flasks at an MOI of 0.001. Flasks were infected with 1 mL of DENVax-4, DENVax-4b-P1, DENVax-4b-P10, or DENVax-2 or other chimeric construct (e.g. DENVax-4e, 4h etc.). Viruses were adsorbed to the monolayers for 90 minutes with rocking every 8 minutes. After adsorption, 10 mL cDMEM without FBS supplemented with 1% F-127 was added to each flask and the samples were incubated at 37° C. with 5% CO2. Samples were collected from the supernatant from each flask on Day 2 and Days 4-12. Vaccines were harvested by collecting the entire amount of the supernatant in the flask, and the growth media was replaced with fresh cDMEM-F127 on Day 4 and Days 6-12. Flasks were washed 3 times with PBS during media changes. Samples were stabilized in 1×FTA (FTA:15% trehalose, 1% F-127, 0.1% human serum albumin, PBS) and plaque titrated as previously described to determine titer.
After DENVax-4 and DENVax-4b were both blindly passaged 10 times in Vero cells, each passage was sequenced to identify mutations between P1 and P10. Sequencing reactions were done on DENVax-4-P2-P1 and P10 and DENVax-4b-P3-P1 and P10 at the CDC. Viral RNA was isolated from virus stocks using a QIAmp viral RNA kit. Reverse transcriptase PCR (RT-PCR) was used to transcribe the RNA into DNA, using primers previously designed by the CDC. The primers are designed from the sequences of DENV-2 16681 and DENV-4 1036. Approximately 7-9 PCR fragments per construct were amplified by RT-PCR. The DNA fragments were then sequenced by Beckman Coulter using an automated sequencing reaction, and aligned for comparison.
Sequencing
After the various constructs are passaged 10 or more times in Vero cells, each passage was sequenced to identify mutations. Sequencing reactions were performed on each sample. Viral RNA was isolated from virus stocks using a QIAmp viral RNA kit. Reverse transcriptase PCR (RT-PCR) was used to transcribe the RNA into DNA, using primers previously designed by the CDC. The primers are designed from the sequences of DENV-2 16681 and DENV-4 1036. Approximately 7-9 PCR fragments per construct were amplified by RT-PCR. The DNA fragments were then sequenced by Beckman Coulter using an automated sequencing reaction, and aligned for comparison.
Non-Human Primate Study
Cynomolgus macaques are place in different study groups and vaccinated with doses of tetravalent DENVax having various DENV-4 constructs. One formulation per group can be tested. Formulation 1 can contained a high dose of DENVax with DENVax-4 1st generation, and primates in Group 1 are primed with 2 doses on Day 0 and given no boost. Formulation 2 contained a high dose of DENVax with a DENV-4 construct included and primates in Group 2 can be primed with 2 doses on Day 0 and given no boost. A vaccine can be administered sub-cutaneous or ID or by other method using a needle or needleless system and syringe. Samples to test for serology can be taken on days 0, 28, 58, 73, 90, 128 or other appropriate timing. Neutralizing antibody responses in sera are measured by plaque reduction neutralization technique.
Plaque Reduction Neutralization Technique
To test for neutralizing antibody production in sera samples, a plaque reduction neutralization assay can be used. Vero 6-well plates are seeded 2 days before inoculation to ensure monolayer confluency. Sera samples were diluted serially two-fold in BA-1 diluent in a 96-well plate and incubated with dengue virus for approximately 20 hours at 4° C. After incubation Vero wells are inoculated with prepared virus/sera dilutions. Samples are adsorbed for 90 minutes with rocking every 8 minutes to prevent drying of the monolayers. After adsorption wells are over-layed with 1:1 solution of BSS and agarose, and incubated at 37° C. for 4 days. On Day 4 cells can be over-layed with a 1:1 ratio of BSS supplemented with neutral red solution and agarose. Plaques visible on wells are counted on for example, Days 5, 6, and 7. A GMT value refers to the average dilution of sera that can neutralize 50% of the virus. This is measured by determining the number of plaques formed in the absence of sera, dividing that value by 2 (to account for dilution), and noting which dilution of sera caused plaque formation equal to or less than that amount.
The higher immunogenicity of DENVax-4h (envelope mutation) compared to DENVax-4e (capsid mutation) suggest that the DENVax-4 envelope protein could be optimized. The envelope protein provides epitopes for the generation of neutralizing antibodies, and modifying the sequence to optimize epitope sites for eliciting a strong antibody response may increase antibody titer. The envelope mutation in DENVax-4h at position 417 is in the conserved portion in the stem region. DENV-4 has a different amino acid in this position compared to other flaviviruses. The stem region is in domain III of the E protein where the strongest neutralizing epitope sites exist. Antibodies that bind and neutralize this site prevent the stem region from fusing with the endosomal membrane after endocytosis. In fact, position 417 is conserved among the flavivirus family including DENV-1, -2, -3, and WNV. A reversion from E to K may change the secondary structure to favor a more robust immune response. A further mutation would be to revert back to the conserved charged Aspartic Acid (D) as seen in the other flaviviruses.
The foregoing discussion of the invention has been presented for purposes of illustration and description. The foregoing is not intended to limit the invention to the form or forms disclosed herein. Although the description of the invention has included description of one or more embodiments and certain variations and modifications, other variations and modifications are within the scope of the invention, e.g., as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative embodiments to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter.

## Claims

1. A nucleic acid molecule comprising a chimeric flavivirus construct comprising nucleic acid sequences encoding nonstructural proteins and one or more structural proteins from a live, attenuated dengue-2 virus and at least encoding one or more structural proteins from a second dengue virus, the second flavivirus is selected from the group dengue-1, dengue-3 or dengue-4 virus wherein the chimeric construct further comprises one or more mutations comprising, a mutation in an envelope (E) protein at an amino acid position synonymous of dengue 4 to amino acid 417 when the E protein is from the second flavivirus wherein the mutation in the envelope (E) protein at the position synonymous to amino acid 417 changes glutamic acid to a positively charged residue; a mutation in the capsid protein at an amino acid position synonymous to amino acid 107 in the attenuated dengue-2 wherein the mutation in the capsid (C) protein at the position synonymous to amino acid 107 changes cysteine to an aromatic amino acid residue in the attenuated dengue-2, and a mutation in NS4A at an amino acid position synonymous to amino acid position 17 in the attenuated dengue-2.

2. The nucleic acid molecule according to claim 1, wherein the second flavivirus is dengue-4.

3. The nucleic acid molecule according to claim 1, wherein the position synonymous to 417 is a lysine instead of the glutamic acid in the at least second flavivirus.

4. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule comprises the mutation in the capsid (C) protein at a position synonymous to amino acid 107 changes cysteine to an aromatic amino acid residue; a mutation in the envelope (E) protein at the position synonymous to amino acid 417 changes glutamic acid to a positively charged residue; and a mutation in NS4A at an amino acid position synonymous to amino acid position 17 in the attenuated dengue-2.

5. The nucleic acid molecule according to claim 1, wherein the aromatic amino acid at the position synonymous to amino acid 107 is a tyrosine.

6. The nucleic acid molecule according to claim 1, wherein the mutation in the NS4A protein at the position synonymous to amino acid 17 changes methionine to a basic amino acid.

7. The nucleic acid molecule according to claim 1, wherein the mutation in the NS4A protein at a position synonymous to amino acid 17 changes methionine to a leucine.

8. The nucleic acid molecule according to claim 1, wherein the live, attenuated dengue-2 contains a mutation at position 57 in the 5′NCR, a mutation at position 53 of NS1 and a mutation at position 250 of NS3.

9. The nucleic acid molecule according to claim 1, wherein the live, attenuated dengue-2 contains a mutation at position 53 of NS1 and a mutation at position 250 of NS3.

10. The nucleic acid molecule according to claim 2, wherein amino acid positions 102-106 of attenuated dengue-2 are substituted with synonymous amino acids of a dengue-4 virus in these positions.

11. The nucleic acid molecule according to claim 1, further comprising a structural protein from a third different flavivirus.

12. The nucleic acid molecule according to claim 11, wherein the third different flavivirus is selected from the group consisting of West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, tickborne encephalitis virus, and yellow fever virus.

13. A composition comprising one or more nucleic acid molecules according to claim 1, and a pharmaceutically acceptable carrier.

14. The composition according to claim 13, wherein the composition further comprises additional dengue-dengue chimeric constructs.

15. The composition according to claim 13, wherein the composition is part of a divalent, trivalent or tetravalent dengue virus composition.

16. The composition according to claim 13, wherein the composition further comprises other live, attenuated flaviviruses.

17. A method of inducing an immune response to dengue virus in a subject comprising administering the composition of claim 13 to the subject.

18. The method according to claim 17, wherein an immune response to at least one of dengue-1, dengue-2, dengue-3 and dengue-4 is induced in the subject.

19. A kit comprising one or more compositions according to claim 13 and a container.

20. A live, attenuated virus comprising the nucleic acid molecule according to claim 1.

21. A live, attenuated dengue-4 chimera comprising DENVax4e, DENVax4i or DENVax4h.

22. The chimera according to claim 21, wherein the chimera comprises SEQ ID NO: 22, SEQ ID NO:9, or SEQ ID NO: 24.

23. A composition comprising one or more nucleic acid chimeras according to claim 21, and a pharmaceutically acceptable carrier.

24. A method of inducing an immune response to dengue virus in a subject comprising administering the composition of claim 23 to the subject.

25. The nucleic acid molecule according to claim 2, wherein dengue-4 is from strain 1036.

26. The nucleic acid molecule according to claim 1, wherein the live, attenuated dengue-2 virus comprises a mutated PDK-53 backbone.

27. The live, attenuated virus of claim 20, wherein the live, attenuated virus is part of an immunogenic composition against two or more dengue virus serotypes.