Nucleic acids encoding chimeric dengue/Zika viruses optimized for growth and stability in vero cells
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Publication Number
US-11344615-B2
Publication Date
2022-05-31
Expiration Date
2037-07-06
Abstract
Chimeric flaviviruses that include non-coding regions, non-structural proteins, a capsid (C) protein and a portion of a premembrane (prM) signal sequence from an attenuated or wild-type dengue serotype 2 virus (DENV-2), and a portion of a prM signal sequence, a prM protein and at least a portion of an envelope (E) protein from a Zika virus (ZIKV) are described. Also described are immunogenic compositions and methods for eliciting an immune response in a subject, such as an immune response directed against ZIKV.
Core Innovation
The invention relates to chimeric flaviviruses that consist of non-coding regions, non-structural proteins, a capsid (C) protein, and a segment of a premembrane (prM) signal sequence derived from a dengue serotype 2 virus (DENV-2), combined with a portion of a prM signal sequence, a prM protein, and at least part of an envelope (E) protein from a Zika virus (ZIKV). These chimeric viruses serve as immunogenic compositions capable of eliciting an immune response against ZIKV in a subject.
The problem addressed by the invention arises from the global spread and increased pathogenicity concerns of ZIKV, including associations with fetal abnormalities and Guillain-Barré syndrome. Existing medical countermeasures are lacking for ZIKV infection, and there is a need for vaccines that can effectively induce immunity. However, while chimeric viruses constructed using a DENV-2 backbone with ZIKV structural proteins can be recovered from mosquito cells, they exhibit poor or unstable replication in Vero cells, which are critical for vaccine production. Hence, the invention aims to optimize chimeric dengue/Zika viruses for improved growth, stability, and immunogenic properties in Vero cells.
The invention further proposes nucleic acid chimeras constructed with nucleotides encoding a modified prM signal sequence derived partly from DENV-2 and partly from ZIKV, and introducing specific Vero cell adaptation mutations that enhance virus replication and stability in Vero cells. These engineered chimeric viruses retain attenuation phenotypes crucial for vaccine safety, demonstrate efficient growth in vaccine production cells, and elicit protective immune responses upon administration.
Claims Coverage
The patent claims 27 inventive features centered on chimeric dengue/Zika virus nucleic acid constructs, their compositions, and methods of eliciting immune responses.
Chimeric dengue/Zika nucleic acid genomic arrangement
The nucleic acid chimera encodes a chimeric virus with the genomic order of DENV-2 5′ non-coding region, DENV-2 capsid gene, ZIKV prM and E genes, followed by DENV-2 non-structural proteins NS1 to NS5, and DENV-2 3′ non-coding region. The prM signal sequence is modified by fusing either the first three amino acids of the DENV-2 prM signal sequence with the last 15 amino acids of the ZIKV prM signal sequence or the first five amino acids of the DENV-2 prM signal sequence with the last 13 amino acids of the ZIKV prM signal sequence. The last 14 amino acids of the envelope protein are from DENV-2.
Incorporation of specific Vero cell adaptation mutations
The nucleic acid chimera comprises Vero cell adaptation mutations that enhance replication and stability in Vero cells. These include: 1) four mutations resulting in glutamine to arginine substitution at E-465, isoleucine to phenylalanine at E-493, lysine to asparagine at NS2A-99, and aspartic acid to asparagine at NS4A-23; 2) four mutations causing glutamine to arginine at E-465, isoleucine to threonine at E-484, isoleucine to phenylalanine at E-493, and lysine to asparagine at NS2A-99; or 3) five mutations combining the previous four mutations with the aspartic acid to asparagine at NS4A-23.
Use of attenuated or wild-type DENV-2 strain backbones
The chimeric nucleic acid can be based on an attenuated DENV-2 strain, such as strain PDK-53, which includes mutations in the 5′ non-coding region at nucleotide 57, at position 2579 resulting in NS1 protein residue 53 as aspartate, or at position 5270 resulting in NS3 protein residue 250 as valine, or on a wild-type DENV-2 strain such as strain 16681.
Encoding ZIKV from specific strains
The chimeric nucleic acid encodes ZIKV prM and E genes from strains such as SPH2015, PRVABC59, or R103451.
Chimeric flavivirus and immunogenic composition
The invention covers chimeric flaviviruses comprising the disclosed nucleic acid chimeras, and immunogenic compositions containing these chimeric viruses along with pharmaceutically acceptable carriers.
Methods of eliciting immune response against ZIKV
Methods involve administering the chimeric flavivirus or immunogenic compositions to a subject, such as a human, to provoke an immune response against ZIKV. Administration can be combined or followed by inactivated ZIKV vaccines.
The claims comprehensively cover chimeric dengue/Zika nucleic acid constructs with precise genomic arrangements and modifications, inclusion of critical Vero cell adaptation mutations for improved growth and stability in production cells, use of specific DENV-2 and ZIKV strains, chimeric virus compositions, and methods for eliciting an immune response against ZIKV in subjects, notably humans.
Stated Advantages
The chimeric dengue/Zika viruses demonstrate improved replication and stability in Vero cells, which are essential for vaccine production.
The chimeric viruses retain attenuation phenotypes, including reduced neurovirulence and limited growth in mosquito cells, contributing to vaccine safety.
The constructs elicit robust protective immune responses against ZIKV when administered to subjects.
The platform allows construction of live-attenuated Zika vaccines compatible with existing dengue vaccine backbones, enabling combination or pentavalent vaccines.
Documented Applications
Development of live-attenuated Zika virus vaccines using chimeric dengue/Zika viruses optimized for growth and stability in Vero cells.
Use of the chimeric dengue/Zika viruses as immunogenic compositions to elicit immune responses against ZIKV in subjects, including humans.
Combination vaccination strategies including live-attenuated chimeric vaccines and inactivated or non-infectious Zika vaccines.
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