Virus-like particles and methods of use
Inventors
Nabel, Gary J. • Rao, Srinivas • Akahata, Wataru
Assignees
US Department of Health and Human Services
Publication Number
US-10138277-B2
Publication Date
2018-11-27
Expiration Date
2032-01-31
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Abstract
The invention features modified alphavirus or flavivirus virus-like particles (VLPs). The invention provides methods, compositions, and kits featuring the modified VLPs. The invention also features methods for enhancing production of modified VLPs for use in the prevention or treatment of alphavirus and flavivirus-mediated diseases. The invention also provides methods for delivering agents to a cell using the modified VLPs.
Core Innovation
The invention features modified alphavirus or flavivirus virus-like particles (VLPs), along with methods, compositions, and kits for their use. It provides methods for enhancing production of modified VLPs to be used in prevention or treatment of diseases mediated by alphaviruses and flaviviruses, and for delivering agents to cells by using such modified VLPs.
The problem being solved relates to the serious public health issues posed by alphaviruses and flaviviruses, which include multiple pathogenic viruses causing severe diseases such as encephalitis, arthritis, and hemorrhages. The evolution and spread of these viruses into new areas combined with a lack of vaccines or antiviral therapies underscores the need for effective preventive or therapeutic approaches.
The invention addresses the difficulty in producing VLPs from certain wild-type alphavirus proteins, particularly for viruses like Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV). It was discovered that specific alterations in amino acid sequences of alphavirus E2 proteins and capsid protein nuclear localization signals (NLS) enhance or enable VLP production. Use of modified VLPs induces high-titer neutralizing antibodies and offers protection against homologous and heterologous alphavirus strains as demonstrated in animal models. Similarly, flavivirus envelope proteins with analogous alterations allow for enhanced production of flavivirus VLPs. The invention provides detailed compositions, nucleic acids, expression vectors, cells, immunogenic compositions, vaccines, and methods of manufacturing VLPs with these modifications.
Claims Coverage
The patent claims focus on isolated polynucleotides encoding altered alphavirus structural proteins and methods for producing virus-like particles, with emphasis on specific mutations that enhance VLP production.
Altered alphavirus E2 protein with specific amino acid substitutions
An isolated polynucleotide encoding alphavirus E2 protein comprising at least one alteration at amino acid positions corresponding to H170, K200, K233, K234, R251, or H256 of Chikungunya virus E2 protein, where the alteration enhances VLP production and the altered protein is capable of self-assembling into a virus-like particle (VLP).
Altered alphavirus capsid protein with modifications in the nuclear localization signal (NLS)
An isolated polynucleotide encoding an alphavirus capsid protein comprising at least one alteration in the Nuclear Localization Signal (NLS) relative to the wild-type sequence, where the alteration enhances VLP production and the altered protein is capable of self-assembling into a VLP.
Alphavirus species coverage
The alphavirus encoded by the polynucleotide can be selected from Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), Venezuelan equine encephalitis virus (VEEV), Semliki Forest virus (SFV), Chikungunya virus (CHIKV), O'nyong-nyong virus, Sindbis virus, Mayaro virus, Ross River virus, Barmah Forest virus, and Ockelbo virus.
Specific NLS alteration sites in alphavirus capsid proteins
Alterations in the NLS involve amino acid positions 67-70 of EEEV capsid protein, 67-70 of WEEV capsid protein, 64-68 of VEEV capsid protein, 62-69 of CHIKV capsid protein, 71-74 of Ross River virus capsid protein, and 64-68 of Barmah Forest virus capsid protein.
Substitution in charged amino acids of the NLS
The at least one alteration in the NLS involves substitution of a charged amino acid with another amino acid to enhance VLP production.
Method for producing VLPs with altered viral proteins
A method comprising introducing into a cell a polynucleotide encoding an altered alphavirus E2 protein (with alterations at specified amino acid positions) or an altered capsid protein with at least one alteration in the NLS, where the alterations enhance VLP production and the protein self-assembles into a VLP.
The claims collectively cover isolated nucleic acids encoding alphavirus E2 proteins with specific amino acid alterations and capsid proteins with nuclear localization signal modifications that enhance virus-like particle production, along with methods for producing VLPs using such nucleic acids, targeting a variety of alphaviruses. The inventive features focus on mutations at defined amino acid positions to improve VLP yield and assembly.
Stated Advantages
High yield production of virus-like particles facilitating cost-effective vaccine and delivery vehicle manufacture.
Improved safety and high immunogenicity of VLP-based vaccines against alphaviruses and flaviviruses.
Ability to induce high-titer neutralizing antibodies against homologous and heterologous virus strains.
Protection against viremia and inflammatory consequences following virus challenge demonstrated in animal models.
Enhanced VLP yield through specific amino acid modifications in E2 proteins and nuclear localization signal changes in capsid proteins.
Feasibility of multivalent VLP vaccines eliciting strong immune responses against multiple alphaviruses without immunologic interference.
Increased stability and longer persistence of VLPs under high pH conditions during production.
Documented Applications
Use of modified alphavirus or flavivirus VLPs as immunogenic compositions or vaccines for prevention or treatment of alphavirus- or flavivirus-mediated diseases.
Delivery of therapeutic or diagnostic agents into cells via packaging into VLPs.
Use of VLPs and DNA vaccines to induce immune responses in mammalian subjects, including humans and non-human primates.
Production of multivalent vaccines combining VLPs from different alphaviruses to protect against multiple viral infections.
Screening for inhibitors of viral entry using recombinant lentiviral vectors expressing reporter genes and alphavirus or flavivirus envelope proteins.
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