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-9487563-B2
Publication Date
2016-11-08
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), compositions, kits, and methods for the prevention or treatment of alphavirus and flavivirus-mediated diseases. It addresses methods for enhancing production of modified VLPs and for delivering agents to cells using these VLPs. The modified VLPs include alterations in alphavirus E2 proteins at amino acid positions corresponding to amino acids 234 or 251 of the Chikungunya virus (CHIKV) E2 protein, or alterations in alphavirus capsid protein Nuclear Localization Signals (NLS), that enhance or allow VLP production.
The problem solved by the invention arises from the significant public health threat posed by alphaviruses and flaviviruses, which are mosquito- or tick-borne viruses causing a range of serious diseases such as encephalitis and flu-like symptoms with chronic arthritis. Currently, there are no effective vaccines or antiviral therapies available to counter the evolution, spread, and disease severity of these viruses. Moreover, for some alphaviruses, such as Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV) CBA, wild-type protein expression does not efficiently produce VLPs, limiting vaccine development and VLP-based delivery strategies.
The innovation includes nucleotide and expression vector constructs encoding alphavirus structural polypeptides where one or more alterations in the E2 protein (notably at positions corresponding to 234 and 251 in CHIKV E2) or capsid protein NLS enhance or enable VLP production. These modified VLPs resemble replication-competent viruses, elicit strong neutralizing antibody responses against homologous and heterologous alphavirus and flavivirus strains, and provide protection from infection and symptoms such as viremia and inflammation. The invention also discloses methods to improve VLP production by expressing the modified proteins in mammalian cells, codon optimization, adding leader sequences, and exposure to higher pH conditions (about pH 7.2 or above) during production or purification to increase yield. The VLPs are useful both as vaccines and as delivery vehicles for agents inside cells.
Claims Coverage
The patent includes eight main independent claims featuring virus-like particles (VLPs) with specific alterations and their methods of use, delivery, and immunogenic compositions.
Alphavirus virus-like particles with specific protein alterations enhancing production
An alphavirus VLP comprising an altered alphavirus E2 protein with one or more amino acid changes at positions corresponding to H170, K200, K233, K234, R251, or H256 of CHIKV E2 protein, and/or an altered alphavirus capsid protein having one or more changes at charged amino acid residues in the Nuclear Localization Signal (NLS). These altered proteins are capable of self-assembling into VLPs and the alterations enhance VLP production.
Species origin of altered viral proteins in VLPs
The altered viral proteins in the VLP are derived from viruses including 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.
Site specificity of alterations in capsid protein NLS enhancing VLP yield
Alterations at charged amino acid residues within NLS regions at amino acids 67-70 of EEEV capsid, 67-70 of WEEV capsid, 64-68 of VEEV capsid, 62-69 of CHIKV capsid, 71-74 of Ross River virus capsid, and 64-68 of Barmah Forest virus capsid.
VLPs comprising agents for cellular delivery
VLPs comprising an agent intended for introduction into cells, facilitating delivery of small molecule compounds, antibodies, nucleic acids, polypeptides, or fragments thereof.
Inducing immune response with altered VLPs
A method for inducing an immune response against an alphavirus in a subject by administering an effective amount of VLPs with the described altered viral proteins.
Pan-viral immunogenic composition with multiple altered VLPs
An immunogenic composition comprising a first VLP as described and a second alphavirus VLP with altered viral proteins capable of self-assembly, enhancing VLP production, and derived from different alphaviruses.
Kit comprising modified VLPs
A kit containing the VLP comprising altered alphavirus proteins as described herein.
Cellular delivery method using altered VLPs
A method for introducing an agent into a cell by packaging the agent into an alphavirus VLP with specific protein alterations that enhance production, then contacting the cell with the VLP under conditions permitting entry and delivery of the agent into the cell.
The claims cover alphavirus VLPs with defined alterations in E2 proteins and capsid protein NLS that enhance VLP production, their use in immunogenic compositions, delivery of agents into cells, and methods to induce immune responses with single or multivalent VLPs derived from various alphaviruses. The claims also encompass kits and methods for packaging and delivering agents into cells using these modified VLPs.
Stated Advantages
Enhanced yield of virus-like particles (VLPs) by specific amino acid alterations in alphavirus E2 proteins and capsid protein nuclear localization signals, reducing the cost of VLP vaccine production.
Use of VLPs and VLP vaccine strategy provides safety and high immunogenicity advantages over other vaccine platforms.
Ability of VLPs to bind and deliver agents to cells facilitates their use as delivery vehicles for therapeutic or diagnostic agents.
Use of high pH exposure during VLP production enhances VLP yield.
Documented Applications
Prevention or treatment of alphavirus-mediated diseases such as infections by Chikungunya virus, Western equine encephalitis virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, Ross River virus, and Barmah Forest virus using VLP vaccines.
Prevention or treatment of flavivirus-mediated diseases by immunization with modified flavivirus VLPs.
Use of VLPs for delivering agents including small molecule chemical compounds, antibodies, nucleic acid molecules, polypeptides, or fragments thereof into cells.
Use of VLPs in immunogenic compositions and vaccines for inducing protective immune responses in mammals including humans.
Multivalent pan-alphavirus VLP vaccines protecting against infections by multiple alphaviruses such as EEEV, WEEV, and VEEV.
Use of DNA vaccines encoding modified alphavirus or flavivirus structural proteins for in vivo VLP production and immunization.
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