Modified foot-and-mouth disease virus 3C proteases and methods for processing FMDV P1 precursor polypeptide
Inventors
Puckette, Michael • Neilan, John
Assignees
US Department of Homeland Security
Publication Number
US-11339387-B2
Publication Date
2022-05-24
Expiration Date
2038-05-22
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Abstract
The disclosure is directed to an isolated polynucleotide encoding a modified picornavirus 3C protease, wherein the modified picornavirus 3C protease includes an altered secondary structure and one or more amino acid substitution(s) located at one or more amino acid position(s) corresponding to positions 16-25, 99-100 and 115-130 of a wild-type Fool-and-Mouth Disease Virus (FMDV) 3C protease, wherein the isolated polynucleotide encoding the modified picornavirus 3C protease, when transformed into and co-expressed in a host cell, enhances transgene expression of a P1 precursor polypeptide in comparison to an amount of P1 precursor polypeptide transgene expression exhibited in a host cell transformed and co-expressed with a control picornavirus 3C protease, wherein the one or more corresponding amino acid position(s) is/are identified by an alignment of the modified picornavirus 3C protease with the one or more of the wild type FMDV 3C protease(s). Methods for processing a picornavirus P1 precursor polypeptide into picornavirus viral proteins and/or virus-like particles using the isolated polynucleotides are also provided.
Core Innovation
The invention is directed to isolated polynucleotides encoding modified picornavirus 3C proteases, particularly those with one or more amino acid substitutions at positions corresponding to 16-25, 99-100 and 115-130 of a wild-type Foot-and-Mouth Disease Virus (FMDV) 3C protease. Such modified proteases have altered secondary structures and, when expressed in host cells, enhance transgene expression of a P1 precursor polypeptide relative to cells expressing a control picornavirus 3C protease. These modified proteases retain proteolytic activity on the picornavirus P1 precursor polypeptide, facilitating processing into viral proteins and/or virus-like particles (VLPs).
The problem addressed is the cytotoxicity and host protein proteolysis caused by the wild-type 3C proteases when expressed in host cells, which limits vaccine production platforms and reduces antigen yields. Existing FMDV 3C protease mutants exist but are limited in their scope and efficacy across different host expression systems. Thus, there is a need for additional modified picornavirus 3C or 3CD protease mutants that reduce host toxicity while maintaining or enhancing processing activity to improve vaccine production outputs in various expression systems including E. coli, mammalian, and insect cells.
Claims Coverage
The claims cover several inventive features related to isolated polynucleotides encoding modified FMDV 3C proteases, including specific amino acid substitutions, vector and host cell compositions, and methods of processing FMDV P1 precursor polypeptides.
Polynucleotide encoding modified FMDV 3C protease with V124P substitution enhancing processing
An isolated polynucleotide encoding a modified FMDV 3C protease comprising a V124P amino acid substitution, which when expressed in host cells increases the accumulated amount of processed FMDV P1 precursor polypeptide fragments compared to wild-type FMDV 3C protease, with at least 95% identity to SEQ ID NO: 6.
Amino acid substitutions at specific positions corresponding to wild-type FMDV 3C protease
The modified FMDV 3C protease includes one or more amino acid substitutions at positions corresponding to 16-25 and/or 99-100 of the wild-type FMDV 3C protease.
Amino acid substitutions at positions I22, L23, and T100
The modified FMDV 3C protease possesses one or more amino acid substitutions specifically at amino acid positions I22 and L23, and/or at position T100.
Reduction in cleavage of host factor eIF4A1
Expression of the modified FMDV 3C protease reduces the amount of proteolytically-cleaved eIF4A1 in host cells compared to wild-type FMDV 3C protease.
Use of proline substitutions
At least one amino acid substitution involves substitution with proline.
Inclusion of nucleotide sequences encoding P1 precursor polypeptide
The isolated polynucleotide additionally encodes at least one P1 precursor polypeptide.
Vectors and host cells comprising modified protease polynucleotide
Vectors comprising the isolated polynucleotide encoding the modified FMDV 3C protease and host cells transformed with such vectors.
Compositions comprising polynucleotide and pharmaceutically acceptable excipients
Compositions including the polynucleotide encoding the modified FMDV 3C protease and pharmaceutically acceptable excipients, adjuvants, buffers, or solutions.
Methods for processing FMDV P1 precursor polypeptide using host cells expressing modified protease
Methods involving culturing host cells expressing the modified FMDV 3C protease and recovering viral proteins VP0, VP1, VP2, VP3, VP4 and/or VLPs processed from the P1 precursor.
The claims primarily protect modified FMDV 3C proteases with specific proline substitutions that enhance processing of P1 precursors with reduced host toxicity, polynucleotides and vectors encoding these proteases, host cells expressing them, compositions including these polynucleotides, and methods for producing viral proteins and VLPs using such host cells.
Stated Advantages
Modified picornavirus 3C proteases exhibit reduced cytotoxicity toward host cells compared to wild-type proteases.
Expression of modified proteases enhances transgene expression of P1 precursor polypeptides, resulting in improved antigen yields.
The modified proteases retain proteolytic activity required for processing P1 precursor polypeptides into viral proteins and VLPs.
Modified proteases show decreased cleavage of host proteins such as eIF4A1, histone H3, NEMO, and SAM68, contributing to increased host cell viability.
Transgene expression output can be increased by up to 20-fold relative to wild-type protease systems.
Reduced toxicity allows broader choice of expression platforms including bacterial and mammalian systems.
Documented Applications
Production of picornavirus viral proteins and virus-like particles (VLPs) in host cells co-expressing modified 3C proteases and P1 precursor polypeptides.
Use in DNA vaccines encoding modified 3C proteases and picornavirus P1 precursors to induce immune responses in subjects.
Development of safe subunit vaccines comprising picornavirus VLPs produced by host cells expressing modified 3C proteases.
Enhanced in vitro or in vivo processing of picornavirus P1 precursor polypeptides to improve vaccine antigen production.
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