Engineered Hansenula fungi efficiently expressing CA10 virus-like particles and uses thereof
Inventors
LI, Guoshun • GU, Meirong • Guo, Lin • Jian, Wei • Liu, Junjie • XIAO, Haifeng • ZHANG, Gaimei • Zhao, Lili • Xie, Xuechao • Chen, Lei • XU, Yingzhi • Liu, Jiankai
Assignees
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Publication Number
US-12251435-B2
Publication Date
2025-03-18
Expiration Date
Abstract
The present disclosure provides an engineered Hansenula fungus that efficiently expresses CA10 (Coxsackievirus A10) virus-like particles and uses thereof. The engineered fungus includes a recombinant vector carrying the P1 and 3CD genes of the CA10 virus optimized according to preferred codons of Hansenula. The present disclosure also provides a preparation method for CA10 virus-like particles and vaccines prepared therefrom.
Core Innovation
An engineered Hansenula cell comprises a PMV-05 vector containing a nucleic acid molecule with a nucleotide sequence of a Coxsackievirus A10 (CA10) P1 gene and a nucleotide sequence of a CA10 3CD gene. The nucleotide sequence of the P1 gene is set forth in SEQ ID NO: 1, and the nucleotide sequence of the 3CD gene is set forth in SEQ ID NO: 2. The engineered Hansenula cell is described as expressing CA10 P1 and CA10 3CD genes.
The document describes Hansenula strains including a uracil auxotroph (AU-0501) and a preferred deposited strain (Hansenula polymorpha CA10-W). The expression of the CA10 P1 and CA10 3CD genes enables 3CD protease cleavage and self-assembly of P1 to form CA10 virus-like particles (VLPs). The document further describes fermentation-based CA10 VLP production using the engineered Hansenula system.
The document describes purification of the CA10 VLPs and a downstream workflow including ultrafiltration followed by three-step chromatography comprising ion exchange chromatography, hydroxyapatite chromatography, and molecular sieve chromatography. The purified CA10 VLPs are described as being used as a vaccine antigen formulated with aluminum hydroxide adjuvant, including a concentration range for the vaccine formulation. The document also describes assay characterizations of the VLPs and reports immunogenicity and protection results associated with vaccine use.
Claims Coverage
The claim set covers one core engineered host-cell inventive concept and four additional refinements.
Engineered Hansenula cell with PMV-05 CA10 P1 and 3CD gene sequences
An engineered Hansenula cell comprising a PMV-05 vector including a nucleic acid molecule having a nucleotide sequence of a Coxsackievirus A10 (CA10) P1 gene and a nucleotide sequence of a CA10 3CD gene, wherein the P1 gene sequence is set forth in SEQ ID NO: 1 and the 3CD gene sequence is set forth in SEQ ID NO: 2.
Specific Hansenula polymorpha CA10-W host strain
The engineered Hansenula cell includes a Hansenula polymorpha CA10-W strain with China General Microbiological Culture Collection accession number 19850.
3CD-mediated cleavage-driven CA10 VLP preparation and purification
A process prepares purified Coxsackievirus A10 virus-like particles by culturing the engineered cell to express 3CD and multiple P1 proteins, using 3CD-mediated cleavage to drive self-assembly of cleaved P1 proteins, and purifying the VLPs.
Adsorption onto aluminum hydroxide adjuvant
The process further comprises adsorbing the purified virus-like particle onto an aluminum hydroxide adjuvant.
Hand-foot-and-mouth disease vaccine with 5 to 40 μg/mL VLP concentration
A hand-foot-and-mouth disease vaccine comprising the virus-like particle has a concentration of 5 to 40 μg/mL.
The claims focus on an engineered Hansenula cell using a PMV-05 vector with CA10 P1 and CA10 3CD gene sequences defined by SEQ ID NO: 1 and SEQ ID NO: 2. The dependent claims further specify a Hansenula polymorpha CA10-W strain, a 3CD-driven cleavage and purification workflow for CA10 VLPs, adsorption of purified VLPs onto aluminum hydroxide adjuvant, and a 5 to 40 μg/mL vaccine antigen concentration for hand-foot-and-mouth disease.
Stated Advantages
Not explicitly described in patent.
Documented Applications
A hand-foot-and-mouth disease vaccine formulated with purified Coxsackievirus A10 (CA10) virus-like particles and aluminum hydroxide adjuvant.
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