Direct rescue of segmented negative-sense RNA viruses from complementary DNA in monkey kidney Vero cells

This technology enables the direct rescue of segmented negative-sense RNA viruses from complementary DNA in monkey kidney Vero cells, facilitating safer and more efficient live-attenuated vaccine production.

Segmented negative-sense RNA viruses pose significant challenges for vaccine development due to the complexity of their genomes and the inefficiency of current methods for recovering infectious viruses from complementary DNA. Traditional approaches often rely on tumorigenic baby hamster kidney (BHK) cells, which raise safety concerns for vaccine manufacturing.

Additionally, existing transfection technologies struggle to deliver viral cDNA plasmids effectively, limiting the production of live-attenuated vaccines for important pathogens such as the influenza virus, rotavirus, and bunyaviruses.

Novel method

This innovation introduces a novel method to rescue segmented negative-sense RNA viruses directly from complementary DNA (cDNA) plasmids within African green monkey kidney Vero cells, which are a World Health Organization (WHO) acceptable cell substrate for human vaccine manufacturing.

The process employs triggered endosome escape technologies to simultaneously deliver viral cDNA plasmids and a bacteriophage T7 DNA-dependent RNA polymerase (DdRp) expression vector. Each viral cDNA plasmid encodes a bacteriophage T7 DdRp promoter that drives transcription of positive-sense antigenomic RNA, which serves as a template for the translation of viral proteins that constitute the viral replication complex.

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A key novelty is replacing the conventional use of tumorigenic BHK cells with non-cancerous Vero cells, thereby aligning with WHO guidelines for vaccine manufacturing and national regulatory authorities. By modifying existing delivery technologies such as VeroFect, this method improves the uptake and expression of viral components required to generate infectious RNA viruses from cDNA. The approach streamlines the development of live-attenuated vaccines for segmented negative-sense RNA viruses, offering a scalable, regulatory-compliant solution that enables rapid response to emerging viral threats.

Advantages:

•    Utilizes non-cancerous Vero cells, reducing the requirement of demonstrating a lack of tumorgenic materials compared to traditional BHK cells.
•    Employs triggered endosome escape transfection technique for efficient delivery of viral cDNA plasmids, improving virus recovery rates.
•    Supports manufacturing of live-attenuated vaccines for important, segmented negative-sense RNA viruses, including influenza virus, rotavirus, and bunyavirus.
•    Reduces reliance on less safe or less efficient cell substrates and transfection methods.
•    Enables a scalable, regulatory-friendly platform suitable for human vaccine production.
•    Leverages a modified existing technology (VeroFect) to enhance performance without requiring entirely new infrastructure.

Applications:

•    Development and production of live-attenuated vaccines targeting segmented negative-sense RNA viruses.
•    Research tool for virologists studying viral rescue and replication using cDNA plasmids in non-cancerious cell lines.
•    Platform technology for rapid vaccine response against emerging segmented negative-sense RNA virus outbreaks.
•    Potential use in improving gene delivery techniques involving RNA virus vectors in the development of candidate vaccines and therapies.
•    Commercial vaccine manufacturing that demands high safety and efficiency standards for cell substrates.