# Oral Immunization with the C488A Live-Attenuated Mutant of Coxsackievirus B4E2 (CVB4E2) Induces Potent Immune Response and Protects Balb/c Mice Against Lethal Infection

## TL;DR

Researchers developed a weakened version of a virus that causes serious diseases in humans and found it can protect mice from lethal infection when used as a vaccine.

## Contribution

A live-attenuated mutant of Coxsackievirus B4E2 was shown to induce strong immunity and protect mice from lethal infection.

## Key findings

- The C488A mutant of CVB4E2 showed reduced replication and translation efficiency in vitro.
- Oral immunization with the C488A mutant protected Balb/c mice against lethal viral challenge.
- The IRES domain V mutation significantly attenuated CVB4 virulence.

## Abstract

Background/Objectives: Coxsackievirus B4 (CVB4), a member of the Enterovirus genus and the Picornaviridae family, is a significant pathogen causing several human diseases such as pancreatitis, myocarditis, cardiomyopathy and type 1 diabetes. Despite its clinical impact, no vaccines or specific antiviral therapies are currently available. This study investigates the attenuation of CVB4 virulence through targeted mutations in the domain V of the IRES (Internal Ribosome Entry Segment) sequence present in the 5′ UTR (Untranslated Region) of the viral genome. Materials and Methods: We engineered six CVB4E2 mutants by introducing single nucleotide mutations in domain V of the IRES sequence using PCR-based site-directed mutagenesis assays. Mutants were rigorously evaluated in vitro for their replicative capacities on HeLa cell culture and for their in vitro translation efficiencies in standard rabbit reticulocyte lysates supplemented with HeLa cell S10 extracts. Using different strategies of immunization and lethal challenges in a Balb/c mice model, we evaluated the immune responses elicited by the most attenuated C488A mutant strain. Results: The obtained results demonstrated that the live-attenuated C488A mutant with the single mutation C to A at nucleotide position 488 of the viral IRES sequence exhibited a significant reduction in vitro of both viral productivity and translation efficiency. The oral immunization with the live-attenuated C488A mutant induced a potent immune response and protected Balb/c mice against lethal infection challenge with a pathogenic strain. Conclusions: These findings underscored the critical role of IRES in CVB4 virulence and highlighted the use of the live-attenuated C488A mutant strain as a promising candidate for developing a live-attenuated vaccine against CVB4 infections.