# Protective antigens hidden in the bovine Clostridium vaccine

## TL;DR

This study identifies two non-toxin proteins, ESBP and YnjE, as promising vaccine candidates for bovine necrotic enteritis, showing strong immune responses and better protection than some commercial vaccines.

## Contribution

The study demonstrates the immunoprotective potential of non-toxin antigens ESBP and YnjE in a mouse model for C. perfringens vaccines.

## Key findings

- ESBP and YnjE proteins induced higher IgG titers and a mixed Th1/Th2 immune response compared to a commercial vaccine.
- Mice immunized with ESBP and YnjE showed better survival and weight recovery than those with Vac3 and comparable results to Vac2.
- Current commercial vaccines show low antibody titers against ESBP and YnjE, indicating their potential is underutilized.

## Abstract

Clostridium perfringens type A is the primary pathogenic bacterium responsible for bovine necrotic enteritis, causing significant economic losses in large-scale livestock farming. Our previous reverse vaccinology screen identified the nontoxin antigen protein of this bacterium as highly immunogenic. However, its immune protection efficacy and practical effectiveness in vaccines remain unclear.

Antigenic epitope analysis, prokaryotic expression, and purification of the C. perfringens Plc, ESBP, and YnjE proteins were performed. Mice were immunized with these proteins to detect serum antibody titers, cytokine levels, and changes in the mouse survival rate and weight. The immunoprotective effects of ESBP and YnjE proteins as well as three commercial vaccines were compared by detecting serum antibody titers against the toxin protein Plc and the non-toxin proteins ESBP and YnjE proteins after three immunizations with the vaccines.

Three proteins with high immunogenicity scores (the classic toxin protein Plc and non-toxin proteins ESBP and YnjE) were successfully expressed and purified. ELISA of mouse sera showed significantly elevated levels of IL-2, IL-4, IL-10, and IFN-γ, indicating the induction of a mixed Th1/Th2 immune response. Indirect ELISA confirmed that all three proteins elicited high IgG titers, with the non-toxin ESBP and YnjE inducing higher titers. Their protective efficacy—based on survival rate and body weight recovery—was superior to Vac3, comparable to Vac2, and lower than Vac1. Notably, sera from mice immunized with the three commercial vaccines exhibited low antibody titers against ESBP and YnjE, suggesting that the immunological potential of these two key non-toxin protective antigens is not fully exploited in current vaccines.

ESBP and YnjE exhibited favorable immunogenicity and immune protection in a mouse model. These findings, derived from the mouse model, highlight the great potential of ESBP and YnjE as key candidate antigens for the development of novel vaccines, and provide important experimental evidence for the antigen design and optimization of C.perfringens vaccines. These conclusions need to be further validated by in-farm animal trials.