# Protective Effect of Nanobodies Targeting Sip Protein Against Streptococcus agalactiae Infection in Tilapia (Oreochromis niloticus)

## TL;DR

Researchers developed a nanobody that targets a key protein in a harmful bacteria affecting tilapia, improving survival rates and offering a sustainable alternative to antibiotics.

## Contribution

The study introduces Nb30, a novel nanobody targeting the Sip protein of Streptococcus agalactiae, demonstrating its efficacy in reducing infection and mortality in tilapia.

## Key findings

- Nb30 significantly reduced Streptococcus agalactiae colonization in tilapia liver, spleen, and brain.
- Treatment with Nb30 increased fish survival rates from 53% to 86%.
- Nb30 modulated immune and metabolic pathways, including upregulating antioxidant enzymes and downregulating inflammation-related genes.

## Abstract

Antibiotics remain the primary method for controlling Streptococcus agalactiae (GBS) in aquaculture environments. However, while antibiotics can eliminate GBS, they may also lead to the release of virulence factors and the emergence of drug-resistant strains. Additionally, GBS can breach the blood–brain barrier in fish and damage the nervous system, accelerating fish mortality. Therefore, this study aims to explore green, virulence-neutralizing, and blood–brain barrier-penetrating strategies for preventing GBS infection. Nanobodies (Nbs), currently the smallest genetically engineered antibodies, possess potential for rapid degradation, toxin neutralization, and barrier system penetration, making them a crucial source for new drug development. Notably, this study utilized phage display technology to screen and obtain a nanobody Nb30 from a camel-derived nanobody phage library, which can target surface immunogenic (Sip) protein and inhibit GBS infection in tilapia (Oreochromis niloticus) tissues, and improve survival rates in infected fish. In summary, Nb30 is a sustainable, effective neutralizing antibody with promising application potential.

Streptococcus agalactiae (GBS) has emerged as one of the most prevalent bacterial pathogens causing severe economic losses in tilapia aquaculture due to its highly contagious and lethal nature. Nanobodies (Nbs), characterized by their small molecular size, enhanced tissue penetration, high tolerance, and exceptional antigen-binding affinity, represent a promising green alternative to conventional antibiotics. In the present study, the objective was to explore the potential of specific Nbs in the treatment of tilapia GBS disease. We first screened specific Nbs targeting the surface immunogenic (Sip) protein of GBS from a naïve phage display library, and a novel nanobody Nb30 was obtained. Nb30 was expressed in Escherichia coli and purified using the Ni-NTA Agarose column. Indirect ELISA showed that Nb30 had a high affinity against Sip and GBS in vitro. Moreover, Nb30 significantly reduced GBS colonization in the liver, spleen, and brain of GBS-infected tilapia. The survival rate in the control groups was 53%, whereas it was increased to 86% after treatment with 100 mg/kg Nb30. Transcriptome profiling revealed that Nb30 could modulate critical biological processes, including antioxidant defense, immune regulation, amino acid/protein synthesis, and energy metabolism in the liver tissues of GBS-infection tilapia. Notably, the expression levels of antioxidant enzymes (cat and gpx) were significantly up-regulated, and the TLR/MyD88/NF-κB pathway-related genes (tlr5, myd88, irak4, traf6, Rela, and NF-κB2) were significantly down-regulated after treatment with Nb30. Collectively, these findings establish a novel therapeutic strategy for controlling GBS infection in tilapia and provide evidence supporting the application of nanobodies as sustainable alternatives to antibiotics in aquaculture disease management.