Cell-penetrating peptide Pep-1-Nb24 inhibits White Spot Syndrome Virus replication and boosts immunity in crayfish

The White Spot Syndrome Virus (WSSV) poses a severe threat to the global crustacean aquaculture industry, causing fatal infections and substantial economic losses. Despite its devastating impact, there are currently no effective commercial therapeutic drugs available to control WSSV spread. Nanobodies (Nbs), due to their small size, high specificity, stable structure, and ease of production, represent a promising antiviral strategy. However, their poor membrane permeability often limits intracellular delivery. Cell-penetrating peptides (CPPs) like Pep-1 offer a solution by acting as vectors to transport proteins into cells, addressing this critical delivery gap for therapeutic nanobodies.

Researchers generated Pep-1-Nb24 by fusing the cell-penetrating peptide Pep-1 with Nb24, a nanobody previously screened for its specific targeting of the WSSV envelope protein VP28. The study evaluated the entry efficiency of Pep-1-Nb24 and its anti-WSSV activity in crayfish (Procambarus clarkii) infected with WSSV. The experimental design compared the effects of Nb24 alone versus Pep-1-Nb24 on WSSV infection. Primary endpoints included WSSV replication inhibition, survival rate, and the expression levels of antioxidant genes (SOD, CAT, GST) and immune genes (Lzm, Crustin, Propo) in infected crayfish. While specific doses or sample sizes were not detailed in the abstract, the study assessed dose-dependent effects.

The nanobody Nb24 alone was found to significantly block WSSV infection in crayfish in a dose-dependent manner, leading to an improved survival rate in WSSV-infected animals. However, Nb24 exhibited poor membrane permeability, hindering its autonomous entry into cells. Intriguingly, the fusion with Pep-1 successfully delivered Nb24 into key tissues, including hemocytes, gills, and hepatopancreas cells of the crayfish. This enhanced cellular uptake translated into a stronger anti-WSSV activity for Pep-1-Nb24 compared to Nb24 alone. Both Nb24 and Pep-1-Nb24 treatments significantly upregulated the expression of antioxidant genes (SOD, CAT, GST) and immune genes (Lzm, Crustin, Propo) in WSSV-infected crayfish. Notably, the expression levels of these crucial genes were consistently higher in the Pep-1-Nb24 treated groups than in the Nb24 only groups, indicating a more robust immune and antioxidant response. While specific numerical data (e.g., percentages of survival, fold-changes in gene expression, or p-values) were not provided in the abstract, the qualitative findings demonstrate a clear and significant improvement with the cell-penetrating peptide fusion.

Pep-1-Nb24 demonstrated stronger anti-WSSV activity in crayfish, effectively inhibiting viral replication and significantly upregulating key antioxidant and immune genes compared to Nb24 alone.