Acute hepatopancreatic necrosis disease (AHPND) is a recently emerged disease in Penaeid shrimp aquaculture that is characterized by high mortality. A binary toxin composed of the PirA and PirB proteins expressed by Vibrio parahaemolyticus (PirABVp) has been shown to be sufficient for the development of the disease. Here we show that nanobodies derived from the heavy chain only antibodies of llamas can neutralize PirABVp activity and reduce shrimp mortality.
Vibrio parahaemolyticus PirA and PirB proteins were recombinantly expressed in Escherichia coli and purified. Together, the recombinantly expressed PirABVp killed specific pathogen free Pacific whiteleg shrimp (Litopenaeus vannamei) and brine shrimp (Artemia), a surrogate model for whiteleg shrimp. To obtain nanobodies targeting PirABVp, llamas were immunized with PirA and PirB, and, using phage-based biopanning techniques, hundreds of nanobodies specific to PirA or PirB were identified. A total of 128 unique PirA-binding nanobodies and 200 unique PirB-binding binding nanobodies were subcloned to an E. coli protein expression system and successfully purified.
The purified nanobodies were tested to confirm their ability to bind the expected antigen in protein-protein pulldown and/or ELISA experiments, to examine their ability to block the interaction of PirA and PirB, and to assess their relative proteolytic stability in gastrointestinal fluid extracts. From these screening experiments, a subset of 52 nanobodies were tested for their ability to protect Artemia from the toxic effects of PirABVp. Shown here are the results for two PirA-binding nanobodies (NBXBG and NBXBH) and two PirB-binding nanobodies (NBXBD and NBXBJ) that can reduce PirABVp -induced death in Artemia (Fig. 1).
Nanobodies represent the smallest functional antibody fragment and have the potential to be used in many applications, including animal feed. With the ability to neutralize PirABVp, our nanobodies provide a new opportunity to reduce or prevent AHPND in shrimp aquaculture.