Passive Immunization with Recombinant Antibody VLRB-PirAvp/PirBvp— Enriched Feeds against Vibrio parahaemolyticus Infection in Litopenaeus vannamei Shrimp

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The causative agent of acute hepatopancreatic necrosis disease (AHPND) is the bacterium, Vibrio parahaemolyticus, which secretes toxins into the gastrointestinal tract of its host. V. parahaemolyticus toxins A and B (PirAvp/PirBvp) have been implicated in the pathogenesis of this disease, and are, therefore, the focuses of studies developing treatments for AHPND. We produced and tested recombinant antibodies based on the hagfish variable lymphocyte receptor B (VLRB) capable of neutralizing some viruses, suggesting that this type of antibody may have a potential application for treatment of AHPND. Results showed significantly higher level of survival in shrimp fed with the PirBvp-9G10 antibody (60%) compared to the group fed the PirAvp-7C12 antibody (3%) and the control group (0%). This suggests that VLRB antibodies may be a suitable alternative to immunoglobulin-based antibodies, as passive immunization treatments for effective management of AHPND outbreaks within shrimp farms.

Acute hepatopancreatic necrosis disease (AHPND), formerly known as early mortality syndrome, was first recognized as an emerging disease in China in 2009, and since being identified has spread to neighboring countries in Southeast Asia, including Vietnam in 2010, Malaysia in 2011, and Thailand in The disease has now reached as far as Mexico in early 2013.

Affected shrimp have an empty gut and an atrophied pale hepatopancreas, which can be reduced in size by more than 50%. AHPND can cause up to 100% mortality within 20–30 days after the pond has been stocked with postlarvae shrimp. The disease has resulted in huge economical losses for shrimp farmers globally.

A unique strain of V. parahaemlyticus is responsible for causing AHPND. V. parahaemlyticus is a Gram-negative, halophilic bacterium found ubiquitously in warm marine and estuarine environments around the world. The strains responsible for causing AHPND possess a 63 to 70 kDa plasmid that encodes binary toxins PirAvp/PirBvp, which are actually homologs of the Photorhabdovirus insect-related (Pir) toxins PirAB.

“These two toxins are secreted by the bacterium and have been associated with the pathogenesis of the disease; they are considered to be the primary virulence factors involved in causing AHPND.”

A variety of methods have been investigated for controlling AHPND, including passive immunization. Here, we report on a VLRB antibody that we developed, which specifically recognizes and neutralizes the binary toxins produced by V. parahaemolyticus that are responsible for inducing the pathogenesis associated with AHPND in shrimp.

Materials and Methods

Construction of Toxin Plasmids Vibrio parahaemolyticus (D2 strain) cells were cultured in brain heart infusion (BHI) broth. DNA was extracted from this bacterial cell culture. PirAvp and PirBvp were amplified using respective primers. To check the veracity of the cloned PirAvp/PirBvp plasmids, each plasmid was sequenced (Solgent, Korea) and the sequences aligned with the original PirAvp/PirBvp sequence.

Expression and Purification of Recombinant Toxin

To induce the expression of PirAvp/ PirBvp proteins, BL21 cells harboring the pet32a- PirAvp or pet28b-PirBvp plasmids were grown overnight in Luria-Bertani (LB) broth with ampicillin (LB amp) and kanamycin (LB kan), respectively. The cells were then subjected to three cycles of freeze-thawing to break the bacterial cell wall and the soluble fractions collected.

The soluble fractions were purified using affinity chromatography columns. The purified recombinant toxins were also subjected to Western blotting to further check their specificity. Large scale preparations of the respective proteins were performed once the correct sizes were verified. The proteins were quantified using PierceTM BCA protein Assay kit. Finally, the quantified proteins were used as antigens in subsequent experiments.

Screening of VLRB Library

The cell line bearing the VLRB cDNA library was seeded into twenty 96-well plates with 200 cells/well and grown to 100% cell confluency. The supernatants containing the recombinant VLRBs was collected and screened by ELISA. The ELISA was performed three times to ensure the specificity of the PirAvp or PirBvp-specific VLRBs, with PirAvp-7C12 and PirBvp-9G10 showing the highest levels of specificity and were subsequently used in for further experiments.

Establishment of Cell Line Secreting Anti-PirAvp/PirBvp Recombinant VLRBs

The cells secreting PirAvp/PirBvp-specific recombinant VLRBs, PirAvp-7C12, and PirBvp-9G10 were collected. Once specificity was established, large scale preparation of the supernatants was performed. These supernatants were designated as PirAvp-7C12 and PirBvp-9G10 antibody from here on.

Bacterial Challenge Test

Litopenaeus vannamei post-larvae (n = 100, 0.1 ± 0.03 g) were transferred into three 250-L tanks corresponding to the two experimental groups (PirAvp-7C12 and PirBvp-9G10 antibody) and one control group fed no antibody (negative control). The shrimp were fed with antibody added to the shrimp diet. The challenged shrimp and bacterial solution were poured and bacterial solution was poured into a new aquarium containing 100 volume of clean water. The final volume was 15 L, which contained 105cfu//mL of V. parahaemolyticus. Mortality data were obtained from two separate trials.

Statistical Analysis

Survival data were statistically analyzed via Kaplan-Meier with the Chi-square test using GraphPad Prism v.5 software. Differences between groups were considered significant when ** p < 0.001.

Results

Of the twenty 96-well plates that that were screened in the first round of ELISA screening, only 19 wells showed high binding with PirAvp and 24 wells with PirBvp. After the third round of screening, only one antibody for each group with the highest binding capacity was selected, namely PirAvp-7C12 and PirBvp-9G10.

Based on the results collected, the level of survival in the first experimental trial in which shrimp were fed with PirBvp-9G10 antibody was 26.7%, which was significantly higher than the group fed with PirAvp-7C12 antibody (3%) and the negative control (6%). In the replicate trial, results were even more pronounced, wherein the group fed with PirBvp-9G10 antibody demonstrated a 60% survival, in contrast to the group fed PirAvp-7C12 or the negative control group that exhibited 3% and 0% survival, respectively.

This particular dataset is statistically significant at p< 0.001, indicating the protective effect of the PirBvp-9G10 antibody.

Discussions

The development of safe and efficacious treatment for diseases such as AHPND has been the subject of increased research in recent years. The use of antibiotics to treat bacterial infections in industrial aquaculture has been opposed despite their effectiveness, due to antibiotic usage giving rise to antibiotic-resistant strains of bacteria. Vaccination is currently considered to be the most effective strategy for controlling infections in aquaculture; however, shrimp do not possess acquired immunity, necessary to induce a memory response to the vaccine.

The potential of using passive immunization to protect shrimp against infections has been explored previously, with varying degrees of success. Gao et al. (2016) showed egg yolk powder containing antibodies against V. harveyi and V. parahaemolyticus orally administered to white shrimp, L. vannamei to be effective for reducing subsequent Vibrio infections. More specifically, the antibodies showed an inhibiting effect on both bacteria in vitro, and in vivo.

The results of several studies, indicate the potential of using an edible antibody as a means of passively immunizing shrimp to help them fight infection. In our current study, we developed VLRB antibodies that specifically recognized PirAvp and PirBvp, which could potentially “neutralize” the effect of these virulence toxins.”

Although the exact mechanism of action of these toxins is still unclear, the presence of the plasmid (pVA1) encoding these toxins in all AHPND-causing strains of V. parahaemolyticus indicates that they are causative factors involved in the disease process. The PirAvp/PirBvp toxins are known to be homologs of the insecticidal Photorhabdus insectrelated (Pir) binary toxin PirAB that exhibits pore-forming activity in insects, thus suggesting that PirAvp and PirBvp might function similarly.

In a previous report, both Pir A and Pir B are needed to be present in insect larvae to induce mortality. Since these binary toxins have been discovered together in V. parahaemolyticus, it would seem reasonable that they are also both essential for the onset of symptoms associated with AHPND.

Several notable studies had demonstrated the potential role of VLRBs in neutralizing certain viruses such as avian influenza virus H9N2, viral hemorrhagic septicemia virus and nervous necrosis virus, and results are compelling enough to promote their usage as a therapeutic agent against bacterial and viral infections.

In the current study, the high level of survival in the group fed with the PirBvp-9G10 antibody after the AHPND-challenge, suggests that the VLRB antibody can provide protection against a V. parahaemolyticus infection in shrimp.

We speculate that the reason behind the effectiveness of the PirBvp-9G10 antibody in our study might be due to the abundant hydrophobic residues in the structure of PirBvp that our VLRB antibody readily recognizes and which the PirAvp noticeably lacks.

“However, in general, our results clearly suggest that the PirBvp-9G10 VLRB antibody can improve shrimp survival against V. parahaemolyticus by simply targeting the virulent toxin PirBvp.”

In summary, the results of a previous report showing that only the PirBvp toxin could induce histological signs of AHPDH, and another stating that the virulence of AHPND relies heavily on the amount of toxins secreted by the bacterial cells, greatly substantiates the him of our study to develop new therapeutic agents for AHPND targeting the PirBvp toxin. Furthermore, the efficacy of VLRB antibodies as immunogenic agents to passively immunize shrimp reared at high stocking densities could significantly help the shrimp industry to combat outbreaks of AHPND

This is a summarized version developed by the editorial team of Aquaculture Magazine based on the review article titled “PASSIVE IMMUNIZATION WITH RECOMBINANT
ANTIBODY VLRB-PirAVP/PirBVP— NRICHED FEEDS AGAINST VIBRIO PARAHAEMOLYTICUS INFECTION IN LITOPENAEUS VANNAMEI SHRIMP” developed by: JASSY MARY S. LAZARTE – Gyeongsang National University, YOUNG RIM KIM- Gyeongsang National University, JUNG SEOK LEE- Gyeongsang National University, JIN HONG CHUNGyeongsang National University, SI WON KIM- Gyeongsang
National University, JAE WOOK JUNG- Gyeongsang National University, JAESUNG KIM- Gyeongsang National University, PATTANAPON KAYANSAMRUAJ – Kasetsart University, KIM D. THOMPSON – Moredun Research Institute, HYEONGSU KIM – National Institute of Fisheries Science, AND TAE SUNG JUNG – Gyeongsang National University. The original article was published on JANUARY 2021, through VACCINES under the use of a creative commons open access license the full version can be accessed freely online through this link: https://doi.org/10.3390/vaccines9010055

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