ISAJ Newsletter - Volume 10, Issue 2 (September 2025)

From Editor’s Desk

Greetings and a warm welcome to this second issue of ISAJ Newsletter in 2025!

We are in the middle of India-Japan Year of Science, Technology and Innovation Exchange (2025-2026).

Our website at https://www.isaj.jp/ was reorganized to bring you all the information with better clarity and simplicity. More pages will be added to it. You may send your suggestions by email.

This year the annual symposium will be held for two days from November 28 (Fri) to 29 (Sat), 2025, in Shizuoka city. It is being co-organized by Shizuoka Institute of Science and Technology (SIST). The first day will be from 12:30 to 17:30 h, which will feature a special symposium on Circular Innovation for Urban Transformation and Beyond. It will have talks by domain experts in Japan’s support for infrastructure development in India over the past few decades. The second full day (8:30 to 17:30 h) will be devoted to symposium on all disciplines of science and technology featuring plenary and invited talks, as well as oral and poster presentations by young researchers and students. The flyer of the symposium is shown on the back cover of this issue. More information and links can be found at https://www.isaj.jp/symposiums/2025/

In this issue, we bring you two research articles from a brilliant scientist couple, both young faculty members, at Faculty of Agriculture in Kyushu University. Drs. Akhil Kizhakkumpat and Harsha Prakash work broadly on immune system in fish and development of vaccine against various fish diseases. These investigations have direct implications on food security.

The Research Highlight 1 is on development of a filter device for prevention of aquatic bacterial disease. In this article, Dr. Harsha Prakash describes use of a revolutionary technique called affinity silk to physically trap and remove specific pathogens from water, to protect fish as a preventive measure against infection of fish disease.

The Research Highlight 2 is on development of a novel antibody in Medaka fish in Japan. Dr. Akhil Kizhakkumpat describes development of a highly reactive and specific antipeptide and antibody against immunoglobulin of the medaka fish, with a goal to create a robust antibody by strategically targeting a specific region of the immunoglobulin heavy chain predicted to be highly antigenic and accessible.

We hope you would find the present issue of our Newsletter interesting. We look forward to receiving your feedback. Any suggestions/ideas for improving the upcoming newsletters are welcome.

Research Highlight 1: Development of a Filter Device for the Prevention of Aquatic Bacterial Disease Using a Single-Chain Variable Fragment (scFv)-Conjugated Affinity Silk

By Dr. Harsha Prakash, Faculty of Agriculture, Kyushu University

Introduction

In aquatic ecosystems, from massive aquaculture farms to the tranquil hobbyist aquarium, a constant and often invisible battle is being waged against disease. Fish, like all living creatures, are susceptible to a wide range of pathogens, including bacteria, viruses, and parasites. These infectious agents can spread rapidly through water, turning a thriving environment into a scene of devastation in a remarkably short time.

The consequences are severe, resulting in billions of dollars in economic losses for the global food industry and heartbreaking loss for ornamental fish enthusiasts.

The traditional approach to managing these diseases has been largely reactive. When an outbreak occurs, antibiotics and other chemical treatments are deployed. However, this strategy has significant drawbacks. It addresses the problem only after it has taken hold, and the widespread use of antibiotics has fueled the global crisis of antimicrobial resistance, creating “superbugs” that are increasingly difficult to treat.

One of the most common fish pathogens, Aeromonas salmonicida (A. salmonicida) serves as a potent example. It is the causative agent of ulcer disease, a condition that causes progressive, disfiguring lesions on fish, including highly prized ornamental koi carp. An infection can decimate a population and render survivors commercially worthless. To combat such threats proactively, a revolutionary new approach has been developed: Affinity Silk. This is a “smart” biomaterial, engineered to physically trap and remove specific pathogens from the water column, preventing disease before it ever starts.

The Concept: Weaving Antibodies into Silk

The remarkable capability of affinity silk stems from the elegant fusion of natural silk protein with the precision of modern antibody engineering. The concept is based on creating a versatile biomaterial with a built-in, highly specific binding function.

1. The Core Components:

• The Scaffold (Silk Fibroin): The foundation of the material is the fibroin L-chain (FibL), a core structural protein in natural silk. Silk is an ideal scaffold due to its biocompatibility, durability, and the ease with which it can be processed into various forms like fibers, films, or coatings.

• The Targeting System (scFv): The specificity is provided by a single-chain variable fragment (scFv). An scFv is the engineered antigen-binding domain of an antibody, containing the precise molecular structure needed to recognize and latch onto a specific target. For this study, an scFv was derived from a monoclonal antibody that shows a high binding affinity for Aeromonas salmonicida.

2. The Creation Process:

The journey from concept to a functional filter involves three key stages of biotechnology:

• Genetic Engineering: Using the piggyBac transposon system, a powerful tool for gene insertion, a new strain of silkworm (designated WS19) was created. These transgenic silkworms carry a DNA construct that fuses the gene for the anti-As scFv directly to the gene for the FibL silk protein.

• Biosynthesis: The genetic construct is designed to be expressed only in the silkworm’s silk glands. As the silkworm spins its cocoon, it naturally produces and weaves the FibL-scFv fusion protein into the silk fibers. The result is a cocoon made of silk that is pre-functionalized to target the pathogen.

• Device Fabrication: The cocoons are harvested and the affinity silk is dissolved to create a liquid solution. This solution is then used to coat an inert, high-surface-area material; in this case, glass wool. The coated glass wool is packed into a standard air-lift water filter, creating a device that actively circulates water through a dense network of pathogen-specific molecular traps.

Results: Proven Efficacy Against Aeromonas salmonicida

A series of rigorous experiments were conducted to validate the performance of the affinity silk filter, confirming its ability to specifically bind and efficiently remove live Aeromonas salmonicida from water.

1. Confirmation of Successful Affinity Silk Production: Before testing the filter, it was crucial to verify that the transgenic silkworms (WS19 strain) were actually producing the correct FibL-scFv fusion protein. Using SDS-PAGE and Western blot analysis, the researchers compared the proteins in the WS19 silk to control silk. The results were clear: the WS19 silk showed a distinct, larger protein band at approximately 57 kD, which was absent in the controls. This new band’s size perfectly matched the expected size of the FibL-scFv fusion protein, confirming that the genetic engineering was successful and the “smart” protein was being expressed at a level sufficient for practical use.

2. Specific and Strong Binding Confirmed: An enzyme-linked immunosorbent assay (ELISA) was first used to confirm that the engineered silk retained its binding function. Wells coated with the affinity silk (WS19) showed a strong, concentration-dependent binding to A. salmonicida, far exceeding the negligible binding of control silks. This proved that the scFv portion of the fusion protein was correctly folded and fully functional.

3. Rapid and Efficient Water Purification: The most critical test involved introducing the affinity silk filter into water contaminated with a high concentration of live A. salmonicida. The results were definitive:

• The filter containing the affinity silk (WS19) began removing bacteria almost immediately, causing a rapid and continuous decrease in the water’s bacterial load over a 120-minute period.
• Filters using control silks showed a significantly smaller and slower reduction in bacteria, highlighting that the capture was not due to simple physical filtration but to the specific biological affinity of the engineered silk.

4. High-Capacity Pathogen Removal: To quantify the filter’s trapping capacity, the glass wool was analyzed after the filtration experiment. The affinity silk-coated wool was found to have captured a vastly greater quantity of A. salmonicida than any of the controls. The study estimated that the filter had sufficient ability to trap a lethal dose of the bacteria, demonstrating that its capacity is high enough to be effective in preventing disease in a real-world aquarium setting. The strong binding ensures that once captured, the pathogens are not released back into the water.

Applications and Future Directions

The success of the A. salmonicida filter is not a singular achievement, but the validation of affinity silk as a powerful and versatile platform technology. The core innovation, the functionalized silk itself opens the door to a wide array of applications beyond simple filtration.

1. Prophylactic Water Filtration: The primary application remains the proactive prevention of disease. Affinity filters provide a continuous, silent defense against specific pathogens, reducing the reliance on antibiotics and creating healthier, more stable aquatic environments for both aquaculture and ornamental fish.

2. A Versatile Library for Disease Control: The true power of the platform lies in its modularity. By changing the scFv, the silk can be tailored to target any pathogen of interest. This work is already expanding:

• Success Against Parasites: The technology has proven effective against entirely different organisms, with a filter successfully designed to capture the parasite Ichthyophthirius multifiliis (Ich).
• Targeting Other Bacteria: Research is underway to create affinity silks for other major bacterial threats, including Aeromonas hydrophila and Edwardsiella tarda.
• “Cocktail” Filters: The ultimate goal is to create multi-target filters coated with a mixture of different affinity silks, capable of capturing a wide spectrum of pathogens simultaneously.

3. Pathogen Detection and Diagnostic Kits: The high specificity and strong binding properties of affinity silk make it an ideal material for developing sensitive diagnostic tools. Instead of a filter, the silk can be used to create:

• ELISA-style Plates: 96-well plates pre-coated with affinity silk could allow for the rapid and accurate detection of specific pathogens in water samples.
• Rapid Diagnostic Tests: The silk could be incorporated into dipsticks or lateral flow assays for quick, on-site screening of ponds, tanks, or environmental water sources, providing an early warning system for farmers and conservationists.

By harnessing the elegance of a natural fiber and the precision of biotechnology, affinity silk offers a future where we can not only prevent disease but also monitor and protect our vital aquatic ecosystems with unprecedented specificity and effectiveness.

Research Highlight 2: Development of a Novel IgM-Specific Antibody in Medaka and its Application to Evaluate the Potential of Microparticles as an Oral Vaccine Carrier

By Dr. Akhil Kizhakkumpat, Faculty of Agriculture, Kyushu University

Introduction: The Need for Precision Tools in Fish Immunology

As aquaculture continues to expand and diversify, the need for species-specific tools to monitor fish health and develop effective vaccines has become paramount. A cornerstone of this research is the ability to accurately measure the antibody response, which is primarily mediated by Immunoglobulin M (IgM) in teleost fish. The development of antibodies that can specifically recognize the IgM of each fish species is therefore essential for conducting reliable immunoassays.

Traditionally, producing such antibodies is a labor-intensive process. However, the increasing availability of whole genome sequences for many teleost species has opened the door to a more efficient method: generating anti-peptide antibodies. This approach uses short, synthesized protein fragments (peptides) based on the known amino acid sequence of the target protein to generate an antibody response. While promising, this technique is often plagued by significant challenges. Anti-peptide antibodies frequently exhibit low specificity, limited reactivity, and, most critically, a failure to recognize the natural, folded structure of the target protein, which severely limits their practical use in biological assays.

A Strategic Approach to Antibody Design

This study aimed to overcome these common hurdles by developing a highly reactive and specific antipeptide antibody against the IgM of the medaka fish (Oryzias latipes), a key model organism for which such immunological tools are underdeveloped. The central goal was to create a robust antibody by strategically targeting a specific region of the IgM heavy chain predicted to be highly antigenic and accessible.

As a practical application of this newly developed tool, the study then sought to evaluate an immune response in medaka. Specifically, it investigated the potential for microplastics to act as an adjuvant or vector for a model antigen, bovine serum albumin (BSA), in a bath immunization experiment. The objective was to use the new antibody to measure both systemic and mucosal antibody production triggered by this antigen delivery system.

A Highly Specific and Reactive Antibody

The strategic design process resulted in an antipeptide antibody that successfully overcame the typical limitations of such tools. Rigorous testing confirmed its high degree of specificity, its reactivity against the native protein, and its sensitivity.

Specificity Confirmed by Western Blot: When tested against medaka serum proteins separated by size, the antibody detected a single, distinct protein band at approximately 80 kDa. This molecular mass corresponds precisely to the IgM heavy chain in teleost fish, demonstrating the antibody’s exceptional specificity for its intended target.

Reactivity Against Native IgM: A crucial test of any anti-peptide antibody is its ability to recognize the protein in its natural, folded state. The antibody developed in this study performed excellently in this regard. A dot blot analysis, which uses native proteins, showed an intense signal when the antibody was applied to both purified medaka IgM and whole medaka serum, confirming its ability to bind the natural antigen effectively.

High Sensitivity for Quantitative Assays: The antibody proved to be a highly sensitive tool suitable for quantitative measurements. In an ELISA format, it was capable of detecting medaka IgM at concentrations as low as 12.2 ng. This level of sensitivity is comparable to, or exceeds, that of other well-established monoclonal antibodies used in fish immunology research, validating the present antibody as powerful tool.

Application: Using the Antibody to Assess Adjuvant Effect of Microplastics in Oral Vaccines

The newly validated antibody was immediately applied to investigate the immune response in medaka following bath immunization. In this experiment, microplastics (1μm) were used as a potential adjuvant to deliver the model antigen, BSA.

Systemic and Mucosal Immune Responses: The results, measured using the new antibody, were clear. The group exposed to BSA-coated microplastics (MP-BSA) by immersion showed a significant systemic (serum) antibody response, which was not seen in the group exposed to BSA alone in the bath. For the mucosal (gut) immune response, the MP-BSA group produced the highest level of antibodies.

Interpretation of Adjuvant Effect: These findings suggest that microplastics can function as an effective adjuvant in bath immunization. The study proposes that the particles may act as a vector for the antigen, protecting the adsorbed BSA protein from digestion and enhancing its uptake by phagocytic cells in the fish’s gut-associated lymphoid tissues. Microplastics successfully crossed the intestinal epithelial barrier and were detected in organs including liver and gills of the fish. This enhanced uptake appears to trigger a more robust antibody-producing response than the antigen alone.

Future Directions

This study’s successful design and application of a specific anti-peptide antibody lays the groundwork for significant advancements in fish immunology and vaccine technology.

Extending the Antibody Design Strategy to Other Species: The most promising future direction is the application of this specific design methodology to other teleost species. The study’s in silico analysis of the IgM heavy chain from seven different fish species revealed that the linker region between the CH3 and CH4 domains is a structurally conserved feature, consistently forming an exposed loop on the protein’s surface. Although the exact amino acid sequence varies, the consistent topology of this region makes it a highly promising epitope for developing species-specific anti-IgM antibodies for a broad range of commercially important and research-model fish. This would provide the aquaculture and research communities with a wealth of desperately needed immunological tools.

Investigating Micro- and Nanoparticles as Vaccine Adjuvants: The clear adjuvant effect observed when microplastics were used to deliver an antigen suggests a powerful new direction for vaccine development. Its certain that usage of microplastics as an adjuvant is not practical in real world scenario but the current study paves a way to explore the potential of micro or nanosized particles which could be used as a carrier for efficient oral vaccine delivery vehicles for fish. Future research should focus on exploring this phenomenon further. This could involve optimizing the adjuvant effect by testing different types, sizes, and materials of biocompatible particles for their ability to deliver vaccine antigens. The ultimate goal would be to leverage this mechanism to create more effective and practical oral or immersion vaccines for aquaculture, which are far less stressful and easier to administer than injections. The mechanisms behind this adjuvant effect, such as the pathways for particle uptake and immune cell activation, also warrant deeper investigation.

Announcement: ISAJ 16th Annual Symposium 2025

The 16th ISAJ Annual Symposium - 2025 will be held on November 28-29, 2025, in Shizuoka City, co-organized by Shizuoka Institute of Science and Technology (SIST).

Day 1 (November 28, 2025, 12:30 – 17:30) Special Symposium on “Circular Innovation for Urban Transformation and Beyond”

• Features talks by domain experts in Japan’s support for infrastructure development in India over the past few decades
• Opening session, Keynote Address, Panel Discussion, and Networking Session

Day 2 (November 29, 2025, 8:30 – 17:30) Full-day symposium on “All disciplines of Science & Technology”

• Keynote Address, Plenary/Invited Talks
• Oral/Poster Presentations by Young Researchers and Students
• Best Poster Award and Concluding Session

Venue: SIST Ekimae Campus, 4th Floor, M20 Building, Shizuoka City (Near North exit of Shizuoka Station)

Registration: https://forms.gle/Mye1GzsdqhDETK8w5 Abstract Submission: Send your abstract (one-page Word document) by October 20th, 2025 (Mon) to [email protected]

Participation Fees: 5,000 円 (Students: Free)

The symposium will provide an opportunity to the participants to listen to the domain experts from both the countries on diverse issues related to the theme of the symposium.

For more information, visit: https://www.isaj.jp/symposiums/2025/