# Preparation and Efficacy Evaluation of Heat-Resistant Freeze-Dried Live-Attenuated Vaccine Formulation of Micropterus salmoides Rhabdovirus

## TL;DR

This study developed a stable freeze-dried vaccine for a fish virus that remains effective for over a year and provides strong immunity.

## Contribution

The development of a thermostable freeze-dried live-attenuated vaccine formulation for Micropterus salmoides rhabdovirus.

## Key findings

- Formulations T1, T9, and T10 maintained stable viral titers at 25 °C and 37 °C.
- The vaccine provided over 80% survival rate in largemouth bass after immersion immunization.
- The vaccine retained immunogenicity and induced IFN-γ and IL-12 expression in fish.

## Abstract

Background/Objectives: An attenuated strain of Micropterus salmoides rhabdovirus (MSRV) 0509 with good immunogenicity has been isolated, showing potential as a candidate for live vaccine development. Methods: To improve the shelf life of attenuated strain of MSRV0509, the virus was formulated using three distinct single-protectant formulations and twelve thermostable protective agent formulations (designated T1–T12). Following lyophilization, the thermostability of each formulation was evaluated. Results: Results indicated that formulations T1, T9, and T10 maintained stable viral titers after storage at 25 °C and 37 °C. Moreover, these formulations retained high viral viability after 12 months at 4 °C, with a titer reduction of less than 0.5 log10. Immunological analyses revealed that the freeze-dried MSRV vaccine elicited both humoral and immune factors responses in largemouth bass. Immersion immunization provided effective protection, yielding a survival rate exceeding 80%. Freeze-dried vaccines maintained their immunogenicity (i.e., the ability to induce antibodies) following 12 months of storage at 4 °C. Additionally, expression of IFN-γ and IL-12 was significantly upregulated in fish post-vaccination. Conclusions: In conclusion, the lyophilized MSRV vaccine developed in this study not only exhibits improved thermostability and extended shelf life, but also effectively preserves its immunogenic properties, supporting its potential for practical aquaculture applications.