Liponanoparticular mRNA and DNA fish vaccines with immunomodulator adjuvants – LipoFishVac

|Sequences of mRNA encoding relevant viral proteins (ie, G proteins of SVCV and VHSV) were optimized. After in vitro transcription, capping and a double purification step, mRNA were formulated onto optimized LNP by a layer-by-layer approach and characterized using standard assays. In parallel, immunomodulators were loaded on micelles: poly(I:C) (a TLR3 ligand); Pam3CSK4, (a TLR2 ligand) and 3M-052, (a TLR7-8 ligand by P4). Micelles from the PLA-b-poly(Nacryloxysuccinimide-co-N-vinylpyrrolidone) block copolymer were prepared and the bioactivity of the loaded ligands was checked by in vitro tests using reporter cell lines. DNA encoding the corresponding Ag was produced in parallel and used as a control. The biotoxicity and biodistribution of mRNA-LNP were assessed in vitro in fish cell lines, then in vivo. Fish Embryo Acute Toxicity (FET) tests were perfomed in zebrafish larvae by analyzing: (1) embryo lethality through coagulation of fertilized eggs, lack of somite formation, lack of detachment of the tail-bud from the yolk sac, and lack of heartbeat and (2) developmental abnormalities. Analysis of the biodistribution of LNP and localization of induced innate responses (especially depending on different micellar adjuvants) was performed in zebrafish larvae: fluorescent LNP will be administered to reporter zebrafish transgenic lines in which specific cell types were fluorescent. In addition to the validation of the protection against a virulent challenge, the characterization of innate and adaptive immune responses induced by LNP mRNA vaccines was performed in comparison to corresponding DNA vaccines, and the modulation by adjuvant micelles evaluated. Responses were studied using individual monitoring based on regular bleeding, thus increasing greatly the power of the analyses. Serum neutralising Abs were quantitated by plaque assay. Selected markers in blood leukocytes (including CD4, CD8, IgM, IL1, TNF, type I IFN, Mx) were determined by QPCR assays. Random-tag 5’RACE approach was used to amplify and sequence IgH V domains expressed in the spleen and other tissues of challenged fish and to determine the impact of vaccination on the structure of the immune repertoires .

|1- Proof of concept for the efficacy of an mRNA vaccine against a viral disease in fish.

We compared different mRNA delivery systems for in vitro expression in cell lines and in vivo in fish. We developed a proof of concept for RNA vaccination in rainbow trout, a salmonid, demonstrating the efficacy of current vaccine delivery systems in fish.

An LNP-mRNA vaccine protects fish against rhabdovirus infection.

Ayad C, et al. .Vaccine. 2025 Apr 19;53:126957. doi: 10.1016/j.vaccine.2025.126957.

2- Characterisation of the composition of the fish B cell response to mRNA, DNA and live attenuated vaccine

We compared B-cell responses induced by an mRNA, a DNA, and an attenuated vaccine, all encoding the same antigen against a fish rhabdovirus. Rainbow trout IgHμ repertoires were examined to investigate how vaccines reshape clonal composition and complexity of the B-cell repertoire. The attenuated virus drove protection through a small number of highly shared public clonotypes encoding neutralizing antibodies. The mRNA vaccine profoundly remodelled the repertoire in some individuals and induces low, but still protective, neutralising Ab titers without public expansions. The DNA vaccine induced high neutralizing Ab titers, providing full protection with minimal impact on B-cell repertoire. These findings highlight profound divergences between fish B-cell responses to nucleic acid and attenuated vaccines whilst all of three vaccines induce protective responses.

Divergent B-cell repertoire remodelling by mRNA, DNA and live attenuated vaccines in fish

Porter D, et al.

NPJ Vaccines. 2025 Jul 24;10(1):166. doi: 10.1038/s41541-025-01232-8.

3- Impact of mRNA modification on the innate response to the vaccine

We evaluated the innate immune responses elicited by four vaccine platforms: a DNA vaccine, a live attenuated viral hemorrhagic septicemia virus (VHSV), an unmodified mRNA, and an N1MΨU-modified mRNA. All four vaccines encode the G protein of VHSV (GVHSV) in rainbow trout. Following intramuscular injection, both mRNA vaccine formats induced robust type I interferon (IFN) responses, comparable to those induced by the DNA and attenuated virus vaccines. Notably, the N1MΨU-modified mRNA vaccine did not suppress IFN induction, as observed in mammalian systems, but instead triggered distinct temporal transcriptional dynamics and stronger enrichment of autophagy, ubiquitination, and transcription-relate

N1MΨU-modified mRNA vaccines break the mold in fish by enhancing innate immune activation.

Porter D, et al. Mol Ther Nucleic Acids. 2026 Feb 7;37(1):102862. doi: 10.1016/j.omtn.2026.102862.

Publications about the biodistribution of mRNA-LNP and effect of micelles adjuvants are in preparation and will be submitted in 2026.

Since this project succeeded to produce a proof of concept protective fish mRNA vaccine, it will be important to design vaccines and delivery systems able to deal with different water T°C. Two main challenges will have to be addressed: (1) mRNA vaccines and their adjuvants will have to be be optimized to express the Ag and to ensure efficient co-stimulation in targeted farmed fish species, at various T°C, while the available systems have been developed for mammals with body T°C around 37°C. (2) Mechanisms of the effect of T°C on fish immune responses remains poorly understood. Global warming affects fish immune responses because they are ectotherms. It also promotes expansion of pathogen ranges and enhances their virulence. It is therefore required to understand basic mechanisms of adaptation of fish immunity to T°C, especially for cooperation between B and T cells and clonal recruitment, to fine-tune mRNA vaccine vehicles and vaccination protocols.

|Since this project succeeded to produce a proof of concept protective fish mRNA vaccine, it will be important to design vaccines and delivery systems able to deal with different water T°C. Two main challenges will have to be addressed: (1)mRNA vaccines and their adjuvants will have to be be optimized to express the Agand to ensure efficient co-stimulation in targeted farmed fish species, at various T°C, while the available systems have been developed for mammals with body T°C around 37°C. (2) Mechanisms of the effect of T°C on fish immune responses remains poorly understood. Global warming affects fish immune responses because they are ectotherms. It also promotes expansion of pathogen ranges and enhances their virulence. It is therefore required to understand basic mechanisms of adaptation of fish immunity to T°C, especially for cooperation between B and Tcells and clonal recruitment, to fine-tune mRNA vaccine vehicles and vaccination protocols.

Aquaculture has become critical for human food production, and is the fastest growing food production sector. It is threatened by multiple diseases especially those caused by viruses, for which there are no treatment available. The sustainable development of aquaculture therefore requires new vaccines against viral diseases. Although messenger RNA vaccines have recently become an integral part of the vaccine arsenal, mRNA vaccines for fish are still in their infancy. However, we just obtained a LipoNanoParticle (LNP)/mRNA vaccine against a carp virus based on the results of our previous ANR funding, FishRNAvax. This vaccine induces an effective protection with a low dose of mRNA. To optimize this first mRNA vaccine candidate, we propose to understand the mechanisms involved as the mRNA vaccine modes of action remain poorly understood, making it difficult to improve them. To this aim,we will focus on two fish diseases, st the Viral Hemorrhagic Septicemia virus – VHSV - in trout, and against the Spring viraemia of carp virus – SVCV - in carp. These two models correspond to notifiable diseases and target representative species of the two main groups of farmed fish, cyprinids and salmonids. Importantly, they allow a comparison with existing DNA vaccines that induce high protection and neutralising antibodies. The proposal aims at characterizing the immune responses, both innate and adaptive, looking for correlates of protection induced by LNP-mRNA vaccinesin carp, and trout, in order to optimize mRNA vaccine formulation with low amount of mRNA. Thus, we will analyse the biodistribution of LNP mRNA after vaccination, and how it can be modulated by three immunomodulators acting as adjuvants. Indeed, we hypothetize thatco-administration of LNP mRNA with micelles bringing TLR agonists will increase the quality and intensity of responses, and will improve their mucosal localization. In particular, we will characterize the effect of the induction of type I IFN at the site of injection, since it increases considerably the intensity of the response to DNA vaccine in Salmon, and leads to much better protection. We will also investigate the structure of the B/T cell responses induced LNP mRNA vaccines or by challenge in vaccinated fish, by Ig/TCR repertoire sequencing. Based on our previous work, we will determine the frequency of public components (present in all individuals) of adaptive responses induced LNP mRNA vaccines, in comparison to what we previously found after immunization with live attenuated vaccine. The consortium gathers four partners with complementary expertise in chemistry, molecular fish immunology and vaccinology, to optimize the nanoformulation of LNP mRNA vaccines and to explore the particularities of fish responses to these innovative vaccines. The recent development of mRNA vaccines against COVID19 has underscored the lack of knowledge about the characteristics of responses induced by such vaccines, either quality and duration, as well as about their biodistribution, and the importance of the doses to ensure strong efficacy. Beside developing optimized mRNA vaccine candidates for carp and trout, this project aims at providing basic knowledge of immune mechanisms to fish mRNA vaccines when packaged in a biodegradable nanocarrier platform. Furthermore, it will explore i) the importance of mRNA doses for ensuring strong protective capacity ii) the importance of co-administration of immunomodulators (TLR ligands) to increase long lasting immunity and mucosal immune responses. This knowledge will be instrumental to guide future developments and design of the next generation of fish mRNA vaccines.