Introduction
Infectious hematopoietic necrosis virus (IHNV , Salmonid novirhabdovirus) poses a major threat to salmon and trout aquaculture around the globe, with the virus nowa days present in America, Asia and Europe (1). Significant efforts have been made to develop an effective vaccine to prevent the spread of the disease. Most of the experimental anti-IHNV vaccine s reported so far are inactivated virus or DNA vaccines, usually administered by injection (2) . The protective e fficacy of injected vaccines has been fairly good in most cases, with one licensed vaccine (Apex IHNV, Canada) available . In contrast , the development vaccines designed for oral administration to fish ( a most desirable way to be implemented in aquaculture facilities) has been challenging (3).
Previous studies by our group demonstrated the efficacy of recombinant vaccines based on the expression of the main antigenic region of the rhabdoviral glycoprotein (G) in E. coli (as nanopellets ) against spring viremia of carp virus (SVCV) in zebrafish and viral hemorrhagic septicemia virus (VHSV) in rainbow trout (4).
In the present work, a fragment of the IHNV glycoprotein (G) al one as well as fused to gamma interferon were expressed in the microalga Chlamydomonas reinhardtii . The final goal of the project is to deliver an oral vaccine to be used in rainbow trout aquaculture as a preventive method to control IHNV outbreaks.
Results
A 190-mer fragment of IHNV G protein was successfully cloned into a Chlamydomonas reinhardtii expression vector, as well as the IHNV-G fused to trout gamma interferon. Positive clones were selected by growing on antibody-agar plates. Next, the p roduction of the antigens (IHNV-G, IFN-IHNG) was verified by western-blot analysis, observing the bands at the expected size (26,1kDa and 45kDa, respectively).
Cultures of clones expressing IHNV-G and IFN-IHNG were concentrated and lyophilized. After reconstitution in PBS the viral antigens were delivered to rainbow trout fingerlings (three 6mg doses per fish) by intra gastric gavage. At 4 and 30 days post-intubation, serum, head kidney, spleen and midgut samples were collected for analysis. When usi ng IFN- IHNG as antigen ELISA OD values in sera were significantly higher at 30 days post intubation than in the control group (fish immunized with non-transformed microalgae). The IHNV-G group showed relatively higher OD values than controls , but the difference was not significant.
Immune-related genes expression was analyzed in the mid gut, spleen and head kidney at three time points after intubation . At early time (4 dp.int.), two interferon-inducible antiviral protein genes (mx and vig) were significantly augmented in head kidney , spleen and intestine of fish intubated with IFN-IHNG. At later times (30 days) up-regulation of mx and vig genes in spleen and intestine was observed. Immunoglobulin M (IgM ) gene expression was not found significantly changed at any time point in immunized fish.
In a follow up experiment, the efficacy of the microalgae -based oral vaccine will be tested on rainbow trout under a diet supplemented with recombinant Chlamydomonas and subsequently challenged with IHNV.
Funding: This work was supported by project PID2021-126710OB-C22 funded by MIVIU/AEI (Spain) and FEDER (UE). We thank Dr. Ralph Bock (Max Planck Institute, Germany) for providing the Chlamydomonas reinhardtti strains.
References