W42:
The aim of the work carried out by W42 was the expression of recombinant variants of the rainbow trout tBAFF-R. As host, the methylotrophic yeast Pichia pastoris (Komagataella phaffii) was selected. The protein to be expressed consists of the receptor domain from Oncorhynchus mykiss (AAK53870.1) fused to an immunoglobulin heavy chain region from Mus musculus domesticus (LOC118938457).
Two different variants were developed:
A) For extracellular production, tBAFF-R was N-term fused with the a-signal peptide from Saccharomyces cerevisiae. The resulting strain was named BAFF1-1.
B) For intracellular production, a variant without any signal peptide was tested. The resulting strain was named BAFF2.
The results obtained were the following:
Variant A) Samples were analyzed for tBAFF-R production and location of the protein. The protein was not exported and was completely present in the non-soluble cell-debris fraction.
Variant B) Samples were analyzed for t-BAFF-R production and location of the protein. The protein was produced completely in the non-soluble cell-debris fraction. Attempts were made to solubilize the protein, but transfer of tBAFF-R into the soluble fraction was not possible.
Therefore, for oral application, broth from fermentation of the strain BAFF1-1 was used to produce freeze-dried extract. For injection, although the tBAFF-R protein seemed to be completely in the insoluble fraction, a preparation of soluble protein by IMAC and subsequent ultrafiltration was carried out.
In summary, the protein tBAFF-R was successfully produced in P. pastoris. Unfortunately, the extracellular production of tBAFF-R with variant BAFF1-1 was not successful. However, this strain could be used to produce samples for oral treatment of infected fish. Unexpectedly, in the variant BAFF2 most of the protein was produced in the insoluble fraction and the overall yield was lower in comparison to BAFF1-1.
CSIC:
With the purified tBAFF-R sent to us by W42, we performed a series of in vitro experiments to determine if the protein had the capacity to block BAFF signaling in rainbow trout. For this, spleen or blood leukocyte populations isolated from rainbow trout were exposed to different doses of tBAFF-R in the presence and absence of recombinant BAFF (also at different doses). After 48 h of incubation at 20ºC, the percentage of IgM B cells and their proliferative activity was determined by flow cytometry. The number of cells secreting IgM was also studied in the cultures by ELISpot. As previously observed, BAFF significantly increased the percentage of IgM B cells in the cultures, their proliferative capacity and their IgM secretion. However, none of the tBAFF-R doses tested were capable of significantly blocking these activities. On the other hand, in some cases, synergistic effects were observed.
Despite these results, we also performed a field experiment to establish if the recombinantly produced tBAFF-R had some effect on the survival to a PKD infection in the field. For this, groups of rainbow trout were either intraperitoneally injected with purified tBAFF-R or mock-injected (BAFF-2). Pichia expressing tBAFF-R or Pichia alone were also administered to some trout by force-feeding (BAFF1-1). These trout were introduced in cages in a farm in Guadalajara (Spain) in the beginning of the summer. During 60 days, fish were naturally exposed to a PKD outbreak and mortalities recorded. We did not observe significant differences in the final mortality rates among groups.
To the light of the results obtained with the recombinant tBAFF-R and the difficulties encountered to produce high amounts of pure protein in Pichia, we decided to undertake more experiments with the tBAFF-R-coding plasmid, as the results seem to point to the plasmid being the easiest way to block BAFF activity and increase the survival during PKD. Hence, we injected fish with the plasmid intramuscularly and at different times post-injection, we obtained blood and muscle to study the transcription of the plasmid. We found that for at least 45 days post-injection, the plasmid was still being effectively transcribed in fish.
Red mark syndrome (RMS) is a disease thought to be provoked by an intracellular bacteria, which affects rainbow trout producing important lesions in the skin. The disease has been seen to go along with a significant proliferation and differentiation of B cells at both systemic and local (in the skin) levels. To determine whether the plasmid coding for tBAFF-R could reduce the symptomatology associated to RMS, we injected fish with the tBAFF-R-coding plasmid or with a control plasmid and at different times post-injection, fish were experimentally exposed to RMS. During several weeks, the progression of the disease was monitored. In this experiment, the plasmid was not capable to reduce the progression of skin lesions in the fish. These results pointed to the tBAFF-R plasmid being exclusively effective against PKD, as this was something that had been broadly demonstrated prior to this project.
