Tailoring mRNA towards future vaccine for lumpfish (Cyclopterus lumpus) – Protein expression and immune responses

Abstract

Lumpfish plays an important role in aquaculture, where it is used to combat sea lice infestations among farmed salmon, acting as a biological alternative to chemical and physical treatment. As a relative newcomer to aquaculture, lumpfish is vulnerable to bacterial and viral infection. Due to a lack of vaccines offering effective protection against pathogens, lumpfish suffers high mortality, and their use has decreased in recent years. Developing vaccines against viral diseases is generally challenging. Currently, available anti-viral vaccine strategies for fish are whole virus, recombinant, and DNA vaccines. While messenger RNA (mRNA) vaccines have been developed for humans, no mRNA vaccine has yet been developed for fish. In vitro transcription (IVT) of mRNA provides a method for synthesising mRNA directly from a sequence template in a cell-free environment. Following delivery, mRNA encoded proteins are synthesised in the target cells, thereby avoiding difficulties in culturing viruses and bacteria in the lab, needed for whole virus/bacteria vaccines, and challenge experiments with misfolded proteins as recombinant vaccines. In this study, various DNA templates were made containing a lumpfish 5’ untranslated region (UTR), an open reading frame encoding enhanced green fluorescent protein (EGFP) as a reporter gene, and one or two lumpfish 3’UTRs, were made for IVT. Additionally, mRNAs containing various modifications possibly beneficial for mRNA protection and translation was synthesised by IVT. Recombinant mRNA was transfected into XL cells, a cell line derived from lumpfish liver, and EGFP expression was monitored by fluorescent microscopy and flow cytometry. Several transfection reagents were tested, revealing lipofectamine 3000 (L3000) to be the most efficient. As IVT mRNA may mimic viral RNA, innate immune responses can be initiated, leading to degradation of mRNA and inhibited translation, resulting in low expression of the encoded protein. Following transfection, innate immune responses, measured by qPCR, showed increased expression of interferons (IFNs) 24 hours post transfection. While successful, transfection efficiency of EGFP in XL cells was low, and should therefore be further optimised. An attempt to transfect lumpfish leukocytes was unsuccessful. Despite this, transfection of mRNA expressed EGFP faster and at a higher transfection efficiency than DNA of the same sequence, highlighting the benefits and potential mRNA vaccines may have with further study. This work is but the first step towards future mRNA vaccines for lumpfish.