In vitro search for alternative promoters to the human immediate early cytomegalovirus (IE-CMV) to express the G gene of viral haemorrhagic septicemia virus (VHSV) in fish epithelial cells

Abstract

Present DNA vaccines against fish rhabdoviruses require intramuscular injection (fish-to-fish vaccination) of their G-protein gene under the control of the human immediate early cytomegalovirus (IE-CMV) promoter, while immersion delivery (mass DNA vaccination), for instance, by using fish epithelial-specific promoters, would be more practical for aquaculture. To find fish epithelial-specific promoters alternative to the IE-CMV, a comparative study of the effectiveness of different fish promoters constitutively expressing the G gene of the viral haemorrhagic septicemia virus (VHSV) in the epithelial papulosum cyprini (EPC) cell line was performed. The study included MCV1.4 (an alternative IE-CMV promoter version), AE6 (a version of the carp β-actin promoter), long terminal repeats (LTR) of zebrafish or walleye retroviruses, trout Mx1, carp myosin-heavy-chain and flatfish pleurocidin promoters and salmonid sleeping beauty (SB)/medaka Tol2 transposon repeats. The G-protein expression in transfected EPC cells was studied by estimating the number of cells expressing the G-protein in their membrane and the average expression level per cell. In addition, in an attempt to reduce their sizes, some regions of the MCV1.4 and AE6 promoters were deleted and expression levels compared to those observed for full-length promoters. Since both zebrafish LTR and carp AE6 promoters were the most effective regulatory sequences for expressing the VHSV G-protein in EPC cells, these sequences might be candidates for new DNA vaccine vectors for fish epithelial tissues avoiding the IE-CMV promoter. Furthermore, known transcription factor binding sites (TFBS) common to most of the fish G-expressing promoters, might enable the future design of fully synthetic or hybrid promoters with improved efficacy of VHSV G-protein expression in epithelial fish cells.

Introduction

Fish rhabdoviruses such as infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic septicemia virus (VHSV) constitute one of the main threats to the development of the worldwide aquaculture industry. Present DNA vaccines against fish rhabdoviruses, including, the one against IHNV licensed in Canada for use in salmonids in 2005 [1], require the intramuscular delivery by injection (fish-to-fish DNA vaccination) of a plasmid coding for their highly immunogenic protein G gene placed under the control of the human immediate early cytomegalovirus promoter (IE-CMV) [1], [2], [3]. However, both immersion delivery to obtain in vivo transfection throughout fish epithelial tissues (mass DNA vaccination) and alternative fish promoters to the human IE-CMV (safer constructs) will be more practical for aquaculture [4].

Rhabdoviral infections begin under fish epidermal mucus [5], [6] and specifically in the fin bases [6], thus suggesting that the dermal-epithelial fish tissue is the main target of natural entrance for rhabdoviruses and, possibly, the best fish body site for immunization. Corroborating these observations, mucosal immunity implicating both intraepithelial lymphocyte T markers (CD8, CD4, CD28, CD3ϵ, TCRξ, TCRγ, TCRβ) [7] and their T cell receptor (TCR) repertoir modifications have been demonstrated in both infected and DNA vaccinated trout [7], further underlining the importance of fish body surfaces in generating immune defenses against rhabdoviral fish infections [8] and immunization. Therefore, DNA vaccination through the epithelial cells present in the fish body surface, such as that achieved through immersion DNA vaccination with ultrasounds reported earlier by our group [9] and later corroborated by others [10], could be an improved system to achieve a practical immunization protocol for fish rhabdoviruses. Because eucaryotic gene transcription is tissue-specific, epithelial-specific sequences in new promoters will be required to increase exogenous gene expression on epithelial tissues. Therefore, a search for alternative regulatory sequences or promoters that are, at least, as effective as IE-CMV and could be active when delivered to epithelial cells by immersion has been undertaken in this work. Moreover, it is believed that the substitution of the human IE-CMV promoter by a non-human analog would increase the security of these vaccines and it constitutes a highly convenient step for their commercialization in regions highly concerned with safety, as it occurs in Europe.

Because the epithelial papulosum cyprini (EPC) cell line is readily transfected [11], [12], [13], [14], we have used EPC cells as a model to study fish epithelial expression of the VHSV G-protein. Up to a certain limit, the more the amount of G gene was provided in DNA vaccination, the best fish protection was obtained by using immersion vaccination [9] and/or injection vaccination [15], [16], therefore optimal G gene expression might improve immunization.

Among the possible alternative fish promoters, we have selected: the inverted terminal repeats (itr) of the sleeping beauty (SB) transposon, reconstructed from salmon and trout inactive copies and improved for use in human cells [17], [18]; the internal inverted repeats (iir) from the Tol2 transposon of medaka (Oryzias latipes) [19], [20]; the long terminal repeats (LTR) from two fish retroviruses, the endogenous from zebrafish (ZFERV) [21] (LTRz) and the epithelial Walleye Dermal Sarcoma virus (WDSV) from Stizostection vitreum [22], [23], [24] (LTRw); the promoter of the pleurocidins from Pleuronectes platessa (antimicrobial short cationic peptides from the mucus of flatfish) [25]; the promoter from the trout Mx1 (interferon inducible proteins) [26]; the 5′ regulatory region of the carp myosin-heavy-chain gene, characterized and used to drive the expression of heterologous proteins[27], [28]; and the AE6 version of the β-actin promoter [29], characterized from several fish like carp and tilapia [30] and widely used to express foreign proteins in fish [31], [32], [33].

To compare the activity of the different promoter containing plasmids, the VHSV G-protein expression was quantified by estimating both the number of cells expressing the G-protein in their membrane and the average expression level per cell. Furthermore, partial deletions were performed in the IE-CMV (MCV1.4 version) and carp β-actin (AE6 version) promoters to explore the possibility to decrease their size or to detect important enhancer sequences. The results suggested that zebrafish LTR and carp AE6 promoters might be used as improved substitutes for the IE-CMV promoter.

Since eucaryotic gene transcription depends on tissue-specific trans-acting transcription factors (TF) recognizing cis-acting DNA transcription factor binding sites (TFBS) [34], [35], for an exogenous gene to be constitutively expressed in epithelial cells, TFBS sequences for epithelial-specific TF need to be present in the promoter under which control the gene would be placed. However, few epithelial specific TF/TFBS have been described in vertebrates and less so in fish. Therefore, as a first step to identify some of the required TFBS for the exogenous expression of the G gene of VHSV in epithelial cells, we studied the presence of known TFBS (TRANSFAC data base) in the higher VHSV G-protein expressing promoters. The results showed that some common TFBS could be found in the higher fish promoters, data which might be used to design new synthetic promoters or combinations of some of their sequences [12], [34], [36], [37], such as the SB itr and MCV1.4 (tested in this work), to improve G-protein expression in epithelial fish cells.