Production of a viral surface protein in Nannochloropsis oceanica for fish vaccination against infectious pancreatic necrosis virus

Abstract

Nannochloropsis oceanica is a unicellular oleaginous microalga of emerging biotechnological interest with a sequenced, annotated genome, available transcriptomic and proteomic data, and well-established basic molecular tools for genetic engineering. To establish N. oceanica as a eukaryotic host for recombinant protein synthesis and develop molecular technology for vaccine production, we chose the viral surface protein 2 (VP2) of a pathogenic fish virus that causes infectious pancreatic necrosis as a model vaccine. Upon stable nuclear transformation of N. oceanica strain CCMP1779 with the codon-optimized VP2 gene, a Venus reporter fusion served to evaluate the strength of different endogenous promoters in transformant populations by qPCR and flow cytometry. The highest VP2 yields were achieved for the elongation factor promoter, with enhancer effects by its N-terminal leader sequence. Individual transformants differed in their production capability of reporter-free VP2 by orders of magnitude. When subjecting the best candidates to kinetic analyses of growth and VP2 production in photobioreactors, recombinant protein integrity was maintained until the early stationary growth phase, and a high yield of 4.4% VP2 of total soluble protein was achieved. The maximum yield correlated with multiple integrations of the expression vector into the nuclear genome. The results demonstrate that N. oceanica was successfully engineered to constitute a robust platform for high-level production of a model subunit vaccine. The molecular methodology established here can likely be adapted in a straightforward manner to the production of further vaccines in the same host, allowing their distribution to fish, vertebrates, or humans via a microalgae-containing diet. Key points • We engineered N. oceanica for recombinant protein production. • The antigenic surface protein 2 of IPN virus could indeed be expressed in the host. • A high yield of 4.4% VP2 of total soluble protein was achieved in N. oceanica.

High-Level Expression of Recombinant VHSV Glycoprotein Using Transformed C. vulgaris and Verification of Vaccine Efficacy

“…For example, in previous studies, the expression level of recombinant basic fibroblast growth factor (bFGF) was 1.61 µg/kg in C. vulgaris compared with 42 g/kg in E. coli, and 185.7 mg/kg in transgenic rice seeds [45][46][47]. In other studies, the expression level of viral surface protein 2 (VP2) of infectious pancreatic necrosis virus in transformed Nannochloropsis oceanica was 1-4.9% of TSP, and those of recombinant proteins in Chlamydomonas reinhardtii were reported to range from 0.1% to 10% of TSP [48,49].…”

“…coli , and 185.7 mg/kg in transgenic rice seeds [ 45 , 46 , 47 ]. In other studies, the expression level of viral surface protein 2 (VP2) of infectious pancreatic necrosis virus in transformed Nannochloropsis oceanica was 1–4.9% of TSP, and those of recombinant proteins in Chlamydomonas reinhardtii were reported to range from 0.1% to 10% of TSP [ 48 , 49 ].…”

“…vulgaris in 1 L culture was produced, representing 27.4% of the TSP. The yields of recombinant proteins from transformed microalgae were reported to range from 0.006 to 6.4 mg/L, corresponding to 0.1% to 10% of the TSP [ 45 , 48 , 49 ]. Therefore, the expression level achieved here by NaCl induction was efficient for the production of recombinant vaccine protein and may also be applicable to the production of other valuable recombinant proteins in the future.…”

Transforming Aquaculture Through Vaccination: A Review on Recent Developments and Milestones

“…By ingesting these organisms, the bioencapsulated antigens are absorbed intact, facilitating the stimulation of both systemic, and mucosal immune responses [96]. This platform enables the oral vaccination of smallersized aquatic organisms, with fish fry being placed in the center of this approach, as it allows vaccine antigens to be incorporated in planktonic organisms that serve as vital food sources for the fry and are rich in polyunsaturated fatty acids (PUFAs), pigments, and antioxidants [82,[205][206][207]. The main organisms utilized in the bioencapsulation of vaccine antigens are mainly crustaceans of planktonic nature, such as brine shrimp nauplii of the genus Artemia, water fleas of the genus Daphnia for freshwater applications, and various small rotifers [10,96].…”

Current status and development prospects of aquatic vaccines

“…Flagellin is also well-studied and effective subunit vaccine candidate ( 185 ). Viral capsid proteins are also regarded as significant possible antigens for the development of subunit vaccines ( 186 , 187 ). It is concerning that subunit vaccines frequently contain a single antigen, resulting in low immunization efficiency, necessitating multiple immunizations and the addition of adjuvants to improve the immunization effect or the development of multiple vaccines ( 188 – 192 ).…”