Surface display of hirame novirhabdovirus (HIRRV) G protein in Lactococcus lactis and its immune protection in flounder (Paralichthys olivaceus)

Abstract

Background Hirame novirhabdovirus (HIRRV) can infect a wide range of marine and freshwater fish, causing huge economic losses to aquaculture industry. Vaccine development, especially oral vaccine, has become an effective and convenient way to control aquatic infectious diseases. HIRRV glycoprotein (G), an immunogenic viral protein is a potential vaccine candidate for prevention of the disease. Here, we aimed to construct a recombinant Lactococcus lactis strain expressing HIRRV-G on the cell surface as an oral vaccine to prevent HIRRV. Results Glycoprotein gene of HIRRV was successfully cloned and expressed in L. lactis NZ9000 in a surface-displayed form, yielding Ll:pSLC-G. An approximately 81 kDa recombinant G protein (containing LysM anchoring motif) was confirmed by SDS-PAGE, western blotting and mass spectrometry analysis. The surface-displayed G protein was also verified by immunofluorescence and flow cytometry assays. Furthermore, to evaluate the potential of Ll:pSLC-G as oral vaccine candidate, flounders were continuously fed with commercial diet pellets coated with 1.0 × 10 9 cfu/g of induced Ll:pSLC-G for 1 week. Four weeks later, booster vaccination was performed with the same procedure. Compared with the controls, Ll:pSLC-G elicited significantly higher levels of specific IgM against HIRRV in flounder gut mucus at the second week and in serum at the fourth week ( p < 0.05). Meanwhile, oral immunization with Ll:pSLC-G could provide 60.7% protection against HIRRV infection and a significantly lower virus load was detected than the controls on the third day post-challenge ( p < 0.01). Moreover, on the first day post 1-week feeding, approximately 10 4 –10 5 recombinant L. lactis cells were detected in every gram of foregut, midgut and hindgut of flounder, which were mainly localized at the bottom of gut mucus layer; and on day 21, 10 2 –10 3 L. lactis cells could still be recovered. Conclusions HIRRV-G protein was successfully expressed on the surface of L. lactis cells, which could trigger mucosal and humoral immune response of flounder and provide considerable immune protection against HIRRV. It suggests that genetically engineered L. lactis expressing G protein can be employed as a promising oral vaccine against HIRRV infection.

Lactococcus lactis Expressing Type I Interferon From Atlantic Salmon Enhances the Innate Antiviral Immune Response In Vivo and In Vitro

“…There, the immune cells could stimulate other cells either by endogenous release of interferon or by the release of interferon produced by MT006, in a mechanism similar to that observed when dendritic cells interact with commensal bacteria in the mammalian intestine and transport them to the lymph nodes (89). This mechanism could explain the immunizing properties of orally administered recombinant Lactic Acid Bacteria that express epitopes from microbial pathogens of fish (33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44). The dosages used in these experiments are between 10 7 to 2x10 8 CFU per fish gram, around 30 to 600 times the used dosage of MT006.…”

“…The oral administration of these improved probiotics produces systemic effects, conferring protection against virus (29), bacterial pathogens (30,31), and parasites (32). In salmonids, oral administration of Lactobacillus casei species expressing IPNV epitopes has shown to confer protection against the virus (33)(34)(35)(36)(37), while Lactococcus lactis strains have been used to orally immunize against Hirame novirhabdovirus in flounder (38), Carp spring viremia in common carps (39), the protozoa Ichthyophthirius multifiliis in Goldfish (40), and against the viral hemorrhagic septicemia in rainbow trout (41). Oral administration of recombinant Lactobacillus casei expressing epitopes of Aeromona veronii confers protection against this pathogen in Cyprinus carpio (42,43), and Carassius carassius (44), while Lactobacillus plantarum expressing G protein of spring viremia of carp virus (SVCV) (45) and the ORF81 protein of koi herpesvirus (KHV) has shown confers protection against both viruses with high titers of IgM after its oral administration to Cyprinus carpio (46).…”

Importance of Probiotics in Fish Aquaculture: Towards the Identification and Design of Novel Probiotics

“…In salmonids, oral administration of L. casei species expressing epitopes from infectious pancreatic necrosis virus (IPNV) has been shown to confer protection against the virus [ 125 , 126 , 127 , 128 , 129 ], while L. lactis strains have been used to orally immunize against viral hemorrhagic septicemia virus (VHSV) [ 130 ]. L. lactis has also been used to immunize against hirame novirhabdovirus (HIRRV) in flounder ( Paralichthys olivaceus ) [ 131 ]. In C. carpio (common carp), oral administration of recombinant L. casei expressing epitopes of A. veronii [ 132 , 133 , 134 , 135 ] or A. hydrophila confers protection against these pathogens [ 136 ], while the administration of L. plantarum expressing G protein of spring viremia of carp virus (SVCV) [ 137 ] and the ORF81 protein of koi herpesvirus (KHV) grants protection against both viruses in challenge assays, with high titers of IgM after its oral administration to C. carpio [ 138 ].…”

Recombinant vaccines in 2022: a perspective from the cell factory

“…Based on the assumption of safety, the development of expression systems in several strains of such species has thrived as of lately. Many and diverse microbial antigens produced in or surface-displayed on lactic acid bacteria have been able to elicit significant immune and often protective responses [96][97][98][99][100][101]. These bacteria do not only serve as microbial cell factories [102,103] but also as vaccine vectors [103,104], for orally and mucosally-administered formulations, as they are able to induce a potent immune response by expressing foreign antigens, while drawing a minimal response against themselves [105][106][107].…”