Full length articleOral immunization with a recombinant Lactobacillus expressing CK6 fused with VP2 protein against IPNV in rainbow trout (Oncorhynchus mykiss)
Introduction
Infection pancreatic necrosis virus (IPNV) is a member of the Aquabirnavirus genus in the Birnaviridae family. It causes pancreatic necrosis disease, which infects wild and cultured salmonid fish causing high mortality with serious economic losses to salmonid aquaculture [[1], [2], [3], [4]].
Among the current vaccines for preventing IPNV, inactivated vaccines stimulate a systemic immune response upon injection causing mechanical injury, and the attenuated vaccine elicits a good immune response, but faces the risk of reversion to virulence and spread of virus [[5], [6], [7], [8], [9]]. However injection cannot be used for small fish, therefore either oral delivery or immersion are more preferable routes for early inoculation [10]. Although several studies have focused on the development of IPNV DNA vaccines, these vaccines which current research are not formulated for oral immunization [[11], [12], [13], [14]].
Lactobacillus are ‘generally regarded as safe (GRAS)’ microorganisms, used in fermenting and preserving food for many years [15,16]. In recent years, Lactobacillus strains have been utilized for drug delivery owing to their ability to present compounds on the mucous membrane, especially immune-modulators and vaccines [10]. Many kinds of lactic acid bacteria (LABs) have the ability to subsist and colonize in the gut. Furthermore, they are an ideal safe bacterial vaccine vector with maximum therapeutic effect and minimal side effects [17,18]. Compared to the traditional injectable vaccine, oral delivery or immersion is more suitable for early vaccination of juvenile fish. In previous studies, we constructed the recombinant Lactobacillus casei VP2 gene expression system of IPNV that has the ability to colonize the intestinal tract and improve the anti-viral response in rainbow trout [10,19]. Disadvantages such as low antigen expression, multiple and long-time immunity, and low levels of specific antibody limit the application of recombinant oral vaccines of lactobacillus [19].
Inclusion of a gene targeting modification in vaccine formulations allows for immunological targeting and improved antigenicity of the vaccine. Several studies have shown that these genes with the capacity to accurately target antigens to a specific site can act as “active molecular adjuvants” [[46], [47], [48]]. Chemokines are low-molecular weight cytokines (mostly 8–10 kDa) that attract and modulate immune cells to migrate to the site of infection especially in response to inflammation. Similar to that seen in mammals, chemokines exist as potential chemical attractants in the process of the migration of macrophages (Mø) and neutrophils in fish [20]. CK6 belonging to the CC family has a strong ability to attract Mø and induce the expression of IL-8, which increases specific antibody levels and promotes the proliferation of Th cells [21].
In this study, we have explored the bioactivity of rainbow trout CK6 fused with target antigen VP2 of IPNV co-expressed in Lactobacillus. After rainbow trout individuals were vaccinated with Lactobacillus-derived CK6-VP2 and challenged with IPNV, there were significant reductions in the viral loads. The mRNA expression profiles of several markers of the innate and adaptive immune responses including CK6, IL-1β, IL-8, TNF-1α, Mx, MHC-Ⅱ, and β-defensin were examined in the spleen and gut of inoculated fishes and compared to the sham-injected ones. The results showed that the recombinant Lactobacillus pPG-612-CK6-VP2/L. casei 393 could induce specific IgM and IgT antibodies against IPNV.
Section snippets
Experimental fish
Healthy rainbow trout individuals without a previous history of IPNV infection and an average weight of 11.5 g were obtained from a cold-water fish experimental station (Mudanjiang, China). Fish were kept at 16 °C in closed recirculating water tanks (120 cm × 50 cm × 60 cm) and fed daily with a diet of commercial dry pellets. The fish tanks were cleaned once a week.
Viruses, bacterial strains, plasmids, and reagents
Chinook salmon embryonic cell line (CHSE-214) was obtained from the American Type Culture Collection (ATCC) and grown in L-15
Expression of CK6-EGFP protein in E. coli and CK6-VP2 protein in Lactobacillus
The immunoreactive band for expression of CK6-EGFP protein was detected by western blotting. The results showed that the proteins were expressed as a soluble protein in the supernatant. An immunoreactive band of 75 kDa (Fig. 2A) protein corresponding to EGFP protein was detected, the results of the supernatant and pellet fractions were the same, but this band was not detected in the un-induced recombinant strain. In the same conditions, CK6-EGFP protein was detected as a 94 kD immunoreactive
Discussion
IPNV is the causative agent of infection pancreatic necrosis disease, which infects wild and cultured salmonid fish, causing high mortality in juvenile trouts and salmons with serious economic losses to salmonid aquaculture. Currently available vaccines employ strategies concentrated on prevention via the parental route but not the mucosal routes [35].
Lactobacillus has been widely used in oral vaccines as an ideal delivery vehicle as lactic acid bacteria are recognized as GRAS bacteria [36,37].
Acknowledgements
The authors wish to thank Jos Seegers for providing the plasmid pPG-612 and Lactobacillus casei ATCC 393. The study was supported by National Science Foundation (NSF) (NO. 31372568, 31672697) and national natural fund international cooperation program(NO. 31511130137).
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2022, Aquaculture Pathophysiology: Finfish Diseases: Volume IOral vaccine against IPNV based on antibiotic-free resistance recombinant Lactobacillus casei expressing CK6-VP2 fusion protein
2021, AquacultureCitation Excerpt :Although these vaccines can provide protective immunity, they do not stimulate the mucosal immune response. Moreover, although the orogastric administration of some IPNV oral vaccines can effectively stimulate intestinal mucosal immunity (Liu et al., 2012; Duan et al., 2018), they are accompanied by serious side effects due to their potentially antibiotics toxic components. Therefore, there is a need to develop a more effective and safe (without side effects) oral vaccines against IPNV that can effectively induce immune responses at both the mucosal and humoral levels.
Oral immunization with recombinant Lactobacillus casei displayed AHA1-CK6 and VP2 induces protection against infectious pancreatic necrosis in rainbow trout (Oncorhynchus mykiss)
2020, Fish and Shellfish ImmunologyCitation Excerpt :Furthermore, L. casei species can enhance innate immune responses, induce anti-inflammatory responses and regulate the balance between T cell subset responses [17]. Duan et al. reported that the rainbow trout chemokine (CK6) fused with VP2 co-expressing in LAB displayed decent immunogenicity against IPNV [18]. In order to improve the ability of antigen adhesion in the intestinal tract, thereby prolong the retention time of the antigen in the intestine tract and increase the accessibility of the antigen and the immune system, which could stimulate the body generate stronger immune response.
Mucosal delivery of fish vaccines: Local and systemic immunity following mucosal immunisations
2020, Fish and Shellfish ImmunologyCitation Excerpt :Furthermore, oral vaccination employing bioencapsulation in live vehicles such as the brine shrimp Artemia [59], rotifers and water fleas [60] has been developed for larval fish that cannot feed on pelleted food. Oral antigen administration, including that of inactivated virus and recombinant viral antigens as well as DNA vaccines, provide effective protection for fishes and result in the upregulation of genes related to adaptive immunity [54–56,60–62] and local and systemic antibody production [63], suggesting induced adaptive immunity in digestive tract. However, recent studies reported that oral administration of alginate encapsulated DNA vaccines did not confer protection against spring viremia of carp virus (SVCV) [64] or koi herpes virus (KHV) in common carp [65].