Oral immunization with a recombinant Lactobacillus expressing CK6 fused with VP2 protein against IPNV in rainbow trout (Oncorhynchus mykiss)

Highlights

• We engineered Lactobacillus casei 393 expressing VP2 protein of IPNV modified by targeted molecules.

• The recombinant strain modified by CK6 could lead to a stronger and quicker immune response.

• The chemokine has the ability to attract immune cells to enhance the function of the innate immune response.

• The pPG-612-CK6-VP2/L. casei 393 is a promising candidate for the development of an oral vaccine against IPNV.

Abstract

Infectious pancreatic necrosis virus (IPNV) infects wild and cultured salmonid fish causing high mortality with serious economic losses to salmonid aquaculture. Ideally, the method of oral immunization should prevent the infection of rainbow trout juveniles with IPNV. In the present study, genetically engineered Lactobacillus casei 393 pPG-612-VP2/L. casei 393 and pPG-612-CK6-VP2/L. casei 393 constitutively expressing VP2 protein of IPNV were constructed. The recombinant strains pPG-612-CK6-VP2/L. casei 393 and pPG-612-VP2/L. casei 393 were orally administrated to juvenile rainbow trouts, and significant titers of IgM and IgT of pPG-612-CK6-VP2/L. casei 393 were observed. The results demonstrate that the recombinants could elicit both local mucosal and systemic immune responses. The proliferation of spleen lymphocytes in trouts immunized with pPG-612-CK6-VP2/L. casei 393 showed that the recombinant strain could induce a strong cellular immune response. The IL-1β, IL-8, CK6, MHC-II, Mx, β-defensin, and TNF-1α levels in the spleen and gut suggest that the target molecular chemokine has the ability to attract relevant immune cells to participate in the inflammatory response and enhance the function of the innate immune response. Additionally, the pPG-612-CK6-VP2/L. casei 393 induced the expression of cytokines, which have the effect of promoting inflammation to drive the differentiation of macrophages and clear target cells. After challenging with IPNV, the reduction in viral load caused by pPG-612-CK6-VP2/L. casei 393 was significantly higher than that of the other groups. Thus, the recombinant pPG-612-CK6-VP2/L. casei 393 is a promising candidate for the development of an oral vaccine against IPNV.

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 T_h 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, M_x, 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.