Oral Immunization of Recombinant Lactococcus lactis and Enterococcus faecalis Expressing Dendritic Cell Targeting Peptide and Hexon Protein of Fowl Adenovirus 4 Induces Protective Immunity Against Homologous Infection

Abstract

Hepatitis-hydropericardium syndrome (HPS) causes severe economic losses in the global poultry industry. The present study aims to explore oral immunization of recombinant Lactococcus lactis and Enterococcus faecalis expressing Hexon protein of fowl adenovirus 4 (FAdV-4). The bacteria L. lactis NZ9000 and E. faecalis MDXEF-1 were, respectively, modified as host strain to deliver truncated Hexon protein (ΔHexon) or ΔHexon protein fusing with dendritic cell (DC) targeting peptide (DC-ΔHexon) on the surface of bacteria. The expression of target protein in L. lactis NZ9000 and E. faecalis MDXEF-1 were detected by western blot. To evaluate the immune responses and protective efficacies provided by the live recombinant bacteria, chickens were immunized with the constructed ΔHexon-expressing bacteria three times at 2-week intervals, then experimentally challenged with hypervirulent FAdV-4/GX01. The results showed that oral immunizations with the four ΔHexon-expressing bacteria (NZ9000/ΔHexon-CWA, NZ9000/DC-ΔHexon-CWA, MDXEF-1/ΔHexon-CWA, and MDXEF-1/DC-ΔHexon-CWA), especially the two bacteria carrying DC-targeting peptide, stimulated higher levels of ΔHexon-specific sera IgG and secretory IgA (sIgA) in jejunal lavage fluid, higher proliferation of peripheral blood lymphocytes (PBLs) and higher levels of Th1/Th2-type cytokines, along with significantly decreased virus loads in liver and more offered protective efficacies against FAdV infection compared with PBS and empty vector control groups (p < 0.01). For chickens in the group MDXEF-1/DC-ΔHexon-CWA, the levels of aspartate transaminase (AST), alanine transaminase (ALT) and lactate dehydrogenase (LDH) in sera, and the virus loads in livers were significantly decreased vs. the other three ΔHexon-expressing bacteria (p < 0.01). The pathological changes in the hearts, livers, spleens and kidneys of chickens in MDXEF-1/DC-ΔHexon-CWA group were relatively slight compared to infection control group and other three ΔHexon-expressing bacteria groups. The rate of protection in MDXEF-1/DC-ΔHexon-CWA group was 90%. The present work demonstrated that cell surface-displayed target protein and immune enhancers in L. lactis and E. faecalis might be a promising approach to enhance immunity and immune efficacy against pathogen FAdV-4 infection.

Development of a Novel Avian Vaccine Vector Derived From the Emerging Fowl Adenovirus 4

“…Previous studies showed that an inactivated FAdV-4 oil-emulsified vaccine ( Pan et al, 2017b ) and subunit vaccines based on fiber 2 and hexon ( Ruan et al, 2018 ; Hu et al, 2021 ) could provide complete protection against FAdV-4. Recombinant Lactococcus lactis and Enterococcus faecalis expressing hexon ( Jia et al, 2021 ) and Newcastle disease virus (NDV) expressing fiber 2 ( Tian et al, 2020 ) were also constructed and evaluated. Given the emergence, high pathogenicity, and lack of knowledge of the non-essential regions of the novel FAdV-4, limited progress has been achieved on the development of the FAdV-4 vector.…”

Probiotics Surface-Delivering Fiber2 Protein of Fowl Adenovirus 4 Stimulate Protective Immunity Against Hepatitis-Hydropericardium Syndrome in Chickens

“…After washing, the immunoreactive bands on the membrane were visualized using ECL Chemiluminescence Detection Kit (P0018S) (Beyotime, Shanghai, China) according to the manufacturer’s instructions. To further demonstrate that Fiber2 protein was displayed on the surface of recombinant probiotics, an indirect immunofluorescence assay (IFA) was carried out as described previously ( 27 ). In short, the probiotics were cultured and induced with nisin (5 ng/ml), and then the bacterial pellets were harvested by centrifugation.…”

“…Likewise, probiotics L. plantarum that display avian pathogen antigenic proteins, such as gp85 of subgroup J avian leukosis virus (ALV-J) ( 23 ), L. lactis that express ORF2 of avian hepatitis E virus (aHEV) ( 24 ), E. faecalis that express 3-1E, and L. plantarum that deliver MIC2 of Eimeria tenella ( 25 , 26 ), have shown protective efficacies against poultry diseases. Meanwhile, our previous studies demonstrated that intestinal mucosal and humoral immune responses elicited by oral immunization with a probiotic vaccine expressing ΔHexon protein provided partial protection against FAdV-4 ( 27 ). FAdV-4 has a typical structure that consists of four structural proteins, namely, Hexon, Penton, Fiber1, and Fiber2 ( 28 ).…”

Immunoprotection of FliBc chimeric fiber2 fusion proteins targeting dendritic cells against Fowl adenovirus serotype 4 infection

“…Dendritic cells (DC) possess a robust capacity for antigen presentation, it is frequently utilized as targets for antigens which making a major determinant of vaccination ( Cohn et al, 2014 ; Macri et al, 2016 ; Matsuda et al, 2022 ). The recombinant Lactococcus lactis expressing FAdV-4-hexon fusion DC targeting peptide was constructed, which resulted in increased levels of serum IgG and secretory IgA ( sIgA ) in jejunal lavage fluid after oral immunization compared to the PBS and empty vector control groups, providing enhanced protection against FAdV infection ( Jia et al, 2021 ). Short peptides (SP) capable of targeting DC can be screened and expressed through fusion with antigen proteins to prepare targeted vaccines, thereby achieving precise and effective delivery of antigens to DC, ultimately enhancing the immunogenicity of antigens ( Chen et al, 2016 ; Shrestha et al, 2018 ; Xia et al, 2022 ).…”