An engineered food-grade Lactococcus lactis strain for production and delivery of heat-labile enterotoxin B subunit to mucosal sites

Abstract

BackgroundRecent researches have been focusing on mucosal immune adjuvants, which play the key roles in mucosal immunization and have become the limitation for non-injected vaccine development. Escherichia coli heat-labile enterotoxin B subunit (LTB) was regarded as a promising mucosal adjuvant for its nontoxicity and potent activity. LTB preparation issues have always been recurring, in part owing to that the recombinant LTB expressed by E. coli does not act as its native form.ResultsWe constructed an engineered Lactococcus lactis strain using a food-grade expression system. The LTB secreted by the engineered strain was detected in the culture supernatant, constituting 10.3% of the supernatant proteins, and recognized by mouse anti-LTB antibodies. The engineered strain, co-administered orally to SPF BALB/c mice with a H. pylori vaccine candidate expressing Lpp20 antigen, could significantly enhance the Lpp20-induced mucosal SIgA antibody responses against H. pylori.ConclusionsThis is the first report that LTB was efficiently produced and delivered via using a food-grade lactococcal expression system, which offers a novel production and utilization mode of this crucial mucosal adjuvant. The engineered L. lactis strain secreting LTB has considerable potential for oral vaccine formulation owing to its outstanding safety, adjuvant activity and high-level production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-017-0345-6) contains supplementary material, which is available to authorized users.

E. coli Enterotoxin LtB Enhances Vaccine-Induced Anti-H. pylori Protection by Promoting Leukocyte Migration into Gastric Mucus via Inflammatory Lesions

“…When OD600 of the culture ≈ 0.35, induction of NapA expression was started by adding nisin at a final concentration of 40 μg/L to the culture, which was maintained for 5 h. L. lactis cell lysate samples were prepared via lysozyme digestion and supersonic sonication, and analyzed through SDS-PAGE and western-blot assays using mouse anti- H. pylori sera and mouse anti-LTB antibody (Abcam, Shanghai, China), respectively, as the primary antibody [27,29]. The supernatant of the bacterial culture was sampled and pretreated for SDS-PAGE using the trichloroacetic acid precipitation method reported previously [22].…”

“…Blood, spleen and intestinal feces of the mice were sampled using the methods reported previously [22,25]. Briefly, seven days after the last vaccination, half of the gavaged mice were taken blood from their orbital sinus, and then slaughtered by cervical dislocation.…”

“…As reported, oral delivery of papillomavirus 16 E7 antigen by L. lactis protected the mice from infection caused by the virus challenges [21]. As for anti- H. pylori immunization, however, most studies showed oral gavages with L. lactis -delivered H. pylori antigens could induce elevated systemic and mucosal immune responses, or reduce gastric bacterial burden, but would rarely be able to prevent H. pylori infection, suggesting the relatively weak adjuvant effect of L. lactis and the necessity of using additional adjuvant in the L. lactis -vectored H. pylori vaccines [19,22,23].…”

Plasmid Replicons for the Production of Pharmaceutical-Grade pDNA, Proteins and Antigens by Lactococcus lactis Cell Factories

“…Secretion of antigens can be achieved by cloning the signal sequence of usp45 gene upstream of the antigen coding gene (ORF) in the pNZ8149 plasmid. Recently, Sun et al [ 102 ] used this approach with L. lactis for producing heat-labile enterotoxin B subunit as adjuvant in oral vaccines formulation. When co-administered to mice with a Helicobacter pylori vaccine candidate expressing Lpp20 antigen, it significantly enhanced the mucosal antibody responses against H. pylori [ 102 ].…”

“…Recently, Sun et al [ 102 ] used this approach with L. lactis for producing heat-labile enterotoxin B subunit as adjuvant in oral vaccines formulation. When co-administered to mice with a Helicobacter pylori vaccine candidate expressing Lpp20 antigen, it significantly enhanced the mucosal antibody responses against H. pylori [ 102 ]. A different study used a recombinant L. lactis with a pNZ8048 vector expressing exendin-4, a receptor agonist that is a therapeutic peptide drug for type 2 diabetes.…”