Expression of a hepatitis A virus antigen in Lactococcus lactis and Escherichia coli and evaluation of its immunogenicity

Abstract

An epidemic shift in Hepatitis A virus (HAV) infection has been observed in recent years in rapidly developing countries, with increasing numbers of severe adult cases which has led to renewed interest in vaccination. Our approach in vaccine development uses recombinant expression of the highly immunogenic HAV antigen VP1-P2a in food-grade lactic acid bacterium Lactococcus lactis and in Escherichia coli. We used genetic constructs that enable nisin-controlled expression of the antigen in L. lactis in three different forms: (a) intracellularly, (b) on the bacterial surface and (c) on the bacterial surface fused with the fragment of the E. coli flagellin molecule that can act as a molecular adjuvant. Expression of the two surface forms of the antigen was achieved in L. lactis, and the resulting antigen-displaying bacteria were administered orally to mice. Half the animals in each of the two groups developed specific IgGs, with titers increasing over time and reaching 1:422 without flagellin and 1:320 with flagellin. A much higher titer 1:25,803 was observed with the parenterally administered antigen, which was purified from E. coli. With the latter, a significant mucosal IgA response was also observed. Despite significant titers, the IgGs elicited with oral or parenteral administration could not prevent HAV from infecting cells in a virus neutralization assay, suggesting that the antibodies cannot recognize viral surface epitopes. Nevertheless, orally administered HAV antigen expressed in L. lactis elicited significant systemic humoral immune response showing the feasibility for development of effective HAV vaccine for mucosal delivery.

Improvement of LysM-Mediated Surface Display of Designed Ankyrin Repeat Proteins (DARPins) in Recombinant and Nonrecombinant Strains of Lactococcus lactis and Lactobacillus Species

“…Several approaches to the display of heterologous proteins on the surfaces of LAB have been established, including the use of LPXTG-type domains (5), lysine motif (LysM) domains (6), surface layer proteins (7), and lipoprotein anchors (8). Display of heterologous proteins on the surfaces of LAB has been exploited for the preparation of mucosal vaccines (9,10), for the delivery of binding molecules to the gastrointestinal tract (11), for the assembly of macromolecular enzyme complexes (12), and for bacterial immobilization (13).…”

Development of Recombinant Lactococcus lactis Displaying Albumin-Binding Domain Variants against Shiga Toxin 1 B Subunit

“…Genes for S1B22, S1B26, ABDwt and H6-ABDwt without tolA spacer or tags were amplified with PCR and cloned into the plasmid pSDLBA3b [17], which had been designed for the surface display of target proteins in fusion with Usp45 secretion signal [41] and the surface anchoring C-terminal domain of AcmA (cA), as reported [17, 18, 20–22, 33]. Fusion proteins were expressed in L .…”

“…lactis NZ9000 in 10 mL cultures. Bacterial suspensions were grown to an A 600 of 0.8, followed by induction with 25 ng/mL nisin (Fluka) for 3 h [17, 18, 33, 34]. Resulting suspensions were stored at 4°C for flow cytometric analysis or whole cell ELISA test.…”

Oral immunization with recombinantLactococcus lactisdelivering a multi-epitope antigen CTB-UE attenuatesHelicobacter pyloriinfection in mice

“…To assess specific mucosal secretory IgA (sIgA) production, c. 150 mg of fecal pellets was suspended in PBS with 0.2% BSA and phenylmethanesulfonyl fluoride (PMSF, Bio Basic Inc., Markham, Canada) and incubated overnight at 4°C. Suspension was centrifuged at 4800 g for 20 min at 4°C and supernatant stored at À80°C (Berlec et al, 2013). ELISA was performed essentially as described for serum samples with minor modifications as follows.…”

“…Because L. lactis does not naturally colonize the intestines of humans or animals, it is perhaps more analogous to inert microparticle vaccine delivery systems (Wells et al, 1996). Recently, a number of research groups have reported that L. lactis can be genetically engineered to express bacterial or viral antigens, including staphylococcal enterotoxin B (Asensi et al, 2013), hepatitis A virus antigen (Berlec et al, 2013) and so on.…”