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Protective Immunity Against Enterotoxigenic Escherichia coli by Oral Vaccination of Engineered Lactococcus lactis.

ABSTRACT: Enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of diarrhea in children globally, and thus suitable vaccines are desired. Antigen display on lactic acid bacteria is a reliable approach for efficient oral vaccination and preventing bowel diseases. To develop an oral vaccine against ETEC, the gene of the binding domain from heat-labile toxin (LTB), a key ETEC virulence factor, was codon-optimized and cloned into a construct containing a signal peptide and an anchor for display on L. lactis. Bioinformatics analysis showed a codon adaptation index of 0.95 for the codon-optimized gene. Cell surface expression of LTB was confirmed by transmission electron microscopy and blotting. White New Zealand rabbits were immunized per os (PO) with the recombinant L. lactis, and the antibody titers were assayed with ELISA. In vitro neutralization assay was performed using mouse adrenal tumor cells and rabbit ileal loop test was performed as the in vivo assay. ELISA results indicated that oral administration of the engineered L. lactis elicited a significant production of IgA in the intestine. In vitro neutralization assay showed that the effect of the toxin could be neutralized with 500 µg/ml of IgG isolated from the oral vaccine group. Furthermore, the dose of ETEC causing fluid accumulation in the ileal loop test showed a tenfold increase in rabbits immunized with either recombinant L. lactis or LTB protein compared to other groups. Our results imply that recombinant L. lactis could potentially be an effective live oral vaccine against ETEC toxicity.

SUBMITTER: Ahmadi Rouzbahani H

PROVIDER: S-EPMC8280578 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundOral immunization with vaccines may be an effective strategy for prevention of Clostridium difficile infection (CDI). However, application of previously developed vaccines for preventing CDI has been limited due to various reasons. Here, we developed a recombinant Lactococcus lactis oral vaccine and evaluated its effect on a C. difficile-infected animal model established in golden hamsters in attempt to provide an alternative strategy for CDI prevention.MethodsRecombinant L. lactis vaccine was developed using the pTRKH2 plasmid, a high-copy-number Escherichia coli-L. shuttle vector: 1) L. lactis expressing secreted proteins was constructed with recombinant pTRKH2 (secreted-protein plasmid) carrying the Usp45 signal peptide (SPUsp45), nontoxic adjuvanted tetanus toxin fragment C (TETC), and 14 of the 38 C-terminal repeats (14CDTA) of nontoxic C. difficile toxin A (TcdA); and 2) L. lactis expressing secreted and membrane proteins was constructed with recombinant pTRKH2 (membrane-anchored plasmid) carrying SPUsp45, TETC, 14CDTA, and the cell wall-anchored sequence of protein M6 (cwaM6). Then, 32 male Syrian golden hamsters were randomly divided into 4 groups (n = 8 each) for gavage of normal saline (blank control) and L. lactis carrying the empty shuttle vector, secreted-protein plasmid, and membrane-anchored plasmid, respectively. After 1-week gavage of clindamycin, the animals were administered with C. difficile spore suspension. General symptoms and intestinal pathological changes of the animals were examined by naked eye and microscopy, respectively. Protein levels of anti-TcdA IgG/IgA antibodies in intestinal tissue and fluid were analyzed by enzyme-linked immunosorbent assay (ELISA). A cell culture cytotoxicity neutralization assay was done by TcdA treatment with or without anti-TcdA serum pre-incubation or treatment. Apoptosis of intestinal epithelial cells was examined by flow cytometry (FL) assay. Expression of mucosal inflammatory cytokines in the animals was detected by polymer chain reaction (PCR) assay.ResultsAfter the C. difficile challenge, the animals of control group had severe diarrhea symptoms on day 1 and all died on day 4, indicating that the CDI animal model was established in hamster. Of the 3 immunization groups, secreted-protein and membrane-anchored plasmid groups had significantly lower mortalities, body weight decreases, and pathological scores, with higher survival rate/time than the empty plasmid group (P < 0.05). The tilter of IgG antibody directed against TcdA was significantly higher in serum and intestinal fluid of secreted-protein and membrane-anchored plasmid groups than in the empty plasmid group (P < 0.05) while the corresponding titer of IgA antibody directed against TcdA had no substantial differences (P > 0.05). The anti-TcdA serum of membrane-anchored plasmid group neutralized the cytotoxicity of 200 ng/ml TcdA with the best protective effect achieved by anti-TcdA serum pre-incubation. The incidences of TcdA-induced death and apoptosis of intestinal epithelial cells were significantly reduced by cell pre-incubation or treatment with anti-TcdA serum of membrane-anchored plasmid group (P < 0.05). MCP-1, ICAM-1, IL-6, and Gro-1 mRNA expression levels were the lowest in cecum tissue of the membrane-anchored groups compared to the other groups.ConclusionRecombinant L. lactis live vaccine is effective for preventing CDI in the hamster model, thus providing an alternative for immunization of C. difficile-associated diseases.

Project description:BackgroundCurrent influenza vaccines need to be annually reformulated to well match the predicated circulating strains. Thus, it is critical for developing a novel universal influenza vaccine that would be able to confer cross-protection against constantly emerging divergent influenza virus strains. Influenza virus A is a genus of the Orthomyxoviridae family of viruses. Influenza virus nucleoprotein (NP) is a structural protein which encapsidates the negative strand viral RNA, and anti-NP antibodies play role in cross-protective immunity. Lactococcus lactis (L. lactis) is an ideal vaccine delivery vehicle via oral administration route. However, L. lactis vectored vaccine exhibits poor immunogenicity without the use of mucosal adjuvant. To enhance the immunogenicity of L. lactis vectored vaccine, cholera toxin B (CTB) subunit, one of mucosal adjuvants, is a safe adjuvant for oral route, when combined with L. lactis vectored vaccine. In this study, we hypothesized that pNZ8008, a L. lactis expression plasmid, encoding NP antigen, would be able to elicit cross-protection with the use of CTB via oral administration route.ResultsTo construct L. lactis vectored vaccine, nucleoprotein (NP) gene of A/California/04/2009(H1N1) was sub-cloned into a L. lactis expression plasmid, pNZ8008. The expression of recombinant L. lactis/pNZ8008-NP was confirmed by Western blot, immunofluorescence assay and flow cytometric analysis. Further, immunogenicity of L. lactis/pNZ8008-NP alone or adjuvanted with cholera toxin B (CTB) subunit was evaluated in a mouse model via oral administration route. Antibodies responses were detected by ELISA. The result indicated that oral administration of L. lactis/pNZ8008-NP adjuvanted with CTB could elicit significant humoral and mucosal immune responses, as well as cellular immune response, compared with L. lactis/pNZ8008-NP alone. To further assess the cross-protective immunity of L. lactis/pNZ8008-NP adjuvanted with CTB, we used L. lactis/pNZ8110-pgsA-HA1 alone or adjuvanted with CTB as controls. Mice that received L. lactis/pNZ8008-NP adjuvanted with CTB were completely protected from homologous H1N1 virus and showed 80% protection against heterologous H3N2 or H5N1 virus, respectively. By contrast, L. lactis/pNZ8110-pgsA-HA1 adjuvanted with CTB also conferred 100% protection against H5N1 virus infection, but indicated no cross-protection against H1N1 or H5N1 virus challenge. As controls, mice vaccinated orally with L. lactis/pNZ8008-NP alone or L. lactis/pNZ8110-pgsA-HA1 alone could not survive.ConclusionThis study is the first to report the construction of recombinant L. lactis/pNZ8008-NP and investigate its immunogenicity with the use of CTB. Compared with L. lactis/pNZ8110-pgsA-HA1 adjuvanted with CTB, our data support 5 × 10(11) CFU of L. lactis/pNZ8008-NP adjuvanted with 1 µg of CTB is a better combination for universal influenza vaccines development that would provide cross-protective immunity against divergent influenza A viruses.

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Protective Immunity Against Enterotoxigenic Escherichia coli by Oral Vaccination of Engineered Lactococcus lactis.

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