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Production and targeting of the Brucella abortus antigen L7/L12 in Lactococcus lactis: a first step towards food-grade live vaccines against brucellosis.

ABSTRACT: Brucella abortus is a facultative intracellular gram-negative bacterial pathogen that infects humans and animals by entry mainly through the digestive tract. B. abortus causes abortion in pregnant cattle and undulant fever in humans. The immunogenic B. abortus ribosomal protein L7/L12 is a promising candidate antigen for the development of oral live vaccines against brucellosis, using food-grade lactic acid bacteria (LAB) as a carrier. The L7/L12 gene was expressed in Lactococcus lactis, the model LAB, under the nisin-inducible promoter. Using different signals, L7/L12 was produced in cytoplasmic, cell-wall-anchored, and secreted forms. Cytoplasmic production of L7/L12 gave a low yield, estimated at 0.5 mg/liter. Interestingly, a secretable form of this normally cytoplasmic protein via fusion with a signal peptide resulted in increased yield of L7/L12 to 3 mg/liter; secretion efficiency (SE) was 35%. A fusion between the mature moiety of the staphylococcal nuclease (Nuc) and L7/L12 further increased yield to 8 mg/liter. Fusion with a synthetic propeptide (LEISSTCDA) previously described as an enhancer for heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895-1903, 1998) raised the yield to 8 mg/liter and SE to 50%. A surface-anchored L7/L12 form in L. lactis was obtained by fusing the cell wall anchor of Streptococcus pyogenes M6 protein to the C-terminal end of L7/L12. The fusions described allow the production and targeting of L7/L12 in three different locations in L. lactis. This is the first example of a B. abortus antigen produced in a food-grade bacterium and opens new perspectives for alternative vaccine strategies against brucellosis.

SUBMITTER: Ribeiro LA

PROVIDER: S-EPMC126665 | biostudies-literature | 2002 Feb

REPOSITORIES: biostudies-literature

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Production and targeting of the Brucella abortus antigen L7/L12 in Lactococcus lactis: a first step towards food-grade live vaccines against brucellosis.

Ribeiro Luciana A LA Azevedo Vasco V Le Loir Yves Y Oliveira Sergio C SC Dieye Yakhya Y Piard Jean-Christophe JC Gruss Alexandra A Langella Philippe P

Applied and environmental microbiology 20020201 2

Brucella abortus is a facultative intracellular gram-negative bacterial pathogen that infects humans and animals by entry mainly through the digestive tract. B. abortus causes abortion in pregnant cattle and undulant fever in humans. The immunogenic B. abortus ribosomal protein L7/L12 is a promising candidate antigen for the development of oral live vaccines against brucellosis, using food-grade lactic acid bacteria (LAB) as a carrier. The L7/L12 gene was expressed in Lactococcus lactis, the mod ...[more]

PMID: 11823235

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Project description:BackgroundWe generated novel, effective candidate vaccine against Brucella abortus based on recombinant influenza viruses expressing the Brucella ribosomal protein L7/L12 or outer membrane protein (Omp)-16 from the NS1 open reading frame. The main purpose of this work was to evaluate the safety, immunogenicity and protectiveness of vaccine candidate in laboratory animals.Methods and resultsFour recombinant influenza A viral constructs of the subtypes Н5N1 or H1N1 expressing the Brucella proteins L7/L12 or Omp16 were obtained by a reverse genetics method: Flu-NS1-124-L7/L12-H5N1, Flu-NS1-124-Omp16-H5N1, Flu-NS1-124-L7/L12-H1N1 and Flu-NS1-124-Omp16-H1N1. Despite of substantial modification of NS1 gene, all constructs replicated well and were retain their Brucella inserts over five passages in embryonated chicken eggs (CE). Administration of the mono- or bivalent vaccine formulation via prime-boost intranasal (i.n.), conjunctival (c.) or subcutaneous (s.c.) immunization was safe in mice; no deaths, body weight loss or pathomorphological changes were observed over 56 days. Moreover, guinea pigs vaccinated i.n. with vaccine vectors did not shed the vaccine viruses through their upper respiratory tract after the prime and booster vaccination. These findings confirmed the replication-deficient phenotype of viral vectors. The highest antibody response to Brucella antigen was obtained with constructs expressing L7/L12 (ELISA, GMT 242.5-735.0); whereas the highest T-cell immune response- with construct expressing Omp16 (ELISPOT, 337 ± 52-651 ± 45 spots/4×105cells), which was comparable (P > 0.05) to the response induced by the commercial vaccine B. abortus 19. Interestingly, c. immunization appeared to be optimal for eliciting T-cell immune response. In guinea pigs, the highest protective efficacy after challenge with B. abortus 544 was achieved with Omp16 expressing constructs in both monovalent or bivalent vaccine formulations; protective efficacy was comparable to those induced by a commercial live B. abortus 19 vaccine.ConclusionThus, influenza vectors expressing Brucella protective antigens can be developed as novel influenza vectored vaccine against B. abortus infection.

Project description:BackgroundMost Brucella infections take place on mucosal membranes. Therefore, creating vaccinations delivered through the mucosa may be crucial for managing brucellosis. Consequently, we assessed the efficacy of a recombinant oral antigen delivery system based on Lactococcus lactis for Brucella abortus omp25 antigen.MethodOral vaccinations with L. lactis transformed with pNZ8148 variants encoding for omp25 (pNZ8148:omp25) and free-pNZ8148 were administered to mice. On day 30, following immunization in animal groups, anti-omp25-specific IgG1 antibodies were assessed by the ELISA test. Additionally, nasal and bronchoalveolar lavages containing omp25-specific secretory IgA (sIgA) were analysed by ELISA. ELISA test and real-time PCR were also used to analyse cytokine responses up to 28 days following the last boost. In addition, the protective potential of L. lactis pNZ8148:omp25 vaccines was assessed in BALB/c mice by exposing them to the B. abortus strain.ResultsBased on the initial screening results, the omp25 protein was identified for immunogenicity because it had the maximum solubility and flexibility and antigenic values of 0.75. The produced plasmid was digested using KpnI and XbaI. By electrophoretic isolation of the digestion fragments at 786 bp, the omp25 gene, the successful production of the recombinant plasmid, was confirmed. Antigen expression at the protein level revealed that the target group generated the 25 kDa-sized omp25 protein, but there was no protein expression in the control group. Fourteen days after priming, there was a considerable amount of omp25-specific IgG1 in the sera of mice vaccinated with pNZ8148-Usp45-omp25-L. lactis (p < 0.001 in target groups compared to the phosphate-buffered saline control group). IFN-γ and TNF-α levels were more significant in samples from mice that had been given the pNZ8148-Usp45-omp25-L. lactis and IRBA vaccinations, in samples taken on days 14 and 28, respectively (p < 0.001). The pNZ8148-Usp45-omp25-L. lactis and IRBA immunization groups had significantly greater IL-4 and IL-10 transcription levels than the other groups. The spleen portions from the pNZ8148-Usp45-omp25-L. lactis and IRIBA vac group had less extensive spleen injuries, alveolar oedema, lymphocyte infiltration and morphological damage due to the inflammatory process.ConclusionOur study offers a novel method for using the food-grade, non-pathogenic and noncommercial bacterium L. lactis as a protein cell factory to produce the novel immunogenic fusion candidate romp25. This method offers an appealing new approach to assessing the cost-effective, safe, sustainable, simple pilot development of pharmaceutical products.

Project description:Brucellosis is a major zoonotic infection in Kazakhstan. However, there is limited data on its incidence in humans and animals, and the genetic diversity of prevalent strains is virtually unstudied. Additionally, there is no detailed overview of Kazakhstan brucellosis control and eradication programs. Here, we analyzed brucellosis epidemiological data, and assessed the effectiveness of eradication strategies employed over the past 70 years to counteract this infection. We also conducted multiple loci variable-number tandem repeat analysis (MLVA) of Brucella abortus strains found in Kazakhstan. We analyzed official data on the incidence of animal brucellosis in Kazakhstan. The records span more than 70 years of anti-brucellosis campaigns, and contain a brief description of the applied control strategies, their effectiveness, and their impact on the incidence in humans. The MLVA-16 method was used to type 94 strains of B. abortus and serial passages of B. abortus 82, a strain used in vaccines. MLVA-8 and MLVA-11 analyses clustered strains into a total of four and seven genotypes, respectively; it is the first time that four of these genotypes have been described. MLVA-16 analysis divided strains into 28 distinct genotypes having genetic similarity coefficient that varies from 60 to100% and a Hunter & Gaston diversity index of 0.871. MST analysis reconstruction revealed clustering into "Kazakhstani-Chinese (Central Asian)", "European" and "American" lines. Detection of multiple genotypes in a single outbreak confirms that poorly controlled trade of livestock plays a crucial role in the spread of infection. Notably, the MLVA-16 profile of the B. abortus 82 strain was unique and did not change during 33 serial passages. MLVA genotyping may thus be useful for epidemiological monitoring of brucellosis, and for tracking the source(s) of infection. We suggest that countrywide application of MLVA genotyping would improve the control of brucellosis in Kazakhstan.

Dataset Information

Production and targeting of the Brucella abortus antigen L7/L12 in Lactococcus lactis: a first step towards food-grade live vaccines against brucellosis.

Publications

Production and targeting of the Brucella abortus antigen L7/L12 in Lactococcus lactis: a first step towards food-grade live vaccines against brucellosis.

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