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Heterologous production of human papillomavirus type-16 L1 protein by a lactic acid bacterium.

Cortes-Perez NG, Kharrat P, Langella P, Bermúdez-Humarán LG - BMC Res Notes (2009)

Bottom Line: The capacity of L. lactis harboring either pCYT:L1 or pSEC:L1 plasmid to accumulate L1 in the cytoplasm and supernatant samples was confirmed by Western blot assays.The presence of conformational epitopes on the L. lactis-derived VLPs was confirmed by ELISA using an anti-HPV16 L1 capsid antigen antibody.Our results support the feasibility of using recombinant food-grade LAB, such as L. lactis, for the production of L1-based VLPs and open the possibility for the development of a new safe mucosal vector for HPV-16 prophylactic vaccination.

View Article: PubMed Central - HTML - PubMed

Affiliation: Equipe Interactions des bactéries commensales et probiotiques avec l'hôte, Unité d'Ecologie et physiologie du Système Digestif, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas, France. naima.cortes-perez@jouy.inra.fr

ABSTRACT

Background: The expression of vaccine antigens in lactic acid bacteria (LAB) is a safe and cost-effective alternative to traditional expression systems. In this study, we investigated i) the expression of Human papillomavirus type 16 (HPV-16) L1 major capsid protein in the model LAB Lactococcus lactis and ii) the ability of the resulting recombinant strain to produce either capsomer-or virus-like particles (VLPs).

Results and conclusion: HPV-16 L1 gene was cloned into two vectors, pCYT and pSEC, designed for controlled intra- or extracellular heterologous expression in L. lactis, respectively. The capacity of L. lactis harboring either pCYT:L1 or pSEC:L1 plasmid to accumulate L1 in the cytoplasm and supernatant samples was confirmed by Western blot assays. Electron microscopy analysis suggests that, L1 protein produced by recombinant lactococci can self-assemble into structures morphologically similar to VLPs intracellularly. The presence of conformational epitopes on the L. lactis-derived VLPs was confirmed by ELISA using an anti-HPV16 L1 capsid antigen antibody. Our results support the feasibility of using recombinant food-grade LAB, such as L. lactis, for the production of L1-based VLPs and open the possibility for the development of a new safe mucosal vector for HPV-16 prophylactic vaccination.

No MeSH data available.


Related in: MedlinePlus

Expression of HPV-16 L1 protein by recombinant L. lactis. A) L. lactis strains were grown and induced with 1 ng/ml of nisin for 1 h. After centrifugation, non-induced (- nis) and induced (+ nis) culture samples were treated as described in materials and methods and L1 production and secretion analyzed by Western blot. L. lactis strain contains pCYT:L1 plasmid encoding for a cytoplasmic form of L1 and L. lactis strain contains pSEC:L1 encoding for the precursor preL1 (i.e. SPUsp45 fused to L1). B) L. lactis (pSEC:L1) strain was grown and induced with 10 ng/ml of nisin for 1 h. After centrifugation, induced (+ nis) culture samples were treated as described in results and discussion and L1 production and secretion analyzed by Western blot. Arrows indicate positions of L1 mature and precursor forms. Abbreviations: C, cell lysates; S, supernatant fraction; M, positions and sizes of molecular mass markers.
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Figure 2: Expression of HPV-16 L1 protein by recombinant L. lactis. A) L. lactis strains were grown and induced with 1 ng/ml of nisin for 1 h. After centrifugation, non-induced (- nis) and induced (+ nis) culture samples were treated as described in materials and methods and L1 production and secretion analyzed by Western blot. L. lactis strain contains pCYT:L1 plasmid encoding for a cytoplasmic form of L1 and L. lactis strain contains pSEC:L1 encoding for the precursor preL1 (i.e. SPUsp45 fused to L1). B) L. lactis (pSEC:L1) strain was grown and induced with 10 ng/ml of nisin for 1 h. After centrifugation, induced (+ nis) culture samples were treated as described in results and discussion and L1 production and secretion analyzed by Western blot. Arrows indicate positions of L1 mature and precursor forms. Abbreviations: C, cell lysates; S, supernatant fraction; M, positions and sizes of molecular mass markers.

Mentions: The capacity of L. lactis to produce and secrete L1 protein was examined using lactococci strains harbouring pCYT:L1 and pSEC:L1 plasmids (Figure 1), respectively. Non-induced and induced culture samples were examined by Western blot using HPV-16 L1-specific monoclonal antibodies. As shown in Figure 2A, no L1 signal was detected in either cell or supernatant fractions of induced cultures of the negative control L. lactis (pGK-). In the absence of nisin, no L1 signal was detected in either L. lactis (pCYT:L1) or L. lactis (pSEC:L1) strain, indicating that NICE system allows tight control of gene expression. Induced cultures of L. lactis (pCYT:L1) strain resulted in a clear band at the expected size for native L1 (~58 kDa) was observed in the cell fraction whereas no signal was detected in the supernatant. Similar analysis of L. lactis (pSEC:L1) resulted in a clear band in the cell fraction corresponding to SPUsp45-L1 precursor (pre-L1, ~60 kDa). Unfortunately, since a major non-specific band (which migrates at the expected size for native L1: ~58 kDa) reacts with anti-L1 antibodies in supernatant samples, L1 secretion cannot be determined. This non-specific band most probably corresponds to Usp45, the predominant L. lactis-secreted protein, and frequently detected by immunoblotting when using protein G-horseradish peroxidase conjugate [10,16].


Heterologous production of human papillomavirus type-16 L1 protein by a lactic acid bacterium.

Cortes-Perez NG, Kharrat P, Langella P, Bermúdez-Humarán LG - BMC Res Notes (2009)

Expression of HPV-16 L1 protein by recombinant L. lactis. A) L. lactis strains were grown and induced with 1 ng/ml of nisin for 1 h. After centrifugation, non-induced (- nis) and induced (+ nis) culture samples were treated as described in materials and methods and L1 production and secretion analyzed by Western blot. L. lactis strain contains pCYT:L1 plasmid encoding for a cytoplasmic form of L1 and L. lactis strain contains pSEC:L1 encoding for the precursor preL1 (i.e. SPUsp45 fused to L1). B) L. lactis (pSEC:L1) strain was grown and induced with 10 ng/ml of nisin for 1 h. After centrifugation, induced (+ nis) culture samples were treated as described in results and discussion and L1 production and secretion analyzed by Western blot. Arrows indicate positions of L1 mature and precursor forms. Abbreviations: C, cell lysates; S, supernatant fraction; M, positions and sizes of molecular mass markers.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2742549&req=5

Figure 2: Expression of HPV-16 L1 protein by recombinant L. lactis. A) L. lactis strains were grown and induced with 1 ng/ml of nisin for 1 h. After centrifugation, non-induced (- nis) and induced (+ nis) culture samples were treated as described in materials and methods and L1 production and secretion analyzed by Western blot. L. lactis strain contains pCYT:L1 plasmid encoding for a cytoplasmic form of L1 and L. lactis strain contains pSEC:L1 encoding for the precursor preL1 (i.e. SPUsp45 fused to L1). B) L. lactis (pSEC:L1) strain was grown and induced with 10 ng/ml of nisin for 1 h. After centrifugation, induced (+ nis) culture samples were treated as described in results and discussion and L1 production and secretion analyzed by Western blot. Arrows indicate positions of L1 mature and precursor forms. Abbreviations: C, cell lysates; S, supernatant fraction; M, positions and sizes of molecular mass markers.
Mentions: The capacity of L. lactis to produce and secrete L1 protein was examined using lactococci strains harbouring pCYT:L1 and pSEC:L1 plasmids (Figure 1), respectively. Non-induced and induced culture samples were examined by Western blot using HPV-16 L1-specific monoclonal antibodies. As shown in Figure 2A, no L1 signal was detected in either cell or supernatant fractions of induced cultures of the negative control L. lactis (pGK-). In the absence of nisin, no L1 signal was detected in either L. lactis (pCYT:L1) or L. lactis (pSEC:L1) strain, indicating that NICE system allows tight control of gene expression. Induced cultures of L. lactis (pCYT:L1) strain resulted in a clear band at the expected size for native L1 (~58 kDa) was observed in the cell fraction whereas no signal was detected in the supernatant. Similar analysis of L. lactis (pSEC:L1) resulted in a clear band in the cell fraction corresponding to SPUsp45-L1 precursor (pre-L1, ~60 kDa). Unfortunately, since a major non-specific band (which migrates at the expected size for native L1: ~58 kDa) reacts with anti-L1 antibodies in supernatant samples, L1 secretion cannot be determined. This non-specific band most probably corresponds to Usp45, the predominant L. lactis-secreted protein, and frequently detected by immunoblotting when using protein G-horseradish peroxidase conjugate [10,16].

Bottom Line: The capacity of L. lactis harboring either pCYT:L1 or pSEC:L1 plasmid to accumulate L1 in the cytoplasm and supernatant samples was confirmed by Western blot assays.The presence of conformational epitopes on the L. lactis-derived VLPs was confirmed by ELISA using an anti-HPV16 L1 capsid antigen antibody.Our results support the feasibility of using recombinant food-grade LAB, such as L. lactis, for the production of L1-based VLPs and open the possibility for the development of a new safe mucosal vector for HPV-16 prophylactic vaccination.

View Article: PubMed Central - HTML - PubMed

Affiliation: Equipe Interactions des bactéries commensales et probiotiques avec l'hôte, Unité d'Ecologie et physiologie du Système Digestif, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas, France. naima.cortes-perez@jouy.inra.fr

ABSTRACT

Background: The expression of vaccine antigens in lactic acid bacteria (LAB) is a safe and cost-effective alternative to traditional expression systems. In this study, we investigated i) the expression of Human papillomavirus type 16 (HPV-16) L1 major capsid protein in the model LAB Lactococcus lactis and ii) the ability of the resulting recombinant strain to produce either capsomer-or virus-like particles (VLPs).

Results and conclusion: HPV-16 L1 gene was cloned into two vectors, pCYT and pSEC, designed for controlled intra- or extracellular heterologous expression in L. lactis, respectively. The capacity of L. lactis harboring either pCYT:L1 or pSEC:L1 plasmid to accumulate L1 in the cytoplasm and supernatant samples was confirmed by Western blot assays. Electron microscopy analysis suggests that, L1 protein produced by recombinant lactococci can self-assemble into structures morphologically similar to VLPs intracellularly. The presence of conformational epitopes on the L. lactis-derived VLPs was confirmed by ELISA using an anti-HPV16 L1 capsid antigen antibody. Our results support the feasibility of using recombinant food-grade LAB, such as L. lactis, for the production of L1-based VLPs and open the possibility for the development of a new safe mucosal vector for HPV-16 prophylactic vaccination.

No MeSH data available.


Related in: MedlinePlus