Biomedical subjects

Philippe Langella

Publications and source records attributed to Philippe Langella.

25 records · Page 2Linked to original sources

PubMed2003

Controlled intra- or extracellular production of staphylococcal nuclease and ovine omega interferon in Lactococcus lactis.

A system for controlled targeting of heterologous protein was developed in the food-grade bacterium Lactococcus lactis. It is composed of the L. lactis strain NZ9000 and of two broad host range expression vectors pCYT:Nuc and pSEC:Nuc for, respectively, cytoplasmic and secreted staphylococcal nuclease (Nuc) nisin-inducible production. The level of intracellular production of Nuc measured with pCYT:Nuc (3 mg x l(-1)) is significantly lower than the one obtained with pSEC:Nuc ( approximately 20 mg x l(-1)). The secretion efficiency (SE) of Nuc is estimated to be approximately 70%, corresponding to approximately 15 mg of secreted Nuc x l(-1). Furthermore, we established that Nuc production continued in L. lactis 10 h after a 1-h nisin-pulse induction. This system was then used for intra- and extracellular production of a protein of therapeutical interest in L. lactis, the ovine interferon-omega (IFN-omega). The SE and the quantity of secreted active IFN-omega were evaluated respectively to be approximately 70% and approximately 1 mg x l(-1) ( approximately two-fold higher than the cytoplasmic form).

Animals↗

PubMed2003

Heterologous protein production and delivery systems for Lactococcus lactis.

Lactic acid bacteria (LAB), widely used in the food industry, are present in the intestine of most animals, including humans. The potential use of these bacteria as live vehicles for the production and delivery of heterologous proteins of vaccinal, medical or technological interest has therefore been extensively investigated. Lactococcus lactis, a LAB species, is a potential candidate for the production of biologically useful proteins. Several delivery systems have been developed to target heterologous proteins to a specific cell location (i.e., cytoplasm, cell wall or extracellular medium). A promising application of L. lactis is its use as an antigen delivery vehicle, for the development of live mucosal vaccines. The expression of heterologous proteins and antigens as well as the various delivery systems developed in L. lactis, and its use as an oral vaccine carrier are discussed.

Animals↗

PubMed2003

Induction of partial protection in mice after oral administration of Lactococcus lactis producing Brucella abortus L7/L12 antigen.

The Brucella abortus ribosomal protein L7/L12 is an immunodominant antigen and an interesting candidate for the development of oral live vaccines against brucellosis. Here, a recombinant Lactococcus lactis strain producing L7/L12 under the control of nisin inducible promoter was orally administered to BALB/c mice. Significant levels of anti-L7/L12 specific IgA detected in feces revealed an induced local humoral immune response. However, serum analysis did not reveal any anti-L7/L12 antibodies suggesting the absence of a systemic response. Nevertheless, the vaccinated mice showed a partial protective immunity against B. abortus virulent strain (S2308) challenged by intraperitoneal inoculation.

Administration, Oral↗

PubMed2003

Characterization of a Lactococcus lactis strain that secretes a major epitope of bovine beta-lactoglobulin and evaluation of its immunogenicity in mice.

Bovine beta-lactoglobulin (Blg) is one of the major cow's milk allergens. Peptide 41-60 of Blg (Blg41-60) was described as a murine T-cell determinant and a murine, rat, and human immunoglobulin E (IgE) epitope. The aim of this study was the expression of Blg41-60 as a fusion protein in the food-grade bacterium Lactococcus lactis and the characterization of its immunogenicity in mice. We constructed a recombinant strain of L. lactis capable of inducible production and secretion of Blg41-60::Nuc, a fusion protein between Blg41-60 and the mature part of the staphylococcal nuclease (Nuc). The highest production yield of Blg41-60::Nuc (32.5 mg/liter) was reached 4 h after induction. At this time, up to 75% of Blg41-60::Nuc was secreted. When monoclonal antibodies specific for Blg41-60 were used, purified Blg41-60::Nuc and synthetic Blg41-60 exhibited very similar immunoreactivities. Subcutaneous coadministration of purified Blg41-60::Nuc and killed nonrecombinant L. lactis resulted in the induction of specific anti-Blg41-60 IgG2a and IgG1. The IgG1/IgG2a ratio and the lack of specific IgE suggest a Th1-type immune response, i.e., a nonallergic response. Similar administrations of the killed Blg41-60::Nuc-producing L. lactis strain did not elicit a specific immune response, whereas a transitory mucosal IgA-specific immune response was induced in mice after oral administration of the live Blg41-60::Nuc-producing L. lactis strain.

Administration, Oral↗

PubMed2003

Intranasal immunization with recombinant Lactococcus lactis secreting murine interleukin-12 enhances antigen-specific Th1 cytokine production.

Interleukin-12 (IL-12), a heterodimeric cytokine, plays an important role in cellular immunity to several bacterial, viral, and parasitic infections and has adjuvant activity when it is codelivered with DNA vaccines. IL-12 has also been used with success in cancer immunotherapy treatments. However, systemic IL-12 therapy has been limited by high levels of toxicity. We describe here inducible expression and secretion of IL-12 in the food-grade lactic acid bacterium Lactococcus lactis. IL-12 was expressed as two separate polypeptides (p35-p40) or as a single recombinant polypeptide (scIL-12). The biological activity of IL-12 produced by the recombinant L. lactis strain was confirmed in vitro by its ability to induce gamma interferon (IFN-gamma) production by mouse splenocytes. Local administration of IL-12-producing strains at the intranasal mucosal surface resulted in IFN-gamma production in mice. The activity was greater with the single polypeptide scIL-12. An antigen-specific cellular response (i.e., secretion of Th1 cytokines, IL-2, and IFN-gamma) elicited by a recombinant L. lactis strain displaying a cell wall-anchored human papillomavirus type 16 E7 antigen was dramatically increased by coadministration with an L. lactis strain secreting IL-12 protein. Our data show that IL-12 is produced and secreted in an active form by L. lactis and that the strategy which we describe can be used to enhance an antigen-specific immune response and to stimulate local mucosal immunity.

Administration, Intranasal↗

PubMed2002

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

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.

Animals↗

PubMedDate not supplied

Fusion to a carrier protein and a synthetic propeptide enhances E7 HPV-16 production and secretion in Lactococcus lactis.

An inducible system to improve and stabilize the production of an extremely labile protein (E7 antigen of human papillomavirus type 16) was developed in the food-grade bacterium Lactococcus lactis. A protein carrier, the staphylococcal nuclease Nuc, was fused either to N- or C-termini of E7 protein, and the resulting hybrid proteins were rescued from intracellular proteolysis but poorly secreted by L. lactis. A synthetic propeptide (LEISSTCDA) was then fused and significantly improved the secretion efficiency of the hybrid protein Nuc-E7 by L. lactis.

Carrier Proteins↗