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Petrovic D.M.,University of Groningen | Leenhouts K.,Mucosis BV | Van Roosmalen M.L.,Mucosis BV | Kleinjan F.,University of Groningen | Broos J.,University of Groningen
Analytical Biochemistry | Year: 2012

The lysin motif (LysM) is a peptidoglycan binding protein domain found in a wide range of prokaryotes and eukaryotes. Various techniques have been used to study the LysM-ligand interaction, but a sensitive spectroscopic method to directly monitor this interaction has not been reported. Here a tryptophan analog fluorescence spectroscopy approach is presented to monitor the LysM-ligand interaction using the LysM of the N-acetylglucosaminidase enzyme of Lactococcus lactis. A three-dimensional model of this LysM protein was built based on available structural information of a homolog. This model allowed choosing the amino acid positions to be labeled with a Trp analog. Four functional single-Trp LysM mutants and one double-Trp LysM mutant were constructed and biosynthetically labeled with 7-azatryptophan or 5-hydroxytryptophan. These Trp analogs feature red-shifted absorption spectra, enabling the monitoring of the LysM-ligand interaction in media with a Trp background. The emission intensities of four of the five LysM constructs were found to change markedly on exposure to either L. lactis bacterium-like particles or peptidoglycan as ligands. The method reported here is suitable to monitor LysM-ligand interactions at (sub)micromolar LysM concentrations and can be used for the detection of low levels of peptidoglycan or microbes in solutions. © 2012 Elsevier Inc. All rights reserved.

de Haan A.,University of Groningen | Haijema B.J.,Mucosis BV | Voorn P.,Mucosis BV | Meijerhof T.,University of Groningen | And 2 more authors.
Vaccine | Year: 2012

Administration of influenza vaccines through the intranasal (IN) route forms an attractive alternative to conventional intramuscular (IM) injection. It is not only a better accepted form of vaccine administration but it also has the potential to induce, in addition to systemic antibodies, local protective antibodies, i.e. S-IgA. Most commercially available vaccines however are inactivated non-replicating vaccines and have a low immunogenicity when administered intranasally. Local administration of these vaccines would therefore need an adjuvant to boost systemic and local antibody responses. Here we explored the use of a safe adjuvant system, i.e. bacterium-like particles (BLPs) derived from the food-grade bacterium in Lactococcus lactis, in the induction of protective antibody responses after intranasal immunization of mice. Supplementation of H1N1 split vaccine with BLPs significantly increased levels of serum influenza-specific IgG and hemagglutination-inhibiting antibodies: this was dependent on the dose of admixed BLPs and number of immunizations. Admixing BLPs further boosted local influenza-specific S-IgA antibody levels at lung and nasal mucosal sites, but also at distant mucosal sites such as the vaginal mucosal tissue. Mice immunized IN with BLP-adjuvanted vaccine and IM with non-adjuvanted vaccine were protected against weight loss upon homologous infection with H1N1 A/PR/8/34. Full protection against weight loss upon heterologous challenge with H1N1 A/PR/8/34 was seen in mice immunized IN with BLP-adjuvanted H1N1 A/New Caledonia-derived split virus vaccine, but not in those receiving the split virus vaccine IM. Mice immunized IN with BLP-adjuvanted vaccine had significantly lower lung viral titers upon homologous and heterologous challenge when compared to titers detected in mice immunized by IM injection of non-adjuvanted vaccine. Thus, adjuvantation of IN-administered influenza vaccines with BLPs effectively enhances systemic and local antibody responses leading to a superior protection against homologous and heterologous influenza infection compared to conventional IM immunization. © 2012 Elsevier Ltd.

Petrovic D.M.,University of Groningen | Leenhouts K.,Mucosis BV | Van Roosmalen M.L.,Mucosis BV | Broos J.,University of Groningen
Amino Acids | Year: 2013

Biosynthetic incorporation of tryptophan (Trp) analogues in recombinant proteins using an E. coli Trp auxotroph expression host is limited to analogues modified with a small substituent like a fluoro atom or a hydroxyl or amine group. We report here the efficient incorporation (>89 %) of chloro- and bromo atoms containing Trp analogues in alloproteins at high expression levels using a Lactococcus lactis Trp auxotroph strain. This result was only obtained after coexpression of the enzyme tryptophanyl-tRNA synthetase (TrpRS) of L. lactis, an enzyme believed to show a more relaxed substrate specificity than TrpRS from E. coli. Chloro- and bromo-Trps are attractive intrinsic phosphorescence probes as these Trp analogues are much less sensitive for quenchers in the medium, like oxygen, than Trp. Coexpression of TrpRS was also essential for the biosynthetic incorporation (94 %) of the Trp analogue 5,6 difluoroTrp. This makes our expression system ideally suited to generate a set of methyl- and fluoro-substituted Trp analogue-containing alloproteins in high yield for investigating the involvement of the Trp residue in cation-pi or pi-pi interactions. Taken together, the presented Trp auxotroph expression system features the most relaxed specificity for Trp analogue structures reported to date and gives a high alloprotein yield. © 2013 Springer-Verlag Wien.

Ramirez K.,University of Maryland, Baltimore | Ditamo Y.,University of Maryland, Baltimore | Rodriguez L.,University of Maryland, Baltimore | Picking W.L.,Oklahoma State University | And 3 more authors.
Mucosal Immunology | Year: 2010

Safe and effective immunization of newborns and infants can significantly reduce childhood mortality, yet conventional vaccines have been largely unsuccessful in stimulating the neonatal immune system. We explored the capacity of a novel mucosal antigen delivery system consisting of non-living, non-genetically modified Lactococcus lactis particles, designated as Gram-positive enhancer matrix (GEM), to induce immune responses in the neonatal setting. Yersinia pestis LcrV, used as model protective antigen, was displayed on the GEM particles. Newborn mice immunized intranasally with GEM-LcrV developed LcrV-specific antibodies, Th1-type cell-mediated immunity, and were protected against lethal Y. pestis (plague) infection. The GEM particles activated and enhanced the maturation of neonatal dendritic cells (DCs) both in vivo and in vitro. These DCs showed increased capacities for secretion of proinflammatory and Th1-cell polarizing cytokines, antigen presentation and stimulation of CD4 and CD8 T cells. These data show that mucosal immunization with L. lactis GEM particles carrying vaccine antigens represents a promising approach to prevent infectious diseases early in life. © 2010 Society for Mucosal Immunology.

Van Braeckel-Budimir N.V.,Mucosis B.V. | Haijema B.J.,Mucosis B.V. | Leenhouts K.,Mucosis B.V.
Frontiers in Immunology | Year: 2013

The successful development of a mucosal vaccine depends critically on the use of a safe and effective immunostimulant and/or carrier system. This review describes the effectiveness and mode of action of an immunostimulating particle, derived from bacteria, used in mucosal subunit vaccines. The non-living particles, designated bacterium-like particles are based on the food-grade bacterium Lactococcus lactis. The focus of the overview is on the development of intranasal BLP-based vaccines to prevent diseases caused by influenza and respiratory syncytial virus, and includes a selection of Phase I clinical data for the intranasal FluGEM vaccine. © 2013 Van Braeckel-Budimir, Haijema and Leenhouts.

The invention relates to the field of immunology and vaccines. In particular, it relates to proteinaceous substances and the uses thereof in vaccines against infections caused by respiratory pathogens.

The invention relates to the field of biology, more specifically to the field of immunology and microbiology. The invention further relates to the field of vaccines against microbial infections and especially bacterial vaccines, in particular to pneumococcal vaccines. More in particular, the invention relates to means and methods to identify, select and isolate a vaccine component for passive and/or active immunisation against a microorganism that can be killed by opsonophagocytic cells. The invention relates to a method to identify an opsonophagocytosis inducing antigen as a vaccine component for immunisation against a microorganism. The invention describes three pneumococcal proteins SlrA, IgA1 proteinase, and PsaA, and their use as a vaccine component with or without PpmA. The invention also discloses the use of antibodies against said proteins for passive immunization and diagnosis.

Heat-stable oligomeric recombinant polypeptides, presenting at least one antigenic epitope of the pre-fusion Respiratory Syncytial Virus (RSV) F protein, comprising the RSV F protein ectodomain, functionally deleted in the HRB region, transmembrane and cytoplasmic domains replaced with a heterologous trimerization domain, and absent two functional multibasic furin cleavage sites, are useful as antigenic components in immunogenic compositions useful in methods of inducing an immune response and vaccinate against RSV infections.

The invention relates to the field of immunology and vaccine development, in particular to the development of vaccines based on native antigen oligomers. Provided is an immunogenic composition in particulate form, comprising oligomers of a surface exposed polypeptide of pathogenic origin or tumour origin, or antigenic part thereof, said oligomers being bound non-covalently to a particulate carrier, and a pharmaceutically acceptable diluent or excipient. Also provided is a recombinant polypeptide comprising (A) an N- or C-terminal antigenic domain, comprising at least one surface exposed polypeptide of pathogenic or tumour origin, or antigenic part thereof, the antigenic domain being fused to (B) an oligomerization domain (OMD), said oligomerization domain being fused via (C) a linker domain to (D) a peptidoglycan binding domain (PBD) consisting of a single copy of a LysM domain capable of mediating the non-covalent attachment of the polypeptide to a non-viable bacterium-like particle (BLP) obtained from a Gram-positive bacterium.

Mucosis B.V. | Date: 2011-04-13

The invention relates to adjuvanted vaccine formulations, in particular influenza vaccines for intranasal delivery. Provided is an adjuvanted influenza vaccine formulation, comprising (i) peptidoglycan microparticles obtained from a Gram-positive bacterium and (ii) at least one influenza virus antigen or antigenic preparation thereof, which antigen or antigenic preparation is not fused or otherwise covalently attached to a proteinaceous peptidoglycan binding moiety.

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