5439808 Method for the high level expression, purification and refolding of the outer membrane group B porin proteins from neisseria meningitidis

1996 ◽  
Vol 14 (4) ◽  
pp. 579
Keyword(s):  
Neisseria Meningitidis ◽  
Outer Membrane ◽  
High Level Expression ◽  
Porin Proteins ◽  
Group B ◽  
High Level

scholarly journals Protection of Mice against Brucellosis by Intranasal Immunization with Brucella melitensis Lipopolysaccharide as a Noncovalent Complex with Neisseria meningitidis Group B Outer Membrane Protein

2002 ◽  
Vol 70 (7) ◽  
pp. 3324-3329 ◽  
Author(s):  
Apurba K. Bhattacharjee ◽  
Lillian Van De Verg ◽  
Mina J. Izadjoo ◽  
Liang Yuan ◽  
Ted L. Hadfield ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Antibody Response ◽  
Neisseria Meningitidis ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Brucella Melitensis ◽  
Noncovalent Complex ◽  
High Titer ◽  
Intranasal Immunization ◽  
Group B

ABSTRACT Intranasal immunization of mice with purified Brucella melitensis lipopolysaccharide (LPS) as a noncovalent complex with Neisseria meningitidis group B outer membrane protein (GBOMP) elicited a high-titer anti-LPS systemic antibody response and a significant mucosal antibody response. The anti-LPS immunoglobulin G (IgG) antibody was predominantly of the IgG1 subtype, although there was some response of the IgG2a, IgG2b, and IgG3 subtypes. The antibody titer remained high for 16 weeks postimmunization. Immunized mice and sham-immunized control mice were challenged intranasally with 104 CFU of virulent B. melitensis strain 16 M 4 weeks after the second dose of vaccine. The numbers of bacteria in lungs, livers, and spleens at 3 days, 9 days, and 8 weeks postchallenge were determined. Bacteria were found in lungs of all mice on day 3, but there was no disseminated infection of liver or spleen. By day 9, 40% of the mice had infected spleens and livers. At 8 weeks postchallenge, spleens of 25 of 62 immunized mice were infected, compared to 61 of 62 control mice (P < 0.0001). The livers of 12 of 43 immunized mice were infected, compared to 22 of 36 control mice (P = 0.005). In contrast, the lungs of 26 of 46 immunized mice were still infected, compared to 27 of 44 control mice. The numbers of bacterial CFU in lungs of immunized and control animals were identical. These studies show that intranasal immunization with B. melitensis LPS-GBOMP subunit vaccine significantly protects mice against intranasal challenge with virulent B. melitensis. Vaccination reduces bacterial dissemination to spleen and liver but has no effect on the course of lung infection.

scholarly journals Immunization with the Recombinant PorB Outer Membrane Protein Induces a Bactericidal Immune Response against Neisseria meningitidis

2002 ◽  
Vol 70 (8) ◽  
pp. 4028-4034 ◽  
Author(s):  
J. Claire Wright ◽  
Jeannette N. Williams ◽  
Myron Christodoulides ◽  
John E. Heckels
Keyword(s):  
Recombinant Protein ◽  
Neisseria Meningitidis ◽  
Outer Membrane ◽  
Lipid A ◽  
Meningococcal Infection ◽  
Native Protein ◽  
Monophosphoryl Lipid A ◽  
Porin Protein ◽  
Life Threatening ◽  
Porin Proteins

ABSTRACT Infections with Neisseria meningitidis are characterized by life-threatening meningitis and septicemia. The meningococcal porin proteins from serogroup B meningococci have been identified as candidates for inclusion in vaccines to prevent such infections. In this study, we investigated the vaccine potential of the PorB porin protein free of other meningococcal components. The porB gene from a strain of Neisseria meningitidis expressing the class 3 outer membrane porin protein (PorB3) was cloned into the pRSETB vector, and the protein was expressed at high levels in a heterologous host Escherichia coli. The recombinant protein was purified to homogeneity by affinity chromatography and used for immunization after incorporation into liposomes and into micelles composed either of zwitterionic detergent or nondetergent sulfobetaine. The immunogenicity of these preparations was compared to recombinant PorB protein adsorbed to Al(OH)3 adjuvant as a control. Although sera raised against the protein adsorbed to Al(OH)3 reacted with the purified recombinant protein, sera raised against liposomes and micelles showed greater activity with native protein, as measured by enzyme immunoassay with outer membranes and by whole-cell immunofluorescence. Reactivity with native protein was considerably enhanced by incorporation of the adjuvant monophosphoryl lipid A into the liposome or micelle preparations. Recognition of the native protein was in a serotype-specific manner and was associated with the ability of the antisera to promote high levels of serotype-specific complement-mediated killing of meningococci. These results demonstrate that the PorB protein should be considered as a component of a vaccine designed to prevent serogroup B meningococcal infection.

Surface antigen analysis of group B Neisseria meningitidis outer membrane by monoclonal antibodies: Identification of bactericidal antibodies to class 5 protein

1995 ◽  
Vol 31 (3) ◽  
pp. 146-151 ◽  
Author(s):  
Maria das Gra�as M. Danelli ◽  
N�dia M. Batoreu ◽  
Maria Diana Lacerda ◽  
Cristiane R. B. Ferreira ◽  
Joana Darc Cardoso ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Neisseria Meningitidis ◽  
Outer Membrane ◽  
Surface Antigen ◽  
Group B

scholarly journals Sequential Immunization with Vesicles Prepared from Heterologous Neisseria meningitidis Strains Elicits Broadly Protective Serum Antibodies to Group B Strains

2002 ◽  
Vol 70 (11) ◽  
pp. 6021-6031 ◽  
Author(s):  
Gregory R. Moe ◽  
Patricia Zuno-Mitchell ◽  
Samantha N. Hammond ◽  
Dan M. Granoff
Keyword(s):  
Neisseria Meningitidis ◽  
Capsular Polysaccharide ◽  
Protein A ◽  
Surface Protein ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Novel Approach ◽  
Bactericidal Antibody ◽  
Porin Proteins ◽  
Group B

ABSTRACT The capsular polysaccharide of Neisseria meningitidis group B is an autoantigen, whereas noncapsular antigens are highly variable. These factors present formidable challenges for development of a broadly protective and safe group B vaccine. Mice and guinea pigs were sequentially immunized with three doses of micovesicles or outer membrane vesicles prepared from three meningococcal strains that were each antigenically heterologous with respect to the two major porin proteins, PorA and PorB, and the group capsular polysaccharide. The resulting antisera conferred passive protection against meningococcal group B bacteremia in infant rats and elicited complement-mediated bactericidal activity against genetically diverse group B strains that were either homologous or heterologous with respect to PorA of the strains used to prepare the vaccine. By using knockout strains, a portion of the bactericidal antibody was directed against the highly conserved protein, neisserial surface protein A (NspA). Further, an anti-NspA monoclonal antibody elicited by the sequential immunization was highly bactericidal against strains that were previously shown to be resistant to bacteriolysis by anti-NspA antibodies produced by immunization with recombinant NspA. Sequential immunization with heterologous vesicle preparations offers a novel approach to eliciting broadly protective immunity against N. meningitidis strains. An NspA-based vaccine prepared from protein expressed by Neisseria also may be more effective than the corresponding recombinant protein made in Escherichia coli.

scholarly journals Functional Activities and Epitope Specificity of Human and Murine Antibodies against the Class 4 Outer Membrane Protein (Rmp) of Neisseria meningitidis

1999 ◽  
Vol 67 (3) ◽  
pp. 1267-1276 ◽  
Author(s):  
Einar Rosenqvist ◽  
Alexis Musacchio ◽  
Audun Aase ◽  
E. Arne Høiby ◽  
Ellen Namork ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Neisseria Meningitidis ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Membrane Vesicle ◽  
Bactericidal Effect ◽  
Outer Membrane Vesicle ◽  
Human Sera ◽  
High Concentrations ◽  
Group B

ABSTRACT Antibodies against the class 4 outer membrane protein (OMP) fromNeisseria meningitidis have been purified from sera from vaccinees immunized with the Norwegian meningococcal group B outer membrane vesicle vaccine. The human sera and purified antibodies reacted strongly with the class 4 OMP in immunoblots, whereas experiments with whole bacteria showed only weak reactions, indicating that the antibodies mainly reacted with parts of the class 4 molecule that were not exposed. The purified human anti-class 4 OMP antibodies and the monoclonal antibodies (MAbs) were neither bactericidal nor opsonic against live meningococci. Three new MAbs against the class 4 OMP were generated and compared with other, previously described MAbs. Three linear epitopes in different regions of the class 4 OMP were identified by the reaction of MAbs with synthetic peptides. The MAbs showed no blocking effect on bactericidal activity of MAbs against other OMPs. However, one of the eight purified human anti-class 4 OMP antibody preparations, selected from immunoblot reactions among sera from 27 vaccinees, inhibited at high concentrations the bactericidal effect of a MAb against the class 1 OMP. However, these antibodies were not vaccine induced, as they were present also before vaccination. Therefore, this study gave no evidence that vaccination with a meningococcal outer membrane vesicle vaccine containing the class 4 OMP induces blocking antibodies. Our data indicated that the structure of class 4 OMP does not correspond to standard β-barrel structures of integral OMPs and that no substantial portion of the OmpA-like C-terminal region of this protein is located at the surface of the outer membrane.

scholarly journals Type IV-Like Pili Formed by the Type II Secreton: Specificity, Composition, Bundling, Polar Localization, and Surface Presentation of Peptides

2003 ◽  
Vol 185 (11) ◽  
pp. 3416-3428 ◽  
Author(s):  
Guillaume Vignon ◽  
Rolf Köhler ◽  
Eric Larquet ◽  
Stéphanie Giroux ◽  
Marie-Christine Prévost ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Type Ii Secretion ◽  
Type Ii ◽  
High Level Expression ◽  
Polar Localization ◽  
Type Iv ◽  
C Terminus ◽  
Type Iv Pilin ◽  
High Level ◽  
K 12

ABSTRACT The secreton or type II secretion machinery of gram-negative bacteria includes several type IV pilin-like proteins (the pseudopilins) that are absolutely required for secretion. We previously reported the presence of a bundled pilus composed of the pseudopilin PulG on the surface of agar-grown Escherichia coli K-12 cells expressing the Klebsiella oxytoca pullulanase (Pul) secreton genes at high levels (N. Sauvonnet, G. Vignon, A. P. Pugsley, and P. Gounon, EMBO J. 19:2221-2228, 2000). We show here that PulG is the only pseudopilin in purified pili and that the phenomenon is not restricted to the Pul secreton reconstituted in E. coli or to PulG. For example, high-level expression of the endogenous E. coli gsp secreton genes caused production of bundled pili composed of the pseudopilin GspG, and the Pul secreton was able to form pili composed of PulG-like proteins from secreton systems of other bacteria. PulG derivatives in which the C terminus was extended by the addition of eight different peptides were also assembled into pili and functioned in secretion. Three of the C-terminal peptides were shown to be exposed along the entire length of the assembled pili. Hence, the C terminus of PulG may represent a permissive site for the insertion of immunogenic epitopes or other peptide sequences. One of these PulG variants, with a six-histidine tag at its C terminus, formed nonpolar, nonbundled pili, suggesting that bundle formation and polar localization are not correlated with the ability of PulG to function in secretion. We propose that the PulG pilus is an artifactual manifestation of a periplasmic “pseudopilus” and that cycles of pseudopilus extension and retraction within the periplasm propel pullulanase through secretin channels in the outer membrane. Abnormally long pili that extend beyond the outer membrane are produced only when pilus length control and retraction are deregulated by overproduction of the major pseudopilus subunit (PulG).

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