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.

scholarly journals Immunization with Recombinant Opc Outer Membrane Protein from Neisseria meningitidis: Influence of Sequence Variation and Levels of Expression on the Bactericidal Immune Response against Meningococci

2001 ◽  
Vol 69 (6) ◽  
pp. 3809-3816 ◽  
Author(s):  
Keith A. Jolley ◽  
Lynn Appleby ◽  
J. Claire Wright ◽  
Myron Christodoulides ◽  
John E. Heckels
Keyword(s):  
Immune Response ◽  
Recombinant Protein ◽  
Neisseria Meningitidis ◽  
Lipid A ◽  
Bactericidal Effect ◽  
Native Protein ◽  
Whole Cells ◽  
Monophosphoryl Lipid A ◽  
Homologous Strain ◽  
Immunoglobulin Subclasses

ABSTRACT The opc gene from Neisseria meningitidiswas cloned into the pRSETA vector, and recombinant protein was expressed at high levels in Escherichia coli. The protein was readily purified by affinity chromatography and used for immunization with conventional Al(OH)3 adjuvant or after incorporation into liposomes and Zwittergent micelles. The resulting sera were analyzed for their ability to recognize purified recombinant protein and “native” protein in an enzyme immunoassay with outer membranes and by whole-cell immunofluorescence. Immunization with Al(OH)3 induced high levels of antibodies which reacted with the purified protein but did not recognize whole cells. In contrast, liposomes and micelles induced antibodies which reacted with the native protein in whole cells. The addition of monophosphoryl lipid A (MPLA) to either liposomes or micelle preparations increased the magnitude of the immune response and induced a wider range of immunoglobulin subclasses. This was associated with the ability of the sera to induce complement-mediated killing of the homologous strain. The most effective bactericidal activity was observed with Opc protein incorporated into liposomes containing MPLA. The magnitude of the bactericidal effect was strongly influenced by the level of expression of the Opc protein and was abolished by limited variation in the sequence of the protein expressed by heterologous strains.

scholarly journals The msbB Mutant of Neisseria meningitidis Strain NMB Has a Defect in Lipooligosaccharide Assembly and Transport to the Outer Membrane

2003 ◽  
Vol 71 (2) ◽  
pp. 647-655 ◽  
Author(s):  
Deborah M. B. Post ◽  
Margaret R. Ketterer ◽  
Nancy J. Phillips ◽  
Bradford W. Gibson ◽  
Michael A. Apicella
Keyword(s):  
Monoclonal Antibody ◽  
Western Blot ◽  
Neisseria Meningitidis ◽  
Outer Membrane ◽  
Immunoelectron Microscopy ◽  
Parent Strain ◽  
Lipid A ◽  
Mass Spectrometric ◽  
Insertion Mutation ◽  
Cytoplasmic Staining

ABSTRACT A deletion-insertion mutation in msbB, a gene that encodes a lipid A acyltransferase, was introduced into encapsulated Neisseria meningitidis serogroup B strain NMB and an acapsular mutant of the same strain. These mutants were designated NMBA11K3 and NMBA11K3cap-, respectively. Neither lipooligosaccharide (LOS) nor lipid A could be isolated from NMBA11K3 although a number of techniques were tried, but both were easily extracted from NMBA11K3cap-. Immunoelectron microscopy using monoclonal antibody (MAb) 6B4, which recognizes the terminal Galβ1-4GlcNAc of LOS, demonstrated that NMB, NMBcap-, and NMBA11K3cap- expressed LOS circumferentially, while MAb 6B4 did not bind to the surface of NMBA11K3. However, cytoplasmic staining of NMBA11K3 with MAb 6B4 was a consistent observation. Mass-spectrometric analyses demonstrated that the relative amounts of the lipid A-specific C12:0 3-OH and C14:0 3-OH present in the membrane preparations (MP) from NMBA11K3 were substantially decreased (25- and 23-fold, respectively) compared to the amount in MP from its parent strain, NMB. Western blot analyses of MP from NMBA11K3 demonstrated that the levels of porin in the outer membrane of NMBA11K3 were also substantially decreased. These studies suggest that the lipid A acylation defect in encapsulated NMBA11K3 influences the assembly of the lipid A and consequently the incorporation of porin in the outer membrane.

scholarly journals Toll-like receptor 4 agonist-based nanoparticles orchestrate protection against sepsis

2022 ◽  
Author(s):  
Yongxiang Zhao ◽  
Xinjing Lv ◽  
Jie Huang ◽  
Huiting Zhou ◽  
Hairong Wang ◽  
...  
Keyword(s):  
Lipid A ◽  
Severe Infection ◽  
Plga Nanoparticles ◽  
Great Promise ◽  
Toll Like Receptor 4 ◽  
E Coli ◽  
Monophosphoryl Lipid A ◽  
Tlr4 Agonist ◽  
Life Threatening ◽  
Plga Nanoparticle

AbstractSepsis, a life-threatening organ dysfunction induced by severe infection and uncontrolled host immune response, threatens the health of people all over the world. Herein, a type of nanoparticle formulation with simple components is synthesized by encapsulating monophosphoryl lipid A (MPLA), a TLR4 agonist, with poly(lactic-co-glycolic acid) (PLGA) nanoparticle. The obtained nanoparticles (MPLA@PLGA) could provide Escherichia coli (E. coli)-induced sepsis protection by regulating the immune system after sepsis challenge, including promoting the levels of various cytokines, boosting the percentage of natural killer cells and accelerating bacterial clearance. Notably, the survival mice pre-treated with these nanoparticles could resist repeated E. coli-induced sepsis. Our work therefore provides the great promise of MPLA@PLGA nanoparticles as a simple yet effective nano-drug for prevention and protection against E. coli-induced sepsis.

scholarly journals Cloning, expression and purification of outer membrane protein PorA of Neisseria meningitidis serogroup B

2011 ◽  
Vol 5 (12) ◽  
pp. 856-862 ◽  
Author(s):  
Fakhri Haghi ◽  
Shahin Najar Peerayeh ◽  
Seyed Davar Siadat ◽  
Mehran Montajabiniat
Keyword(s):  
Recombinant Protein ◽  
Neisseria Meningitidis ◽  
Outer Membrane ◽  
Prokaryotic Expression ◽  
Membrane Vesicle ◽  
Outer Membrane Proteins ◽  
Expression System ◽  
Sds Page ◽  
Prokaryotic Expression System ◽  
Prokaryotic Expression Vector

Introduction: Neisseria meningitidis is a major causative agent of bacterial septicemia and meningitis in humans. Currently, there are no vaccines to prevent disease caused by strains of N. meningitidis serogroup B. PorA is a major component of the outer membrane of N. meningitidis and functions as a cationic porin. This study aimed to clone and determine the expression of PorA. Methodology: A 1200 bp fragment of porA gene was amplified by PCR from serogroup B N. meningitidis and then cloned into prokaryotic expression vector pET-32a. For expression of recombinant protein, pET32a-porA plasmid was transformed into competent Origami B (DE3) cells. Recombinant protein was overexpressed with isopropythio-beta-D-galctoside (IPTG) and affinity purified by Ni-NTA agarose. SDS-PAGE and western blotting were performed for protein determination and verification. Results: Cloning of porA was confirmed by colony-PCR and enzymatic digestion. In comparison with the corresponding sequences of original genes, the nucleotide sequence homology of the cloned porA gene was 97%. IPTG with a dosage of 1.0 mmol/L could efficiently induce protein expression. SDS-PAGE analysis showed that our constructed prokaryotic expression system pET32a-PorA-Origami efficiently produces a target recombinant protein with a molecular weight of 65 kDa. The recombinant PorA was overexpressed as inclusion bodies and reacted with the serum from a rabbit previously immunized with native outer membrane vesicle. Conclusion: This prokaryotic expression system provides an easy method for producing recombinant PorA and may also be useful for the production of other bacterial outer membrane proteins for vaccine studies.

scholarly journals Immunogenicity of Outer Membrane Proteins in a Lipopolysaccharide-Deficient Mutant of Neisseria meningitidis: Influence of Adjuvants on the Immune Response

1999 ◽  
Vol 67 (10) ◽  
pp. 4988-4993 ◽  
Author(s):  
Liana Steeghs ◽  
Betsy Kuipers ◽  
Hendrik Jan Hamstra ◽  
Gideon Kersten ◽  
Loek van Alphen ◽  
...  
Keyword(s):  
Immune Response ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Lipid A ◽  
Outer Membrane Proteins ◽  
Protective Mechanism ◽  
Deficient Mutant ◽  
Adjuvant Activity ◽  
Monophosphoryl Lipid A ◽  
Meningococcal Strain

ABSTRACT The immunogenicity of outer membrane complexes (OMCs) or heat-inactivated bacteria of a lipopolysaccharide (LPS)-deficient mutant derived from meningococcal strain H44/76 was studied. The immune response in BALB/c mice to the major outer membrane proteins was poor compared to the immune response elicited by wild-type immunogens. However, addition of external H44/76 LPS to mutant OMCs entirely restored the immune response. By using an LPS-deficient mutant, it may be possible to substitute a less toxic compound as adjuvant in meningococcal outer membrane vaccines. Therefore, a broad panel of adjuvants were tested for their potential to enhance the immunogenicity of LPS-deficient OMCs. AlPO4, Rhodobacter sphaeroides LPS, monophosphoryl lipid A and alkali-hydrolyzed meningococcal LPS showed significantly lower adjuvant activity than did H44/76 LPS. Adjuvant activity similar to H44/76 LPS was found forEscherichia coli LPS, meningococcal icsB andrfaC LPS, QuilA, subfractions of QuilA, and MF59. Good adjuvant activity was also found with meningococcal htrB1LPS, containing penta-acylated lipid A. Antisera elicited with the less active adjuvants showed relatively high immunoglobulin G1 (IgG1) titers, whereas strong adjuvants also induced high IgG2a and IgG2b responses in addition to IgG1. Antisera with the IgG2a and IgG2b isotypes showed high bactericidal activity, indicating that adjuvants promoting the IgG2a and IgG2b response contribute most to the protective mechanism. Thus, this study demonstrates that the immunogenicity of meningococcal LPS-deficient OMCs can be restored by using less toxic adjuvants, which opens up new avenues for development of vaccines against meningococcal disease.

Immunogenic properties of the Plasmodium vivax vaccine candidate MSP119 expressed as a secreted non-glycosylated polypeptide from Pichia pastoris

Parasitology ◽  
2002 ◽  
Vol 124 (3) ◽  
pp. 237-246 ◽  
Author(s):  
I. S. SOARES ◽  
M. M. RODRIGUES
Keyword(s):  
Pichia Pastoris ◽  
Recombinant Protein ◽  
Plasmodium Vivax ◽  
Lipid A ◽  
Surface Protein ◽  
Methylotrophic Yeast ◽  
Merozoite Surface Protein ◽  
Gene Encoding ◽  
Monophosphoryl Lipid A ◽  
Antibody Titres

The 19 kDa C-terminal region of the merozoite surface protein 1 (MSP119) is one of the most promising vaccine candidates against the erythrocytic forms of malaria. In the present study, a gene encoding the Plasmodium vivax MSP119 epitope (PvMSP119) and the Pan-Allelic DR epitope (PADRE) was expressed in the methylotrophic yeast Pichia pastoris. A non-glycosylated form of the recombinant protein rPvMSP119-PADRE was purified from culture supernatants. This recombinant protein maintains its antigenicity, being recognized by a very high percentage (85·6%) of sera from Brazilian individuals naturally exposed to P. vivax. The antibody immune response elicited by rPvMSP119-PADRE was compared in C57BL/6 mice immunized with different adjuvant formulations. After 3 immunizing doses, antibody titres induced in the presence of the adjuvants monophosphoryl lipid A, trehalose dicorynomycolate and cell wall skeleton or alum plus CpG ODN 1826 were as high as titres generated by Complete Freund's Adjuvant. Based on these immunological studies, we concluded that rPvMSP119-PADRE deserves further evaluation in pre-clinical immunizations against P. vivax in non-human primates.

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