Characterisation of the Protein Content of a Meningococcal Outer Membrane Vesicle Vaccine by Polyacrylamide Gel Electrophoresis and Mass Spectrometry

Abstract

Previously published online as a Human Vaccines ACKNOWLEDGEMENTSWe wish to thank Philipp Oster for critical reading of the manuscript. Research Paper Characterization of the Protein Content of a Meningococcal Outer Membrane Vesicle Vaccine by Polyacrylamide Gel Electrophoresis and Mass Spectrometry ABSTRACTThe development and evaluation of outer membrane vesicles as vaccines against meningococcal disease has been carried out for more than two decades. Although such vaccines have limitations and are not widely licensed, they continue to be used to disrupt clonal outbreaks caused by group B meningococci and a wealth of information is now available from large-scale clinical studies. One dimensional polyacrylamide gel electrophoresis and semi-quantitative measurement of the major proteins is one method used to evaluate and control these products. However, it is often difficult to determine exactly which bands on a one dimensional gel correspond to the key antigens whose presence must be demonstrated for control and lot release. We have therefore carried out mass spectrometric analyses of outer membrane vesicle vaccine samples to definitively identify the bands containing seven key antigens: Omp85, FetA, PorA, PorB, RmpM, OpcA and NspA. An additional 33 proteins present in the vaccine were also identified and this information will be useful both for future quality control and for the interpretation of data from vaccine trials.

Functional and Specific Antibody Responses in Adult Volunteers in New Zealand Who Were Given One of Two Different Meningococcal Serogroup B Outer Membrane Vesicle Vaccines

“…Antibodies to NspA were determined with OMVs from E. coli cells that expressed or that did not express rNspA. Although MeNZB contained a distinct amount of NspA protein, as previously shown on Coomassie blue-stained polyacrylamide gels (24,56) and as probed in our study with a specific antibody on immunoblots, most vaccinees showed no increase in IgG to this protein, nor was there any significant difference in the responses between the two vaccine groups (data not shown). MenBvac contained only trace amounts of this antigen.…”

Immunoproteomic Analysis of the Development of Natural Immunity in Subjects Colonized by Neisseria meningitidis Reveals Potential Vaccine Candidates

“…The second ABC transporter, NMB0634 (FbpA), is a virulence factor with a role in iron uptake (42); however, the role for the third ABC transporter, NMB1612, is unknown despite its presence in OMV and OM preparations (59). The other hypothetical protein associated with the periplasm, NMB0928, has been reported to be present as a constituent of OMV derived from N. meningitidis (52,53,55,59) and N. lactamica (53) and also to be conserved across many meningococcal serogroups and capable of inducing murine bactericidal antibodies (8). The thiol-disulfide interchange protein DsbC is potentially interesting as a vaccine candidate due to function, as it catalyzes the formation of disulfide bonds essential for the conformational stability and folding of proteins, and recently, the DsbC-encoding open reading frame has been reported to become upregulated in meningococci during infection of HeLa cells (47).…”

Functional Significance of Factor H Binding toNeisseria meningitidis

“…The pellet was resuspended in 100 l of SDS-PAGE sample buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 100 M 2-ME, 10% glycerol, 0.1% bromophenol blue). Outer membrane vesicles (OMVs) were prepared as described previously (35). Proteins were separated under reducing conditions by SDS-12% PAGE, then the proteins transferred to polyvinylidene difluoride membranes (Millipore).…”