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

Abstract

The opc gene from Neisseria meningitidis was 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.

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

“…For IgG3, a biotin-conjugated rat monoclonal anti-mouse IgG3 antibody (Pharmingen/Becton Dickinson, Crawley, United Kingdom) and HRP-streptavidin (Zymed) were used. All conjugates were used at a 1 in 1,000 dilution (22).…”

“…After being washed in PBS, the meningococci were counterstained with propidium iodide (25 g ml Ϫ1 ; Sigma) for 5 min before a rewashing in PBS. Slides were viewed by using a fluorescent microscope (22).…”

“…The generally accepted correlate of protection against meningococcal infection is the presence of antibodies with the ability to activate complement-mediated killing of meningococci (15). Several studies with recombinant meningococcal PorA and Opc proteins, as well as with purified PI porin from Neisseria gonorrhoeae, have clearly demonstrated that the production of bactericidal antibodies was dependent on refolding of the protein to induce native conformation, which could be achieved by incorporation either into artificial membranes (liposomes) (8,20,22,30,29,42,44) or detergent micelles (21,22,43).…”

A cross-reactive neisserial antigen encoded by the NMB0035 locus shows high sequence conservation but variable surface accessibility

“…Ethanol damages bacterial outer structures, partially eliminating the capsule and LOS outer residues, exposing surface antigens and facilitating their accessibility to antibodies (Jolley et al, 2001;Michaelsen et al, 2001). In N. meningitidis, capsular structure and/or the amount and length of LOS can significantly vary among strains, and all commensal Neisseria lack a capsule and have particular LOS due to the absence of lgt genes (which encode proteins involved in glycosyltransferase synthesis) in most species (Zhu et al, 2002;Kahler et al, 2005).…”

Septic Shock by Gram-Negative Infections: Role of Outer Membrane Proteins

“…The role of porins in pathogenesis is also confirmed by studies on the development of an effective vaccine against serogroup B Neisseria meningitides. The nonimmunogenicity of serogroup B capsular polysaccharide has led to the development of outer membrane vesicle (OMV) vaccines, based on the presence of PorB (Jolley, 2001;Wright, 2002). The porin proteins adopt a β-sheet structure within the outer membrane with surface exposed loops (Van der Ley, 1991).…”