Functional activity of antibodies against the recombinant OpaJ protein from Neisseria meningitidis

Abstract

The opacity proteins belong to the major outer membrane proteins of the pathogenic Neisseria and are involved in adhesion and invasion. We studied the functional activity of antibodies raised against the OpaJ protein from strain H44/76. Recombinant OpaJ protein was obtained from Escherichia coli in two different ways: cytoplasmic expression in the form of inclusion bodies followed by purification and refolding and cell surface expression followed by isolation of outer membrane complexes (OMCs). Immunization with purified protein and Quillaja saponin A (QuilA) induced high levels of Opa-specific antibodies, whereas the E. coli OMC preparations generally induced lower levels of antibodies. Two chimeric Opa proteins, hybrids between OpaB and OpaJ, were generated to demonstrate that the hypervariable region 2 is immunodominant. Denatured OpaJ with QuilA induced high levels of immunoglobulin G2a (IgG2a) in addition to IgG1, whereas refolded OpaJ with QuilA induced IgG1 exclusively. These sera did not induce significant complement-mediated killing. However, all sera blocked the interaction of OpaJ-expressing bacteria to CEACAM1-transfected cells. In addition, cross-reactive blocking of OpaB-expressing bacteria to both CEACAM1- and CEA-transfected cells was found for all sera. Sera raised against purified OpaJ and against OpaJ-containing meningococcal OMCs also blocked the nonopsonic interaction of Opa-expressing meningococci with human polymorphonuclear leukocytes.

FIG. 1.

Amino acid sequences of the variable regions of OpaB128 and OpaJ129. Minimal epitopes of MAbs MN20E12.70 and 15-1-P5.5 are underlined.

FIG. 2.

(A) Total IgG response measured in whole-cell ELISA with E. coli strain CE1265 expressing OpaJ (CE1265-opaJ) as the immobilized antigen and wild-type CE1265 as the negative control (CE1265).The data shown represent the geometric means and standard errors of 8 mice per group. (B) Total IgG response measured in whole-cell ELISA with H44/76 expressing Opa proteins detectable with 15-1-P5.5 MAb (H44/76 P5.5+) and H44/76 expressing Opa proteins detectable with MN20E12.70 MAb (H44/76 D2+) and H44/76 not expressing any Opa protein as a negative control (H44/76 Opa−). (C) Total IgG response measured in ELISA with purified OpaB and OpaJ. The data shown represent the geometric means and standard errors of 8 mice per group. Mice immunized with OMCs containing Opa protein giving lower titers than mice immunized with OMCs without Opa proteins are defined as nonresponders. Two of eight mice immunized with OMCs containing OpaJ did not respond; the titers from these nonresponders were included.

FIG. 3.

Antibody reaction against chimeric Opa proteins measured in whole-cell ELISA. The construction of the chimeric opa genes is depicted in panel A. The antibodies present in the sera of mice, immunized with Opa-negative and OpaJ-positive meningococcal OMCs and with purified refolded (Ref.) and denatured (Den.) OpaJ with QuilA, were tested for reaction with E. coli bacteria expressing OpaBj and OpaJb (B). The data shown represent the geometric means and standard errors of 8 mice per group.

FIG. 4.

Antibody subclass-specific response to meningococcal OMCs and purified OpaJ plus QuilA with whole cells of CE1265-OpaJ as the immobilized antigen (A) and E. coli OMCs containing OpaJ with whole cells of H44/76 meningococci as the immobilized antigen (B). The data shown represent the geometric means and standard errors of 8 mice per group.

FIG. 5.

Antibody blocking activity in an in vitro infection assay with sera raised against refolded and denatured OpaJ and meningococcal OMCs containing OpaJ. Two dilutions (1/100 and 1/1,000) of all sera were tested for blocking the binding between OpaJ and CEACAM1 (A), OpaB and CEACAM1 (B), and OpaB and CEA (C). The data shown represent the geometric means and standard errors of three independent experiments.

FIG. 6.

(a) Characterization by FACS analysis of PorA P1.16, Opc, OpaB, and OpaD (MAb MN20E12.70) and OpaA and OpaJ expression (MAb 15-1-P5.5) of the meningococcal H44/76 Opa⁺ and Opa⁻ variants used in the opsonophagocytosis assay. α, anti. (b) Opsono- and nonopsonophagocytosis of heat-killed Alexa 488-labeled meningococcal H44/76 Opa⁻ (A and C) and Opa⁺ variants (B and D) by human PMN in the presence of antisera raised against purified refolded and denatured OpaJ (A and B) and against H44/76 Opa⁺ and Opa⁻ OMCs (C and D). As a positive control for opsonophagocytosis, the two meningococcal H44/76 variants were incubated with PMN in the presence of MAb HMN12H2 IgG1, a humanized MAb specific for PorA. The experiments were repeated two times with similar results.