Gonococcal Outer Membrane Protein Research Articles

Development of flow cytometry based adherence assay for Neisseria gonorrhoeae using 5\u2032-carboxyfluorosceinsuccidyl ester

BackgroundNeisseria gonorrhoeae is an obligate human pathogen and its adherence to host cells is essential for its pathogenesis. Gonococcal adherence assays are based on the enumeration of bacteria attached to human cells on solid media. Because conventional adherence assays are based on bacterial counts, they are often time consuming to perform and prone to observer bias. A flow cytometry based method, using the cell-permeable fluorescent dye 5′-carboxyfluoroscein succidyl ester (CFSE), was developed to dramatically increase the number of adherent N. gonorrhoeae quantified per assay while improving repeatability and removing observer bias. Piliated N. gonorrhoeae F62 were stained with CFSE then the staining reaction was quenched with foetal bovine serum. Human cervical ME-180 cells were infected with CFSE-stained N. gonorrhoeae (multiplicity of the infection 100:1) for 2 h. Infected cells were washed to remove loosely adhered bacteria. Flow cytometry was used to quantify the percentage of ME-180 cells associated with CFSE-stained N. gonorrhoeae and a minimum of 30,000 events were recorded. Real time-PCR analysis targeting opa gene (encoding N. gonorrhoeae opacity associated gonococcal outer membrane protein) was performed on infected ME-180 cells to confirm the flow cytometric adherence assay results. A rabbit was immunized with heat-killed N. gonorrhoeaeF62 to generate hyperimmune serum. The functional compatibility of the assay was confirmed by studying the effect of N. gonorrhoeae F62 antiserum on blocking adherence/invasion of CFSE-stained bacteria to ME-180 cells.ResultsWe observed that 20.3% (+/− 1.0) ME-180 cells were associated with CFSE-stained N. gonorrhoeae. Heat-inactivated hyperimmune serum, at 1:10 to 1:80 dilutions, significantly inhibited gonococcal adherence by 6 and 3 fold, respectively. Real time-PCR analysis targeting opa gene confirmed that hyperimmune serum blocked adherence/invasion of N. gonorrhoeae to the ME-180 cells in a dilution-dependent manner.ConclusionsFlow cytometric analysis was amenable to quick, easy and high-throughput quantification of the association of N. gonorrhoeae with ME-180 cells and was functionally confirmed using PCR analysis. These approaches may be adapted for in vitro and in vivo adherence studies related to gonococcal pathogenesis.

Phage proteins are expressed on the surface of Neisseria gonorrhoeae and are potential vaccine candidates.

All Neisseria gonorrhoeae strains whose DNA sequences have been determined possess filamentous phage sequences representing their full genomes. The presence of filamentous phage DNA sequences in all sequenced N. gonorrhoeae strains suggest that purified phage particles might be used as a gonococcal vaccine. To test this hypothesis, we purified filamentous NgoΦfil phages and immunized rabbits subcutaneously. The elicited sera contained large quantities of anti-phage IgG and IgA antibodies that bound to the surface of N. gonorrhoeae cells, as shown by ELISA and flow cytometry. The elicited sera bound to the structural NgoΦ6fil proteins present in phage particles and to N. gonorrhoeae cells. The sera did not react with gonococcal outer membrane proteins. The sera also had bactericidal activity and blocked adhesion of gonococci to tissue culture cells. These data demonstrate that NgoΦfil phage particles can induce antibodies with anti-gonococcal activity and may be a candidate for vaccine development.

Neisseria gonorrhoeae porin modifies the oxidative burst of human professional phagocytes.

A hallmark of infection with the gram-negative bacterium Neisseria gonorrhoeae is the local infiltration and subsequent activation of polymorphonuclear neutrophils. Several gonococcal outer membrane proteins are involved in the interaction with and the activation of these phagocytes, including gonococcal porin, the most abundant protein in the outer membrane. Previous work suggests that this porin plays a role in various cellular processes, including inhibiting neutrophils activation and phagosome maturation in professional phagocytes. Here we investigated the ability of porin to modify the oxidative metabolism of human peripheral blood neutrophils and monocytes in response to particulate stimuli (including live gonococci) and soluble agents. The activation of the oxidative metabolism was determined by chemiluminescence amplified with either luminol or lucigenin. We found that treatment of the phagocytes with porin inhibits the release of reactive oxygen species measured as luminol-enhanced chemiluminescence in response to zymosan, latex particles, and gonococci. The engulfment of these particles was not, however, affected by porin treatment. Similar effects of porin on the chemiluminescence response were observed in cytochalasin B-treated neutrophils exposed to the soluble chemotactic peptide N-formylmethionyl-leucyl-phenylalanine. This indicates that porin selectively inhibits granule fusion with those cellular membranes that are in direct contact with porin, namely, the phagosomal and plasma membranes. This porin-induced downregulation of oxidative metabolism may be a potent mechanism by which gonococci modulate oxygen-dependent reactions by activated phagocytes at inflammation sites.

Acid sphingomyelinase is involved in CEACAM receptor-mediated phagocytosis of Neisseria gonorrhoeae

The interaction with human phagocytes is a hallmark of symptomatic Neisseria gonorrhoeae infections. Gonococcal outer membrane proteins of the Opa family induce the opsonin-independent uptake of the bacteria that relies on CEACAM receptors and an active signaling machinery of the phagocyte. Here, we show that CEACAM receptor-mediated phagocytosis of Opa 52-expressing N. gonorrhoeae into human cells results in a rapid activation of the acid sphingomyelinase. Inhibition of this enzyme by imipramine or SR33557 abolishes opsonin-independent internalization without affecting bacterial adherence. Reconstitution of ceramide, the product of acid sphingomyelinase activity, in imipramine- or SR33557-treated cells restores internalization of the bacteria. Furthermore, we demonstrate that CEACAM receptor-initiated stimulation of other signalling molecules, in particular Src-like tyrosine kinases and Jun N-terminal kinases, requires acid sphingomyelinase. These studies provide evidence for a crucial role of the acid sphingomyelinase for CEACAM receptor-initiated signalling events and internalization of Opa 52-expressing N. gonorrhoeae into human neutrophils.

Expression of AniA, the major anaerobically induced outer membrane protein of Neisseria gonorrhoeae, provides protection against killing by normal human sera.

Anaerobically grown Neisseria gonorrhoeae has previously been shown to have elevated serum resistance in the absence of exogenous CMP-N-acetylneuraminic acid or detectable sialylation. We hypothesized that the anaerobically induced gonococcal outer membrane protein AniA might have a role in this phenomenon, as it is the only known gonococcal protein that is absent under aerobic conditions. An N. gonorrhoeae F62 derivative, RUG7035, in which aniA is under control of the tac promoter, was used to examine the effect of AniA expression on serum resistance. In this study, we found that expression of AniA enhanced the serum resistance of N. gonorrhoeae and may account for these earlier observations.

Incorporation of bacterial membrane proteins into liposomes: factors influencing protein reconstitution

Meningococcal and gonococcal outer membrane proteins were reconstituted into liposomes using detergent-mediated dialysis. The detergents octyl glucopyranoside (OGP), sodium cholate and Empigen BB were compared with respect to efficiency of detergent removal and protein incorporation. The rate of OGP removal was greater than for cholate during dialysis. Isopycnic density gradient centrifugation studies showed that liposomes were not formed and hence no protein incorporation occurred during dialysis from an Empigen BB containing reconstitution mixture. Cholate-mediated reconstitution yielded proteoliposomes with only 75% of the protein associated with the vesicles whereas all of the protein was reconstituted into the lipid bilayer during OGP-mediated reconstitution. Essentially complete protein incorporation was achieved with an initial protein-to-lipid ratio of 0.01:1 (w/w) in the reconstitution mixture; however, at higher initial protein-to-lipid ratios (0.02:1) only 75% protein incorporation was achieved. Reconstituted proteoliposomes were observed as large (>300 nm), multilamellar structures using cryo-electron microscopy. Size reduction of these proteoliposomes by extrusion did not result in significant loss of protein or lipid. Extruded proteoliposomes were unilamellar vesicles with mean diameter of about 100 nm.

Biophysical and antigenic characterization of gonococcal protein I incorporated into liposomes

The major gonococcal outer membrane protein, protein I (Por), was reconstituted into liposomes composed of either 1-palmitoyl, 2-oleoyl phosphatidylcholine (POPC) or POPC:1-palmitoyl, 2-oleoyl phosphatidylethanolamine (POPE) (1:1 weight ratio) and the resulting proteoliposomes characterized with respect to their biophysical and antigenic properties. Isopycnic density gradient centrifugation studies established that essentially all of the protein was reconstituted into the lipid bilayer with no significant differences in incorporation seen as a function of lipid composition. Examination of Por orientation in these proteoliposomes revealed that over 80% of the protein was oriented facing outwards in the same ‘hairpin loop’ fashion found in the native bacterial membrane. Reconstituted Por proteoliposomes exhibited a mean vesicle diameter of >0.5 μm but could be reduced by extrusion without significant loss of protein or lipid. These extruded systems were suitable for sterilization by terminal filtration. The antibody binding activities of various Por liposome formulations were determined using both anti-Por monoclonal antibodies and an immunized rabbit sera. No significant differences in antibody binding were observed as a function of proteoliposome lipid composition. However, consistently higher levels of antibody binding were obtained for Por liposomes prepared in this way compared with reconstituted systems prepared as described in earlier publications.

Neisserial porins inhibit human neutrophil actin polymerization, degranulation, opsonin receptor expression, and phagocytosis but prime the neutrophils to increase their oxidative burst

Porins are trimeric proteins that constitute water-filled pores that allow transmembrane diffusion of small solutes through the outer membrane layer of gram-negative bacteria. The porins are capable of inserting into the membranes of eucaryotic cells, and in the present study we have examined the in vitro effects on neutrophil functions of the following purified porins: meningococcal outer membrane protein classes 1 and 3 and gonococcal outer membrane protein 1B (P1B). The neisserial porins inhibited human neutrophil chemoattractant-induced actin polymerization and degranulation of both primary and secondary granules. The neutrophil expression of immunoglobulin G (IgG) Fc receptors II (Fc gamma RII; CDw32) and III (Fc gamma RIII; CD16), as well as the activation-dependent downregulation of Fc gamma RIII, were reduced by the meningococcal and gonococcal porins. The neisserial porins impaired the upregulation of complement receptors 1 (CD35) and 3 (CD11b) and inhibited the phagocytic capacity of neutrophils, as evaluated by the uptake of meningococci (strain 44/76) in the presence of patient serum containing known amounts of IgG against meningococcal porins. The porins also primed neutrophils to increase their intracellular hydrogen peroxide production in response to FMLP, whereas no such priming was observed if the neutrophil protein kinase C was stimulated directly with phorbol myristate acetate. The neisserial porins influenced neutrophil functions in a time- and concentration-dependent manner. The meningococcal class 1 outer membrane protein and the gonococcal P1B tended to alter neutrophil functions more than the meningococcal class 3 protein. Thus, the neisserial porins inhibited human neutrophil actin polymerization, degranulation, opsonin receptor expression, and phagocytosis but primed the neutrophils to increase their oxidative burst. It remains to be determined whether these in vitro observations reflect mechanisms that may be of importance for the interaction between neutrophils and Neisseria species in vivo.

Antibodies to opacity proteins (Opa) correlate with a reduced risk of gonococcal salpingitis.

Acute salpingitis complicating cervical gonococcal infection is a significant cause of infertility. Relatively little data are available concerning the pathophysiologic mechanisms of this disease. A cohort of 243 prostitutes residing in Nairobi were followed between March 1985 and April 1988. Gonococcal cultures were performed at each visit, and acute salpingitis was diagnosed clinically. Serum at enrollment was tested by immunoblot for antibody to gonococcal outer membrane proteins. 8.6% (146/1689) of gonococcal infections were complicated by salpingitis. Increased risk of salpingitis was associated with younger age, shorter duration of prostitution, HIV infection, number of gonococcal infections, and episodes of nongonococcal salpingitis. Rmp antibody increased the risk of salpingitis. Antibody to Opa decreased the risk of salpingitis. By logistic regression analysis, antibody to Opa was independently associated with decreased risk of gonococcal salpingitis (adjusted odds ratio [OR], 0.35; 95% confidence interval [95%CI], 0.17-0.76); HIV infection (adjusted OR, 3.5; 95% CI, 0.96-12.8) and episodes of nongonococcal salpingitis (adjusted OR, 3.4; 95% CI, 1.8-6.4) were independently associated with an increased risk of salpingitis. Antibody to Opa appears to protect against ascending gonococcal infection, perhaps by interfering with Opa mediated adherence and endocytosis. The demonstration of natural immunity that protects against upper genital tract infection in women suggests that a vaccine to prevent gonococcal salpingitis is possible.

Antibody to Rmp (outer membrane protein 3) increases susceptibility to gonococcal infection.

The severe adverse effects of gonococcal infection on human fertility suggests that Neisseria gonorrhoeae would exert powerful selection for the development of a protective immune response in humans. N. gonorrhoeae is an obligate human pathogen and must persist in humans to survive. Since it is an ecologically successful organism, it must have evolved strategies to evade any human immune response it elicits. In a longitudinal study among 243 women working as prostitutes and experiencing frequent gonococcal infection, younger women, women with HIV infection, and women with antibody to the gonococcal outer membrane protein 3 (Rmp) were at increased risk of infection (adjusted odds ratio 3.4, CI95% 1.1-10.4, P < 0.05). Rmp is highly conserved in N. gonorrhoeae and the blocking of mucosal defences may be one of its functions. As similar proteins occur in many gram negative mucosal pathogens, the enhancing effect of such proteins may be a general strategy whereby bacteria evade human immune responses.

Interactions of Neisseria gonorrhoeae with human neutrophils: studies with purified PII (Opa) outer membrane proteins and synthetic Opa peptides

We investigated the role of gonococcal outer membrane protein PII (also called Opa protein) in nonopsonic adherence to human neutrophils. Gonococcal outer membranes, purified Opa in detergent (Opa), purified Opa in liposomes (Opa+ lips), and peptides composing the second hypervariable (HV2) region of OpaB (strain FA1090) in liposomes (pepHV2 lips) were tested for their abilities to inhibit subsequent gonococcal adherence to human neutrophils. Outer membranes from gonococci possessing adherent Opa, liposomes containing adherent Opa, purified adherent Opa, and two of three liposome preparations (pepHV2 lips) containing peptides from the HV2 region of an adherent Opa inhibited subsequent adherence to neutrophils of homologous Opa+ gonococci. On the other hand, outer membranes from Opa- gonococci, outer membranes containing a nonadherent Opa (OpaA from strain FA1090), purified OpaA, and OpaA lips had little or no inhibitory effect. Outer membranes containing adherent Opas, purified adherent Opas, and liposomes containing such Opas all bound to neutrophils, whereas preparations containing OpaA or no Opa protein did not. The results indicate that (i) Opa proteins can bind to neutrophils in a partially purified or purified form and (ii) the HV2 region of Opa appears to at least partially mediate Opa's biological role.

Distribution of a protein antigenically related to the major anaerobically induced gonococcal outer membrane protein among other Neisseria species

The Pan 1 protein of Neisseria gonorrhoeae is a novel 54-kDa outer membrane protein expressed only when gonococci are grown in the absence of oxygen. It is a major antigen recognized by sera from patients with gonococcal infection. We raised mouse monospecific polyclonal antiserum to gel-purified Pan 1 from gonococcal strain F62. The antiserum was broadly cross-reactive among gonococcal strains; all strains tested reacted in immunoblot analysis proportionate to the amount of Pan 1 visible in silver-stained sodium dodecyl sulfate (SDS)-polyacrylamide gels. In immunoblot experiments, N. lactamica and N. cinerea reacted very strongly to the anti-Pan 1 antiserum, whereas N. sicca, N. flava, and N. mucosa did not react at all. The other commensals tested, N. subflava and N. perflava, exhibited only a minor reaction. These results correlated with the apparent amount of Pan 1 seen on SDS-polyacrylamide gels of outer membranes. SDS-polyacrylamide gel analysis of six meningococcal strains revealed no visible anaerobically induced outer membrane proteins, and the subsequent immunoblots showed only slight or no reaction to the anti-Pan 1 antibody. In the four meningococcal strains that did react slightly with the antiserum, a Pan 1-like protein was seen only in anaerobically grown cells. Thus, meningococci did not express Pan 1 at levels comparable to that found in gonococci; however, when Pan 1 was expressed in meningococcal strains, it was oxygen regulated. This is the first example of a protein found in the gonococcal outer membrane that, under identical growth conditions, is not expressed at similar levels in the meningococcus.

Gonococcal outer-membrane protein PIB: comparative sequence analysis and localization of epitopes which are recognized by type-specific and cross-reacting monoclonal antibodies

Comparison of the inferred amino acid sequence of outer-membrane protein PIB from gonococcal strain P9 with those from other serovars reveals that sequence variations occur in two discrete regions of the molecule centred on residues 196 (Var1) and 237 (Var2). A series of peptides spanning the amino acid sequence of the protein were synthesized on solid-phase supports and reacted with a panel of monoclonal antibodies (mAbs) which recognize either type-specific or conserved antigenic determinants on PIB. Four type-specific mAbs reacted with overlapping peptides in Var1 between residues 192-198. Analysis of the effect of amino acid substitutions revealed that the mAb specificity is generated by differences in the effect of single amino acid changes on mAb binding, so that antigenic differences between strains are revealed by different patterns of reactivity within a panel of antibodies. The variable epitopes in Var1 recognized by the type-specific mAbs lie in a hydrophilic region of the protein exposed on the gonococcal surface, and are accessible to complement-mediated bactericidal lysis. In contrast, the epitope recognized by mAb SM198 is highly conserved but is not exposed in the native protein and the antibody is non-bactericidal. However, the conserved epitope recognized by mAb SM24 is centred on residues 198-199, close to Var1 , and is exposed for bactericidal killing.

Vaccination against gonorrhoea: the potential protective effect of immunization with a synthetic peptide containing a conserved epitope of gonococcal outer membrane protein IB

Monoclonal antibody SM24 recognizes a protective, highly conserved but non-immunogenic epitope on outer membrane protein PIB of Neisseria gonorrhoeae. A series of overlapping synthetic peptides, spanning the deduced amino acid sequence of PIB from strain R10, have been synthesized on solid phase supports. Monoclonal antibody SM24 reacted with two adjacent decapeptides corresponding to residues 191-200 and 196-205, containing the common sequence TYSIP. Following localization of the epitope recognized, a peptide was synthesized corresponding to residues 193-204. The peptide was coupled to a carrier protein (KLH) and both the free peptide and peptide-KLH conjugate were used for immunization of rabbits. The resulting antisera reacted with the immunizing peptide, with denatured PIB on Western blots and, in addition, with native PIB in outer membranes of both the homologous and a heterologous strain. In the presence of human complement the sera were bactericidal for both the homologous and the heterologous strain. Thus synthetic peptides may be used to induce a protective polyclonal immune response against epitopes on gonococcal PI which are normally only weakly or non-immunogenic.

Identification and characterization of a Neisseria gonorrhoeae gene encoding a glycolipid-binding adhesin.

We recently identified a set of mammalian cell receptors for Neisseria gonorrhoeae that are glycolipids. These receptors, lactosylceramide [Gal(beta 1-4)Glc(beta 1-1)Cer], gangliotriosylceramide [GalNAc( beta 1-4)Gal(beta 1-4)Glc(beta 1-1)Cer], and gangliotetraosylceramide [Gal(beta 1-3)GalNAc(beta 1-4)Gal(beta 1-4)Glc(beta 1-1)Cer], were shown to be specifically bound by a gonococcal outer membrane protein distinct from pilin and protein II. Here we report the isolation of the gene encoding the gangliotetraosylceramide-binding adhesin from a N. gonorrhoeae MS11 gene bank in Escherichia coli. Transposon mutagenesis studies in E. coli indicate that the adhesion is a protein with a molecular mass of 36,000 Da. The gene encoding the 36-kDa protein is duplicated in MS11 since two transposon insertions were required to abolish expression of the gene in this bacterium. This protein is present on the surface of the gonococcus and is not associated with the pilus.

The construction and characterization of Neisseria gonorrhoeae lacking protein III in its outer membrane.

Protein III (PIII) is a highly conserved, antigenically stable gonococcal outer membrane protein that is closely associated with the major outer membrane protein, protein I (PI). We have previously reported the cloning of the PIII gene. This gene was inserted into the Eco RI site of the runaway plasmid pMOB45. The beta-lactamase (beta la) Bam HI restriction fragment from the gonococcal plasmid pFA3 was inserted at the Xba I site in the PIII gene. The plasmid construct was Hae III methylated and the PIII/beta la insert was excised with Eco RI and used to transform gonococcal strain F62. One beta la+, ampicillin-resistant transformant was isolated and designated 2D. A Western blot of 2D whole cell lysate was probed with affinity-purified polyclonal PIII antisera. No PIII reactivity was detected. Southern blot analysis was performed on F62 and 2D chromosomal DNA that were cut with Eco RI or Cla I. A PIII DNA probe hybridized with fragments 2.2 kb larger in strain 2D than strain F62. This corresponds to the size of the beta la insert. A beta la-specific probe hybridized with the same 2D restriction fragments as above, but did not react with any F62 fragments, confirming that homologous recombination had occurred. There were minimal phenotypic changes between 2D and its parent strain, F62. Chromosomal DNA from 2D was able to transform gonococcal strains F62, UU1, and Pgh 3-2, rendering these PIII-. 2D and other PIII- transformants can now be used to study the role of PIII in gonococcal physiology, metabolism, membrane structure, and pathogenesis. Moreover, we now have organisms from which we can purify gonococcal proteins without PIII contamination.

Presence of antibodies to the major anaerobically induced gonococcal outer membrane protein in sera from patients with gonococcal infections

Anaerobically grown Neisseria gonorrhoeae induces and represses the sythesis of outer membrane proteins. One of the anaerobically induced proteins, Pan 1, reacted strongly on Western blots with sera from patients with uncomplicated gonococcal infection, pelvic inflammatory disease, and disseminated gonococcal infection, but not with normal human serum. The pattern of reactivity of the sera against Pan 1 from several gonococcal strains suggested that the protein was antigenically heterogeneous, containing both common and unique epitopes. Staphylococus aureus V8 protease digestion of Pan 1 from four gonococcal strains revealed the presence of common peptides, with one strain also containing some unique peptides and lacking others. The class of the antibody reactive with gonococcal outer membrane antigens was examined; anti-Pan 1 antibody was found to be IgG or IgM, but not IgA. The IgM antibody present reacted predominantly with Pan 1. These data indicate that the Pan 1 protein is expressed in vivo and strongly suggest that N. gonorrhoeae can grow anaerobically in vivo.

Gonococci possessing only certain P.II outer membrane proteins interact with human neutrophils.

We investigated the role of the protein II (P.II) family of gonococcal outer membrane proteins in the interaction of seven single P.II variants of Neisseria gonorrhoeae FA1090 with human neutrophils in vitro. The abilities of nonpiliated gonococci to adhere to and be killed by neutrophils and to stimulate luminol-dependent chemiluminescence (CL) depended on the possession of at least one P.II. Gonococci lacking P.II (i.e., P.II-) adhered poorly to and were not killed by neutrophils and induced only minimal CL. Although most P.II-containing (i.e., P.II+) variants adhered to, stimulated, and were readily killed by neutrophils, one variant, containing P.IIa, possessed none of these characteristics; it acted just like a P.II- variant. No correlation was found between the colony opacity phenotype and the interaction of gonococci with neutrophils. Data from CL experiments suggest that the stimulatory effect of P.II was dominant over that of pili; i.e., piliated P.II+ gonococci were much more stimulatory than piliated P.II- gonococci. The results indicate that most but not all P.II proteins mediate, in part or in full, the interaction of N. gonorrhoeae with human neutrophils, including adherence, stimulation of the neutrophil respiratory burst, and phagocytic killing.

Lipopolysaccharide masking of gonococcal outer-membrane proteins modulates binding of bacterial cathepsin G to gonococci.

Human polymorphonuclear leucocyte (PMN) lysosomal cathepsin G exerts potent bactericidal action against Neisseria gonorrhoeae in vitro, independent of its serine esterase activity. The results presented demonstrate that (1) bactericidal, diisopropylfluorophosphate-treated cathepsin G binds in a specific and saturable manner to the surface of gonococci, (2) loss of carbohydrates in gonococcal LPS due to mutation increases total and specific binding of cathepsin G, and (3) at least three outer-membrane proteins (OMPs) (PIA, PIII, and a 45 kDa OMP) interact with cathepsin G. Taken together, the results suggest that gonococcal susceptibility to the lethal action of cathepsin G, and perhaps susceptibility of gonococci to oxygen-independent killing by PMNs, is controlled by LPS-masking of cathepsin-G-binding OMPs.

The Potential Protective Effect of Monoclonal Antibodies to Gonococcal Outer Membrane Protein IA

Hybrid cell lines were derived which produced monoclonal antibodies directed against gonococcal outer membrane protein IA. One antibody, SM101, recognized 24 P.IA-expressing strains out of 25 tested--the rest exhibited relatively less cross-reactivity. Competitive radioimmunoassays revealed that each antibody could effectively inhibit binding of the others, suggesting close proximity of the epitopes recognized. The antibodies were used in assays in vitro to investigate their potential efficacy in protection against gonococcal infection. In a cytotoxicity assay, the antibodies afforded some protection to epithelial cells challenged with gonococci. They were very effective at bactericidal killing in the presence of complement and, in addition, were opsonic for homologous and heterologous strains. The cross-reacting antibody, SM101, was one of the most effective in both assays. The results show that the conserved epitope on P.IA recognized by antibody SM101 is potentially an effective target on the gonococcal surface for immunoprophylaxis.