Localization of Protein I and H.8 Antigens

INFECTION AND IMMUNITY, May 1987, p. 1190-11970019-9567/87/051190-08$02.00/0Copyright ©D 1987, American Society for Microbiology

Probing the Surface of Neisseria gonorrhoeae: SimultaneousLocalization of Protein I and H.8 Antigens

EDWARD N. ROBINSON, JR.,1t ZELL A. McGEE,l* THOMAS M. BUCHANAN,2 MILAN S. BLAKE,3AND PENNY J. HITCHCOCK4

Center for Infectious Diseases, Diagnostic Microbiology, and Immunology, Division ofInfectious Diseases, Departmentof Internal Medicine, University of Utah School of Medicine, Salt Lake City, Utah 841321; Division ofInfectious

Diseases, University of Washington School of Medicine, Seattle, Washington 981952; Department of Microbiology,Rockefeller University, New York, New York 100213; National Institutes of Health, Rocky Mountain Laboratories,

Hamilton, Montana 598404

Received 20 August 1986/Accepted 4 February 1987

Gonococcal outer membrane protein I and the neisserial antigen H.8 are being investigated for inclusion ina gonococcal vaccine. To determine the distribution of immunoaccessible protein I and H.8 molecules on thesurface of viable gonococci and to approximate the accessibility of these antigens to vaccine-elicited antibodies,immunologic probes composed of protein I- and H.8-specific antibodies linked to gold spheres were developed.When whole gonococci were exposed to the protein I and H.8 immunologic probes and examined bytransmission electron microscopy, gold spheres clearly marked the surface of some of the gonococci, but not thesurface of other gonococci from the same culture. The immunologic accessibility of gonococcal protein I orneisserial H.8 varied among gonococci. This diversity may affect the efficacy of a vaccine composed of thesesurface antigens.

Efforts to prevent infections due to Neisseria gonorrhoeaehave focused on the production of a vaccine comprisingisolated, purified, or synthesized gonococcal surface com-ponents (5, 11, 33). The gonococcal macromolecules mostlikely to elicit protective antibodies are those that are surfaceexposed and therefore accessible to antibodies when theyare in their native state on viable gonococci (28). Twogonococcal surface components currently being evaluatedand characterized are gonococcal major outer membraneprotein I and the neisserial antigen H.8.

Interest in gonococcal major outer membrane protein I asa vaccine candidate stems from a number of observations:the low number of antigenic serotypes of protein I, thepropensity of gonococci possessing certain protein Iserotypes to cause disseminated or complicated gonococcalinfections, and the ability of protein I to function as an anionpore (porin) in artificial lipid bilayers or host cell membranes(7, 9, 21, 41). Native protein I appears to be surface exposed.It is accessible to proteolytic enzymes and radioactivetracers (2, 6, 12, 17, 36). Results of previous immunologicstudies of protein I in which either purified protein I orstructurally altered organisms were used have demonstratedthe uniformity of protein I within a given strain of N.gonorrhoeae and that the immunologic variation betweenstrains is limited to nine serotypes (32). Thus, a polyvalentprotein I vaccine potentially could elicit antibodies against abroad range of protein I serotypes, which might preventsevere gonococcal infections.The neisserial H.8 antigen is a recently detected surface

macromolecule that has been found in all strains of N.gonorrhoeae and N. meningitidis tested (8, 16). Although the

* Corresponding author.t Present address: Division of Infectious Diseases, Department of

Medicine, School of Medicine, University of Louisville, Louisville,KY 40292.

role of H.8 in the pathogenic process has yet to be eluci-dated, it has been demonstrated that in humans antibody iselicited to H.8 following naturally acquired systemic gono-coccal and meningococcal infections (3). Like protein I, H.8appears to be antigenically homogeneous within a givenstrain, irrespective of piliation or opacity phenotype. Al-though there is variation of H.8 among strains based on itselectrophoretic mobility on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), all strains haveone or more common surface-exposed epitopes (16). Resultsof recent studies have also shown that monoclonal antibod-ies directed toward H.8 are bactericidal in strains that aresusceptible to killing by normal human serum (P. J. Hitch-cock and K. Joiner, manuscript in preparation). A vaccinecomposed of the H.8 antigen, if effective, theoretically mightabrogate not only gonococcal but also meningococcal infec-tions.

Technical advances have allowed the visualization ofantibody binding to the surface of whole bacteria through theuse of gold sphere immunologic probes (30). Thus, theimmunologic accessibility of candidate vaccine antigens canbe determined by using whole bacteria. To obtain directevidence that specific native gonococcal macromolecules areaccessible to antibody, we constructed immunologic probescomposed of specific antibodies linked to gold spheres thatare visible in transmission electron micrographs (30). Usingthese immunologic probes, we detected major differences inthe accessibility to antibody of protein I and H.8 on thesurface of intact gonococcal cells.

MATERIALS AND METHODS

Microorganisms. The microorganisms used in this studyincluded two individual passages of N. gonorrhoeae MEL.The first passage (henceforth designated MEL-I) that waspropagated and examined demonstrated nonpiliated (P-) and

1190

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PROBING THE SURFACE OF N. GONORRHOEAE 1191

opaque (Op) colony morphology (18, 19, 35). The uniformand consistent immunologic accessibility of protein I on thesurface of MEL-I organisms has been reported previously(29). The second passage (henceforth designated MEL-II)that was propagated and examined demonstrated heavilypiliated (P++) and Op colony morphology. P- organismswere derived from MEL-II by single-colony isolation andpassage. P- progeny of MEL-II behaved similarly to P+MEL-II progeny in their marking by anti-protein I and H.8gold probes. Other microorganisms used in this study in-cluded N. gonorrhoeae R1OT (P+ and transparent [Tr]colony phenotypes); N gonorrhoeae MS11 (P+, Tr); and themore recent clinical isolates N. gonorrhoeae P-21CS (P+,Tr), N. gonorrhoeae P-35Cx (P+, Tr), and N. gonorrhoeaeUU1018 (P+, very opaque [Op++]). Organisms were frozenin defibrinated sheep blood at -70°C. On the day prior touse, samples were thawed and cultivated on an agar mediumconsisting of GC medium base (Difco Laboratories, Detroit,Mich.) containing 2% (vol/vol) IsoVitaleX (BBL Microbiol-ogy Systems, Cockesville, Md.). The organisms were incu-bated for 18 to 20 h at 37°C in 2% CO2 in air before they wereharvested for use in immunoelectron microscopic studies.

Buffers and media. Phosphate-buffered saline and stabiliz-ing buffer were prepared as described previously (30).Media for the dilution of albumin and the suspension of

microorganisms consisted of 0.05 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; SigmaChemical Co., St. Louis, Mo.)-buffered Eagle minimal es-sential medium (MEM) (pH 7.45) containing Earle salts andL-glutamine (GIBCO Laboratories, Grand Island, N.Y.;HEPES-MEM).

Antibody. Polyclonal rabbit antisera to purified R1OTprotein I (serogroup II, serotype 5 [4, 35]) antibody wasprepared as described previously (29).

Protein I-specific monoclonal antibody (Seattle #81) wasprepared, purified, and characterized as described previ-ously (25, 38). This antibody recognizes protein I ofserogroups II and III (protein IB).

H.8-specific monoclonal antibody 10 was prepared andcharacterized as described previously (16).SDS-PAGE. Whole-cell lysates of gonococci were exam-

ined by the electrophoresis method described by Laemmli(20), with the use of modifications described previously (14).Low-molecular-weight markers (Bio-Rad Laboratories,Richmond, Calif.) were used in some gels. The proteinstandards (and their molecular weights) included phosphor-ylase (94,000), bovine serum albumin (68,000), ovalbumin(43,000), carbonic anhydrase (30,000), soybean trypsin in-hibitor (21,000), and lysozyme (14,300). Some gels werestained with 0.2% (wt/vol) Coomassie brilliant blue R250(Fisher Scientific Co., Fairlawn, N.J.) in 25% (vol/vol)isopropanol-75% (vol/vol) acetic acid. Silver staining of gelswas performed as described previously (15).

Immunoprecipitation. Immunoprecipitation with monoclo-nal anti-protein I antibody was performed by the methoddescribed by Swanson et al. (37).

Electroblotting. Electroblotting was performed with thebuffer system described by Towbin et al. (39) and Renart etal. (27), as modified by Barbour et al. (1). Electrophoretictransfer conditions were 15 V and <100 mA for 12 h at 18°C(16). After incubation with antisera, '25I-radiolabeled proteinA was used to identify antibody (immunoglobulin G)-antigencomplexes (also described by Barbour et al. [1]).

Preparation of gold sphere-protein A-antibody conjugates.Gold sphere-protein A-antibody conjugates were manufac-tured and purified as described previously in detail (30).

When reduced to its elemental form with heat and mild acid,gold chloride forms electron-dense spheres (10, 34). The sizeof the spheres depends on the initial concentration of acid,with the result that spheres of different and distinguishablesizes can be produced. The addition of staphylococcalprotein A to the suspension of gold spheres coats the surfaceof the spheres because of electrostatic forces (31). Theresultant gold sphere-protein A complexes were purified andconcentrated by using repetitive ultracentrifugations. Thecomplexes were then stored at 4°C. On the day of eachexperiment, 100-,u quantitites of the complexes wereultracentrifuged at 50,000 x g for 2 min (Airfuge; BeckmanInstruments, Inc., Fullerton, Calif.). The resultant pelletswere suspended with 100 ,ul of protein A-column-purifiedprotein I-specific mouse monoclonal antibody, rabbit poly-clonal strain RlOT-protein I antibody, or supernatant fluidcontaining H.8-specific mouse monoclonal antibody. Eachof these combinations was diluted 1:100 in a stabilizingbuffer. After 30 min of incubation, the gold sphere-antibodyconjugates were purified by using repetitive washings andultracentrifugations.

Preparation of electron microscopic specimens. N. gonor-rhoeae organisms were transferred from the surface of agarplates and suspended in a drop of HEPES-MEM. Carbon-stabilized, Formvar-coated copper electron microscopicgrids were floated on the suspensions of gonococci (22). Thegrids were transferred to drops of reagents in the followingsequence (the duration of incubation at room temperature isgiven in parentheses): (i) HEPES-MEM (brief wash), (ii) 1%bovine serum albumin in HEPES-MEM (15 min), (iii) 1%bovine serum albumin in stabilizing buffer (brief wash), (iv)gold sphere-protein A-antibody conjugates (30 min), (v)successive drops of phosphate-buffered saline and aggitatedin a beaker containing phosphate-buffered saline (wash), and(vi) 1% phosphotungstic acid (1 min). When double-labelingexperiments were performed, a second 30-min incubationwith the larger sized gold probes were included after step iv.Examination of electron microscopic specimens. The grids

were allowed to air dry and then were examined by trans-mission electron microscopy (Siemens Elmiskop). All pho-tographs were processed with photographic paper(Polycontrast rapid II RC; Eastman Kodak Co., Rochester,N.Y.).

RESULTS

Marking of native protein I with gold sphere-protein A-antibody conjugates. The immunologic accessibility of pro-tein I in its native state on the surface of intact gonococcalorganisms was studied by using specific probes consisting ofmonoclonal anti-protein I antibodies linked to electron-dense gold spheres (25, 38) (Fig. 1). Unfixed N. gonorrhoeaeMEL-I and MEL-II organisms were removed from thesurface of the agar plates, suspended in HEPES-MEM,exposed to the anti-protein I immunologic probes, and thennegatively stained. All specimens were then examined bytransmission electron microscopy.

In contrast to the previously reported uniform and heavymarking by the anti-protein I probe of virtually all organismsfrom MEL-I cultures (29), in cultures of MEL-II containingmorphologically homogeneous colonies there weregonococci that were marked heavily with the anti-protein Iimmunologic probes and gonococci that were marked mini-mally or not at all (Fig. 2). For example, in experimentsperformed on 5 separate days, a mean of 40 of 100 cells(range, 19 to 71 of 100 cells) counted on each of 12 grids

VOL. 55, 1987

1192 ROBINSON ET AL.

failed to be marked with the anti-protein I probes. Althoughthere was variability in the percentage of MEL-II gonococcithat failed to be marked with the anti-protein I probe fromexperiment to experiment, in many experiments more thanhalf of the cells were not marked with the probes. In allexperiments there were MEL-II gonococci that were notmarked with the protein I immunologic probes. Althoughthere were rare exceptions, gonococcal daughter pairsshowed similar markings by the anti-protein I probes.The same pattern of protein I marking was observed for an

intact organism and its associated outer membrane blebs;i.e., cells that lacked accessible protein I were associatedwith outer membrane blebs that lacked accessible protein I.To ensure that the colonies that were examined originated

from one diplococcus and therefore comprised isogenicprogeny, a suspension of gonococci of strain MEL-II waspassed through a membrane filter (pore size, 0.8 ,um;

1 2 3 1 2 3

A

P.I

MW 1

D

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P.N 'w

B

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FIG. 1. The specificity of monoclonal antibodies to gonococcalprotein I (P.I) and neisserial H.8 antigen. Displayed in 12.5%SDS-polyacrylamide gels, the Coomassie blue (A) and silver-stained(B) profiles of three gonococcal strains are shown. In all gels, lanes1, 2, and 3 contain R1OT, MEL-II, and MS11, respectively. The Mrof the protein I, indicated by a dot, is larger in strain MS11. All otherstrains used in this study had Mrs of protein I identical to those ofR1OT and MEL-II by SDS-PAGE (data not shown). (C) Autoradio-gram of a Western blot with monoclonal anti-H.8 antibody (16). (D)Autoradiogram of paired lanes containing 1251-surface-labeledgonococci (left) and radioimmunoprecipitated antigens with theprotein I-specific monoclonal antibody (36). In strains R1OT andMEL-II, the monoclonal antibody precipitated large amounts ofprotein I (P.1) and lesser amounts of protein III (P.III) (see below).The reactivity with strain MS11 was considerably less in thisexperiment, and the protein I antigen was more apparent with a

longer exposure of the film. However, there was some variability inthe amount of protein I that was precipitated with the monoclonalantibody; in one instance, the reactivity with strain MS11 wasequivalent to that with R1OT and MEL-II. Note that Swanson andco-workers (37) demonstrated coprecipitation of protein I andprotein III outer membrane proteins with a protein III-specificmonoclonal antibody. MW, Molecular weight.

Nalgene Labware Div., Nalge/Sybron Corp., Rochester,N.Y.), and the effluent was cultured. The resultant colonieswere selected and serially passaged. Gonococci from thesecultures also exhibited heterogeneity of marking; i.e., theycontained cells that were or were not marked with theimmunologic probe.

Six other gonococcal strains representing both laboratorystrains (RiOT and MS11) and more recent clinical isolates(P-21CS, P-35Cx, and UU1018) were passaged to obtainuniform colony phenotypes. Gonococci from each of thesestrains were examined for the immunoaccessibility of pro-tein I. Within these homogeneous colonies were cells thatwere and were not marked with the protein I probes to thedegree seen in MEL-II. Among the strains examined, MEL-Iwas unique in the uniform accessibility of protein I to theanti-protein I gold probes. There was no correlation betweenpiliation or opacity phenotype to the marking or failure ofmarking of the component gonococci. The inaccessibility ofprotein I of some organisms to antibody was not limited to asingle gonococcal strain, but appeared to be a commonphenomenon.Organisms that failed to be marked with gold spheres were

also evident when polyclonal rabbit antibody elicited bypurified gonococcal protein I was used as the immunologicreagent (4, 29). The surface differences that were responsiblefor the inaccessibility of protein I to antibody were detect-able by both monoclonal and polyclonal reagents and there-fore do not appear to be the result of minor antigenicdifferences in protein I or structural differences detected bymonoclonal antibodies alone.Marking of H.8 with gold sphere-protein A-antibody conju-

gates. To determine the accessibility of H.8 to antibody,gonococcal organisms of the MEL-I, MEL-II, R1OT, andMS11 strains were exposed to gold spheres that werecomplexed to H.8-specific monoclonal antibody and wereexamined with the electron microscope. In these strains, 5 to20% of the gonococcal organisms from a given culture weremarked with the H.8 probe. Those that were marked weremarked heavily. In contrast to the striking differences seenin the immunologic accessibility of protein I in organismsfrom the two passages of the MEL strain, no differences inthe percentage of cells that were marked by the anti-H.8 goldprobes were evident in MEL-I and MEL-II. Unlike proteinI, outer membrane blebs elaborated by organisms that werenot marked with the H.8 probe occasionally containedimmunoaccessible H.8.

Simultaneous marking of protein I and H.8 with goldsphere-protein A-antibody conjugates. To assess the relation-ship, if any, between H.8 and protein I on the surface of thecells, gonococci from cultures of the previously examinedstrains (RiOT, MS11, MEL-TI, P-21CS, P-35Cx, andUU1018) were exposed in sequence to small gold spheresthat were complexed to anti-protein I antibody and largegold spheres that were complexed to anti-H.8 antibody. Ineach culture examined there were cells that were markedpredominantly with anti-protein I probes; cells that weremarked predominantly with anti-H.8 probes; cells that weremarked heavily with both probes; and, most importantly,cells that were not marked with either immunologic probe(Fig. 3 and 4). For example, in the MEL-II strain, of 100 cellsthat were examined randomly, 29 cells were marked solelywith the anti-protein I probes, 3 cells were marked with theanti-H.8 probe alone, 12 cells were marked with the bothprobes, and 56 cells were unmarked. The number of cellsthat were labeled by either probe during double-labelingexperiments was similar to the number labeled during exper-

INFECT. IMMUN.


FIG. 2. Uneven distribution of accessible outer membrane protein I on the surface of MEL-II organisms of N. gonorrhoeae, as detectedby gold spheres complexed with a monoclonal antibody against gonococcal protein I. Note the heavy attachment of gold sphere-antibodycomplexes to one gonococcal organism but the sparse marking of the adjacent organism. Over half the organisms from this culture failed tobe marked with the anti-protein I immunologic probe. These two cells appear to be adjacent to one another and are not true daughter cells.Individual cells within diplococcal pairs rarely showed such dramatic differences in protein I accessibility. Negative stain was used.Magnification, x51,160.

iments in which a single probe was used, indicating that theprobes themselves do not interfere with each other inbinding to whole organisms. Thus, exposure of protein I orH.8 on the surface of whole gonococci appears to occurindependently. The presence of immunoaccessible protein Idid not preclude or predict the presence of immunoaccess-ible H.8.

DISCUSSION

By using gold sphere immunologic probes, we determinedthat the surface accessibility of protein I and H.8 varieswithin otherwise homogeneous populations of gonococci.Within the strains of gonococci studied, there were cells thathad either antigen, both antigens, or neither antigen exposedon their surfaces. Three possible explanations for the dimin-ished or absent marking of individual gonococcal cells by theprotein I and H.8 immunologic probes are absence, varia-tion, or sequestration of the antigens.

First, some gonococcal cells may totally lack protein I orH.8 on their surfaces. Mutant gram-negative organisms thatlack porin protein have been described previously (13, 23,24). However, porin proteins are thought to serve an impor-tant cellular function by chaneling low-molecular-weightsubstances across the hydrophobic lipid outer membrane.Although porinless organisms would not be expected to existin large numbers in any given population of gonococci andalthough diminished protein I has not been detected inCoomassie brilliant blue-stained gels containing lysates ofthe organisms used in this study (data not shown), we cannotexclude such a possibility.

Results of previous studies by enzyme-linked immunosor-bent, slide agglutination, and immunofluorescence assays todetect H.8 have indicated that H.8 is surface exposedthroughout populations of gonococcal organisms (8, 16).More recently, some variation in the immunoaccessibilityamong strains has been shown by immunofluorescence andslide agglutination assays (P. J. Hitchcock, J. Boslego,

VOL. 55, 1987


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FIG. 3. N. gonorrhoeae MEL-II exposed simultaneously to two immunologic probes. Small gold spheres were complexed to monoclonalanti-protein I antibody. Larger gold speres were complexed to monoclonal anti-H.8 antibody. Note the attachment of small gold spheres(anti-protein I) predominantly to the surface of one organism (left) and the attachment of large gold spheres (anti-H.8) to the surface of thediplococcus (right). Also note the presence of large gold spheres (anti-H.8) in the outer membrane bleb elaborated by one organism (left)without appreciable large gold on the rest of its surface. Negative stain was used. Magnification, x45,860.

K. A. Joiner, and E. N. Robinson, Jr., Proceedings of theFifth International Pathogenic Neisseria Conference, inpress.) Thus, the absence of accessible H.8 on gonococcalcells within a given strain examined with gold immunologicprobes appears least likely to be due to an absence of themolecule on the surface of the cells. The unexpected de-crease in the percentage of gonococci marked by the anti-H.8 gold probes when compared with gonococci marked byimmunofluorescence is unexplained at this time. Steric hin-drance of antigen-antibody binding by the gold sphere maylower the percentage of cells that are labeled by the anti-H.8probes. However, attempts to diminish that interference (byfirst exposing the bacteria to the H.8 antibody, followed byexposing them to the gold spheres conjugated to anti-antibody) did not significantly increase the percentage ofcells that were labeled. Interestingly, anti-H.8 probes wereoccasionally present on the surface of the outer membraneblebs, but not on the surface of the cell from which theypresumably arose (Fig. 3). Although we cannot explain thediscrepancies between the results generated by the variousmethods, consistent and reproducible results were generatedby all of these methods. Results of biologic assays such asopsonization, complement binding, and bactericidal activitywith H.8-specific antibodies may be helpful in determiningthe significance of these apparent contradictions.

Second, antigenic variability in the surface-exposed por-tion of protein I or H.8 would explain the lack of antibodybinding to some of the gonococcal cells. Antigenic variabilityof protein I within a given strain of gonococcus has not beendescribed. However, in the immunologic tests that wereused to examine such variability (i.e., enzyme-linked immu-nosorbent assay or the coagglutination test), large popula-tions of cells, and not individual gonococci, were examined.

Antigenic changes in protein I from cell to cell might remainundetected by these tests. Antigenic differences in H.8among strains or within a strain have not been described(16).

Finally, there may be an adjacent macromolecule on thesurface of some gonococci that sterically hinders the acces-sibility of protein I or H.8 to gold immunologic probes.There is variation with respect to the accessibility of proteinI to the anti-protein I gold probe, as evidence by the uniformmarking of organisms from the culture of MEL-I (29) versusthe diversity of labeling seen with the other strains andMEL-II. So far, as candidate molecules that are responsiblefor the sequestration of these antigens, protein I has beendemonstrated to be closely associated with protein III (Fig.1) and lipopolysaccharide (LPS) (14) on the surface ofgonococcal cells. Structural changes in either of these adja-cent molecules might result in the shielding of protein I fromantibodies. Changes in the length of the LPS side chainshave been observed and correlated with differences in anti-body binding to the major outer membrane protein of N.meningitidis and to the porin proteins of other gram-negativebacteria (26, 40). Results of these previous studies promptedus to examine the LPS phenotype of MEL-I and MEL-II.Whole-cell lysates were prepared with and without protein-ase K digestion and were subjected to SDS-PAGE (14, 20).The gels were silver stained by a procedure that is selectivefor LPS (15). Results of these preliminary experimentsinclude marked differences, without overlap, in the electro-phoretic mobility of proteinase K-digested lysates; the re-sults were consistent with differing LPS phenotypes inMEL-I compared with MEL-I1. However, there was noevidence of LPS heterogeneity among MEL-II organisms.We would expect that if LPS heterogeneity was responsible

INFECT. IMMUN.

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FIG. 4. N. gonorrhoeae MEL-II exposed simultaneously to two immunologic probes. Small gold spheres were complexed to proteinI-specific monoclonal antibody. Larger gold spheres were complexed to H.8-specific monoclonal antibody. Note the attachment of small goldspheres (anti-protein I) predominantly to the surface of one diplococcus (upper left), the attachment of both large (anti-H.8) and small(anti-protein I) gold spheres to the surface of another diplococcus (right), and the absence of attachment above the background of either sizedgold sphere to other organisms (upper and lower middle). The presence of immunoaccessible H.8 did not predict or exclude the presence ofimmunoaccessible protein I. Negative stain was used. Magnification, x42,640.

for the immunoinaccessibility of .40% of the organisms, itwould have been apparent in the SDS-PAGE results. Inaddition, the LPS phenotypes of the other strains examinedwere not heterogeneous. The significance of the LPS differ-ences in MEL-I and MEL-II is unknown.

Antibodies elicited to purified antigens may not bind thoseantigens in their native configuration on the surface of intactgonococcal organisms. The gold immunologic probes pro-vided information regarding the accessibility of antigen toantibody and detected a subpopulation of microorganismsthat were, for unknown reasons, structurally or antigenicallydifferent from the rest. Immunologic probes containing an-tibodies against protein I or H.8 did not bind to allgonococci. Such information is crucial to the construction ofan effective gonococcal vaccine. Unless it is hypothesizedthat there are differences in the virulence of individualgonococci related to differences in the surface accessibilityof these antigens, the heretofore unsuspected microheter-

ogeneity with respect to protein I and H.8 antigens suggeststhat multiple antigens may have to be included in an effectivegonococcal vaccine.

ACKNOWLEDGMENTSN. gonorrhoeae P-21CS and P-35Cx were the generous gifts of

Deborah Draper, University of California, San Francisco. Weacknowledge Christopher M. Clemens, Janis K. Larson, and TeresaBrown for excellent technical assistance and David Low and JohnSwanson for critical review of the manuscript. Z.A.M. is grateful forthe excellent help of Nancy Inaba, Jan Aprin, and Holly Shurtleffthat made his participation in these studies possible.

This study was supported by Public Health Service research grantAI-20265 from the National Institute of Allergy and InfectiousDiseases (to E.N.R. and Z.A.M.) and by a grant from InstitutMerieux, Lyons, France (T.M.B.). The electron microscope facili-ties of the University of Utah School of Medicine are under thecapable supervision of Elizabeth M. Hammond and are supportedby a grant from the R. J. Reynolds Foundation.

VOL. 55, 1987


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INFECT. IMMUN.


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VOL. 55, 1987

Localization of Protein I and H.8 Antigens

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Transcript of Localization of Protein I and H.8 Antigens