Vol. 35, No. 3INFECTION AND IMMUNITY, Mar. 1982, p. 800-8080019-9567/82/030800-09$02.00/0

Evaluation of Streptococcus pneumoniae Type XIV Opsoninsby Phagocytosis-Associated Chemiluminescence and a

Bactericidal AssaySUSAN E. GARDNER,l t* DONALD C. ANDERSON,1 BETTE J. WEBB,1 ANN E. STITZEL,2

MORVEN S. EDWARDS,1 ROGER E. SPITZER,2 AND CAROL J. BAKER1Myers-Black Section of Infectious Diseases of the Department of Pediatrics, Baylor College of Medicine,Houston, Texas 77030,1 and Department of Pediatrics, Upstate Medical Center, State University ofNew

York, Syracuse, New York 132102

Received 31 August 1981/Accepted 20 October 1981

The relative roles of serum factors required for opsonization of type XIVStreptococcus pneumoniae were investigated by means of luminol-enhancedchemiluminescence (CL), bactericidal, and immunofluorescence assays employ-ing adult sera containing high (>1,000 ng of antibody nitrogen per ml) or low(<200 ng of antibody nitrogen per ml) antibody concentrations as determined byradioimmunoassay. Specific antibody concentration correlated directly with bothtotal and heat-labile CL activity (P < 0.005) and with the bactericidal index (P <0.05) at a serum concentration of 10%. The importance of specific antibody as anopsonin was confirmed by the abolition of CL activity and immunoglobulinimmunofluorescence observed after absorption of heated sera with type XIVpneumococcal cells and by the dose response in CL and bactericidal activityobserved with the addition of immunoglobulin G to hypogammaglobulinemicserum. A role for the classical complement pathway in opsonization was indicatedby significantly greater CL integrals for high-antibody sera than for low-antibodysera depleted of factor D and by the bactericidal activity noted for untreated, butnot magnesium ethylene glycol-bis(3-aminoethyl ether)-N,N-tetraacetic acid-chelated low-antibody sera. The alternative pathway contributed more than halfof the CL activity of both high- and low-antibody sera. However, after magnesiumethylene glycol-bis(P-aminoethyl ether)-N,N-tetraacetic acid chelation, only serawith high antibody concentrations or agammaglobulinemic serum reconstitutedwith immunoglobulin G with high specific antibody levels supported significantbactericidal activity. Therefore, type-specific antibody and complement promoteopsonization of type XIV S. pneumoniae, and this may occur via either comple-ment pathway. These results suggest that CL is a suitable tool to delineate serumfactors and their contribution to opsonization, but results must be related to otherfunctional assays.

The virulence of Streptococcus pneumoniaehas been attributed traditionally to its polysac-charide capsule, which enables the organism toresist phagocytosis by host cells (38). One hostdefense which abrogates this capsule-mediatedvirulence is opsonization of the organism byserum constituents (immunoglobulin and com-plement) which render it suitable for ingestionand intracellular killing by phagocytes (38). Defi-ciencies of critical serum factors appear to ac-count for the increased attack rate of pneumo-coccal infection among certain patients (e.g.,those with sickle cell disease, childhood nephro-sis, or asplenia). In vitro methods which allow

t Reprint request address: Texas Children's Hospital, Pedi-atric Infections Diseases, Houston, TX 77030.

definition of the functional role of opsonins ofpneumococci in sera from normal and selectcompromised hosts are tools by which thepathogenesis of human infection may be com-prehended more fully.Luminol-enhanced chemiluminescence (CL)

methods for the detection of serum opsoninshave been reported for several microorganisms(3, 21, 25). We developed a CL assay to evaluatethe opsonic requirements of normal human serafor type XIV S. pneumoniae and to correlateresults with a bactericidal assay employing hu-man polymorphonuclear leukocytes (PMNL)and endogenous complement. It is believed thatthese methods may be useful for the study of theinteraction between this organism and sera frompatients with epidemiologically proved en-

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S. PNEUMONIAE TYPE XIV OPSONINS 801

hanced susceptibility to this common childhoodpathogen (4, 9, 11, 18, 20).

MATERIALS AND METHODS

Isolation of PMNL for CL assays. Venous bloodsamples collected from a single adult volunteer weresedimented in 0.1 ml of 6% dextran in 0.87% NaCl per10 ml of blood. Leukocyte-rich plasma was centri-fuged at 800 x g for 5 min, and leukocytes wereresuspended in Dulbecco phosphate-buffered saline(DPBS), pH 7.4, containing 0.2% dextrose (GIBCOLaboratories, Grand Island, N.Y.). Cells were layeredover a solution of Ficoll-Hypaque containing 10 partsof 33.9% Hypaque (Winthrop Laboratories, N.Y.) and24 parts of 9o Ficoll (Sigma Chemical Co., St. Louis,Mo.), centrifuged at 800 x g for 30 min, and washed inDPBS. Erythrocyte contamination was eliminated byhypotonic lysis. Purified PMNL suspensions wereadjusted to a final concentration of 4 x 106 PMNL perml of DPBS.

Preparation of leukocytes for bactericidal assay. Leu-kocyte-rich plasma was obtained by sedimenting pe-ripheral venous blood in a 6% dextran-citrate buffersolution (10). The leukocytes were removed by centrif-ugation and washed twice in minimal essential medium(MEM; Microbiological Associates, Walkersville,Md.) containing 3% bovine serum albumin (BSA).Lysis of erythrocytes was achieved by treatment with0.84% ammonium chloride for 3 min. Cells werewashed twice in MEM with 3% BSA and adjusted to afinal concentration of approximately 1.5 x 107 PMNLper ml.

Fractionation of IgG. A purified immunoglobulin G(IgG) fraction was prepared from human serum con-taining a high concentration (1,185 ng of antibodynitrogen [Ab N] per ml) of antibody to the type XIVpolysaccharide antigen of S. pneumoniae by octanoicacid precipitation followed by dialysis and chromatog-raphy on DEAE-cellulose (24, 35). Column fractionscontaining IgG were detected by monospecific goatantiserum to human IgG prepared in our laboratory,pooled, and concentrated. Purity of the IgG fractionwas assessed employing goat antibody to IgG, IgM,IgA, and properdin by double diffusion in agar and byimmunoelectrophoresis. The final concentration ofantibody to type XIV polysaccharide in this prepara-tion was 9,831 ng of Ab N per ml as determined by aradioimmunoassay (32).

Preparation of factor D and RD serum. Samples (20ml) of human sera were passed over Sephadex G-75Superfine. The first two peaks, constituting factor D-deficient (RD) serum, were combined and concentrat-ed to the original volume. A third peak containingfactor D was also concentrated to the original volume(22, 27). Specificity of the RD sera was tested byreacting the depleted sera with cobra venom factor at37°C to determine the extent of C3-C9 consumption.The specificity of the factor D preparation was shownby its ability to allow cobra venom factor activation ofRD sera, but not of factor B-deficient serum.

Sera. Whole blood from adult volunteers was al-lowed to clot at 25°C for 20 min. Sera were separatedby centrifugation and frozen at -70°C. Samples werethawed immediately before testing in opsonic or bacte-ricidal assays. Complement activity in selected serawas inactivated by heating to 56°C for 30 min.

Sera from 10 adult volunteers were selected torepresent a range in concentrations of specific anti-body to the type XIV pneumococcal polysaccharide.Determination of antibody concentrations in sera waskindly performed by Gerald Schiffman (State Univer-sity of New York Downstate Medical Center, Brook-lyn, N.Y.) (32). Six of these sera had low (<200 ng ofAb N per ml) and four had high (>1,000 ng ofAb N perml) concentrations of type-specific antibody. Thesesera were tested in both CL and bactericidal assays.The classical complement pathway of selected sera

was inhibited by chelating test sera at 37°C for 5 minwith 3 mM MgCl2 and 8 mM ethylene glycol-bis(13-aminoethyl ether)-N,N-tetraacetic acid (EGTA) (Sig-ma). Preliminary experiments indicated that a 3 mMconcentration of MgCl2 allowed maximum alternativecomplement pathway (AP) activity as determined bythe CL assay. AP activity was assessed in the samemanner for RD sera, except that factor D first wasadded to restore normal serum concentrations. Thecontrol sera for chelation experiments were incubatedin isotonic (pH 7.5) 0.1% gelatin Veronal buffer madeto 0.15 mM Ca2" and 0.5 mM Mg2" (GVB2+; CordisLaboratories, Miami, Fla.).Serum from an adult with common variable immu-

nodeficiency was employed as a complement source inCL experiments requiring exogenous complement.This serum contained normal 50%o hemolytic comple-ment activity, a total IgG concentration of 156 mg/dl,and no detectable antibody to pneumococcal type XIVpolysaccharide. Agammaglobulinemic serum from achild with severe combined immunodeficiency (IgG,14 mg/dl; specific antibody, 0 ng of Ab N per ml) wasemployed as a complement source in bactericidalassays requiring exogenous complement.Preparation of bacteria. Type XIV S. pneumoniae

Robles strain, isolated from the blood of a child withminimal lesion nephrotic syndrome, and type III strain3065, a blood isolate received from Barry Gray (Uni-versity of Alabama, Birmingham, Ala.) were em-ployed as test organisms. The strains were stored insamples frozen at -70°C in Todd-Hewitt broth con-taining 20%o glycerol and were thawed and inoculatedonto blood agar plates before each assay. After over-night incubation at 34°C, organisms were inoculatedinto 30 ml of Todd-Hewitt broth. For CL assays, thebacterial suspensions were incubated at 34°C until anoptical density of 0.21 at 540 nm (Spectronic 20;Bausch & Lomb, Inc., Rochester, N.Y.) was achieved(log phase of growth). Bacterial suspensions werecentrifuged at 12,000 x g for 10 min and adjusted to afinal concentration of 1 x 108 to 2 x 108 colony-forming units (CFU) per ml. For bactericidal assays,bacteria were grown to an optical density of 0.15(Coleman Junior Spectrophotometer, model 6C; Cole-man Instruments, Maywood, Ill.). Bacterial suspen-sions then were centrifuged, and the pellet was sus-pended in MEM with 3% BSA to achieve a finalconcentration of 3 x 107 CFU/ml.

Absorption procedure. Pneumococcal strains, typeXIV Robles, or type III 3065, were inoculated intoTodd-Hewitt broth and incubated at 37°C until anoptical density of 0.3 to 0.4 at 540 nm was achieved(approximately 109 CFU/ml). The broth suspensionswere centrifuged at 12,000 x g for 10 min, and thepellets were added to heat-inactivated sera containinga high concentration of specific antibody in a ratio of

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1:5 (vol/vol). These were placed at 37°C in a shakingwater bath for 30 min, followed by an ice bath withintermittent mixing for 1 h. Bacteria first were re-moved by centrifugation (12,000 x g for 10 min) andthen by filtration through a 0.22-,um filter unit (Milli-pore Corp., Bedford, Mass.).Luminol-enhanced CL assay. Suspensions of type

XIV S. pneumoniae (approximately 4 x 10' CFU)were centrifuged at 1,600 x g for 10 min, and superna-tants were removed. Pelleted organisms were mixedwith serum (0.5 ml), opsonized for 45 min at 37°C in ashaking water bath, centrifuged, washed twice, andsuspended in 0.2 ml of DPBS. All opsonic mixtureswere prepared in duplicate.

Phagocytic reaction mixtures containing opsonizedbacteria (0.5 ml), 5 x 10' M luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) (0.05 ml), 1% humanserum albumin in DPBS (0.05 ml), and PMNL (0.25ml) were prepared in 1.5-ml conical polyethylenemicrovials in a partially darkened room. The finalreaction mixture contained 1 x 106 PMNL, 4 x 107CFU of bacteria per ml, 0.5 mg of human serumalbumin, and 5 x 10-8 M luminol (bacterium/PMNLratio, approximately 40:1).CL was measured in a liquid scintillation counter

(model C 2425 Tri-Carb; Packard Instruments, Hou-ston, Tex.) in the out-of-coincidence mode. CL evolu-tion was quantitated over 0.4-min intervals during fivesequential 15-min cycles. Baseline CL counts andresting CL values were determined as previouslydescribed (3) and subtracted from phagocytic CLvalues. Values for duplicate vials were within 10% ofeach other. Data were reported as timed intervalrecordings of CL intensity expressed in counts perminute x 10-3. The area under the curve or integral ofthe CL response between 0 and 60 min after theaddition of PMNL to the reaction mixture was deter-mined by planimetry (summation of trapezoids). Theinitial slope from 0 to 30 min was determined by usingthe method of least squares. CL activities (initial slopeand integral) of test sera were compared statisticallyby using Student's t-test.

Visual determination of phagocytosis. To provideadditional evidence that CL evolution related tophagocytosis, samples from reaction mixtures wereexamined by light microscopy. At peak CL activity byhigh-antibody serum, reaction mixtures were removedfrom the scintillation counter and centrifuged at 400 xg (5 min, 4°C); cell pellets were washed twice withDPBS. Samples of each mixture were placed on micro-scope slides, air dried, and fixed with 100% methanolfor 10 min. Slides were stained with Giemsa reagent(Fisher Scientific Co., Fair Lawn, N.J.). The percent-age of cells containing ingested bacteria and the num-ber of organisms ingested by each PMNL were record-ed for _50 cells. Phagocytic indexes were calculatedas previously described (3).

Indirect immunofluorescence (IF) procedure. Fluo-rescein isothiocyanate-conjugated anti-human IgG,IgA, IgM, and C3 (Meloy, Springfield, Va.) wereadded to pellets of washed opsonized or unopsonizedbacteria. Appropriate dilutions of conjugated antiserain 0.87% NaCl ranged from 1:7 to 1:9. Mixtures ofconjugates (0.1 ml) and organisms were incubated at4°C for 30 min and washed twice with DPBS. Organ-isms then were suspended in a solution of 90%o glycer-ine in 0.87% NaCl (0.1 ml) and examined by fluores-

cent microscopy. The intensity of fluorescence wasgraded from 0 to 4+ by two independent observers andrecorded as the mean of values.

Bactericidal assay. The opsonophagocytic assay de-scribed by Edwards et al. (10) was modified as follows.Reaction mixtures contained bacteria (0.1 ml, approxi-mately 3 x 10' CFU/ml), MEM with 3% BSA (0.1 ml),leukocytes (0.1 ml, approximately 106 PMNL), andserum (0.1 ml). For select experiments, 40 ,ul ratherthan 0.1 ml of serum was employed with 60 p. ofMEMwith 3% BSA. Mixtures were incubated for 60 min at37°C in an end-over-end rotating apparatus (FisherRoto-Rack). The bacterium/PMNL ratio was approxi-mately 6:1 in all experiments. Control tubes lackingleukocytes, serum, or complement were included ineach experiment. The bactericidal index (BI) wascalculated by the following formula: BI (%) = 100 -[(CFU at 60 min/CFU at 0 min) x 100]. Greater than orequal to 90% reduction in CFU at the end of the 1-hincubation represented significant bactericidal activi-ty.For experiments in which the complement pathway

was inactivated by magnesium-EGTA (MgEGTA)chelation, the procedure was modified to include apreopsonization step to avoid toxicity to leukocytes.Sera diluted 1/2 in MgEGTA or GVB2' as a controlwere incubated for 5 min at 37°C to allow chelation ofcalcium. Bacteria then were added, and mixtures wereopsonized for 30 min at 37°C with end-over-end rota-tion, chilled, and centrifuged. The bacteria were sus-pended in MEM with 3% BSA with or without addedleukocytes for continuation of the phagocytic killingassay as described above. Experiments with chelatedsera employed a serum concentration of 25% of thereaction mixture.

RESULTS

CL assay conditions. To determine conditionsoptimal for the assessment of serum opsonins toS. pneumoniae type XIV, serum from a singledonor (1,185 ng of type XIV Ab N/ml) wastested. Maximal CL values for both untreatedand heat-inactivated sera occurred when opson-ic reaction mixtures had a serum concentrationof 50%. The bacterium/PMNL ratio in phagocyt-ic mixtures also influenced CL; maximal re-sponses were elicited when ratios of 40:1 to 80:1were employed. A final luminol concentration of5 x 10-8 M allowed ready differentiation be-tween the opsonic CL activity from sera con-taining high compared with low concentrationsof type-specific antibody. Under these condi-tions, peak CL responses occurred after approx-imately 30 min of incubation.

Relationship of CL and bactericidal activity toantibody concentration. As shown in Fig. 1, high-antibody serum (1,185 ng of Ab N per ml)produced significantly greater CL evolution thanlow-antibody serum (168 ng of Ab N per ml) (P< 0.001 for CL integral and initial slope). Afterheat inactivation, CL activity was diminishedfor both sera, but CL activity of high-antibodyserum was significantly greater than that of low-

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S. PNEUMONIAE TYPE XIV OPSONINS 803

CL

E - - High (n-22)400

300 I-ILow (n -7)

200

100 _i/ _Heated High (n-15)

00 Heated Low (n -6)0 115 30 45 60

Time (minutes)

FIG. 1. Mean chemiluminescence values for S.pneumoniae type XIV of untreated or heated serumcontaining high (1,185 ng of Ab N per ml) or low (168ng of Ab N per ml) specific antibody concentrations.Outer bars indicate + 1 standard deviation from meanvalues; inner bars indicate ± 1 standard error of themean from mean values. Results represent the mean ofn experiments, as indicated.

antibody serum (P < 0.001 for CL integral orinitial slope). Approximately 80 to 90% of totalopsonic CL activity in both sera was heat labile.When tested in a bactericidal assay, both serapromoted significant opsonophagocytosis (bac-tericidal index, _90%) when employed in opson-ic mixtures at a serum concentration of 25%(bactericidal indices, 97.5 and 95.3%, respec-tively). However, when these sera were testedin a concentration of 10o, significant bactericid-al activity was observed in high-antibody serum(mean of four experiments, 93.5%; range, 87 to99%o), but not in low-antibody serum (mean,34%; range, 0 to 58%) (P < 0.05). Bacterialgrowth was always observed in the absence ofleukocytes or when heat-inactivated sera weretested.For the 10 sera containing high or low concen-

trations of type-specific antibody, a significantcorrelation between opsonic CL integrals andantibody concentration was demonstrated forboth untreated sera (r = 0.9590, P < 0.005) andheat-inactivated sera (r = 0.8396, P < 0.01). Theinfluence of specific antibody on opsonic CLand bactericidal activity was assessed further byadding various concentrations of the purifiedIgG to opsonic mixtures. Serial dilutions of thisIgG preparation resulted in diminishing opsonicCL values. A dilution containing 1,229 ng of AbN per ml allowed for opsonic CL activity com-parable to that of heated high-antibody serum,whereas no CL activity was detected at a dilu-

tion containing approximately 178 ng of Ab Nper ml. When the IgG preparation was added toagammaglobulinemic sera as a source of comple-ment, there was a stepwise increase in bacteri-cidal activity which was related directly to thefinal concentration of specific antibody (Fig. 2).Bacterial growth occurred in all sera in theabsence of complement or leukocytes.

Heat-inactivated serum containing a high con-centration of antibody (1,185 ng of Ab N per ml)was absorbed with type XIV or type III S.pneumoniae. The concentration of antibody tothe type XIV pneumococcal polysaccharide inheterologously absorbed serum was similar tothat of unabsorbed serum, whereas antibodywas not detected in sera absorbed with homolo-gous organisms. Essentially no opsonic CL ac-tivity was noted in serum absorbed with typeXIV organisms, but sera absorbed with a typeIII strain produced opsonic CL activity compa-rable to that of unabsorbed sera (Fig. 3). Theaddition of untreated antibody-deficient serumas a complement source to heat-inactivated ab-sorbed sera resulted in CL values similar tothose of untreated serum (Fig. 3; P > 0.05 forinitial slope and integral values).

Interaction of antibody and complement opso-nins (role of the AP). Test sera chelated withMgEGTA were employed in CL opsonic mix-tures. The mean CL integrals, expressed as apercentage of values for untreated serum, were90%o (73 to 100lo) for four high-antibody sera,and 78% (53 to 109%) for five low-antibody sera(Table 1). If the hypogammaglobulinemic andagammaglobulinemic sera were excluded, CLvalues for the three remaining sera with lowantibody were a mean of 94% of untreatedvalues. Both CL integrals and initial slopes weresignificantly (P < 0.01) greater for sera contain-

1o0

an-

x

c 60-3

.r 40

*Dc 20

o59 118 237 473 946 1889

Specific Antibody Concentration (xng Ab N/ ml)

FIG. 2. Bactericidal activity of agammaglobulin-emic serum after the addition of purified IgG contain-ing various concentrations of specific antibody. Testsera were untreated (hatched bars) or chelated withMgEGTA (solid bars).

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500

400

,UntreatedE

iHeated + C'

> Heated, Absorbed (itt+ C,

300

200

100

,HeatediAbsorbed ll),Absorbed (X IV)

15 30 45 60Ti me

FIG. 3. Opsonic activity of serum containing a highconcentration of specific antibody. The test sera were

untreated, heat inactivated, or absorbed with S. pneu-moniae type XIV or III. Selected sera had hypo-gammaglobulinemic serum added as a source ofcomplement (C'). Curves display the mean of twoexperiments.

ing high antibody concentrations than for thosewith low antibody concentrations. The additionof the purified IgG preparation to chelated anti-body-deficient serum resulted in stepwise in-creases in opsonic CL activity (Fig. 4).

Chelated sera with high antibody levels alsowere compared with low-antibody and hypo-gammaglobulinemic sera in the bactericidal as-

say. Sera with high levels of antibody promotedsignificant opsonophagocytosis with the classi-cal pathway blocked (mean bactericidal index ofthree experiments, 96.7%; range, 93.5 to 98.7%;P < 0.001). Significant bactericidal activity alsowas observed in chelated agammaglobulinemicserum when a high concentration of specificantibody (purified IgG) was added (Fig. 2). Incontrast, low-antibody sera caused significantopsonophagocytosis only when both comple-ment pathways were intact. Bacterial growthwas observed in chelated sera with low levels ofantibody.

iMgEGTA + IgG (350ng Ab N)

UntreatedMgEGTA + IgG (178ng Ab N)

MgEGTA + IgG 118ng Ab N)

Heated + IgG (350ng Ab N)Heated

15 30 45Time (Minutes)

60

FIG. 4. Opsonic activity of serum lacking specificantibody. Serum samples were untreated, heat inacti-vated, or MgEGTA chelated. The addition of purifiedIgG at a specific dilution of a preparation containing9,831 ng of Ab N per ml of type-specific antibody was

made. Curves display the mean of two experiments.

In additional CL experiments (Fig. 5), high-and low-antibody RD sera were tested. OpsonicCL activity of high-antibody RD serum was

significantly (P < 0.01) greater than that of low-antibody RD serum, both before and after theaddition of purified factor D. The opsonic activi-ty of chelated high-antibody RD serum withadded factor D (RD+D serum) was approxi-mately 80% of whole serum CL values. Theaddition of purified IgG to chelated low-anti-body RD+D serum produced a striking increasein CL activity with a CL slope comparable tothat of chelated high-antibody RD+D serum.

Visual determination of phagocytosis. Samplesof phagocytic reaction mixtures were examinedby light microscopy after 30 min of incubation.Differentiation of ingested from cell-associatedorganisms was often impossible, and only cellsin which there were obvious intracellular organ-isms were counted. Both the percentage of cellscontaining organisms and the phagocytic indicesof phagocytosing cells were higher in mixtures

TABLE 1. CL scores of sera with high and low concentrations of specific antibody before and after chelation

Mean CL scores (SD)Serum antibody concn

No. (ng of Ab N/ml) Untreated sera MgEGTA-chelated sera

Sera testedCL Maximum CL % Maximum %

Mean Range SD integral slope integral Integral slope Slope(x106) (x102) (x 106) untreated (x 102) untreated

High 4 1,155 1,125-1,185 31.8 26.2 (5.1) 19.6 (3.4) 23.1 (1.1) 90 (12.4) 16.5 (0.8) 85.3 (10.4)antibody

Low 7 86.4 0-168 65.9 10.2 (4.1) 7.2 (2.6) 6.8 (2.7) 78 (25) 4.4 (1.9) 68.4 (27.7)antibodya

a Includes sera from a patient with hypogammaglobulinemia (antibody concentration, 0) and from a child withcombined immune deficiency (antibody concentration, 0).

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700

600

500

ECL0

400

300

200

100

a_ RD + D +MgEGTA+*4IgG (614ng Ab N)

RD + D +MgEGTA

RD +DI RD

_-

RD + EGTA

0 15 30 45 60Time (Minutes)

FIG. 5. Opsonic activity of sera containing high (1,185 ng of Ab N per ml; left) or low (168 ng of Ab N per ml;right) concentrations of antibody. Sera were heat inactivated, RD, chelated with EGTA, or chelated withMgEGTA. Additions to test sera were provided by donor-specific factor D and purified IgG containing specificantibody. Curves display results of a single experiment for high-antibody sera and mean of two experiments forlow-antibody sera.

with sera containing high compared with lowspecific antibody concentrations; 98% (untreat-ed serum) and 36% (heated serum) of cellscontained ingested organisms when high-anti-body sera were used, whereas these values were88 and 33%, respectively, for low-antibody sera.Phagocytic indices of 2.3 and 0.56 were deter-mined for untreated high- and low-antibodysera, respectively; indices of 0.66 and 0.46,respectively, were observed for these same seraafter heat inactivation.

IF of opsonized organisms. Opsonized organ-isms were examined for fluorescence after expo-sure to fluorescein-conjugated anti-human IgG,IgA, IgM, and C3. Strong IgG IF (3.0 to 4.0+)was noted in untreated, heat-inactivated, andchelated sera with high concentrations of specif-ic antibody, whereas lower IF scores (1.5 to2.0+) were found in sera with low antibodyconcentrations. Absorption of heated high-anti-body serum with homologous organisms abol-ished detectable IgG IF, whereas absorption ofthe same serum with a heterologous strain didnot diminish IgG disposition. Neither IgA norIgM IF was detected in untreated or heat-inacti-vated serum regardless of antibody concentra-tion.

Deposition of C3 on opsonized organisms wasalso related to specific antibody content of testsera. Higher C3 IF scores were observed inhigh-antibody sera (3.5+) when compared withvalues for low-antibody sera (2.5+). The addi-tion of untreated antibody-deficient serum toabsorbed heated serum restored C3 IF to values

comparable to those of untreated serum. Whenchelated with MgEGTA, both high- and low-antibody sera were associated with C3 deposi-tion comparable to that in untreated sera. HigherC3 IF scores were observed in high-antibodysera (4.0+) than in low-antibody sera (2.5+).

DISCUSSIONA variety of experimental techniques have

been employed in efforts to delineate the opson-ic requirements for serotypes of S. pneumoniae.In vivo studies by Hosea et al. (19) have shownthat both the classical complement pathway andantibody are required for clearance from thebloodstream of serotype VII S. pneumoniae,and studies in humans have defined opsonicdeficiencies in some patients (14). Several inves-tigators (13, 17, 25, 31, 36, 39) have employedcomplement consumption, CL, or phagocyticassays which have indicated that both the classi-cal complement pathway and the AP can func-tion to promote opsonization of different pneu-mococcal serotypes. However, although CLassays have been used to evaluate the relativeroles of specific serum factors required for op-sonization of other microorganisms (3, 21, 37),the relationship between CL and other function-al assays of serum opsonic requirements has notbeen previously investigated for S. pneumoniae.Although CL assays allow precise evaluations ofthe opsonic factors, CL generation does notnecessarily imply intracellular killing of the in-gested organism. Therefore, the present studywas designed to compare a CL assay for type

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XIV S. pneumoniae with a bacterial opsonicassay and two nonfunctional assays of opsonins(IF, radioimmunoassay).Our results indicate that sera containing en-

dogenous complement and high concentrationsof antibody to the type XIV pneumococcal poly-saccharide have more opsonic actvity than thosewith low antibody concentrations. Use of seracontaining high antibody concentrations for op-sonization of test organisms promoted more IgGdeposition (measured by IF) and higher phago-cytic indices than did sera containing low anti-body concentrations. CL integrals correlateddirectly with antibody concentration of test sera.Although not apparent at a serum concentrationof 25%, a striking difference in bacterial activitybetween high- and low-antibody sera was ob-served at 10% serum concentration. The impor-tance of specific antibody as a serum opsoninwas confirmed by the stepwise increments inbactericidal activity observed when purified IgGcontaining specific antibody was added to agam-maglobulinemic serum. These results are consis-tent with the reported efficacy of pneumococcalpolysaccharide vaccines in prevention of bacter-emic pneumococcal disease by the induction oftype-specific antibodies (5, 33).

In contrast, sera containing either high or lowantibody concentrations which had been heatedto inactivate complement resulted in lowerphagocytic indices for PMNLs, generated signif-icantly less CL activity than untreated sera, andfailed to promote bactericidal activity. Thesefindings suggest that antibody in the absence ofcomplement does not function efficiently as anopsonin. Clinical experience among compro-mised patients who can respond to polyvalentpneumococcal vaccines with rises in serum anti-body concentration (2, 12), but who may none-theless develop vaccine-type pneumococcal in-fections, supports the concept that heat-labileopsonins in addition to antibody may be re-quired for efficient bactericidal activity (1, 16,26).Our assays allowed assessment of the interac-

tion of specific antibody with the classical com-plement pathway in promoting opsonization oftype XIV S. pneumoniae in three ways. HigherC3 IF scores were observed when high- ratherthan low-antibody sera were used to opsonizetest organisms. In the CL assay, when only theclassical pathway could function, integrals weresignificantly greater for high- than for low-anti-body sera. Finally, an important role for theclassical complement pathway in opsonizationwas demonstrated by the finding that untreatedbut not MgEGTA-chelated low-antibody serawere able to promote intracellular bacterial kill-ing.

Chelation of sera with MgEGTA to inhibit

selectively the classical pathway provided ameans for evaluating the contribution of thealternative pathway to opsonization of type XIVS. pneumoniae. After chelation, the AP ac-counted for more than half of the CL activitygenerated in the presence of both high- and low-antibody sera. However, our results suggest thatthe concentration of specific antibody can mod-ulate AP function for opsonizing type XIV pneu-mococci. C3 IF scores were higher for chelatedhigh-antibody sera than for chelated low-anti-body sera. Increases in CL response were ob-served when purified IgG containing specificantibody was added to chelated hypogamma-globulinemic or RD+D sera. In the bactericidalassay, chelated high- but not low-antibody seradisplayed significant bactericidal activity, as didchelated agammaglobulinemic serum, after theaddition of a high concentration of specific anti-body provided by a purified IgG preparation.These data demonstrate that specific antibodycan augment AP activation in bactericidal killingof type XIV S. pneumoniae. They also suggestthat a critical concentration of antibody may berequired for this augmentation to occur; thespecific antibody concentration required for APfacilitation cannot be determined from the pre-sent studies since the IgG preparation employedalso contained non-immunospecific but poten-tially opsonically active globulin (36, 40). Anti-body facilitation of the AP has been shownpreviously in hemolytic systems (28, 30) and forspecific microorganisms including pneumococci(40), group B Streptococcus (10), Bacteroides(6), Pseudomonas (8), and virus particles (29).Our IF results and the work of Giebink et al.

in which a radiolabeled bacterial uptake methodwas employed to assess phagocytosis (17) sug-gest that the CL activity generated by heat-inactivated or MgEGTA-chelated (or both) low-antibody sera reflects surface deposition of IgGand subsequent ingestion of organisms. Thedichotomy between the CL and the bactericidalassays which results when heated or MgEGTA-chelated low-antibody sera were employed hasbeen discussed elsewhere (3) and may relate torequirements of extracellular factors (such asIgG and complement) for bacterial killing (23,34). These findings emphasize the need for cor-relation ofCL results with those of other assays.

In summary, we have shown that complementgenerates the major portion of opsonic CL activ-ity for type XIV S. pneumoniae and that bothcomplement pathways participate in this activi-ty. Specific antibody also has an important rolein opsonization of these organisms. The meth-ods employed to define the opsonic require-ments of normal human sera for type XIV pneu-mococci should be useful for study of sera fromcompromised hosts. Previous investigators have

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found that sera from such patients may havedefective opsonic activity (7), and that improvedopsonic activity may not correlate with in-creased concentrations of specific antibodiesafter immunization with pneumococcal polysac-charides (15). The assays described in this reportmay provide a means of delineating the underly-ing opsonic defects among patients at specialrisk for serious pneumococcal infections.

ACKNOWLEDGMENTS

We thank Ralph D. Feigin for critical review of the manu-

script, Edward 0. Mason, Jr., for laboratory advice, andGeraldine Hughes for help in preparing the manuscript.

S.E.G. is a Research Fellow of the National Kidney Foun-dation. M.S.E. was supported in part by the Myers-BlackMellon Enterprises Pediatric Infectious Diseases ResearchFund. C.J.B. is the recipient of Public Health Service Re-search Career Development Award 1 K04 AI00323 from theNational Institute of Allergy and Infectious Diseases.

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