JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1988, p. 755-7590095-1137/88/040755-05$02.00/0

Prevention of Pneumococcal Otitis Media in Chinchillas with HumanBacterial Polysaccharide Immune Globulin

PAUL A. SHURIN,lt* G. SCOTT GIEBINK,2 DARA L. WEGMAN,3 DONNA AMBROSINO,4 JAMES RHOLL,2MARY OVERMANS THOMAS BAUER,6 AND GEORGE R. SIBER4

Department of Pediatrics, Cleveland Metropolitan General Hospital, Case Western Reserve University School ofMedicine, Cleveland, Ohio 441091; Department of Pediatrics, University of Minnesota School of Medicine, Minneapolis,Minnesota 554552; University ofDelaware, Newark, Delaware 197163; Laboratory of Infectious Disease, Dana-FarberCancer Institute, Harvard Medical School, Boston, Massachusetts 021154; Euclid Clinic, Cleveland, Ohio 441195; and

Department of Pathology, Cleveland Clinic Foundation, Cleveland, Ohio 441066

Received 15 July 1987/Accepted 14 December 1987

Clinical and experimental observations suggest that immune globulin may prevent otitis media (OM) inchildren. We performed experiments in chinchillas to test the hypothesis that human bacterial polysaccharideimmune globulin (BPIG) might prevent OM caused by Streptococcus pneumoniae. Animals were given BPIGor saline intraperitoneally on day 0. On day 3 the epitympanic bulla was inoculated with S. pneumoniae type7F. Ali 12 saline-treated and none of 12 BPIG-treated animals developed pneumococcal OM by day 7 (P <0.0001). Bacteremia developed in 6 of 12 saline- and 0 of 12 BPIG-treated animals (P = 0.007). Death withpneumococcal OM occurred within 28 days in 5 of 12 saline- and 0 of 12 BPIG-injected animals (P = 0.02). Achinchilla-specific immunoassay was used to show that surviving saline-injected animals developed serumanticapsular antibody; BPIG-treated animals had no detectable response. At levels of anticapsular immuno-globulin G similar to those of human adults, BPIG given systemically prevented pneumococcal OM anddisseminated infection in chinchillas. BPIG may be of value in preventing human bacterial infection and mayalso inhibit development of antibody if it affects local infection or colonization. Specific immunoglobulin Gantibody may provide an important antibacterial defense of mucosal surfaces of the respiratory tract.

Development of effective prevention for otitis media (OM)in infancy is a public health priority. The disease is promi-nent among causes of morbidity in children and is diagnosedin 1/3 of illness-associated office visits to pediatricians (25).Presently available vaccines are not effective in reducinginfection of young children with the bacterial and viralagents associated with acute OM (7, 12). Improved vaccinesare under development but may not protect high-risk infantswithin the first months of life. Therefore, alternative meth-ods of immunoprophylaxis deserve careful evaluation.Recent studies have shown that chinchillas are protected

from experimental middle ear infection with nontypeablestrains of Haemophilus influenza after either recovery fromhomologous-strain infection (14) or by systemic administra-tion of immune chinchilla serum (2). Streptococcus pneumo-niae is the most important bacterial agent of OM; newapproaches to prevention of pneumococcal OM are of par-ticular interest.We have postulated that passive immunoprophylaxis

might protect the middle ear from infection with S. pneumo-niae and have performed experiments in chinchillas to testthis hypothesis. To provide data relevant to protection ofhigh-risk human infants, we used a bacterial polysaccharideimmune globulin (BPIG) which was prepared from thepooled plasma of human donors immunized with polyvalentpneumococcal, meningococcal, and H. influenza type bvaccines (23).(A preliminary abstract has been published [Pediatr. Res.

20:321A, 1986].)

* Corresponding author.t Present address: Medical Research Division; Lederle Laborato-

ries, Pearl River, NY 10965.

MATERIALS AND METHODS

Passive immunoprophylaxis in chinchillas. BPIG producedat the Massachusetts Public Health Biological Laboratorieswas used for these experiments. Two lots of BPIG were usedand had similar concentrations of total protein and antibodyto S. pneumoniae type 3. However, lot 3 contained morespecific antibody to the capsule of S. pneumoniae type 7F(type 51 by American nomenclature) than did lot 1R, asmeasured by enzyme immunoassay (EIA) and radioimmu-noassay (RIA) (Table 1). The globulin used contained 2%immunoglobulin M (IgM) and 0.5% IgA as assayed bynephelometry (23).

Chinchillas were used because this species is well charac-terized as an experimental model for OM, has publishedstandards for otologic evaluation (8), and has been used instudies of the efficacy of active immunization against S.pneumoniae (6, 8, 9). Two identical experiments were per-formed with six BPIG- and six saline-injected animals each.Experiment 1 used lot 1R, and experiment 2 used lot 3 ofBPIG. Healthy outbred chinchillas 1 to 2 years old andweighing approximately 500 g each were obtained from localbreeders. Animals used in these experiments were housed inindividual cages in a separate room. Standards for animalcare conformed to those in the Guide for Care and Use ofLaboratory Animals published by the Public Health Service.Each had normal otoscopic and tympanometric examina-tions prior to study (8). Ketamine anesthesia (15 mg intra-muscularly) was given before each examination or proce-dure.By random allocation, animals were given either BPIG or

saline (1 ml/kg of body weight) intraperitoneally (i.p.) on day0. The injections were given by an investigator who did notparticipate in the subsequent experimental procedures. Thei.p. route was used because of its efficacy in transferring

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TABLE 1. Protein and type-specific pneumococcal antibodyconcentrations in two BPIG lots

Pneumococcal IgG antibody (ng of

IgG protein antibody protein/ml)Expt Lot concentration

no. (g/100 mI) EIA RIA

Type 7F Type 3 Type 7F Type 3

1 1R 14.1 99,770 27,540 126,450 140,6302 3 15.8 229,150 27,810 199,270 128,640

passive immunity in rats (G. Schiffman, Clin. Immunol.Newsl. 3:33-37, 1982).Three days after BPIG or saline administration, the right

middle ear space of each animal was inoculated by percuta-neous puncture of the epitympanic bulla (9). The inoculumcontained 20 CFU of S. pneumoniae type 7F in 0.1 ml ofsterile phosphate-buffered saline. Virulence was enhancedby mouse passage as previously described (9).

Otoscopic examination and tympanometry with a Grason-Stadler model GSI-28 Auto Tymp (Grason-Stadler, Inc.,Littleton, Mass.) were performed twice weekly during thefollowing 28 days. Middle ear abnormalities were defined bypreviously published criteria for otoscopy and tympan-ometry (8). Aspiration of each abnormal middle ear was

performed via needle puncture of the epitympanic bulla. Themiddle ear effusion (MEE) was cultured on 5% sheep bloodagar (SBA); when effusion was not obtained, the aspiratingneedle tip was cultured. Once aspirated, an ear was retappedat weekly intervals.Blood (0.1 ml) for culture was drawn aseptically via

cardiac puncture at 2- to 5-day intervals and inoculated ontoSBA. Additional samples of blood (2.0 ml) were obtainedbefore and at weekly intervals following pneumococcalinoculation; sera were separated by centrifugation andstored at -70°C for antibody assay.

All cultures were incubated for 72 h at 37°C in a candle jar

and examined for growth at 24-h intervals. Pneumococciwere identified and serotyped by capsular swelling reactionwith antisera from the Statens Seruminstitut (Copenhagen).Contaminating organisms were not present in MEE culturesand only rarely present in blood cultures.Postmortem examinations were performed on animals that

died during the course of the study. At sacrifice or postmor-tem, middle ear cavities were opened aseptically, and any

MEE present was aspirated and cultured on SBA and inbroth. Cerebrospinal fluid and blood were also cultured atthis time.Four additional chinchillas received BPIG lot 3 (1 ml/kg

i.p.) and were bled daily for 1 week and every 7 days for thesubsequent 3 weeks to measure the elimination of passivelyadministered pneumococcal antibody. These animals were

maintained separately from the infected group and receivedno other examinations or procedures.

Determination of human and chinchilla antipneumococcalIgG antibodies. The concentration of type-specific pneumo-

coccal anticapsular antibodies was measured in serum by a

modification of an EIA previously described by Carlson etal. (3). A type-specific capture antibody was used to bindhighly purified pneumococcal capsular polysaccharide to thewells of polystyrene microtiter plates (Nunc-immuno Plate I;GIBCO Laboratories, Grand Island, N.Y.). Test serum was

incubated in these antigen-coated wells. Bound antibodywas quantitated by using peroxidase-conjugated antibodyagainst human or chinchilla IgG.

Reagents for the EIA were prepared by previously de-scribed methods (3). Immune rabbit sera against type 3 andtype 7F pneumococci (R-anti-3 and R-anti-7F; Laboratory ofthe State Department of Health, Albany, N.Y.) were used toprepare the IgG F(ab)2 capture fragments. Purified type 3and type 7F pneumococcal capsular polysaccharides (Le-derle Laboratories, Pearl River, N.Y.) were reconstituted to4 mg/ml in phosphate-buffered saline and stored at 4°C. TheIgG fraction of Fc fragment-specific goat anti-human IgG(G-anti-HG; Cappel Laboratories, Westchester, Pa.) waspurified in a diethylaminoethyl column (DEAE Affi-GelBlue; Bio-Rad Laboratories, Richmond, Calif.) and conju-gated with horseradish peroxidase (HRPO; type VI RZ3.0;Sigma Chemical Co., St. Louis, Mo.) by a modification ofthe method of Mathiesen et al. (16), as described previously(9). Rabbit anti-chinchilla IgG (R-anti-CG) was obtained byimmunizing rabbits with chinchilla IgG and purified andconjugated as described above (13). Because whole chin-chilla IgG was used as the immunizing antigen, rabbitantibodies were produced against both heavy- and light-chain determinants; the resulting antiserum was cross-reac-tive with all classes of chinchilla immunoglobulin. Requireddilutions ofR-anti-3 and R-anti-7F F(ab)2 capture antibodies,antigen, and HRPO-conjugated antibodies were determinedby the checkerboard technique.To measure human antipneumococcal IgG, dilutions of

test sera were added to antigen-coated and antigen-freewells. HRPO-conjugated G-anti-HG was added to all wells.After incubation, plates were washed, H202 substrate incitrate phosphate buffer containing o-phenylenediaminedihydrochloride was added, plates were reincubated for 30min, and the reaction was stopped with 2.5 N H2SO4. Plateswere read at 450 nm on a microplate reader (MR600;Dynatech, Alexandria, Va.). The net optical density for eachserum dilution was calculated as the difference between theantigen-positive and corresponding antigen-negative wells.To measure chinchilla antipneumococcal antibodies, dilu-tions of chinchilla test serum were added to the antigen-coated and antigen-free wells. R-anti-CG conjugated withHRPO was used to complete the reaction.Net optical densities of the serum dilutions were con-

verted into quantitated antibody protein concentrations bythe method described by Zollinger and Boslego (26). Forhuman IgG antibodies, a standard curve relating opticaldensity to IgG antibody protein concentration was derivedfrom an IgG standard (human gamma-globulin; Calbiochem-Behring, La Jolla, Calif.). To quantitate chinchilla antibody,pooled serum from chinchillas immunized with formalinizedwhole type 7F S. pneumoniae was used as the antibodystandard. Individual antibody titers were assigned by usingthe net optical density of the serum dilutions adjusted on thebasis of the pooled serum standard; results were expressedas EIA units. This pool contained a high titer of antibodyagainst type 7F capsular polysaccharide (2,303 ng of type 7Fpneumococcal antibody nitrogen per ml by RIA).

Histopathology of temporal bones. Temporal bones wereremoved from the six saline- and six BPIG-treated chinchil-las in experiment 2, either at sacrifice on day 31 or at autopsyof animals that died earlier. Specimens were fixed in 10%buffered Formalin, decalcified for approximately 48 h, anddehydrated in ethyl alcohol and xylene prior to embedmentin paraffin.

Sagitally oriented serial sections were cut on a rotarymicrotome at a thickness of 4 to 5 ,um. Every 10th section ofmiddle ear and cochlear regions for 10 sections was stainedwith hematoxylin and eosin. These 10 sections of each

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TABLE 2. Clinical observations in chinchillas inoculated with S. pneumoniae type 7F

No. of animals with abnormal result/no. tested at day postinjection: Cumulative P vs salineGroup and testresulttoagru0 5 7 10 13 17 20 24 31 total group

Saline group (n = 12)Abnormal otoscopy

Right ear 0/12 12/12 12/12 10/10 7/7 5/6 6/6 6/6 3/6 12/12Left ear 0/12 0/12 5/12 5/10 3/8 3/8 5/7 5/7 5/7 11/12

Abnormal tympanometryRight ear 0/12 8/12 12/12 10/10 8/8 6/8 6/7 5/7 1/6a 12/12Left ear 0/12 0/12 3/12 4/10 2/8 3/8 3/7 4/7 3/7 9/12

BPIG group (n = 12)Abnormal otoscopy

Right ear 0/12 3/12 3/12 3/12 2/12 2/11 2/11 2/11 3/10 3/12 <0.001Left ear 0/12 0/12 0/12 0/12 0/12 0/11 0/11 0/11 1/11 1/12 <0.001

Abnormal tympanometryRight ear 0/12 2/12 3/12 2/12 2/12 2/11 2/11 2/11 1/11 3/12 <0.001Left ear 0/12 0/12 0/12 0/12 1/12 0/11 0/11 0/11 0/11 1/12 0.003

a One ear not done due to excessive drainage.

temporal bone were submitted for interpretation; the pres- bacteremia, five had or developed left OM. The onset ofence of middle ear inflammation and labyrinthitis was re- bacteremia in each case followed onset of OM in thecorded by a pathologist unaware of treatment history. inoculated right ear (Table 3).

Statistical analysis. Results were expressed in fourfold Death associated with pneumococcal OM occurred in 5 ofcontingency tables and analyzed by Fisher's exact test (1). 12 saline-treated and in none of the BPIG-injected animals (P

= 0.019). One BPIG-treated animal died on day 17; hemo-RESULTS pericardium was found at autopsy, and postmortem cultures

Passive immunoprophylaxis in chinchillas. All 12 saline of the middle ear cavities, lungs, and peritoneal,'pleural, andcontrols developed otoscopic or tympanometric evidence of subdural cavities were sterile. This presumably was causedright OM by day 5 (2 days after inoculation of the right by the cardiac puncture. In contrast, five saline-treatedmiddle ear cavity with pneumococci) (Table 2). In each animals died between 7 and 18 days after saline administra-animal, infection of the right middle ear with S. pneumoniae tion; postmortem cultures yielded S. pneumoniae from thetype 7F was confirmed by culture (Table 3). In contrast, right middle ear in five of five, from the left middle ear in fourwhile 3 of 12 BPIG-treated animals had transient otoscopic of four, and from cerebrospinal fluid in three of four.or tympanometric abnormalities involving the right ear, none Pneumococci were cultured from the right middle ear at thehad pneumococcal infection or MEE (P < 0.0001). Results time of sacrifice (day 31) in four of seven saline controls andwith BPIG lots containing high and low concentrations of in zero of six BPIG-treated animals.type 7F pneumococcal antibody were identical. The elimination of passively administered human pneumo-Among saline-treated animals, otoscopic or tympanomet- coccal antibodies was monitored in four healthy chinchillas

ric abnormalities of the uninoculated left ear were seen in 11 given BPIG lot 3. Type 7F and type 3 human IgG pneumo-of 12; 6 of these had proven pneumococcal infection. One coccal antibodies declined during the 4 weeks followingBPIG-treated animal had transient tympanometric abnor- inoculation with half-lives of 6.2 and 6.5 days, respectivelymality of the left ear; none had effusion or pneumococcal (Fig. 1). There was little variation in antibody concentrationsinfection (P = 0.014). Bacteremia developed in 6 of 12 observed in the four animals; mean human anti-7F IgG onsaline-treated and in none of the BPIG-treated animals (P = day 3 (corresponding to the day of inoculation of experimen-0.014) (Table 3). The occurrence of pneumococcal infection tal animals with pneumococci) was 2,935 ng of antibodyof the uninoculated left middle ear tended to be concordant protein per ml (range, 2,842 to 3,178 ng). The estimatedwith the occurrence of bacteremia. Of six saline-treated mean anti-7F antibody level on day 3 for animals whichanimals which developed left OM, five were or became received lot 1R was 1,278 ng of antibody protein per ml. Thebacteremic. Of six saline-treated animals which developed rate of decline of human type 7F IgG pneumococcal anti-

TABLE 3. Cultures of middle ear exudates and blood from chinchillas inoculated with S. pneumoniae type 7F

Group and cultureNo. of animals with positive culture/no. tested at day postinjection: Cumulative P vs saline

5 7 10 13 17 20 24 31 total group

Saline group (n = 12)Right middle ear 11/11 1/1 a - 2/3 2/3 12/12Left middle ear 5/6 0/3 1/1 1/3 6/9Blood 1/12 3/12 3/10 1/8 0/8 0/7 0/7 0/7 6/12

BPIG group (n = 12)Right middle ear 0/3 - 0/3 0/2 0/3 <0.0001Left middle ear - - - 0.014Blood 0/12 0/12 0/12 0/12 0/11 0/11 0/11 0/11 0/12 0.014a -, No cultures obtained.

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FIG. 1. BPIG antibii.p. administered'humfected chinchillas. Calc7F and type 3 antibodyconfidence intervals.

body was also stuanimals. The observantibody decline in;

Following type 7type 7F antibody le)control group but ditreated control grouantibody showed thhad a specific resporincreasing 2.6- to 32No increases were c

Histopathologic retion of the middle eathree of six saline-tirinthitis was also obrwith middle ear inf

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DayFIG. 2. Chinchilla;

course of experiment.chinchilla antibody dirtype 7F in saline-treatebars indicate 95% conf

infection during the 31-day observation period. The otherthree saline-treated animals had recovered'by day 31 and had

TsType 7 F normal temporal bone histology. Of six BPIG-treated ani-pe 7F

mals, only one presented any abnormal middle ear histology.In this animal, acute inflammation of the right middle earmucosa was observed with no involvement of the left middleear or either cochlea. Postmortem cultures taken on day 17were negative for S. pneumoniae type 7F; both otoscopy andtympanometry were repeatedly normal in this BPIG-treated

3 Type 3 animal, including observations made hours before death.

DISCUSSION

Animal and human experiments with pneumococcal vac-, , , , , , cines have previously demonstrated the importance of ac-

4 1 2 20 28 quired immunity in protection against pneumococcal OM (6,7). In an experiment which was analogous to the present

Days after BPIG Injection study, Barenkamp immunized chinchillas with killed cells of

dy decline without challenge. Elimination of nontypeable Haemophilus influenza. The immune serum,an antipneumococcal IgG in healthy nonin- given intravenously, prevented infection of the middle earelated haif-!ives are 6.2 and 6.5 days for type with this organism (2). Local secretion of IgA, IgG, and IgMies, respectively. Vertical bars indicate 95% by the middle ear mucosa has been considered important

(19, 24), but previous experimental studies have not sepa-rately evaluated the role of local and systemic antibody.Effects of local and systemic antibodies are difficult to

died in six pneumococcus-challenged separate in studies with vaccines because active immuniza-ed half-life of 8.0 days was similar to the tion may stimulate both systemic and local antibody produc-animals not challenged. tion (21).IF pneumococcal challenge, chinchilla Diamant and co-workers in 1961 provided evidence of avels increased significantly in the saline possible role for systemically administered immune serumid not change appreciably in the BPIG- globulin in preventing acute OM (4). In their randomizedp (Fig. 2). Assays of chinchilla type 7F study, children who presented with acute OM were giveniat all surviving saline-treated animals monthly injections of immune serum globulin or were ob-ise, with serum antibody concentrations served without treatment. Globulin-treated children had an!.5-fold in individual animals by day 24. apparent 57% reduction of OM incidence compared withbserved in BPIG-treated chinchillas. control subjects (P < 0.02) during a 7-month period ofCults revealed bilateral acute inflamma- clinical observation. Our findings in chinchillas indicate thattr mucosa at time of death or sacrifice in systemically administered IgG antibody can prevent bothreated animals. Bilateral purulent laby- pneumococcal OM and bacteremic dissemination of thisserved in two of the three saline animals infection.lammation; these animals had died of Schiffman (Clin. Immunol. Newsl.) has suggested that

antibody levels found in most normal adults protect againstsystemic pneumococcal infection. In contrast, infants havehigh attack rates for OM and very low serum antibody levels(11). Giebink et al. reported mean concentrations of type 7F

* and type 3 antipneumococcal antibody in' 10 healthy, unim-munized adults' to be 2,308 (range, 330 to 11,550) and 163

_/,* (range, 26 to 1,767) ng of antibody protein per ml, respec-tively (10). These antibody levels are remarkably similar tothose we achieved in BPIG-treated chinchillas.The present experiments suggest a role for passive immu-

noprophylaxis of OM in high-risk infants. They may alsohave general applicability in defining the role of circulating

1kV,_ __ ~____IgG in antibacterial defense of the respiratory tract mucosa.As reviewed by McDermott et al. (17), IgG is present insignificant quantities in various mucosal secretions, mayenter the respiratory tract by transudation from serum, and

._______.__.___.___,_ may promote local phagocytosis. Furthermore, studies re-8 16 24 32 ported to date have not clearly documented a role for

secretory IgA in protecting against bacterial colonization ors after Pneumococcal Inoculation invasion (17). Recent studies by Oxelius et al. (20) of patientsantipneumococcus type 7F antibody during with selective IgA deficiency also support the concept thatEIA was used to measure species-specific IgG2 is a major host defense against respiratory infection.

erected against the capsule of S. pneumoniae Among patients with selective IgA deficiency, recurrentd (-) and BPIG-treated (O) animals. Vertical respiratory infections were a clinical problem only in thosefidence limits. *, P < 0.05. deficient in both IgA and IgG2. If these and comparable

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observations of children with recurrent OM (5) are con-firmed, we would anticipate a resurgence of interest in theuse of immune globulin for prevention of respiratory infec-tion in children too young to have naturally acquired circu-lating antibody or to respond to currently available pneumo-coccal vaccines. Prophylactic use of BPIG might inhibitdevelopment of immunity in children as it did in the chin-chilla. Alternatively, the immune stimulus of natural coloni-zation might be sufficient to stimulate infection; an ex-tremely small number of viable organisms was used to infectchinchillas. Furthermore, high-risk children (15) might ben-efit from a period of protection given in circumstances whenavailable vaccines are not effective (7). The need to docu-ment the effects of BPIG administration has been greatlyaccentuated by the recent demonstration that, in children,this preparation is highly effective in preventing bacteremicinfections with H. influenza type b (21a). Clinical trials areneeded to validate the efficacy of this approach in reducingmorbidity from OM.

ACKNOWLEDGMENTS

This work was supported in part by Public Health Service grantsNS-14538 from the National Institute of Neurological and Commu-nicative Disorders and Stroke, AI-18125 and AI-17160 from theNational Institute ofAllergy and Infectious Diseases, and HD-18753from the National Institute of Child Health and Human Develop-ment.We thank Gerald Schiffman, Department of Microbiology and

Immunology, State University of New York Downstate MedicalCenter, for performing the RIAs and Crystal E. Galbreath andMaryann Kaczur for expert assistance with the manuscript.

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