CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY,1071-412X/97/$04.0010

Nov. 1997, p. 778–782 Vol. 4, No. 6

Copyright © 1997, American Society for Microbiology

Western Blot Analysis of Antibody Response to PneumococcalProtein Antigens in a Murine Model of Pneumonia

HYAM MOUNEIMNE,1 MANETTE JUVIN,1 JEAN-LUC BERETTI,1 ESTHER AZOULAY-DUPUIS,2

ERIC VALLEE,2 PIERRE GESLIN,3 PATRICK PETITPRETZ,4 PATRICK BERCHE,1

AND JEAN-LOUIS GAILLARD1*

Laboratoire de Microbiologie, INSERM U411, Faculte Necker-Enfants Malades,1 and INSERM U13,Hopital Bichat,2 Paris, Centre National de Reference des Pneumocoques, Creteil,3 and

Service de Pneumologie, Hopital Andre Mignot, Versailles,4 France

Received 12 May 1997/Returned for modification 5 June 1997/Accepted 23 July 1997

To detect new antigen candidates for serological tests, we studied the antibody response to pneumococcalprotein antigens in mice infected intratracheally with various Streptococcus pneumoniae strains. Sera weretested by Western blotting against whole-cell protein extracts. Mice developed a detectable immunoglobulinG-type response against a small number of polypeptides. The antibody response was strain dependent: serafrom individuals infected with the same strain gave similar banding patterns on immunoblots. The bandingpatterns varied with the strain used for infection. However, a band at 36 to 38 kDa was recognized by allreactive sera. This band appeared to correspond to a polypeptide that was antigenically well conserved amongthe different S. pneumoniae serotypes. An antibody response to this antigen developed in mice irrespective ofthe capsular type, the virulence, and the susceptibility to penicillin G of the infecting strain. Thus, this 36- to38-kDa protein antigen may be of value for the development of a serological test for humans.

Streptococcus pneumoniae is a major etiological agent ofcommunity-acquired pneumonia (14). An estimated 150,000 to270,000 cases of pneumococcal pneumonia occur annually inthe United States, with a fatality rate of approximately 5%(15). The emergence of multiple-antibiotic-resistant strains is agrowing problem and complicates the therapeutic strategy forthis disease (5).

Diagnosis of pneumococcal pneumonia is difficult. The iso-lation of pneumococci from blood is definitive proof of thedisease’s presence (14), but it is estimated that only 20 to 25%of the cases of pneumococcal pneumonia are bacteremic (15,17). The demonstration of pneumococci in sputum or in thenasopharynx is of little diagnostic value, as S. pneumoniae canbe found as a commensal organism in the upper respiratorytracts of healthy individuals (17). Moreover, bronchial coloni-zation with S. pneumoniae is common in patients with chronicobstructive lung disease. Invasive diagnostic techniques, suchas transtracheal aspiration, are both specific and sensitive.However, these techniques are unpleasant and not suitable fornonhospitalized patients. The detection of pneumococcal an-tigens in body fluids is rapid and specific but lacks sensitivity (12).

Serodiagnosis of pneumococcal pneumonia could be a use-ful approach for establishing diagnosis retrospectively andfor large-scale epidemiological studies. To date, interest hasmainly been focused on the antibody response to pneumococ-cal capsular polysaccharides, which are major virulence factors(10). Other nonprotein pneumococcal components, such as Cpolysaccharide and phosphorylcholine, have also been evalu-ated as antigens in serological tests (4, 7). Among pneumococ-cal proteins, only pneumolysin has been the subject of exten-sive studies (4, 9). The sensitivity of serological tests based onthis protein does not exceed 50% for patients with bacterio-logically proven pneumococcal pneumonia.

Using a Western blot technique, Renneberg et al. recentlydetected serum antibodies against pneumococcal polypeptidesin apparently healthy individuals (18). The numbers of differ-ent polypeptides recognized by these sera were consistent withthe levels of antibodies against type-specific polysaccharidesand C polysaccharide, suggesting the possible value of thisapproach for serological analysis. We used a similar Westernblot technique to study the antibody response to pneumococcalprotein antigens in a murine model of pneumonia. This exper-imental model has the following advantages. Intratracheal per-oral inoculation is highly reproducible. Blood and lung culturesof samples collected 48 h after inoculation allow the degree ofinfection to be assessed (21). Comparable antigenic burdenscan be obtained irrespective of the intrinsic virulence of thestrains by adjusting the dose given to animals. Finally, experi-mental pneumococcal pneumonia in mice is a good model ofprimary infection, as mice do not naturally carry S. pneu-moniae.

The S. pneumoniae strains used for infecting mice are listedin Table 1. The other S. pneumoniae strains used in this study,all obtained from the Centre National de Reference des Pneu-mocoques (Creteil, France), were strains 40492 (serotype 4),40336 (serotype 7), 40500 (serotype 9V), 40527 (serotype 14),and 40421 (serotype 18). Strains were stored at 280°C in brainheart infusion (BHI) broth (BioMerieux, Marcy l’Etoile,France) supplemented with 5% filtered horse serum (SanofiDiagnostics Pasteur, Marnes-la-Coquette, France). The MICof penicillin G was determined by E-test (BMD, Marne laVallee, France). The intraperitoneal 50% lethal dose was es-timated by the probit method. Groups of eight mice werechallenged intraperitoneally with various doses of bacteria, andthe mortality rates were monitored for 3 weeks. For infectionof mice, pneumococci were grown in BHI broth for 5 to 6 h at37°C in a 5% CO2 atmosphere until the optical density at 600nm reached 0.4. Ten-milliliter aliquots were centrifuged, andthe resulting pellets were resuspended in the same volume ofphosphate-buffered saline (PBS; pH 7.2). For inoculations, thisbacterial suspension was diluted 1:100 in PBS (virulent strains

* Corresponding author. Mailing address: Laboratoire de Microbi-ologie, INSERM U411, Faculte Necker-Enfants Malades, 156 rue deVaugirard, 75730 Paris Cedex 15, France. Fax: 33 (1) 44 49 49 60.

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4241 and 6254) or used undiluted (all other strains). The titersof the inocula were determined by plating 0.1 ml each of serial10-fold dilutions on Columbia agar containing 5% sheep blood(BioMerieux).

Female Swiss mice (Iffa-Credo Laboratories, St. Germainsur l’Arbresle, France), aged 7 weeks (20 to 22 g each), wereinfected by the intratracheal peroral route as described indetail elsewhere (6). Animals were anesthetized intraperitone-ally with 0.2 ml of 0.65% sodium pentobarbital (Dolethal;Vetoquinol, Lure, France). They were suspended vertically byplacing the upper incisor teeth on a wire hook, with the ventralside of the mouse facing the experimenter. The trachea wasthen cannulated with a blunt metal needle (23 gauge) by theoral route. The bacterial inoculum was delivered in a volume of0.05 ml via a Microliter syringe (Hamilton, Reno, Nev.). Micewere kept vertical for 5 min to allow distal alveolar migrationof bacteria. Fifteen mice per group were inoculated. Amoxi-cillin (50 mg/kg of body weight) was administered intraperito-neally 48 and 72 h after pneumococcal inoculation, irrespectiveof the susceptibility of the infecting strain to penicillin G.Blood and lung cultures were initiated 48 h after bacterialinoculation, before amoxicillin injection. Five mice per groupwere sacrificed by cervical dislocation. The thorax was opened,and heart blood was collected and cultured for 18 h at 37°C inBHI broth. Cultures turbid to the naked eye were consideredpositive and were subcultured to confirm the presence of pneu-mococci. The lungs were dissociated from the trachea andother structures and homogenized in 1 ml of PBS. Bacterialtiters were determined by plating 0.1 ml each of serial 10-folddilutions of the homogenates on Columbia agar.

Western blots were prepared as follows. Bacteria weregrown in BHI for 15 h at 37°C in a 5% CO2 atmosphere.Ten-milliliter cultures were centrifuged, and the resulting pel-lets were washed once in PBS and resuspended in 500 ml ofsodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE) sample buffer (100 mM Tris-HCl [pH 6.8], 2%SDS, 5% 2-mercaptoethanol, 10% sucrose, 0.01% bromophe-nol blue). SDS-PAGE was carried out as described by Laemmli(11) in 11% polyacrylamide minigels (Mini Protean II; Bio-Rad, Ivry sur Seine, France). Protein concentrations were mea-sured by the method of Bradford (3): 10 to 15 mg was loadedinto each lane. The banding patterns obtained after stainingwith Coomassie brilliant blue were superimposable for allstrains (not shown). The proteins were transferred to nitrocel-lulose membranes (BA 85; Schleicher & Schuell, Dassel, Ger-many) with a Mini TransBlot cell (Bio-Rad) in transfer buffer(25 mM Tris [pH 8.5], 0.2 M glycine, 20% [vol/vol] isopropa-nol). Transfer of the proteins was verified by staining withPonceau S (Sigma). The membranes were stored at 220°Cuntil use.

Blood samples to be analyzed were obtained by retro-orbitalpuncture (individual kinetics of antibody response) or heartpuncture. After centrifugation, serum samples were stored at280°C until use. Antipneumococcal antibodies were detectedby probing the Western blots with the sera. Immunoglobulin G(IgG) was removed from the sera prior to IgM detection withprotein G columns (HITRAP columns; Pharmacia Biotech, Or-say, France). The nitrocellulose membranes were first blockedby incubation with washing buffer (0.15% Tween 20 in PBS[pH 7.2]) containing 5% skim milk for 1 h at room tempera-ture. The membranes were then incubated for 45 min at roomtemperature with murine serum samples diluted 1:100 (IgG) or1:10 (IgM). After being washed, the membranes were incubat-ed with peroxidase-conjugated goat anti-mouse IgG or IgM(Organon Teknika, West Chester, Pa.) for 45 min at roomtemperature. All sera and labeled antibody dilutions were pre-pared in washing buffer containing 5% skim milk. Antibodybinding was revealed by adding 0.05% diaminobenzidine-tetra-hydrochloride (Sigma) and 0.03% hydrogen peroxide. No anti-pneumococcal response was detected in serum samples ob-tained from noninfected control mice.

FIG. 1. Kinetics of the antipneumococcal IgG-type response in a mousechallenged with 5 3 105 CFU of strain 4241. Western blot strips of S. pneumoniae4241 whole-cell extracts were probed with sera collected from an individualmouse on days 0 (lane 1), 3 (lane 2), 10 (lane 3), 16 (lane 4), and 30 (lane 5).Arrowheads indicate the main labeled bands, at 36 to 38 and 75 kDa. Numberson the right are molecular masses (in kilodaltons).

FIG. 2. Antipneumococcal IgG-type antibodies 30 days after challengingmice with strain 4241 at doses of 5 3 103 (a), 5 3 104 (b), and 5 3 105 (c) CFUper animal. Ten mice per group were infected for Western blot analysis. Westernblot strips of S. pneumoniae 4241 whole-cell extracts were probed with seracollected on day 30 from surviving mice. Arrowheads indicate the 36- to 38-kDapolypeptide. Numbers on the right are molecular masses (in kilodaltons).

TABLE 1. S. pneumoniae strains used for infection

Strain Serotype SourceMIC of

penicillin G(mg/liter)

Intraperitoneal 50%lethal dose

(CFU/mouse)

6254 1 Blood 0.008 1 3 103

4241 3 Blood 0.008 ,2 3 103

26772 6 Blood 0.032 ,2.8 3 104

60600 6 Ear 1 ,3.4 3 104

15986 19 Ear 4 6.3 3 106

54988 23 Sinus 4 1.8 3 106

TABLE 2. Results of blood and lung cultures 48 h after infectionwith various doses of strain 4241

Inoculum(CFU/mouse)

No. of bacteremic mice(n 5 5)

Bacterial count in thelungs (log10 CFU)a

5 3 105 5 6.6 6 1.05 3 104 2 5.1 6 2.05 3 103 1 2.2 6 3.35 3 102 1 1.7 6 2.9

a Values are means 6 standard deviations.

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Antibody response to inoculation with strain 4241 at a doseof 5 3 105 CFU/mouse. Strain 4241 (serotype 3) was tested firstbecause its behavior in the mouse pneumonia model is welldocumented (1, 21). Mice were inoculated by the intratrachealroute with 5 3 105 CFU per animal, a dose at which strain 4241has been reproducibly shown to cause pneumonia with bacte-remia in Swiss mice (21). Blood was collected by retro-orbitalpuncture on days 0, 3, 10, 16, and 30, and sera were tested byWestern blotting for the presence of IgM and IgG antibodiesagainst crude protein extracts prepared from strain 4241.

A group of 15 mice was challenged. The five mice sacrificedon day 2 were all bacteremic, and the bacterial count in theirlungs (mean log10 CFU 6 standard deviation) was 6.9 6 1.1.Of the 10 mice kept for analysis of the antibody response, 5survived until completion of the study protocol. The IgG-typeresponses were similar in all five. No response was observed

until day 10, when the sera recognized two bands at 37 and 75kDa (Fig. 1). On days 16 and 30, these bands became muchstronger, while several fainter bands appeared at 40, 46, 48,and 66 kDa. Overall, the sera recognized very few polypep-tides. No IgM-type response was detected in any of the micetested.

Dose-response effect in mice challenged with strain 4241.Groups of 15 mice were challenged with various doses of strain4241 (5 3 102 to 5 3 105 CFU per animal). Five mice pergroup were killed on day 2. The results of the blood and lungcultures for these animals are shown in Table 2.

Blood was collected on days 10 and 16 from five animals pergroup and on day 30 from all the animals still living. Only theIgG-type response was studied. No response to the smallestinoculum (5 3 102 CFU per mouse) was detected at any time.Mice inoculated with 5 3 104 or 5 3 103 CFU did not developdetectable responses until day 30. The reactivities of sera col-lected at this time from mice inoculated with 5 3 103, 5 3 104,or 5 3 105 CFU are shown in Fig. 2. Three of four, two of four,and four of four serum samples obtained from mice inoculatedwith, respectively, doses of 5 3 103, 5 3 104, and 5 3 105 CFUgave positive reactions. Interestingly, the patterns of responsewere similar with the three doses. As previously found, thestrongest response was directed against a 37-kDa polypeptide.Fainter bands were detected at 40, 48, 75, and .90 kDa.

Antibody response after infection with various S. pneu-moniae strains. Using the same murine model of pneumonia,we studied the IgG-type responses induced by S. pneumoniaestrains of various serotypes and with various susceptibilities to

FIG. 3. Antipneumococcal IgG-type antibodies 30 days after challenging mice with S. pneumoniae 6254 (a), 26772 (b), 60600 (c), 15986 (d), and 54988 (e). Ten miceper group were infected for Western blot analysis. Sera collected on day 30 from surviving mice were tested at a dilution of 1:20 (strain 6254) or 1:100 (other strains)against the whole-cell protein extracts from the corresponding infecting strains. Lanes 5, 9, 8, 11, and 9 in panels a to e, respectively, are Western blot strips probedwith a serum sample obtained 30 days after inoculation of strain 4241. Arrowheads indicate the 36- to 38-kDa polypeptide. Numbers on the right are molecular masses(in kilodaltons).

TABLE 3. Results of blood and lung cultures 48 h after infectionwith various S. pneumoniae strains

Strain(serotype)

Inoculum(CFU/mouse)

No. of bacteremicmice/no. studied

Bacterial count in thelungs (log10 CFU)a

6254 (1) 3.5 3 105 0/3 4.5 6 0.226772 (6) 6.5 3 105 3/5 5.4 6 1.260600 (6) 3.0 3 105 2/5 4.8 6 0.515986 (19) 2.0 3 107 0/5 4.7 6 0.554988 (23) 2.9 3 107 1/5 5.5 6 1.4

a Values are means 6 standard deviations.

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penicillin G and intrinsic virulences (Table 1). Groups of 15mice were each challenged with a different strain. The infectingdose was adjusted according to the virulence of the strain sothat similar bacterial burdens were obtained: on day 2, themean bacterial counts in the lungs (for five mice per group)were between 4.5 and 5.5 log10 CFU for all strains (Table 3).

Sera collected on day 30 were analyzed by Western blottingwith the infecting strain as the source of protein antigens (Fig.3). Serum samples diluted 1:100 from surviving animals (4 to10 mice) were tested for each strain. Strains 26772, 60600,15986, and 54988 elicited detectable IgG-type responses inmost animals. Sera from mice challenged with the same straingave similar staining patterns. The response was strain depen-dent: the banding patterns varied according to the infectingstrain. However, for all infecting strains, sera consistently gavepositive signals at 36 to 38 kDa. In most cases, the banddetected at this position was the most pronounced. Serumsamples from mice challenged with strain 6254 were negativeat a dilution of 1:100 (not shown). At a dilution of 1:20, anti-pneumococcal IgGs were detected in one of four mice (Fig. 3).Again, the strongest response was directed against a 36- to38-kDa polypeptide.

Recognition of conserved antigens by sera. We studiedwhether the antigens recognized by the sera, especially the 36-to 38-kDa antigen, were type specific. For this purpose, immu-noblots of protein extracts prepared from a panel of S. pneu-moniae strains belonging to the most common serotypes wereprobed with sera obtained on day 30 of infection with strains

4241, 26772, 60600, 6254, and 15986. Each serum gave similarbanding patterns with all antigenic preparations (Fig. 4). Inparticular, the 36- to 38-kDa antigen was recognized in allcases. This suggests that the antibodies elicited in the animalsreacted with antigens found in all of the serotypes tested.

In the model of pneumonia used in this study, mice devel-oped detectable antibody responses against a small number ofpneumococcal polypeptides. Sera collected 30 days postinfec-tion gave a maximum of four or five strong bands on immu-noblots, irrespective of the infecting strain. This contrastedwith the large number of polypeptides stained by Coomassiebrilliant blue after separation of the pneumococcal extracts bySDS-PAGE. Although outbred mice were used in this study,sera from animals challenged with a given S. pneumoniae straingave very similar banding patterns on immunoblots. The band-ing patterns varied substantially among the strains. Thus, in themurine model of pneumonia, the antibody response to pneu-mococcal proteins appears to be strain dependent. However,sera from mice infected with one S. pneumoniae strain showedstrong cross-reactivity with extracts of S. pneumoniae strains ofother serotypes. This suggests that most antipneumococcal an-tibodies elicited during murine infection recognize well-con-served protein antigens. Rennenberg et al. also found a con-siderable degree of similarity in the banding patterns whenusing a given human serum to probe protein extracts from S.pneumoniae strains of different serotypes (18). This is consis-tent with previous studies showing that most S. pneumoniae

FIG. 4. IgG-type reactivities of sera obtained 30 days after inoculation of strains 4241 (a), 26772 (b), 15986 (c), and 54988 (d) against protein extracts from S.pneumoniae strains of the most common serotypes. Extracts were from strains 54988, serotype 23 (lanes 1); 15986, serotype 19 (lanes 2); 40421, serotype 18 (lanes 3);40527, serotype 14 (lanes 4); 40500, serotype 9V (lanes 5); 40336, serotype 7 (lanes 6); 26772, serotype 6 (lanes 7); 40492, serotype 4 (lanes 8); 4241, serotype 3 (lanes9); and 6254, serotype 1 (lanes 10). Arrowheads indicate the 36- to 38-kDa antigen; triangles indicate the protein extracts from infecting strains. Numbers on the rightare molecular masses (in kilodaltons).

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protein antigens are common to the different capsular sero-types (16).

Among the polypeptides recognized by the sera, a polypep-tide with an apparent mobility of 36 to 38 kDa might beparticularly useful as a diagnostic antigen. An antibody re-sponse to this polypeptide developed in all mice, irrespective ofthe capsular type, the virulence, and the susceptibility to pen-icillin G of the infecting strain. This response was the earliestand, for most strains, the strongest that was observed. Anti-bodies elicited after infection with one strain recognized a 36-to 38-kDa polypeptide in the antigenic extracts prepared fromS. pneumoniae strains of the most common serotypes. Thus,this 36- to 38-kDa polypeptide appears to be both stronglyimmunogenic and highly conserved among the different S.pneumoniae serotypes. Two other findings suggest that thispolypeptide may be suitable for serological tests with humans.First, the antibody response to the 36- to 38-kDa polypeptidewas not abrogated by early antibiotic treatment. Second, theresponse was detectable with small bacterial burdens in thelungs. The 36- to 38-kDa polypeptide remains to be identified.However, it may be the pneumococcal protein PsaA, whosegene has been recently cloned and sequenced (20). PsaA is a37-kDa adhesin present in virtually all S. pneumoniae strains(19). It is probably exposed on the pneumococcal surface (19)and therefore may be a preferred target for the humoral im-mune response in infected hosts. Alternatively, the 36- to 38-kDa polypeptide may be the pneumococcal autolysin LytA,whose molecular mass is 35 to 36 kDa (8), or a previouslyundescribed protein.

There are some limitations to this study. First, we used crudebacterial extracts as antigenic preparations and consequentlymay not have detected antibodies against pneumococcal pro-teins present in small amounts in these extracts. This is prob-ably why we did not find an antibody response against a 53-kDaantigen that could correspond to the pneumococcal toxinpneumolysin (2). Secondly, we detected IgG- but not IgM-typeantipneumococcal antibodies. This is unlikely to be due to theinability of mice to mount an IgM-type antibody response topneumococcal antigens. A recent study reported that mice asyoung as 2 weeks old produced antipneumococcal IgM anti-bodies (13). Possibly, the Western blot technique may fail toreveal low levels of IgM-type antibodies. Other techniques, forexample, immunocapture assays, are much better suited to thedetection of IgMs. Third, the local immune response is poten-tially important in pneumococcal pneumonia. This fact shouldbe considered in future studies. Finally, the antibody responseto pneumococcal infection may be different in humans. We arecurrently assessing the relevance of our findings for humans.

Financial support was received from Institut Beecham, Nanterre,France.

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