Serotypes and penicillin susceptibility of pneumococci isolated from blood.

To learn the prevalence of penicillin-resistant pneumococci and the distribution of serotypes in proved pneumococcal infections, we studied 98 pneumococci recovered from blood over a 4-year period. Penicillin susceptibility was determined by the agar dilution method. Serotyping was done by the capsular swelling (quellung) test. Only one strain showed diminished susceptibility to penicillin (minimal inhibitory concentration, 0.12 micrograms/ml). Twenty-three different serotypes were identified; types 3, 4, 6, 8, 9, 14, and 19 were the most frequent. Type 4 was the most common serotype. Sixty-two percent of strains were serotypes included in a recently licensed 14-type pneumococcal polysaccharide vaccine (Pneumovax, Merck), and an additional 16% were antigenically related serotypes. Even though penicillin-resistant strains of pneumococci may rarely cause bacteremic pneumococcal infection, we suggest that isolates from cerebrospinal fluid, blood, and other normally sterile body fluids be tested for penicillin susceptibility. Inclusion of additional serotypes in future pneumococcal polysaccharide vaccines should be based on the current distribution of serotypes in large series of proved pneumococcal infections.     

Stability of serotypes during nasopharyngeal carriage of Streptococcus pneumoniae

Serotype changes among natural isolates of Streptococcus pneumoniae are well documented and occur by recombinational exchanges at the capsular biosynthetic locus. However, the frequency with which this phenomenon occurs within the nasopharynx of children is not clear and is likely to be highest in the nasopharynx of children, who have high rates of pneumococcal carriage. A birth cohort of 100 infants was studied, and pneumococci were recovered from nasopharyngeal samples taken at monthly intervals during the first 6 months of life and then at 2-monthly intervals until the age of 2 years. Among the 1,353 nasopharyngeal samples were 523 that contained presumptive pneumococci, and three colonies from each were serotyped. A total of 333 isolates, including all isolates of differing serotypes from the same child, were characterized by multilocus sequence typing. Sixty-eight children carried multiple serotypes during the first 2 years of life. Two children carried a typeable and a nonserotypeable pneumococcus of identical genotype, and five children carried genetically indistinguishable isolates of serotypes 15B and 15C. These isolates were considered, respectively, to be due to loss of capsule expression and the known ability of serotype 15B and 15C pneumococci to interconvert by loss or gain of an acetyl group on the capsular polysaccharide. In all other cases, isolates from the same children that differed in serotype also differed in genotype, indicating the acquisition of a different pneumococcal strain rather than a change in capsular type. There was therefore no evidence in this study for any change of serotype due to recombinational replacements at the capsular locus among the pneumococci carried within the nasopharynges of the children.     

Genomics Reveals the Worldwide Distribution of Multidrug-Resistant Serotype 6E Pneumococci

The pneumococcus is a leading pathogen infecting children and adults. Safe, effective vaccines exist, and they work by inducing antibodies to the polysaccharide capsule (unique for each serotype) that surrounds the cell; however, current vaccines are limited by the fact that only a few of the nearly 100 antigenically distinct serotypes are included in the formulations. Within the serotypes, serogroup 6 pneumococci are a frequent cause of serious disease and common colonizers of the nasopharynx in children. Serotype 6E was first reported in 2004 but was thought to be rare; however, we and others have detected serotype 6E among recent pneumococcal collections. Therefore, we analyzed a diverse data set of ∼1,000 serogroup 6 genomes, assessed the prevalence and distribution of serotype 6E, analyzed the genetic diversity among serogroup 6 pneumococci, and investigated whether pneumococcal conjugate vaccine-induced serotype 6A and 6B antibodies mediate the killing of serotype 6E pneumococci. We found that 43% of all genomes were of serotype 6E, and they were recovered worldwide from healthy children and patients of all ages with pneumococcal disease. Four genetic lineages, three of which were multidrug resistant, described ∼90% of the serotype 6E pneumococci. Serological assays demonstrated that vaccine-induced serotype 6B antibodies were able to elicit killing of serotype 6E pneumococci. We also revealed three major genetic clusters of serotype 6A capsular sequences, discovered a new hybrid 6C/6E serotype, and identified 44 examples of serotype switching. Therefore, while vaccines appear to offer protection against serotype 6E, genetic variants may reduce vaccine efficacy in the longer term because of the emergence of serotypes that can evade vaccine-induced immunity.     

Diagnosis of invasive pneumococcal infection by serotype-specific urinary antigen detection

Widespread use of conjugate pneumococcal polysaccharide-protein vaccines may alter the spectrum of pneumococci producing invasive disease. Novel sensitive diagnostic methods would be valuable for monitoring the epidemiology of pneumococcal disease within populations and vaccine recipients. Ideally, these methods should allow determination of the serotype of the infecting clone. Serotype-specific enzyme-linked immunosorbent assays (ELISA) for 13 capsular polysaccharides (types 1, 3, 4, 5, 6A, 6B, 7A, 9 V, 14, 18C, 19 A, 19F, and 23 F) were developed. Experiments with pure capsular polysaccharide demonstrated that the assays were sensitive (0.01 to 1.0 ng/ml) and specific. These assays were used to detect capsular polysaccharide in urine from 263 adult patients with proven (blood culture-positive) invasive pneumococcal disease and pneumonia of unknown etiology and from patients with positive blood cultures yielding bacteria other than pneumococci (control group). Among 76 patients with invasive pneumococcal disease from whom blood culture isolates had been serotyped, 62 (82%) had infections with pneumococci of serotypes represented in the ELISA panel. Capsular antigen matching the serotype of the blood culture isolate was detected in the urine of 52 of these patients, giving a sensitivity of 83.9% for the target serotypes. The tests were significantly more sensitive for urine from patients with pneumococcal pneumonia (89.8%) than for urine from patients with non-pneumonic invasive infection (61.5%; P<0.05). Data from the control group indicated a specificity of 98.8%. These assays should prove valuable in epidemiological investigation of invasive pneumococcal infection in adults, particularly if combined with a sensitive C-polysaccharide detection assay to screen for positive samples.     

Lung Bacterial Clearance in Murine Pneumococcal Pneumonia

We studied the bactericidal capacity of the rat lung during the development of pneumococcal pneumonia. Pneumonia was produced in a lower lobe by the intrabronchial instillation of 10(4)Streptococcus pneumoniae cells in buffer. Lung bacterial counts progressively increased, reaching 10(7) per lung within 48 h, and the increase was associated with localized atelectasis and consolidation. Bacterial multiplication was inhibited with tetracycline at various intervals after infection, and the subsequent clearance of pneumococci was determined. Viable pneumococci were rapidly killed by lung defenses if bacterial multiplication was inhibited within 12 h of the onset of infection. No change occurred in the bacterial populationif tetracycline was delayed until 24 h after infection, indicating that pneumococcal killing by lung defenses had ceased. This effect could be reproduced with the addition of pneumococcal capsular polysaccharide to the inoculum, which produced a dose-related inhibition of pneumococcal clearance. The clearance of S. epidermidis was not impaired in the presence of pneumococcal pneumonia or by administration of exogenous capsular polysaccharide. These data indicate that pneumococcal pneumonia causes a marked impairment in lung antipneumococcal defenses within 24 h of the onset of infection. This acquired defect in antibacterial defenses may be due to the accumulation of pneumococcal capsular material in the lungs of infected animals.     

Upper and lower respiratory tract infection by Streptococcus pneumoniae is affected by pneumolysin deficiency and differences in capsule type

Pneumococci frequently colonize the upper respiratory tract, and these pneumococci are believed to act as a reservoir for infection of the lower respiratory tract and bacteremia. We investigated how the pneumococcal toxin pneumolysin affects the capacity of pneumococci to infect the upper and lower respiratory tract of the mouse. Wild-type Streptococcus pneumoniae serotype 2 and 3 strains, a serotype 2 pneumolysin-deficient mutant, and a serotype 2 mutant with the pneumolysin gene reinserted were used to study differences in colonization and disease. In addition, we also examined a pneumococcal chimeric mutant (capsule type switched from serotype 2 to serotype 3) to gain further insight into the role that capsule plays in nasopharyngeal infection. Absence of pneumolysin was found to be associated with significantly lower numbers of pneumococci in the nasopharynx, trachea, and lungs. Differences in pneumococcal capsule type were found to have significant effects on pneumococcal infection of the nasopharynx, trachea, and lungs. However, it was the combination of capsule type and genetic background that was important, and the influence of this combination varied with the site of infection. For example, in the nasopharynx the wild-type serotype 3 strain and the capsule-switched mutant behaved similarly, whereas in the lungs the mutant that was switched to serotype 3 survived less well than the wild-type serotype 3 strain. The combination of capsule type and genetic background also determined virulence. Thus, the wild-type serotype 3 strain was virulent, whereas the capsule-switched mutant was avirulent.     

A comparison of antibody concentration measured by mouse protection assay and radioimmunoassay in sera from patients at high risk of developing pneumococcal disease

The radioimmunoassay (RIA) of pneumococcal capsular polysaccharide antibodies is dependent on the association of radiolabeled antigen and pneumococcal antibody. However, it is not known whether the ability of the antibody to complex with antigen correlates with in vivo protection against infection. A method for evaluating protective ability of antibody vis-à-vis binding ability is to passively transfer a measured quantity of antibody into recipient mice followed by a lethal challenge with virulent pneumococci. Protection against a fatal outcome is then correlated with the amount of antibody (as measured by RIA) passively transferred. This comparison of quantitation by RIA and biological protection in mice was performed on 30 sera from humans. The sera were obtained from vaccinated healthy persons and vaccinated persons at high risk of developing pneumococcal infection, including people with nephrotic syndrome, chronic obstructive pulmonary disease and various forms of cancer. The results of these studies indicate that antibody as measured by RIA correlates with protective antibody against pneumococcal infection. These studies were conducted on pneumococcal serotype 3.     

Modified Opsonization,Phagocytosis, and Killing Assays To Measure Potentially Protective Antibodies against Pneumococcal Surface Protein A

The standard opsonophagocytosis killing assay (OPKA) for antibodies to pneumococcal capsular polysaccharide was modified to permit an evaluation of the protection-mediating antibodies to pneumococcal surface protein A (PspA). We found that by increasing the incubation time with the complement and phagocytes from 45 min to 75 min, the protective activity was readily detected. In another modification, we used a capsule type 2 target strain that expressed PspA but not pneumococcal surface protein C (PspC). With these modifications separately or in combination, rabbit antisera to the recombinant α-helical or proline-rich domains of PspA mediated >50% killing of the target strain. The ability of normal human sera to mediate the killing of pneumococci in this modified OPKA correlated with their levels of antibodies to PspA and their ability to protect mice against fatal infection with a type 3 strain. Passive protection of mice against pneumococci and killing in the modified OPKA were lost when normal human sera were adsorbed with recombinant PspA (rPspA) on Sepharose, thus supporting the potential utility of the modified OPKA to detect protective antibodies to PspA. In the standard OPKA, monoclonal antibodies to PspA were strongly protective in the presence of subprotective amounts of anti-capsule. Thus, the currently established high-throughput OPKA for antibodies to capsule could be modified in one of two ways to permit an evaluation of the opsonic efficacy of antibodies to PspA.     

The Nonserotypeable Pneumococcus: Phenotypic Dynamics in the Era of Anticapsular Vaccines

Nonserotypeable pneumococci (NSP) are commonly carried by Australian Indigenous children in remote communities. The purpose of this study was to characterize carriage isolates of NSP from Indigenous children vaccinated with the seven-valent pneumococcal conjugate vaccine (PCV7) and to use these data to guide decisions on reporting of NSP. A total of 182 NSP were characterized by BOX typing, antibiogram analysis, and multilocus sequence typing (MLST) of common BOX types. NSP positive for the wzg capsule gene were analyzed by a multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB-H) targeting capsule genes to determine the serotype. Among 182 NSP, 49 BOX types were identified. MLST of 10 representative isolates found 7 STs, including ST448 (which accounted for 11% of NSP). Non-penicillin susceptibility was evident in 51% of the isolates. Pneumococcal wzg sequences were detected in only 23 (13%) NSP, including 10 that contained an ∼1.2-kb insert in the region. mPCR/RLB-H identified serotype 14 wzy sequences in all 10 NSP, and 1 also contained a serotype 3-specific wze sequence. Among the remaining 13 wzg-positive NSP, few belonged to the serotypes represented in PCV7. It appears that most NSP identified in Australian Indigenous children are from a true nonencapsulated lineage. Few NSP represented serotypes in PCV7 that suppress capsular expression. High rates of carriage and penicillin resistance and the occasional presence of capsule genes suggest a role for NSP in the maintenance and survival of capsulated pneumococci. To avoid the inflation of pneumococcal carriage and antibiotic resistance rates, in clinical trials, we recommend separate reporting of rates of capsular strains and NSP and the exclusion of data for NSP from primary analyses.Australian Indigenous children in remote communities of the Northern Territory experience dense, persistent nasopharyngeal colonization with Streptococcus pneumoniae (pneumococcus) from early infancy (13, 25). Cross-sectional studies report pneumococcal carriage rates of over 80% for these children (15). Pneumococcal serotype diversity contributed to swift serotype replacement following introduction of the seven-valent pneumococcal conjugate vaccine (PCV7) in 2001. The vaccine had no significant impact on the incidence of overall pneumococcal carriage or otitis media (14).To date, 91 pneumococcal serotypes have been described (21). In addition, a population of nonserotypeable pneumococcal (NSP) isolates does not react with the capsular polysaccharide typing sera. Molecular typing has identified three categories of NSP: (i) pneumococci that lack capsule genes, (ii) pneumococci that possess capsule genes but that are phenotypically nonencapsulated, and (iii) atypical pneumococci that are phenotypically NSP but that are genetically divergent from pneumococci (8,18). Pneumococci that lack capsule genes make up a highly diverse population that includes strains that have spread globally (8). These strains have been associated with a variety of mucosal (and, occasionally, invasive) diseases (for a review, see reference 24). Phenotypically nonencapsulated pneumococci that possess capsule genes may represent pneumococci that have lost the ability to express the capsule or strains that have temporarily ceased or downregulated capsule expression. Understanding the distinction between these populations is particularly important in the context of widespread immunization with anticapsular vaccines.NSP frequently colonize Indigenous Australian children. In our studies with young Indigenous children, NSP were detected in up to 18% of nasopharyngeal swab specimens. In a recent cross-sectional carriage study, NSP were the 3rd most common pneumococcal serotype (unpublished data from reference 16). It is also likely that we underestimate NSP carriage rates because of their morphological differences from their capsular counterparts; NSP tend to be smaller and dryer than capsular pneumococci, and the dimple is less conspicuous. The purpose of this study was to characterize NSP carriage isolates collected from Indigenous children after the introduction of PCV7. Our aim was to understand the potential importance of this population, particularly with regard to the presence of the capsule genes and antibiotic resistance. Importantly, we require evidence to guide reporting of NSP carriage and resistance in pneumococcal surveillance studies and as microbiological outcomes in clinical trials for otitis media.     

Filamentous Capsulated Streptococci from the Human Respiratory Tract: Chemical and Immunochemical Characterization of a Glycoprotein Capsular Antigen of Provisional Binary Capsular Type 87

A filamentous alpha-hemolytic streptococcus of provisional capsular type 87 isolated from the human respiratory tract has been shown to be binary capsulated. One of the capsular antigens appears to be a glycoprotein; the other appears to be a polysaccharide. Transformation reactions with deoxyribonucleic acid from streptococcus type 87 and a number of noncapsulated pneumococci yielded transformed pneumococci with either a glycoprotein capsule or a polysaccharide capsule, but not with both. Capsular precipitin (quellung) reactions were observed when streptococcus type 87 was treated with homologous antiserum or with antisera to either of the two distinct capsular transformants. Each of the transformed pneumococci gave a quellung reaction with its homologous antiserum or with antiserum to streptococcus type 87, but neither reacted with antiserum to the heterologous transformant. Chemical analysis showed the glycoprotein antigen of streptococcus type 87 to contain, in addition to amino acids, glucose, galactose, glucosamine, and phosphate. The amino acid composition of the glycoprotein capsular antigens from streptococcus type 87 and of those from transformed pneumococci were similar, showing only minor differences. The glycoprotein capsular antigen from streptococcus type 87 gave two closely associated precipitin bands with homologous antiserum or antisera to transformed pneumococci with the glycoprotein capsule. That the two precipitin bands represent two unrelated proteins is precluded largely on the basis of the unlikely probability of 100% cotransformation of the genes coding for both proteins in the pneumococcal transformants that were isolated. Chemical analyses of the various fractions of the glycoprotein indicate that the two precipitin bands may represent a glycoprotein and its corresponding apoprotein.     

Role of Pht Proteins in Attachment of Streptococcus pneumoniae to Respiratory Epithelial Cells

Pneumococcal adherence to mucosal surfaces is a critical step in nasopharyngeal colonization, but so far few pneumococcal adhesins involved in the interaction with host cells have been identified. PhtA, PhtB, PhtD, and PhtE are conserved pneumococcal surface proteins that have proven promising as vaccine candidates. One suggested virulence function of Pht proteins is to mediate adherence at the respiratory mucosa. In this study, we assessed the role of Pht proteins in pneumococcal binding to respiratory epithelial cells. Pneumococci were incubated with human nasopharyngeal epithelial cells (Detroit-562) and lung epithelial cells (A549 and NCI-H292), and the proportion of bound bacteria was measured by plating viable counts. Strains R36A (unencapsulated), D39 (serotype 2), 43 (serotype 3), 4-CDC (serotype 4), and 2737 (serotype 19F) with one or more of the four homologous Pht proteins deleted were compared with their wild-type counterparts. Also, the effect of anti-PhtD antibodies on the adherence of strain 2737 to the respiratory epithelial cells was studied. Our results suggest that Pht proteins play a role in pneumococcal adhesion to the respiratory epithelium. We also found that antibody to PhtD is able to inhibit bacterial attachment to the cells, suggesting that antibodies against PhtD present at mucosal surfaces might protect from pneumococcal attachment and subsequent colonization. However, the relative significance of Pht proteins to the ability of pneumococci to bind in vitro to epithelial cells depends on the genetic background and the capsular serotype of the strain.     

Peptide mimics of two pneumococcal capsular polysaccharide serotypes (6B and 9V) protect mice from a lethal challenge with Streptococcus pneumoniae

Anti‐polysaccharide immunity is a key facet of protection against several bacterial pathogens. Problems exist with current polysaccharide vaccines and alternative strategies that deliver a protective response are needed. We have identified immunological peptide mimics of type 6B and 9V pneumococcal capsular polysaccharides that could be used as vaccine antigens. Peptides mimicking antigenic properties of serotype 6B capsular polysaccharide were obtained from a phage‐displayed peptide library expressing dodecameric peptides, using a human monoclonal antibody (Db3G9). A murine monoclonal antibody (206, F‐5) against the serotype 9V capsular polysaccharide identified three peptide mimotopes from the dodecameric peptide library and one from a random pentadecameric peptide library. In ELISA, binding of 206, F‐5 and Db3G9 to phage displaying the selected mimotopes was significantly inhibited by type‐specific pneumococcal polysaccharide. Peptides were conjugated to keyhole limpet haemocyanin and were used to immunise mice. Two peptides, MP13 and MP7, induced specific anti‐6B and 9V polysaccharide antibodies, respectively. Mice immunised with MP7‐keyhole limpet hemocyanin or MP13‐keyhole limpet hemocyanin conjugate were significantly and specifically protected against a lethal challenge with pneumococci of the appropriate serotype. This study provides strong in vivo evidence that peptide mimics are alternatives to polysaccharide vaccines.     

Pneumococcal serotypes causing pneumonia with pleural effusion in pediatric patients

To determine the prevalence of serotypes of Streptococcus pneumoniae responsible for pneumonia with pleural effusion, we determined the capsular polysaccharide (PS) type directly on 49 pleural fluid specimens collected from pediatric patients during 2007 to 2009 with laboratory-confirmed pneumococcal pneumonia by using monoclonal antibodies and a multiplex, bead array immunoassay. Because the fluids had to be heated to remove nonspecific reactivity before being tested in the immunoassay and type 19A PS is heat labile, the pleural fluid samples were also tested for serotype 19A capsule gene locus by PCR. Use of the multiplex immunoassay combined with type-specific 19A PCR allowed for serotype determination on 40 of 49 pleural fluids. Pneumococcal pneumonia with pleural effusion was associated with a limited number of serotypes, with types 1, 3, 7F/A, and 19A accounting for 75% of the typeable cases. The concentration of capsular PS in the pleural fluids was often greater than 1 μg/ml and sufficient to inhibit the opsonic capacity of sera from individuals who had received the 23-valent pneumococcal PS vaccine. Based on the serotypes observed before and after introduction of the 7-valent pneumococcal conjugate vaccine, the recently licensed 13-valent pneumococcal conjugate vaccine may reduce the incidence of pneumonia with pleural effusions.     

Adhesion of Streptococcus pneumoniae to human pharyngeal epithelial cells in vitro: differences in adhesive capacity among strains isolated from subjects with otitis media, septicemia, or meningitis or from healthy carriers.

A method was developed to study the adhesion of Streptococcus pneumoniae to human pharyngeal epithelial cells. Epithelial cells from healthy persons, pneumococcal strains from patients with otitis media, meningitis, or septicemia, and pneumococcal cells from the nasopharynx of healthy carriers were used. Adhesion was found to be influenced by changes in the bacterial incubation medium and growth phase, the concentration of bacteria and epithelial cells, the epithelial cell donor, the incubation time and temperature, and the pH and osmolarity of the incubation medium. Pretreatment of bacteria with heat, Formalin, or trypsin decreased adhesion. The highest adhesion was obtained when 10(9) bacteria cultivated for 18 h in streptococcus cultivation broth were added to 10(4) pharyngeal cells and incubated at 37 degrees C for 30 min. S. pneumoniae strains from patients with frequent episodes of otitis media and strains from healthy carriers had the highest adhesion values; septicemia and meningitis strains had the lowest. The capsular polysaccharide type did not determine the adhesive capacity of the strains, but otitis strains belonging to the capsular types often associated with otitis media adhered in high numbers. Adhesion may be important for pneumococci colonizing the nasopharynx or inducing otitis media.     

A successful, diverse disease-associated lineage of nontypeable pneumococci that has lost the capsular biosynthesis locus

Streptococcus pneumoniae strains which fail to produce a polysaccharide capsule are commonly isolated from carriage and disease contexts. Here we use a multilocus approach to distinguish genuine nontypeable pneumococci from closely related nontypeable streptococcal isolates in a data set of 121 untypeable pneumococci from nasopharyngeal swabs and middle ear fluid of Finnish children and demonstrate that 70 of these belong to a pneumococcal lineage which has lost its capsular locus. Strains of this relatively old lineage include sequence types 344, 448, and 449. Comparison with the multilocus sequence typing database shows that strains of this lineage have spread intercontinentally and have been isolated from carriage, mucosal, and invasive disease. Furthermore we note a particular association of this nontypeable lineage with outbreaks of conjunctivitis. The diversification and geographic spread of this lineage suggest that loss of capsule is not inconsistent with long-term persistence and raise questions about the capsule's role in pneumococcal transmission.     

Simple, rapid latex agglutination test for serotyping of pneumococci (Pneumotest-Latex)

The "gold standard" for epidemiological typing of Streptococcus pneumoniae (pneumococcus) is the capsular reaction test (Neufeld test) with antisera against the 90 pneumococcal polysaccharide capsules, i.e., serotyping. The method is labor intensive and requires a certain level of experience to be performed satisfactory, and thus it has been restricted for use in specialized reference or research laboratories. Surveillance of the serotype distribution of pneumococci that cause infections is important to secure an optimal composition of pneumococcal vaccines and to monitor antibiotic resistance in pneumococci. At Statens Serum Institut, a simple latex agglutination test for serotyping of pneumococci has been developed. The Pneumotest-Latex kit consists of 14 different pooled pneumococcus antisera (pools A to I and pools P to T) applied to latex particles. In a blind test of 352 isolates (with all 90 serotypes represented), 336 (95.5%) were typed or grouped correctly by the Pneumotest-Latex; in addition, 2 (7%) of 30 strains regarded as nontypeable or rough strains were serotyped, and the serotypes of these two isolates were confirmed by the capsular reaction test with type-specific antisera. The Pneumotest-Latex seems to be a sensitive method for serotyping or grouping of the majority of pneumococcal strains. By use of this ready-to-perform latex agglutination kit (Pneumotest-Latex), serotyping of pneumococci can gain more ground as a tool in prevention of pneumococcal diseases.     

Specific IgG Subclass Antibody Levels and Phagocytosis of Serotype 14 Pneumococcus Following Immunization

Complement and specific antibody directed against capsular polysaccharide are necessary for efficient phagocytosis of pneumococci. In normal adults, specific antibody to pneumococci is predominantly of the IgG2 subclass. However, the role of IgG2 in bacterial clearance is debatable. We therefore decided to investigate the relationship between specific IgG subclass antibody levels and phagocytosis of serotype 14 pneumococcus, before and after immunization with a pneumococcal capsular polysaccharide vaccine. Specific IgG subclass antibody was measured by an ELISA technique and the effect of serum on phagocytosis of radiolabelled pneumococci by normal polymorphs was determined. We found that in the presence of complement, phagocytosis correlated significantly with both specific IgGl and IgG2 antibody titres( r = 0.547, P = 0.002 and r = 0.464. P = 0.009, respectively). However, in decomplemented sera, the correlation with IgGl antibody was lost, whereas that with IgG2 antibody was strengthened ( r = 0.641. P = < 0.001). The possibility that IgG2 binds to receptors on polymorphs should be considered.     

Prolonged and preferential production of polymeric immunoglobulin A in response to Streptococcus pneumoniae capsular polysaccharides.

Streptococcus pneumoniae is an invasive mucosal pathogen for which host defense is dependent on capsular polysaccharide-specific antibody. Capsule-specific immunoglobulin G (IgG), IgM, and IgA are produced following pneumococcal vaccination and infection. Serum IgA has two molecular forms, polymeric and monomeric. These forms may modulate the avidity of antigen binding and evolve over time as the immune response matures. Therefore, we sequentially characterized the molecular forms of serum IgA to three serotypes of pneumococcal capsular polysaccharides (types 8, 12F, and 14) after pneumococcal vaccination and after natural infection with type 14 S. pneumoniae. Although typically the form of IgA in antigen-specific systemic responses to protein antigens is predominantly polymeric in sera of patients shortly after exposure and shifts to the monomeric form in sera obtained several weeks later, the form of IgA in response to each pneumococcal capsular polysaccharide remained predominantly polymeric 1 month after natural infection and up to I year following vaccination. In contrast, IgA to pneumococcal cell wall polysaccharide was both polymeric and monomeric. Moreover, the form of IgA in response to polyribosyl-ribitol-phosphate (PRP), the capsular polysaccharide of Haemophilus influenzae type b, was predominantly monomeric in the sera of 8 of 10 subjects tested 1 to 3 months after vaccination with either PRP alone or the diphtheria toxoid conjugate of PRP. We conclude that systemic responses to pneumococcal capsular polysaccharides are distinct in the production of predominantly polymeric IgA over time. The persistence of polymeric IgA may facilitate binding and clearance of pneumococci from the systemic circulation or reflect limited maturation of the immune response to pneumococcal capsular polysaccharides.     

Linkage Units in Cell Walls of Gram-Positive Bacteria

Abstract

Pneumococcal infection persists as a major cause of pneumonia, otitis media, and meningitis in infants. Children less than 2 years of age show the highest incidence of pneumococcal diseases.

Production of monoclonal antibody (MAb) to polysaccharide (PS) and binding characteristics to PS epitopes were studied. Removal of the O-acetyl group from 9V PS by alkali hydrolysis resulted in a decreased binding with rabbit 9V antiserum (AS). However, the binding reaction with 9V MAb was less affected by the loss of O-acetyl content. Type 9V IgG MAb provided passive protection and enhanced the opsonophagocytic activity of polymorphonuclear (PMN) leukocytes to kill type 9V pneumococci.

The pathogenecity of pneumococci is attributed to various virulence factors distributed on the cell surface, including capsular polysaccharide and protein antigens, for example, pneumolysin, autolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesion (PsaA), and hemin binding protein. Some of these protein antigens may be used as a component to combine with pneumococcal PS vaccine or as a carrier of conjugate vaccine.

Clinical trials of pneumococcal conjugate vaccines showed that covalent linkage of capsular PS to protein carriers improved the immunogenicity of the PS. Development of glycoconjugate vaccine for selected pneumococcal types will help solve the problem of poor immunogenecity of PS vaccine in young children used for prevention of pneumococcal infection.     

Ultrastructural localization of capsules, cell wall polysaccharide, cell wall proteins, and F antigen in pneumococci.

The localization of pneumococcal capsular and cell wall antigens was examined by immunoelectron microscopy. C polysaccharide (C-Ps), a common component of all pneumococci, was uniformly distributed on both the inside and outside of the cell walls. The thickness of the C-Ps varied with the strain. Encapsulated strains were covered by varied amounts of capsular polysaccharide concealing the C-Ps of the bacteria so as to render it inaccessible to anti-C-Ps antibodies. In addition to C-Ps, protein antigens were demonstrable on the surface of nonencapsulated pneumococci. The proteins were not masked by the C-Ps layer. An extra layer on the cell walls was conspicuous on electron micrographs of both rough and encapsulated pneumococci. The nature of this extra layer has not been disclosed. F antigen, another common antigen of pneumococci, was uniformly distributed on the surface of the plasma membranes. During the course of the experimental work a reproducible method of gold labeling immunoglobulins was developed.