Pneumococcal Vaccines.

Streptococcus pneumoniae is a Gram-Positive pathogen that is a major causative agent of pneumonia, otitis media, sepsis and meningitis across the world. The World Health Organization estimates that globally over 500,000 children are killed each year by this pathogen. Vaccines offer the best protection against S. pneumoniae infections. The current polysaccharide conjugate vaccines have been very effective in reducing rates of invasive pneumococcal disease caused by vaccine type strains. However, the effectiveness of these vaccines have been somewhat diminished by the increasing numbers of cases of invasive disease caused by non-vaccine type strains, a phenomenon known as serotype replacement. Since, there are currently at least 98 known serotypes of S. pneumoniae, it may become cumbersome and expensive to add many additional serotypes to the current 13-valent vaccine, to circumvent the effect of serotype replacement. Hence, alternative serotype independent strategies, such as vaccination with highly cross-reactive pneumococcal protein antigens, should continue to be investigated to address this problem. This chapter provides a comprehensive discussion of pneumococcal vaccines past and present, protein antigens that are currently under investigation as vaccine candidates, and other alternatives, such as the pneumococcal whole cell vaccine, that may be successful in reducing current rates of disease caused by S. pneumoniae.

Human antibodies to pneumococcal surface protein A in health and disease.

BACKGROUND: Diseases caused by Streptococcus pneumoniae have a high impact in young children whose ability to mount antibodies to capsular polysaccharides is impaired. Pneumococcal surface protein A (PspA) is a potential vaccine candidate for this age group. METHODS: We used Western blot analysis and enzyme immunoassay to study human sera of healthy adults from Alabama (n = 20) and from Finland (n = 21), healthy children from Finland (n = 20) and ill children from Finland, those with pneumococcal invasive infection (n = 26) and those with nonpneumococcal invasive infection (n = 26). RESULTS: Human antibodies to PspA exhibited strong cross-reactivity among different pneumococcal strains. The geometric mean titer of IgG antibody to PspA in sera from 21 healthy adults was 4,040, from ten 3-year-old healthy children 1,080 and from ten 2-month-old healthy children 1,650. The geometric mean titer of PspA antibody of acute phase sera of children with invasive pneumococcal disease was 140, significantly (P < 0.001) lower than the respective value, 1,020, for children with infection caused by other bacteria. CONCLUSIONS: We demonstrate for the first time the existence of antibodies to PspA in human sera in health and disease. The findings in ill children suggest that antibodies to PspA might play a role in protection against pneumococcal disease.

Risk factors for nasopharyngeal carriage of resistant Streptococcus pneumoniae and detection of a multiply resistant clone among children living in the Yukon-Kuskokwim Delta region of Alaska.

BACKGROUND: Children < 2 years old living in the Yukon-Kuskokwim Delta (YKD) region of Alaska have one of the highest pneumococcal bacteremia rates of in the world. METHODS: To determine the prevalence of and risk factors for infection with intermediate or resistant Streptococcus pneumoniae in the YKD, we cultured nasopharyngeal secretions of healthy children < or = 5 years old, reviewed their hospital records and administered questionnaires to accompanying parents. RESULTS: Of 185 children evaluated we obtained 95 pneumococcal isolates; drug susceptibility patterns and serotyping results were available for 92. Of these, 33 (36%) were intermediate or resistant to at least one drug class tested; 27 isolates were intermediate (minimum inhibitory concentration 0.1 to 1.0 mg/l) and none were resistant to penicillin. Compared with other isolates, capsular serotype 6B isolates were more likely to be intermediate or resistant to at least one drug (relative risk, 5.3; P < 0.001) and to more than one drug (relative risk, 17.0; P < 0.001). The majority of 6B isolates had identical pneumococcal surface protein A patterns. Carriage of intermediate or resistant pneumococcus was associated with age < 2 years (relative risk, 3.0; P < 0.001) but not with antibiotic use or other evaluated risk factors. CONCLUSIONS: Young age but not antibiotic use was associated with carriage of intermediate or resistant S. pneumoniae in the YKD region of Alaska. Much of the intermediate or resistant pneumococcus in the YKD may have resulted from the proliferation of a single capsular serotype 6B clone.

Patients with Wiskott-Aldrich syndrome have normal IgG2 levels.

The Wiskott-Aldrich Syndrome (WAS) in humans has a number of similarities to the immunodeficiencies found in CBA/N mice, including X-chromosome-linked inheritance, inability to produce antibodies to various carbohydrate antigens, susceptibility to various bacterial infections, and an imbalance in B lymphocyte subpopulations. Moreover, in both man and mice, IgG antibodies to polysaccharides are predominantly, but not exclusively, restricted to a single IgG subclass--IgG2 in man, and IgG3 in the mouse. Because CBA/N mice have a deficiency of IgG3 antibodies and because human IgG2 subclass deficiencies have been generally associated with inability to produce antibodies to carbohydrate antigens, it would seem likely that patients with WAS would have greatly reduced levels of IgG2. Quite to the contrary, the data presented here demonstrate that WAS patients have normal levels of the different IgG subclasses, including IgG2. Thus, inability to produce antibodies to carbohydrates is not always associated with IgG2 subclass deficiency.

Analysis of IgG subclass production in cell cultures from IgA deficient patients and in normal controls as a function of age.

IgA deficient individuals may also have low serum levels of IgG subclasses, especially IgG2. In the present study we examined the development of plasma cells producing IgM, IgA or IgG, and the IgG1 and IgG2 subclasses, following lipopolysaccharide (LPS) and pokeweed mitogen (PWM) stimulation of mononuclear cells (MNC) from normal and IgA deficient individuals as a function of age. Studies of blood MNC from 38 normal donors (age range 2-44 years) revealed an age-related distribution pattern of mu, gamma, alpha, gamma 1 and gamma 2 plasma cells produced in mitogen-stimulated and control cultures. Decreased IgA responses to both LPS and PWM were consistently observed in cultures of MNC from all of the nine children with IgA deficiency. When compared with age-matched controls the IgG response was also diminished in PWM stimulated cultures, whereas the IgM responses were normal. The IgG deficit was due to reduced responses for the gamma 1 and gamma 2 subclasses, and was most pronounced for IgG2; IgG2 plasma cell differentiation was particularly depressed in LPS cultures. In contrast to normal adult cells, blood MNC from the nine children with IgA deficiency and age-matched controls (2-17 years) yielded more IgG1 than IgG2 plasma cells in both control and LPS cultures, while the pattern of response to PWM was similar in all groups (gamma 1 greater than gamma 2). A good concordance was found between the level of secreted Ig in the culture supernatants and the relative number of IgM or, IgG and IgA plasma cells identified by immunofluorescence staining of cytoplasmic immunoglobulins.

Characterization of a multidrug-resistant clone of invasive Streptococcus pneumoniae serotype 6B in Alaska by using pulsed-field gel electrophoresis and PspA serotyping.

Antimicrobial susceptibility, pneumococcal surface protein A (PspA) serotyping, and pulsed-field gel electrophoresis (PFGE) were used to evaluate clonal relatedness among 66 invasive isolates of Streptococcus pneumoniae serotype 6B collected during 1982-1996 from patients in Alaska. Thirty-seven (56%) of the isolates had penicillin minimal inhibitory concentration values >/=0.125 microgram/mL and were resistant to at least 1 other antibiotic. Fourteen PspA serotypes were observed; PspA 16 was the most common (35%). Forty-five (68%) of the 66 isolates shared common and highly related PFGE patterns using 3 enzymes. Twenty-six (58%) of the isolates with common PFGE patterns were from Native Alaskan children

Epstein-Barr virus preferentially induces proliferation of primed B cells.

EBV can induce human B cells to proliferate, differentiate, and undergo transformation into continuously growing lymphoblastoid cell lines. The EBV responsiveness appears to be confined to a very limited subpopulation of B cells, the nature of which is still unclear. In these studies, we sorted tonsillar B cells on the basis of their expression of the early surface activation Ag, Bac-1, and compared their proliferative responses to EBV. Bac-1+ cells responded to EBV with a relatively high level of DNA synthesis, whereas the Bac-1- cells did not. Both large and small Bac-1+ cells were responsive to EBV and the responsiveness was unrelated to the level of Bac-1 immunofluorescence intensity. Bac-1+ cells were relatively enriched for surface IgM and IgD expression. When the Bac-1- population was enriched for IgM+ cells, the proliferative response was still significantly lower than that of the Bac-1+ population. B cells acquire the ability to bind IgM relatively late after activation, and this feature did not distinguish the EBV-responsive B cells. The results suggest B cells become responsive to EBV after an early activation signal.

Strong association between capsular type and virulence for mice among human isolates of Streptococcus pneumoniae.

The relationship between capsular type and virulence for mice was examined with 69 fresh human isolates of Streptococcus pneumoniae. These isolates represented eight capsular types or groups. Serologic and molecular weight differences in PspA (pneumococcal surface protein A) indicated that the strains were clonally distinct. Mice were infected intravenously with washed bacteria of all 69 isolates in sterile salt solutions. Twenty-eight of the isolates were also injected intraperitoneally to permit comparisons between the intravenous and intraperitoneal routes. With a few exceptions, there was concordance between the ability of strains to cause fatal infections by the two routes. About 30% of the 69 isolates were virulent for mice. The abilities of the isolates to kill mice and the length of time between inoculation and death were strongly associated with capsular type. All type 4 isolates, 40% of type 3 isolates, and 60% of group 6 isolates were virulent for mice; type 1 isolates were marginally virulent; and all type or group 14, 19, and 23 isolates were avirulent. Times to death were generally longer for mice infected with group 6 or type 1 than for those infected with type 3 or 4 pneumococci. There was no relationship between clinical diagnosis or tissue source of the isolates and virulence for mice.

Evaluation of lymphocyte differentiation in primary and secondary immunodeficiency diseases.

The differentiation status of T and B cells was evaluated in patients with common variable immunodeficiency (CVI), selective IgA deficiency (IgA), X-linked agammaglobulinemia (XLA), and the acquired immune deficiency syndrome (AIDS) with the use of conventional lymphocyte markers and four new monoclonal antibodies that identify lymphocyte subpopulations. These antibodies are HB 4, which identifies a subpopulation of resting B cells; HB 5, which identifies the C3d/EBV receptor on mature B cells; HB 7, which identifies immature B lymphocytes; and HB 10, which reacts with virgin but not activated or memory T cells. T and B cells from the IgA patients typically had normal phenotypic profiles, whereas diverse patterns of lymphocyte maturation were observed in CVI. In 11 of 16 CVI patients, B cells had normal antigenic phenotypes. Although B cells from four other CVI patients had normal frequencies of HB 5 and HB 7 antigen expression, few expressed the HB 4 antigen, suggesting that they were activated. In contrast, a large percentage of B cells from one CVI patient were of an immature phenotype. The expression of the HB 10 antigen by T cells in CVI patients was also variable, being normal in 10 of 16 patients, yet significantly decreased in six others. The vast majority of the limited numbers of IgM B cells from five XLA patients (greater than 100-fold reduction) has an immature phenotype (HB 4-5-7+). Interestingly, the circulating T cells in XLA patients were phenotypically similar to those in normal newborns, suggesting that T cell immaturity or defective T cell activation may occur in these B cell-deficient individuals. Circulating B cells from AIDS patients were mostly HB 7-, with variable expression of the HB 4 antigen and significantly decreased expression of the HB 5 antigen. Most of the T cells from AIDS patients were HB 10-, and thus appeared to be activated.

Molecular localization of variable and conserved regions of pspA and identification of additional pspA homologous sequences in Streptococcus pneumoniae.

PspA is anchored to the surface of all pneumococci by the C-terminal end of the molecule. The N-terminal half of PspA is known to be serologically variable and to be able to elicit protective immune responses. Molecular analysis with DNA probes spanning different regions of pspA was carried out to identify homologous sequences among pneumococcal isolates. At high stringency, DNA probes derived from the 3'-half of pspA (encoding the C-terminal half of PspA) hybridized to all of 37 pneumococcal isolates tested, representing 20 capsular serotypes and 12 PspA serotypes. Most strains had two sequences highly homologous to this region of pspA. Using derivatives of strain Rx1, with insertion mutations in pspA, it was possible to identify the functional pspA sequence. At 50% stringency, the 3' pspA probes also detected lytA and additional sequences. lytA encodes autolysin and shares homology with the 3' portion of pspA. A probe derived from the 5'-half of pspA (encoding the N-terminal half of PspA) hybridized with only 75% of strains and generally detected only one of the two sequences recognized by the 3' probes. Thus, the 3'-half of pspA appears to contain more highly conserved sequences than the 5'-half of pspA and shares homology with several additional sequences, suggesting that the pneumococcus might make several proteins that interact with the surface by the same mechanism as PspA.

Clones of Streptococcus pneumoniae isolated from nasopharyngeal carriage and invasive disease in young children in central Tennessee.

To determine whether nasopharyngeal carriage isolates of Streptococcus pneumoniae are of the same genetic background as isolates that caused invasive disease in one community, IS1167 and boxA genotypes were obtained for 182 pneumococcal isolates from children living in central Tennessee. The isolates represented 70 combined IS1167-boxA genotypes. The genotypic diversity of the invasive isolates was significantly less than that of the total population (P=.003). Most of the carriage isolates belonged to genotypes unique to carriage, whereas most of the invasive isolates belonged to genotypes common to carriage and disease (P=.02). Monte Carlo simulations showed a greater number of genotypes unique to carriage than can be explained by chance (P<.05 in all cases). Two genotypes were identified by multilocus sequence typing as members of globally disseminated clones, and one such genotype that was strictly carriage in this sample caused disease in other studies. Thus, clones can have different propensities for carriage and invasion.

The IS1167 insertion sequence is a phylogenetically informative marker among isolates of serotype 6B Streptococcus pneumoniae.

The phylogenetic utility of the IS1167 insertion sequence was examined with restriction fragment length polymorphism (RFLP) analyses of a sample of 50, predominantly invasive, capsular serotype 6B Streptococcus pneumoniae isolates previously characterized by multilocus enzyme electrophoresis (MLEE). The strains represented a genetically diverse assemblage of 34 distinct clonotypes composed of 26 restriction fragment types and 23 multilocus enzyme types. All isolates carried the IS1167 insertion sequence, with an average of 9.5 copies. The cross-classification of isolates based on RFLP and MLEE typing schemes was 81% concordant. Phylogenetic analyses demonstrated a significant (P < 0.0001) association between strains of a given RFLP lineage with those of a given MLEE lineage. A significant correlation (P < 0.00004) was also found between the proportion of restriction fragments shared by any given pair of isolates and their genetic distances estimated from the MLEE data. Parity between the two genetic markers implied that the sampled isolates were in linkage disequilibrium. The existence of nonrandom associations among genetic loci was confirmed by Monte Carlo analyses of the MLEE data. These studies, thus, demonstrated that invasive pneumococcal isolates of a single capsule type recovered on a regional scale can retain a largely clonal population structure over a period of 8 years. The ability to detect linkage disequilibrium and generate relatively congruent dendrograms based on distance and parsimony methods suggested that the restriction fragment data were robust to phylogenetic analysis.

Naturally occurring antibodies to phosphocholine as a potential index of antibody responsiveness to polysaccharides.

Naturally occurring antibodies reactive with the phosphocholine (PC) determinant of pneumococcal teichoic acids may be useful for evaluating the potential of patients to make antibodies to polysaccharides. Antibodies to PC are present in most adults under the age of 60 years, are absent in very young children, and are present at low levels in Wiscott-Aldrich patients and in IgG2-deficient adults. These last three groups respond very poorly to polysaccharide antigens. Antibodies to PC are also found at low levels in the elderly, a group that has previously been shown to have low levels of antibody to blood groups A and B carbohydrates. The levels of antibody to PC over time were constant in most individuals and, in adults, seemed to show slightly less variation than did titers of antibody to blood group B. Our findings suggest that titers of antibody to PC may be superior to titers of antibody to blood group A or B for monitoring responsiveness to carbohydrate antigens.

Evidence for a clonal origin of relative penicillin resistance among type 9L pneumococci in northwestern Canada.

The relationships among capsular type, protein type, and penicillin resistance for capsular group 9 Streptococcus pneumoniae collected in northwestern Canada between 1974 and 1987 were examined. The group 9 relatively penicillin-resistant (RPR) isolates were of the rare 9L capsular type. Of 47 penicillin-susceptible (PS) group 9 isolates that were typed for capsule, only 1 was 9L. Among 29 PS group 9 isolates that were protein typed, 9 protein types were observed. Of the 70 RPR isolates, 51 were protein type P23, 1 was P19, and 18 could not be typed (P0). Protein types P23 differed from P0 by a single epitope on pneumococcal surface protein A. These results suggest that the Canadian P23 and P0 capsular group 9 isolates are likely subclones of a primordial 9L RPR strain.

Phase I evaluation of zidovudine administered to infants exposed at birth to the human immunodeficiency virus.

This study evaluated the safety, tolerability, and pharmacokinetics of zidovudine administered intravenously and orally to infants born to women infected with the human immunodeficiency virus. Thirty-two symptom-free infants were enrolled before 3 months of age. The pharmacokinetics of zidovudine were evaluated in each infant after single intravenously and orally administered doses of zidovudine on consecutive days, and during long-term oral administration of the drug for 4 to 6 weeks. As new patients were enrolled, doses of zidovudine were progressively increased from 2 to 4 mg/kg. Therapy was continued for up to 12 months in 7 of the infants proved to be infected with human immunodeficiency virus. Zidovudine was generally well tolerated; 20 children (62.5%) had anemia (hemoglobin level < 10.0 gm/dl) during therapy and 9 (28.1%) had neutropenia (neutrophil count < or = 750 cells/mm3); these hematologic abnormalities usually resolved spontaneously. The total body clearance of zidovudine increased significantly with age, from an average of 10.9 ml/min per kilogram in infants < or = 14 days of age to 19.0 ml/min per kilogram in older infants (p < 0.0001). Concurrently, there was a significant decrease in serum half-life from 3.12 hours in infants < or = 14 days to 1.87 hours in older infants (p = 0.0002). Oral absorption was satisfactory and bioavailability decreased significantly with age, from 89% in infants < or = 14 days to 61% in those > 14 days of age (p = 0.0002). Plasma concentrations of zidovudine were calculated to be in excess of 1 mumol/L (0.267 micrograms/ml) for 4.12 +/- 1.86 hours and 2.25 +/- 0.78 hours after oral doses of 2 mg/kg in infants younger than 2 weeks and 3 mg/kg in older infants, respectively. We conclude that zidovudine administered at oral doses of 2 mg/kg every 6 hours to infants aged less than 2 weeks and 3 mg/kg every 6 hours to infants older than 2 weeks resulted in plasma concentrations that are considered virustatic against human immunodeficiency virus. Zidovudine was well tolerated by infants at these doses.

Evidence for the simultaneous expression of two PspAs by a clone of capsular serotype 6B Streptococcus pneumoniae.

Pneumococcal surface protein A (PspA) has been shown to be a serologically variable virulence factor of Streptococcus pneumoniae. In mice, PspA can elicit antibodies capable of protecting them against otherwise fatal infections with encapsulated pneumococci. In previous studies it has been reported that almost all isolates have two apparently unlinked genomic sequences that are highly homologous to the 5' and 3' halves of Rx1 pspA, although out MAbs to PspA have not detected more than one PspA in any given isolate of S. Pneumoniae. Recently, we have identified four isolates from a clone of capsular serotype 6B pneumococci (MC25-28) that simultaneously express two distinct PspAs. Each of the isolates (MC25-28) exhibited the same two Kpn I fragments (each containing a Hind III site) that hybridized with Rx1 pspA. MAbs specific for PspA detected two PspAs characterized by different molecular weights and different serologic patterns of reactivity (PspA type 6 detected by MAbs XiR278 and 2A4, and PspA type 34 detected only by MAb 7D2) in each of the four isolates. In previous studies XiR278 and 2A4 frequently have been observed to react with PspA epitopes of the same strain. Based on molecular weight data both epitopes were always present on the same molecule. Our present findings raise the possibility that pneumococci make a second serologically variable PspA which is generally not detected by currently available MAbs to PspA.

DNA polymorphisms and variant penicillin-binding proteins as evidence that relatively penicillin-resistant pneumococci in western Canada are clonally related.

Previous studies have suggested that relatively penicillin-resistant (RPR) capsular group 9L strains in western Canada may be clonally related. To test this hypothesis, restriction fragment length polymorphisms (RFLPs) were examined using DNA probes for pspA and a newly recognized pneumococcal genetic element, IS1167. Penicillin-binding proteins (PBPs) and PBP genes from representative strains were also studied. All RPR type 9L strains demonstrated an identical RFLP when probed with IS1167, and 12 of 14 RPR strains had the same RFLP when examined with pspA. Amplification of pspA by polymerase chain reaction and restriction endonuclease digestion showed that the 9L strains had common DNA fragments not identified in any of the penicillin-susceptible strains. The 9L strains apparently have a low-affinity PBP 2B distinct from those of other capsular types. These data derived from new genetic markers and PBP analysis strongly support a clonal origin of RPR type 9L pneumococci of western Canada.

Pneumococcal surface protein A (PspA) is serologically highly variable and is expressed by all clinically important capsular serotypes of Streptococcus pneumoniae.

Pneumococcal surface protein A (PspA) has been shown previously to elicit antibodies protective against pneumococcal infection and to be necessary for full pneumococcal virulence in mice. The protein was originally defined by the two mouse monoclonal antibodies Xi64 and Xi126, which together recognized PspA on 14% of pneumococcal isolates. Some PspA molecules reacted with both antibodies, but most reacted with only one or the other. In the present study we demonstrated that PspA is produced by all pneumococci, confirming our hypothesis that there are variants of PspA which are not detected by Xi64 and Xi126. We produced a rabbit antiserum and five additional monoclonal antibodies specific for PspA for these studies. The rabbit antiserum reacted with each of 95 pneumococcal isolated tested, comprising 16 capsular serotypes. One or more of the seven monoclonal anti-PspA antibodies reacted with 95% (53 of 57) of pneumococcal isolates tested. The specificity of the monoclonal and polyclonal antibodies to PspA was confirmed in two ways: (i) by detection of molecules on wild-type pneumococci that are identical in molecular weight to those detected in Western blots (immunoblots) with Xi64 and Xi126 and (ii) by the use of mutants of Streptococcus pneumoniae that fail to produce PspA or that produce a truncated form of PspA. By using the seven monoclonal antibodies, we observed 31 PspA types among the 57 isolates. When the 53 strains reactive with the monoclonal antibodies were analyzed by capsular type as well as by serologic type and molecular weight of PspA, we observed 50 different clonotypes of pneumococci.

Molecular characterization of a globally distributed lineage of serotype 12F Streptococcus pneumoniae causing invasive disease.

These studies have identified a major genetic lineage of capsule serotype 12F Streptococcus pneumoniae, which has maintained two different types of the pneumococcal surface protein A (PspA) virulence factor and caused invasive disease in geographically disjoint locations. Twenty outbreak strains from a Texas jail and Maryland day care center and 16 reference strains from Texas, Maryland, Washington, Michigan, Oklahoma, Missouri, Alaska, and Australia were examined. Although the Texas and Maryland outbreak strains were indistinguishable by IS1167 and boxA genotyping procedures, all strains examined were members of a genetically similar lineage. The microevolutionary history of pspA differed from that of the overall genetic background of the strains. Taken together, these findings suggested that the Texas and Maryland outbreaks were caused by different clones of a major genetic lineage of serotype 12F pneumococci, within which at least one PspA has been acquired via localized genetic recombination.