Immunogenicity of a Heptavalent Conjugate Pneumococcal Vaccine Administered Concurrently with a Combination Diphtheria,Tetanus, Five-Component Acellular Pertussis,Inactivated Polio,and Haemophilus influenzae Type b Vaccine and a Meningococcal Group C Conjugate Vaccine at 2, 3, and 4 Months of Age

The immunogenicities of conjugate pneumococcal vaccines have been demonstrated when they are administered at 2, 3, and 4 months of age. There is a paucity of data on the immunogenicity of this vaccine when it is administered concurrently with other vaccines in the primary immunization schedule of the United Kingdom. We immunized 55 term infants at 2, 3, and 4 months of age with the seven-valent pneumococcal conjugate vaccine (PCV7), the meningococcal group C conjugate (MCC) vaccine, and the diphtheria, tetanus, five-component acellular pertussis, inactivated polio, and Haemophilus influenzae type b (DTaP₅/IPV/Hib-TT) vaccine. The immune responses to the H. influenzae type b (Hib), MCC, and tetanus vaccines were measured at 2, 5, and 12 months of age; and the immune responses to PCV7 were measured at 2 and 5 months and then either at 12 months or following a 4th dose of PCV7. There were increases in the geometric mean concentrations (GMCs) of all antigens postimmunization. Greater than or equal to 90% of the infants achieved putatively protective levels postimmunization for all vaccine antigens except pneumococcal serotype 6B and Hib. The GMCs of the PCV7 serotypes increased following a 4th dose, although one infant had not reached putative levels of protection against serotype 6B. In conclusion, when infants were vaccinated according to the schedule described above, they had lower postprimary immunization responses to Hib, meningococcus group C capsular polysaccharide, and pneumococcal serotype 6B than the responses demonstrated by use of the other schedules. Despite this finding, there was a good response following a 4th dose of PCV7.The primary immunization schedule of the United Kingdom is continually evolving. While a vaccine may have been demonstrated to be immunogenic when it was administered according to one schedule, minor changes to that schedule can have an adverse impact on the response to the vaccine (2). In 2002, the chief medical officer of the United Kingdom recommended that infants considered to be at increased risk of invasive pneumococcal disease receive the seven-valent pneumococcal conjugate vaccine (PCV7) at 2, 3, and 4 months of age with their primary immunizations as well as a booster dose in the second year of life (8). In September 2004, the combined diphtheria, tetanus, five-component acellular pertussis, inactivated polio, and Haemophilus influenzae type b conjugate (DTaP₅/IPV/Hib-TT) vaccine was introduced into the United Kingdom primary immunization schedule.PCV7 has previously been demonstrated to have good immunogenicity when it is administered at 2, 4, and 6 months of age (3) and at 2, 3, and 4 months of age (7). There is a paucity of data examining the immunogenicity of PCV7 when it is administered concurrently with the DTaP₅/IPV/Hib-TT vaccine and a meningococcal group C conjugate (MCC) vaccine. Additionally, at the time of this study, no other immunizations were boosted during the second year of life, and a questionnaire survey of neonatal units suggested that many at-risk infants were not receiving the recommended booster dose (14).We therefore recruited healthy term infants to determine the immunogenicity of PCV7 when it was administered at 2, 3, and 4 months of age with the other vaccines in the primary immunization schedule of the United Kingdom in effect at that time. Additionally, we examined the effect of a booster dose in infants who responded poorly to the primary schedule and examined the antibody response at 12 months of age in infants who had had a good response to the primary immunization schedule.     

Immunogenicity of a Reduced Schedule of Meningococcal Group C Conjugate Vaccine Given Concomitantly with the Prevenar and Pediacel Vaccines in Healthy Infants in the United Kingdom

This study investigated the use of two doses of three different meningococcal group C conjugate (MCC) vaccines when given for primary immunization with a seven-valent pneumococcal conjugate vaccine (PCV7) and Pediacel, a combination product containing five acellular pertussis components, diphtheria and tetanus toxoids, Haemophilus influenzae type b (Hib) conjugate, and inactivated-poliovirus vaccine. The immune response after a single dose of MCC is also presented. Infants were randomized to receive two doses of one of the MCC vaccines and PCV7 at 2 and 3 months or at 2 and 4 months of age. Meningococcal group C serum bactericidal antibody (SBA) geometric mean titers, Hib-polyribosylribitol phosphate (PRP) immunoglobulin G (IgG) geometric mean concentrations (GMCs), and diphtheria and tetanus antitoxin GMCs, together with the proportions of infants achieving putative protective levels, were determined. A total of 393 infants were recruited. Following the first dose of NeisVac-C (MCC conjugated to tetanus toxoid), 97% of infants achieved protective levels (SBA titer of ≥8), compared with 80% and 53%, respectively, for Menjugate and Meningitec (both of which are conjugated to CRM₁₉₇). SBA responses to MCC vaccines were not significantly different when administered at 2 and 3 or 2 and 4 months of age. Following two doses of each MCC, 98 to 100% of infants achieved protective levels. Both PRP IgG and tetanus responses were significantly enhanced when Pediacel was coadministered with NeisVac-C. This study demonstrates that NeisVac-C and Menjugate generate good immunogenicity after the first dose at 2 months of age when coadministered with PCV7 and Pediacel and merit further investigation in single-dose priming strategies.In autumn 1999, the United Kingdom was the first country to introduce meningococcal group C conjugate (MCC) vaccines in the primary immunization schedule (18). Infants were vaccinated at 2, 3, and 4 months of age, receiving a combined diphtheria (D) and tetanus toxoid (TT), whole-cell pertussis (wP), and Haemophilus influenzae type b (Hib) conjugate vaccine (DTwP/Hib-TT) concomitantly with an MCC vaccine and an oral polio vaccine. Three different manufacturers'' monovalent MCC vaccines were used, two conjugated to CRM₁₉₇, a nontoxigenic natural variant of diphtheria toxin, and one conjugated to TT, all of which showed good immunogenicity under a 2-, 3-, and 4-month schedule (13, 22, 23). In 2004, wP was replaced by an acellular pertussis vaccine (aP) on the grounds of the reduced reactogenicity of the latter but subject to the availability of a combined DTaP/Hib-TT vaccine with pertussis efficacy equivalent to that of United Kingdom wP (19) and with a satisfactory Hib response (15). At the same time, the oral polio vaccine was replaced with an inactivated-poliovirus vaccine (IPV) since by then the risk of importation of polio into the United Kingdom from India or Africa had been greatly reduced by the efforts to achieve global eradication. The combination vaccine of choice was a DTaP₅/IPV/Hib-TT vaccine (Pediacel; Sanofi Pasteur). When given concomitantly with either MCC-TT or MCC-CRM₁₉₇, Pediacel was shown to give Hib responses within the range seen with the DTwP/Hib-TT vaccine (11).At this time, consideration was also being given to the inclusion of a seven-valent pneumococcal conjugate vaccine (PCV7) in the United Kingdom infant immunization program as a two-dose infant schedule with a booster after 12 months of age. PCV7 (Prevenar; Wyeth Vaccines) was licensed in the United Kingdom on a three-dose infant schedule with a booster dose to be given in the second year of life, based on an efficacy study in the United States that used a 2-, 4-, and 6-month schedule with a booster at 18 months (2). However, a recent United Kingdom study in which a nine-valent pneumococcal conjugate vaccine (PCV9) was given either at 2, 3, and 4 months or at 2 and 4 months, with concomitant DTaP₃/Hib-TT and MCC-CRM₁₉₇ vaccines (9), showed pneumococcal serotype-specific responses that were equivalent to those reported with the two- and three-dose courses and similar in magnitude to those reported after the third dose of PCV7 at 6 months in U.S. infants (2, 21, 24).There are similar data suggesting that a reduced number of doses of MCC are adequate for priming (4, 22, 26), and all three MCC vaccines are now licensed on a two-dose primary schedule from 2 months of age, with at least 2 months between doses. However, none of the MCC immunogenicity studies using a reduced schedule have included coadministration of PCV7 and Pediacel. This study was designed to determine whether a two-dose schedule of each of three MCC vaccines is acceptable when given with concomitant PCV7 and Pediacel and to investigate the optimal interval between doses. The study design also permitted the evaluation of the immunogenicity of a single dose of each of the three MCC vaccines in the hope of reducing the need for three injections to be given at the 4-month visit when Pediacel, PCV7, and MCC are coadministered.     

Impact of a Pneumococcal Conjugate Vaccination Program on Carriage among Children in Norway

In July 2006, the seven-valent pneumococcal conjugate vaccine (PCV7) was introduced in Norway with a reduced (2 doses + 1 boost) dose schedule. Post-PCV7 shifts in pneumococcal reservoirs were assessed by two point prevalence studies of nasopharyngeal colonization among children in day care centers, before (2006) and after (2008) widespread use of PCV7. Nasopharyngeal swabs were obtained from 1,213 children, 611 in 2006 and 602 in 2008. A total of 1,102 pneumococcal isolates were recovered. Serotyping, multilocus sequence typing, and antimicrobial drug susceptibility testing were performed on all isolates. Although carriage of PCV7 serotypes decreased among both vaccinated and unvaccinated children, the overall prevalence of pneumococcal carriage remained high (80.4%) after vaccine introduction. The pneumococcal populations were diverse, and in the shift toward non-PCV7 serotypes, expansion of a limited number of established clonal complexes was observed. While non-antimicrobial-susceptible clones persisted among PCV7 serotypes, antimicrobial resistance did not increase among non-PCV7 serotypes. Direct and indirect protection of PCV7 against nasopharyngeal colonization was inferred from an overall decrease in carriage of PCV7 serotypes. No preference was found for nonsusceptible clones among the replacing non-PCV7 serotypes.Streptococcus pneumoniae is a leading cause of otitis media, sinusitis, pneumonia, and meningitis worldwide (35). S. pneumoniae colonizes the nasopharynx and is considered a part of the normal flora in early childhood (1). Following the implementation of childhood vaccination with the seven-valent conjugated pneumococcal vaccine (PCV7), reports from several locations have described declines in carriage of the seven serotypes included in the vaccine, i.e., serotypes 4, 6B, 9V, 14, 18C, 19F and 23F (4, 6, 13, 19, 24). Due to reduced transmission of PCV7 serotypes, the incidence of invasive pneumococcal disease (IPD) declines also among the unvaccinated, which is an indirect effect of conjugate pneumococcal vaccination. However, the effect of PCV7 may in part be eroded over time as non-PCV7 serotypes emerge as a more frequent cause of IPD (11). In the United States, non-antimicrobial-susceptible clones seem to have an advantage among the emerging and expanding non-PCV7 serotypes, both in asymptomatic colonization and IPD (10, 20). This is primarily demonstrated by increasing incidence rates of drug-resistant clones of serotype 19A (23).PCV7 was introduced into the Norwegian childhood vaccination program in a dose schedule of two doses and one boost (2 + 1 dose schedule) in July 2006 and has been offered free of charge to all children born in 2006 and since. A high level of effectiveness of the vaccination program among children was demonstrated quickly after vaccine introduction, and the effect included a decline in IPD caused by erythromycin-resistant S. pneumoniae (34).As part of the Norwegian surveillance of PCV7 introduction, a cross-sectional study of nasopharyngeal carriage of Streptococcus pneumoniae among children attending day care centers (DCC) was performed in the early autumn of 2006. Data from this study, with exception of data regarding 38 vaccinated participants, have been reported previously (33). To assess the impact of the 2 + 1 dose schedule PCV7 vaccination program on carriage of S. pneumoniae, a follow-up was performed in 2008. Serotyping, antimicrobial susceptibility testing, and genotyping of the isolates from 2008 were performed, and the results were compared to those from analyses of the previous collection. Shifts in clonal compositions of the pneumococcal populations were analyzed and are reported here.Limited outpatient use of antimicrobial agents is recommended in Norway, and the levels of nonsusceptibility to antimicrobials among S. pneumoniae isolates from both local infections and IPD are low (25). Hence, emphasis has been put on post-PCV7 changes in nonsusceptibility to antimicrobials and nonsusceptible clones in a setting with limited antimicrobial use and resistance.     

Cellular Immune Responses of Preterm Infants after Vaccination with Whole-Cell or Acellular Pertussis Vaccines

Based on studies reporting specific antibody titers, it is recommended to vaccinate preterm infants against Bordetella pertussis according to their chronological age. However, as specific T-cell responses also are involved in the protection against B. pertussis, we have determined whether highly preterm infants (<31 weeks) are able to mount these immune responses during vaccination. Forty-eight premature infants were vaccinated at 2, 3, and 4 months of their chronological age with an acellular (Pa; n = 24) or a whole-cell (Pw; n = 24) tetravalent diphtheria-tetanus-pertussis-polio vaccine, and blood samples were collected at 2, 3, and 6 months of age. Most of the Pa- and Pw-vaccinated infants developed at 3 or 6 months of age a gamma interferon (IFN-γ) response to the B. pertussis antigens, accompanied by an interleukin-5 (IL-5) and IL-13 secretion for the Pa-vaccinated infants. No association was found between a very low infant birth weight, the occurrence of severe infections, and corticosteroid treatment or the administration of gammaglobulins with a low level of antigen-induced IFN-γ secretion. We conclude that like full-term infants, most preterm infants are able to mount a specific cellular immune response to the administration of the first doses of an acellular or a whole-cell pertussis vaccine.Preterm infants are at high risk of Bordetella pertussis infections, and this infection is more severe and is associated with a higher morbidity and mortality than it is in infants born at full term (11, 26). The American Academy of Pediatrics therefore recommends immunizing preterm infants at their chronological age with the same vaccine schedule as that recommended for full-term infants (22). These recommendations initially were based on antibody titers measured in studies performed on small numbers of infants who had received whole-cell pertussis vaccines (Pw) (2, 3, 10). Recent studies with the acellular pertussis vaccines (Pa) have confirmed that preterm infants mount antibody responses to the B. pertussis vaccine antigens, with titers in preterm infants reported to be lower (23) or similar (17, 24) to those obtained in full-term infants. However, these studies assessed only the humoral immune responses, although protection against pertussis relies both on humoral and on cellular Th1-type immune responses (1, 15, 16, 18).Until recently, doubt existed about the ability of infants to develop a specific Th1-type immune response and therefore adequate immune responses to an early administration of a B. pertussis vaccine. Infants most often are considered relatively deficient in their capacity to secrete gamma interferon (IFN-γ) (19, 25). However, some exceptions have been reported, including the ability to secrete IFN-γ in response to the major B. pertussis antigens during infection (14) or after vaccination (13, 5), indicating that infants are able to mount both antibody-specific and antigen-specific IFN-γ responses upon the infection or administration of pertussis vaccines.To our knowledge, in contrast to infants born at term, no data on the early cellular immune responses of preterm infants after the administration of the primary series of pertussis vaccines have been reported yet. Therefore, we do not know whether preterm infants are able to mount adequate specific immune responses to an early administration of a B. pertussis vaccine. In the context of the current resurgence of B. pertussis infections, such knowledge should help to offer the best vaccination strategy for preterm infants. We therefore assessed here the specific cellular immune responses, together with the humoral responses, in preterm infants with very low gestational age (VLGA; born at <31 weeks) that have received their first three doses of pertussis vaccines at their chronological ages. Immune responses induced by a Pa vaccine were compared to those induced by a Pw vaccine.     

Association of Serotypes of Streptococcus pneumoniae with Age in Invasive Pneumococcal Disease

A total of 7,764 isolates from patients with invasive pneumococcal disease (IPD) were collected from 1992 to June 2006. Data on serotypes were available for 5,022 isolates (64.7% of all invasive isolates). Some 54.0% of the isolates originated from adults ≥16 years of age, and 46.0% were from children <16 years of age. The leading serotypes were 14, 23F, 1, 6B, 7F, 3, and 4. The serotypes significantly more common in children were 14, 6B, 19F, and 18C, while among adults, serotypes 3 and 4 were predominant. Serotype 7F was statistically more prevalent among children <4 months old than among the other age groups. Among children aged ≥4 months and <1 year, serotype 19F occurred statistically more frequently; and among children aged ≥1 year to <5 years, serotypes 14, 6B, and 18C were overrepresented. The serotypes predominantly affecting patients younger than the remaining collective of patients were 14, 6B, 19F, and 18C, while patients with IPD caused by serotypes 3, 4, and 9V were older than the collective, on average.Streptococcus pneumoniae is among the most important pathogens in bacterial pneumonia, sepsis, and meningitis worldwide (1). The capsular polysaccharide of S. pneumoniae is known to be an important factor in the pathogenicity of the organism (7), and associations of the capsular serotypes with the severity of invasive pneumococcal diseases (IPDs) have been described (2, 14). IPDs are known to be much more frequent among young children and elderly persons than among older children and middle-aged adults (25). The results of several studies of the frequencies of pneumococcal serotypes and pneumococcal vaccine coverage have been published, but only few data on the serotype distribution among children and adults or different age groups are available. Studies from the United States (10, 19, 25), Canada (18), England and Wales (21), and Denmark (9), as well as the available meta-analyses (4, 15), refer to a diversity of age intervals. Although some studies contain statistics on the serotypes covered by the seven-valent pneumococcal conjugate vaccine (PCV7) and those not covered by the vaccine, no information on the association of individual serotypes with age are given. Another meta-analysis reported on the relative risk (odds ratio) of IPD caused by the various serotypes among all age groups subdivided into 10-year bands (26). Furthermore, in a 19-year nationwide surveillance study from Denmark, statistical analyses of the serotype distribution with age have been performed, but that study included only children aged 0 to 6 years (16). Some statistical data about individual serotypes and their association with age can also be found in the report of a study from the United States on the increased prevalence of pediatric pneumococcal serotypes in elderly adults. However, the study contained data only for adults aged ≥35 years (5).The aim of the study described here was to evaluate the association of serotypes of S. pneumoniae with age in IPD among isolates from all age groups sent to the German National Reference Center for Streptococci (NRCS) between 1992 and 2006.     

Kinetics of Antibody Persistence following Administration of a Combination Meningococcal Serogroup C and Haemophilus influenzae Type b Conjugate Vaccine in Healthy Infants in the United Kingdom Primed with a Monovalent Meningococcal Serogroup C Vaccine

The kinetics of antibody persistence following the administration of a combination meningococcal serogroup C and Haemophilus influenzae type b (Hib) conjugate vaccine (Menitorix) in the second year of life in children primed with two doses of one of three monovalent meningococcal serogroup C (MCC) vaccines was investigated. The study subjects were administered either Menitorix at 12 to 15 months of age, followed by the seven-valent pneumococcal conjugate vaccine (PCV7) and the measles, mumps, and rubella vaccine 4 to 6 weeks later, or all three vaccines concomitantly at 12 to 15 months of age. Blood samples were collected before and 1, 2, 12, and 24 months after the boosting. Sera were analyzed for meningococcal serogroup C serum bactericidal antibody (SBA) and IgG as well as Hib-polyribosylribitol phosphate (PRP)-specific IgG. The antibody persistence data from this study were compared to those of a prior study of Southern et al. (Clin. Vaccine Immunol. 14:1328-1333, 2007) in which children were given three primary doses of a vaccine containing both the MCC and the Hib vaccines but were boosted only with a Hib conjugate vaccine. The magnitude of the meningococcal SBA geometric mean titer was higher for those subjects primed with the MCC vaccine conjugated to tetanus toxoid (NeisVac-C) than for those primed with one of two MCC vaccines conjugated to CRM₁₉₇ (Menjugate or Meningitec) up to 1 year following boosting. Two years after boosting, the percentages of subjects with putatively protective SBA titers of ≥8 for children primed with NeisVac-C, Menjugate, and Meningitec were 43%, 22%, and 23%, respectively. Additional booster doses of the MCC vaccine may be required in the future to maintain good antibody levels; however, there is no immediate need for a booster during adolescence, as mathematical modeling has shown that persisting herd immunity is likely to control disease for a number of years.A booster dose of meningococcal serogroup C conjugate (MCC) and Haemophilus influenzae type b (Hib) conjugate vaccine was introduced in September 2006 in England and Wales for children in the second year of life in the form of a combined vaccine, Menitorix (GlaxoSmithKline [GSK]), which has tetanus toxoid (TT) as the carrier protein. In England and Wales, infants receive a combined diphtheria toxoid (D), TT, acellular pertussis (aP₅), inactivated poliovirus (IPV), and Hib-TT conjugate vaccine (DTaP₅/IPV/Hib-TT; Pediacel; Sanofi Pasteur) at 2, 3, and 4 months of age; MCC vaccination at 3 and either 4 or 5 months of age; and a seven-valent pneumococcal conjugate vaccine (PCV7; Prevenar; Wyeth Vaccines) at 2 and 4 months of age. Three different MCC vaccine manufacturers'' vaccines are available: two are conjugated to CRM₁₉₇, a nontoxigenic natural variant of diphtheria toxin (Meningitec [Wyeth Vaccines] and Menjugate [Novartis Vaccines]), and one is conjugated to TT (NeisVac-C; Baxter Bioscience). Although the data obtained following the administration of the current primary vaccine series in the United Kingdom have been reported (16), antibody persistence following boosting with Menitorix and priming with two doses of each of the licensed MCC vaccines has not been reported. This report details the immunogenicity data for those receiving the MCC and Hib vaccines, by primary MCC vaccine, for before and at 1, 2, 12, and 24 months after a booster dose of Menitorix administered at 12 to 15 months of age. The rates of antibody decline for those receiving the Hib and MCC vaccines are also compared.     

Temporal Analysis of Invasive Pneumococcal Clones from Scotland Illustrates Fluctuations in Diversity of Serotype and Genotype in the Absence of Pneumococcal Conjugate Vaccine

In September 2006, the seven-valent pneumococcal conjugate vaccine (PCV7; Prevenar) was introduced into the childhood vaccination schedule in the United Kingdom. We monitored the population of invasive pneumococci in Scotland in the 5 years preceding the introduction of PCV7 by using serogrouping, multilocus sequence typing (MLST), and eBURST analysis. Here, we present a unique analysis of a complete national data set of invasive pneumococci over this time. We observed an increase in invasive pneumococcal disease (IPD) caused by serotypes 1, 4, and 6 and a decrease in serogroup 14-, 19-, and 23-associated disease. Analysis of sequence type (ST) data shows a significant increase in ST306, associated with serotype 1, and a decrease in ST124, associated with serotype 14. There have also been increases in the amounts of IPD caused by ST227 (serotype 1) and ST53 (serotype 8), although these increases were not found to reach significance (P = 0.08 and 0.06, respectively). In the course of the study period preceding the introduction of PCV7, we observed considerable and significant changes in serogroup and clonal distribution over time.Streptococcus pneumoniae (the pneumococcus) is regarded as an opportunistic pathogen, as it may exist asymptomatically as part of the normal flora of the nasopharynx but is also considered an important global pathogen, causing otitis media, pneumonia, septicemia, and meningitis. Pneumococcal pneumonia is a major cause of childhood mortality in the developing world and of adulthood mortality worldwide. Pneumococci can be divided into more than 90 serotypes on the basis of the immunohistochemistry of their polysaccharide capsule. The heptavalent pneumococcal conjugate vaccine (PCV7) is now in use in many countries and contains seven of these serotypes (serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F). Although serotype is important in determining invasiveness (3), several studies have demonstrated that genotype also plays a significant role (19, 44, 45, 48); virulence factors of the pneumococcus have now been found to vary in presence and sequence according to serotype (28, 30) and genotype (38, 47). Multilocus sequence typing (MLST) is a sequence-based typing method that allows division of bacterial species by genotyping of housekeeping genes (13). This method has resulted in the recognition of over 3,000 pneumococcal sequence types (STs), or clones (http://www.mlst.net/).Fluctuations in the prevalences of serotypes and genotypes may occur naturally in pneumococcal populations in the absence of conjugate vaccine pressure (43). We and others have previously reported the clonal expansion of the serotype 1 clone ST306 in recent years (22, 30). In order to investigate changes in the pneumococcal population prior to introduction of PCV7 in the United Kingdom, we carried out temporal analysis of the invasive pneumococci for the 5 years leading up to the introduction of PCV7. In the United States, clonal expansion (the increase in the number of previously rare clones expressing nonvaccine serotypes) has also been documented (2). Nonvaccine serotypes have been reported in a number of postvaccine studies (11, 23, 27). It is not clear whether such changes are directly driven by vaccine pressure or are due in some part to the natural fluctuations that occur in this naturally transformable species.Here, we show, for the first time, changes in the epidemiology of pneumococcal disease immediately prior to the introduction of PCV7 in a whole country. This has the advantage of providing longitudinal data rather than a population snapshot. We used the eBURST (based upon related sequence types) algorithm to analyze clonal and serotype changes in pneumococcal isolates causing invasive disease in Scotland during the period from April 2001 to April 2006. We observed that trends in incidence of invasive pneumococcal disease (IPD) due to certain serotypes and clones were already occurring prior to introduction of PCV7, namely, that IPD associated with the nonvaccine serotype 1 ST306 clone has increased significantly over the 5-year period and that IPD associated with serotype 14 has decreased. eBURST analysis of each yearly data set has shown that the majority of clones isolated from IPD patients occur transiently and that a small number of stable clones, expressing serotypes represented in PCV7, cause the majority of invasive disease. eBURST analysis has also shown that most clonal complexes (CCs) are associated with one major serotype but that there is evidence for a small number of serotype switch events even in the absence of any conjugate vaccine. It should be noted that the 23-valent polysaccharide antipneumococcal vaccine (PPV23) was recommended for all those aged 65 and over in Scotland midway through the study period (winter 2003-2004) and therefore that the use of this vaccine may have affected the phenotypic and genotypic distributions of IPD-associated pneumococci observed here (37).     

Priming of Immunological Memory by Pneumococcal Conjugate Vaccine in Children Unresponsive to 23-Valent Polysaccharide Pneumococcal Vaccine

Pneumococcal polysaccharide vaccine (PPV) is of limited immunogenicity in infants and immunocompromised patients. Our prospective randomized controlled trial investigated whether priming with pneumococcal conjugate vaccine (PCV) induced specific immunological memory in previously nonresponders to PPV. Of a total of 33 children (2 to 18 years) with polysaccharide-specific immunodeficiency (PSI), group A (n = 16) received two doses of 7-valent PCV in a 4- to 6-week interval, and a booster dose of 23-valent PPV after one year. Group B (n = 17) received two doses of PPV in a 1-year interval exclusively. Specific antibody concentrations for serotypes 4, 5, 6B, 9V, 14, 18C, 19F, and 23F were determined (enzyme-linked immunosorbent assay) before and at 7 and 28 days after administration of the PPV booster and compared to an opsonophagocytosis assay. Of group A, 64 to 100% had antibody concentrations of ≥1 μg/ml on day 28 after the booster versus 25 to 94% of group B. Group A had significantly higher antibody concentrations for all PCV-containing serotypes already on day 7, indicating early memory response. Antibody concentrations were in accordance with functional opsonic activity, although opsonic titers varied among individuals. Pneumococcal vaccination was well tolerated. The incidence of airway infections was reduced after priming with PCV (10/year for group A versus 15/year for group B). Following a PPV booster, even patients primarily not responding to PPV showed a rapid and more pronounced memory response after priming with PCV.     

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.     

Serotype and Genotype Replacement among Macrolide-Resistant Invasive Pneumococci in Adults: Mechanisms of Resistance and Association with Different Transposons

The aim of this study was to analyze trends in adult invasive pneumococcal disease (IPD) due to macrolide-resistant strains and to study the evolution of serotypes, genotypes, and macrolide-resistant determinants of strains collected in a prospective study between 1999 and 2007 in Barcelona, Spain. IPD due to macrolide-resistant strains of serotypes included in the 7-valent conjugate vaccine (PCV7) decreased from 2.16/100,000 (pre-PCV7 period, 1999 to 2001) to 0.80/100,000 (late-PCV7 period, 2005 to 2007) (P = 0.001), whereas IPD due to macrolide-resistant strains of non-PCV7 serotypes increased from 1.08/100,000 to 2.83/100,000 (P < 0.001). These changes were related to a fall of clones of PCV7 serotypes (ST81 [P < 0.05], ST90, ST315, and ST17) and an increase in new clones of serotypes 19A and 24F (ST230) and 33F (ST717) in the late-PCV7 period. The most common phenotype was MLS_B (90.9%), related to the erm(B) gene. The frequent association between MLS_B phenotype and tetracycline resistance [tet(M) gene], was related to transposons of the Tn916-family such as Tn6002 or Tn3872. In conclusion, overall adult IPD rates due to macrolide-resistant pneumococci stabilized between 1999 and 2007 in Barcelona. The decrease in macrolide-resistant PCV7 pneumococci was balanced by the increase in macrolide-resistant non-PCV7 pneumococci.The worldwide increase in the prevalence of macrolide resistance (MR) in Streptococcus pneumoniae is a matter for concern, because of the risk of treatment failure in infections caused by these microorganisms (16). A global international surveillance project (PROTEKT, 2003 to 2004) found that the overall rate of erythromycin resistance was 37.2%, although significant geographical differences in these rates were observed (11). MR in S. pneumoniae is mediated mainly by two major mechanisms, namely, target site modification [encoded by the erm(B) gene and related to the MLS_B phenotype] and an efflux pump (encoded by mef genes and related to the M phenotype). Other, less-common resistance mechanisms are mutations in the 23S rRNA and/or alterations in ribosomal proteins (L4 and L22) (18). Most macrolide-resistant S. pneumoniae strains are also resistant to tetracycline. This association is due to the insertion of the erm(B) gene into conjugative and composite transposons of the Tn916 family (Tn1545, Tn3872, Tn6002, and Tn6003) which carry the tet(M) gene (32).Since the introduction of the pediatric heptavalent pneumococcal conjugate vaccine (PCV7), major changes in the epidemiology of invasive pneumococcal disease have been described. In the United States, the incidence of invasive pneumococcal disease (IPD) has decreased since PCV7 introduction, even in the nontarget population due to herd immunity (33). Since most PCV7 serotypes (6B, 9V, 14, 19F, and 23F) are usually penicillin and multidrug resistant, the rates of penicillin-resistant disease have decreased (17). Moreover, a decrease in macrolide-resistant IPD was observed in the United States (29), mainly due to a reduction of serotype 14 pneumococci harboring mef(E).In Spain, since the introduction of voluntary PCV7 vaccination in June 2001, the rates of IPD due to PCV7 serotypes disease have decreased in both children and adults (13). However, an overall increase in IPD due to non-PCV7 serotypes was observed between 2005 and 2007 in our geographical area (2, 24).The aim of the present study was to analyze trends in macrolide-resistant IPD and their relationship to serotypes, genotypes, and MR genes.     

Maternal Antibodies to Pneumolysin but Not to Pneumococcal Surface Protein A Delay Early Pneumococcal Carriage in High-Risk Papua New Guinean Infants

Immunization of pregnant women can be an efficient strategy to induce early protection in infants in developing countries. Pneumococcal protein-based vaccines may have the capacity to induce pneumococcal serotype-independent protection. To understand the potential of maternal pneumococcal protein-specific antibodies in infants in high-risk areas, we studied the placental transfer of naturally acquired antibodies to pneumolysin (Ply) and pneumococcal surface protein A family 1 and 2 (PspA1 and PspA2) in relation to onset of pneumococcal nasopharyngeal carriage in infants in Papua New Guinea (PNG). In this study, 76% of the infants carried Streptococcus pneumoniae in the upper respiratory tract within the first month of life, at a median age of 19 days. Maternal and cord blood antibody titers to Ply (ρ = 0.824, P < 0.001), PspA1 (ρ = 0.746, P < 0.001), and PspA2 (ρ = 0.631, P < 0.001) were strongly correlated. Maternal pneumococcal carriage (hazard ratio [HR], 2.60; 95% confidence interval [CI], 1.25 to 5.39) and younger maternal age (HR, 0.74; 95% CI, 0.54 to 1.00) were independent risk factors for early carriage, while higher cord Ply-specific antibody titers predicted a significantly delayed onset (HR, 0.71; 95% CI, 0.52 to 1.00) and cord PspA1-specific antibodies a significantly younger onset of carriage in PNG infants (HR, 1.57; 95% CI, 1.03 to 2.40). Maternal vaccination with a pneumococcal protein-based vaccine should be considered as a strategy to protect high-risk infants against pneumococcal disease by reducing carriage risks in both mothers and infants.Every year approximately 1 million children under 5 years of age die of pneumococcal pneumonia, meningitis, or sepsis, mostly in developing countries (4). Despite the efficacy of pneumococcal conjugate vaccines, Streptococcus pneumoniae remains an important cause of serious morbidity and mortality in young infants in developing countries (5, 8, 44), where the age of onset of disease is often younger than the recommended vaccination age of 6 weeks old and many of the serotypes causing serious disease are not included in currently available conjugate vaccines. Alternative vaccines and vaccine strategies are therefore needed to induce the earliest protection possible in high-risk infants.Early onset of pneumococcal colonization and prolonged carriage in the upper respiratory tract are believed to play important roles in the high incidence and early onset of pneumococcal diseases in children in developing countries (10, 25). In the highlands of Papua New Guinea (PNG), where this study was performed, all infants carry pneumococci in the upper respiratory tract by the age of 3 months old, and 60% of them are already carriers during the neonatal period at a median age of 17 days old (14). This is in contrast to high-income countries, where less than half of the children experience pneumococcal colonization within the first year of life (3, 10, 43). Besides children, pneumococcal carriage rates remain higher in adulthood in developing countries, including PNG, where approximately half of the adults carry pneumococci in the upper respiratory tract (17, 39), compared to 1 to 13% of adults in low-risk countries (12, 15, 20). Consequently, maternal pneumococcal carriage may be an important risk factor for early colonization in infants in high-risk areas, in particular considering the frequent and close contact between mother and child in the critical early period of life when infants are highly susceptible.In the first few months of life, when the human immune system is still highly immature (27), infants largely depend on passively acquired maternal immunoglobulin G (IgG) antibodies to protect themselves against invading pathogens. Immunization of pregnant women is a strategy that has been proven to reduce infection risks in both mothers and infants (9, 13, 45). This includes the potential to reduce acute lower respiratory illnesses in infants in high-risk areas, as shown with maternal immunization with the 23-valent pneumococcal polysaccharide vaccine (36, 37). However, the efficacy of pneumococcal polysaccharide vaccines on reducing nasopharyngeal colonization is limited, whereas the protective effect of pneumococcal conjugate vaccines is restricted by the number of pneumococcal serotypes that can be included. On the other hand, novel vaccines based on conserved pneumococcal proteins may offer better, serotype-independent protection against pneumococcal carriage and disease. This may include maternal immunization strategies, as supported by findings in mice (21).Pneumolysin (Ply) and pneumococcal surface protein A (PspA) are two conserved proteins that are expressed by virtually all S. pneumoniae isolates and that are being considered as vaccine candidates. Pneumolysin is the thiol-activated cytolysin produced by S. pneumoniae that enables the bacterium to penetrate the host''s physical defenses through its cytotoxic effect on epithelial cells, thus facilitating carriage and disease (28). PspA is a cell wall-associated protein that plays a role in inhibiting complement-mediated opsonization (7, 33) and can prevent lactoferrin-mediated clearance (19). In contrast to Ply, PspA shows structural diversity between pneumococcal strains and has been classified into three families based on the sequence variability of the most C-terminal 100 amino acids of the N-terminal domain of PspA. Although S. pneumoniae strains expressing family 1 or 2 PspA proteins account for 98% of clinical isolates, protective PspA-specific IgG antibodies binding to this highly variable region are family dependent (7).Both Ply and PspA have been shown to be highly immunogenic and to protect mice against disease and colonization following pneumococcal challenge (2, 6, 7, 11, 34). There is evidence that in humans naturally acquired IgA and IgG antibodies to PspA and Ply can mediate protection against subsequent pneumococcal carriage and disease (22, 24, 30, 31, 38, 46). Moreover, naturally acquired antibodies to Ply and PspA have been shown to be transferred from mother to child and to protect against early pneumococcal carriage and infection, at least in populations in low-risk areas (18, 38). It is not known whether these findings hold true for areas of high endemicity, where infants are at a considerably higher risk for early carriage and disease.In order to understand the role of maternal antibodies to Ply and PspA in protecting high-risk infants against early carriage, we studied antibody titers in paired maternal and cord blood samples in relation to the infant''s age of first pneumococcal nasopharyngeal carriage. We hypothesized that, compared to lower-risk settings, maternal Ply- and PspA-specific antibody titers would be higher and would be associated with a delay in the age of first pneumococcal carriage in the offspring.     

Age-Specific Cluster of Cases of Serotype 1 Streptococcus pneumoniae Carriage in Remote Indigenous Communities in Australia

Seven-valent pneumococcal conjugate vaccination commenced in 2001 for Australian indigenous infants. Pneumococcal carriage surveillance detected substantial replacement with nonvaccine serotypes and a cluster of serotype 1 carriage. Our aim was to review Streptococcus pneumoniae serotype 1 carriage and invasive pneumococcal disease (IPD) data for this population and to analyze serotype 1 isolates. Carriage data were collected between 1992 and 2004 in the Darwin region, one of the five regions in the Northern Territory. Carriage data were also collected in 2003 and 2005 from four regions in the Northern Territory. Twenty-six cases of serotype 1 IPD were reported from 1994 to 2007 in the Northern Territory. Forty-four isolates were analyzed by BOX typing and 11 by multilocus sequence typing. In the Darwin region, 26 children were reported carrying serotype 1 (ST227) in 2002 but not during later surveillance. Scattered cases of serotype 1 carriage were noted in two other regions. Cocolonization of serotype 1 with other pneumococcal serotypes was common (34% serotype 1-positive swabs). In conclusion, pneumococcal carriage studies detected intermittent serotype 1 carriage and an ST227 cluster in children in indigenous communities in the Northern Territory of Australia. There was no apparent increase in serotype 1 IPD during this time. The rate of serotype 1 cocolonization with other pneumococcal serotypes suggests that carriage of this serotype may be underestimated.Seven-valent pneumococcal conjugate vaccine (PCV7) immunization for infants and catch-up immunization programs commenced in 2001 for indigenous Australian children. The incidence of invasive pneumococcal disease (IPD) in indigenous children less than 2 years of age in the Northern Territory fell from approximately 600 cases/100,000 individuals to 264 cases/100,000 individuals after the introduction of PCV7 (9). Serotype replacement in IPD is not confirmed in this population as reported elsewhere (21). However, replacement with nonvaccine serotypes is evident in carriage, and it is responsible for continued pneumococcal carriage rates in excess of 80% among young indigenous children (15). Following the introduction of PCV7, our carriage surveys of children detected a shift to a serotype hierarchy led by serotype 16F (14) and a cluster of serotype 1 carriage.Streptococcus pneumoniae serotype 1 is an important contributor to IPD in children and adults in much of the world (6). There can be substantial variations in the proportion of serotype 1 IPD in studies from the same population in different years and from the same country at different sites. This is due to the serotype''s propensity to cause outbreaks of disease (7). Serotype 1 is associated with complicated pneumonia, pulmonary empyema, peritonitis, and salpingitis (reviewed in reference 8) and was identified in a highly lethal pneumococcal meningitis epidemic (27).Serotype 1 is not commonly detected in studies of colonization, presumably due to its short duration and low density of carriage (7). However, serotype 1 carriage can be evident during outbreaks of serotype 1 IPD. For example, a central Australian community outbreak of predominantly adult serotype 1 bacteremic pneumonia was related to nasopharyngeal (NP) colonization of 13 of 75 (17.3%) children with acute lower respiratory tract infection (4). Furthermore, in a closed community in southern Israel, serotype 1 carriage in 26 of 650 (4%) residents across all age groups was reported during an outbreak of serotype 1 disease in adults and children (2). We found few reports of NP serotype 1 carriage in the absence of a reported serotype 1 IPD outbreak, for example, among young children at an outpatient clinic in Mozambique (0.5% of 192 pneumococcal isolates) (24), among outpatients presenting with acute otitis media or pneumonia in Switzerland (1.7% of 1,540 isolates) (10), and among healthy children 7 years old or older in Turkey (4.3% of 117 isolates) (1).The aim of this study was to determine the molecular characteristics of serotype 1 pneumococci detected in various carriage studies and those from cases of IPD in indigenous Australians in remote communities in the Northern Territory of Australia. This included a 2002 cluster of serotype 1 carriage in children.     

Open-Label Trial of Immunogenicity and Safety of a 13-Valent Pneumococcal Conjugate Vaccine in Adults ≥50 Years of Age in Mexico

This open-label multicenter clinical trial conducted in Mexico assessed the immunogenicity and safety of a 13-valent pneumococcal conjugate vaccine (PCV13) in adults ≥50 years of age not previously vaccinated with the 23-valent pneumococcal polysaccharide vaccine (PPSV23). The PCV13 elicited a robust immune response in this study population, as reflected by the magnitude of fold rises in functional antibody levels measured by serotype-specific opsonophagocytic activity (OPA) assays before and 1 month after vaccination. Although the prevaccination OPA geometric mean titers (GMTs) for the majority of the serotypes were significantly lower in the 50- to 64-year age group than those in the ≥65-year age group, the postvaccination immune responses were generally similar. The overall immune responses were higher for the majority of the serotypes in the Mexican study population than those in similar adult study populations who received the PCV13 in Europe and the United States. PCV13 was well tolerated, and there were no vaccine-related serious adverse events. In conclusion, PCV13 is safe and immunogenic when administered to adults ≥50 years of age in Mexico and has the potential to protect against vaccine-type pneumococcal disease. (This study has been registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT01432262","term_id":"NCT01432262"}}NCT01432262.)     

Serotype distribution and antimicrobial resistance of invasive Streptococcus pneumoniae isolates among Croatian adults during a fifteen-year period (2005-2019)

AimTo assess serotype distribution, antibiotic resistance, and vaccine coverage against Streptococcus pneumoniae causing invasive infections in Croatian adults from 2005 to 2019.MethodsIn this retrospective study, invasive pneumococcal strains were collected through a microbiological laboratory network with country coverage >95%. Capsular typing was performed with the Quellung reaction. In vitro susceptibility testing was carried out according to the European Committee on Antimicrobial Susceptibility Testing guidelines. In macrolide-resistant isolates, the presence of ermB and mefA genes was evaluated.ResultsDuring the fifteen-year study period, 1123 invasive pneumococcal isolates were obtained. The most prevalent serotypes were 3, 14, 19A, 9V, 7F, and 23F, comprising 60% of all invasive pneumococcal isolates. Serotype 3 was the dominant serotype, with the highest prevalence in patients ≥65 years of age. Penicillin susceptibility, increased exposure was 18.6%, mostly associated with serotypes 14 and 19A. Resistance to penicillin was low (<1%). Macrolide resistance was 23%, mostly associated with serotypes 14, 19A, and 19F. The coverage with 13-valent conjugate vaccine (PCV13) and 23-valent polysaccharide vaccine (PPV23) was 80.2% and 93.6%, respectively.ConclusionsThe incidence of invasive pneumococcal disease in adults is highest in patients ≥65 years of age. Penicillin susceptibility, increased exposure and macrolide resistance were mostly associated with serotypes 14 and 19A. PCV13 and PPV23 provide very high serotype coverage. Future studies should evaluate the effects of the 10-valent vaccine, introduced in the Croatian National Immunization Program in June 2019, on serotype distribution and antibiotic resistance rates.

Streptococcus pneumoniae is among the most concerning human pathogens, with high morbidity and mortality rates worldwide. Pneumococcal infections range from non-invasive mucosal diseases (including acute otitis media, acute sinusitis, and pneumonia) to invasive, life-threatening infections (such as meningitis, sepsis, and bacteremic pneumonia) (1). Invasive pneumococcal disease (IPD) mostly affects children younger than 5 years and patients ≥65 years old (2). Every year, 500 000 children under 5 years of age die of IPD (3). Both morbidity and mortality rates are higher in developing countries (3). Community-acquired pneumonia is the most common pneumococcal disease worldwide, being responsible for more than 1.5 million of deaths annually. A significant fraction of these deaths are caused by Streptococcus pneumoniae (4,5).Antimicrobial resistance of Streptococcus pneumoniae is a growing global health problem, mostly affecting penicillin and macrolides. The patterns of antimicrobial susceptibility differ among serotypes and geographic regions (6). Penicillin resistance has emerged within a few decades after penicillin introduction and has spread worldwide (7). The prevalence of antibiotic-resistant Streptococcus pneumoniae has been increasing (8,9). In Europe, the resistance rate in France, Spain, and Eastern European countries is concerning (10). Worldwide, some regions, such as South Africa, have antibiotic non-susceptibility rates of up to 79% (11). However, in the past several years some countries have reported decreased resistance rates (12-15).Macrolide resistance is commonly present among invasive and non-invasive Streptococcus pneumoniae isolates. The main mechanisms are drug efflux system encoded by mef genes (M phenotype) and target modification mainly due to ermB genes, (MLS_B phenotype) (16,17).The new fluoroquinolones or respiratory quinolones (levofloxacin, gatifloxacin, and moxifloxacin) have enhanced in vitro activity against Streptococcus pneumoniae and are used to treat respiratory tract infections in adults. Increasing resistance to fluoroquinolones has been reported in Asia and Africa (18,19). In addition, ineffectiveness of fluoroquinolones in the treatment of pneumococcal infections is associated with acquired resistance of Streptococcus pneumoniae to this group of antibiotics (20,21).Increased resistance of Streptococcus pneumoniae to routinely used antibiotics warrants pneumococcal vaccine introduction as a tool for IPD prevention. In Europe, two pneumococcal vaccines are registered for use in adults: a 13-valent pneumococcal conjugate vaccine (PCV13, including serotypes 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F, 3, 6A, and 19A) and a 23-valent pneumococcal polysaccharide vaccine (PPV23, including PCV13 serotypes plus 1, 2, 5, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F) (22,23). In June 2019, a 10-valent pneumococcal conjugate vaccine (PCV10) was introduced in the Croatian National Immunization Program (NIP) for children only (scheme: 8 weeks – 16 weeks – 12 months) (24). In January 2021, the Croatian Public Health Institute revised its recommendations for pneumococcal vaccination of adults. Immunocompetent adults are now advised to be vaccinated with PPV23 only, while immunocompromised and asplenic patients are recommended to receive both vaccines, starting with PCV13 as the first dose (25). The aim of this study was to analyze the serotype distribution and antibiotic resistance of invasive Streptococcus pneumoniae isolates before the introduction of PCV10 in the childhood vaccination schedule, together with the coverage of currently available vaccines (PCV13 and PPV23). This study is the first and the most comprehensive so far in Croatia, analyzing invasive pneumococcal isolates collected during 15 consecutive years. These data will help us assess the impact of different vaccines in the IPD prevention among adults, especially those ≥65 years old.     

Serum Opsonic Activity in Infants with Sickle-Cell Disease Immunized with Pneumococcal Polysaccharide Protein Conjugate Vaccine

Pneumococcal infections are an important cause of morbidity and mortality in children with sickle-cell disease (SCD). Pneumococcal conjugate vaccines (PCVs) are immunogenic in healthy infants <2 years of age but have not been evaluated in young children with SCD. Infants with SCD were immunized with a 7-valent PCV (Wyeth-Lederle Vaccines & Pediatrics) at 2, 4, and 6 months of age. A booster dose of 23-valent pneumococcal polysaccharide vaccine (PPV; Pnu-Immune) was administered at 24 months of age. Antipneumococcal type 6B and 14 serum opsonic activity was measured to assess the biologic function of the antibody. Following the administration of three doses of PCV, opsonic activity against serotype 6B increased from 4.8% at 2 months to 33.5% at 7 months, with a subsequent decline to 8.1% at 12 months and 7.5% at 24 months and with an increase to 30.7% at 25 months after administration of a booster dose of PPV. Similar trends were seen with serotype 14 (opsonic activities were 9.4% at 2 months, 24.9% at 7 months, 16.5% at 12 months, and 12.6% at 24 months, and the opsonic activity was 27.3% 1 month after the administration of PPV). Serum opsonic activity correlated with antibody levels for both serotypes. PCV induces serum opsonic activity in infants with SCD. Antipneumococcal serum opsonic activity correlates with antibody levels.     

Optimization and Application of a Multiplex Bead-Based Assay To Quantify Serotype-Specific IgG against Streptococcus pneumoniae Polysaccharides: Response to the Booster Vaccine after Immunization with the Pneumococcal 7-Valent Conjugate Vaccine

We describe the optimization and application of a multiplex bead-based assay (Luminex) to quantify antibodies against polysaccharides of 13 pneumococcal serotypes. In the optimized multiplex immunoassay (MIA), intravenous immune globulin was introduced as an in-house reference serum, and nonspecific reacting antibodies were adsorbed with the commercial product pneumococcal C polysaccharides Multi. The antibody concentrations were assessed in 188 serum samples obtained pre- and post-booster vaccination at 11 months after administration of a primary series of the pneumococcal seven-valent conjugate vaccine (PCV-7) at 2, 3, and 4 months of age. The results of the MIA were compared with those of the ELISA for the serotypes included in the seven-valent conjugated polysaccharide vaccine and for a non-vaccine serotype, serotype 6A. The geometric mean concentrations of the antibodies determined by MIA were slightly higher than those determined by ELISA. The correlations between the assays were good, with R² values ranging from 0.84 to 0.91 for all serotypes except serotype 19F, for which R² was 0.70. The concentrations of antibody against serotype 6A increased after the administration of PCV-7 due to cross-reactivity with serotype 6B. The differences between the results obtained by ELISA and MIA suggest that the internationally established protective threshold of 0.35 μg/ml should be reevaluated for use in the MIA and may need to be amended separately for each serotype.In 2006, the pneumococcal 7-valent conjugate vaccine (PCV-7; Prevenar; Wyeth Vaccines, Pearl River, NY) was introduced into the National Immunization Program (NIP) in the Netherlands. The vaccine is administered to children at the ages of 2, 3, and 4 months and a booster is given at 11 months of age. During the prevaccination era, the seven serotypes covered by the vaccine accounted for approximately 60% of the cases of invasive pneumococcal disease among children 0 to 4 years in age in the Netherlands (14). Recently, new pneumococcal conjugate vaccines that protect against more serotypes than PCV-7 have been developed, including a 10-valent vaccine (PCV-10; Synflorix; GSK, Middlesex, United Kingdom) and a 13-valent vaccine (PCV-13; Wyeth Vaccines) It is anticipated that one of these vaccines will replace the current PCV-7.Efficacy and immunogenicity studies with PCV-7 demonstrated that it has 97% efficacy against invasive disease (3). A Finnish trial evaluated the efficacy of the vaccine against otitis media and found 57% efficacy against the vaccine serotypes (9). Most immunogenicity studies were performed by enzyme-linked immunosorbent assay (ELISA) (3, 4, 6), the “gold standard” for quantifying the concentrations of antibodies to pneumococcal serotype-specific polysaccharides. In 2000, guidelines for the pneumococcal ELISA were described in an international standard protocol, referred to as the WHO protocol (http://www.vaccine.uab.edu). This protocol was initially developed for evaluation of the immunogenicities of pneumococcal vaccines. On the basis of a meta-analysis of the results of the various vaccine trials (3, 8, 12, 15, 19), WHO recommends the use of an antibody concentration of 0.35 μg/ml as a correlate of protection if it is assessed by ELISA (29). This recommended protective concentration is identical for all serotypes.The standard reference serum used in the ELISA is lot 89S serum. This comprises a pool of serum samples from 17 adults immunized with the 23-valent pneumococcal polysaccharide vaccine (23). Quantification of the serotype-specific IgG concentrations present in lot 89S was performed by an antibody-capture reference ELISA (24, 25). This serum can be obtained through the FDA; however, the supply of this serum is finite, and the current stock is running low. A replacement for the current reference serum, 007sp, is currently being produced and characterized (10).The polysaccharides used in the ELISA also contain cell wall polysaccharides (CWPSs) that are covalently bound to the serotype-specific capsular polysaccharide by peptidoglycan (5, 28). Antibodies reacting to CWPSs are present in serum samples, but these antibodies do not offer functional protection against the pneumococcus and may cause a nonspecific signal in the assay (18, 30). To remove CWPS antibodies that may react with CWPS in the ELISA, CWPS is added as a sorbent (18). Additionally, polysaccharide 22F is added as a sorbent to remove antibodies against a second type of CWPS, designated CWPS2 (27). A mixture of CWPS and CWPS2, CWPS Multi, is now commercially available and is evaluated in this study.The Luminex technology is an upcoming method for quantification of pneumococcal antibodies and, in time, may replace the ELISA. In 2002, Pickering et al. first described the use of this technology for use with the pneumococcus (20). It allows the simultaneous measurement of the concentrations of antibodies directed against a large number of different capsular polysaccharides in a single assay. Here, we describe a multiplex bead-based assay that uses the Luminex technology to quantify antibodies against 13 pneumococcal polysaccharides simultaneously. The results obtained by this multiplex immunoassay (MIA) were compared with those obtained by ELISA for eight serotypes. In the assay, we used an in-house reference serum, intravenous immune globulin (IVIG). Furthermore, we used the newly available CWPS Multi to adsorb the non-type-specific anti-CWPS antibodies. For evaluation of the performance of the MIA developed, sera from children taken pre- and post-booster vaccination at 11 and 12 months of age were used.     

Immunization with a Combination of Three Pneumococcal Proteins Confers Additive and Broad Protection against Streptococcus pneumoniae Infections in Mice

Pneumococcal polysaccharide-based vaccines are effective in preventing pneumococcus infection; however, some drawbacks preclude their widespread use in developing and undeveloped countries. Here, we evaluated the protective effects of ATP-dependent caseinolytic protease (ClpP), pneumolysin mutant (ΔA146 Ply), putative lipoate-protein ligase (Lpl), or combinations thereof against pneumococcal infections in mice. Vaccinated mice were intraperitoneally and/or intranasally challenged with different pneumococcal strains. In intraperitoneal challenge models with pneumococcal strain D39 (serotype 2), the most striking protection was obtained with the combination of the three antigens. Similarly, with the intranasal challenge models, (i) additive clearance of bacteria in lungs was observed for the combination of the three antigens and (ii) a combination vaccine conferred complete protection against intranasal infections of three of the four most common pneumococcal strains (serotypes 14, 19F, and 23F) and 80% protection for pneumococcal strain 6B. Even so, immunity to this combination could confer protection against pneumococcal infection with a mixture of four serotypes. Our results showed that the combination vaccine was as effective as the currently used vaccines (PCV7 and PPV23). These results indicate that system immunization with the combination of pneumococcal antigens could provide an additive and broad protection against Streptococcus pneumoniae in pneumonia and sepsis infection models.Streptococcus pneumoniae (pneumococcus) commonly colonizes the upper respiratory tract asymptomatically and was estimated, in 2005, to kill 1.6 million people every year, most of whom were children aged <5 years in developing and undeveloped countries (36). As far as we know, 91 capsular polysaccharide serotypes have been identified in S. pneumoniae (33); among these, serotypes 23F, 19F, 14, and 6B are the four most epidemic strains worldwide (2, 5, 15, 17, 25, 26, 29). Moreover, and of recent concern, the widespread use of antibiotics, leading to the development of antibiotic resistance or multidrug resistance against S. pneumoniae, is increasing (9, 12, 26).Heptavalent protein-polysaccharide conjugate vaccine (PCV7) and 23-valent pneumococcal polysaccharide vaccine (PPV23) are the two vaccines currently being used against S. pneumoniae. Both of these vaccines are polysaccharide-based formulations and effective in preventing invasive pneumococcal infections; however, some drawbacks, such as high cost, the limited polysaccharides covered, poor immunogenicity in the very young and the very old, and serotype replacement (22, 24, 26, 36), limit their wider use.Alternatively, in an attempt to overcome the disadvantages of polysaccharide-based vaccines, a number of studies have been focusing on the screening and evaluation of protein-based vaccine candidates. Pneumococcal protein vaccine candidates, such as nontoxic pneumolysin derivates, pneumococcal surface proteins (PspA and PspC), pneumococcal surface adhesin (PsaA), and ATP-dependent caseinolytic proteases (ClpP), have been studied and shown to provide protection against S. pneumoniae. In addition, another surface protein, putative lipoate-protein ligase (Lpl), has been suggested to be a vaccine candidate, which could effectively elicit a high IgG titer and reduce the blood bacterial load (30). These vaccine candidates are shared by all S. pneumoniae. Of note, it is generally recognized that pneumolysin localized in the cytoplast in a soluble monomer, and its release was dependent on or independent of autolysin (3, 4, 18, 19). A recent study, which showed that pneumolysin was also partially localized on the cell wall (34), strengthened its utility as a vaccine candidate. Additive protections were obtained with combinations of these protein candidates. However, previous evaluations have been based only on intraperitoneal challenge models of pneumococcal disease and were not, to our knowledge, been performed in pneumonia models (7, 10, 13, 20, 23, 30, 37).In the present study, putative lipoate-protein ligase (Lpl), ClpP, and Ply toxoid were expressed, purified, and confirmed to express on all of the pneumococcal strains used here. A focal pneumococcal pneumonia model, mimicking the natural pneumococcal infection, was used to evaluate pneumococci on lung colonization. We also set up models of invasive diseases, which were used to evaluate their systemic protective effects against pneumococcal infections. System vaccination with the combination of three antigens was sufficient to provide complete protection against pneumococcal serotypes 14, 19F, and 23F. In addition, this vaccination regimen conferred protection against the intranasal infection of a mixture of serotypes 14, 6B, 19F, and 23F. We now report the details of the protective effects elicited by ΔA146 Ply, ClpP, Lpl, and combinations thereof against pneumococcal infections.     

Concentration and High Avidity of Pneumococcal Antibodies Persist at Least 4 Years after Immunization with Pneumococcal Conjugate Vaccine in Infancy

To provide more extensive evidence of long-term effects of vaccination on immunity against Streptococcus pneumoniae, a follow-up study of the Finnish Otitis Media (FinOM) Vaccine Trial was conducted. One of the objectives was to assess the persistence and avidity of pneumococcal antibodies 4 years after pneumococcal vaccination given in infancy. Children with complete follow-up in the FinOM trial up to 24 months of age were invited to a single visit in their fifth year of life. A blood sample was taken from all children for determination of anticapsular antibody concentrations to vaccine serotypes and avidity of antibodies to three serotypes. Children had been vaccinated at 2, 4, 6, and 12 months of age with 7-valent pneumococcal capsular polysaccharide, CRM197 conjugate vaccine (PCV7), or a control vaccine. Serum IgG antibody concentrations to vaccine serotypes remained significantly higher in children who had received PCV7 than in control children for 4 years after the fourth PCV7 dose. Concentrations of antibodies to frequently carried serotypes (6B and 19F) declined less than those of antibodies to a rarely carried serotype (4), suggesting that natural boosting contributed to antibody persistence. Furthermore, antibody avidity was significantly higher in PCV7 than control vaccine recipients. Four doses of PCV7 given in infancy elicit long-lasting antibody responses with high avidity. (This study has been registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT00378417","term_id":"NCT00378417"}}NCT00378417.)     

Evaluation of Pneumococcal Polysaccharide Immunoassays Using a 22F Adsorption Step with Serum Samples from Infants Vaccinated with Conjugate Vaccines

The history of the pneumococcal polysaccharide enzyme-linked immunosorbent assay (ELISA) is characterized by a continuous search for increased specificity. A third-generation ELISA that uses 22F polysaccharide inhibition has increased the specificity of the assay, particularly at low antibody concentrations. The present work compared various 22F ELISAs and non-22F ELISAs. The comparisons involved three different laboratories, including a WHO reference laboratory, and included sera from subjects from different geographic areas immunized with different pneumococcal conjugate vaccines, including the licensed 7-valent Prevenar vaccine and the 10-valent Synflorix vaccine. All comparisons led to the same conclusion that the threshold defined as 0.35 μg/ml for the WHO non-22F ELISA is lower when any 22F ELISA is used. The use of highly purified polysaccharides for coating further improved the specificity of the assay. In conclusion, we confirm that the 22F ELISA can be recommended as a reference method for the determination of antibodies against pneumococcal polysaccharides.It has been amply demonstrated that protection against the various manifestations of pneumococcal disease is mediated by antibodies induced to the type-specific pneumococcal polysaccharides (PSs) (10, 19). The biological function of these antibodies is to bind onto the surface of the pneumococcal cell and in so doing induce complement activation, leading to the uptake and killing of the pneumococcal cells by human phagocytic cells, particularly polymorphonuclear leukocytes (26). Such antibodies are referred to as being opsonic, and the whole process of uptake and killing is referred to as opsonophagocytosis. With the development of new pneumococcal conjugate vaccines (PCVs), it is critical that highly specific immunoassays be used to measure the vaccine-induced antibodies that are associated with protective immunity. This implies that the widely used anti-PS enzyme-linked immunosorbent assay (ELISA) should selectively measure those antibodies with opsonic activity. For that purpose, several improvements have been made to the initial ELISA, principally through the recognition of the role played by the antibodies directed against pneumococcal determinants other than the PSs.One of these determinants is the common cell wall polysaccharide (CWPS). CWPS is covalently linked to the serotype-specific capsular PS through as yet to be determined linkages (4, 9). Thus, when the pneumococcal serotype PS is purified, the CWPS is copurified. The PSs distributed by the American Type Culture Collection (ATCC) to laboratories that perform ELISA for the quantitation of antipneumococcal antibodies are the 23 PSs included in the 23-valent PS vaccine manufactured by Merck (Whitehouse Station, NJ) and contain CWPS as a contaminant.The preadsorption of postimmunization sera from adults and children with CWPS alone may not be sufficient for the measurement of antibody concentrations that are predictive of vaccine efficacy (24, 31), the reason being that there is a poor correlation between the antibody concentration and opsonic activity (coefficient of correlation [r] = about 0.5 rather than 0.8 or higher). Yu et al. reported in 1999 (31) that many heterologous pneumococcal PSs reduced the level of antibody binding of sera from PS vaccine-immunized adults to the serotype 6B PS over that removed by CWPS alone. They concluded that the antibodies removed by the heterologous pneumococcal PSs were against a novel epitope not found on CWPS. Importantly, they found that these antibodies against novel epitopes were not opsonic. They went on to suggest that the specificity of the pneumococcal PS ELISA could be improved by preadsorption with an unrelated pneumococcal PS, in addition to the CWPS. Soininen at al. (24) examined PSs obtained from the ATCC and from three manufacturers. They observed that the amount of cross-reactive antibody removed by preadsorption with heterologous pneumococcal PS in sera from both adults and infants differed depending upon the manufacturer of the purified pneumococcal PS used in the ELISA. This is in line with the observation that ATCC PS batches have varing degrees of contaminants (30). Xu et al. reported that CWPS is present in two forms: one form is bound and the other form is unbound to the capsular PS (29). Thus, it would be expected that those methods of PS purification that disassociate unbound CWPS would yield purer capsular PS.From the observations made above, Concepcion and Frasch recommended that each serum be doubly adsorbed: with CWPS, as previously recommended for the second-generation pneumococcal PS ELISA, and with a heterologous pneumococcal serotype PS (2). They recommended that the 22F PS be used for the second adsorption, because the 22F PS was not being considered for use in any of the pneumococcal conjugate vaccines and it was available from the ATCC. The use of 22F as the second adsorbent has now been evaluated in a number of laboratories (5, 7, 21, 23) and is included in the WHO-recommended third-generation pneumococcal ELISA (25).Some countries that have introduced the pneumococcal conjugate vaccine into their routine immunization schedules had instituted a reduced, accelerated schedule. For example, the United Kingdom has instituted two primary doses at 2 and 4 months of age and a booster at about 12 months of age. As noted above, adults have rather high levels of IgG to CWPS, and maternal antibodies will be more evident in the infant when a compressed or shorter immunization schedule is used. This means that the added 22F adsorption will be required for the measurement of serotype-specific antibodies after primary immunization when such schedules are used.Recommendations for the serological criteria to be considered for the licensure of new pneumococcal conjugate vaccines for use in infants were given as an appendix in volume 927 of the WHO Technical Report Series, published in 2005 (28). The serological criteria included the demonstration of noninferiority against a registered vaccine, with the primary end point being the IgG antibody concentration, as measured by ELISA, in sera from infants collected 4 weeks after a three-dose primary series, with a single threshold of IgG antibody concentration of 0.35 μg/ml for all serotypes. Of note, the threshold value was defined by using pooled immunogenicity and efficacy data from three different efficacy trials with invasive disease end points. The reference value was first proposed to be 0.20 μg/ml, on the basis of a vaccine efficacy trial conducted in Northern California (1, 8). When pooled data from three different efficacy trials with invasive disease end points performed in Northern California, in South Africa, and among Navajo Indians were taken into account (1, 8, 11, 14), an antibody concentration of 0.35 μg/ml, aggregated across the seven serotypes, was determined. However, the WHO agreed that alternative methods may be used for the validation of new conjugate vaccine formulations, and this may eventually lead to the establishment of an alternative threshold (28), but no guidance for bridging was provided. It was further agreed that other clinical end points (e.g., pneumonia or otitis media) would not be taken into account. Nevertheless, this reference threshold value, determined by using data from an ELISA without 22F adsorption, does not necessarily predict protection in an individual subject. A possible correlation between antibody concentrations in the range of 0.20 to 0.35, as measured by ELISA, and a titer of 1:8, as measured by an opsonophagocytosis assay (OPA), was mentioned. At last, the demonstration of the functional capacity of the antibody through determination of opsonophagocytic activity, as measured by OPA after a three-dose priming series, was defined as an important secondary end point. These recommendations published in 2005 have recently been confirmed in a 2009 WHO publication, based upon a consensus meeting held in July 2008 in Ottawa, Ontario, Canada (3).There is an important inconsistency in the 2003 WHO recommendations for serological criteria. The WHO-recommended third-generation ELISA, approved by WHO in 2000 and set up in two WHO reference laboratories, includes the routine use of the 22F adsorbent, as described by Wernette et al. (25). However, as noted above, the recommended threshold concentration was derived without the use of the 22F adsorbent.While the 22F adsorbent did not affect the antibody concentrations in sera from infants in the Northern California efficacy trial (20), 22F adsorption reduced the antibody concentrations over 30%, on average, in vaccinated children in the South African trial (13). In the case in which a reduced primary immunization schedule is used, it will be important to remove maternal unspecific nonopsonizing antibody. Henckaerts et al. (5) made observations comparable to those derived from the South African study.In order to further investigate the impact of 22F adsorption and improved ELISA coating conditions, a series of comparison ELISA measurements was carried out. These involved three different laboratories and used serum samples from pediatric subjects after immunization with different PCVs, including the Prevenar and Synflorix vaccines.     

PsrP,a Protective Pneumococcal Antigen,Is Highly Prevalent in Children with Pneumonia and Is Strongly Associated with Clonal Type

Invasive pneumococcal disease (IPD) is a major health problem worldwide. Due to ongoing serotype replacement, current efforts are focused in an attempt to identify the pneumococcal antigens that could be used in a next-generation multivalent protein vaccine. The objective of our study was to use real-time PCR to determine the distribution and clonal type variability of PsrP, a protective pneumococcal antigen, among pneumococcal isolates from children with IPD or healthy nasopharyngeal carriers. psrP was detected in 52.4% of the 441 strains tested. While no differences were determined when the prevalence of psrP in colonizing strains (n = 89) versus that in all invasive strains (n = 352) was compared, a strong trend was observed when the prevalence of psrP in all pneumonia isolates (n = 209) and colonizing isolates (P = 0.067) was compared, and a significant difference was observed when the prevalence in all pneumonia isolates and those causing bacteremia (n = 76) was compared (P = 0.001). An age-dependent distribution of psrP was also observed, with the incidence of psrP being the greatest in strains isolated from children >2 years of age (P = 0.02). Strikingly, the presence of psrP within a serotype was highly dependent on the clonotype, with all isolates of invasive clones such as clonal complex 306 carrying psrP (n = 88), whereas for sequence type 304, only 1 of 19 isolates carried psrP; moreover, this was inversely correlated with antibiotic susceptibility. This finding suggests that inclusion of psrP in a vaccine formulation would not target resistant strains. We conclude that psrP is highly prevalent in strains that cause IPD but is most prevalent in strains isolated from older children with pneumonia. These data support the potential use of PsrP as one component in a multivalent protein-based vaccine.Invasive pneumococcal disease (IPD), defined herein as the isolation of Streptococcus pneumoniae from normally sterile sites during a clinical syndrome of infection such as bacteremia/sepsis, pneumonia, or meningitis, is an important health problem worldwide. In the year 2000, it is estimated that there were 11 million to 18 million episodes/cases of IPD and 0.7 million to 1 million deaths in children younger than 5 years of age as a result (17). Streptococcus pneumoniae is a Gram-positive commensal that colonizes the nasopharynx of healthy children and, less frequently, adults. From the upper respiratory tract, the bacteria can be aspirated into the lungs and can translocate through mucosal cell barriers to the bloodstream and lead to development of IPD (18). This primarily occurs in young children, elderly individuals, and those who are immunocompromised.The ability of S. pneumoniae to cause IPD is dependent on the presence of a polysaccharide capsule that prevents phagocytosis (1). At least 92 chemically and immunologically distinct capsular types (i.e., serotypes) can be produced by the pneumococcus, with certain serotypes more frequently being associated with invasive disease (23). Importantly, while the capsule is requisite for IPD, it is insufficient alone to confer virulence; and an assortment of additional determinants such as adhesins, proteases, toxins, transport systems, and enzymes that modify the extracellular milieu are also required (25). This requirement for noncapsular virulence determinants is proven by human epidemiological studies that show that invasive and noninvasive clonotypes exist within the most invasive serotypes, comparative genomic analyses that find an unequal distribution of noncapsular genes between invasive and noninvasive isolates within the same serotype, and scores of studies that show that deletion of noncapsular genes impact pneumococcal virulence in animal models of pneumonia, sepsis, and meningitis (7, 11, 19, 22).One recently identified pneumococcal virulence determinant is the pneumococcal serine-rich repeat protein (PsrP), a lung cell and intraspecies bacterial adhesin that is encoded within the 37-kb pathogenicity island called psrP-secY2A2 (16). PsrP is an extremely large glycosylated cell surface protein that belongs to the serine-rich repeat protein (SRRP) family of Gram-positive bacteria (22). For the pneumococcus, the presence of PsrP has been positively correlated with strains that cause human disease, and PsrP has been shown to mediate adhesion to keratin 10 on lung cells and to mediate the formation of bacterial aggregates in the nasopharynges and lungs of infected mice (21, 22). Antibodies against PsrP neutralize bacterial adhesion to cells in vitro and inhibit biofilm formation (20, 21). Furthermore, passive immunization of mice with PsrP antiserum or active immunization with recombinant protein protected mice against pneumococcal challenge (20). Thus, PsrP is an important virulence factor by which S. pneumoniae is able to cause IPD and is potentially a vaccine candidate.At this time, considerable resources are being spent in an attempt to identify the pneumococcal antigens that would be used in a next-generation multivalent protein vaccine designed against the pneumococcus. The advantage of such a vaccine is that it would have a lower cost and potentially expanded global coverage compared with the cost and coverage of existing conjugate vaccines. It is generally accepted that multiple antigens will be necessary due to the fact that not all protein determinants are conserved or found within all pneumococcal strains and on their own are not able to confer sufficient protection. To this end, knowledge of the real prevalence of a protein in different clones and serotypes of Streptococcus pneumoniae is necessary to consider any protein as a candidate vaccine antigen. Therefore, the objective of our study was to determine the distribution and clonal type variability of PsrP among pneumococcal isolates from children with IPD or healthy nasopharyngeal carriers.