Pneumococcal Surface Adhesin Research Articles

Anti-PsaA and the risk of pneumococcal AOM and carriage

Pneumococcal surface adhesin A (PsaA) is one of the common protein antigens of Streptococcus pneumoniae investigated as a possible vaccine candidate on the basis of studies in experimental animal models. The relation between the serum anti-PsaA concentration collected at 6, 12 and 18 months of age and the risk of pneumococcal carriage and acute otitis media (AOM) in the following 6 months was evaluated in 329 children of the Finnish Otitis Media (FinOM) Cohort Study. A higher anti-PsaA concentration at all three time points studied was found to predict a higher risk of pneumococcal carriage 6 months later. A higher anti-PsaA concentration at 6 months also predicted a higher risk of pneumococcal AOM during the following 6 months (RR 1.51, 95% CI 1.24–1.83), whereas a higher anti-PsaA concentration at 12 or 18 months seemed to decrease the risk of pneumococcal AOM (RR 0.94 [95% CI 0.80–1.09] and RR 0.88 [95% CI 0.73–1.07], respectively). These relations remained the same when concomitant risk factors for pneumococcal AOM were included in the models. Previous pneumococcal AOM was the most important risk factor for a subsequent pneumococcal AOM (RR 5.93 [95% CI 2.87–12.3], RR 2.2 [95% CI 1.21–4.00], and RR 3.3 [95% CI 1.72–6.32] during the three periods).

Immunogenic protein contaminants in pneumococcal vaccines.

Currently available pneumococcal vaccines were examined for contamination by pneumococcal surface protein A (PspA) and pneumococcal surface adhesin A (PsaA). PspA could be detected in some (but not all) lots of 23-valent polysaccharide vaccine. Many lots of pneumococcal vaccines, including the heptavalent conjugate vaccine, were found to elicit small (but variable) antibody responses to PspA, PsaA, or both. The degree of contamination was highly variable, and this should be considered in pneumococcal vaccine evaluations or when capsular polysaccharide is used for pneumococcal antibody assays.

Inhibition of pneumococcal adherence to human nasopharyngeal epithelial cells by anti-PsaA antibodies.

The role of pneumococcal (Pnc) surface adhesin A (PsaA) in the adherence of Streptococcus pneumoniae (pneumococcus) to host cells is not well defined. We examined the effect of anti-PsaA antibodies in an inhibition of adherence assay using Detroit 562 nasopharyngeal human epithelial cells. Rabbit polyclonal (Pab) anti-recombinant PsaA (rPsaA) sera, a purified mouse monoclonal antibody (MAb) (MAb 6F62G8E12), and 22 healthy adult sera with known anti-PsaA IgG levels (obtained by enzyme-linked immunosorbent assay) were evaluated for their abilities to inhibit Pnc adherence to confluent monolayers (measured as percent reduction in CFU counts compared to those of uninhibited controls). Pnc adherence was dependent on capsular phenotype (no or low adherence for opaque strains). With an inoculum of 10(4) to 10(5) bacteria/well, the mean +/- standard deviation count in controls was 163 +/- 32 CFU/well for transparent strains. Low adherence was observed for a PsaA-minus mutant even at higher inoculum doses. Mean percent inhibitions of adherence with Pab and MAb were 54 and 50%, respectively. Adult sera showed inhibition in a dose-response fashion with a range of 98 to 8%, depending on the serum anti-PsaA antibody concentration. Absorption of Pab with rPsaA restored Pnc adherence to control levels. Absorption of sera with a PsaA-minus mutant did not result in a significant decrease (P >0.05) of inhibition of adherence activity. Additionally, nearly 100% of Pnc adherence was inhibited by lipidated rPsaA at 2.5 micro g/ml. Our data support the argument that PsaA is an adhesin that mediates Pnc adherence to human nasopharyngeal cells. This functional assay may be useful in evaluating antibodies elicited in response to PsaA vaccination.

Enhanced immunogenicity of pneumococcal surface adhesin A by genetic fusion to cytokines and evaluation of protective immunity in mice.

Immunization of mice with pneumococcal surface adhesin A (PsaA) emulsified in complete Freund's adjuvant (CFA) provides protection against systemic infection with Streptococcus pneumoniae. Because the use of CFA is not acceptable in humans, we sought to develop alternative means of enhancing the immunogenicity of protein antigens of potential use in pneumococcal vaccines. We designed a series of genetic constructs in which coding sequences for PsaA were linked to sequences encoding either murine interleukin-2 (mIL-2), mIL-4, or two copies of an immunostimulatory nonapeptide derived from mIL-1beta. The PsaA-cytokine constructs were cloned and expressed in Escherichia coli. Mice immunized twice with PsaA-IL-2, or PsaA-IL-4 responded with PsaA-specific antibody production comparable in magnitude to that of mice primed with PsaA in CFA and boosted with PsaA in incomplete Freund's adjuvant (PsaA-Adj). Antibodies elicited by PsaA-Adj were predominantly of the immunoglobulin G1 (IgG1) subclass, while PsaA-IL-2 and PsaA-IL-4 elicited substantial amounts of IgG2a in addition to IgG1. Mice immunized with PsaA-Adj or PsaA-IL-4 were partially protected against intraperitoneal challenge with virulent S. pneumoniae (30% overall survival beyond 15 days postchallenge). Mice immunized with PsaA and no adjuvant or PsaA-IL-2 exhibited 0 or 5% survival rates, respectively, following challenge. In contrast, mice immunized twice with capsular polysaccharide were 100% protected. The modest levels of protection seen in mice immunized with PsaA and its more immunogenic derivatives may be explained in part by the relative inaccessibility of antibody to PsaA on the surface of encapsulated S. pneumoniae.

Validation of immunoglobulin G enzyme-linked immunosorbent assay for antibodies to pneumococcal surface adhesin A in the diagnosis of pneumococcal pneumonia among adults in Kenya.

Epidemiologic studies of pneumococcal pneumonia, including vaccine efficacy trials, are hampered by a lack of sensitive and specific diagnostic tests. Pneumococcal surface adhesin A (PsaA) is a genetically conserved, surface-expressed protein common to all serotypes of Streptococcus pneumoniae and is highly immunogenic. Detection of anti-PsaA immunoglobulin G by recombinant PsaA enzyme-linked immunosorbent assay was evaluated for diagnosis of pneumococcal pneumonia in paired serum samples from 4 adult populations: 47 healthy control subjects, 56 clinic control subjects without pneumococcal disease syndromes, 109 patients with pneumococcal pneumonia, and 93 pneumonia patients with no evidence of pneumococcal etiology. By considering a 2-fold increase in antibody concentration as positive, sensitivity was 0.70, and specificity was 0.98. With a 1.3-fold increase, these were 0.89 and 0.98, respectively. The test's performance was not affected by the patients' human immunodeficiency virus status or by the pneumococcal serotype. The combination of high sensitivity and high specificity makes this an ideal assay for epidemiologic studies of pneumococcal pneumonia.

Cross-protective immunity of mice induced by oral immunization with pneumococcal surface adhesin a encapsulated in microspheres.

The global use of a capsular polysaccharide-based pneumococcal vaccine has been limited because of serotype-specific protection and poor effectiveness in individuals with low immunocompetency. The mucosal immune system develops earlier in infants and lasts longer in the elderly than does the systemic immune system. Furthermore, mucosal immunization is beneficial for AIDS patients, because human immunodeficiency virus-infected subjects can develop normal mucosal antibody responses even in late clinical phases. For these reasons, we evaluated recombinant pneumococcal surface adhesin A (rPsaA) of Streptococcus pneumoniae in terms of cross-protective immune responses after oral delivery. Encapsulated rPsaA provided higher immunogenicity than naked rPsaA. Coencapsulation or codelivery of the cholera toxin (CT) B subunit (CTB) and CT also increased the immunogenicity of rPsaA. Cross-protective immunities against five strains of S. pneumoniae (types 4, 6B, 14, 19F, and 23F) were induced after oral immunization with microencapsulated rPsaA. Lung colonization and septicemia caused by the five serotypes were significantly inhibited by oral immunization with microencapsulated rPsaA. These results suggest that rPsaA coencapsulated with CTB can be used as an oral vaccine to induce cross-protective immunity for the prevention of pneumococcal infection.

Inhibition of pneumococcal carriage in mice by subcutaneous immunization with peptides from the common surface protein pneumococcal surface adhesin a.

Pneumococcal surface adhesin A (PsaA), a common protein expressed on all 90 pneumococcal serotypes, is a vaccine candidate. Three anti-PsaA monoclonal antibody phage display-expressed monopeptides (15 mers), in various formulations as lipidated or nonlipidated multiantigenic peptides or as bi- or tripeptide constructs, were studied in a mouse nasopharyngeal carriage model to determine the inhibitory effect of induced antibodies on carriage of pneumococcal serotypes 2, 4, and 6B. Antibodies to each of the various peptides tested reduced carriage of the 3 serotypes. Reduction in carriage by nonlipidated multiantigenic peptide antibodies was highly variable (39%-94%; mean, 59%; standard deviation [SD], 20.2%); however, more-consistent results were observed in mice immunized with lipidated (56%-98%; mean, 69%; SD, 13.6%) and combination or bipeptide (55%-91%; mean, 76%; SD, 13.1%) formulations. These peptides are immunogenic, and their induced antibodies reduce carriage in mice. PsaA peptides demonstrate potential for being important new vaccines against pneumococcal carriage, otitis media, and invasive pneumococcal disease.

PsaA (pneumococcal surface adhesin A) and PspA (pneumococcal surface protein A) DNA vaccines induce humoral and cellular immune responses against Streptococcus pneumoniae

Streptococcus pneumoniae is one of the most important human pathogens and improvement of the currently used polysaccharide vaccines is being pursued. We constructed DNA vaccine vectors containing either the full-length psaA (pneumococcal surface adhesin A) or a truncated pspA (pneumococcal surface protein A — pspA′) gene. Both constructs showed transient expression of the antigens in vertebrate cells and induced significant antibody response to the pneumococcal antigens in BALB/c mice injected intramuscularly (i.m.). Fusion with an N-terminal cytoplasmatic SV40 T-antigen (CT-Ag), which was previously shown to stabilize poorly expressed antigens through association with Hsp73, also induced anti-PspA antibody response. The induction of antibodies with a low IgG1:IgG2a ratio and elevated gamma interferon (IFN-γ) production by spleen cells elicited by DNA vaccination indicate preferential priming of Th1 immunity. Since induction of antibodies against both PsaA and PspA was previously shown to correlate with protection against fatal infection with S. pneumoniae and cell-mediated immune responses could contribute to protection, further evaluation of PsaA and PspA as antigens for a DNA vaccine against S. pneumoniae could be promising.

Identification of thepsaAGene, Coding for Pneumococcal Surface Adhesin A, in Viridans Group Streptococci other thanStreptococcus pneumoniae

The gene encoding the pneumococcal surface adhesin A (PsaA) protein has been identified in three different viridans group streptococcal species. Comparative studies of the psaA gene identified in different pneumococcal isolates by sequencing PCR products showed a high degree of conservation among these strains. PsaA is encoded by an open reading frame of 930 bp. The analysis of this fragment in Streptococcus mitis, Streptococcus oralis, and Streptococcus anginosus strains revealed a sequence identity of 95, 94, and 90%, respectively, to the corresponding open reading frame of the previously reported Streptococcus pneumoniae serotype 6B strain. Our results confirm that psaA is present and detectable in heterologous bacterial species. The possible implications of these results for the suitability and potential use of PsaA in the identification and diagnosis of pneumococcal diseases are discussed.

Do antibodies to pneumococcal surface adhesin a prevent pneumococcal involvement in acute otitis media?

Antibodies to the pneumococcal (Pnc) surface protein PsaA are immunogenic and protective in experimental animal models, but their role in protection from Pnc disease in humans is not known. In the present study, the ability of antibodies to PsaA to prevent the progression of Pnc carriage to Pnc acute otitis media (Pnc AOM) was evaluated. Antibodies to PsaA were measured in acute-phase serum samples of children with AOM and with Streptococcus pneumoniae cultured from the nasopharynx. The risk of Pnc AOM was evaluated by a logistic regression model with anti-PsaA concentration as the predictive variable. Higher concentrations of antibodies to PsaA were associated with lower risk of the Pnc nasopharyngeal carriage progression to Pnc AOM. This was true in children 9-24 months old (odds ratio [OR], 0.49; 95% confidence interval [CI], 0.31-0.78) but not in children <9 months old (OR, 0.81; 95% CI, 0.48-1.35).

Antibody response to the pneumococcal proteins pneumococcal surface adhesin A and pneumolysin in children with acute otitis media.

Pneumococcal surface adhesin A (PsaA) and pneumolysin (Ply) are common to virtually all Streptococcus pneumoniae isolates, and they are immunogenic and protective against pneumococcal challenge in experimental animals. We have recently shown production of antibodies to PsaA and Ply in young children, but data on the immune response to these antigens during culture-confirmed pneumococcal infection are lacking. To evaluate whether young children respond to S. pneumoniae by producing antibodies to PsaA and Ply during acute otitis media (AOM). A cohort of 329 children was followed prospectively from the age of 2 months to the age of 2 years. Paired sera were obtained during episodes of AOM and used to measure antibodies to PsaA and Ply by enzyme-linked immunosorbent assay. S. pneumoniae cultured from the middle ear fluid was taken as evidence of pneumococcal AOM. The presence of S. pneumoniae in the nasopharyngeal aspirate collected in connection of AOM or any other respiratory infection or in the nasopharyngeal swab collected at scheduled visits was taken to indicate pneumococcal carriage and thus a history of previous contact with S. pneumoniae. Children with previous pneumococcal contacts had high anti-PsaA and anti-Ply concentrations in the acute phase sera regardless of the nature (AOM or carriage) of the current pneumococcal contact. Of the children with no previous pneumococcal contact, those with current pneumococcal AOM had lower antibody concentrations than those with current pneumococcal carriage only. Anti-PsaA and anti-Ply responses were found in children with current pneumococcal contact. The antibody response was strongly associated with low acute phase antibody concentration, but not significantly with age and the nature of the current pneumococcal contact. We showed that infants are capable of developing a specific antibody response to the pneumococcal proteins PsaA and Ply during AOM.

Streptococcus pneumoniae serotype 4 outbreak in a home for the aged: report and review of recent outbreaks.

To describe a pneumonia outbreak caused by Streptococcus pneumoniae among residents of a home for the aged and to review contemporary pneumococcal outbreaks. Epidemiological investigation. S pneumoniae isolates were serotyped and analyzed by pulsed-field gel electrophoresis. Paired sera were tested for antibodies to pneumococcal surface adhesin A protein (PsaA, a 37-kDa cell-wall protein). Pneumococcal outbreaks reported in the last decade in English were reviewed. Pneumonia developed in 18 of 200 residents. In 11 (61%), a pneumococcal etiology was demonstrated. S pneumoniae, serotype 4, was isolated from the blood cultures of 3 patients; all isolates were indistinguishable by pulsed-field gel electrophoresis. Pneumococcal involvement was established in 2 by sputum culture and latex agglutination of parapneumonic fluid and in 6 others by a twofold rise in optical density of serum antibody reactive to PsaA. Pneumococcal immunization had not previously been received by any patient; mortality was 22%. No additional cases were noted following administration of pneumococcal vaccine and antibiotic prophylaxis with penicillin or erythromycin. Twenty-six outbreaks of invasive pneumococcal disease since 1990 were reviewed. Twelve occurred in the United States, and serotypes 23F, 14, and 4 accounted for 8 (67%) of 12 outbreaks. All confirmed serotypes in US outbreaks are included in the 23-valent vaccine. More than one half of pneumococcal outbreaks worldwide involved elderly persons in hospitals or long-term-care facilities. A pneumococcal pneumonia outbreak occurred among unvaccinated residents of a residential facility for the aged. Institutionalized elderly persons are at risk of outbreaks of pneumococcal disease and should be vaccinated.

Pneumococcal surface adhesin A antibody concentration in serum and nasopharyngeal carriage of Streptococcus pneumoniae in young African infants

Pneumococcal surface adhesin A antibody concentration in serum and nasopharyngeal carriage of Streptococcus pneumoniae in young African infants

Natural Development of Antibodies to Pneumococcal Surface Protein A, Pneumococcal Surface Adhesin A, and Pneumolysin in Relation to Pneumococcal Carriage and Acute Otitis Media

Pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), and pneumolysin (Ply) are common to virtually all Streptococcus pneumoniae isolates. They are immunogenic and protective against pneumococcal challenge in animals and are the major candidates for a protein-based pneumococcal vaccine for humans. However, little is known of the natural development of antibodies to these proteins in humans. The objective of this study was to evaluate the natural development of antibodies to PspA, PsaA, and Ply in relation to pneumococcal infection and carriage in young children. Serum antibodies to these proteins were measured by EIA in children at ages 6, 12, 18, and 24 months and in their mothers. All age groups were capable of producing antibodies to the 3 proteins. The antibody concentrations increased with age and were strongly associated with pneumococcal exposure, whether by carriage or infection (acute otitis media).

Purification and characterization of Streptococcus pneumoniae palmitoylated pneumococcal surface adhesin A expressed in Escherichia coli

All Streptococcus pneumoniae isolates tested to date express a species-common lipoprotein designated as pneumococcal surface adhesin A (PsaA). This protein is cell-associated, hydrophobic, immunogenic, and genetically conserved. It is currently under investigation as a potential component in third-generation pneumococcal vaccine formulations. To overcome the problem of low-level expression of native hydrophobic PsaA in S. pneumoniae, and also of the recombinant PsaA (rPsaA) in Escherichia coli, we generated a stable E. coli construct expressing functional palmitoylated rPsaA (∼10 mg/l of fermentation culture) using Borrelia burgdorferi outer surface protein A (OspA, a hydrophobic lipoprotein) signal peptide. By Western blot analysis, the chimeric rPsaA (∼34 kDa) was detected in the cell lysate using anti-PsaA antibodies. It was partially purified by extracting the cell pellet with PBS/Triton X R-114 buffers, followed by anion exchange filter chromatography. A trypsin digestion profile of rPsaA closely resembled that of the native protein, as revealed by SDS–PAGE/silver staining. Lipidation of rPsaA was confirmed by labeling recombinant E. coli cells with [ 3H] palmitic acid and analyzing the labeled E. coli cells by Western blotting coupled with autoradiography. Further, analysis of purified rPsaA by mass spectrometry (MALDI-TOF) revealed a heterogenous spectrum with a major peak (M+H) +1 of mass 33,384 Da (theoretical mass of palmitoylated rPsaA=33,361 Da). Purified rPsaA was immunogenic in CBA/NCAHN-XID female mice following intranasal immunization with or without adjuvant, as determined by measurement of anti-PsaA serum IgG levels. These anti-PsaA antibodies reacted with both native and rPsaA polypeptides. Our data strongly suggest that E. coli-expressed rPsaA is palmitoylated and closely resembles the native protein in structure and immunogenicity. It was also observed to elicit measurable protection against nasopharyngeal carriage with S. pneumoniae.

Confirmation of psaA in all 90 serotypes of Streptococcus pneumoniae by PCR and potential of this assay for identification and diagnosis.

The gene encoding the pneumococcal surface adhesin A (PsaA) protein, psaA, was confirmed in all Streptococcus pneumoniae serotypes by a newly developed PCR (psaA PCR) assay. Eighty-nine of the 90 serotypes amplified produced an 838-bp fragment; the exception was a serotype 16F strain acquired from the American Type Culture Collection (ATCC). Analysis of 20 additional 16F strains from the United States and Brazil showed that the gene was amplified in all 16F strains, implying that the serotype 16F ATCC strain must be a variant. The specificity of the assay was verified by the lack of signal from analysis of heterologous bacterial species (n = 30) and genera (n = 14), including viridans group streptococci. The potential of the assay for clinical application was shown by its ability to detect pneumococci in culture-positive nasopharyngeal specimens. Demonstration of psaA in all 90 serotypes and lack of amplification of heterologous organisms suggest that this assay could be a useful tool for detection of pneumococci and diagnosis of disease.

Baculovirus Expression, Purification and Evaluation of Recombinant Pneumococcal Surface Adhesin A of Streptococcus pneumoniae

Pneumococcal surface adhesin A (PsaA), with a molecular mass of ∼37 kD by SDS-PAGE, is a common surface protein expressed by all 90 serotypes of Streptococcus pneumoniae. S. pneumoniae serotype 6B genomic DNA was amplified to generate a DNA fragment carrying the full-length psaA sequence and was cloned into a baculovirus expression system. We expressed either cell-associated or cell-free nonfusion PsaA polypeptides using two insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni 5B1-4 (High-Five). Recombinant PsaA (rPsaA) polypeptides were partially purified by partitioning in PBS/Triton X-114 buffers and by weakly basic ion exchange filter chromatography. Membrane-bound ‘hydrophobic rPsaA’ (hrPsaA) expressed by either Sf9 or High-Five cells had a molecular mass of ∼38 kD by SDS-PAGE and partitioned in a Triton X-114 phase, it reacted with both rabbit polyclonal and five monoclonal anti-PsaA antibodies by dot blot or Western blot analysis. High-Five-cell-expressed ‘soluble rPsaA’ (srPsaA) with a molecular mass of ∼37 kD by SDS-PAGE, was isolated from the serum-free culture medium and did not partition in the Triton X-114 phase; it reacted with anti-PsaA rabbit polyclonal and mouse monoclonal antibodies by ELISA and Western blot analysis. Both rPsaA polypeptide forms were immunogenic in Swiss-Webster adult female mice. In an infant mouse model of bacteremia, survival rates for mice given mouse anti-rPsaA immune serum (from mice immunized with High-Five-expressed srPsaA; 20 µl, 1:50,000 titer) 24 h before bacteremic challenge were greater than for the control group (48 h postchallenge, 20 vs. 90% survival rates) when challenged with S. pneumoniae serotype 6B. These results indicate that rPsaA is immunogenic and elicits protective antibody in mice similar to native protein.

Immunoreactivity of five monoclonal antibodies against the 37-kilodalton common cell wall protein (PsaA) of Streptococcus pneumoniae.

Five monoclonal antibodies (MAbs) were produced against the Streptococcus pneumoniae pneumococcal surface adhesin A (PsaA) 37-kDa common cell wall protein. These antibodies were used in a dot immunoblot and Western blot study of clinical isolates of S. pneumoniae to detect the presence of the protein. By both assays, the MAbs reacted with clinical isolates representing the 23 type-specific serotypes present in the licensed pneumococcal polysaccharide vaccine. Western blot analysis confirmed the presence of a protein migrating in the gel with a molecular mass of 37 kDa. An extension of the study by using dot immunoblot analysis that included an analysis of the 90 serotypes of S. pneumoniae showed that all five MAbs reacted with 89 of the 90 serotypes tested. MAb 1B6, the exception, did not react with S. pneumoniae serotype 16F. Dot immunoblot analysis of the MAbs with Enterococcus faecalis and viridans streptococci showed varied reactivity patterns, depending on the species. The MAbs against the 37-kDa antigen did not react with Escherichia coli, respiratory pathogens, or nonpathogens representing 22 genera and 29 species of bacteria. All five MAbs also reacted with five multidrug-resistant strains of S. pneumoniae. In summary, these MAbs may be useful for detection of pneumococcal antigen and may lead to the development of diagnostic assays for pneumococcal disease.

Limited diversity of Streptococcus pneumoniae psaA among pneumococcal vaccine serotypes.

The pneumococcal surface adhesin A (PsaA) is a surface-exposed protein of the gram-positive bacterium Streptococcus pneumoniae. It belongs to a group of proteins designated the lipoprotein receptor I antigen family. The gene encoding PsaA from an encapsulated strain of pneumococcal serotype 6B was cloned and sequenced. The peptide sequence was compared to that of homologs found in S. pneumoniae serotype 2, viridans streptococci, and Enterococcus faecalis. Identity values among the deduced peptides ranged from 57 to 98%. The polymorphism of psaA was examined among the 23 encapsulated vaccine serotypes by using PCR-restriction fragment length polymorphism analysis. Ten different enzymes were used to analyze 80 strains representing the 23 serotypes in a 23-valent polysaccharide vaccine. This analysis showed that restriction sites within the gene were highly conserved, with only a minor variation occurring in 10% of the strains, the result of an additional Tsp509I site. The lack of variation for the other restriction sites within the gene examined here indicates that psaA is genetically conserved, an important characteristic necessary for a candidate common protein vaccine.

Molecular analysis of putative pneumococcal virulence proteins.

Although the polysaccharide capsule has been recognized as a sine qua non of virulence, recent attention has focused on the role of pneumococcal proteins in pathogenesis, particularly in view of their potential as vaccine antigens. The contribution of pneumolysin, two distinct neuraminidases, autolysin, hyaluronidase, and the 37 kDa pneumococcal surface adhesin A has been examined by specifically mutagenizing the respective genes in the pneumococcal chromosome and examining the impact on virulence in animal models. The vaccine potential of these proteins has also been assessed by immunization of mice with purified antigens, followed by challenge with virulent pneumococci.