A murine model for the study of immune memory in response to pneumococcal conjugate vaccination.

We developed a murine model for assessment of immunological memory and antibody-induced protection to nasopharyngeal (NP) challenges. BALB/c female mice (n = 10 mice per study parameter) were immunized with two priming doses of the licensed 7-valent pneumococcal (Pnc) conjugate vaccine and immune responses [antibody immunoglobulin G (IgG) levels, avidity and opsonophagocytic activity] were monitored for 26 weeks until IgG levels decreased to nearly baseline. A booster dose of either 2 microg conjugate or 5 microg polysaccharide vaccine was given at week 26. The ability of these two treatments to recall immune memory established by the conjugate vaccine was determined for types 4 and 14 for up to 63 days post-booster. The ability of challenge with pneumococcal type 14 to recall the immune response was also evaluated, as well as, the number of antibody secreting cells (ASC) specific to polysaccharide (Ps) 4, 6B, and 14. A higher dose of conjugate vaccine (2 microg) was necessary to elicit a significant increase in IgG levels after priming with one dose. Priming with lower doses (0.5 and 1.0 microg) only elicited modest increases in IgG levels. Recall of the immune response was found with either conjugate or Ps vaccines. NP challenge with type 14 at week 26 did not recall the immune response, although reduction in NP Pnc load was seen post-primary immunization at 5, 10 and 26 weeks. ASCs were detected in response to either conjugate or Ps booster doses. This model allows for the screening and determination of potential alternative vaccination regimens and the study of immunological markers of memory following Pnc vaccination.

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

OBJECTIVE: To describe a pneumonia outbreak caused by Streptococcus pneumoniae among residents of a home for the aged and to review contemporary pneumococcal outbreaks. DESIGN: Epidemiological investigation. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Analysis of immunoreactivity to a Streptococcus equi subsp. zooepidemicus M-like protein To confirm an outbreak of poststreptococcal glomerulonephritis, and sequences of M-like proteins from isolates obtained from different host species.

The etiologic agent of a large 1998 outbreak of poststreptococcal acute glomerulonephritis (PSGN) in Nova Serrana, Brazil, was found likely to be a specific strain of Streptococcus equi subsp. zooepidemicus from contaminated cheese (S. Balter et al., Lancet 355:1776-1780, 2000). In the present study, we used a serologic screen for a known surface-exposed virulence factor to confirm the epidemiologic findings. Using primers flanking a previously characterized M-like protein gene (J. F. Timoney et al., Infect. Immun. 63:1440-1445, 1995), we amplified and sequenced the M-like protein (designated Szp5058) gene and found it to be identical among four independent acute-phase PSGN patient isolates. Convalescent-phase sera from 33 of 44 patients in the PSGN outbreak were found to contain antibodies highly reactive to a purified Szp5058 fusion protein, compared with 1 of 17 control sera (P < 0. 0001), suggesting that Szp5058 was expressed during infection and further implicating this strain as the cause of the PSGN outbreak. The predicted signal sequence and cell wall association motif of Szp5058 were highly conserved with the corresponding sequence from S. equi subsp. zooepidemicus SzpW60, while the predicted surface-exposed portions differed markedly between these two proteins. The 5' end of the szp5058 gene, including its variable region, was identical to the szp gene from another strain associated with a previous PSGN outbreak in England (M. Barham et al., Lancet i:945-948, 1983), and the corresponding szp sequence found from the Lancefield group C type strain isolated from a guinea pig. In addition, the hypervariable (HV) portion of szp5058 was identical to a previously published HV sequence from a horse isolate (J. A. Walker and J. F. Timoney, Am. J. Vet. Res. 59:1129-1133, 1998). Three other strains of S. equi subsp. zooepidemicus, including another strain previously associated with a PSGN outbreak, were each found to contain a distinct szp gene. Two of these szp genes had HV regions identical to szp regions from isolates recovered from different host species.

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).

Selection of an immunogenic and protective epitope of the PsaA protein of Streptococcus pneumoniae using a phage display library.

Streptococcus pneumoniae is an important pathogen that causes disease in young and elderly individuals. The currently available polysaccharide vaccines have limited efficacy in those age groups most susceptible to pneumococcal infections. This study focuses on mapping the epitopes of a surface protein of S. pneumoniae by biopanning a 15 mer phage display library using 5 different monoclonal antibodies (MAbs) against the Pneumoccal surface adhesin A (PsaA). PsaA is a component of the bacterial cell wall that is highly species specific and is involved in bacterial adherence and virulence. Biopanning of the phage display library reveals three distinct epitopes on the PsaA protein. The sequence homology of these epitopes ranges from two to six amino acids when compared to the native PsaA protein type 2. Two of these epitopes have been evaluated for their immunogeneicity in mice. The peptide selected by the MAbs 8G12, 6F6, and 1B7 is referred to as the consensus peptide and is immunogenic in mice. Optimal anti-PsaA response is observed in mice immunized with 50microg of the consensus peptide complexed to proteosomes in 1:1 ratio. The anti-PsaA response is significantly lower than the response to the PsaA native protein. The peptide selected by monoclonal antibody 4E9 in its lipidated form is significantly protective in mice challenged with S. pneumoniae serotype 2 when compared to mice immunized with the native protein. These results show that the selected epitopes of PsaA protein are immunogenic and protective in mice. These epitopes need to be evaluated further as alternatives to currently available vaccines.

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 ( approximately 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 ( approximately 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 [(3)H] 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.

Intranasal immunization of mice with a mixture of the pneumococcal proteins PsaA and PspA is highly protective against nasopharyngeal carriage of Streptococcus pneumoniae.

Acquisition of pneumococci is generally from carriers rather than from infected individuals. Therefore, to induce herd immunity against Streptococcus pneumoniae it will be necessary to elicit protection against carriage. Capsular polysaccharide-protein conjugates, PspA, and PsaA are known to elicit some protection against nasopharyngeal carriage of pneumococci but do not always completely eliminate carriage. In this study, we observed that PsaA elicited better protection than did PspA against carriage. Pneumolysin elicited no protection against carriage. Immunization with a mixture of PsaA and PspA elicited the best protection against carriage. These results indicate that PspA and PsaA may be useful for the elicitation of herd immunity in humans. As PspA and pneumolysin are known to elicit immunity to bacteremia and pneumonia, their inclusion in a mucosal vaccine may enable such a vaccine to prevent invasive disease as well as carriage.

Baculovirus expression, purification and evaluation of recombinant pneumococcal surface adhesin A of Streptococcus pneumoniae.

Pneumococcal surface adhesin A (PsaA), with a molecular mass of approximately 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 approximately 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 approximately 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 microl, 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.

Comparison of a pneumococcal common protein (PsaA) antibody ELISA and a PsaA immune complex ELISA for detection of pneumococcal serum antibody.

We examined and compared results from three assays, an enzyme-linked immunosorbent assay (ELISA) and two immune complex ELISAs for analysis of the serum antibody response to a native pneumococcal 37-kD common cell-wall protein by using acute- and convalescent-phase sera from 56 patients with community-acquired pneumonia. The sensitivities of the ELISA, the undissociated and dissociated immune complex assays were 85% (23 of 27), 78% (21 of 27) and 67% (18 of 27), respectively. To determine specificity, paired sera from patients with pneumonia of other bacterial etiologies were tested. The specificities were 83, 83 and 72% for the ELISA, undissociated immune complex, and dissociated immune complex, respectively. Based on this study, the sensitivities of the three assays were not statistically different. These tests could be used retrospectively to confirm invasive pneumococcal disease.

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.

Cloning and nucleotide sequence analysis of psaA, the Streptococcus pneumoniae gene encoding a 37-kilodalton protein homologous to previously reported Streptococcus sp. adhesins.

Gene psaA, which encodes the Streptococcus pneumoniae 37-kDa protein, was cloned in Escherichia coli, and its complete nucleotide sequence was determined. Analysis of the sequence of the 2.4-kb cloned fragment revealed three open reading frames (ORFs). ORF2, which is 933 bp long, was identified as psaA. The two other ORFs identified flank psaA. ORF1, located upstream of psaA, is 836 nucleotides long and encodes a protein with a calculated molecular mass of 29,843 Da. The sequence for ORF3, located downstream of psaA, was only partially determined. Northern (RNA) blot analysis of pneumococcal RNA suggests that psaA is transcribed as part of a polycistronic message. Analysis of the primary structure of the protein encoded by this gene indicated significant similarity to two previously reported streptococcal proteins, SsaB (80% similarity) and FimA (92.3% similarity), from S. sanguis and S. parasanguis, respectively. These two homologous proteins have been shown to be associated with bacterial adhesion, and the possibility of a similar role for PsaA is hypothesized.

Immunologic characterization and specificity of three monoclonal antibodies against the 58-kilodalton protein of Legionella pneumophila.

Three monoclonal antibodies against the Legionella pneumophila 58-kDa protein were produced. By using immunoblot analysis, the percentages of reactivity against 47 serogroups of Legionella representing 29 species were determined to be 80.9, 87.2, and 95.6 for monoclonal antibodies GB5BE8, GB5AF6, and CA4AF5, respectively. Specificities obtained from testing 63 heterologous organisms representing 22 genera and 46 species were 90.7, 92.2, and 95.3% for monoclonal antibodies GB5BE8, GB5AF6, and CA4AF5, respectively. No single heterologous strain was reactive with all three monoclonal antibodies. These monoclonal antibodies successfully identified all 10 clinical isolates of Legionella examined in a dot blot assay and should be excellent reagents for use in genuswide diagnostic immunoassays.

Nucleotide sequence of htpB, the Legionella pneumophila gene encoding the 58-kilodalton (kDa) common antigen, formerly designated the 60-kDa common antigen.

Gene htpB, which encodes the 58-kilodalton protein of Legionella pneumophila, was cloned in Escherichia coli and its complete nucleotide sequence was determined. Analysis of this sequence revealed an open reading frame of 1,644 nucleotides encoding a protein with a predicted molecular mass of 57,952 daltons. Data obtained by amino-terminal sequencing of the purified 58-kilodalton protein agreed, except for one amino acid residue, with the predicted amino acid sequence, identifying this open reading frame as htpB. A comparison of the primary structure of this protein to other proteins of similar molecular weights from E. coli, Mycobacterium leprae, M. tuberculosis, and Coxiella burnetii revealed significant regions of sequence similarity, which are discussed.

Evaluation of sera from patients with Lyme disease in the fluorescent treponemal antibody-absorption test for syphilis.

To determine whether the cross-reactivity between Treponema pallidum and Borrelia burgdorferi affects the specificity of the fluorescent treponemal antibody-absorption (FTA-Abs) test for syphilis, sera from patients with Lyme disease or syphilis were examined in a quantitative FTA-Abs test. Sera were diluted serially in phosphate-buffered saline, then in sorbent, and were tested with T. pallidum and B. burgdorferi antigens. Nine of 40 sera from patients with known Lyme disease were reactive at the 1:5 dilution with antigen from T. pallidum; only one serum was reactive at the 1:10 dilution. When both antigens were tested, the titer against B. burgdorferi was always higher than that against T. pallidum. Similarly, sera from patients with syphilis showed cross-reactivity with B. burgdorferi. Although reactivity could be absorbed with Treponemal phagedenis (Reiter strain), simultaneous titration with both antigens was easily performed and designated the etiologic agent.

Evaluation of a commercial gene probe for identification of Legionella cultures.

Confirmation of a culture as Legionella when it is unreactive with available serologic reagents involves tests that are impractical in most clinical laboratories. A nucleic acid probe that hybridizes only to members of the genus Legionella was recently prepared for marketing by Gen-Probe, Inc., San Diego, Calif. We tested 215 Legionella strains, representing 22 species, and 84 non-Legionella strains, representing 17 bacterial genera, with the Gen-Probe kit. All but four Legionella strains (L. bozemanii, less than 2% of total) and no heterologous strains gave positive test results. We conclude that the Legionella gene probe is a valuable addition to existing diagnostic tests for Legionella organisms.

Immunologic response of patients with legionellosis against major protein-containing antigens of Legionella pneumophila serogroup 1 as shown by immunoblot analysis.

Major protein-containing antigens of Legionella pneumophila serogroup 1 were were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis with rabbit antisera to 14 different Legionella species or serogroups. Fourteen bands were observed in immunoelectropherograms of whole-cell, sonicated cell, and heated cell preparations, seven of which appeared in the supernatant fluid from the heated cells and three of which were shown in an outer membrane fraction. Immunoblots of whole-cell antigen preparations of 14 Legionella species or serogroups revealed seven major Legionella proteins: antigens with molecular weights of 58,000, 79,000, and 154,000 were present in all Legionella sp. strains, antigens with molecular weights of 44,000 and 97,000 occurred in multiple species, and antigens with molecular weights of 14,000 and 25,000 were present only in L. pneumophila strains. All sera from 15 patients with culture-confirmed L. pneumophila serogroup 1 disease and 14 of 18 (78%) sera from serologically diagnosed patients reacted with the 58-kilodalton (kDa) common antigen. In contrast, less than one-half of the sera reacted with the L. pneumophila-specific proteins (14 and 25 kDa). Absorption of sera with Escherichia coli cells had no effect on their reactivity with the 58-kDa antigen, whereas absorption with L. pneumophila serogroup 1 cells removed reactivity. These data suggest that the 58-kDa antigen may prove useful in serodiagnostic tests for legionellosis.