Jacquelyn S. Sampson

Evaluation and improvement of real-time PCR assays targeting lytA, ply, and psaA genes for detection of pneumococcal DNA.

ABSTRACT The accurate diagnosis of pneumococcal disease has frequently been hampered not only by the difficulties in obtaining isolates of the organism from patient specimens but also by the misidentification of pneumococcus-like viridans group streptococci (P-LVS) as Streptococcus pneumoniae. This is especially critical when the specimen comes from the respiratory tract. In this study, three novel real-time PCR assays designed for the detection of specific sequence regions of the lytA, ply, and psaA genes were developed (lytA-CDC, ply-CDC, and psaA, respectively). These assays showed high sensitivity (<10 copies for lytA-CDC and ply-CDC and an approximately twofold less sensitivity for psaA). Two additional real-time PCR assays for lytA and ply described previously for pneumococcal DNA detection were also evaluated. A panel of isolates consisting of 67 S. pneumoniae isolates (44 different serotypes and 3 nonencapsulated S. pneumoniae isolates from conjunctivitis outbreaks) and 104 nonpneumococcal isolates was used. The 67 S. pneumoniae isolates were reactive in all five assays. The new real-time detection assays targeting the lytA and psaA genes were the most specific for the detection of isolates confirmed to be S. pneumoniae, with lytA-CDC showing the greatest specificity. Both ply PCRs were positive for all isolates of S. pseudopneumoniae, along with 13 other isolates of other P-LVS isolates confirmed to be non-S. pneumoniae by DNA-DNA reassociation. Thus, the use of the ply gene for the detection of pneumococci can lead to false-positive reactions in the presence of P-LVS. The five assays were applied to 15 culture-positive cerebrospinal fluid specimens with 100% sensitivity; and serum and ear fluid specimens were also evaluated. Both the lytA-CDC and psaA assays, particularly the lytA-CDC assay, have improved specificities compared with those of currently available assays and should therefore be considered the assays of choice for the detection of pneumococcal DNA, particularly when upper respiratory P-LVS might be present in the clinical specimen.

Intranasal Immunization of Mice with a Mixture of the Pneumococcal Proteins PsaA and PspA Is Highly Protective against Nasopharyngeal Carriage of Streptococcus pneumoniae

ABSTRACT Acquisition of pneumococci is generally from carriers rather than from infected individuals. Therefore, to induce herd immunity againstStreptococcus 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.

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).

Pneumococcal Surface Adhesin A (PsaA): A Review

Pneumococcal surface adhesin A (PsaA) is a surface-exposed common 37-kilodalton multi-functional lipoprotein detected on all known serotypes of Streptococcus pneumoniae. This lipoprotein belongs to the ABC-type transport protein complex that transports Mn2+; it is also an adhesin that plays a major role in pneumococcal attachment to the host cell and virulence. PsaA is immunogenic and natural nasopharyngeal colonization of pneumococci elicits an increase in antibody towards PsaA. Hence, PsaA is being actively evaluated as a component of a vaccine in formulations composed of pneumococcal common proteins. PsaA has been expressed as an E. coli recombinant protein, purified, and evaluated in a phase one clinical trial. This article reviews PsaA, its structure and role in pneumococcal virulence, immunogenicity, and potential to reduce nasopharyngeal colonization (a major prerequisite for pneumococcal pathogenesis) as a component of a common pneumococcal protein vaccine.

Inhibition of Pneumococcal Adherence to Human Nasopharyngeal Epithelial Cells by Anti-PsaA Antibodies

ABSTRACT 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 104 to 105 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 μg/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.

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.

Differentiation of Streptococcus pneumoniae Conjunctivitis Outbreak Isolates by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

ABSTRACT Streptococcus pneumoniae (pneumococcus [Pnc]) is a causative agent of many infectious diseases, including pneumonia, septicemia, otitis media, and conjunctivitis. There have been documented conjunctivitis outbreaks in which nontypeable (NT), nonencapsulated Pnc has been identified as the etiological agent. The use of mass spectrometry to comparatively and differentially analyze protein and peptide profiles of whole-cell microorganisms remains somewhat uncharted. In this report, we discuss a comparative proteomic analysis between NT S. pneumoniae conjunctivitis outbreak strains (cPnc) and other known typeable or NT pneumococcal and streptococcal isolates (including Pnc TIGR4 and R6, Streptococcus oralis, Streptococcus mitis, Streptococcus pseudopneumoniae, and Streptococcus pyogenes) and nonstreptococcal isolates (including Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus) as controls. cPnc cells and controls were grown to mid-log phase, harvested, and subsequently treated with a 10% trifluoroacetic acid-sinapinic acid matrix mixture. Protein and peptide fragments of the whole-cell bacterial isolate-matrix combinations ranging in size from 2 to 14 kDa were evaluated by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Additionally Random Forest analytical tools and dendrogramic representations (Genesis) suggested similarities and clustered the isolates into distinct clonal groups, respectively. Also, a peak list of protein and peptide masses was obtained and compared to a known Pnc protein mass library, in which a peptide common and unique to cPnc isolates was tentatively identified. Information gained from this study will lead to the identification and validation of proteins that are commonly and exclusively expressed in cPnc strains which could potentially be used as a biomarker in the rapid diagnosis of pneumococcal conjunctivitis.

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.

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.

Differential PsaA-, PspA-, PspC-, and PdB-specific immune responses in a mouse model of pneumococcal carriage.

ABSTRACT Larger numbers of pneumococci were detected in the nasal tract compared to the lung, cervical lymph nodes, and spleen 1, 2, 4, 7, 14, and 21 days after nasal challenge with Streptococcus pneumoniae strain EF3030. In this mouse model of pneumococcal carriage, peripheral S. pneumoniae pneumococcal surface adhesin A (PsaA)-specific humoral responses (immunoglobulin G2a [IgG2a] ≫ IgG1 = IgG2b > IgG3) were significantly higher than pneumococcal surface protein A (PspA)-specific, genetic toxoid derivative of pneumolysin (PdB)-specific, or pneumococcal surface protein C (PspC)-specific serum antibody levels. However, PspA-specific mucosal IgA antibody levels were significantly higher than those against PsaA, PdB, and PspC. In general, both PsaA- and PspA-specific lung-, cervical lymph node-, nasal tract-, and spleen-derived CD4+ T-cell cytokine (interleukin-4, interleukin-6, granulocyte-macrophage colony-stimulating factor, gamma interferon, and tumor necrosis factor alpha) and proliferative responses were higher than those for either PspC or PdB. Taken together, these findings suggest that PsaA- and PspA-specific mucosal responses as well as systemic humoral and T helper cell cytokine responses are predominantly yet differentially induced during pneumococcal carriage.