Scott E. Johnson

Epidemic Listeriosis — Evidence for Transmission by Food

The bacterium Listeria monocytogenes is a motile, gram-positive coccobacillus that can frequently be isolated from soil, water, and vegetation. It is a common cause of meningoencephalitis and abort...

Specific, sensitive, and quantitative enzyme-linked immunosorbent assay for human immunoglobulin G antibodies to anthrax toxin protective antigen.

The bioterrorism-associated human anthrax epidemic in the fall of 2001 highlighted the need for a sensitive, reproducible, and specific laboratory test for the confirmatory diagnosis of human anthrax. The Centers for Disease Control and Prevention developed, optimized, and rapidly qualified an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum. The qualified ELISA had a minimum detection limit of 0.06 µg/mL, a reliable lower limit of detection of 0.09 µg/mL, and a lower limit of quantification in undiluted serum specimens of 3.0 µg/mL anti-PA IgG. The diagnostic sensitivity of the assay was 97.8%, and the diagnostic specificity was 94.2%. A competitive inhibition anti-PA IgG ELISA was also developed to enhance diagnostic specificity to 100%. The anti-PA ELISAs proved valuable for the confirmation of cases of cutaneous and inhalational anthrax and evaluation of patients in whom the diagnosis of anthrax was being considered.

Competition among Streptococcus pneumoniae for intranasal colonization in a mouse model

Widespread use of conjugate vaccines against Streptococcus pneumoniae, by reducing carriage of S. pneumoniae serotypes included in the vaccine, may result in an increase in nasopharyngeal carriage of - and disease from - nonvaccine serotypes of the same species. Mathematical models predict that the extent of such replacement will depend positively on the degree to which carriage of vaccine-type S. pneumoniae inhibits acquisition of nonvaccine-type pneumococci, and may depend negatively on the inhibition of vaccine-type pneumococci by nonvaccine-type pneumococci. We used a mouse model of intranasal carriage of pneumococci to test whether such inhibition occurs between different pneumococcal strains. Mice carrying a streptomycin-resistant derivative of S. pneumoniae BG9163 (serotype 6B) as a resident strain showed reduced levels of colonization when challenged intranasally by optochin-resistant derivatives of the same strain and of a serotype 23F pneumococcus, BG8826. Inhibition could be overcome by increasing the dose of the challenge strain. Carriage of optochin-resistant BG9163 did not inhibit acquisition of the streptomycin-resistant variant. Colonization by a challenge strain did not significantly affect the level of colonization with the resident strain. These results provide evidence that is consistent with several hitherto untested assumptions of mathematical models of serotype replacement and suggest that a biological mechanism exists that could account for serotype replacement that is observed in clinical trials. The findings provide a basis for further studies of in vivo interactions between strains of S. pneumoniae.

Correlation of Opsonophagocytosis and Passive Protection Assays Using Human Anticapsular Antibodies in an Infant Mouse Model of Bacteremia for Streptococcus pneumoniae

An infant mouse assay system for assessment of protective concentrations of human serum pneumococcal anticapsular antibodies is described. Passive immunization of anticapsular antibodies was evaluated for protection of infant mice challenged with Streptococcus pneumoniae serotypes 1, 4, 5, 6B, 18C, and 23A, with bacteremia as an end point. Protection was defined as no detectable bacteremia in 70% of mice 48 h after challenge. Type-specific anticapsular concentrations required for protection varied with serotype (</=0.05 to >0.4 microg/mL). Across serotypes, there was no significant correlation between human IgG concentration in mouse serum and protection from bacteremia or between IgG concentration and opsonophagocytic titer. Significant correlation (r=.84, P<.001) was observed between opsonophagocytic titer of human IgG antibody in mouse sera and protection from bacteremia. Thus, protective concentrations of anticapsular antibodies against bacteremia are serotype dependent. Opsonophagocytosis is a better predictor of in vivo protective capacity of pneumococcal anticapsular antibodies than are ELISA IgG antibody concentrations.

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.

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.

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.

Predictors of Immunity after a Major Serogroup W-135 Meningococcal Disease Epidemic, Burkina Faso, 2002

BACKGROUND The African meningitis belt undergoes recurrent epidemics caused by Neisseria meningitidis serogroup A. During 2002, Burkina Faso documented the first large serogroup W-135 (NmW-135) meningococcal disease epidemic. To understand the emergence of NmW-135, we investigated meningococcal carriage and immunity. METHODS Immediately after Burkina Fasos epidemic, we conducted a cross-sectional survey of meningococcal carriage and seroprevalence in an epidemic and a nonepidemic district. We identified predictors of elevated NmW-135 serum bactericidal activity (SBA), a functional correlate of protection, using multivariate logistic regression. RESULTS The NmW-135 carriage rate was 25.2% in the epidemic district and 3.4% in the nonepidemic district (P<.0001). Compared with residents of the nonepidemic district, those of the epidemic district had higher geometric mean titers of NmW-135 SBA (P<.0001). NmW-135 SBA titers>or=1:8, an estimated protective threshold, were observed in 60.4% and 34.0% of residents of the epidemic and nonepidemic district, respectively (P=.0002). In a multivariate model, current NmW-135 carriage, age, and residence in the epidemic district were independent predictors of having an NmW-135 SBA titer>or=1:8. CONCLUSIONS Extensive NmW-135 carriage and transmission in the epidemic area caused residents to acquire natural immunity. Serial carriage and seroprevalence surveys could establish the duration of immunity in the population. The persistent circulation of NmW-135 underscores the potential for periodic NmW-135 epidemics in Africa.

Effects of Long-Term Storage on Plasmid Stability in Bacillus anthracis

ABSTRACT The plasmid profiles of 619 cultures of Bacillus anthracis which had been isolated and stored between 1954 and 1989 were analyzed using the Laboratory Response Network real-time PCR assay targeting a chromosomal marker and both virulence plasmids (pXO1 and pXO2). The cultures were stored at ambient temperature on tryptic soy agar slants overlaid with mineral oil. When data were stratified by decade, there was a decreasing linear trend in the proportion of strains containing both plasmids with increased storage time (P < 0.001). There was no significant difference in the proportion of strains containing only pXO1 or strains containing only pXO2 (P = 0.25), but there was a statistical interdependence between the two plasmids (P = 0.004). Loss of viability of B. anthracis cultures stored on agar slants is also discussed.

Concomitant administration of recombinant PsaA and PCV7 reduces Streptococcus pneumoniae serotype 19A colonization in a murine model.

A murine colonization model was used to determine the effect of co-administering 7-valent polysaccharide-protein conjugate vaccine and pneumococcal surface adhesin A. Mice were challenged intranasally with either PCV7 serotypes, 4 or 14, or a non-PCV7 serotype, 19A. Post-challenge samples were evaluated for IgG antibody levels, opsonophagocytic activity, and nasopharyngeal colonization. No interference was observed between immune responses from the concomitant and individual immunizations. Concomitant immunizations reduced carriage for tested serotypes; largest reduction was observed for 19A. From these mouse studies, co-administering pneumococcal antigens appear to expand coverage and reduce colonization against a non-PCV7 serotype without inhibiting immunogenicity to other serotypes.