Lea-Ann S. Kirkham

Construction and Immunological Characterization of a Novel Nontoxic Protective Pneumolysin Mutant for Use in Future Pneumococcal Vaccines

ABSTRACT Pneumolysin, the pore-forming toxin produced by Streptococcus pneumoniae, may have an application as an immunogenic carrier protein in future pneumococcal conjugate vaccines. Most of the 90 S. pneumoniae serotypes identified produce pneumolysin; therefore, this protein may confer non-serotype-specific protection against pneumococcal infections such as pneumonia, meningitis, and otitis media. However, as pneumolysin is highly toxic, a nontoxic form of pneumolysin would be a more desirable starting point in terms of vaccine production. Previous pneumolysin mutants have reduced activity but retain residual toxicity. We have found a single amino acid deletion that blocks pore formation, resulting in a form of pneumolysin that is unable to form large oligomeric ring structures. This mutant is nontoxic at concentrations greater than 1,000 times that of the native toxin. We have demonstrated that this mutant is as immunogenic as native pneumolysin without the associated effects such as production of the inflammatory mediators interleukin-6 and cytokine-induced neutrophil chemoattractant KC, damage to lung integrity, and hypothermia in mice. Vaccination with this mutant protects mice from challenge with S. pneumoniae. Incorporation of this mutant pneumolysin into current pneumococcal vaccines may increase their efficacy.

Identification of Invasive Serotype 1 Pneumococcal Isolates That Express Nonhemolytic Pneumolysin

ABSTRACT Recently, there has been an increase in invasive pneumococcal disease (IPD) caused by serotype 1 Streptococcus pneumoniae throughout Europe. Serotype 1 IPD is associated with bacteremia and pneumonia in Europe and North America, especially in neonates, and is ranked among the top five most prevalent pneumococcal serotypes in at least 10 countries. The currently licensed pediatric pneumococcal vaccine does not afford protection to this serotype. Upon screening of 252 clinical isolates of S. pneumoniae, we discovered mutations in the pneumolysin gene of two out of the four serotype 1 strains present in the study group. Analysis of an additional 28 serotype 1 isolates from patients with IPD from various Scottish Health Boards, revealed that >50% had mutations in their pneumolysin genes. This resulted in the expression of nonhemolytic forms of pneumolysin. All of the strains producing nonhemolytic pneumolysin were sequence type 306 (ST306), whereas those producing “wild-type” pneumolysin were ST227. The mutations were in a region of pneumolysin involved in pore formation. These mutations can be made in vitro to give the nonhemolytic phenotype. Pneumolysin is generally conserved throughout all serotypes of S. pneumoniae and is essential for full invasive disease; however, it appears that serotype 1 ST306 does not require hemolytically active pneumolysin to cause IPD.

Molecular surveillance of true nontypeable Haemophilus influenzae: An evaluation of PCR screening assays

Background Unambiguous identification of nontypeable Haemophilus influenzae (NTHi) is not possible by conventional microbiology. Molecular characterisation of phenotypically defined NTHi isolates suggests that up to 40% are Haemophilus haemolyticus (Hh); however, the genetic similarity of NTHi and Hh limits the power of simple molecular techniques such as PCR for species discrimination. Methodology/Principal Findings Here we assess the ability of previously published and novel PCR-based assays to identify true NTHi. Sixty phenotypic NTHi isolates, classified by a dual 16S rRNA gene PCR algorithm as NTHi (n = 22), Hh (n = 27) or equivocal (n = 11), were further characterised by sequencing of the 16S rRNA and recA genes then interrogated by PCR-based assays targeting the omp P2, omp P6, lgtC, hpd, 16S rRNA, fucK and iga genes. The sequencing data and PCR results were used to define NTHi for this study. Two hpd real time PCR assays (hpd#1 and hpd#3) and the conventional iga PCR assay were equally efficient at differentiating study-defined NTHi from Hh, each with a receiver operator characteristic curve area of 0.90 [0.83; 0.98]. The hpd#1 and hpd#3 assays were completely specific against a panel of common respiratory bacteria, unlike the iga PCR, and the hpd#3 assay was able to detect below 10 copies per reaction. Conclusions/Significance Our data suggest an evolutionary continuum between NTHi and Hh and therefore no single gene target could completely differentiate NTHi from Hh. The hpd#3 real time PCR assay proved to be the superior method for discrimination of NTHi from closely related Haemophilus species with the added potential for quantification of H. influenzae directly from specimens. We suggest the hpd#3 assay would be suitable for routine NTHi surveillance and to assess the impact of antibiotics and vaccines, on H. influenzae carriage rates, carriage density, and disease.

Presence of Nonhemolytic Pneumolysin in Serotypes of Streptococcus pneumoniae Associated with Disease Outbreaks

Pneumolysin is an important virulence factor of the human pathogen Streptococcus pneumoniae. Sequence analysis of the ply gene from 121 clinical isolates of S. pneumoniae uncovered a number of alleles. Twenty-two strains were chosen for further analysis, and 14 protein alleles were discovered. Five of these had been reported previously, and the remaining 9 were novel. Cell lysates were used to determine the specific hemolytic activities of the pneumolysin proteins. Six strains showed no hemolytic activity, and the remaining 16 were hemolytic, to varying degrees. We report that the nonhemolytic allele reported previously in serotype 1, sequence type (ST) 306 isolates is also present in a number of pneumococcal isolates of serotype 8 that belong to the ST53 lineage. Serotype 1 and 8 pneumococci are known to be associated with outbreaks of invasive disease. The nonhemolytic pneumolysin allele is therefore associated with the dominant clones of outbreak-associated serotypes of S. pneumoniae.

Predominance of nontypeable Haemophilus influenzae in children with otitis media following introduction of a 3 + 0 pneumococcal conjugate vaccine schedule

In Australia the 7-valent pneumococcal conjugate vaccine (PCV7) is administered at 2, 4 and 6 months of age, with no booster dose. Information on bacterial carriage and the aetiology of recurrent acute otitis media (rAOM) after introduction of PCV7 using the 3+0 schedule is required to evaluate the potential impact of second generation pneumococcal vaccines. We found that 2-4 years after introduction of PCV7 in the National Immunisation Program, nontypeable Haemophilus influenzae (NTHi) was the predominant pathogen isolated from the nasopharynx and middle ear of children with a history of rAOM. Compared with healthy controls (n=81), NTHi and Streptococcus pneumoniae carriage rates were significantly higher in children with a history of rAOM (n=186) (19% vs. 56% p<0.0001 and 26% vs. 41%, p=0.02, respectively). Carriage of PCV7 pneumococcal serotypes was rare, whereas PCV7-related and non-PCV7 serotypes were isolated of 38% of cases and 24% of controls. Serotype 19A was the most common serotype isolated from the nasopharynx and middle ear and accounted for 36% (14/39) of total pneumococcal isolates with reduced susceptibility to cotrimoxazole. Of the 119 children carrying NTHi, 17% of isolates were β-lactamase positive. The scarcity of PCV7 serotypes in children with and without a history of rAOM indicates that the 3+0 PCV7 schedule is preventing carriage and rAOM from PCV7 serotypes. Introduction of new vaccines in Australia with increased pneumococcal serotype and pathogen coverage, including 19A and NTHi, should decrease the circulation of antibiotic-resistant bacteria and reduce the burden of rAOM.

Neutrophil Extracellular Traps and Bacterial Biofilms in Middle Ear Effusion of Children with Recurrent Acute Otitis Media – A Potential Treatment Target

Background Bacteria persist within biofilms on the middle ear mucosa of children with recurrent and chronic otitis media however the mechanisms by which these develop remain to be elucidated. Biopsies can be difficult to obtain from children and their small size limits analysis. Methods In this study we aimed to investigate biofilm presence in middle ear effusion (MEE) from children with recurrent acute otitis media (rAOM) and to determine if these may represent infectious reservoirs similarly to those on the mucosa. We examined this through culture, viability staining and fluorescent in situ hybridisation (FISH) to determine bacterial species present. Most MEEs had live bacteria present using viability staining (32/36) and all effusions had bacteria present using the universal FISH probe (26/26). Of these, 70% contained 2 or more otopathogenic species. Extensive DNA stranding was also present. This DNA was largely host derived, representing neutrophil extracellular traps (NETs) within which live bacteria in biofilm formations were present. When treated with the recombinant human deoxyribonuclease 1, Dornase alfa, these strands were observed to fragment. Conclusions Bacterial biofilms, composed of multiple live otopathogenic species can be demonstrated in the MEEs of children with rAOM and that these contain extensive DNA stranding from NETs. The NETs contribute to the viscosity of the effusion, potentially contributing to its failure to clear as well as biofilm development. Our data indicates that Dornase alfa can fragment these strands and may play a role in future chronic OM treatment.

Trivalent influenza vaccine and febrile adverse events in Australia, 2010: Clinical features and potential mechanisms

INTRODUCTION Increased numbers of children presenting with febrile adverse events following trivalent influenza vaccine (TIV) were noted in Australia in 2010. We describe the epidemiology and clinical features of the adverse events and explore the biological basis for the adverse events using an in vitro model. MATERIALS AND METHODS Children presenting to a tertiary paediatric hospital in 2010 with adverse events within 72 h of TIV were retrospectively reviewed. Demographics, clinical features, physiological variables and outcomes were examined. Plasma cytokine and chemokine levels were examined in a subgroup of children with vaccine-related febrile convulsions. Peripheral blood mononuclear cells of age-matched children were stimulated with different TIV preparations. Inflammatory cytokine and chemokine analysis was performed on cultured supernatants. RESULTS Vaccine-related febrile adverse events were identified in 190 children. Most occurred in healthy children (median age: 1.5 years) within 12 h of vaccination. Twenty-eight (14.7%) required hospital admission. High temperature ≥39.0 °C (101/190; 53%), vomiting (120/190; 63%) and convulsions (38/190; 20%) were common. All children presenting had received Fluvax(®) or Fluvax Junior(®). In the in vitro model, IFN-α, IL-1β, IL-6, IL-10, IP-10 and MIP-1α levels were significantly higher when measured at 6 and 24 h in cultures stimulated with Fluvax(®) compared with alternative 2010 TIV preparations. CONCLUSIONS Numerous febrile adverse events (including febrile seizures) were observed following Fluvax(®) or Fluvax Junior(®) in 2010. Clear differences in cytokine production were observed when peripheral blood mononuclear cells were stimulated with Fluvax(®) compared with alternate TIV preparations. Increased awareness of these potential adverse events is required to ensure earlier detection and prevention in the future.

Identification of a Secreted Cholesterol-Dependent Cytolysin (Mitilysin) from Streptococcus mitis

We have detected a cholesterol-dependent cytolysin, which we have named mitilysin, in a small number of Streptococcus mitis isolates. We have sequenced the mitilysin gene from seven isolates of S. mitis. Comparisons with the pneumococcal pneumolysin gene show 15 amino acid substitutions. S. mitis appear to release mitilysin extracellularly. Certain alleles of mitilysin are not recognized by a monoclonal antibody raised to the related toxin pneumolysin. Based on enzyme-linked immunosorbent assay and neutralization assay results, one isolate of S. mitis may produce a further hemolytic toxin in addition to mitilysin. As genetic exchange is known to occur between S. mitis and Streptococcus pneumoniae, this finding may have implications for the development of vaccines or therapies for pneumococcal disease that are based on pneumolysin.

High detection rates of nucleic acids of a wide range of respiratory viruses in the nasopharynx and the middle ear of children with a history of recurrent acute otitis media

Both bacteria and viruses play a role in the development of acute otitis media, however, the importance of specific viruses is unclear. In this study molecular methods were used to determine the presence of nucleic acids of human rhinoviruses (HRV; types A, B, and C), respiratory syncytial viruses (RSV; types A and B), bocavirus (HBoV), adenovirus, enterovirus, coronaviruses (229E, HKU1, NL63, and OC43), influenza viruses (types A, B, and C), parainfluenza viruses (types 1, 2, 3, 4A, and 4B), human metapneumovirus, and polyomaviruses (KI and WU) in the nasopharynx of children between 6 and 36 months of age either with (n = 180) or without (n = 66) a history of recurrent acute otitis media and in 238 middle ear effusion samples collected from 143 children with recurrent acute otitis media. The co‐detection of these viruses with Streptococcus pneumoniae, nontypeable Haemophilus influenzae, and Moraxella catarrhalis was analyzed. HRV (58.3% vs. 42.4%), HBoV (52.2% vs. 19.7%), polyomaviruses (36.1% vs. 15.2%), parainfluenza viruses (29.4% vs. 9.1%), adenovirus (25.0% vs. 6.1%), and RSV (27.8% vs. 9.1%) were detected significantly more often in the nasopharynx of children with a history of recurrent acute otitis media compared to healthy children. HRV was predominant in the middle ear and detected in middle ear effusion of 46% of children. Since respiratory viruses were detected frequently in the nasopharynx of both children with and without a history of recurrent acute otitis media, the etiological role of specific viruses in recurrent acute otitis media remains uncertain, however, anti‐viral therapies may be beneficial in future treatment and prevention strategies for acute otitis media. J. Med. Virol. 83:2008–2017, 2011.

Nasopharyngeal Carriage of Haemophilus haemolyticus in Otitis-Prone and Healthy Children

ABSTRACT Haemophilus haemolyticus is often incorrectly categorized as nontypeable Haemophilus influenzae (NTHI) upon culture. PCR analyses of 266 NTHI-like nasopharyngeal isolates from children with and without recurrent acute otitis media (rAOM) revealed that 11.7% were H. haemolyticus and 9.4% gave equivocal results. Children with rAOM were more likely to carry H. haemolyticus.