Construction and immunological characterisation of a non-toxic form of pneumolysin for use in pneumococcal vaccines

There are 90 different serotypes of Streptococcus pneumoniae and current pneumococcal vaccines are somewhat limited in their protection against invasive pneumococcal disease. The adult pneumococcal vaccine is composed of capsule polysaccharide from 23 of the most common disease causing serotypes. Infants are the major 'at risk' group from pneumococcal disease; however, polysaccharide-based vaccines are not protective in this age group. This has led to the development of a paediatric conjugate vaccine, which is composed of polysaccharide from seven of the most prevalent disease causing serotypes individually conjugated to a carrier protein. Although the conjugate vaccine is highly efficacious, it only elicits protection against disease caused by the seven homologous pneumococcal serotypes. Serotype specific immunisation is only a short-term solution to combating pneumococcal disease. Problems with serotype replacement have already arisen within five years since licensure of the paediatric vaccine, with non-vaccine serotypes replacing the eliminated vaccine serotypes. A solution to this is the development of pneumococcal vaccines containing a species wide pneumococcal protein to elicit nonserotype specific protection, Pneumolysin, the pore-forming toxin produced by S. pneumoniae, may have an application in fixture pneumococcal vaccines as it is produced by all invasive isolates, is a major virulence factor and has been demonstrated to confer non-serotype specific protection against pneumococcal disease. The toxicity of pneumolysin is problematic in terms of vaccine use and existing pneumolysin mutants possess residual cytotoxicity. By mutating a region involved in protein oligomerisation, pneumolysin pore formation was abolished. This resulted in a non-toxic form of pneumolysin that retained the immunogenicity of wild type pneumolysin without the associated effects such as hypothennia, inflammatory cytokine production and vascular leakage following intranasal administration to mice. Vaccination of mice with this pneumolysin toxoid elicited high titres of neutralising antibody, which significantly protected animals from pneumococcal infection. Conjugation of the pneumolysin toxoid to capsule polysaccharide from serotype 4 S. pneumoniae elicited full protection against infection from the homologous serotype. This indicated that the pneumolysin toxoid was as effective as the carrier protein used in the current pneumococcal conjugate vaccine. Active vaccination with free pneumolysin toxoid significantly increased survival times in mice following challenge with a non-vaccine serotype. This research implies that combining conjugated and free pneumolysin toxoid may be more efficacious than current vaccines in protection against pneumococcal disease. The importance of the pore-forming property of pneumolysin in pathogenesis of pneumococcal disease was investigated by construction of a pneumococcal strain carrying the same mutation used to construct the pneumolysin toxoid for vaccination. Pore formation was found not to be important for pathogenesis during murine pneumococcal pneumonia. This was further supported by the identification of serotype 1 strains from clinical disease that expressed non pore-forming pneumolysin yet were isolated from patients with invasive pneumococcal disease

Production of IgG2 Antibodies to Pneumococcal Polysaccharides After Vaccination of Treated HIV Patients May Be Augmented by IL-7Rα Signaling in ICOS+ Circulating T Follicular-Helper Cells

Greater understanding of factors influencing the maturation of antibody responses against pneumococcal polysaccharides (PcPs) may improve pneumococcal vaccination strategies. Although PcPs are type 2 T cell-independent antigens thought not to induce follicular immune responses, we have previously shown that IgG2 antibody responses against antigens in the 23-valent unconjugated PcP vaccine (PPV23) are associated with expansion of ICOS+ circulating T follicular helper (cTFH) cells in HIV seronegative subjects but not HIV patients. As IL-7Rα signaling in CD4+ T cells may affect TFH cell function and is adversely affected by HIV-1 infection, we have examined the relationship of IL-7Rα expression on ICOS+ cTFH cells with PcP-specific IgG2 antibody responses. PPV23 vaccination was undertaken in HIV patients receiving antiretroviral therapy (n = 25) and HIV seronegative subjects (n = 20). IL-7Rα expression on ICOS+ and ICOS− cTFH cells was assessed at day(D) 0, 7, and 28. Fold increase between D0 and D28 in serum IgG1 and IgG2 antibodies to PcP serotypes 4, 6B, 9V, and 14 and the frequency of IgG1+ and IgG2+ antibody secreting cells (ASCs) at D7 were also assessed. Decline in IL-7Rα expression on ICOS+ cTFH cells between D0 and D7 occurred in 75% of HIV seronegative subjects and 60% of HIV patients (Group A), with changes in IL-7Rα expression being more pronounced in HIV patients. Group A patients exhibited abnormally high IL-7Rα expression pre-vaccination, an association of serum IgG2, but not IgG1, antibody responses with a decline of IL-7Rα expression on ICOS+ cTFH cells between D0 and D7, and an association of higher IgG2+ ASCs with lower IL-7Rα expression on ICOS+ cTFH cells at D7. As decline of IL-7Rα expression on CD4+ T cells is an indicator of IL-7Rα signaling, our findings suggest that utilization of IL-7 by cTFH cells affects production of IgG2 antibodies to PPV23 antigens in some HIV patients

Understanding the aetiology and resolution of chronic otitis media from animal and human studies

Inflammation of the middle ear, known clinically as chronic otitis media, presents in different forms, such as chronic otitis media with effusion (COME; glue ear) and chronic suppurative otitis media (CSOM). These are highly prevalent diseases, especially in childhood, and lead to significant morbidity worldwide. However, much remains unclear about this disease, including its aetiology, initiation and perpetuation, and the relative roles of mucosal and leukocyte biology, pathogens, and Eustachian tube function. Chronic otitis media is commonly modelled in mice but most existing models only partially mimic human disease and many are syndromic. Nevertheless, these models have provided insights into potential disease mechanisms, and have implicated altered immune signalling, mucociliary function and Eustachian tube function as potential predisposing mechanisms. Clinical studies of chronic otitis media have yet to implicate a particular molecular pathway or mechanism, and current human genetic studies are underpowered. We also do not fully understand how existing interventions, such as tympanic membrane repair, work, nor how chronic otitis media spontaneously resolves. This Clinical Puzzle article describes our current knowledge of chronic otitis media and the existing research models for this condition. It also identifies unanswered questions about its pathogenesis and treatment, with the goal of advancing our understanding of this disease to aid the development of novel therapeutic interventions

Pneumococcal carriage, serotype distribution, and antimicrobial susceptibility in Papua New Guinean children vaccinated with PCV10 or PCV13 in a head-to-head trial

Background: Children in Papua New Guinea (PNG) are at high risk of pneumococcal infections. We investigated pneumococcal carriage rates, serotype distribution, and antimicrobial susceptibility in PNG children after vaccination with 10-valent or 13-valent pneumococcal conjugate vaccines (PCV10; PCV13). Methods: Infants (N = 262) were randomized to receive 3 doses of PCV10 or PCV13 at 1-2-3 months of age, followed by pneumococcal polysaccharide vaccination (PPV) or no PPV at 9 months of age. Nasopharyngeal swabs (NPS) collected at ages 1, 4, 9, 10, 23 and 24 months were cultured using standard bacteriological procedures. Morphologically distinct Streptococcus pneumoniae colonies were serotyped by the Quellung reaction. Antimicrobial susceptibility was determined by Kirby-Bauer disc diffusion and minimum inhibitory concentration (MIC). Results: S. pneumoniae was isolated from 883/1063 NPS collected at 1–23 months of age, including 820 serotypeable (64 different serotypes) and 144 non-serotypeable isolates. At age 23 months, 93.6% (95%CI 86.6–97.6%) of PCV10 recipients and 88.6% (95%CI 80.1–94.4%) of PCV13 recipients were pneumococcal carriers, with higher carriage of PCV10 serotypes by PCV10 recipients (19.8%, 95%CI 12.2–29.5) than PCV13 recipients (9.3%, 95%CI 4.1–17.3) (p = 0.049). There were no other statistically significant differences between PCV10 and PCV13 recipients and children receiving PPV or no PPV. Nearly half (45.6%) of carried pneumococci were non-susceptible to penicillin based on the meningitis breakpoint (MI

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. pneumonia

Safety and immunogenicity of pneumococcal conjugate vaccines in a high-risk population : A randomized controlled trial of 10-valent and 13-valent pneumococcal conjugate vaccine in Papua New Guinean infants

Background. There are little data on the immunogenicity of PCV10 and PCV13 in the same high-risk population. Methods. PCV10 and PCV13 were studied head-to-head in a randomized controlled trial in Papua New Guinea in which 262 infants received 3 doses of PCV10 or PCV13 at 1, 2, and 3 months of age. Serotype-specific immunoglobulin G (IgG) concentrations, and pneumococcal and nontypeable Haemophilus influenzae (NTHi) carriage were assessed prevaccination and at 4 and 9 months of age. Infants were followed up for safety until 9 months of age. Results. One month after the third dose of PCV10 or PCV13, 80% of infants had IgG concentrations ≥0.35µg/mL for vaccine serotypes, and 6 months postvaccination IgG concentrations ≥0.35 µg/mL were maintained for 8/10 shared PCV serotypes in > 75% of children vaccinated with either PCV10 or PCV13. Children carried a total of 65 different pneumococcal serotypes (plus nonserotypeable). At 4 months of age, 92% (95% confidence interval [CI] 85–96) of children vaccinated with PCV10 and 81% (95% CI 72–88) vaccinated with PCV13 were pneumococcal carriers (P = .023), whereas no differences were seen at 9 months of age, or for NTHi carriage. Both vaccines were well tolerated and not associated with serious adverse events. Conclusions. Infant vaccination with 3 doses of PCV10 or PCV13 is safe and immunogenic in a highly endemic setting; however, to significantly reduce pneumococcal disease in these settings, PCVs with broader serotype coverage and potency to reduce pneumococcal carriage are needed. Clinical Trials Registration. NCT01619462

The contribution of geogenic particulate matter to lung disease in indigenous children

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. Indigenous children have much higher rates of ear and lung disease than non-Indigenous children, which may be related to exposure to high levels of geogenic (earth-derived) particulate matter (PM). The aim of this study was to assess the relationship between dust levels and health in Indigenous children in Western Australia (W.A.). Data were from a population-based sample of 1077 Indigenous children living in 66 remote communities of W.A. (>2,000,000 km2), with information on health outcomes derived from carer reports and hospitalisation records. Associations between dust levels and health outcomes were assessed by multivariate logistic regression in a multi-level framework. We assessed the effect of exposure to community sampled PM on epithelial cell (NuLi-1) responses to non-typeable Haemophilus influenzae (NTHi) in vitro. High dust levels were associated with increased odds of hospitalisation for upper (OR 1.77 95% CI [1.02–3.06]) and lower (OR 1.99 95% CI [1.08–3.68]) respiratory tract infections and ear disease (OR 3.06 95% CI [1.20–7.80]). Exposure to PM enhanced NTHi adhesion and invasion of epithelial cells and impaired IL-8 production. Exposure to geogenic PM may be contributing to the poor respiratory health of disadvantaged communities in arid environments where geogenic PM levels are high

Immunogenicity of a Third Scheduled Dose of Rotarix in Australian Indigenous Infants: A Phase IV, Double-blind, Randomized, Placebo-Controlled Clinical Trial

BackgroundRotarix (GlaxoSmithKline) oral rotavirus vaccine is licensed as 2 doses in the first 6 months of life. In settings with high child mortality rates, clinical protection conferred by 2 doses of Rotarix is reduced. We assessed vaccine immune response when an additional dose of Rotarix was given to Australian Aboriginal children 6 to MethodsORVAC is a 2-stage, double-blind, randomized, placebo-controlled trial. Australian Aboriginal children 6 to ResultsBetween March 2018 and August 2020, a total of 253 infants were enrolled. Of these, 178 infants (70%) had analyzable serological results after follow-up; 89 were randomized to receive Rotarix, and 89 to receive placebo. The proportion with seroresponse was 85% after Rotarix compared with 72% after placebo. There were no occurrences of intussusception or any serious adverse events.ConclusionsAn additional dose of Rotarix administered to Australian Aboriginal infants 6 to Clinical trials registrationNCT02941107