Karli J. Corscadden

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.

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.

IgG Responses to Pneumococcal and Haemophilus Influenzae Protein Antigens Are Not Impaired in Children with a History of Recurrent Acute Otitis Media

Background Vaccines including conserved antigens from Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi) have the potential to reduce the burden of acute otitis media. Little is known about the antibody response to such antigens in young children with recurrent acute otitis media, however, it has been suggested antibody production may be impaired in these children. Methods We measured serum IgG levels against 4 pneumococcal (PspA1, PspA 2, CbpA and Ply) and 3 NTHi (P4, P6 and PD) proteins in a cross-sectional study of 172 children under 3 years of age with a history of recurrent acute otitis media (median 7 episodes, requiring ventilation tube insertion) and 63 healthy age-matched controls, using a newly developed multiplex bead assay. Results Children with a history of recurrent acute otitis media had significantly higher geometric mean serum IgG levels against NTHi proteins P4, P6 and PD compared with healthy controls, whereas there was no difference in antibody levels against pneumococcal protein antigens. In both children with and without a history of acute otitis media, antibody levels increased with age and were significantly higher in children colonised with S. pneumoniae or NTHi compared with children that were not colonised. Conclusions Proteins from S. pneumoniae and NTHi induce serum IgG in children with a history of acute otitis media. The mechanisms in which proteins induce immunity and potential protection requires further investigation but the dogma of impaired antibody responses in children with recurrent acute otitis media should be reconsidered.

Haemophilus haemolyticus Interaction with Host Cells Is Different to Nontypeable Haemophilus influenzae and Prevents NTHi Association with Epithelial Cells

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that resides in the upper respiratory tract and contributes to a significant burden of respiratory related diseases in children and adults. Haemophilus haemolyticus is a respiratory tract commensal that can be misidentified as NTHi due to high levels of genetic relatedness. There are reports of invasive disease from H. haemolyticus, which further blurs the species boundary with NTHi. To investigate differences in pathogenicity between these species, we optimized an in vitro epithelial cell model to compare the interaction of 10 H. haemolyticus strains with 4 NTHi and 4 H. influenzae-like haemophili. There was inter- and intra-species variability but overall, H. haemolyticus had reduced capacity to attach to and invade nasopharyngeal and bronchoalveolar epithelial cell lines (D562 and A549) within 3 h when compared with NTHi. H. haemolyticus was cytotoxic to both cell lines at 24 h, whereas NTHi was not. Nasopharyngeal epithelium challenged with some H. haemolyticus strains released high levels of inflammatory mediators IL-6 and IL-8, whereas NTHi did not elicit an inflammatory response despite higher levels of cell association and invasion. Furthermore, peripheral blood mononuclear cells stimulated with H. haemolyticus or NTHi released similar and high levels of IL-6, IL-8, IL-10, IL-1β, and TNFα when compared with unstimulated cells but only NTHi elicited an IFNγ response. Due to the relatedness of H. haemolyticus and NTHi, we hypothesized that H. haemolyticus may compete with NTHi for colonization of the respiratory tract. We observed that in vitro pre-treatment of epithelial cells with H. haemolyticus significantly reduced NTHi attachment, suggesting interference or competition between the two species is possible and warrants further investigation. In conclusion, H. haemolyticus interacts differently with host cells compared to NTHi, with different immunostimulatory and cytotoxic properties. This study provides an in vitro model for further investigation into the pathogenesis of Haemophilus species and the foundation for exploring whether H. haemolyticus can be used to prevent NTHi disease.

Children with otitis media mount a pneumococcal serotype specific serum IgG and IgA response comparable to healthy controls after pneumococcal conjugate vaccination

It has been suggested that otitis-prone children have an impaired antibody response. To investigate this in the context of pneumococcal vaccination, we used a multiplex bead-based assay to measure serum IgG and IgA levels against pneumococcal serotypes included in the 7-valent pneumococcal conjugate vaccine (PCV7; serotypes 4, 6B, 9V, 14, 18C, 19F and 23F) and 4 non-PCV7 serotypes (1, 5, 7F and 19A) in healthy (n=43) and otitis-prone children (n=75) before, 6 weeks after and 1 year after vaccination with one dose of PCV7. Pre-vaccination, otitis-prone children had significantly higher serum IgG levels against serotypes 4, 9V and 23F and against all non-PCV7 serotypes. One year following vaccination, there was no difference in IgG or IgA levels between healthy and otitis-prone children. The effect of the administration of one or two doses of PCV7 was investigated in otitis-prone children. After a second dose of PCV7, pneumococcal serotype specific IgG levels, but not IgA titres, were higher compared to the levels measured after the initial dose of PCV7. One year post PCV7 vaccination there was no difference in either IgG or IgA antibody levels to any of the PCV7 serotypes between children who received either one or two doses of PCV7. The finding that otitis-prone children do not have an impaired pneumococcal serotype-specific serum IgG or IgA response suggests that new pneumococcal conjugate vaccines may be immunogenic in otitis-prone children, however, further investigations are necessary to determine the clinical impact of such vaccines against the development of recurrent acute otitis media.

High pneumococcal serotype specific IgG, IgG1 and IgG2 levels in serum and the middle ear of children with recurrent acute otitis media receiving ventilation tubes

Recurrent acute otitis media (AOM), frequently caused by Streptococcus pneumoniae, is a major paediatric health problem. A reduced antibody response against pneumococcal polysaccharides may contribute to an increased susceptibility to AOM. Using a multiplex bead-based assay we measured IgG, IgG1 and IgG2 levels against 11 pneumococcal polysaccharides in serum samples from 166 children below 3 years of age with a history of at least 3 episodes of acute otitis media receiving ventilation tubes, and 61 healthy controls. Pneumococcal serotype specific IgG was also determined in 144 middle ear effusion samples. Pneumococcal serotype specific IgG, IgG1 and IgG2 levels were similar in children with or without AOM, except for IgG and IgG1 levels against serotype 5, which were significantly higher in children with a history of frequent AOM (IgG: 137.5 μg/ml vs. 84.0 μg/ml; p=0.02; IgG1: 24.5 μg/ml vs. 18.2 μg/ml; p=0.05). The age-related development of pneumococcal serotype-specific IgG, IgG1 and IgG2 levels was similar in children with or without a history of AOM. Pneumococcal serotype specific IgG was present in middle ear effusion and these levels correlated significantly with serum titres. Children with a history of frequent AOM receiving ventilation tubes do not have a deficient IgG, IgG1 or IgG2 response against pneumococcal polysaccharides, either induced by vaccination or due to natural exposure. The strong correlation between IgG levels in serum and the middle ear suggests parenteral pneumococcal conjugate vaccination induces antibodies in the middle ear which may therefore contribute to reducing the burden of AOM.

Chronic HIV-1 infection induces B-cell dysfunction that is incompletely resolved by long-term antiretroviral therapy

Objectives:To determine the effect of long-term antiretroviral therapy (ART) on HIV-1–induced B-cell dysfunction. Design:Comparative study of ART-naive and ART-treated HIV-infected patients with non-HIV controls. Methods:B-cell dysfunction was examined in patients with HIV-1 infection (n = 30) who had received ART for a median time of 9.25 years (range: 1.3–21.7) by assessing proportions of CD21lo/- B cells (a marker of B-cell exhaustion) and proportions of tumor necrosis factor-related apoptosis-inducing ligand+ or B and T lymphocyte attenuator+ B cells, and serum levels of immunoglobulin free light chains (markers of B-cell hyperactivation). The association of these markers with serum levels of IgG1 and IgG2, and production of IgG antibodies after vaccination with pneumococcal polysaccharides were also examined. ART-naive patients with HIV (n = 20) and controls (n = 20) were also assessed for comparison. Results:ART-treated patients had increased proportions of CD21lo/- and tumor necrosis factor-related apoptosis-inducing ligand+ B cells and, furthermore, although proportions of B and T lymphocyte attenuator+ B cells were not significantly different from controls, they correlated negatively with CD21lo/- B cells. Proportions of CD21lo/- B cells also correlated negatively with current CD4+ T-cell counts. In ART-naive patients with HIV, free light chains correlated with CD21lo/- B cells and IgG1, but not IgG2. Serum IgG2:IgG1 ratios were substantially lower than normal in patients with HIV and did not resolve on ART. In ART-treated patients, IgG antibody responses to pneumococcal polysaccharides after vaccination were not associated with markers of B-cell dysfunction. Conclusions:B-cell dysfunction persists in patients with HIV receiving long-term ART. The causes and consequences of this require further investigation.

Production of IgG antibodies to pneumococcal polysaccharides is associated with expansion of ICOS+ circulating memory T follicular-helper cells which is impaired by HIV infection

Dysfunction of T follicular-helper (TFH) cells is a possible cause of impaired germinal centre (GC) and IgG antibody responses in individuals with human immunodeficiency virus-1 (HIV-1) infection and might contribute to decreased magnitude and isotype diversification of IgG antibodies to pneumococcal polysaccharides (PcPs). We examined the production of IgG1 and IgG2 antibodies to PcPs 4, 6B, 9V and 14 by enumerating antibody secreting cells (ASCs) at day (D) 7 and determining fold-increase in serum antibody levels at D28 after vaccination with unconjugated PcPs in HIV seronegative subjects (n = 20) and in HIV patients who were receiving antiretroviral therapy (ART) (n = 28) or who were ART-naive (n = 11) and determined their association with ICOS+ and ICOS- circulating memory TFH (cmTFH) cells (CD4+CD45RA-CD27+CXCR5+PD-1+) and short lived plasmablasts (SPBs) at D7, and with PcP-specific and total IgM+ and IgG+ memory B cells at D0. In HIV seronegative subjects, production of IgG1+ and IgG2+ ASCs was consistently associated with the frequency of ICOS+ cmTFH cells but not ICOS- cmTFH cells or memory B cells. In contrast, post-vaccination ASCs in HIV patients, regardless of ART status, were lower than in HIV seronegative subjects and not associated with ICOS+ cmTFH cells, the expansion of which was absent (ART-naive patients) or much lower than in HIV seronegative subjects (ART-treated patients). Production of SPBs was also lower in ART-naive patients. Fold-increase in IgG2 antibodies at D28 also correlated with ICOS+ cmTFH cells at D7 in HIV seronegative subjects but not in HIV patients. These novel findings provide evidence that ICOS+ cmTFH cells contribute to the regulation of PcP-specific IgG antibody responses, including isotype diversification, and that TFH cell dysfunction may be a cause of impaired PcP-specific IgG antibody responses and increased susceptibility to pneumococcal disease in HIV patients.

Australian Aboriginal children with otitis media have reduced antibody titers to specific nontypeable Haemophilus influenzae vaccine antigens

ABSTRACT Indigenous populations experience high rates of otitis media (OM), with increased chronicity and severity, compared to those experienced by their nonindigenous counterparts. Data on immune responses to otopathogenic bacteria in these high-risk populations are lacking. Nontypeable Haemophilus influenzae (NTHi) is the predominant otopathogen in Australia. No vaccines are currently licensed to target NTHi; however, protein D (PD) from NTHi is included as a carrier protein in the 10-valent pneumococcal polysaccharide conjugate vaccine (PHiD10-CV), and other promising protein vaccine candidates exist, including outer membrane protein 4 (P4) and protein 6 (P6). We measured the levels of serum and salivary IgA and IgG against PD, P4, and P6 in Aboriginal and non-Aboriginal children with chronic OM who were undergoing surgery and compared the levels with those in healthy non-Aboriginal children (controls). We found that Aboriginal cases had lower serum IgG titers to all NTHi proteins assessed, particularly PD. In contrast, serum IgA and salivary IgA and IgG titers to each of these 3 proteins were equivalent to or higher than those in both non-Aboriginal cases and healthy controls. While serum antibody levels increased with age in healthy controls, no changes in titers were observed with age in non-Aboriginal cases, and a trend toward decreasing titers with age was observed in Aboriginal cases. This suggests that decreased serum IgG responses to NTHi outer membrane proteins may contribute to the development of chronic and severe OM in Australian Aboriginal children and other indigenous populations. These data are important for understanding the potential benefits of PHiD10-CV implementation and the development of NTHi protein-based vaccines for indigenous populations.

Otitis-prone children produce functional antibodies to pneumolysin and pneumococcal polysaccharides

ABSTRACT The pneumococcus is a major otitis media (OM) pathogen, but data are conflicting regarding whether otitis-prone children have impaired humoral immunity to pneumococcal antigens. We and others have shown that otitis-prone and healthy children have similar antibody titers to pneumococcal proteins and polysaccharides (vaccine and nonvaccine types); however, the quality of antibodies from otitis-prone children has not been investigated. Antibody function, rather than titer, is considered to be a better correlate of protection from pneumococcal disease. Therefore, we compared the capacities of antibodies from otitis-prone (cases) and healthy (controls) children to neutralize pneumolysin, the pneumococcal toxin currently in development as a vaccine antigen, and to opsonize pneumococcal vaccine and nonvaccine serotypes. A pneumolysin neutralization assay was conducted on cholesterol-depleted complement-inactivated sera from 165 cases and 61 controls. A multiplex opsonophagocytosis assay (MOPA) was conducted on sera from 20 cases and 20 controls. Neutralizing and opsonizing titers were calculated with antigen-specific IgG titers to determine antibody potency for pneumolysin, pneumococcal conjugate vaccine (PCV) polysaccharides, and non-PCV polysaccharides. There was no significant difference in antibody potencies between cases and controls for the antigens tested. Antipneumolysin neutralizing titers increased with the number of episodes of acute OM, but antibody potency did not. Pneumolysin antibody potency was lower in children colonized with pneumococci than in noncarriers, and this was significant for the otitis-prone group (P < 0.05). The production of functional antipneumococcal antibodies in otitis-prone children demonstrates that they respond to the current PCV and are likely to respond to pneumolysin-based vaccines as effectively as healthy children.