Evolutionary and functional relationships among the nontypeable Haemophilus influenzae HMW family of adhesins

Nontypeable Haemophilus influenzae (NTHi) is a common cause of localized respiratory tract disease and initiates infection by colonizing the nasopharynx. Approximately 75 to 80% of NTHi clinical isolates produce proteins that belong to the HMW family of adhesins, which are believed to facilitate colonization. The prototype HMW adhesins are designated HMW1 and HMW2 and were identified in NTHi strain 12. HMW1 and HMW2 are 71% identical and 80% similar overall, yet display differing cellular binding specificities. In the present study we set out to define more clearly the relationships between HMW1 and HMW2 and other members of the HMW family of adhesins. PCR analysis of 49 epidemiologically distinct isolates revealed that all strains possessing hmw genes as determined by Southern analysis contain two hmw loci in conserved, unlinked physical locations on the chromosome. Functional analysis of the HMW adhesins produced by three unrelated strains demonstrated that each isolate possesses one protein with HMW1-like adherence properties and another with HMW2-like adherence properties. These findings suggest that the hmw1 and hmw2 loci may have arisen via a gene duplication event in an ancestral strain. In addition, they support the hypothesis that the distinct binding specificities of HMW1 and HMW2 emerged early and have persisted over time, suggesting an ongoing selective advantage

Panel 4: Report of the Microbiology Panel

Objective. To perform a comprehensive review of the literature from July 2011 until June 2015 on the virology and bacteriology of otitis media in children.Data Sources. PubMed database of the National Library of Medicine.Review Methods. Two subpanels comprising experts in the virology and bacteriology of otitis media were created. Each panel reviewed the relevant literature in the fields of virology and bacteriology and generated draft reviews. These initial reviews were distributed to all panel members prior to meeting together at the Post-symposium Research Conference of the 18th International Symposium on Recent Advances in Otitis Media, National Harbor, Maryland, in June 2015. A final draft was created, circulated, and approved by all panel members.Conclusions. Excellent progress has been made in the past 4 years in advancing our understanding of the microbiology of otitis media. Numerous advances were made in basic laboratory studies, in animal models of otitis media, in better understanding the epidemiology of disease, and in clinical practice.Implications for Practice. (1) Many viruses cause acute otitis media without bacterial coinfection, and such cases do not require antibiotic treatment. (2) When respiratory syncytial virus, metapneumovirus, and influenza virus peak in the community, practitioners can expect to see an increase in clinical otitis media cases. (3) Biomarkers that predict which children with upper respiratory tract infections will develop otitis media may be available in the future. (4) Compounds that target newly identified bacterial virulence determinants may be available as future treatment options for children with otitis media.</p

Severe Community-acquired Pneumonia Due to Staphylococcus aureus, 2003–04 Influenza Season

S. aureus community-acquired pneumonia has been reported from 9 states

Panel 4 : Report of the Microbiology Panel

Objective. To perform a comprehensive review of the literature from July 2011 until June 2015 on the virology and bacteriology of otitis media in children. Data Sources. PubMed database of the National Library of Medicine. Review Methods. Two subpanels comprising experts in the virology and bacteriology of otitis media were created. Each panel reviewed the relevant literature in the fields of virology and bacteriology and generated draft reviews. These initial reviews were distributed to all panel members prior to meeting together at the Post-symposium Research Conference of the 18th International Symposium on Recent Advances in Otitis Media, National Harbor, Maryland, in June 2015. A final draft was created, circulated, and approved by all panel members. Conclusions. Excellent progress has been made in the past 4 years in advancing our understanding of the microbiology of otitis media. Numerous advances were made in basic laboratory studies, in animal models of otitis media, in better understanding the epidemiology of disease, and in clinical practice. Implications for Practice. (1) Many viruses cause acute otitis media without bacterial coinfection, and such cases do not require antibiotic treatment. (2) When respiratory syncytial virus, metapneumovirus, and influenza virus peak in the community, practitioners can expect to see an increase in clinical otitis media cases. (3) Biomarkers that predict which children with upper respiratory tract infections will develop otitis media may be available in the future. (4) Compounds that target newly identified bacterial virulence determinants may be available as future treatment options for children with otitis media.Peer reviewe

Panel 6 : Vaccines

Objective. To review the literature on progress regarding (1) effectiveness of vaccines for prevention of otitis media (OM) and (2) development of vaccine antigens for OM bacterial and viral pathogens. Data Sources. PubMed database of the National Library of Science. Review Methods. We performed literature searches in PubMed for OM pathogens and candidate vaccine antigens, and we restricted the searches to articles in English that were published between July 2011 and June 2015. Panel members reviewed literature in their area of expertise. Conclusions. Pneumococcal conjugate vaccines (PCVs) are somewhat effective for the prevention of pneumococcal OM, recurrent OM, OM visits, and tympanostomy tube insertions. Widespread use of PCVs has been associated with shifts in pneumococcal serotypes and bacterial pathogens associated with OM, diminishing PCV effectiveness against AOM. The 10-valent pneumococcal vaccine containing Haemophilus influenzae protein D (PHiD-CV) is effective for pneumococcal OM, but results from studies describing the potential impact on OM due to H influenzae have been inconsistent. Progress in vaccine development for H influenzae, Moraxella catarrhalis, and OM-associated respiratory viruses has been limited. Additional research is needed to extend vaccine protection to additional pneumococcal serotypes and other otopathogens. There are likely to be licensure challenges for protein-based vaccines, and data on correlates of protection for OM vaccine antigens are urgently needed. Implications for Practice. OM continues to be a significant health care burden globally. Prevention is preferable to treatment, and vaccine development remains an important goal. As a polymicrobial disease, OM poses significant but not insurmountable challenges for vaccine development.Peer reviewe

Human Antibodies Specific for the High-Molecular-Weight Adhesion Proteins of Nontypeable Haemophilus influenzae Mediate Opsonophagocytic Activity

The HMW1- and HMW2-like adhesion proteins of nontypeable Haemophilus influenzae are expressed by 75% of these strains, and antibodies directed against these proteins are protective in animal models of infection. The purpose of the present study was to define the functional activity of human antibodies specific for these proteins in an in vitro complement-dependent opsonophagocytic assay. Human promyelocytic cell line HL-60 served as the source of phagocytic cells, and a commercial preparation of intravenous immunoglobulin (IVIG) served as the source of human antibodies. High-molecular-weight (HMW) proteins were purified from four prototype nontypeable H. influenzae strains and used to prepare solid-phase affinity columns. IVIG was adsorbed on each column to remove strain-specific anti-HMW antibodies and to allow recovery of affinity-purified anti-HMW antibody fractions. Unadsorbed IVIG killed each of the prototype strains at titers of 1:80 to 1:320. HMW-adsorbed sera demonstrated fourfold decreases in opsonophagocytic titer against the homologous strains compared to unadsorbed IVIG. Affinity-purified anti-HMW antibody preparations demonstrated opsonophagocytic titers of 1:20 to 1:80 against the respective homologous strains and opsonophagocytic titers as high as 1:80 against heterologous strains. None of the affinity-purified anti-HMW antibody preparations was opsonophagocytic for a representative nontypeable H. influenzae strain that did not express HMW1- or HMW2-like proteins. These data demonstrate that human antibodies specific for the HMW1/HMW2-like adhesion proteins of nontypeable H. influenzae are opsonophagocytic and that such antibodies recognize epitopes shared by the HMW proteins of unrelated nontypeable H. influenzae strains. These results argue for continued investigation of the HMW1/HMW2-like proteins as potential vaccine candidates for prevention of disease due to nontypeable H. influenzae

Antibodies Specific for the High-Molecular-Weight Adhesion Proteins of Nontypeable Haemophilus influenzae Are Opsonophagocytic for both Homologous and Heterologous Strains

The HMW1/HMW2-like adhesion proteins of nontypeable Haemophilus influenzae (NTHI) are expressed by 75% of NTHI strains. Antibodies directed against these proteins are opsonophagocytic in vitro and are protective in an animal model of infection. The objective of the present study was to determine the opsonophagocytic activity of high-titer anti-HMW1/HMW2 immune sera against both homologous and heterologous NTHI strains. Chinchillas were immunized with purified HMW1/HMW2-like proteins from five prototype NTHI strains. Serum opsonophagocytic activity was monitored in an assay that uses a human promyelocytic cell line, HL-60, as the source of phagocytic cells. Preimmune sera did not demonstrate opsonophagocytic killing of any strains. In contrast, the immune sera demonstrated killing of the five homologous NTHI strains at titers ranging from 1:320 to 1:640. The immune sera also demonstrated killing of eight heterologous NTHI strains that express HMW1/HMW2-like proteins at titers ranging from 0 to 1:640. Killing of heterologous strains sometimes demonstrated a prozone phenomenon. None of the immune sera killed NTHI strains that did not express HMW1/HMW2-like proteins. Adsorption of immune sera with HMW1/HMW2-like proteins purified from either homologous or heterologous NTHI strains eliminated opsonophagocytic killing of homologous strains in most cases. These data demonstrate that antibodies produced following immunization with the HMW1/HMW2-like proteins are opsonophagocytic for both homologous and heterologous NTHI and strongly suggest that common epitopes recognized by functionally active antibodies exist on the HMW1/HMW2-like proteins of unrelated NTHI strains. The results argue for the continued investigation of the HMW1/HMW2-like proteins as potential vaccine candidates for the prevention of NTHI disease

Construction and Immunogenicity of Recombinant Adenovirus Vaccines Expressing the HMW1, HMW2, or Hia Adhesion Protein of Nontypeable Haemophilus influenzae▿

The objective of the present study was to construct and assess the immunogenicity of recombinant adenovirus vectors expressing the HMW1, HMW2, or Hia protein of nontypeable Haemophilus influenzae (NTHi). These proteins are critical adhesins and potential protective antigens expressed by NTHi. Segments of the hmw1A and hmw2A structural genes that encode the distal one-half of mature HMW1 or HMW2 were cloned into the T7 expression vector pGEMEX-2. These constructs encoded stable HMW1 or HMW2 recombinant fusion protein that expresses B-cell epitopes common to most NTHi strains. A segment of the hia gene that encodes the surface-exposed portion of mature Hia was also cloned into pGEMEX-2. The resulting T7 gene 10 translational fusions were excised from the parent plasmids and cloned into the shuttle plasmid pDC316. Cotransfection of HEK 293 cells with the pDC316 derivatives and pBHGloxΔE1,3Cre resulted in the production of viral plaques from which recombinant adenoviruses expressing fusion proteins were recovered. Chinchillas immunized intraperitoneally with a single 108-PFU dose of either the HMW2 or Hia adenoviral construct developed high anti-HMW2 or anti-Hia serum antibody titers within 4 weeks of immunization. Chinchillas immunized intranasally with a single 107- to 109-PFU dose of the Hia adenoviral construct also developed high anti-Hia serum antibody titers within 8 weeks of immunization. Recombinant adenoviruses represent a promising system to induce mucosal and systemic immunity and protection against mucosal diseases such as otitis media. Recombinant adenoviruses expressing recombinant HMW1, HMW2, or Hia protein will be important new tools in NTHi vaccine development efforts