Lipidation of Haemophilus influenzae Antigens P6 and OMP26 Improves Immunogenicity and Protection against Nasopharyngeal Colonization and Ear Infection

Nontypeable Haemophilus influenzae (NTHi) causes respiratory infections that lead to high morbidity and mortality worldwide, encouraging development of effective vaccines. To achieve a protective impact on nasopharyngeal (NP) colonization by NTHi, enhanced immunogenicity beyond that achievable with recombinant-protein antigens is likely to be necessary. ABSTRACT Nontypeable Haemophilus influenzae (NTHi) causes respiratory infections that lead to high morbidity and mortality worldwide, encouraging development of effective vaccines. To achieve a protective impact on nasopharyngeal (NP) colonization by NTHi, enhanced immunogenicity beyond that achievable with recombinant-protein antigens is likely to be necessary. Adding a lipid moiety to a recombinant protein would enhance immunogenicity through Toll-like receptor 2 signaling of antigen-presenting cells and Th17 cell response in the nasal-associated lymphoid tissue (NALT). We investigated effects of lipidation (L) of recombinant proteins P6 and OMP26 compared to nonlipidated (NL) P6 and OMP26 and as fusion constructs (L-OMP26ϕNL-P6 and L-P6ϕNL-OMP26) in a mouse model. After intraperitoneal or intranasal vaccination, antibody responses were compared and protection from NP colonization and middle ear infection were assessed. L-P6 and L-OMP26 induced approximately 10- to 100-fold-higher IgG antibody levels than NL-P6 and NL-OMP26. Fusion constructs significantly increased IgG antibody to both target proteins, even though only one of the proteins was lipidated. NP colonization and middle ear bullae NTHi density was 1 to 4 logs lower following vaccination with L-P6 and L-OMP26 than with NL-P6 and NL-OMP26. Fusion constructs also resulted in a 1- to 3-log-lower NTHi density following vaccination. NALT cells from mice vaccinated with lipidated protein constructs had higher levels of interleukin-17 (IL-17), IL-22, and CD4+ T-cell memory. Passive transfer of sera from L-OMP26ϕNL-P6-vaccinated mice to recipient infant mice reduced NP colonization and ear bulla NTHi density. We conclude that L-P6, L-OMP26, and fusion constructs generate enhanced antibody responses and protection from NP colonization and middle ear infection by NTHi in mice.

[1]  J. Alcorn,et al.  Influenza Suppresses Neutrophil Recruitment to the Lung and Exacerbates Secondary Invasive Pulmonary Aspergillosis , 2020, The Journal of Immunology.

[2]  D. Schwudke,et al.  Lipidation of Pneumococcal Antigens Leads to Improved Immunogenicity and Protection , 2020, Vaccines.

[3]  L. Hammitt,et al.  Panel 8: Vaccines and immunology. , 2019, International journal of pediatric otorhinolaryngology.

[4]  K. O'Brien,et al.  Efficacy, safety and immunogenicity of a pneumococcal protein-based vaccine co-administered with 13-valent pneumococcal conjugate vaccine against acute otitis media in young children: A phase IIb randomized study. , 2019, Vaccine.

[5]  C. Brightling,et al.  Non-typeable Haemophilus influenzae protein vaccine in adults with COPD: A phase 2 clinical trial. , 2019, Vaccine.

[6]  K. Jolley,et al.  High diversity of invasive Haemophilus influenzae isolates in France and the emergence of resistance to third generation cephalosporins by alteration of ftsI gene. , 2019, The Journal of infection.

[7]  P. van Damme,et al.  Safety and immunogenicity of non-typeable Haemophilus influenzae-Moraxella catarrhalis vaccine. , 2019, Vaccine.

[8]  J. Vallejo,et al.  Invasive Haemophilus influenzae Disease at Texas Children's Hospital, 2011 to 2018. , 2019, The Pediatric infectious disease journal.

[9]  E. Tanaka,et al.  Isolation of multidrug-resistant Haemophilus influenzae harbouring multiple exogenous genes from a patient diagnosed with acute sinusitis. , 2019, Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy.

[10]  S. Michalik,et al.  Intranasal Vaccination With Lipoproteins Confers Protection Against Pneumococcal Colonisation , 2018, Front. Immunol..

[11]  M. Pichichero,et al.  Intranasal coinfection model allows for assessment of protein vaccines against nontypeable Haemophilus influenzae in mice , 2018, Journal of medical microbiology.

[12]  O. Tichá,et al.  Lipid moieties on lipoproteins of commensal and non-commensal staphylococci induce differential immune responses , 2017, Nature Communications.

[13]  M. Pichichero,et al.  Epidemiology of Acute Otitis Media in the Postpneumococcal Conjugate Vaccine Era , 2017, Pediatrics.

[14]  Nicole R. Luke-Marshall,et al.  Serum antibody response to Moraxella catarrhalis proteins in stringently defined otitis prone children. , 2017, Vaccine.

[15]  T. Mrkvan,et al.  Impact of protein D-containing pneumococcal conjugate vaccines on non-typeable Haemophilus influenzae acute otitis media and carriage , 2017, Expert review of vaccines.

[16]  S. Schoonbroodt,et al.  Efficacy of a novel, protein-based pneumococcal vaccine against nasopharyngeal carriage of Streptococcus pneumoniae in infants: A phase 2, randomized, controlled, observer-blind study. , 2017, Vaccine.

[17]  M. Pichichero,et al.  Protection against Streptococcus pneumoniae Invasive Pathogenesis by a Protein-Based Vaccine Is Achieved by Suppression of Nasopharyngeal Bacterial Density during Influenza A Virus Coinfection , 2016, Infection and Immunity.

[18]  T. Carvalhanas,et al.  Effect of 10-valent pneumococcal conjugate vaccine on nasopharyngeal carriage of Streptococcus pneumoniae and Haemophilus influenzae among children in São Paulo, Brazil. , 2016, Vaccine.

[19]  A. Almudevar,et al.  Functional Immune Cell Differences Associated With Low Vaccine Responses in Infants. , 2016, Journal of Infectious Diseases.

[20]  P. van Damme,et al.  Phase I, randomized, observer-blind, placebo-controlled studies to evaluate the safety, reactogenicity and immunogenicity of an investigational non-typeable Haemophilus influenzae (NTHi) protein vaccine in adults. , 2016, Vaccine.

[21]  M. Pichichero,et al.  Developing a vaccine to prevent otitis media caused by nontypeable Haemophilus influenzae , 2016, Expert review of vaccines.

[22]  M. Pichichero,et al.  Otitis-prone Children Have Immunologic Deficiencies in Naturally Acquired Nasopharyngeal Mucosal Antibody Response after Streptococcus pneumoniae Colonization , 2015, The Pediatric infectious disease journal.

[23]  P. Chong,et al.  Recombinant bacterial lipoproteins as vaccine candidates , 2015, Expert review of vaccines.

[24]  R. Malley,et al.  TH17-Mediated Protection against Pneumococcal Carriage by a Whole-Cell Vaccine Is Dependent on Toll-Like Receptor 2 and Surface Lipoproteins , 2015, Clinical and Vaccine Immunology.

[25]  T. Murphy.  Vaccines for Nontypeable Haemophilus influenzae: the Future Is Now , 2015, Clinical and Vaccine Immunology.

[26]  S. Ladhani,et al.  Non-typeable Haemophilus influenzae, an under-recognised pathogen. , 2014, The Lancet. Infectious diseases.

[27]  John L. Perez,et al.  A randomized, phase 1/2 trial of the safety, tolerability, and immunogenicity of bivalent rLP2086 meningococcal B vaccine in healthy infants. , 2014, Vaccine.

[28]  Todd M. Gierahn,et al.  Toll-Like Receptor 2-Dependent Protection against Pneumococcal Carriage by Immunization with Lipidated Pneumococcal Proteins , 2014, Infection and Immunity.

[29]  F. E. Lee,et al.  Understanding nontypeable Haemophilus influenzae and chronic obstructive pulmonary disease , 2014, Current opinion in pulmonary medicine.

[30]  M. Pichichero,et al.  Relationship With Original Pathogen in Recurrence of Acute Otitis Media After Completion of Amoxicillin/Clavulanate: Bacterial Relapse or New Pathogen , 2013, The Pediatric infectious disease journal.

[31]  R. Veenhoven,et al.  Effects of the 10-Valent Pneumococcal Nontypeable Haemophilus influenzae Protein D–Conjugate Vaccine on Nasopharyngeal Bacterial Colonization in Young Children: A Randomized Controlled Trial , 2012, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[32]  M. Pichichero,et al.  Bactericidal antibody response against P6, protein D, and OMP26 of nontypeable Haemophilus influenzae after acute otitis media in otitis-prone children. , 2012, FEMS immunology and medical microbiology.

[33]  J. Bengoechea,et al.  Nontypeable Haemophilus influenzae biofilms: role in chronic airway infections , 2012, Front. Cell. Inf. Microbio..

[34]  P. Chong,et al.  The Immunomodulatory Activity of Meningococcal Lipoprotein Ag473 Depends on the Conformation Made up of the Lipid and Protein Moieties , 2012, PloS one.

[35]  Ly-Mee Yu,et al.  Immunogenicity and tolerability of recombinant serogroup B meningococcal vaccine administered with or without routine infant vaccinations according to different immunization schedules: a randomized controlled trial. , 2012, JAMA.

[36]  J. Gilsdorf,et al.  Invasive disease caused by Haemophilus influenzae in Sweden 1997-2009; evidence of increasing incidence and clinical burden of non-type b strains. , 2011, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[37]  M. Pichichero,et al.  Serum Antibody Response to Five Streptococcus pneumoniae Proteins During Acute Otitis Media in Otitis-prone and Non–otitis-prone Children , 2011, The Pediatric infectious disease journal.

[38]  M. Pichichero,et al.  Serum antibody response to three non-typeable Haemophilus influenzae outer membrane proteins during acute otitis media and nasopharyngeal colonization in otitis prone and non-otitis prone children. , 2011, Vaccine.

[39]  A. Almudevar,et al.  Antibody response to Haemophilus influenzae outer membrane protein D, P6, and OMP26 after nasopharyngeal colonization and acute otitis media in children. , 2010, Vaccine.

[40]  Chen Dong,et al.  Toll-like receptor 2 signaling in CD4(+) T lymphocytes promotes T helper 17 responses and regulates the pathogenesis of autoimmune disease. , 2010, Immunity.

[41]  M. Pichichero,et al.  New Patterns in the Otopathogens Causing Acute Otitis Media Six to Eight Years After Introduction of Pneumococcal Conjugate Vaccine , 2009, The Pediatric infectious disease journal.

[42]  J. Campos,et al.  Nontypeable Haemophilus influenzae as a Pathogen in Children , 2009, The Pediatric infectious disease journal.

[43]  M. Lipsitch,et al.  Interleukin-17A Mediates Acquired Immunity to Pneumococcal Colonization , 2008, PLoS pathogens.

[44]  L. Wetzler,et al.  Toll-like receptor 2-mediated human B cell differentiation. , 2006, Clinical immunology.

[45]  C. Snapper,et al.  Both Innate Immunity and Type 1 Humoral Immunity to Streptococcus pneumoniae Are Mediated by MyD88 but Differ in Their Relative Levels of Dependence on Toll-Like Receptor 2 , 2005, Infection and Immunity.

[46]  L. Bakaletz,et al.  Efficacy of the 26-Kilodalton Outer Membrane Protein and Two P5 Fimbrin-Derived Immunogens To Induce Clearance of Nontypeable Haemophilus influenzae from the Rat Middle Ear and Lungs as Well as from the Chinchilla Middle Ear and Nasopharynx , 2003, Infection and Immunity.

[47]  A. Forsgren,et al.  Developing a nontypeable Haemophilus influenzae (NTHi) vaccine. , 2000, Vaccine.

[48]  A. Cripps,et al.  Nontypeable Haemophilus influenzae: Pathogenesis and Prevention , 1998, Microbiology and Molecular Biology Reviews.

[49]  J. Klein.  Role of nontypeable Haemophilus influenzae in pediatric respiratory tract infections. , 1997, The Pediatric infectious disease journal.

[50]  L. van Alphen,et al.  Mechanism of antibody-mediated reduction of nasopharyngeal colonization by Haemophilus influenzae type b studied in an infant rat model. , 1996, The Journal of infectious diseases.

[51]  A. Cripps,et al.  Enhanced respiratory clearance of nontypeable Haemophilus influenzae following mucosal immunization with P6 in a rat model , 1995, Infection and immunity.

[52]  M. Leinonen,et al.  Reduction of oropharyngeal carriage of Haemophilus influenzae type b (Hib) in children immunized with an Hib conjugate vaccine. , 1991, The Journal of infectious diseases.

[53]  B. Green,et al.  Biologic activities of antibody to a peptidoglycan-associated lipoprotein of Haemophilus influenzae against multiple clinical isolates of H. influenzae type b , 1987, Infection and immunity.

[54]  Manmohan J. Singh,et al.  Acceptable levels of endotoxin in vaccine formulations during preclinical research. , 2011, Journal of pharmaceutical sciences.

[55]  S. Akira,et al.  Toll-like receptors. , 2003, Annual review of immunology.

[56]  J. Bernstein,et al.  Recurrent otitis media with non-typable Haemophilus influenzae: the role of serum bactericidal antibody. , 1992, International journal of pediatric otorhinolaryngology.

[57]  G. Beeson,et al.  in adults with , 2019 .