Intranasal Immunization with Nontypeable Haemophilus influenzae Outer Membrane Vesicles Induces Cross-Protective Immunity in Mice

Sandro Roier,Jeremy Iwashkiw,Georg Krohne,Deborah R Leitner,Stefan Schild,Kristina Schild-Prüfert,Joachim Reidl,Mario F Feldman,Lisa A Morici

doi:10.1371/journal.pone.0042664

Abstract

Haemophilus influenzae is a Gram-negative human-restricted bacterium that can act as a commensal and a pathogen of the respiratory tract. Especially nontypeable H. influenzae (NTHi) is a major threat to public health and is responsible for several infectious diseases in humans, such as pneumonia, sinusitis, and otitis media. Additionally, NTHi strains are highly associated with exacerbations in patients suffering from chronic obstructive pulmonary disease. Currently, there is no licensed vaccine against NTHi commercially available. Thus, this study investigated the utilization of outer membrane vesicles (OMVs) as a potential vaccine candidate against NTHi infections. We analyzed the immunogenic and protective properties of OMVs derived from various NTHi strains by means of nasopharyngeal immunization and colonization studies with BALB/c mice. The results presented herein demonstrate that an intranasal immunization with NTHi OMVs results in a robust and complex humoral and mucosal immune response. Immunoprecipitation revealed the most important immunogenic proteins, such as the heme utilization protein, protective surface antigen D15, heme binding protein A, and the outer membrane proteins P1, P2, P5 and P6. The induced immune response conferred not only protection against colonization with a homologous NTHi strain, which served as an OMV donor for the immunization mixtures, but also against a heterologous NTHi strain, whose OMVs were not part of the immunization mixtures. These findings indicate that OMVs derived from NTHi strains have a high potential to act as a vaccine against NTHi infections.

Highlights

Haemophilus influenzae is a Gram-negative coccobacillus that commonly colonizes the human respiratory tract as a commensal or pathogen
nontypeable H. influenzae (NTHi) strains release outer membrane vesicles (OMVs) To investigate whether the NTHi strains used in this study are able to release OMVs, we adapted an established method to isolate V. cholerae OMVs derived from culture supernatants [34]
We identified the allelic profile and sequence types (ST) for the NTHi strains 1479, 3198, 5657 and 7502, which subsequently allowed a comparison of all six NTHi strains (Table 2)

Summary

Introduction

Haemophilus influenzae is a Gram-negative coccobacillus that commonly colonizes the human respiratory tract as a commensal or pathogen. This bacterium can be differentiated into typeable and nontypeable strains based on the presence or absence of a polysaccharide capsule. Encapsulated strains are divided into six capsular serotypes (a–f), with serotype b (Hib) being the most common associated with human disease. Nonencapsulated and nontypeable H. influenzae (NTHi) strains generally cause pneumonia, sinusitis, and otitis media [1,2,3]. NTHi strains are one of the most common bacterial cause of exacerbations in patients suffering from chronic obstructive pulmonary disease (COPD) [4,5]. According to the latest WHO estimates, over 3 million people died of COPD in 2004 and it is predicted that COPD will become the third leading cause of death worldwide by 2030 [6]

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