Variability of Outer Membrane Protein P1 and Its Evaluation as a Vaccine Candidate against Experimental Otitis Media due to NontypeableHaemophilus influenzae: an Unambiguous, Multifaceted Approach

Abstract

Candidate vaccine antigens for preventing otitis media caused by nontypeable Haemophilus influenzae (NTHI) should possess one or more conserved epitopes. We sought to evaluate the candidacy of P1, a surface-expressed outer membrane protein knowing that this antigen is subject to diversifying selection. Therefore, we selected NTHI strains from among >500 phylogenically variant isolates representative of the diversity found in natural populations of H. influenzae. Twenty-three variants of P1 (<95% similarity) were identified among 42 strains. When chinchillas were immunized with recombinant P1 (rP1) obtained from one of these isolates (BCH-3), all animals developed antibodies specific for rP1. Immunized animals were protected against disease when challenged with BCH-3, but not with an ompP1 mutant of BCH-3 or a strain (BCH-2) possessing a heterologous P1 (91% identity). We conclude that (i) while P1 induces protection against NTHI-mediated otitis media, development of a polyvalent vaccine reflecting the variability of P1 would be necessary to construct an efficacious vaccine and (ii) use of a phylogenically characterized collection of representative isolates in concert with gene sequencing, cloning, gene inactivation, and animal testing offers an efficient, rational, and rigorous strategy for evaluating the potential problems associated with variability of vaccine targets and specificity of related immune responses.

Analysis of Genetic Relatedness of Haemophilus influenzae Isolates by Multilocus Sequence Typing

“…Determination of the distribution of some of these islands across the H. influenzae phylogenetic tree is in progress (A. L. Erwin et al, unpublished data) and will add to our understanding of the differences between genetic groups. While in this study we have scored genetic islands simply as present or absent, we recognize that there is substantial sequence heterogeneity for many islands and for several other genes encoding outer membrane proteins (3,6,19,24). Our goal in studying these islands was not to characterize differences between b Phylogenetic groups are included if more than two NTHI strains in the group were examined for the indicated loci.…”

Host-derived sialic acid is incorporated into Haemophilus influenzae lipopolysaccharide and is a major virulence factor in experimental otitis media

“…1). Details of the dendrogram and its validation using multilocus sequence typing have been published (12)(13)(14). Hi strains were cultured at 37°C in brain-heart infusion (BHI) broth supplemented with 10 g͞ml haemin and 2 g͞ml NAD (sBHI); BHI solidified with agar (1% wt͞vol) and supplemented with 10% Levinthals reagent; or chocolate agar, supplemented with 10 g͞ml kanamycin when appropriate.…”

Comparison of usefulness of randomly amplified polymorphic DNA and amplified-fragment length polymorphism techniques in epidemiological studies on nasopharyngeal carriage of non-typable Haemophilus influenzae

“…DNA for AFLP analysis was prepared according to the method of Bolduc et al (2000). Two millilitres of overnight culture of H. influenzae was centrifuged at 5000 g for 10 min at 4 8C, resuspended in 3 ml 50 mM Tris/HCl, 20 mM EDTA (pH 8·0) and recentrifuged.…”