Variability of outer membrane protein P1 and its evaluation as a vaccine candidate against experimental otitis media due to nontypeable Haemophilus 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.

FIG. 1

Alignment of deduced amino acid sequences of ompP1 alleles carried by the 42 phylogenically variant typeable H. influenzae and NTHI clinical isolates, listed as in Table 1. Dotted rows, isolates BCH-1, BCH-2, and BCH-3 used in the animal challenges; shaded residues, those that differ from the majority consensus sequence (not shown); black bars, variable regions. Numbers to the right of sequences indicate amino acid residue positions. Arrows above residues 97 (variable region I) and 395 (nonvariable region) identify the only two positions at which strains BCH-1 and BCH-3 differ. Smaller arrowheads indicate positions at which phylogenically variant strain BCH-2 differs from BCH-1, BCH-3, or both. Amino acid sequence alignment was performed with the LASERGENE software (DNASTAR), using the CLUSTAL multiple-alignment algorithm (30).

FIG. 2

Construction of pBJG1-A, a suicide plasmid vector carrying a 969-bp fragment of ompP1 inactivated by insertional mutagenesis with a Km^r cassette. All depicted plasmids and constructs are to the scale noted at bottom right. Ap, ampicillin resistance; Tc, tetracycline resistance; Km, kanamycin resistance; ompP1′ and ompP1", ompP1 fragments of the larger 969-bp ompP1 fragment insertionally inactivated with a Km^r cassette at the indicated, unique BglII site. Note that products of two of the plasmid digests shown appear as base pair size markers in Fig. 4A, lanes 1 and 2. This includes (i) the HindIII/PstI digest of pFRG100 yielding five fragments among which is the 969-bp ompP1 fragment and (ii) the BamHI digest of pUC4K yielding the 1,264-bp Km^r cassette as well as the 2,650-bp plasmid backbone.

FIG. 3

Western blot analysis of outer membrane preparations from the ompP1⁺ strains wt BCH-1 (lanes 1 to 4) and wt BCH-3 (lanes 5 to 8), and the putative mutant ompP1 BCH-3 Km^r transformant (lanes 10 to 13). AD4 MAb and serum from a chinchilla immunized with a P1_BCH-1-enriched preparation were used to probe unheated (20°C) and heated (100°C) outer membrane preparations. P1 and ΔP1, non-heat-modified and heat-modified forms of OMP P1, respectively (25). Molecular mass standards appear in lane 9.

FIG. 4

Characterization of the putative P1-deficient isogenic mutant of NTHI BCH-3 by Southern blot analysis. (A) Agarose gel electrophoretic separation of digests and PCR products, stained with ethidium bromide. Lane 1, HindIII/PstI double digest of the ompP1-carrying vector plasmid pFRG100; the bottom band is the predicted 969-bp ompP1 fragment (see Fig. 2); lane 2, BamHI digest of pUC4K; the bottom band is the predicted 1,264-bp Km^r cassette, and the upper band is the predicted 2,650-bp backbone of pUC4K (see Fig. 2); lane 3, PCR amplification of the ompP1 gene (1,371 bp) plus flanks (109 bp upstream and 59 bp downstream) from wt BCH-3 (; unpublished data) using flanking primers P1-5 and P1-6 (see Materials and Methods) to generate a 1,539-bp product; lane 4, PCR amplification of ompP1 from the putative, insertionally inactivated ompP1 BCH-3 Km^r transformant using flanking primers P1-5 and P1-6 (Materials and Methods); when this product is compared with the ompP1 PCR product in the adjacent lane 3, the size increase is found to correlate with that predicted for insertion of the 1,264-bp Km^r cassette as indicated in Fig. 2. (B) Southern blot transfer of the panel A gel electrophoretic separation to a Zeta Probe nylon membrane (Bio-Rad), followed by hybridization with a ³²P-labeled (2) ompP1 969-bp fragment probe (the bottom HindIII/PstI fragment shown in panel A, lane 1, as isolated from an equivalent digest and electrophoretic separation). (C) Duplicate electrophoretic separation of the identical digests and PCR products in panel A, transferred by Southern blotting to a Zeta Probe nylon membrane and hybridized with a ³²P-labeled (2) Km^r cassette probe (the smaller, 1,264-bp BamHI fragment shown in panel A, lane 2, as isolated from an equivalent digest and electrophoretic separation). The lower band in lane 4 of panel C is likely a PCR breakdown product.

FIG. 5

Western blot analysis of recombinant H. influenzae OMP P1_BCH-3 (rP1) purified from cytoplasmic inclusion bodies of an E. coli cloning host. The rP1 was probed with serum from a chinchilla immunized with a P1_BCH-1-enriched preparation (lane 1), the Invitrogen anti-Xpress MAb (lane 2), the AD4 MAb (lane 3), and normal preimmune chinchilla serum (lane 4). Coomassie blue-stained rP1 and molecular mass standards are shown in lane 5 and lane 6, respectively. As described in Materials and Methods, the rP1 generated from expression vector pTrcHisB is a translated fusion product with 30 additional amino acids at the peptide N terminus containing the epitope AspLeuTyrAspAspAspAspLys recognized by the Invitrogen anti-Xpress MAb (lane 2).

FIG. 6

Western blot analysis of outer membrane preparations with rP1_BCH-3 immune serum. Outer membrane preparations from NTHI wt BCH-3 (P1⁺) and the otherwise isogenic BCH-3 ompP1 mutant (P1⁻) were probed with preimmune chinchilla serum (lanes 2 to 5) and post-rP1_BCH-3-immune chinchilla serum (lanes 6 to 9). The wt BCH-3 outer membrane was also probed with the P1-specific AD4 MAb as a control (lane 10). Lane 1, molecular mass standards; P1 and ΔP1, non-heat-modified and heat-modified forms of OMP P1, respectively (25).