Antigenic analysis of Bordetella pertussis filamentous hemagglutinin with phage display libraries and rabbit anti-filamentous hemagglutinin polyclonal antibodies

Abstract

Although substantial advancements have been made in the development of efficacious acellular vaccines against Bordetella pertussis, continued progress requires better understanding of the antigenic makeup of B. pertussis virulence factors, including filamentous hemagglutinin (FHA). To identify antigenic regions of FHA, phage display libraries constructed by using random fragments of the 10-kbp EcoRI fragment of B. pertussis fhaB were affinity selected with rabbit anti-FHA polyclonal antibodies. Characterization of antibody-reactive clones displaying FHA-derived peptides identified 14 antigenic regions, each containing one or more epitopes. A number of clones mapped within regions containing known or putative FHA adhesin domains and may be relevant for the generation of protective antibodies. The immunogenic potential of the phage-displayed peptides was assessed indirectly by comparing their recognition by antibodies elicited by sodium dodecyl sulfate (SDS)-denatured and native FHA and by measuring the inhibition of this recognition by purified FHA. FHA residues 1929 to 2019 may contain the most dominant linear epitope of FHA. Clones mapping to this region accounted for ca. 20% of clones recovered from the initial library selection and screening procedures. They are strongly recognized by sera against both SDS-denatured and native FHA, and this recognition is readily inhibited by purified FHA. Given also that this region includes a factor X homolog (J. Sandros and E. Tuomanen, Trends Microbiol. 1:192-196, 1993) and that the single FHA epitope (residues 2001 to 2015) was unequivocally defined in a comparable study by E. Leininger et al. (J. Infect. Dis. 175:1423-1431, 1997), peptides derived from residues of 1929 to 2019 of FHA are strong candidates for future protection studies.

FIG. 1

Phage display vectors. (A) Sequence of fd-tet, corresponding to the last residue of the pIII preprotein sequence and the first three residues of mature pIII, shown for comparison with fDRW70 and fDRW8nn vectors. (B and C) Amber vector fDRW70 was designed to allow construction of libraries free of nonrecombinants. An amber (TAG) codon within a stuffer fragment (B) can be removed with FspI and PvuII, creating a linear fragment for cloning blunt-end inserts (C) of length 3n + 2 (where n is an integer). Peptides are displayed near the N terminus of mature pIII and are flanked by Gly-Ala-rich linker sequences. The vector is propagated in an amber-suppressing host strain such as E. coli LE392 (SupE SupF), while libraries are constructed in a non-amber-suppressing host. Vector self-ligation yields a frameshift in gene III; since pIII is required for virion morphogenesis and infectivity, cells infected with self-ligated vector produce few, noninfectious virions. If the stuffer is not excised, the amber codon similarly prevents production of pIII. In principle, only recombinants possessing inserts of appropriate length contribute to the phage library. (D and E) fDRW8nn vectors were designed to display foreign peptides flanked by N-terminal Gly-Pro and variable C-terminal Gly-containing linker peptides. Each vector incorporates a rare SrfI restriction site (D) for receiving inserts of length 3n (E). Ligation products can be digested with SrfI prior to transforming E. coli to reduce or eliminate recombinants from a library. Vectors used were fDRW836 (inserts followed by GVGTGA in one-letter amino acid code), fDRW863 (GVGSGA), fDRW864 (GAGTGA), fDRW867 (GAGSGA), and fDRW861 (GAGA).

FIG. 2

Output from a single round of biopanning. Each library (70-A, 70-B, etc.) was biopanned with the indicated quantities of pooled FN2/4-FS1/4. FHA-70 libraries (A) were biopanned with ∼10⁹ virions; FHA-80 libraries (B) were biopanned with ∼10¹¹ virions. Note a, no virions (titered as transducing units) were detected; the lower limit of detection was 750 transducing units.

FIG. 3

Enrichment for antibody-reactive clones. (A) Fractions of clones recognized in plaque lifts (with a 1:8,000 dilution of PAb FN2/4) of libraries 70-A and 70-B before (unenriched library) and after a single round of biopanning with 3.6 μg and 360 ng of pooled FN2/4-FS1/4. Each bar represents a single plaque lift. The fraction over each bar represents the number of antibody-reactive plaques as a fraction of the total number of plaques; these values are expressed as a percentage on the y axis. (B and C) Fractions of clones recognized in plaque lifts (with the indicated dilutions of antibodies) after a single round of biopanning libraries 70-A to -D (B) and library 80-A (C) with 3.6 μg and 360 ng of pooled FN2/4-FS1/4 and combining the enriched virions. The fraction placed over each bar represents the number of antibody-reactive plaques as a fraction of the total number of plaques; these values are expressed as a percentage on the y axis. Superscripts: a, plaque lifts were probed with a 1:8,000 dilution of pooled FN2/4-FS1/4; b, plaque lifts were probed with the indicated dilution of FN2/4. n.d., not determined.

FIG. 4

Positional distribution of antibody-reactive clones and comparison with other studies. (A) Phage-displayed antibody-reactive clones identified in the present study, mapped according to their position within the primary amino acid sequence of FhaB. (B) Antibody-reactive clones identified in a similar study (35a) that used a Pseudomonas aeruginosa OprF expression system. (C) Recognition of FhaB-derived recombinant proteins by nine anti-FHA MAbs allowed the approximate mapping of their epitopes within a 1,200-residue immunoreactive domain (9). (D) Antigenic domains identified (18) by using 23 MAbs directed against FHA and mapped by using FHA, its proteolytic fragments, and recombinant FHA proteins. Although epitopes within domains IA, IB, IIA, and IIC were readily isolated to the identified fragments, MAbs that nominally mapped to region IIB cross-reacted with several recombinant FHA proteins that together spanned the entire region of domain II, including the region identified with dashed lines, possibly because of the repeat-rich nature (F to H) of much of FHA. (E) Epitopes identified in the same study (; described above for panel D) by PepScan analysis. Both domain IIB antibodies recognized sequences that can best be defined in terms of the indicated consensus sequence. Three domain IA antibodies recognized a common sequence corresponding to FHA_2001–2015. (F to H). Repeating sequence motifs. (F) Imperfect repeats of ∼37 (“A” repeats) and ∼41 (“B” repeats) amino acid residues (9). (G) Proposed structural domain rich in β strands and turns, comprised of 38 repeats of a 19-residue compositional motif (21). (H) Direct repeats identified using the SAPS (statistical analysis of protein sequences) algorithm (3). (i), repeats of SGGGAVN (in one-letter amino acid code); (ii), imperfect repeats of GRDAVR, GRDAVRV, and GKDAVRV; (iii), QAVALGSASSNALSVRAGGALKAGKLSAT and QAVQLGAASSRQALSVNAGGALKADKLSAT; (iv), repeats of SAHGAL; (v), repeats of GAVEAA; (vi), DVDGKQAVALGSASSNALSVRAGG and DVDGKQAVTLGSVASDGALSVSAGG; (vii), GAIGVQGGEAVS and GAIGVQAGGSVS; (viii), SAGAMTVNGRD and SAGAMTVRD.

FIG. 5

Dot blots of antibody-reactive clones. (A) Relative positions within FhaB of antibody-reactive clones assayed by means of dot blotting with anti-FHA antibodies and serum. (B) Thirty unique clones (I-a, I-b, etc.) were assayed. In many cases (e.g., clone ID I-a), more than one sibling (e.g., clones 43A, 46, and 52) were assayed to control for variability. Controls included clone 30 (see Table 3) and two variants (pseudorevertants) of vector fDRW70, prA and prB. Triplicate 2-μl samples (800 ng of protein) applied to nitrocellulose were probed with the indicated dilutions of E. coli-absorbed sera FS1/4 and FN2/4 and crude serum FN1/4; protein A-purified antiphage (α-f1) antibodies, as a control to assess virion quantities bound to nitrocellulose; and no primary antibody, as a control for recognition of virions by secondary antibody alone. Only the first of the triplicates are shown for these latter controls. After blot development, scanned computer images were imported into the format shown here. Two sets of nonconcurrent dot blots, (i) and (ii), are shown.

FIG. 6

ELISA and BCA protein assay of antibody-reactive clones. (A) ELISA of 30 unique clones (I-a through XIV). In some cases (e.g., clone ID III-a), more than one sibling (e.g., clones 2, 9, and 25) were assayed to control for variability. Controls included clone 30 (see Table 3) and vector fDRW70 variant (pseudorevertants) prA. Each clone was probed with E. coli-absorbed sera FN2/4 and FS1/4, prepared by preincubating 1:5,000 dilutions of antibodies with affinity-purified FHA at final concentrations of 0, 0.8, 5, and 30 μg ml⁻¹ for 2.75 h at 37°C and subsequently diluting these mixtures twofold before use in ELISA. Values shown are means of duplicate wells. (B) BCA protein assay of virions bound to plates after washing as for ELISA. Values shown are means of triplicate wells ± 2 standard errors.

FIG. 7

Sequence overlaps in region I and XI clones. Sequences correspond to those in Table 3 and are abbreviated for convenience; numbers in brackets indicate the numbers of residues omitted. Uppercase letters are FhaB sequences; lowercase letters are vector-derived sequences. Boxed regions of overlap are discussed in the text.