Polymorphism in the Bordetella pertussis virulence factors P.69/pertactin and pertussis toxin in The Netherlands: temporal trends and evidence for vaccine-driven evolution

Abstract

The Bordetella pertussis proteins P.69 (also designated pertactin) and pertussis toxin are important virulence factors and have been shown to confer protective immunity in animals and humans. Both proteins are used in the new generation of acellular pertussis vaccines (ACVs), and it is therefore important to study the degree of antigenic variation in these proteins. Sequence analysis of the genes for P.69 and the pertussis toxin S1 subunit, using strains collected from Dutch patients in the period 1949 to 1996, revealed three P.69 and three S1 variants which show differences in amino acid sequence. Polymorphism in P.69 was confined to a region comprised of repeats and located proximal to the RGD motif involved in adherence to host tissues. Variation in S1 was observed in two regions previously identified as T-cell epitopes. P.69 and S1 variants, identical to those included in the Dutch whole-cell pertussis vaccine (WCV), were found in 100% of the strains from the 1950s, the period when the WCV was introduced in The Netherlands. However, nonvaccine types of P.69 and S1 gradually replaced the vaccine types in later years and were found in approximately 90% strains from 1990 to 1996. These results suggest that vaccination has selected for strains which are antigenically distinct from vaccine strains. Analysis of strains from vaccinated and nonvaccinated individuals indicated that the WCV protects better against strains with the vaccine type P.69 than against strains with non-vaccine types (P = 0.024). ACVs contain P.69 and S1 types which are found in only 10% of recent Dutch B. pertussis isolates, implying that they do not have an optimal composition. Our findings cast a new light on the reemergence of pertussis in highly vaccinated populations and may have major implications for the long-term efficacy of both WCVs and ACVs.

FIG. 1

Structure of the prn gene, coding for the P.69 precursor. The regions coding for the N- and C-terminal parts of the P.69 precursor, which are removed, are indicated in gray. The central white region codes for P.69, which is exposed at the cell surface. Regions 1 and 2, which code for the repeats GGxxP and PQP, respectively, are indicated in black. The small arrows show the approximate positions of primers used for PCR and sequencing. The figure is based on data in reference .

FIG. 2

Variants of P.69 observed in the Dutch B. pertussis population. Depicted are the DNA sequence and corresponding amino acid sequence of the single polymorphic region observed. In P.69C and P.69A, only differences with respect to P.69B are indicated. Dots and dashes indicate identical bases and gaps, respectively. GGxxP repeats are underlined. The RGD sequence, which is involved in attachment to mammalian cells, is indicated by double underlining. The number 778 refers to the position of the first base of the indicated sequence in the prn gene.

FIG. 3

Temporal trends in the frequency of P.69 variants in the Dutch B. pertussis population. The percentage of strains harboring distinct P.69 variants was determined in Dutch strains collected between 1949 and 1996 for each period indicated on the x axis. Strains collected between 1949 and 1980 contained P.69A only. Insufficient strains from 1991 were available. The Dutch WCV contains P.69A.

FIG. 4

Variants of the pertussis toxin S1 subunit. Depicted are the DNA and corresponding amino acid sequences of the two polymorphic regions observed. In S1B and S1D, only differences with respect to S1A are indicated. Dots indicate identical bases. The numbers refer to positions in the s1 gene.

FIG. 5

Temporal trends in the frequencies of the pertussis toxin S1 subunit in the Dutch B. pertussis population. The percentages of each variant in three periods were determined. The strains used for the Dutch WCV produce S1B and S1D.