The results of the strain characterization by means of serotype, prn
type, and PFGE type indicate that there have been shifts in the B. pertussis
population over the years. Similar observations of polymorphism and variation in virulence factors and strains have been made in many other countries using Pw vaccines and in which nonvaccine strains are circulating (2
Detoxified fimbriae, pertactin, and toxin are used as components in acellular vaccines. It is therefore of particular interest to study these single markers separately over time in relation to the vaccination program used.
We have previously documented that serotype Fim2 strains were only seldom seen among our isolates before 1977 but increased continuously thereafter during the vaccine-free era (36
). In the Stockolm trial I, the proportion of serotype Fim2 was 88% for the placebo group (Table ). Earlier studies also showed serotype Fim2 to be most prevalent in unvaccinated populations, whereas serotype Fim3 and serotype Fim2,3 seem to be most common in vaccinated populations, leaving better conditions for expressing serotype Fim3 (32
). It may be that Fim2 is a superior antigen to Fim3 and that the Swedish whole-cell vaccine used up to 1979 was efficient against serotype Fim2 strains. The highly developed fimbriae 2 may also confer a colonization advantage, explaining their predominance during the period without vaccination.
An immunity-driven selection is further supported by the significant differences in serotypes between those vaccinated with vaccines containing fimbriae and unvaccinated children observed in the Stockholm trial I (1992 to 1995) (17
). In that vaccine trial, it was shown that there was a lower rate of the serotype Fim2 type in both the DTPw and the DTPa5 vaccine groups (both containing fimbriae), 72.4% and 72.5%, respectively, compared to the rate of Fim2 in the DT and DTPa2 groups (neither containing fimbriae), 84.2% and 83.3%, respectively. This difference was also confirmed in the present study when isolates from two of the groups from the Stockholm trial I were reanalyzed using microplate agglutination with monoclonal antibodies (Table ). There was no such difference when the DTPa2 group was compared with the placebo group, either in the 1992-to-1995 study or in the 1986-to-1987 study (36
Interestingly, this difference in serotype Fim2 expression between vaccinated and unvaccinated individuals was also present during the first years after the introduction of the Pa vaccination program, indicating an effect of vaccination, although the mechanism is unclear. The selection of Fim3 may be indirectly due to an effect of the immune system on the expression of genes, or possibly gene combinations, not all of which are known.
More remarkable is the fact that serotype Fim3 gradually replaced serotype Fim2 after the renewal of vaccination in 1996, with the use of three- or two-component acellular vaccines. None of the vaccines contain fimbriae; the two-component vaccine does not even contain pertactin. It may be that impurities are present in the vaccines. In fact, at least one two-component vaccine similar to that used in parts of Sweden since 1998 was shown to induce a booster response to agglutinins in a Dutch study, indicating that a small amount of fimbriae may be present in the vaccine (4
In some studies, there are indications that vaccines containing fimbriae are more efficient in the serotype Fim2 environment. In the Stockholm trial I from 1992 to 1995 (17
), the efficacy of a DTPa5 vaccine was 85% and that of a DTPw vaccine was 48%. In a parallel Italian study (11
), the efficacy of the same DTPw vaccine was only 36% with a different, and perhaps less favorable, bacterial population. In Sweden, the proportion of serotype Fim2 strains was above 80%, and in Italy, 16%. The corresponding figures for serotype Fim3 were 13% and 76%, respectively (12
). During the Stockholm trial II from 1993 to 1996 (30
), the incidences per 100,000 person years for another DTPw, a DTPa3, and a DTPa5 vaccine were 61, 155, and 85, respectively. In a follow-up study after 6 years, the incidences were 25, 40, and 42 (16
). The earlier advantage of the DTPa5 over the DTPa3 vaccine was leveled out, perhaps due to the shift from serotype Fim2 to serotype Fim3 strains.
From the host perspective, it would be of great interest to follow anti-Fim2 and anti-Fim3 antibody profiles over the years. Unfortunately however, it has not been possible to obtain separate Fim2 and Fim3 antigens for this purpose. In the Swedish vaccine trials (17
), antibody response against fimbriae was measured using a combined Fim2-Fim3 antigen. Alternatively, an inhibition antibody enzyme-linked immunosorbent assay could be used if anti-Fim2 and anti-Fim3 monoclonal antibodies were available in sufficient amounts.
As for pertactin, the circulating population of B. pertussis
has regularly presented pertactins of nonvaccine type. Based on the polymorphism of pertactin and toxin, Mooi et al. in 1998 (27
) suggested a vaccine-driven evolution of new subtypes in The Netherlands, referring to temporal trends. This seems to hold true for Sweden also. The Swedish whole-cell vaccine contained prn10
. During the Pw period, prn1/7
was most frequent and predominated over prn2
. After 1979, there has been an expansion of B. pertussis
strains with prn2
that did not change with the introduction of DTPa2 and DTPa3 after 1996, a finding corresponding to the lack of prn2
in the DTPa vaccines. However, the DTPa3 vaccine contains prn1
. After 1996, there was a decrease in prn1/7
until after 2001 to 2003, when this pertactin type was no longer seen. For pertactin, no significant difference was seen between vaccinated and unvaccinated individuals, supporting the Italian results (24
). Isolates showing prn3
appeared more often shortly after 1996 and reached a peak in 1999, a trend that was discontinued in 2001. It may be that pertactin 1-based vaccines give some protection against pertactin 3. Results by He et al. (21
) suggest that the specificities of the immune responses induced by strains harboring prn2
do not induce antibodies against the conformational epitopes that are induced by vaccine strains expressing prn1
. This might explain the emergence of B. pertussis
strains with prn2
Besides the three common prn
types, we found only four other prn
types in single isolates; one of them was prn4
, which was seen in 9% of Finnish isolates from the 1990s (26
) and has also been reported from Poland (19
For toxin, there has been no selective pressure from vaccines. Most acellular vaccines contain ptxA
(2) or ptxA
(3), but the vast majority of circulating clones now produce ptxA
). Also, in our material over the three decades, almost all strains were of ptxA
(1) type from the beginning of the study in the 1970s, when a whole-cell vaccine expressing ptxA
(4) was used. However, this divergence between the vaccine toxin type and the type of toxin produced by clinical isolates was not seen in the United Kingdom. All of the most recently circulating 105 strains (from 1998 and 1999) were of the same pertussis toxin type as those present in the United Kingdom DTPw, i.e., ptxA
(1) and ptxA
Chromosomal fingerprinting by means of PFGE has been shown to be a very discriminating way to discover changes in the circulating bacterial population (2
). Most changes seen in the DNA structure may be due to gain or loss of restriction sites or rearrangement of the fluid genome. These changes may also reflect an antigenic shift most evident for pertactin (2
). This may happen independently, but new variants may be selected by the immune status of the population, due to natural infections, together with vaccination. It is therefore of specific interest to study how the PFGE profiles, along with vaccine-related markers, change over time.
Our study demonstrates that as time has gone by the strain variants have come in waves, gradually peaking over time, disappearing, and being replaced by new profiles (Fig. ). The total number of PFGE profiles seen in Sweden during the years 1970 to 2003 may be large, but there have been only a few predominant ones. A general observation was that the main patterns changed with the period of vaccination policy. During the Pw period (1970 to 1977), BpSR25 (serotype Fim3 prn1
) and BpSR18 (serotype Fim3 prn2
) were predominant. After the cessation of general vaccination in 1979, the PFGE profile BpSR1 (serotype Fim2 prn2
) took over, to be replaced in turn by BpSR11 (serotype Fim3 prn2
) during an incidence peak after the reintroduction of general vaccination in 1996. The BpSR11 PFGE profile had already appeared in Europe (IVβ) before 1996 (41
It has been argued that the changes over time may well be due to natural variation rather than to immunity-driven selection. In Japan, the first country to introduce acellular pertussis vaccines in 1981, it was suggested that if acellular pertussis vaccine-induced antigenic divergence exists, it is likely to be a slow or rare process, but the number of studied isolates were few (13
On the other hand, immunity-driven selection may well explain changes in the Swedish B. pertussis populations over time, as characterized by serotype, prn, and PFGE profiles. Adapted B. pertussis variants may appear due to vaccination and/or boosting of immunity by natural infection. At least, this seems very obvious when the serotypes during the Pw- and the vaccine-free periods are compared, in addition to the observations recorded during the vaccine trials during the vaccine-free period.
Swedish experience so far does not indicate that the divergence between toxin and pertactin subtypes used in acellular vaccines and those found in clinical isolates has had significant influence on the effectiveness of the acellular vaccines against disease. Other parts of the antigen molecules probably contribute to the human immune response. The reported incidence of pertussis has dropped from 120 to 150 cases per 100,000 person years of follow-up during the years 1993 through 1995 to 11, 15, and 7/100,000 in 2001, 2002, and 2003, respectively.
In conclusion, shifts in the B. pertussis population coincide with changes in vaccination policy, from the use of DTPw vaccine in the 1970s to a 17-year period without general vaccination starting in 1979 and once more after 1996, when vaccination with Pa-containing vaccines was introduced.
It is too early to predict whether bacterial polymorphism will have a negative effect on the vaccination program against pertussis in the long run and how or if there will be an immunity-driven selection of new clones or adapted mutants. It may even be that the selected variants are less virulent. Interestingly, serotype Fim2 strains seem to cause more severe illness in terms of cough duration and hospital admission, as reported in the United Kingdom (37
). It would be interesting to compare isolates from regions using DTPa2 with isolates from regions using DTPa3 and to relate profiles to clinical outcome (studies are ongoing). For the future, it is important to follow the bacterial population and study the impact of changes in relation to vaccination policy and incidence of disease. In Sweden, it is of particular interest to follow a possible reappearance of variants expressing serotype Fim2 due to waning immunity, as serotype Fim2 is not present in the acellular vaccines used after 1996 or prevalent among circulating strains.