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The molecular basis for the pathogenicity of Bordetella pertussis is becoming clear. Pertussis is characterized by growth of B. pertussis on the respiratory mucosa, resulting in local damage and production of toxins that cause systemic symptoms. B. pertussis can attach to human cells using several adhesins (15), including filamentous hemagglutinin (FHA), pertactin, BrkA (Bordetella resistance to killing), pili, and tracheal colonization factor, with other potential adhesins appearing in the genome sequence. As a result of the redundancy of adhesins, with the exception of BrkA, which also promotes resistance to complement (25), mutants deficient in production of a single adhesin are often as virulent as the wild-type strain in animal models of disease (7, 11, 25, 26). Only mutants lacking more than one adhesin are reduced in virulence. B. pertussis produces potent toxins (pertussis toxin and adenylate cyclase toxin) that can poison the host immune response, and this may allow it to escape detection even with only a limited number of adhesins. Pertussis toxin is required for long-term persistence of the bacteria, but its absence makes no difference in the first few weeks of infection (7). In contrast, adenylate cyclase toxin is critical for establishment of infection, since mutants lacking adenylate cyclase toxin were unable to survive past the first week of infection (7).

The new acellular pertussis vaccines contain inactivated pertussis toxin and various combinations of the bacterial adhesins (FHA, pertactin, and fimbrial antigens). Antibodies to these antigens should block adherence and neutralize the effects of pertussis toxin. These vaccines are highly effective at preventing the severe manifestations of pertussis but are less effective at preventing bacterial colonization (1, 4, 8, 17, 20). This is consistent with animal experiments which suggest that adhesins not targeted by the vaccine may permit a bit of colonization and that neutralization of pertussis toxin would limit the severity of the disease but would not have an impact on the initial stages of infection.

From a public health point of view there are many advantages to a vaccine that would prevent the organisms from becoming established in the respiratory tract. In this study we have examined the ability of acellular vaccines to promote two immune defenses that could kill the bacteria and prevent colonization: (i) complement mediated lysis and (ii) opsonization and phagocytosis by neutrophils.

Human immune serum. Human immune serum from an individual with occupational exposure to B. pertussis has been described previously (12, 22-24, 27). This sample has antibodies to B. pertussis lipopolysaccharide (LPS) and several surface-localized protein virulence factors, including FHA (27). Antibodies to adenylate cyclase were not present in this serum sample, as determined by Western blot analysis using purified adenylate cyclase toxin obtained from List Biologicals. Using this technique, we observed antibodies to adenylate cyclase toxin in sera from some, but not all, convalescent individuals (data not shown). Sera from adults participating in an acellular vaccine trial (10) are described in Table 1. Pre- and postvaccination serum samples were selected based on patterns of high and low responses to the four antigens in the acellular vaccines (Table 1). All of the preimmunization samples had low titers of antibodies to the four vaccine antigens, which were pertussis toxin, FHA, pertactin, and fimbriae. The individual receiving a placebo vaccine had low postimmunization titers of antibodies to all of the vaccine antigens. Four samples were chosen because each individual had very high postimmunization titers of antibodies to a single vaccine antigen (the four individuals, designated PT-Hi, FHA-Hi, Prn-Hi, and Fim-Hi, displayed high levels of antibody to pertussis toxin, FHA, pertactin, and fimbriae, respectively, in serum), although a rise in titers of antibodies to other antigens occurred in some cases. One individual (designated 4-Hi) had high postimmunization titers of antibodies to all four antigens, and another individual (designated 3-Hi) had high postimmunization titers of antibodies to pertussis toxin, FHA, and pertactin. All human serum samples were heat-inactivated at 56°C for 30 min to eliminate complement activity.

Antibody-dependent complement-mediated bacterial lysis. Complement is an important defense in the lungs and is also extruded to mucosal surfaces. Intact mucosal surfaces have about 10% as much complement as serum, and the amount increases during infection (16). B. pertussis expressing the surface protein, BrkA, resists killing by complement (5, 6, 27). However, some individuals mount an immune response that can overcome the BrkA defense (27). In one study, sera from adults with different exposure to B. pertussis were characterized for bactericidal activity against a wild-type strain and a BrkA complement-sensitive mutant (27). All of the sera killed the complement-sensitive mutant, suggesting that adults often have preexisting immune responses to B. pertussis, resulting from childhood immunization or from exposure to B. pertussis or cross-reactive organisms. However, only the sera collected from the convalescent and occupationally exposed individuals activated complement to kill the wild-type strain. Individuals receiving a two-component acellular vaccine (pertussis toxoid and FHA) lacked antibodies that promoted complement-mediated lysis (27). Pertussis toxin is secreted and FHA is easily detached from the bacterial surface, making these poor targets for complement fixation and bactericidal killing. Pertactin (an outer membrane protein) and fimbriae remain attached to the bacteria and would be better targets for complement killing. In this study we examined the ability of acellular vaccines containing pertactin and fimbriae as antigens to promote complement killing.

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Antibody-dependent complement-mediated killing of B. pertussis. Bacterial survival was determined after a 1-h incubation with guinea pig complement. The control samples included a no-serum control (checked bar) and a heat-inactivated human immune serum (27) with known bactericidal activity (closed bar immediately to right of checked bar). The bactericidal activity of preimmunization (Pre) and postimmunization (Post) samples from an adult acellular vaccine trial are plotted. The preimmunization antibody titers were low for all vaccine antigens tested, and the individuals from whom the samples were collected were assigned designations on the basis of the pattern of their postvaccination response to the vaccine antigens pertussis toxin, FHA, pertactin, and fimbriae (Fim). 4-Hi displayed a high response to all of the vaccine antigens, and 3-Hi displayed a high response to pertussis toxin, FHA, and pertactin, as indicated in Table 1. Data were analyzed by the Student t test. *, samples with statistically significant activity compared to the no-serum control (P of <0.05 to 0.002).

Phagocytosis by human neutrophils. Neutrophils are an important part of the innate immune response, and opsonizing antibodies enable neutrophils to contribute to microbial clearance in the presence of an acquired immune response. The early phases of inflammation due to infectious agents are characterized by an infiltration by leukocytes, especially neutrophils. Neutrophils are recruited in response to inflammatory mediators such as tumor necrosis factor alpha and interleukin-1 or bacterial products, such as LPS or formylated peptides (e.g., formylmethionyl-leucine-phenylalanine) and a recent study confirmed that neutrophil recruitment occurs in human airways in response to such agents (9). Neutrophil infiltration was observed in the lungs of mice following aerosol challenge with B. pertussis (13).

View larger version (25K): [in this window] [in a new window]   FIG. 2.   Effect of opsonization by human serum on bacterial attachment to neutrophils. Bacteria were not opsonized (checked bar) or were opsonized by the addition of either a control serum characterized in previous studies (12, 22-24) or sera from human volunteers participating in an acellular pertussis vaccine trial and incubated with human neutrophils for 1 h. One hundred consecutive neutrophils were examined microscopically for the number of adherent extracellular bacteria. Data were analyzed by the Student t test. Each bar represents the mean (+ standard error of the mean [error bars]) from seven to eight independent experiments. An asterisk above a bar indicates a result that is significantly different from that of the nonopsonized control (P of <0.05 to 0.00001). An asterisk placed after a label indicates a result that is significantly different from that of the preimmunization control (P of <0.05 to 0.002). Pre, preimmunization; Post, postimmunization.

View larger version (22K): [in this window] [in a new window]   FIG. 3.   Effect of opsonization by human serum on bacterial phagocytosis by neutrophils. Bacteria were not opsonized (checked bar) or were opsonized by addition of human sera as described in the legend to Fig. 2, and phagocytosis was allowed to occur. One hundred consecutive neutrophils were examined for the number of intracellular bacteria. Data were analyzed by the Student t test. Each bar represents the mean (+ standard error of the mean [error bar]) from seven to eight independent experiments. *, significantly different from nonopsonized control result (P < 0.04). Pre, preimmunization; Post, postimmunization.