Characterization of Serological Responses to Pertussis

Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati,^{Division of Infectious Disease, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio}²

^{Corresponding author. Mailing address: Department of Molecular Genetics, Biochemistry, and Microbiology, College of Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267-0524. Phone: (513) 558-2820. Fax: (513) 558-8474. E-mail:}ude.cu@ssiew.nosila.

^{Present address: The Kitasato Institute, Research Center for Biologicals, 6-111 Arai, Kitamoto, Saitama 364-0026, Japan.}

Received 2005 Oct 13; Revised 2005 Dec 1; Accepted 2005 Dec 15.

Abstract

We have compared the use of five nonvaccine antigens to the use of conventional vaccine antigens, pertussis toxin (PT), and filamentous hemagglutinin (FHA) for the serological diagnosis of pertussis by enzyme-linked immunosorbent assay (ELISA). The nonvaccine antigens included the catalytic region of adenylate cyclase toxin (CatACT), the C-terminal region of FHA (C-FHA), lipooligosaccharide (LOS), the peptidoglycan-associated lipoprotein (PAL), and the BrkA protein. The serological responses of individuals with culture-confirmed pertussis were compared to those of adults with no recent history of a coughing disease. An immunoglobulin G (IgG) ELISA for PT was the most sensitive (92.2%) test for the serodiagnosis of pertussis. Of the nonvaccine antigens, ELISA for IgG responses to CatACT (sensitivity, 62.8%), C-FHA (sensitivity, 39.2%), and LOS IgA (sensitivity, 29.4%) were less sensitive but could also distinguish culture-positive individuals from control individuals. The use of a combination of multiple ELISA targets improved the sensitivity of the assay for serological diagnosis. Elevated IgG and IgA antibody titers persisted for more than a year in the individuals with culture-confirmed pertussis.

Abstract

Bordetella pertussis, the causative agent of whooping cough, is a strict human pathogen with no known animal or environmental reservoir. Pertussis vaccination has dramatically decreased the incidence of disease, but the organism continues to circulate in the human population. Severe disease is primarily seen in infants and children who have not been adequately immunized. The World Health Organization definition of pertussis is based both on clinical symptoms (a minimum of 2 weeks of paroxysmal cough) and on confirmation of infection by laboratory tests (36). The clinical symptoms of pertussis are less severe in vaccinated individuals; however, individuals with mild disease can serve as the bacterial reservoirs and act as important sources of transmission to the highly susceptible pediatric population (21, 31, 32).

The diagnosis of pertussis is challenging, and the true incidence of pertussis infection is unknown. The “gold standard” for diagnosis is isolation of B. pertussis from the nasopharynx. The bacterium grows very slowly and is extremely fastidious, and many laboratories cannot successfully culture the microorganism (15). PCR-based detection is more rapid and sensitive than culture-based detection (33). However, the carriage of the organism (and the presence of DNA) decreases with the duration of symptoms and with antibiotic treatment; thus, the sensitivities of both culture and PCR decrease with time (33). Serological methods can also be used for diagnosis. Enzyme-linked immunosorbent assay (ELISA) can detect B. pertussis infection even after the organisms are gone and is especially useful for epidemiological studies. Currently, most of the commercial ELISAs for the diagnosis of pertussis are based on antibody responses to pertussis toxin (PT) or filamentous hemagglutinin (FHA). Since both PT and FHA are included in the acellular pertussis vaccines, high levels of antibodies to these antigens could persist following vaccination, hindering serodiagnosis in vaccinated individuals. For pertussis control, immunization of adolescents is now being introduced and immunization of adults is being considered (19). Given the limitations of culture and PCR, serodiagnostic tests which can discriminate between vaccine responses and infection by B. pertussis will be important for monitoring the efficacies of these additional vaccine strategies to discern their impacts on disease transmission.

To address this issue we compared the performance characteristics of ELISA-based serodiagnostic tests based on five antigens not included in acellular vaccines to those of standard assays based on the vaccine antigens, PT and FHA. The nonvaccine antigens included two factors associated with the bacterial cell wall and three virulence factors. Peptidoglycan-associated lipoprotein (PAL) stabilizes the bacterial outer membrane (7). Carbonetti et al. (5) found that PAL was highly immunogenic in mice infected with a B. pertussis mutant lacking PT expression but was less so in mice infected with wild-type B. pertussis. Lipopolysaccharide (LPS) is a component of the bacterial outer membrane. The B. pertussis LPS has a highly branched core structure, a unique trisaccharide, and no repeating O antigen (6). Due to the lack of repeating O antigen, the LPS of B. pertussis has been termed lipooligosaccharide (LOS). The closely related species Bordetella bronchiseptica and Bordetella parapertussis express LOS but add a repeating O antigen, producing a conventional LPS structure (6). There are unique and shared antigenic determinants between the LOSs and LPSs of the Bordetella species.

The antigens derived from virulence factors of B. pertussis include BrkA, the C-terminal region of FHA (C-FHA), and adenylate cyclase toxin (ACT). BrkA is an adhesin and has been shown to mediate resistance to complement killing (18). FHA is a major adhesion of B. pertussis and is a component of most acellular pertussis vaccines. FHA is produced from a 367-kDa precursor. The first 71 amino acids comprise a signal peptide, which is removed by proteolysis. FHA is further processed by the SphB1 protease (13, 14), resulting in the 220-kDa N-terminal region that is traditionally known as FHA. Much of this form of FHA is secreted from the cell and is the source of FHA for the acellular vaccines. The C-terminal 130-kDa portion (which we term C-FHA) remains associated with the B. pertussis cell (13, 28) and is not included in the pertussis vaccine.

ACT is an essential virulence factor of B. pertussis which functions by inhibiting neutrophil defenses (35). Antibody responses to ACT have been detected in healthy individuals (1, 11), compromising its utility as a diagnostic antigen. The presence of antibodies in uninfected individuals could be due to cross-reactivity with other members of the RTX toxin family, including the Escherichia coli hemolysin (1, 3, 17, 22), which lacks adenylate cyclase enzymatic activity. The 400 amino acids of the N terminus of ACT (termed CatACT) comprise the catalytic domain and are unique to ACT.

Several of the nonvaccine antigens displayed good sensitivity and specificity for the serodiagnosis of pertussis and are promising candidates for the serological diagnosis of pertussis in vaccine recipients.

Acknowledgments

We thank Scott Millen for invaluable assistance.

This research was supported by NIH/NIAID grants R01AI45715, AI25459, and R01AI23695.

Acknowledgments

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