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| **          | **Background:** Despite the high vaccination coverage  |
| Abstract:** | in the United States, pertussis continues to cause     |
|             | substantial morbidity and has the highest mortality    |
|             | among vaccine-preventable diseases of children.        |
|             | Although only children aged \<7 years currently        |
|             | receive pertussis vaccines, an adolescent/adult        |
|             | pertussis vaccine is likely to be made available in    |
|             | the next few years. Because vaccines are not 100%      |
|             | effective in preventing pertussis, cases are expected  |
|             | to occur among vaccinated persons.                     |
|             |                                                        |
|             | **Unmet need:** Currently available seroassay targets  |
|             | (e.g. IgG antibodies to pertussis toxin) do not        |
|             | diagnose pertussis in persons tested within 3 years of |
|             | vaccination. Moreover, the conventional technology     |
|             | used for pertussis serodiagnosis is problematic.       |
|             |                                                        |
|             | **Objective:** We propose to overcome limitations to   |
|             | serodiagnostic assays by identifying alternative       |
|             | antigenic targets or host antibody responses and by    |
|             | developing alternative assays to diagnose pertussis in |
|             | recently vaccinated persons.                           |
|             |                                                        |
|             | **Methods:** The lipooligosaccharide (LOS) of *B*.     |
|             | *pertussis* will be the primary alternative target     |
|             | evaluated. Other potential targets will be identified  |
|             | from proteins that are elaborated by the agent only    |
|             | during infection. Computer analyses of the *B*.        |
|             | *pertussis* genome may identify additional novel       |
|             | diagnostic targets for evaluation; the presence of     |
|             | antibodies to these targets will initially be          |
|             | determined by Western analysis of select serum         |
|             | specimens. The diagnostic potential of non-IgG         |
|             | antibodies to conventional targets will also be        |
|             | determined. The principal alternative technology to be |
|             | evaluated uses color-coded microscopic beads as the    |
|             | assay platform, allowing for high throughput and       |
|             | multiple analyses in a single tube.                    |
|             |                                                        |
|             | **Analysis:** The frequencies of specific antibodies   |
|             | in available serum specimens representing vaccinees,   |
|             | pertussis patients, and persons \> 11 years of age     |
|             | without recent cough illness will be calculated to     |
|             | determine their diagnostic potential.                  |
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| **Specific  | To identify and evaluate alternative targets, host     |
| aims:**     | responses, and assay methods to diagnose pertussis in  |
|             | recently vaccinated persons, including adolescents and |
|             | adults.                                                |
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| **Ba        | Despite high vaccination compliance in the United      |
| ckground:** | States, pertussis causes substantial morbidity and     |
|             | exhibits the highest mortality among vaccine           |
|             | preventable diseases of childhood. Moreover, reported  |
|             | incidence rates are increasing among children and      |
|             | adolescents aged 10-19 years and in infants aged 0-2   |
|             | months, who are most vulnerable to death and           |
|             | complications requiring hospitalization from           |
|             | pertussis. Only children aged \<7 years currently      |
|             | receive pertussis vaccines in the U.S., so in order to |
|             | further reduce *Bordetella pertussis* transmission and |
|             | morbidity, a new recommendation for an                 |
|             | adolescent/adult pertussis vaccine is likely to be     |
|             | made in the near future. But \>50% of children with    |
|             | pertussis had received at least one pertussis          |
|             | vaccination, so these vaccines do not prevent          |
|             | pertussis in 100% of recipients and numerous cases can |
|             | be expected among future vaccinees. Consequently,      |
|             | improved surveillance is vital to evaluate the impact  |
|             | of proposed vaccination programs and to monitor the    |
|             | changing epidemiology of pertussis in an increasingly  |
|             | vaccinated population. However, surveillance is        |
|             | compromised by problematic diagnostic tests; culture   |
|             | is insufficiently sensitive and available seroassay    |
|             | targets (e.g. IgG antibodies to pertussis toxin) do    |
|             | not diagnose pertussis in persons tested within 3      |
|             | years of vaccination. Moreover, there are              |
|             | technological disadvantages to the conventional        |
|             | serologic assays.                                      |
|             |                                                        |
|             | Enzyme-Linked Immunoassays (ELISA) have provided the   |
|             | most sensitive and specific measurements of immune     |
|             | responses to pertussis. Conventional ELISAs quantitate |
|             | levels of serum antibody directed against filamentous  |
|             | hemagglutinin (FHA) or pertussis toxin (PT), two       |
|             | protein antigens of *B*. *pertussis*. Some antigenic   |
|             | fragments of FHA are shared with other bacteria, so    |
|             | only antibodies against PT are specific for *B*.       |
|             | *pertussis*. Responses to antigens are usually         |
|             | reported by immunoglobulin (Ig) subclass, with IgG     |
|             | levels being most useful for pertussis diagnosis.      |
|             | However, the materials and methods reported for ELISAs |
|             | are not uniform, reference standards are critically    |
|             | limited, and no technique has been FDA-approved for    |
|             | diagnostic use in the U.S.                             |
|             |                                                        |
|             | In addition, there are technological disadvantages to  |
|             | the conventional ELISAs used for pertussis serology;   |
|             | the antigen-antibody reaction product is not measured  |
|             | linearly with respect to the amount of antibody; and   |
|             | the interpretation of ELISA results is confounded by   |
|             | the lack of a quantitative diagnostic cutoff.          |
|             | Consequently, the prototypic ELISA requires multiple   |
|             | dilutions of specimens, complex data transformation,   |
|             | and collection of acute and convalescent specimens.    |
|             | Overcoming these limitations requires identification   |
|             | of alternative antigenic targets or host antibody      |
|             | responses and the development of alternative assays to |
|             | conventional ELISAs. These improvements will meet the  |
|             | need for the serologic diagnosis of pertussis in       |
|             | recently vaccinated persons.                           |
|             |                                                        |
|             |                                                        |
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| **Study     | To overcome the limitations of the conventional ELISAs |
| des         | that negatively impact pertussis surveillance and to   |
| ign/Plan:** | meet the need for serodiagnosis of pertussis among     |
|             | persons recently vaccinated, we propose to:            |
|             |                                                        |
|             | 1.  Identify alternative antigenic targets or          |
|             |     antibodies that will maximize the sensitivity of   |
|             |     pertussis serodiagnosis in a vaccinated            |
|             |     population;                                        |
|             |                                                        |
|             | 2.  Develop alternative assay methods to prototypic    |
|             |     ELISAs.                                            |
|             |                                                        |
|             | The lipooligosaccharide (LOS) of *B*. *pertussis* will |
|             | be the primary alternative antigenic target, as this   |
|             | component is highly immunogenic, available in purified |
|             | form, absent from the current acellular vaccine        |
|             | formulations, and contains epitopes specific for *B*.  |
|             | *pertussis*. Other potential targets include recently  |
|             | identified proteins which are produced by *B*.         |
|             | *pertussis* only during infection. Computer analysis   |
|             | of the *B*. *pertussis* genome and of the amino acid   |
|             | sequence of known antigenic proteins will be used to   |
|             | identify unrecognized, but potentially diagnostically  |
|             | valuable epitopes. The presence of antibodies to these |
|             | targets in selected serum specimens available at the   |
|             | CDC will initially be determined by ELISA (LOS) or     |
|             | Western blot (novel antigens). The diagnostic          |
|             | implications of dimeric serum IgA and high-avidity IgG |
|             | antibodies directed against currently recognized       |
|             | antigens will also be evaluated by ELISA using         |
|             | specific anti-human Ig antibodies and reaction buffer  |
|             | with a disrupting agent, respectively.                 |
|             |                                                        |
|             | The principal alternative technology to be evaluated   |
|             | has the advantage of high throughput and is based on   |
|             | specific antigen-antibody interactions occurring on    |
|             | the surfaces of color-coded microscopic beads. In this |
|             | format, the color-code of the microsphere identifies   |
|             | individual reactions throughout the test, and the      |
|             | magnitude of the reaction is monitored with advanced   |
|             | optics and laser illumination via a reporter molecule. |
|             | The various color signals allow for multiplexing       |
|             | different rapid assays in a single tube. This approach |
|             | uses reagents and an instrument (Luminex) available at |
|             | the Meningitis and Special Pathogens Branch, CDC.      |
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