Use of a Toxin Neutralization Assay To Characterize the Serologic Response to Adenylate Cyclase Toxin after Infection with Bordetella pertussis

Abstract

Adenylate cyclase toxin (ACT) is an essential virulence factor of Bordetella pertussis, and antibodies to ACT protect against B. pertussis infection in mice. The toxin is therefore a strong candidate antigen for addition to future acellular pertussis vaccines. In order to characterize the functionality of the immunologic response to ACT after infection, we developed an assay for testing the ability of serum samples from subjects infected with B. pertussis to neutralize ACT-induced cytotoxicity in J774 macrophage cells. Baboons develop neutralizing anti-ACT antibodies following infection with B. pertussis, and all sera from baboons with positive anti-ACT IgG enzyme-linked immunosorbent assay (ELISA) results neutralized ACT cytotoxicity. The toxin neutralization assay (TNA) was positive in some baboon sera in which ELISA remained negative. Of serum samples obtained from humans diagnosed with pertussis by PCR, anti-ACT IgG ELISA was positive in 72%, and TNA was positive in 83%. All samples positive for anti-ACT IgG ELISA were positive by TNA, and none of the samples from humans without pertussis neutralized toxin activity. These findings indicate that antibodies to ACT generated following infection with B. pertussis consistently neutralize toxin-induced cytotoxicity and that TNA can be used to improve understanding of the immunologic response to ACT after infection or vaccination.

Evaluation of Adenylate Cyclase Toxoid Antigen in Acellular Pertussis Vaccines by Using a Bordetella pertussis Challenge Model in Mice

“…ACT has potent toxic effects against CD-11b/CD-18-expressing phagocytes, such as macrophages or neutrophils (32)(33)(34). To determine the functional capacity of the serum antibodies induced via immunization, we utilized the AC toxin neutralization assay with J774A.1 cells (35). Only sera obtained from mice vaccinated with aP plus ACT or aP plus RTX were able to neutralize the toxicity of ACT in J774A.1 macrophages (Fig.…”

“…ACT neutralization assay. ACT is, through a combination of mechanisms, cytotoxic for J774A.1 (ATCC TIB-67) macrophage-like cells, and protection against this effect is the basis for the toxin neutralization assay (35). J774A.1 cells were cultured in Dulbecco's modified Eagle's medium with 25 mM glucose (Gibco).…”

No pain no gain? Adjuvant effects of alum and monophosphoryl lipid A in pertussis and HPV vaccines

“…Whole fixed B. pertussis cells contain many more protein antigens than the 3–5 purified pertussis proteins present in various manufacturer’ formulations of DTaP. Some investigators have proposed that additional pertussis antigens are needed [33] such as the B. pertussis virulence factor adenylate cyclase toxin (ACT) which is protective in mice and immunogenic in baboons [34,35 • ]. DTP also contained ligands for several Toll-like receptors, TLR1, 2, 4, 5, 6 and 9 [36] whereas subunit DTaP has only the conventional vaccine adjuvant alum, often faulted for its Th2-bias.…”

Bordetella Adenylate Cyclase-Hemolysin Toxins

“…Bp AC-Hly is highly immunogenic. Antibodies are detected in serum of infected non-vaccinated subjects [ 51 , 52 , 53 , 54 ]. Since none of the actual pertussis acellular vaccines contain Bp AC-Hly, it was proposed to use Bp AC-Hly as an antigen for diagnosis.…”

“…Recently, Eby et al [ 54 ] demonstrated that anti AC-Hly antibodies developed by humans following infection with B. pertussis consistently neutralize toxin-induced cytotoxicity. They suggest that toxin-neutralization assay can be used to characterize the immunological response to AC-Hly after infection and vaccination [ 54 ].…”