Abstract
ABSTRACTDespite high vaccination rates, the incidence of whooping cough has steadily been increasing in developing countries for several decades. The current acellular pertussis (aP) vaccines all include the major protective antigen pertussis toxin (PTx) and are safer, but they appear to be less protective than infection or older, whole-cell vaccines. To better understand the attributes of individual antibodies stimulated by aP, we isolated plasmablast clones recognizing PTx after booster immunization of two donors. Five unique antibody sequences recognizing native PTx were recovered and expressed as recombinant human IgG1 antibodies. The antibodies all bind different epitopes on the PTx S1 subunit, B oligomer, or S1-B subunit interface, and just one clone neutralized PTx in an in vitro assay. To better understand the epitopes bound by the nonneutralizing S1-subunit antibodies, comprehensive mutagenesis with yeast display provided a detailed map of the epitope recognized by antibodies A8 and E12. Residue R76 is required for antibody A8 binding and is present on the S1 surface but is only partially exposed in the holotoxin, providing a structural explanation for A8's inability to neutralize holotoxin. The B-subunit-specific antibody D8 inhibited PTx binding to a model receptor and neutralized PTx in vitro as well as in an in vivo leukocytosis assay. This is the first study, to our knowledge, to identify individual human antibodies stimulated by the acellular pertussis vaccine and demonstrates the feasibility of using these approaches to address outstanding issues in pertussis vaccinology, including mechanisms of accelerated waning of protective immunity despite repeated aP immunization.
Highlights
Despite high vaccination rates, the incidence of whooping cough has steadily been increasing in developing countries for several decades
Plasmablast cells were isolated by flow cytometry and sorted into single wells of 96-well plates (50 plates per donor), and the antibody-containing supernatants were screened by a custom Luminex assay for pertussis toxin (PTx) reactivity
The sequences of antibodies isolated from plasmablasts, precursors to the mature bone marrow plasma cells responsible for the bulk of the serum antibodies, correlate with those of serum antibodies observed by use of proteomics, indicating that these approaches identify the specific sequences present in the serum responsible for enzyme-linked immunosorbent assay (ELISA) signals observed during serological analyses [35]
Summary
The incidence of whooping cough has steadily been increasing in developing countries for several decades. The B-subunit-specific antibody D8 inhibited PTx binding to a model receptor and neutralized PTx in vitro as well as in an in vivo leukocytosis assay. This is the first study, to our knowledge, to identify individual human antibodies stimulated by the acellular pertussis vaccine and demonstrates the feasibility of using these approaches to address outstanding issues in pertussis vaccinology, including mechanisms of accelerated waning of protective immunity despite repeated aP immunization. PTx belongs to the AB5 class of toxins, which are characterized by a catalytically active A subunit ( called S1; 26 kDa) and a receptor-binding B oligomer. The PTx holotoxin is directly responsible for leukocytosis, which is predictive of clinical outcomes
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