Browsing by Subject "Bordetella pertussis"
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Item Biochemical characterization of monoclonal antibodies to the Bordetella pertussis Filamentous hemagglutinin (FHA) and Pertussis toxin (PTx) : implications for improved acellular pertussis vaccine design(2015-08-28) Acquaye, Edith Abena; Maynard, Jennifer Anne, 1974-; Hoffman, David; Russell, Rick; Barrick, Jeffrey E.; Mukhopadhyay, SomshuvraIncidence rates of whooping cough were dramatically decreased by immunization with whole cell pertussis (wP) vaccines in the 1940s. However, concerns about the safety of the wP vaccine resulted in development of new pertussis vaccines using acellular components of B. pertussis. However, B. pertussis continues to circulate and cause whooping cough disease, even with the safer acellular pertussis (aP) vaccines. Over the last decade, in spite of the high vaccination coverage in many countries, there has been a significant rise in whooping cough infection in industrialized countries. The filamentous hemagglutinin (FHA) and pertussis toxin (PTx) are two antigens included in all currently licensed aP vaccines. Both FHA and PTx induce an immune response sufficient for protection, although anti-PTx antibodies correlate more with protection. Most studies assessing the efficacy of aP vaccines have used serum titers as the primary means to measure an antibody response to a given antigen. Single cell techniques however offer an opportunity to better understand the biochemical attributes of individual antibodies induced upon aP vaccination. In this study, we characterized the antibody responses to FHA and PTx after adult aP booster vaccination. Monoclonal antibodies derived from single B cells which responded to FHA and PTx were purified, the binding affinities to the antigens evaluated, mechanisms of neutralization of FHA and PTx toxicity evaluated, and the binding epitopes of the antibodies analyzed. This study is the first to provide insight into the anti-FHA and PTx antibody repertoire after pertussis vaccination, and also identifies useful antibodies for further elucidation of the structure and function of these antigens. In addition, the mechanisms of neutralization of two potently neutralizing anti-PTx monoclonal antibodies, 1B7 and 11E6 are presented. Antibody binding to live B. pertussis, inhibition of PTx binding to a model receptor fetuin and effects on PTx trafficking in CHO-K1 cells were evaluated. Further understanding of the molecular mechanisms of 1B7 and 11E6 neutralization provides a platform for engineering or isolating such unique anti-PTx antibodies for passive immunization therapies.Item The potently neutralizing monoclonal antibody 1B7 : its unique epitope, effects on intracellular trafficking, and elicitation upon infection with pertussis(2010-08) Sutherland, Jamie Nicole; Maynard, Jennifer Anne, 1974-; Georgiou, George; Alper, Hal; Brown, Katherine; Truskett, Thomas M.Disease caused by Bordetella pertussis persists with rates increasing over the past decade in industrialized countries. A hindrance to vaccine development has been the lack of a clear serological correlate of protective immunity. Pertussis toxin (PTx), an AB-type toxin, is one of the bacteria’s major virulence factors and among the lead candidates for potential correlates. Of the numerous monoclonal antibodies (mAbs) binding PTx, the murine IgG2a mAb 1B7 is potently neutralizing in all in vitro assays and in vivo murine models of infection. 1B7 binds an epitope on the enzymatic S1-subunit of PTx with some linear elements but previous work was unable to more precisely define the epitope or determine its exact mechanism of protection. We characterize the epitope bound by 1B7 on PTx-S1 in molecular detail and define energetically important interactions between residues at the interface including six residues on PTx-S1 and six residues on 1B7. Using this information, a model of the 1B7-S1 interaction was developed, indicating a predominantly conformational epitope located on the base of S1 near S4. The location of this epitope is consistent with previous data and is shown to be conserved across several naturally occurring strain variants including PTx-S1A, B, D, and E in addition to the catalytically inactive 9K/129G variant. Using immunofluorescent microscopy, it was determined that 1B7’s unique mode of action lies in its ability to bind to the toxin and co-traffic into target cells. Upon endocytosis, 1B7 protects from PTx intoxication by redirecting its intracellular retrograde trafficking. In order to determine whether antibody responses are differently induced by infection or acellular vaccination, we analyzed sera from 30 adults with confirmed exposure to pertussis and 30 recent vaccinees. Natural infection resulted in significantly higher titers of anti-PTx-S1, 1B7-like, and 11E6-like antibodies, while overall anti-PTx titers were similar to vaccinated samples. We also observed a direct correlation between in vitro protection and the presence of 1B7-like and 11E6-like antibodies. Thus, natural infection elicits higher titers of protective antibodies indicating that the use of detoxified PTx in current acellular vaccines although highly immunogenic results in the elicitation of predominantly non-neutralizing antibodies.Item The adenylate cyclase toxin as a target for antibody therapeutics and vaccination against whooping cough(2015-08) Wang, Xianzhe; Maynard, Jennifer Anne, 1974-; Georgiou, George; Payne, Shelley M; Keatinge-Clay, Adrian T; Ehrlich, LaurenWhooping cough, also known as pertussis, is caused by the bacterium Bordetella pertussis. Since widespread vaccination with heat-killed whole cell vaccines (wP) in the 1950s, the number of cases dropped dramatically. However, there has been a consistent resurgence in the past two decades, coinciding with the switch from wP vaccines to acellular vaccines (aP). US CDC estimates 16 million cases and 195,000 deaths worldwide per year. Accumulating evidence show that aP vaccines provide short protection against the symptoms but not against subclinical infection and transmission of the disease. Changing the adjuvant to induce more protective immunity or inclusion of additional protective antigens are some of the strategies to improve the efficacy of aP vaccines. Adenylate cyclase toxin (ACT) is a 177 kDa protein produced by B. pertussis and related species. ACT mainly targets leukocytes through [alpha] [subscript M] [beta] ₂ integrin and translocates its N-terminal cyclase domain into the cytosol, generating supraphysiological level of cAMP. Studies have shown that ACT-deficient stains are less pathogenic and passive immunization with polyclonal antibodies or active immunization with ACT protected mice against bacterial challenges. However, ACT is not included in any of the current aP vaccines, due to a lack of understanding of its protective epitopes and its poor solubility and stability. We aimed to identify potent neutralizing antibodies (nAbs) as therapeutic candidates, and map their epitopes to guide the design of vaccine antigen overcoming the solubility and stability issues. Two nAbs, M2B10 and M1H5, were discovered from antibody phage display libraries from ACT-immunized mice. They bind non-overlapping conformational epitopes in the C-terminal RTX domain of ACT. Our data suggest their mode of action is interrupting the interaction between the toxin and its cellular receptor. On the other hand, individual domains of ACT were expressed in E. coli and purified. The catalytic and RTX domains retained antigenicity but are biophysically superior to full-length ACT. We further showed that the RTX domain elicited similar level of neutralizing antibody response to ACT in mice. These antibodies, together with those neutralizing other major B. pertussis toxins, may constitute a therapy for severe pertussis infection, and the epitopes provide the basis for structure-based antigen design for superior stability and immunogenicity.