Mapping of a Protective Epitope of Pertussis Toxin by In Vitro Refolding of Recombinant Fragments

Bartoloni, Antonella; Pizza, Mariagrazia; Bigio, Massimo; Nucci, Daniele; Ashworth, L.A.E.; Irons, L.I.; Robinson, A.; Burns, Drusilla L; Manclark, C R; Sato, Hiroko; Rappuoli, Rino

doi:10.1038/nbt0688-709

Cited by 53 publications

(56 citation statements)

References 15 publications

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“…The recognition of the epitope in Western blotting under denaturing conditions is probably due to the strong tendency of the protein to refold in the Western blotting membrane. Generation of epitopes by mixing fragments containing part of the epitope is rare, but it has been described previously in the case of a neutralizing antibody against pertussis toxin (2). In that case, the epitope described was a stable immunodominant conformational epitope that was important for a vaccine.…”

Section: Discussionmentioning

confidence: 99%

The Region Comprising Amino Acids 100 to 255 ofNeisseria meningitidisLipoprotein GNA 1870 Elicits Bactericidal Antibodies

et al. 2005

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GNA 1870 is a novel surface-exposed lipoprotein, identified by genome analysis of Neisseria meningitidis strain MC58, which induces bactericidal antibodies. Three sequence variants of the protein were shown to be sufficient to induce bactericidal antibodies against a panel of strains representative of the diversity of serogroup B meningococci. Here, we studied the antigenic and immunogenic properties of GNA 1870, which for convenience was divided into domains A, B, and C. The immune responses of mice immunized with each of the three variants were tested using overlapping peptides scanning the entire protein length and using recombinant fragments. We found that while most of the linear epitopes are located in the A domain, the bactericidal antibodies are directed against conformational epitopes located in the BC domain. This was also confirmed by the isolation of a bactericidal murine monoclonal antibody, which failed to recognize linear peptides on the A, B, and C domains separately but recognized a conformational epitope formed only by the combination of the B and C domains. Arginine in position 204 was identified as important for binding of the monoclonal antibody. The identification of the region containing bactericidal epitopes is an important step in the design of new vaccines against meningococci.

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Section: Discussionmentioning

confidence: 99%

The Region Comprising Amino Acids 100 to 255 ofNeisseria meningitidisLipoprotein GNA 1870 Elicits Bactericidal Antibodies

et al. 2005

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“…Furthermore, in highly vaccinated populations, circulating strains have emerged with increased virulence, correlating with increased PTx production due to promoter mutations (23). Antibodies specific to PTx have been analyzed in detail, revealing four or more nonoverlapping immunodominant epitopes on the catalytically active S1 subunit, of which only one is highly protective (2,21). The Sato group performed a comparison of 32 anti-PTx monoclonal antibodies in several protection assays, including inhibition of catalytic activity, CHO cell clustering, and murine intracerebral and aerosol challenge models (34).…”

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confidence: 99%

Antibodies Recognizing Protective Pertussis Toxin Epitopes Are Preferentially Elicited by Natural Infection versus Acellular Immunization

Sutherland

Chang

Yoder

et al. 2011

Clin. Vaccine Immunol.

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Despite more than 50 years of vaccination, disease caused by the bacterium Bordetella pertussis persists, with rates increasing in industrialized countries over the past decade. This rise may be attributed to several factors, including increased surveillance, emergence of vaccine escape variants, waning immunity in adults, and the introduction of acellular subunit vaccines, which include chemically detoxified pertussis toxin (PTd). Two potently protective epitopes on pertussis toxin (PTx) are recognized by the monoclonal antibodies 1B7 and 11E6, which inhibit catalytic and cell-binding activities, respectively. In order to determine whether the PTx exposure route affects antibody responses to these epitopes, we analyzed sera from 30 adults with confirmed pertussis exposure and from 30 recently vaccinated adults for specific anti-PTx antibody responses and in vitro CHO cell neutralization titers. While overall titers against PTx and the genetically detoxified variant, PTg, containing the R9K and E129G substitutions, were similar in the two groups, titers against specific epitopes depended on the exposure route. Natural infection resulted in significantly higher titers of anti-PTx-subunit 1, 1B7-like, and 11E6-like antibodies, while acellular vaccination resulted in significantly higher titers of antibodies recognizing PTd. We also observed a correlation between in vitro protection and the presence of 1B7-like and 11E6-like antibodies. Notably, chemical detoxification, as opposed to genetic inactivation, alters the PTx tertiary and quaternary structure, thereby affecting conformational epitopes and recognition of PTx by 1B7 and 11E6. The lower levels of serum antibodies recognizing clinically relevant epitopes after vaccination with PTd support inclusion of PTg in future vaccines.Pertussis vaccines, widely introduced as an inactivated whole-cell vaccine in 1950, have been responsible for a dramatic decline in pertussis-related morbidity and mortality but have been unable to eradicate disease despite 95% coverage in many areas. Disturbingly, rates of confirmed pertussis cases in industrialized countries have increased steadily

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“…The S-1 subunit contains 235 amino acids (18) and catalyzes the covalent transfer of the ADP-ribose portion of NAD to Gi protein, the negative regulatory component of the adenylate cyclase complex (12). The remaining five subunits (S-2 through S-5) compose a pentamer which is responsible for delivering the S-1 subunit to the Gi protein.We and others (1,3,5,6,20) have begun to define the functional residues of the S-1 subunit of PT with the goal of generating noncatalytic but immunologically conserved forms of the subunit amenable for administration as an acellular vaccine. The subunits of PT have been expressed in E. coli (2,13,17) and Bacillus slibtilis (21) showed that residues 2 through 180 possessed ADP-ribosyltransferase activity (1,13,20).…”

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confidence: 99%

“…We and others (1,3,5,6,20) have begun to define the functional residues of the S-1 subunit of PT with the goal of generating noncatalytic but immunologically conserved forms of the subunit amenable for administration as an acellular vaccine. The subunits of PT have been expressed in E. coli (2, 13,17) and Bacillus slibtilis (21) showed that residues 2 through 180 possessed ADP-ribosyltransferase activity (1, 13,20).…”

mentioning

confidence: 99%

“…DISCUSSION The inability of E. (oli to transcribe the PT promoter (19) has prompted several laboratories to express the subunits of PT as gene fusions under the regulation of E. coli promoters (2, 5, 13, 17). The gene products of these constructions have been used to localize functional regions of the S-1 subunit (1, 3,13,20). In this study, we expressed the genes encoding for rS-1 and the C180 peptide under the regulation of the tac promoter as a nonfusion gene product.…”

mentioning

confidence: 99%

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Expression and secretion of the S-1 subunit and C180 peptide of pertussis toxin in Escherichia coli

1989

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The structural gene of the S-1 subunit of pertussis toxin (rS-1) and the catalytic C180 peptide of the S-1 subunit (C180 peptide) were independently subcloned downstream of the tac promoter in Escherichia coli. Both constructions included DNA encoding for the predicted leader sequence of the S-1 subunit which was inserted between the tac promoter and the structural gene. E. coli containing the plasmids encoding for rS-1 and C180 peptide produced a peptide that reacted with anti-pertussis toxin antibody and had a molecular weight corresponding to that of the cloned gene; some degradation of rS-1 was observed. Extracts of E. coli containing plasmids encoding for rS-1 and the C180 peptide possessed ADP-ribosyltransferase activity. Subcellular fractionation showed that both rS-1 and the C180 peptide were present in the periplasm, indicating that E. coli recognized the pertussis toxin peptide leader sequence. The protein sequence of the amino terminus of the C180 peptide was identical to that of authentic S-1 subunit produced by Bordetella pertussis, which showed that E. coli leader peptidase correctly processed the pertussis toxin peptide leader sequence. Two single amino acid substitutions at residue 26 (C1801-26) and residue 139 (C180S-139) which were previously shown to reduce ADP-ribosyltransferase activity were introduced into the C180 peptide. C1801-26 possessed approximately 1% of the NAD-glycohydrolase activity of the C180 peptide, suggesting that tryptophan 26 functions in the interaction of NAD with the C180 peptide. In contrast, C180S-139 possessed essentially the same level of NAD-glycohydrolase activity as the C180 peptide, suggesting that glutamic acid 139 does not function in the interaction of NAD but plays a role in a later step in the ADP-ribosyltransferase reaction.Bordetella pertussis, the etiologic agent of whooping cough, produces several factors which contribute to its pathogenesis (26). Two of these factors, pertussis toxin (PT) and filamentous hemagglutinin, also act as immunogens which stimulate production of protective antibodies against the bacterium (22). Present investigations have focused on the development of an acellular vaccine to replace the whole cell vaccine currently administered in the United States. One approach to the development of an acellular vaccine would be to use a nontoxic form of PT and filamentous hemagglutinin, alone or in concert, as the immunogen. The cloning of the PT operon in Escherichia coli (14,18) has allowed the use of recombinant DNA techniques to attempt to modify PT such that the protein is nontoxic but retains immunogenic properties, allowing its use as an immunogen.PT (molecular weight, 105,060 [18]) is composed of six noncovalently bound subunits designated S-1 through S-5 (24). There is one copy of each subunit, except for S-4, which is present in two copies. The S-1 subunit contains 235 amino acids (18) and catalyzes the covalent transfer of the ADP-ribose portion of NAD to Gi protein, the negative regulatory component of the adenylate cyclase compl...

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Mapping of a Protective Epitope of Pertussis Toxin by In Vitro Refolding of Recombinant Fragments

Cited by 53 publications

References 15 publications

The Region Comprising Amino Acids 100 to 255 ofNeisseria meningitidisLipoprotein GNA 1870 Elicits Bactericidal Antibodies

The Region Comprising Amino Acids 100 to 255 ofNeisseria meningitidisLipoprotein GNA 1870 Elicits Bactericidal Antibodies

Antibodies Recognizing Protective Pertussis Toxin Epitopes Are Preferentially Elicited by Natural Infection versus Acellular Immunization

Expression and secretion of the S-1 subunit and C180 peptide of pertussis toxin in Escherichia coli

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