Epitope Mapping of the Diphtheria Toxin and Development of an ELISA-Specific Diagnostic Assay

De-Simone, Salvatore Giovanni; Gomes, Larissa Rodrigues; Napoleão-Pêgo, Paloma; Lechuga, Guilherme Curty; Pina, Jorge Soares de; Silva, Flavio Rocha

doi:10.20944/preprints202103.0328.v1

Cited by 11 publications

References 51 publications

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Relative positioning of B and T cell epitopes drives immunodominance

Biavasco

Giovanni

2022

Preprint

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Humoral immunity is crucial for protection against invading pathogens. Broadly neutralizing antibodies (bnAbs) provide sterilizing immunity by targeting conserved regions of viral variants and represent the goal of most vaccination approaches. While antibodies can be selected to bind virtually any region of a given antigen, consistent induction of bnAbs in the context of influenza and HIV has been representing a major roadblock. Many possible explanations have been considered, however, none of the arguments proposed so far seems to fully recapitulate the observed counter-selection for broadly protective antibodies. Antibodies can influence antigen presentation by enhancing the processing of CD4 epitopes adjacent to the binding region while suppressing the overlapping ones. We analyzed the relative positioning of dominant B and T cell epitopes in published antigens that elicit strong and poor humoral responses. In strong immunogenic antigens, regions bound by immunodominant antibodies are frequently adjacent to CD4 epitopes, potentially boosting their presentation. Conversely, poorly immunogenic regions targeted by bnAbs in HIV and influenza overlap with clusters of dominant CD4 epitopes, potentially conferring an intrinsic disadvantage for bnAb-bearing B cells in germinal centers. Here we propose the theory of immunodominance relativity, according to which relative positioning of immunodominant B and CD4 epitopes within a given antigen drives immunodominance. Thus, we suggest that relative positioning of B-T epitopes may be one additional mechanism that cooperates with other previously described processes to influence immunodominance. If demonstrated, this theory can improve the current understanding of immunodominance, provide a novel explanation on HIV and influenza escape from humoral responses, and pave the way for new rational design of universal vaccines.

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Relative positioning of B and T cell epitopes drives immunodominance

Biavasco

Giovanni

2022

Preprint

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A Pencil-Lead Immunosensor for the Rapid Electrochemical Measurement of Anti-Diphtheria Toxin Antibodies

et al. 2021

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Diphtheria is a vaccine-preventable disease, yet immunization can wane over time to non-protective levels. We have developed a low-cost, miniaturized electroanalytical biosensor to quantify anti-diphtheria toxin (DTx) immunoglobulin G (anti-DTx IgG) antibody to minimize the risk for localized outbreaks. Two epitopes specific to DTx and recognized by antibodies generated post-vaccination were selected to create a bi-epitope peptide, biEP, by synthesizing the epitopes in tandem. The biEP peptide was conjugated to the surface of a pencil-lead electrode (PLE) integrated into a portable electrode holder. Captured anti-DTx IgG was measured by square wave voltammetry from the generation of hydroquinone (HQ) from the resulting immunocomplex. The performance of the biEP reagent presented high selectivity and specificity for DTx. Under the optimized working conditions, a logarithmic calibration curve showed good linearity over the concentration range of 10−5–10−1 IU mL−1 and achieved a limit of detection of 5 × 10−6 IU mL−1. The final device proved suitable for interrogating the immunity level against DTx in actual serum samples. Results showed good agreement with those obtained from a commercial enzyme-linked immunosorbent assay. In addition, the flexibility for conjugating other capture molecules to PLEs suggests that this technology could be easily adapted to the diagnoses of other pathogens.

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Microsphere Peptide-Based Immunoassay for the Detection of Recombinant Bovine Somatotropin in Injection Preparations

et al. 2022

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The use of peptides in immunoassays can be favored over the use of the full protein when more cost effective or less toxic approaches are needed, or when access to the full protein is lacking. Due to restricted access to recombinant bovine somatotropin (rbST), a protein enhancing growth and lactating performances of livestock, which use has been banned in the EU, Canada and Australia (amongst others), we developed a peptide-based biorecognition assay on an imaging planar array analyzer. For this, we identified the rbST epitope that is responsible for binding to the rbST-targeting monoclonal antibody 4H12 (MAb 4H12) to be 115DLEEGILALMR125. This linear peptide was synthesized and coupled to microspheres, after which it was tested in a biorecognition competitive inhibition assay format. We observed IC50 values of approximately 0.11 μg mL−1, which are lower than observed for the full rbST protein (IC50 = 0.20 μg mL−1). Importantly, there was no binding with the scrambled peptide. Preliminary results of directly coupled peptides in a microsphere biorecognition assay for detection of rbST are presented. Real-life applicability for detection of somatotropins (STs) in injection preparations of bovine-, porcine- and equine ST are shown. This newly developed immunoassay strongly supports future developments of peptide-based immunoassays to circumvent the limited access to the full protein.

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Epitope Mapping of the Diphtheria Toxin and Development of an ELISA-Specific Diagnostic Assay

Cited by 11 publications

References 51 publications

Relative positioning of B and T cell epitopes drives immunodominance

Relative positioning of B and T cell epitopes drives immunodominance

A Pencil-Lead Immunosensor for the Rapid Electrochemical Measurement of Anti-Diphtheria Toxin Antibodies

Microsphere Peptide-Based Immunoassay for the Detection of Recombinant Bovine Somatotropin in Injection Preparations

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