Dataset Information

Epitope Mapping of Neutralizing Monoclonal Antibodies to Human Interferon-? Using Human-Bovine Interferon-? Chimeras.

ABSTRACT: Our aim was to identify conformational epitopes, recognized by monoclonal antibodies (mAbs) made against human (h) interferon (IFN)-?. Based on the mAbs' (n?=?12) ability to simultaneously bind hIFN-? in ELISA, 2 epitope clusters with 5?mAbs in each were defined; 2?mAbs recognized unique epitopes. Utilizing the mAbs' lack of reactivity with bovine (b) IFN-?, epitopes were identified using 7?h/bIFN-? chimeras where the helical regions (A-F) or the C terminus were substituted with bIFN-? residues. Chimeras had a N-terminal peptide tag enabling the analysis of mAb recognition of chimeras in ELISA. The 2?mAb clusters mapped to region A and E, respectively; the epitopes of several mAbs also involved additional regions. MAbs in cluster A neutralized, to various degrees, IFN-?-mediated activation of human cells, in line with the involvement of region A in the IFN-? receptor interaction. MAbs mapping to region E displayed a stronger neutralizing capacity although this region has not been directly implicated in the receptor interaction. The results corroborate earlier studies and provide a detailed picture of the link between the epitope specificity and neutralizing capacity of mAbs. They further demonstrate the general use of peptide-tagged chimeric proteins as a powerful and straightforward method for efficient mapping of conformational epitopes.

SUBMITTER: Zuber B

PROVIDER: S-EPMC5011633 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Publications

Epitope Mapping of Neutralizing Monoclonal Antibodies to Human Interferon-γ Using Human-Bovine Interferon-γ Chimeras.

Zuber Bartek B Rudström Karin K Ehrnfelt Cecilia C Ahlborg Niklas N

Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research 20160623 9

Our aim was to identify conformational epitopes, recognized by monoclonal antibodies (mAbs) made against human (h) interferon (IFN)-γ. Based on the mAbs' (n = 12) ability to simultaneously bind hIFN-γ in ELISA, 2 epitope clusters with 5 mAbs in each were defined; 2 mAbs recognized unique epitopes. Utilizing the mAbs' lack of reactivity with bovine (b) IFN-γ, epitopes were identified using 7 h/bIFN-γ chimeras where the helical regions (A-F) or the C terminus were substituted with bIFN-γ residues. ...[more]

PMID: 27336613

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Project description:In an attempt to generate broadly cross-reactive, neutralizing monoclonal antibodies (MAbs) to simian immunodeficiency virus (SIV), we compared two immunization protocols using different preparations of oligomeric SIV envelope (Env) glycoproteins. In the first protocol, mice were immunized with soluble gp140 (sgp140) from CP-MAC, a laboratory-adapted variant of SIVmacBK28. Hybridomas were screened by enzyme-linked immunosorbent assay, and a panel of 65 MAbs that recognized epitopes throughout the Env protein was generated. In general, these MAbs detected Env by Western blotting, were at least weakly positive in fluorescence-activated cell sorting (FACS) analysis of Env-expressing cells, and preferentially recognized monomeric Env protein. A subset of these antibodies directed toward the V1/V2 loop, the V3 loop, or nonlinear epitopes were capable of neutralizing CP-MAC, a closely related isolate (SIVmac1A11), and/or two more divergent strains (SIVsmDeltaB670 CL3 and SIVsm543-3E). In the second protocol, mice were immunized with unfixed CP-MAC-infected cells and MAbs were screened for the ability to inhibit cell-cell fusion. In contrast to MAbs generated against sgp140, the seven MAbs produced using this protocol did not react with Env by Western blotting and were strongly positive by FACS analysis, and several reacted preferentially with oligomeric Env. All seven MAbs potently neutralized SIVmac1A11, and several neutralized SIVsmDeltaB670 CL3 and/or SIVsm543-3E. MAbs that inhibited gp120 binding to CD4, CCR5, or both were identified in both groups. MAbs to the V3 loop and one MAb reactive with the V1/V2 loop interfered with CCR5 binding, indicating that these regions of Env play similar roles for SIV and human immunodeficiency virus. Remarkably, several of the MAbs generated against infected cells blocked CCR5 binding in a V3-independent manner, suggesting that they may recognize a region analogous to the conserved coreceptor binding site in gp120. Finally, all neutralizing MAbs blocked infection through the alternate coreceptor STRL33 much more efficiently than infection through CCR5, a finding that has important implications for SIV neutralization assays using CCR5-negative human T-cell lines.

Dataset Information

Epitope Mapping of Neutralizing Monoclonal Antibodies to Human Interferon-? Using Human-Bovine Interferon-? Chimeras.

Publications

Epitope Mapping of Neutralizing Monoclonal Antibodies to Human Interferon-γ Using Human-Bovine Interferon-γ Chimeras.

Similar Datasets

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