Epitope mapping by random peptide phage display reveals essential residues for vaccinia extracellular enveloped virion spread

Abstract

BackgroundA33 is a type II integral membrane protein expressed on the extracellular enveloped form of vaccinia virus (VACV). Passive transfer of A33-directed monoclonal antibodies or vaccination with an A33 subunit vaccine confers protection against lethal poxvirus challenge in animal models. Homologs of A33 are highly conserved among members of the Orthopoxvirus genus and are potential candidates for inclusion in vaccines or assays targeting extracellular enveloped virus activity. One monoclonal antibody directed against VACV A33, MAb-1G10, has been shown to target a conformation-dependent epitope. Interestingly, while it recognizes VACV A33 as well as the corresponding variola homolog, it does not bind to the monkeypox homolog. In this study, we utilized a random phage display library to investigate the epitope recognized by MAb-1G10 that is critical for facilitating cell-to-cell spread of the vaccinia virus.ResultsBy screening with linear or conformational random phage libraries, we found that phages binding to MAb-1G10 display the consensus motif CEPLC, with a disulfide bond formed between two cysteine residues required for MAb-1G10 binding. Although the phage motif contained no linear sequences homologous to VACV A33, structure modeling and analysis suggested that residue D115 is important to form the minimal epitope core. A panel of point mutants expressing the ectodomain of A33 protein was generated and analyzed by either binding assays such as ELISA and immunoprecipitation or a functional assessment by blocking MAb-1G10 mediated comet inhibition in cell culture.ConclusionsThese results confirm L118 as a component of the MAb-1G10 binding epitope, and further identify D115 as an essential residue. By defining the minimum conformational structure, as well as the conformational arrangement of a short peptide sequence recognized by MAb-1G10, these results introduce the possibility of designing small molecule mimetics that may interfere with the function of A33 in vivo. This information will also be useful for designing improved assays to evaluate the potency of monoclonal and polyclonal products that target A33 or A33-modulated EV dissemination.

Active vaccination with vaccinia virus A33 protects mice against lethal vaccinia and ectromelia viruses but not against cowpoxvirus; elucidation of the specific adaptive immune response

“…The results also agree with previous work by He et al [27], substantiating the essential role of L118 in binding of protective antibodies to A33. In that work, an important role in antibody binding to the epitope is attributed to the D115 residue, yet D115 is conserved between VACV and CPXV excluding its contribution to the mechanism utilized by CPXV to evade A33 immunity.…”

“…Vaccination with a A33 VACV , modified to contain each of these substitutions, revealed the critical role of L118 in protection against VACV-WR and in blocking of EV spread by antibodies. These results support previous studies which mapped the critical role of L118 in binding the monoclonal neutralizing antibody MAb-1G10 and the consequence of each of these substitutions on binding of this antibody [26,27]. Since A33 is considered an important contributor to future smallpox subunit vaccines which might also be used against other orthopoxvirus infections such as MPXV, and antibodies to A33 are developed for treatment of orthopoxvirus infections, unraveling the basis for this protective ability at the single aa level is of major importance.…”

“…Using this system we show that A33 vaccination protected mice against VACV-WR and ECTV but failed to protect against CPXV challenge. By introducing A33 CPXV domains and residues into A33 VACV , we show that protection with A33 VACV based vaccination depends on a single protective epitope in A33 VACV , previously demonstrated in-vitro to serve as a target for the neutralizing antibody MAb-1G10 [26,27]. We further map the role of protective residues and elaborate on the mechanism of cross-protection against orthopoxviruses.…”

“…We identified a single protective region in A33 VACV located between residues 104–120 that harbors a putative H-2Kd CD8+ T cell epitope and a B cell epitope. This B cell epitope was shown to be a target for the neutralizing antibody MAb-1G10 that blocks spreading of extracellular virions [26,27]. Both epitopes are affected in A33 CPXV by 3 amino acids (aa) substitutions L112F, Q117K and L118S and shown to be non-functional.…”

High throughput functional epitope mapping: Revisiting phage display platform to scan target antigen surface

“…47 Others mimic the tridimensional array of physicochemical properties behind recognition. 48 Making an unequivocal connection between peptide sequences and the original antigenic determinants is not always simple, 49 despite the availability of computational tools designed to look for such correspondence. 50 The emergence of multiple theoretical solutions pointing to diverse possible epitopes complicates the task.…”