Selection and characterization of human recombinant antibodies against Orthopoxviruses from an immunoglobulin library and mapping of functional epitopes of Vaccinia virus surface proteins
von Henrike Ahsendorf
Datum der mündl. Prüfung:2019-11-04
Erschienen:2020-04-07
Betreuer:Prof. Dr. Dr. Bertram Brenig
Gutachter:Prof. Dr. Dr. Bertram Brenig
Gutachter:Dr. Christiane Stahl-Hennig
Gutachter:Dr. Ahmed Abd El Wahed
Einzelne Artikel / Kapitel dieser Arbeit wurden auch separat publiziert:
2.2. Chapter II: "Characterization of an In Vivo Neutralizing Anti-Vaccinia Virus D8 Single-Chain Fragment Variable (scFv) from a Human Anti-Vaccinia Virus-Specific Recombinant Library" DOI: https://doi.org/10.3390/vaccines9111308
2.3. Chapter III: "Characterization of an Anti-Vaccinia Virus F13 Single Chain Fragment Variable from a Human Anti-Vaccinia Virus-Specific Recombinant Immunoglobulin Library“ DOI: https://doi.org/10.3390/v14020197
Dateien
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Zusammenfassung
Englisch
The genus Orthopoxvirus (OPXV) contains a group of large (130-380 kb) and closely related double-stranded DNA viruses within the Poxviridae family, which replicates in the cytoplasm of vertebrate or invertebrate cells. Vaccinia virus (VACV), the prototype of the OPXV genus, was applied as a vaccine against the closely related Variola virus (VARV). VARV, the causative agent of smallpox, elicited more fatalities than all other human dis-eases taken together. However, strict VACV-vaccination campaign led to the eradication of smallpox. Another advantage of the vaccination is the achievement of cross protection against all the other OPXVs. Unfortunately, due to the termination of the vaccination campaign, the majority of humans is not protected anymore. Therefore, there is considera-ble concern regarding the use of VARV and monkeypox virus (MPXV) as potential bio-logical weapons, especially after recurrent outbreaks of MPXV in Africa, America and Europe. Moreover, reservoirs for other closely related OPXVs, e.g. cowpox viruses (CPXV), exist in the environment and may also endanger human health, especially in im-muno-compromised humans. Therefore, it is crucial to join forces in the development of safer vaccines, antiviral agents, and protective human recombinant antibodies for passive immunization. Morphogenesis of VACV results mainly in two distinct virus particle forms. The majority (>90%) consists of the “intracellular mature virus” (IMV), which mediates host-to-host transmission. “Extracellular enveloped virus” (EEV) on the other hand is important for direct cell-to-cell transmission inside the host and is surrounded by an additional golgi-derived envelope. Another difference between IMVs and EEVs is the distribution of envelope proteins, as several structural proteins of immunological rele-vance were identified on the IMV (A10, A13, A14, A17, A25, A26, A27, A28, C3, D8, D13, H3 and L1), and on the EEV (A33, A56, B5 and F13). In my PhD thesis, studies on antibody-viral interaction with focus on the VACV proteins A27, D8 and F13 were conducted, because of their important functions in the virus repli-cation cycle. One of the best characterized envelope proteins is the A27, which is encoded by the open reading frame (ORF) A27L. This conserved protein is present in all members of OPXVs. A27 is important for virus attachment, by binding to the glycosaminoglycan (GAG) heparan sulfate on the surface of mammalian cells. In this study, the binding sites of six specific A27 monoclonal antibodies (mAbs) were identified by peptide SPOT syn-thesis and peptide microarray technology. In the region of amino acids (aa) 26 to 39, a complex of four antigenic sites was identified (epitope #1A: aa 32-39, #1B: aa 28-33, #1C: aa 26-31, #1D: 28-34), and another two at the N-terminus (epitope #4: aa 9-14) and C-terminus (epitope #5: aa 68-71). Binding affinities were determined using ELISAs with different purified OPXV reference strains. Interestingly, all mAbs directed to epitope complex #1 showed strong binding activities to VACV, CPXV and camelpox virus (CMLV) but either did not react or only bound weakly to ectromelia virus (ECTV) and MPXV. These differences are caused by amino acid exchanges of the epitope regions. To determine the sequence variability of the six antigenic sites, 391 published sequences of A27 protein homologs were compared. Epitope #4 was conserved among almost all OPXVs with the exception of three buffalopox viruses (BPXV), three skunkpox viruses (SkPXV), 12 truncated OPXV sequences and one VACV sequence, while epitope #5 was constant among 389 of the 391 sequences. The sequential epitope complex #1A-D was more variable and, therefore, responsible for species-specific epitope characteristics, which is in correspondence to the ELISA results. Moreover, the neutralization capabilities of A27 specific mAbs were tested, whereby the mAbs detecting epitopes #1A-D and #4 neutralized VACV Elstree in the presence of 1% complement (50% plaque-reduction: 12.5-200 µg/ml). Another crucial IMV protein is the D8 type 1 membrane protein, which is highly con-served in poxviruses. It plays an important role in virus attachment to the host cell via binding to the GAG chondroitin sulfate (CS). For neutralization studies, specific human anti-D8 antibodies were generated. Therefore, the IgG repertoire from four donors vac-cinated intracutaneously with live vaccinia virus vaccine was amplified, cloned and dis-played onto M13K07ΔpIII phage. This library displayed a diversity of ≥4x108 independ-ent colonies. Different immuno-screening protocols against VACV Elstree revealed a predominant selection of scFv-clones specifically binding to the D8 protein, which is known to induce strong antibody responses. To improve the binding affinity and the im-mune response, the obtained scFv-1.2.2.H9 was also engineered into the larger human scFv-Fc-1.2.2.H9 and IgG1-1.2.2.H9 formats. Similar binding affinities were shown by scFv-1.2.2.H9 and scFv-Fc-1.2.2.H9 (1.61 nM and 7.68 nM, respectively), whereas, IgG1-1.2.2.H9 was much more efficient (43.82 pM). However, none of the purified re-combinant 1.2.2.H9 antibodies were able to neutralize 100 pfu of VACV Elstree in vitro. Interestingly, after addition of 1% human complement, the neutralization abilities of the larger antibody formats scFv-Fc-1.2.2.H9 and IgG1-1.2.2.H9 could be improved (0.0776 µM and 0.01324 µM, respectively). In an in vivo passive immunization NMRI-mouse-model, 100 µg of scFv-1.2.2.H9 and the IgG1-1.2.2.H9 partially protected the mice against the challenge with 4LD50 VACV Munich 1 as 3/6 animals survived. In con-trast, the mice inoculated with scFv-Fc-1.2.2.H9 showed no protection. Moreover, the existing OPXV phage library was screened against the F13 protein of VACV, which is the major envelope protein of EEV particles. The nonglycosylated F13 membrane protein is encoded by the ORF F13L gene. The F13 protein has no transmem-brane domain, instead, its N- and C-terminus are both directed towards the inner side within the EEV membrane. Because of its location in the TGN membrane, it plays an im-portant role in the virion wrapping progress as well as the EEV production. After apply-ing immuno-screening protocols against F13, one anti-F13 scFv was isolated and charac-terized. Interestingly, two antigenic binding sites (139-GSIHTIKTLGVYSDY-153 and 169-AFNSAKNSWLNL-188) were mapped using a cellulose membrane encompassing 372 15-mere peptides with 12 overlaps, therefore covering the whole F13 protein. Be-cause of the inner location of the protein, scFv 3E2 showed no capability of VACV neu-tralization. In conclusion, more research on poxvirus replication is crucial. The epitope mapping on immuno-protective proteins such as the A27 and D8 proteins of VACV provides more insights into host-pathogen interaction. Moreover, data on virus species-specific epitope variations will enable the future development of safer vaccines or antivirals. The construc-tion of recombinant scFv phage libraries is a promising strategy to produce target specific antibodies which are useful to investigate the replication cycle of poxviruses. Moreover, these libraries are of high interest because they enable generating specifically engineered human recombinant scFv antibodies, which might be a helpful tool for controlling any future eruption of zoonotic OPXV infections.
Keywords: Vaccinia virus; epitope mapping; antibody engineering; recombinant proteins; immunoglobulin library; recombinant antibodies; Orthopoxvirus