key: cord-0265406-wv30ubbf authors: de Oliveira França, Renato Kaylan Alves; Silva, Jacyelle Medeiros; Rodrigues, Lucas Silva; Maranhão, Andrea Queiroz; Brigido, Marcelo Macedo title: New broad-spectrum anti-Flavivirus human antibodies with Zika virus-neutralizing potential date: 2022-04-01 journal: bioRxiv DOI: 10.1101/2022.04.01.486666 sha: 43f847addce9bcbdcd8ead6dc155ed5a4ba04ca3 doc_id: 265406 cord_uid: wv30ubbf Flavivirus infections show recurrent outbreaks and can be responsible for disease complications such as Hemorrhagic Dengue Fever and Congenital Zika Virus Syndrome. Effective therapeutic interventions are still a challenge. Antibodies can provide significant protection, although antibody response may fail due to ADE (Antibody-Dependent Enhancement) reactions or immune escape mutations. To generate effective neutralizing antibodies, the choice of the target antigen is a crucial part of the process. Human anti-Flavivirus antibodies were selected from a combinatorial library displayed on a phage surface. The antibodies were selected against a mimetic peptide based on the fusion loop region in Domain II of the protein E, which is highly conserved among different Flavivirus. Four rounds of selection were performed using the synthetic peptide in two strategies: the first was using acidic elution of bound phages, and the second was applying a competing procedure. After panning, the selected VH and VL domains were determined by combining NGS and bioinformatic approaches. Three different human monoclonal antibodies were expressed as scFvs and further characterized. All showed binding capacity to Zika (ZIKV), Yellow Fever (YFV), and Dengue (DENV) viruses. Two of these antibodies, AZ1p and AZ6m, could neutralize the ZIKV infection in a PRNT assay. These new antibodies have the potential to be used in therapeutic intervention against different Flavivirus illnesses and, due to the conservation of the fusion loop region, they may be resistant to scape mutations. Author summary The central idea of this work is to present a possible unique therapeutic approach to combat different diseases that cause health problems annually, such as Dengue and yellow fever infections and Zika congenital syndromes. The viruses that cause these diseases, of the Flavivirus genus, typically have disease amplification reactions and evasion mechanisms of the immune response that hinder the success of specific therapies and vaccines and require new forms of effective and safe treatments. We have developed new neutralizing human antibodies so that they bind to a highly conserved region of Flavivirus, called the fusion loop, and in such a way as to avoid adverse effects associated with anti-Flavivirus antibodies. We showed that the antibodies have high cross-reactivity against different Flaviviruses and we exemplified their neutralizing capacity for Zika virus infection in an ex vivo assay. New monoclonal antibodies such as those presented here may contribute to the control of important tropical diseases in a safer and more efficient way. 168 On the next day, 100 µL of input phages were added to the blocked plate, incubated for 169 one and a half hours at 37°C. Non-binding phages were removed with a variable number of washes at each 171 selection round (5 and 10 washes for rounds 1and 2, respectively; 15 washes for rounds 172 3 and 4). The binding phages were eluted (output) through two elution strategies: one, (Fig 1A) and 319 was conceived to contain the FL linked, by a disulfide bridge, to a contacting loop in the 320 domain II region, the SRCPT peptide. The use of the cysteine-bound peptide aims to 321 bring the conformation of the peptide closer to that which occurs in the viral particle (Fig 322 1B) . 356 indicating a successful selection process (S1 Table) . IGHK4-1*01 366 a The letters "p", "ac" and "m" indicate variable domain from competitive selection, acid selection or both 367 selections, respectively. 368 c FC values correspond to frequency variation of an unique VH peptide along selection procedure. 369 c V gene family of each sequence was identified based on IgBlast annotation. 370 Both selections observed top enriched heavy chain variable domains, except for 372 the most enriched VH, Hp1, and Hac1, which were selection exclusive. Hac1 shows an 373 FC de 1,318 after acidic elution and Hp1 with FC de 3,827 after competitive elution. 374 Unlike VH, none of the most enriched VL was shared by both selection procedures (Table 375 1) . The two selections shared the majority of the most enriched VH gene families 377 except for IGHV1-8 and IGHV3-21, exclusive to the acidic selection, and IGHV1-2, 378 exclusive to competitive selection (Fig 2A) . The IGKV1-33 gene family, 379 overrepresented among top enriched VLs in the competitive selection, was not selected 380 after acidic elution. On the other hand, the IGKV4-1 gene family, the most frequent 381 family among the VLs of acidic selection, only appears in this selection (Fig 2B) . Antigen-selected VH sequences were more divergent to germline than VL 393 sequences (Fig 2C and Fig 3) change (FC) value represents the changing the frequency of each sequence at the end of 200 selection compared to its respective frequency before selection. ATTILA also assign V The degree of dissimilarity was determined as the number of variations in the 204 amino acid sequences of the most enriched antibodies. The length and the hydrophobicity 205 of the amino acid CDR3 sequences of selected VH and VL were also analysed. Kyte and 206 Doolittle hydrophobicity scale was used to calculate the GRAVY hydrophobicity scores Recombinant antibody design, cloning and expression The most enriched VH and VL in the selection were combined to construct single-chain 211 antibody fragments (scFv) Additional incubation at 200 rpm, 25°C 216 for 4 hours was carried out. Cells were sonicated, and the clarified supernatant was 217 purified by immobilised metal affinity chromatography on 1 ml HISTRAP HP (Cytiva, 218 cat: 17524701) column in AKTA system (Cytiva). The eluted fractions were cleaned and 219 concentrated in Amicon Ultra-2 30 kDa Antibody expression and purification were 222 evaluated using polyacrylamide gel electrophoresis stained with Coomassie Brilliant Blue G-250 and Western-blot using a mouse anti-HA (Santa Cruz Biotechnology, cat: sc-7392) 400 sequences with both high and low degrees of dissimilarity compared to their germinal 401 counterpart The use of either elution strategy leads to VH selection with a varying degree of V gene maturation, CDR length, and hydrophobicity (Fig 3). However, for VL the degree 404 of divergence in relation to the germinal sequences is lower in antibodies selected after Properties of the most enriched VH and VL. The characteristics of the ten most 408 frequent variable domains in the acid selection (Acid) and competitive selection (CP) 409 were analyzed. The characteristics of VH and VL studied were: divergence to the 410 germline V gene (number of non-identical residues) 411 and GRAVY scores of hydrophobicity of the CDR3 amino acid sequence. Data are 412 represented as the means and SEM in each group (N=10/group). * Means of groups were 413 statistically different at P < 0.05 Construction of the recombinant anti-Flavivirus antibodies Top enriched VH and VL domains were strategically combined in scFv (single-chain 417 variable fragment) format, considering their enrichment, presence in both selections or 418 just one (prioritising the competitive selection), and their germinal dissimilarity degree containing Hp1; AZ3m, 420 with Hm1, and AZ6m, with Hm2. The exceptionally enriched Lp1 was chosen as the VL 421 counterpart of all three scFv (Fig 4). Another set of scFv molecules was constructed 422 utilising another VL Three anti-FL monoclonal antibodies, AZ1p, 426 AZ3m and AZ6m were constructed, by combining the enriched VHs (Hp1, Hm1 and 427 Hm2) with the most enriched VL (Lp1), respectively Characteristics of the three scFvs, considering the variable domain of the heavy chain (B) Profiles of VH amino acid divergence (compared to their germline sequence) and length 430 and hydrophobicity of the CDR3 protein sequence show differences between tested 431 antibodies The three scFvs generated remained the ability to bind to the synthetic antigen 434 used in the Phage Display selection, showing different binding profiles On the left, the 439 binding of the recombinant scFvs to the fusion loop used in the selection was analyzed 440 by immunoassay (ELISA). The normalized area under curve for each antibody FL 441 binding is on the right. Data are presented as mean +/− SD. An unspecific scFv To determine whether the generated antibodies reacted to FL in their native conformation 446 in different Flavivirus, they were screened for binding to ZIKV, YFV and the 4 DENV 447 serotypes using viral particles. All antibodies showed some binding capacity to all 448 Flavivirus tested, following the same order of binding activity among Flavivirus AZ1p scFv showed the highest virus binding 450 activity, with EC 50 values of 14.67; 16.24 and 545.8 ηΜ, followed by AZ6m, with EC 50 451 values of 361.7; 256.4 and 740.0 ηΜ, and finally AZ3m In these 457 experiments, the plates were sensitized with the whole viral particles. The half effective 458 concentration (EC 50 ) for each scFv against Data are presented as mean +/− SD. An unspecific scFv was used as a negative antibody 460 The differences between 463 antibody binding to DENV serotypes were minimal and were smaller than that observed 464 for ZIKV and YFV. The highly divergent AZ3m presented a very low binding capacity 465 to ZIKV. However, the antibody AZ1p, with a germinal VH, showed expressive binding 466 to ZIKV and YFV (Fig 6A and 6B) whether the constructed anti-FL scFv has neutralizing capacity. The AZ1p and 471 AZ6m monoclonal antibodies showed similar neutralizing activity, with half-maximal 472 inhibitory concentration AZ3m antibody did not show neutralizing capacity (IC 50 > 100 µΜ), corroborating the On the right, half-maximum inhibitory 480 concentrations (IC 50 ) represent means of replicates from at least two independent 481 experiments. The representation of the IC 50 values was limited to 1 µΜ Antibodies specific to FL may have important neutralizing potential to infections of 487 different strains of Flavivirus [24]. In this work, we reported the productive selection of 488 anti-FL antibodies, reducing the library diversity throughout the rounds, warranting the 489 selection process. The selection leads to the identification of unrelated VH and VL 490 enriched in response to the selection. Interestingly common set of VH among the top selected, except for the first most enriched VH: Hp1 492 and Hac1. Unlike heavy chain domains, none of the most enriched VLs were selected in 493 both schemes. The most significant differences between the selections are the V gene families, and the degree of 495 dissimilarity to the germinal sequences. This corroborates the idea that distinct elution 496 schemes lead to the selection of particular antibodies and also shows that different 497 pressure determinants drive the selection of VHs and VLs. Thus, the elution protocol 498 impacts Some of the selected VH and VL showed a high dissimilarity with their germline 500 sequences. Likewise, studies of the antibody-mediated immune response to Influenza 501 virus and HIV have shown that it is possible to accumulate a vastly hypermutated VH in 502 highly neutralizing antibodies [25,26], which may explain the nature of these highly 503 hypermutated sequences. However, a naive phage library is not supposed to contain 504 antigen-driven hypermutated antibodies, and it is considered a limitation for using such 505 libraries. Even though the library used in this work may be regarded as naive for viral 506 infection, the library used here was assembled from donors from an endemic area for 507 DENV. 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Emerg Microbes Infect Bi-specific Antibody against Zika Virus with High Therapeutic Potential Analysis 723 of a Therapeutic Antibody Cocktail Reveals Determinants for Cooperative and 724 Combination 727 therapy with anti-HIV-1 antibodies maintains viral suppression A combination of two human monoclonal antibodies limits fetal damage by Zika 731 virus in macaques A Combination of Two Human Monoclonal Antibodies Prevents Zika Virus 735 Escape Mutations in Non-human Primates A combinatorial library 740 was used to generate fusion phages expressing human antibody fragments on their 741 surfaces. Four selection rounds were performed, increasing the stringency at each round 742 by raising the number of washes. Two strategies eluted the specific phages. One used an 743 acidic solution to disfavour antibody-antigen binding Phagemid pools from the 748 original library and the fourth round of selection were used as templates for PCR using 749 specific primers for VH (capital letters) and VL (lowercase letters) domains 750 amplification. The amplicons were obtained through antibody library before selection 751 (A, a) and from the fourth round of selection using acid Analysis of IMAC purified recombinant scFvs. The scFv were expressed in 755 bacteria and purified by affinity chromatography in nickel columns. The production of 756 antibodies AZ1p, AZ3m and AZ6m were analysed by denaturing SDS-PAGE (A) and 757 detected with anti-HA tag antibody by western-blot (B) 567 We are grateful to Prof. Connie McManus for English revision. 568 569