with Dengue virus (DENV). Site-directed mutagenesis identified two residues 36 within  chain, N31 and S91 that are essential to the functional maturation. The 37 dynamic features unveiled here will assist us to better understand the 38 pathogenesis of ZIKV infection and inform rational design of vaccines. In this study, we applied long-read next-generation sequencing (NGS) and separate NGS runs on the Ion S5 GeneStudio platform (S1 Table) . The
Antibodyomics 2.0 pipeline was used to process, annotate, and analyze the 141 NGS data, rendering 1.3 to 2.9 million reads per time point (S1 Table) . Of these 142 sequences, 55.3% to 71.2% are high-quality, full-length antibody variable 143 regions which were used for the analyses of B cell repertoire profiles (S1 Table) . left). In contrast, some specific germline genes were noticed with low frequency, 151 such as IGHV1-8, the V H germline gene of ZK2B10, ranging from 0.98% to 4.80% 152 in seven time points (Fig 1B, left) . The V L germline gene of ZK2B10, 153 corresponding to IGLV1-47, ranging from 2.58% to 5.34% (Fig 1B, right) . which with many derived from common memory B cell clones [19, 47] . 178 Interestingly, the similar pattern has also been reported for an HIV-1 patient 179 undergoing chronic infection in response to a rapidly mutating envelope spike 180 [44]. 181 We next determined changes in the CDR3 loop length. Due to the diversity 182 of the D gene, a rather dispersed distribution in HCDR3 loop length was 183 observed as compared to a steady, canonical CDR3 loop length distribution 184 obtained for  and  chains ( Fig 1D) . The HCDR3 loops were mainly distributed 185 at the range of 9-aa to 15-aa (Fig 1D, left) . As for the light chain, 9-aa KCDR3 186 loops accounted for 79.3% to 88.2% of the  chain repertoire, while 9-aa to 11- Nonetheless, our data suggests that ZK2B10 lineage antibodies were induced 211 rapidly and transiently at the end of acute phase during ZIKV infection.
Interestingly, the majority of ZK2B10 somatic variants showed a germline 213 divergence of less than 5.0% in both heavy and  chain repertoires ( confirming that this germline-like mAb lineage emerged at the peak of 265 plasmablast response. According to the alignment compared to IGHV1-8 266 germline sequence, the functional loss of 2H-2, -3 and -14 could be explained 267 potentially by the mutation of D39 located toward the end of HCDR1 (Fig 3A) . 268 As for 11 synthesized ZK2B10-like  chains, 7 (2L-1, -2, -3, -6, -8, -9, and -11) 269 were expressible when paired with WT ZK2B10 heavy chain. Surprisingly, 5 270 (2L-1, -3, -8, -9, and -11) of these 7  variants failed to bind ZIKV E or E DIII 271 combined with undetectable neutralizing activities against ZIKV (Fig 4B) . (Fig 4D) . The RNA load in these groups was suppressed in blood 307 with distinguishable level from the MERS-4 group (Fig 4F) . Exceptionally, 2H-15 308 2, -3 and -14 failed to offer any protection with a median survival time of 7.25 309 to 8 days after ZIKV challenge (Fig 4D) . The viral RNA levels measured in mice 310 treated by them were 3.72 to 4.45 log 10 greater on average than the ZK2B10 311 group on day 5 after ZIKV challenge, respectively (Fig 4F) . In contrast, all of Critical residues for ZK2B10 functional maturation 319 To the further investigation of the maturation pathway of the ZK2B10 lineage, 320 we performed reverse mutagenesis and structural analysis to identify 'hotspot' 321 residues. Before that, we aligned the amino acid sequences of representative 322 ZK2B10 heavy and  chain variants with their putative germline genes, IGHV1-323 8 and IGLV1-47, respectively (Fig 3) . For heavy chain, 2H-2, 2H-3, and 2H-14 324 lose their potency to ZIKV both in vitro and in vivo. These 3 heavy chains 325 possess a single substitution mutation at the D39 with respect to their germline 326 gene (Fig 3A) . To test that, we conducted reverse mutagenesis on 2H-2 (A39D), (Fig 3B) . One such mutation, located on the 343 LCDR1 loop, is N31, which is shared by the weak-functional variants 2L-2 and 344 2L-6, as well as WT ZK2B10  chain (Fig 3B) . The other is at position 91 on 345 LCDR3 loop, which is S91 in WT ZK2B10  chain but predominantly A91 in all 346 these weak-or non-functional  chain variants (Fig 3B) . Thus, N31N and S91 347 could be the critical mutations for ZK2B10  chain maturation. We first 348 examined the effect of these two mutations individually by performing site-349 directed mutagenesis on 2L-1, which is 100% identical to germline gene 350 IGLV1-47. Neither S31N nor A91S could effectively render the germline 351 antibody functional (Fig 5B) . We then introduced a double mutation 17 352 (S31N+A91S) into 2L-1, which, as expected, bound to ZIKV E and E DIII with 353 high affinity and potently neutralized ZIKV at the same level of WT ZK2B10 (Fig   354   5B ). As shown by the crystal structure, 5 residues within ZK2B10  chain (N31, 355 N32, Y33, R51, D94 and L96) directly involved in the in the contact interface 356 [31]. For the two identified critical residues, N31 directly interacts with T309 on 357 ZIKV E DIII, while S91 forms a hydrogen bond with N32, which interacts with 358 T335 on ZIKV E DIII (Fig 5D) . In brief, our combined analyses of NGS data, 359 antibody functions, and complex structure confirm that residues N31 and S91 Committee of Tsinghua University (16-ZLQ9).
Acknowledgements 569 We are grateful to the ZIKV convalescent patient for donating his blood samples 570 from which mAbs were isolated and Drs. Jiang Wang, Wenxin Hong, and 571 Lingzhai Zhao for providing the patient with treatment and care. We thank Drs. The authors declare that they have no competing interests. 