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Single B cell transcriptomics identifies multiple isotypes of broadly neutralizing antibodies against flaviviruses [1]

['Jay Lubow', 'Vaccine', 'Infectious Disease Division', 'Fred Hutchinson Cancer Center', 'Seattle', 'Washington', 'United States Of America', 'Lisa M. Levoir', 'Duncan K. Ralph', 'Computational Biology Program']

Date: 2023-11

Sequential dengue virus (DENV) infections often generate neutralizing antibodies against all four DENV serotypes and sometimes, Zika virus. Characterizing cross-flavivirus broadly neutralizing antibody (bnAb) responses can inform countermeasures that avoid enhancement of infection associated with non-neutralizing antibodies. Here, we used single cell transcriptomics to mine the bnAb repertoire following repeated DENV infections. We identified several new bnAbs with comparable or superior breadth and potency to known bnAbs, and with distinct recognition determinants. Unlike all known flavivirus bnAbs, which are IgG1, one newly identified cross-flavivirus bnAb (F25.S02) was derived from IgA1. Both IgG1 and IgA1 versions of F25.S02 and known bnAbs displayed neutralizing activity, but only IgG1 enhanced infection in monocytes expressing IgG and IgA Fc receptors. Moreover, IgG-mediated enhancement of infection was inhibited by IgA1 versions of bnAbs. We demonstrate a role for IgA in flavivirus infection and immunity with implications for vaccine and therapeutic strategies.

A central challenge for developing clinical interventions for dengue virus or the closely related Zika virus is the ability of IgG antibodies to enhance, rather than neutralize infection under certain conditions. When present prior to infection, as in the case of vaccination, these antibodies can worsen disease outcome. In this study, we analyzed B cells of individuals who experienced dengue or Zika infection to identify those expressing antibodies that can potently neutralize these viruses with minimal potential to enhance infection. We used a method that captured a larger number and wider variety of antibodies than previous approaches. We discovered several potent antibodies that simultaneously neutralized dengue and Zika viruses, including those of IgG isotype, which are common, and one of IgA isotype, which had never been described against this group of viruses. Although IgG antibodies enhanced infection in certain cases, the IgA antibody did not. We further showed that modifying a region of IgG antibodies to convert them to IgA antibodies eliminated their ability to enhance infection. Moreover, the modified IgA versions inhibited the ability of IgG versions to enhance infection. These results suggest that inducing IgA antibodies may be an attractive goal for safe and effective vaccines.

Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: LG, JL, FAM, DKR, LML are inventors on a patent application filed by Fred Hutchinson Cancer Center relating to newly discovered antibodies described in this paper. SK, ED, BJD are employees of Integral Molecular, Inc.; BJD is also a shareholder of the company.

Funding: This work was supported by the Fred Hutchinson Cancer Center Translational Data Science Integrated Research Center New Collaborations Award (LG, FAM, JL, LML, DKR); NIH R01 AI146028 (DKR, FAM); the Howard Hughes Medical Institute (FAM); Viral Pathogenesis and Evolution Training Grant T32 AI083203 (LB); Fred Hutchinson Cancer Center Diverse Trainee Fund (MC); an Investigator Initiated Award W81XWH1910235 from the Department of Defense Office of the Congressionally Directed Medical Research Programs (SE); Catalyst and Transformational Awards from Dr. Ralph & Marian Falk Medical Research Trust (SE); NIH U19 AI057229 supplement (SE); the Chan Zuckerberg Biohub (SE); the Antibody Technology (RRID:SCR_022608), Flow Cytometry (RRID:SCR_022613), and the Genomics & Bioinformatics (RRID:SCR_022606) Shared Resource Facilities of the Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium (P30 CA015704); and the Scientific Computing Infrastructure at Fred Hutch (ORIP grant S10OD028685). FAM is an Investigator of the Howard Hughes Medical Institute. SE is a Chan Zuckerberg Biohub - San Francisco Investigator. VD was supported by a Chan Zuckerberg Biohub Collaborative Postdoctoral Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2023 Lubow et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here, we have improved upon our scRNAseq-based method to discover new bnAbs by systematically profiling the antibody response in 4 individuals whose sera potently cross-neutralized DENV1-4 and ZIKV. We identified 23 new bnAbs, of which a subset displayed neutralization breadth and potency comparable or superior to leading bnAbs in the field but with distinct epitopes. Moreover, one of our newly identified bnAbs neutralized DENV1-4 and ZIKV and is derived from the IgA1 isotype, thus representing the first non-IgG bnAb described against flaviviruses. Notably, monomeric IgA1 versions of newly and previously characterized bnAbs not only retained IgG neutralization capacity, but also inhibited IgG-mediated enhancement of infection in cells expressing both IgG and IgA Fc receptors.

The above antibodies were discovered by sorting hundreds of single B cells from individuals infected with DENV and/or ZIKV, followed by either immortalization or PCR amplification of variable heavy and light chain genes for recombinant IgG production and characterization [ 38 ]. Although these approaches have successfully identified bnAbs against many viruses, they are laborious, typically requiring robots and/or large teams to increase throughput. As an alternative high-throughput method, we previously provided proof-of-principle for a single cell RNA sequencing (scRNAseq)-based approach to identify multiple DENV1-4 bnAbs, of which two somatic IgG variants, J8 and J9, were the most potent [ 39 ]. Single cell transcriptomics also allows unbiased profiling of multiple antibody isotypes unlike previous methods, which were largely restricted to isolation of IgG antibodies [ 28 , 33 – 35 , 40 ].

In contrast to primary DENV exposure, secondary exposure to a different DENV serotype typically elicits broadly neutralizing antibody responses associated with protection against subsequent disease [ 8 , 21 – 26 ]. Studying the antibody repertoire in individuals who have experienced multiple DENV infections can thus provide insight into the properties of cross-reactive neutralizing antibody responses that an effective vaccine seeks to mimic. A handful of monoclonal broadly neutralizing antibodies (bnAbs) that can potently neutralize DENV1-4 and in some cases, ZIKV, have been isolated from naturally infected individuals living in endemic regions [ 22 , 27 – 29 ]. The most well-characterized class of flavivirus bnAbs targets a quaternary E-dimer epitope (EDE) spanning both E protein monomers within the dimer subunit [ 28 , 30 ]. There are two subclasses of EDE bnAbs, of which EDE1 but not EDE2 antibodies can potently neutralize ZIKV in addition to DENV1-4 [ 31 ]. A few antibodies that can cross-neutralize ZIKV and some DENV serotypes have also been described [ 32 – 35 ], but other than those of the EDE1 subclass, SIgN-3C is the only known naturally occurring antibody that can potently neutralize ZIKV and all four DENV serotypes [ 27 , 36 , 37 ].

Zika virus (ZIKV) and the four circulating serotypes of dengue virus (DENV1-4) are mosquito-borne flaviviruses with overlapping geographic distributions [ 1 ]. Climate change is predicted to further expand the geographic range of mosquito vectors [ 2 – 4 ], highlighting the need for effective clinical interventions to curb epidemics. The complex antibody response to DENV1-4 has hampered the development of safe and effective vaccines. A first exposure to a given DENV serotype generates potently neutralizing antibodies that typically provide long-term, though sometimes incomplete protection against reinfection by that serotype [ 5 – 7 ]. However, antibodies that are cross-reactive in binding but not neutralizing activity against other DENV serotypes are also elicited [ 8 – 11 ] and pre-existing non-neutralizing antibodies predict the risk of severe disease following secondary exposure to a different DENV serotype [ 12 – 16 ]. This phenomenon is attributed to a process called antibody-dependent enhancement (ADE), in which non-neutralizing IgG antibodies [ 12 , 17 ] facilitate the uptake of bound DENV particles into relevant myeloid target cells via Fc-Fc gamma receptor (FcɣR)-dependent pathways [ 18 ]. ADE-related safety concerns derailed the widespread use of the first licensed DENV vaccine, which increased the risk of severe dengue disease following subsequent infection in previously DENV-naive recipients [ 19 , 20 ]. As pre-existing IgG antibodies from one prior exposure to ZIKV can also enhance subsequent dengue disease risk [ 21 ], a safe vaccine would ideally induce durable antibodies that can broadly and potently neutralize DENV1-4 and ZIKV.

Results

Identifying donors with broadly neutralizing antibody responses We previously identified bnAbs against DENV1-4 [39] via secondary analyses of relatively few (~350) B cells from an existing scRNAseq dataset of bulk peripheral blood mononuclear cells (PBMCs). This dataset was generated in an unrelated study with the primary goal of identifying biomarkers of severe dengue [41] in a cohort of individuals with acute DENV or ZIKV infection [42,43]. Here, we initiated a new study to specifically leverage scRNAseq for bnAb discovery by focusing our analysis on B cells (instead of bulk PBMCs) from individuals whose serum broadly neutralized DENV1-4 and ZIKV (Fig 1). [42,43]. To identify such individuals, we screened longitudinal serum samples from 38 cohort participants for their ability to neutralize commonly used DENV1-4 and ZIKV strains in two independent experiments. S1 Fig summarizes the serum neutralization profile of cohort participants, along with demographic and clinical information. When tested at a single dilution, no serum sample reproducibly neutralized West Nile virus (WNV), a more distantly related flavivirus included as a control. In contrast, even at the earliest available time point (range: 0 to 7 days after fever onset), serum samples from 26/38 individuals inhibited infection by two or more DENV serotypes by >50% in both experiments (S1 Fig). This high prevalence of cross-serotype neutralizing activity likely reflects repeated DENV exposures, as confirmed by IgG avidity testing of these samples in prior studies [42,43]. In addition to broad neutralizing activity against DENV1-4, serum samples from 11/38 individuals reproducibly neutralized >50% infection by ZIKV. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Workflow to identify broadly neutralizing antibodies (bnAbs) from donor samples. (A) Serum neutralization profile of 4 cohort participants chosen for downstream analysis based on potent neutralizing activity against DENV1-4 and ZIKV. The mean reciprocal serum dilution that neutralized 50% of virus infection (NT50) in 3 independent experiments is depicted as a heatmap with a darker color indicating greater potency according to the key. (B) B cells isolated from the peripheral blood mononuclear cells (PBMCs) of donors selected in (A) were processed for (C) single-cell RNA sequencing of both global gene expression (GEX) and B cell receptor (BCR)-specific libraries. (D) We analyzed BCR libraries using the software package partis [44], which groups antibodies into clonal families and infers their shared ancestry. (E) Antibody sequences most likely to encode broadly neutralizing antibodies (bnAbs) were bioinformatically downselected for functional characterization. (F) We recombinantly expressed selected antibodies as IgG1 and screened them for the ability to neutralize DENV1-4 and ZIKV. This figure was created with Biorender.com. https://doi.org/10.1371/journal.ppat.1011722.g001

Functional screens for broadly neutralizing antibodies We performed screening in two rounds. In the first round, our goal was to identify clonal families encoding bnAbs. To do this, we selected 1–3 antibodies from each of ~20 clonal families per donor according to the above criteria (Fig 1E). These antibodies were recombinantly expressed initially as IgG1 by transfection of mammalian cells and the antibody-containing supernatant screened at a single dilution (1:10) for neutralization of DENV1-4, ZIKV, and West Nile Virus (WNV). As controls, we produced and screened previously published antibodies, EDE1-C10 [28,31] and CR4354 [54] in parallel. Consistent with their known specificities, EDE1-C10 broadly neutralized DENV1-4 and ZIKV, but not WNV, while CR4354 specifically neutralized WNV (S2A Fig). Although our downselected antibodies had little to no neutralizing activity (<50%) against WNV, several potently neutralized DENV and/or ZIKV (S2B–S2G Fig; antibodies screened in this first round are left aligned). The number and neutralization profile of clonal families encoding neutralizing antibodies against DENV and/or ZIKV varied by donor. For example, of 14 total families tested from donor 001 (S2B Fig), only two (F05, F07) encoded neutralizing antibodies: F05 displayed ZIKV-specific neutralization, while F07 neutralized DENV1-3 and ZIKV, but not DENV4. Similarly, of the 18 selected families from donor 012 only two (F12, F15) encoded neutralizing antibodies (S2C Fig). In contrast, almost all 25 families from donor 002 neutralized DENV1 and DENV3, and one (F09) broadly neutralized DENV1-4 and ZIKV (S2D Fig). Donor 014 antibodies displayed the broadest neutralization profile (S2E–S2G Fig): almost all 27 selected clonal families neutralized multiple serotypes of DENV and, in some cases, ZIKV with varying potencies. Of these, antibodies from two families (F05 and F09) neutralized DENV1-4 by a mean of 97% and one family (F25) neutralized DENV1-4 and ZIKV by a mean of 92%. Having identified clonal families encoding bnAbs (bolded in S2B–S2G Fig), we initiated a second round of screening to identify additional bnAbs within those families. Antibodies screened in round two are italicized and indented in S2B–S2G Fig. In general, antibodies within a given family displayed similar neutralization breadth. For example, all 10 antibodies selected from family F07 of donor 001 neutralized DENV1, DENV2, DENV3, and ZIKV, but not DENV4 (S2B Fig). Similarly, all tested antibodies from donor 014 family F09 neutralized DENV1-4 but not ZIKV (S2F Fig), while 6/8 antibodies from family F25 broadly neutralized DENV1-4 and ZIKV (S2G Fig). These results demonstrate that our bioinformatics-based approach successfully identified clonal families encoding multiple bnAbs.

Functional characterization of the broadest neutralizing antibodies Based on the above crude screens performed with transfection supernatant (S2 Fig), we purified 23 IgG1 antibodies that inhibited DENV1-4 and in some cases ZIKV by >50% for further characterization. All but one (F15.S01 from donor 012) of these antibodies were from donor 014. We confirmed their neutralizing activities in dose-response assays and calculated the concentration at which they inhibited 50% of virus infection (IC50) (S1 Table). As comparison, we expressed and tested previously identified bnAbs in parallel. These include: EDE1 [28,31] and SIgN-3C [27,37] antibodies, all of which potently neutralize DENV1-4 and ZIKV; EDE2 antibodies, which are distinguished from EDE1 by their weak potency against ZIKV [31]; MZ4, which neutralizes ZIKV and some DENV serotypes [33]; and J9, an antibody we previously isolated from a different donor in the same cohort, which potently neutralizes DENV1-4, but not ZIKV [39]. We assigned antibodies into two categories based on neutralization breadth: 1) those that neutralized DENV1-4 and ZIKV, and 2) those that neutralized DENV1-4 but not ZIKV. Antibodies in each category were ranked based on geometric mean IC50 (S1 Table). Among all category 1 antibodies tested, the top-ranking was F25.S02 from donor 014 (geometric mean IC50 value of 69 ng/mL). Compared to previously published category 1 bnAbs, the potency of F25.S02 against ZIKV was similar to EDE1-C10 (IC50 of 18 and 14 ng/ml, respectively) but was ~39 times higher than that of SIgN-3C (IC50 of 694 ng/ml). The geometric mean potency of F25.S02 against DENV1-4 was also ~2-fold higher than that of EDE1-C10 (IC50 of 96 ng/ml versus 207 ng/ml, respectively). Family F25 contained 3 other antibodies that broadly neutralized DENV1-4 and ZIKV. These antibodies (F25.S03, F25.S04, F25.S06) neutralized DENV1, DENV2, DENV3, and ZIKV with relatively similar potency as F25.S02, but they were less potent against DENV4 (IC50 > 1 μg/ml). Among newly identified category 2 antibodies, F09.S05 was most potent; its geometric mean IC50 against DENV1-4 was comparable to the previously identified J9 [39] (36 ng/ml and 33 ng/ml, respectively). Additional high-ranking category 2 antibodies include others from family F09 and antibody F05.S03 from family F05. Even within the same donor, bnAbs were derived from multiple germline genes and did not display unusually high levels of somatic hypermutation (S2 Table), as has been reported for some bnAbs against other viruses [55, 56]. For subsequent detailed characterization, we chose the top-ranking antibody from each clonal family of donor 014, namely F25.S02, F09.S05, and F05.S03. Fig 3A shows representative dose-response neutralization assays demonstrating that these new bnAbs were roughly as potent as, and in some cases, more potent than previously published bnAbs (Fig 3B and S1 Table). PPT PowerPoint slide

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TIFF original image Download: Fig 3. Neutralization profile of top bnAbs expressed as IgG1. (A) Representative dose-response neutralization curves of each antibody against DENV1 WP-74, DENV2 16681, DENV3 CH53489, DENV4 TVP376, and ZIKV H/PF/2013 reporter virus particles performed in at least 3 biological replicates in duplicate wells. The data points and error bars represent the mean and range of the duplicates, respectively. (B) Mean IC50 values for antibody-virus pairs shown in (A) and compiled from S1 Table. *The final column displays the geometric mean IC50 values against neutralized viruses. (C) IC50 values against additional DENV variants selected due to known antigenic divergence from the panel in (B). Values shown are means from at least two biological replicates. (D) Mean IC50 values against fully infectious DENV clinical isolates from 2004–2007. Values were obtained from at least two biological replicates. *The final column displays the geometric mean IC50 of each antibody against the four viruses. In (B-D), IC50 values are displayed as heatmaps according to the key. Gray indicates that 50% neutralization was not observed at the highest antibody concentration tested (10,000 ng/ml). https://doi.org/10.1371/journal.ppat.1011722.g003

Effect of antibody valency on neutralizing activity To gain additional insight into the epitope specificities, we compared the neutralization potency of F25.S02, F09.S05, and F05.S03 tested as bivalent IgG1 or monovalent Fab against DENV2 and ZIKV (S4 Fig). Except for F09.S05, the Fab versions of all antibodies tested, including known bnAb controls EDE1-C10 and SIgN-3C, failed to neutralize DENV2 by at least 50% at the highest antibody concentration tested (400 nM), suggesting that bivalent engagement is important for potent DENV2 neutralization by these antibodies [82]. Although SIgN-3C IgG1 neutralized ZIKV with moderate potency, no neutralization was detected with Fab, consistent with previous findings [37]. In contrast, EDE1-C10 and F25.S02 retained the ability to completely neutralize ZIKV as Fab. Although IgG1 versions of EDE1-C10 and F25.S02 neutralized ZIKV with similar potency, their Fab neutralization profiles were more distinct; unlike EDE1-C10 Fab, which retained relatively potent neutralization consistent with previous findings (<10-fold increase in IC50 compared to IgG) [82], F25.S02 neutralized ZIKV with much reduced potency as Fab (64-fold increase in IC50 compared IgG). These results suggest that EDE1-C10, SIgN-3C, and F25.S02 target distinct epitopes on ZIKV.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1011722

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