For the immune system, practice makes perfect. Previous exposure to an infection elicits a stronger, faster memory response. But how does the immune system respond to a similar but not identical infection? Rogers et al. tracked the neutralizing antibody response to zika virus infection in individuals with and without previous exposure to the closely related dengue viruses. They found that zika virus infection primed the preexisting dengue virus response, but that this cross-reactive response was poorly neutralizing. In contrast, de novo zika virus responses were potently neutralizing. These data suggest that zika virus vaccines should target epitopes not present in dengue virus subtypes.

Zika virus (ZIKV) shares a high degree of homology with dengue virus (DENV), suggesting that preexisting immunity to DENV could affect immune responses to ZIKV. We have tracked the evolution of ZIKV-induced B cell responses in three DENV-experienced donors. The acute antibody (plasmablast) responses were characterized by relatively high somatic hypermutation and a bias toward DENV binding and neutralization, implying the early activation of DENV clones. A DENV-naïve donor in contrast showed a classical primary plasmablast response. Five months after infection, the DENV-experienced donors developed potent type-specific ZIKV neutralizing antibody responses in addition to DENV cross-reactive responses. Because cross-reactive responses were poorly neutralizing and associated with enhanced ZIKV infection in vitro, preexisting DENV immunity could negatively affect protective antibody responses to ZIKV. The observed effects are epitope-dependent, suggesting that a ZIKV vaccine should be carefully designed for DENV-seropositive populations.

In this study, we have longitudinally tracked the ZIKV-specific B cell response in three DENV-experienced donors using single B cell cloning and large-scale antibody isolation. The acute-phase (plasmablast) response was dominated by somatically mutated clones that showed preferential binding and neutralization of DENV, providing evidence for original antigenic sin (OAS) during the early B cell response to ZIKV in DENV-experienced donors. However, by 5 months after infection, the memory B cell responses were composed of both OAS-phenotype antibodies that were broadly cross-reactive and poorly neutralizing and de novo generated antibodies that were ZIKV-specific and potently neutralizing. Collectively, the results have implications for the development of ZIKV vaccines intended for DENV-immune populations and provide insight into the role that preexisting B cell memory plays in modulating the antibody specificities induced by antigenically variable viruses.

Previous studies have shown that nonstructural protein 1 (NS1), envelope (E), and precursor membrane (prM) proteins are dominant targets for the human B cell response to flaviviruses. NS1 is secreted by infected cells and functions in pathogenesis and immune evasion ( 15 ), the surface E protein mediates viral entry and is the primary target for nAbs ( 7 , 16 , 17 ), and prM is a 166–amino acid protein that is associated with E on immature and partially mature viruses ( 18 ). The E protein consists of three domains: domain I (DI), which participates in conformational changes required for viral entry; domain II (DII), which contains the conserved fusion loop (FL); and domain III (DIII), which is the putative receptor binding domain ( 19 ). Previous studies have established that monoclonal antibodies (mAbs) targeting epitopes within DIII are typically type-specific and potently neutralizing, whereas mAbs targeting the conserved FL are cross-reactive and poorly neutralizing ( 7 , 16 , 17 , 20 ).

Zika virus (ZIKV) is a mosquito-borne flavivirus that has been linked to microcephaly and severe neurological complications, such as Guillain-Barré syndrome ( 1 , 2 ). The virus is closely related to the four serotypes of dengue virus (DENV) (DENV1, 2, 3, and 4), as well as other circulating flaviviruses including West Nile virus (WNV), resulting in substantial immunological cross-reactivity ( 3 – 7 ). Although neutralizing antibodies (nAbs) play an important role in protection against flavivirus infection, they can also contribute to severe disease through a phenomenon termed antibody-dependent enhancement (ADE) ( 8 – 10 ). In the case of DENV, subneutralizing concentrations of preexisting heterotypic nAbs have been implicated in promoting viral replication by facilitating the interaction of the virus with Fc receptor–bearing target cells ( 8 ). Enhancement of ZIKV infection by cross-reactive DENV-specific antibodies and vice versa has been demonstrated in vitro, and ADE of ZIKV pathogenesis by preexisting anti-flavivirus immunity has been observed in mouse models ( 5 – 7 , 11 – 14 ). Because ZIKV is currently circulating in regions that are highly endemic for DENV, an understanding of how previous DENV exposure affects the B cell response to ZIKV will be critical for the design of vaccines and therapies intended for DENV-immune populations.

RESULTS

Potent plasmablast induction in ZIKV-infected, DENV-experienced donors To study the early ZIKV-induced B cell response in DENV-experienced individuals, three ZIKV-infected donors (donors 2, 3, and 4) living in a highly DENV endemic region of Colombia were sampled during acute infection (within 8 days after symptom onset) (table S1) (21). A single donor (donor 1) from the United States who was infected with ZIKV while overseas was also sampled for comparison (table S1). As expected, the acute sera from donors 2, 3, and 4 showed strong immunoglobulin G (IgG) binding reactivity to whole DENV1–4 particles and recombinant DENV NS1 proteins (fig. S1). In contrast, the acute serum from donor 1 showed little to no such binding (fig. S1). Although the acute serum reactivity to DENV particles and NS1 proteins could, in principle, be due to cross-reactive ZIKV-induced antibodies, previous studies have shown that NS1-specific IgG antibodies are not present in the serum during the first week after the onset of symptoms of ZIKV infection (22, 23), implying that the acute serum reactivity to DENV NS1 observed for donors 2, 3, and 4 is largely mediated by preexisting antibodies that were induced by previous DENV exposure. Furthermore, the acute serum from these three donors showed similar or more potent neutralizing activity against DENV compared with ZIKV, whereas the acute serum from donor 1 showed little to no neutralizing activity against DENV1–4 and relatively weak neutralization of ZIKV. These serological and epidemiological data provide convincing evidence that donors 2, 3, and 4 were previously exposed to DENV, whereas donor 1 was DENV-naïve at the time of infection. The appearance of antigen-specific plasmablasts in peripheral blood during acute viral infection is typically the first indication of a B cell response (24–26). Therefore, we first measured the plasmablast frequency in peripheral blood about 1 week after symptom onset (table S1). In all three DENV-experienced donors, a massive plasmablast population (CD3−CD20−/loCD19+CD27hiCD38hi) that accounted for 39 to 63% of peripheral B cells was observed (Fig. 1A and fig. S2). In contrast, the plasmablast population in the ZIKV-infected, DENV-naïve donor was relatively small, accounting for only 2.5% of peripheral B cells (Fig. 1A). To deconstruct the acute ZIKV-induced B cell response, we single cell–sorted between 100 and 300 plasmablasts from each donor sample and rescued the antibody variable heavy (V H ) and variable light (V L ) chain sequences by single-cell polymerase chain reaction (PCR). Between 42 and 119 cognate V H and V L pairs from each donor were cloned and expressed as full-length IgGs. Sequence analysis revealed that the plasmablasts sorted from the three DENV-experienced donors were somatically mutated—with per-donor averages ranging between 15 and 20 nucleotide substitutions in V H —and clonally expanded (Fig. 1, B and C). This level of somatic hypermutation (SHM) is comparable with that observed in typical IgG+ memory B cells as well as in plasmablasts induced by influenza vaccination (25), supporting a memory B cell origin for these acutely induced cells. In contrast, most of the plasmablasts isolated from the DENV-naïve donor sample showed little to no SHM and somewhat more limited clonal expansion (Fig. 1, B and C), supporting their emergence via activation of naïve B cells. We conclude that ZIKV infection induces a rapid and robust plasmablast response in DENV-experienced donors and that the large majority of these acutely induced cells appear to originate from the memory B cell compartment. Fig. 1 Analysis of plasmablast responses in one naïve and three DENV-experienced donors. (A) Flow cytometric analysis of plasmablast responses in one naïve and three DENV-experienced donors during ongoing ZIKV infection. Plasmablasts are defined herein as CD19+CD3−CD20−/loCD38hiCD27hi cells. Plots shown are gated for CD3−CD20−/lo cells, with the CD27hiCD38hi plasmablasts marked by a box. The frequency of plasmablasts within the CD3−CD20−/lo population is shown next to the gate. The numbers in parentheses show the percentage of peripheral B cells that are plasmablasts. Flow cytometry data were analyzed using FlowJo software (version 10.0.8). (B) Load of somatic mutations [expressed as the number of nucleotide (NT) substitutions] in V H and V L in plasmablast-derived mAbs isolated from a DENV-naïve (donor 1) and three DENV-experienced donors (donors 2 to 4). All V H and V L chains are shown, regardless of whether the paired antibody chain was recovered. (C) Clonal lineage analysis. Heavy-chain sequences were assigned to lineages using Clonify (52). Each lineage is represented as a segment proportional to the lineage size. The total number of recovered heavy chains is shown in the center of each pie. All lineages that contain only a single sequence are combined and shown as a single gray segment.

A large proportion of plasmablast-derived mAbs isolated from ZIKV-infected, DENV-experienced donors bind the ZIKV E protein Enzyme-linked immunosorbent assay (ELISA) binding studies showed that between 50 and 75% of the mAbs isolated from the DENV-experienced donors bound to whole ZIKV (fig. S3), which is similar to the percentage of antigen-specific mAbs recovered from plasmablasts induced by influenza vaccination and DENV infection (25, 27). In contrast, only 13% of the plasmablast-derived mAbs isolated from the DENV-naïve donor bound with detectable affinity to whole ZIKV particles. To further map the antigenic specificities of the 303 plasmablast-derived mAbs, we performed biolayer interferometry (BLI) binding experiments with recombinant ZIKV NS1 and E proteins. The percentage of mAbs isolated from the three DENV-experienced donors that showed reactivity with ZIKV NS1 was low at 8 to 12% and higher for recombinant E at 15 to 39% (Fig. 2A and table S2). In contrast, 17% of the mAbs isolated from the DENV-naïve donor bound to recombinant NS1, whereas only a single mAb bound with detectable affinity to recombinant E (Fig. 2A and table S2). The remaining ZIKV-specific mAbs, which accounted for 13 to 32% of the corresponding donor responses, bound to epitopes expressed on whole virus but not on recombinant E. Seventy percent of the plasmablast mAbs isolated from donor 1 and about 20 to 50% of the plasmablast mAbs isolated from donors 2, 3, and 4 could not be associated with any ZIKV reactivity (Fig. 2A). Because anti-prM antibodies have been shown to dominate the antibody response during secondary DENV infection (28), we next investigated the specificities of the whole virus–specific mAbs isolated from the DENV-experienced donors by Western blot. None of the mAbs recognized prM, 30% showed reactivity with E protein from viral lysates, and the remaining mAbs failed to bind to ZIKV in this assay (fig. S4). Hence, most of these mAbs likely bind to E-specific epitopes expressed only on intact virions. Similar specificities have been previously described for both DENV and ZIKV (4, 7, 27, 29). Fig. 2 Binding characteristics of mAbs isolated from plasmablasts during acute ZIKV infection. (A) Specificities of the mAbs isolated from one DENV naïve donor and three DENV-experienced donors. Distribution of mAbs that bind to NS1, recombinant E, epitopes expressed only on whole ZIKV, and those for which no specificity could be assigned are shown. The number at the top of each bar represents the total number of mAbs cloned from each donor. (B) Epitope mapping of recombinant E–specific antibodies isolated from three DENV-experienced donors. Pie charts show the distribution of antibodies that bind to epitopes within DIII, within or proximal to the FL on DII, and defined by mAbs ADI-24314 and ADI-24247. 4G2-like FL: Competition with 4G2, WNV E–reactive, and E-FL–sensitive; non-WNV FL: 4G2-competitive, E-FL–sensitive, and WNV E–nonreactive; FL proximal: 4G2 competitive, E-FL–reactive, and WNV E–nonreactive; DIII: Recombinant DIII–reactive; ADI-24314 competitor: 4G2-noncompetitive, ADI-24314–competitive, and E-FL–reactive; ADI-24247 competitor: 4G2-noncompetitive, ADI-24247–competitive, and E-FL–reactive. NC, not characterized because of low binding affinity. (C) Heat map of mAb binding reactivity to ZIKV and DENV1–4 E proteins or whole viral particles (left) and ZIKV and DENV1–4 NS1 proteins (right). The E-specific mAbs shown were isolated from DENV-experienced donors. Apparent binding affinities for recombinant E and NS1 proteins were determined by BLI measurements, and apparent binding affinities for whole virus were determined by ELISA. To further define the epitopes targeted by the recombinant E–reactive mAbs isolated from the DENV-experienced donors, we (i) compared the apparent binding affinities of the mAbs for recombinant DIII, WNV E, and a previously described ZIKV E protein (E-FL) that contains substitutions within and proximal to the FL that abolish binding by most FL-specific antibodies (30) (fig. S5) and (ii) performed competitive binding experiments using two previously characterized mAbs, 4G2 and ZV-67, that bind to the FL on DII and the lateral ridge on DIII, respectively (table S2) (30, 31). Because the FL-specific mAb 4G2 reacts with WNV E and fails to react with E-FL (table S3), we classified mAbs that competed with 4G2, bound to WNV E, and failed to bind to E-FL as “4G2-like FL binders.” 4G2 competitor mAbs that failed to bind to E-FL but did not cross-react with WNV E were classified as “non-WNV FL binders,” and 4G2 competitor mAbs that did not cross-react with WNV E but did bind to E-FL were classified as “FL proximal binders.” Together, mAbs that bound within or proximal to the FL comprised up to 50% of the recombinant E–reactive responses (Fig. 2B and table S2). DIII-specific mAbs were absent from the donor 3 and donor 4 responses but comprised about 30% of the donor 2 response (Fig. 2B and table S2). Six of eight DIII-reactive mAbs competed with ZV-67, suggesting that they likely recognize epitopes within or proximal to the lateral ridge on DIII (table S2). This class of antibodies showed preferential VH3-23 germline gene usage and contained convergent sequence signatures in both CDRH3 and CDRL3 (table S4), suggesting that these mAbs may share a common mode of antigen recognition. To estimate the number of different antigenic sites recognized by the remaining E-specific mAbs, we performed competitive binding experiments with two broadly cross-reactive mAbs from the panel (ADI-24247 and ADI-24314) that did not compete with 4G2, ZV-67, or each other (tables S2 and S5). Between 12 and 35% of the E-specific donor responses bound to epitopes overlapping that of ADI-24247 or ADI-24314 (Fig. 2B). Overall, we conclude that substantial fractions of the acute ZIKV-induced B cell response in DENV-experienced donors are directed against quaternary epitopes expressed only on whole virus, and those that are reactive with recombinant E are largely directed to the FL and other highly conserved epitopes.

The ZIKV-induced plasmablast response in DENV-experienced donors is dominated by DENV cross-reactive clones The E proteins of ZIKV and DENV share more than 50% sequence identity, resulting in substantial immunological cross-reactivity (3, 6, 7). To determine the degree to which mAbs generated from plasmablasts during acute ZIKV infection cross-reacted with DENV, we measured the apparent binding affinities of the mAbs for recombinant DENV1–4 E and NS1 proteins. Most of the E-specific mAbs from the three DENV-experienced donors bound with higher affinity to at least one of the four DENV E proteins than to ZIKV E, consistent with a recall response dominated by reactivated DENV-induced memory B cells (Fig. 2C, table S2, and fig. S6). Selected mAbs isolated from donors 2, 3, and 4 that targeted epitopes expressed only on whole ZIKV showed similar DENV-biased binding profiles (fig. S7). In contrast, the whole virus–specific mAbs isolated from donor 1 bound exclusively to ZIKV (fig. S7). About 50% of the ZIKV E–reactive mAbs isolated from the DENV-experienced donors cross-reacted with all four DENV serotypes (Fig. 2C and table S2). Most of these broadly cross-reactive mAbs targeted epitopes within or proximal to the FL or unknown epitopes defined by ADI-24247 and ADI-24314 (Fig. 2C and table S2). In contrast, the DIII-specific mAbs only showed cross-reactivity with DENV1 E (Fig. 2C and table S2). One hundred percent of the NS1-specific mAbs isolated from donors 2 and 3 showed cross-reactivity with DENV1 and DENV3 NS1, whereas the NS1-specific mAbs from donor 4 were exclusively ZIKV-specific (Fig. 2C and table S6). Sequence analysis revealed that the ZIKV NS1-specific mAbs from donor 4 lacked SHM, supporting a naïve B cell origin (fig. S8). The reasons for this are unclear but may be due to the lack of preexisting cross-reactive NS1-specific memory B cells in this donor. As expected, all of the NS1-specific mAbs isolated from donor 1 were ZIKV-specific and lacked SHM (Fig. 2C). The affinities of these mAbs were comparable with the affinities of the NS1-specific mAbs isolated from the DENV-experienced donors (Fig. 2C and table S6), which is perhaps because the plasmablast-derived antibodies from the DENV-experienced donors had not undergone affinity maturation toward ZIKV. Together, the sequencing and binding results provide evidence for OAS during the acute-phase B cell response to ZIKV in DENV-experienced donors.

Most of the plasmablast-derived mAbs isolated from DENV-experienced donors are poorly neutralizing and potently enhancing To assess the functional properties of the plasmablast-derived mAbs isolated from the DENV-experienced donors, we next performed neutralization and ADE assays. Because of the large number of mAbs, initial neutralization screening was performed using a single concentration of purified IgG. At a concentration of 1 μg/ml, about 20% of mAbs from each donor reduced ZIKV-Paraiba (Para) infectivity by ≥50%, and all of these nAbs cross-neutralized at least one of the four DENV serotypes (Fig. 3A). Additionally, the majority of nAbs (53 of 63 or 84%) showed more potent neutralizing activity against one of the four DENV serotypes than ZIKV-Para (Fig. 3A). We next performed neutralization titration experiments on a subset of mAbs to evaluate neutralization potency. Consistent with the binding and single-point neutralization results, a large proportion of mAbs showed preferential neutralization of DENV, further supporting a DENV-induced memory B cell origin for these recently activated cells (Fig. 3B, table S7, and fig. S9). Of significance, most of the plasmablast-derived mAbs showed poorly neutralizing activity against ZIKV-Para (Fig. 3, B and C). The notable exceptions were the four DIII-specific mAbs, which showed highly potent neutralizing activity against both ZIKV-Para and DENV1 (Fig. 3, B and C, and table S7). These nAbs displayed IC 50 s (half-maximal inhibitory concentrations) between 0.9 and 2.7 ng/ml against ZIKV-Para and between 0.2 and 27 ng/ml against DENV1 (Fig. 3, B and C, and table S7). Most of the mAbs that bound to quaternary epitopes on the E protein showed little to no neutralizing activity against ZIKV. Previous studies have described two classes of quaternary mAbs (EDE1 and EDE2), which are defined on the basis of differing sensitivity to removal of the N-glycan at Asp153 (4). Given that mAbs directed against EDE2 have been shown to be substantially less potent against ZIKV than mAbs targeting EDE1, it is likely that the quaternary mAbs described here recognize the EDE2 epitope. Fig. 3 Neutralizing activity of mAbs isolated from plasmablasts during acute ZIKV infection in DENV-experienced donors. (A) Heat map showing mAb neutralization of ZIKV-Para, DENV1, DENV2, DENV3, and DENV4 at a concentration of 1.0 μg/ml. (B) Heat map showing neutralization IC 50 s for selected mAbs against ZIKV-Para, DENV1, DENV2, DENV3, and DENV4. Epitope assignments are indicated on the left. (C) Neutralization IC 50 s for selected mAbs against ZIKV-Para. IC 50 values represent the concentration of IgG required to reduce viral infectivity by 50%. Neutralization assays were performed using a live virus plaque reduction assay. N.N., non-neutralizing. We next tested the abilities of selected mAbs to enhance ZIKV infection using nonpermissive K562 cells. Consistent with previous studies, all of the mAbs enhanced ZIKV infection, but peak enhancement was observed at lower concentrations for the potently neutralizing DIII-specific mAbs compared with the poorly neutralizing cross-reactive mAbs (fig. S10) (6, 7, 16, 32). Additionally, there was a linear correlation between neutralization potency and the sum of the enhancement activity over the range of mAb concentrations tested, as defined by the area under the curve (fig. S10). Together, the results show that the acute B cell response to ZIKV in DENV-immune donors is dominated by cross-reactive antibodies that show poorly neutralizing activity and a propensity to enhance ZIKV infection in vitro.

Isolation of E-specific mAbs from longitudinal samples obtained from the DENV-experienced donors To track the evolution of the B cell response to ZIKV in DENV-experienced donors, we collected blood samples from donors 2, 3, and 4 about 5 months after infection (table S1). To assess the magnitude of the E-specific memory B cell response at this time point, we stained peripheral B cells with a fluorescently labeled ZIKV E protein and analyzed them by flow cytometry (Fig. 4A and fig. S11). Robust E-specific memory B cell responses were observed in donors 3 and 4, whereas E-specific memory B cells were undetectable in donor 2 (Fig. 4A). The reasons for this are unclear, but given that donor 2 showed the highest frequency of plasmablasts during acute infection, one explanation is that the virus was rapidly cleared by the plasmablast-derived antibodies, which prevented the formation of a germinal center (GC) response. Four hundred ZIKV E–specific memory B cells were single cell–sorted from donors 3 and 4, and about 200 V H and V L pairs were cloned and expressed as full-length IgGs for further characterization. To assess the extent of affinity maturation that occurred over this time period, the apparent binding affinities of the E-specific memory B cell–derived mAbs were measured and compared with the affinities of the E-specific plasmablast-derived mAbs. As expected, the average apparent affinity for ZIKV E was significantly higher in the memory B cell subset, suggesting that the ZIKV-induced B cells underwent affinity maturation over time (Fig. 4B). Clonal expansions were observed in both donor response repertoires (Fig. 4C), but there were few to no clonal lineages shared between the plasmablast and memory B cell–derived subsets (table S9 and fig. S12). Although the sampling size is too small to determine the degree of overlap between the two B cell populations, these results suggest that the most dominant clonal lineages likely differ between the two compartments. Fig. 4 Longitudinal analysis of ZIKV E–specific memory B cell responses in the DENV-experienced donors. (A) ZIKV E–specific memory B cell sorting. Fluorescence-activated cell sorting (FACS) plots show ZIKV E reactivity of IgG+ B cells from the three convalescent DENV-experienced donors and a ZIKV seronegative healthy donor control. B cells in quadrant 2 (Q2) were single cell–sorted for mAb cloning. ZIKV E was labeled with two different colors to reduce background binding. (B) Apparent binding affinities of plasmablast and memory B cell–derived mAbs to recombinant ZIKV E. Red bars indicate the median IC 50 s. (C) Clonal lineage analysis of memory B cell–derived mAbs. Heavy-chain sequences were assigned to lineages using Clonify (52). Each lineage is represented as a segment proportional to the lineage size. The total number of recovered heavy chains is shown in the center of each pie. All lineages that contain only a single sequence are combined and shown as a single gray segment. (D) Heat map showing apparent binding affinities of the memory B cell–derived mAbs to recombinant ZIKV E and DENV1–4 E proteins. Top: mAbs cloned from donor 3. Bottom: mAbs cloned from donor 4. 023, ADI-30023 competitor; 056, ADI-30056 competitor; 314, ADI-24314 competitor; 247, ADI-24247 competitor; FL, 4G2 competitor; DIII, recombinant DIII binder; UK, unknown. (E) Load of somatic mutations (expressed as the number of nucleotide substitutions in the V H ) in mAbs isolated from plasmablasts and memory B cells in DENV-experienced donors. Each point represents an individual mAb. Red bars indicate the median number of nucleotide substitutions. (F) Binding of plasmablast and memory B cell–derived mAbs to recombinant ZIKV E. Apparent binding affinities are shown in the plot. Red bars indicate median apparent IC 50 s. Statistical comparisons were made using Mann-Whitney test (***P < 0.001, **P < 0.01; n.s., not significant).