Potent Zika and dengue cross-neutralizing antibodies induced by Zika vaccination in a dengue-experienced donor

Abstract

Zika virus (ZIKV) has caused significant disease, with widespread cases of neurological pathology and congenital neurologic defects. Rapid vaccine development has led to a number of candidates capable of eliciting potent ZIKV-neutralizing antibodies (reviewed in refs. 1,2,3). Despite advances in vaccine development, it remains unclear how ZIKV vaccination affects immune responses in humans with prior flavivirus immunity. Here we show that a single-dose immunization of ZIKV purified inactivated vaccine (ZPIV)4,5,6,7 in a dengue virus (DENV)-experienced human elicited potent cross-neutralizing antibodies to both ZIKV and DENV. Using a unique ZIKV virion-based sorting strategy, we isolated and characterized multiple antibodies, including one termed MZ4, which targets a novel site of vulnerability centered on the Envelope (E) domain I/III linker region and protects mice from viremia and viral dissemination following ZIKV or DENV-2 challenge. These data demonstrate that Zika vaccination in a DENV-experienced individual can boost pre-existing flavivirus immunity and elicit protective responses against both ZIKV and DENV. ZPIV vaccination in Puerto Rican individuals with prior flavivirus experience yielded similar cross-neutralizing potency after a single vaccination, highlighting the potential benefit of ZIKV vaccination in flavivirus-endemic areas.

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Fig. 1: ZPIV vaccination boosts pre-existing immunity and elicits cross-neutralizing antibodies in a flavivirus-experienced individual.
Fig. 2: Crystal structure of human antibody MZ1 and MZ4 in complex with ZIKV E glycoprotein.
Fig. 3: MZ4 protects mice in vivo from ZIKV and DENV-2 replication.
Fig. 4: A single ZPIV vaccination elicited potent MZ4-like antibodies.

Data availability

The associated accession numbers for the coordinates and structure factors reported in this paper have the following Protein Data Bank IDs: 6MTX, 6MTY, 6NIP, 6NIU and 6NIS. The sequences for MZ4, MZ1, MZ2, MZ20, MZ24, MZ54, MZ56 heavy chains and MZ4, MZ1, MZ2, MZ20, MZ24, MZ54, MZ56 light chains have been deposited in GenBank under ID codes MN523667, MN523668, MN523669, MN523670, MN523671, MN523672, MN523673, MN523674, MN523675, MN523676, MN523677, MN523678, MN523679 and MN523680, respectively. The data that support the findings of this study are available from the corresponding authors upon request. The interim aggregate data of the ZPIV trial in Puerto Rico (NCT03008122) are available with permission from the Division of Microbiology and Infectious Disease, NIAID, NIH, as this is currently an active, ongoing Phase I study.

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Acknowledgements

We sincerely thank the clinical trial participants and staff. In addition, we thank M. Creegan, M. Eller and the MHRP FlowCore facility for help with FACS sorting and C. Kuklis, Q. Chen, D. Barvir, A. Srikanth, T. Li, C. Fung, B. Yadav, B. Sumlin, G. Ballarini, N. Burrell, R. Olson and A. Dean for technical support. X-ray diffraction data were collected at beamlines at the Advanced Photon Source, Argonne National Laboratory, and the National Synchrotron Light Source II. This work was primarily funded by the US Department of the Army and the Defense Health Agency (0130602D16) to K.M. Work at BIDMC under D.B. was performed with support from the US Department of Defense, Defense Health Agency (0130602D16), the Henry M. Jackson Foundation and the Harvard Catalyst, Harvard Clinical and Translational Science Center (National Center for Research Resources and the National Center for Advancing Translational Sciences, National Institutes of Health Award UL1 TR001102) and with financial contributions from Harvard University and its affiliated academic healthcare centers. The ZPIV vaccine trial in Puerto Rico was funded by the Vaccine Treatment Evaluation Unit (VTEU) at Saint Louis University (contract no. HHSN2722013000021I) under S.L.G. The network of VTEUs is supported by the National Institute of Allergy and Infectious Diseases, part of the National Institutes of Health. The funders of the clinical trials were involved in the clinical study design, clinical study operations and approval of the clinical protocols. The ZPIV program leads (K.M. and N.L.M.) and the study sponsors had final responsibility for the decision to submit for publication. This work was supported by a cooperative agreement (W81XWH-07-2-0067) between the Henry M. Jackson Foundation for the Advancement of Military Medicine and the US Department of Defense (DoD) under the leadership of N.L.M. and M.R. The Structural Biology Center (SBC) and Northeastern Collaborative Access Team (NE-CAT) beamlines are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P41 GM103403) at the Advanced Photon Source, Argonne National Laboratory. SBC-CAT is operated by UChicago Argonne for the US Department of Energy, Office of Biological and Environmental Research under contract no. DE-AC02-06CH11357. This research used ID-17-1 (AMX) beamline of the National Synchrotron Light Source II, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under contract no. DE-SC0012704. In addition, this work was supported by NIH contract no. HHSN272201400058C to B.J.D. Material has been reviewed by the Walter Reed Army Institute of Research. The content is solely the responsibility of the authors and does not necessarily represent the official views of Harvard Catalyst, Harvard University and its affiliated academic healthcare centers or the National Institutes of Health. The opinions or assertions contained herein are the private views of the authors, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense. The investigators have adhered to the policies for protection of human subjects as prescribed in AR 70–25.

Author information

Conceptualization was provided by K.E.S., D.H.B., E.B., S.L.G., K.M., S.J.K., M.G.J. and N.L.M. Investigations were carried out by V.D., R.S.S., G.D.G., G.D., R.A.D.L.B., R.A.L., W.Z., L.M.-R., M.C., E.D., M.K.M., J.D.B., P.A., H.B., A.L.B., C.H.B., I.M.B., N.B., T.C., N.A.D.-R., A.G.i.E., J.A.F., A.G., M.H., B.S.H., N.J., K.K., D.J.L., J.L., A.K.P., W.R., I.S., U.T., S.T., M.G.J. and S.J.K. Data curation was performed by V.D., R.S.S., G.D.G., G.D., R.A.D.L.B., R.A.L., M.G.J. and S.J.K. The original draft was written by V.D., G.D., R.S.S., M.G.J. and S.J.K. Review and editing was carried out by all authors. Visualization was provided by V.D., R.S.S., G.D.G., G.D., R.A.L., M.G.J. and S.J.K. Supervision was provided by B.J.D., M.R., A.B.M., I.S.G., R.T., M.L.R., K.H.E., M.K., D.R.S., R.G.J., S.L.G., K.E.S., D.H.B., K.M., N.L.M., M.G.J. and S.J.K. Funding acquisition was carried out by M.L.R., R.G.J., B.J.D., D.H.B., K.M. and N.L.M.

Correspondence to M. Gordon Joyce or Shelly J. Krebs.

Ethics declarations

Competing interests

B.J.D. is a shareholder of Integral Molecular. D.H.B. has received grants from Novavax and personal fees from IGM Biosciences. Patent application no. PCT/US19/28952 containing the mAbs described in this publication has been filed for authors S.J.K., N.L.M., V.D., D.H.B., K.M., R.G.J., R.S.S., G.D., M.G.J. and K.E.S. The status of the patent is pending, not yet published. The other authors declare no competing interests.

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Peer review information Alison Farrell was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Flow cytometry gating strategy.

a, Gating strategy and flow cytometry plots of peripheral blood mononuclear cells from Participant A at week 8. CD19+IgG+IgD-IgM- B cells reactive to whole ZIKV virions, ZIKV E, and DENV-2 E were sorted into lysis buffer for B cell Receptor (BCR) sequencing. b, Flow cytometry plots of antigen-positive B cells from a known ZIKV-naïve participant and week 0, week 2, and week 8 PBMCs from Participant A. PBMCs at week 8 were sorted on a different day compared to PBMCs from week 0 and week 2 and the ZIKV-naïve participant. Increased frequencies of antigen-specific and cross-reactive B cells were detected against all antigens (whole ZIKV virions, ZIKV E, and DENV-2 E) between week 0 and week 2 in Participant A. A single sort was performed for each time point.

Extended Data Fig. 2 Cross-neutralization characteristics of isolated antibodies.

Heat map of binding (shades of blue) and neutralization (shades of red) titrations of all antibodies isolated from Participant A that bound to ZIKV and/or DENV-2 virions. Binding data are reported as follows: - (below background), + (OD<0.5), ++ (OD 0.5-1.0), +++ (OD 1.0-1.5) and ++++ (OD>1.5). Shown are geometric mean neutralizing titers of ZIKV and DENV1-4 from at least 2 independent experiments with the IC50 values indicated (ng ml-1).

Extended Data Fig. 3 Antibody isolation and neutralizing characteristics of MZ4 family antibodies.

a, Isolation of whole ZIKV virion, ZIKV E and DENV-2 E reactive CD19+/IgG+ B cells 4 weeks following the second ZPIV immunization (week 8) from Participant A. Individual B cells encoding ZIKV-neutralizing mAbs are indicated in the flow cytometry plots. b, Neutralization heat map of the MZ4 family and control mAbs against ZIKV PR, DENV1-4, JEV, WNV and YFV, with the microneutralization IC50 values indicated (ng ml-1). c,d, FlowNT neutralization of MZ4 against (c) ZIKV (Paraiba_01) and (d) DENV-2 (S16803) compared to other potent ZIKV/DENV cross-neutralizing and specific antibodies. All mAbs were tested in at least 2 independent experiments. Where shown, error bars indicate mean ± s.e.m. for mAbs tested in 3 independent experiments. e, PRNT neutralization of MZ4 against ZIKV strains of American, Asian and African lineages, indicated by country of origin and date of isolation. Shown is the mean % neutralization from a single experiment using triplicates. The IC50 neutralization titers (ng ml-1) are indicated in parentheses for each mAb. Source data

Extended Data Fig. 4 In vitro antibody dependent enhancement (ADE).

MZ4, MZ4 harboring the Fc mutations abolishing binding to Fcγ receptors (MZ4 LALA) and the pan flavivirus FLE antibody 4G2 were tested in a flow cytometry-based assay for their ability to enhance infection in K562 cells. ADE is reported as fold change in percent of infected cells relative to baseline percent infection of K562 cells (in absence of antibody, dotted line). The HIV-1 specific antibody VRC01 served as negative control. Shown is the mean from 2 independent experiments performed in duplicates. Source data

Extended Data Fig. 5 Antibody binding characteristics and epitope mapping.

a-d, Binding of ZIKV-neutralizing mAbs to ZIKV and DENV-2 monomeric E proteins, and whole ZIKV and DENV-2 virions by ELISA. a, Binding to monomeric ZIKV E (left) and virions (right). Shown is the mean from 3 (± s.e.m as indicated by error bars) or 2 independent experiments. b, Relative binding ratio of monomeric ZIKV E to ZIKV whole virions calculated from (a). Antibodies with low ratio values were characteristic of quaternary epitopes, such as EDE1-C8, whereas ratios closer to 1 were characteristic of monomeric recognition similar to an FLE antibody, such as 2A10G6, which binds to both monomeric E and ZIKV. c, Binding to monomeric DENV-2 E (left) and whole DENV-2 virions (right). Shown is the mean of 2 independent experiments. d, Relative binding ratio of monomeric DENV-2 E to DENV-2 whole virions calculated from (c). e, Binding of mAbs to ZIKV E recombinant DIII domain assessed by ELISA. Shown is the mean of 2 independent experiments. f, Shotgun mutagenesis ZIKV E epitope analysis. Relative binding to ZIKV prM/E for individual mutations is plotted. Residues from which substitution to alanine causes >60% loss in binding (dotted line) were considered important for binding. Shown is the mean of duplicates from a single experiment. Source data

Extended Data Fig. 6 Structural analysis of MZ4 family antibodies.

a, MZ1-ZIKV E crystal structure asymmetric unit contents. ZIKV E is shown in surface representation (DII and DIII [blue] and DI [dark green]), and MZ1 Fab is shown in ribbon representation (heavy chain [red] and light chain [salmon]). Zoom-in window shows the 2Fo-Fc electron density for the MZ1-DI/DIII linker interaction (gray mesh representation contoured at 1.5σ). b, MZ4-ZIKV E crystal structure asymmetric unit contents. Four MZ4 Fv molecules bound to four molecules of ZIKV E were observed in the asymmetric unit. ZIKV E protomers within a dimer are shown in surface representation (blue and white with DI dark green). MZ4 antibody heavy and light chains are shown in ribbon representation (dark and light orange). Zoom-in window shows the 2Fo-Fc electron density for the MZ4-DI/DIII linker interaction (gray mesh contoured at 1.0σ). c, Crystal structure of MZ1, MZ4, and MZ24 Fab molecules are shown in ribbon representation with light and heavy chain CDRs indicated. d, Overlay of MZ1 and MZ4 (left), and MZ4 and MZ24 (right) Fab structures. Antigen-contacting residues that differ between MZ4 and the other family members are shown in stick representation and amino acid changes are indicated. e, Model of MZ4 Fv on the ZIKV (PDB: 5IRE). MZ4 is shown in surface representation (dark [heavy chain] and light [light chain] orange). E protomers on the ZIKV virion are colored blue, gray, and green, which indicates dimeric, trimeric, and pentameric vertex interfaces, respectively. Close-up view of the modelled MZ4 interactions at the five-fold vertex (1) and inter-raft interface (2) are shown.

Extended Data Fig. 7 Epitope recognition by antibody MZ4.

a, Flavivirus-neutralizing antibodies that recognize DIII and adjacent regions. Flavivirus E molecules are shown in ribbon representation with the DIII, DI/DIII linker region, and lateral ridge loop architecture shown on the top left for reference. Antibody contact residues (defined using PISA) are shown in stick representation and colored as follows: MZ4 (orange), 5H2 (dark blue), 2C8 (magenta), Ab513 (red), Z004 (green), 3H5 (yellow), and ZV-67 (light orange). ZIKV E – MZ4 contact residues G182 and S368 (and DENV equivalent residues), are shown as black spheres. b, Antibody epitopes are indicated as colored solid lines on the surface of ZIKV E (blue, with the DI/DIII linker in brown). c, Sequence alignment of flavivirus E proteins (residues 160-400, ZIKV numbering). Antibody contact residues on flavivirus E proteins are highlighted. d, MZ4 antibody binding competitions. Competition between the indicated first mAb and MZ4 for binding to the relevant E protein were performed as described in Fig. 1h. Shading from dark to light red indicates competition strength ranging from strong (0-30%), to intermediate (31-69%), to weak/none (70-100%).

Extended Data Fig. 8 In vivo titration of MZ4 prophylactic dose.

Forty naïve Balb/c mice were infused intravenously with the indicated dose (n=5/dose) of MZ4 or saline (sham). Two hours later, mice were challenged with 105 viral particles (102 plaque-forming units) of ZIKV BR/2015 intravenously. Following infusion with the indicated dose or saline (sham), ZIKV viral loads were measured post-challenge by RT–PCR.

Extended Data Fig. 9 Characteristics of MZ2, isolated two weeks after the first ZPIV vaccination.

a, Binding of MZ4 and MZ2 to ZIKV (left) and DENV-2 (right) virions, assessed by ELISA. Shown is the mean from 2 independent experiments. b, Binding kinetics of MZ4 and MZ2 antibodies to ZIKV (top) and DENV-2 (bottom) E proteins as measured by BLI. Affinity constants (KD) were calculated from binding curves (red [MZ4] and orange [MZ2] obtained from 4 serial dilutions of Fabs and fitted (grey curves) from a single experiment using a 1:1 binding model. Below, summary table of binding kinetic constants and fit parameters. c, Microneutralization heat map of MZ4 and MZ2 against ZIKV, DENV1-4, JEV, WNV and YFV, with the IC50 values indicated (ng ml-1). d, Shotgun mutagenesis ZIKV and DENV-2 E epitope analysis. Residues critical for binding are indicated by check marks. Below, alignment of ZIKV and DENV-2 DI/DIII linkers with respective residue numbering. Source data

Extended Data Fig. 10 Additional characterization of polyclonal antibody responses in flavivirus-experienced and flavivirus-naïve vaccinated participants.

a, b, Polyclonal antibody binding, off-rates and neutralization titers (FlowNT) to ZIKV (left) or DENV-2 (right) of (a, red) Participant A (n=1) and (b, blue) flavivirus-naïve vaccinees (5 donors with the highest responses) at baseline (week 0), 2 weeks following the first vaccination (week 2) and 4 weeks following the second vaccination (week 8). For (a), shown is the mean titer or off-rate and range from at least 2 independent experiments. For (b), shown is the mean titer per individual with s.e.m. indicated by the error bars per assay. Plasma binding responses and affinity off-rates to the indicated E protein (nm) were measured by BLI; NB=no binding detected. Plasma dilution series were used to calculate off-rates or dissociation constants (kd, s-1) against the indicated E protein. Lower off-rates indicate slower dissociation and higher affinity. Dotted line indicates lower limit of detection. As no DENV-2 neutralization was observed for naïve donors at week 8, week 0 and 2 were not tested (NT). Source data

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Dussupt, V., Sankhala, R.S., Gromowski, G.D. et al. Potent Zika and dengue cross-neutralizing antibodies induced by Zika vaccination in a dengue-experienced donor. Nat Med 26, 228–235 (2020). https://doi.org/10.1038/s41591-019-0746-2

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