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PreprintCited by 14Mosquito-borne diseases and controlViral Infections and VectorsSARS-CoV-2 and COVID-19 Research

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Broadly neutralizing antibodies against Zika virus from dengue patients with prior exposure to Japanese encephalitis virus

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Salem, Gielenny, Galula, Jedhan, Wu, Shang-Rung, Liu, Jyung, Chen, Yen-Hsu, Wang, Wen-Hung, Wang, Sheng-Fan, Song, Cheng-Sheng, Chen, Fan-Chi, Wang, Cheng-Yi, Chao, Day-Yu·2023·Communications Biology
DOI 10.21203/rs.3.rs-2969700/v1PMC PMC10808242

Preprint — published as 10.1038/s42003-023-05661-w · Paper page

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Abstract

Exposure to multiple mosquito-borne flaviviruses within a lifetime is not uncommon; however, how sequential exposures to different flaviviruses shape the cross-reactive (CR) humoral response against an antigen from a different serocomplex has yet to be explored. Here, we report that dengue-infected individuals initially primed with the Japanese encephalitis virus (JEV) showed broadly highly neutralizing potencies against the Zika virus (ZIKV). We also identified a rare class of ZIKV CR human monoclonal antibodies (huMAbs) with increased somatic hypermutation and broad neutralization against multiple flaviviruses. One huMAb, K8b, binds quaternary epitopes with heavy and light chains separately interacting with overlapping E dimer units spanning domains I, II, and III through cryo-electron microscopy and structure-based mutagenesis. Using JEV virus-like particle (VLP) immunization in mice further confirmed that such CR antibodies, mainly IgG3 isotype, can be induced and proliferate through heterologous DENV-2 VLP stimulation. Our findings provide novel insights for future vaccination strategies.

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Fig. 1 Dengue-immune donors with prior JEV exposure have elevated ZIKV-neutralizing titers. a Neutralization antibody profile against the four serotypes of DENV, JEV, and ZIKV of healthy (n = 80) and dengue-infected febrile (DF, n = 60) individuals. Two-fold serially diluted plasma samples from healthy and DF individuals were evaluated for neutralization against the prototype strains of DENV-1 to -4 serotypes, JEV, and ZIKV using FRμNT90. Empty and shaded circles represent the FRμNT90 values of the healthy and DF individuals, respectively. b Comparison of the neutralization antibody profile of DF individuals against DENV-1 to -4 serotypes, JEV, and ZIKV prototype virions based on age classifications: 20–30 years old (n = 30) and 50–70 years old (n = 30). Empty and shaded circles represent the FRμNT90 values of the 20–30 and 50–70 age groups, respectively. c Assessment of the breadth of ZIKV neutralization among the four groups of donors dichotomized according to the presence (pos) or absence (neg) of protective or high neutralizing FRμNT90 titers against JEV and DENV, respectively. JEV-immune individuals with high (JEVposDENVhigh; n = 39) or low DENV immunity (JEVposDENVlow; n = 24) are represented by red filled or empty squares, respectively. JEV-negative donors with high (JEVnegDENVhigh ; n = 12) or low DENV (JEVnegDENVlow; n = 65) immunity are shown in blue filled or empty squares, respectively. d Effect of JEV and DENV immunity in DF individuals to ZIKV-neutralizing antibody titers. Fisher’s exact test was performed in n = 60 with a p-value of 0.000013. All data are representative of three independent experiments. In (a–c), the black solid horizontal bars represent the geometric mean titers (GMT) of samples with SD analyzed within each group. FRμNT90 titer <10 was represented with 5 for graphic display and statistical analysis. Significant differences were tested using one-way ANOVA followed by Tukey’s multiple comparisons post-test, with levels of significance defined by ***P < 0.0001; **P < 0.001. Each point shows the mean of data from two independent experiments. DENV dengue virus, JEV Japanese encephalitis virus, ZIKV Zika virus, FRμNT focus-reduction microneutralization test, VLP virus-like particle, ANOVA analysis of variance, SD standard deviation.
Fig. 1 Dengue-immune donors with prior JEV exposure have elevated ZIKV-neutralizing titers. a Neutralization antibody profile against the four serotypes of DENV, JEV, and ZIKV of healthy (n = 80) and dengue-infected febrile (DF, n = 60) individuals. Two-fold serially diluted plasma samples from healthy and DF individuals were evaluated for neutralization against the prototype strains of DENV-1 to -4 serotypes, JEV, and ZIKV using FRμNT90. Empty and shaded circles represent the FRμNT90 values of the healthy and DF individuals, respectively. b Comparison of the neutralization antibody profile of DF individuals against DENV-1 to -4 serotypes, JEV, and ZIKV prototype virions based on age classifications: 20–30 years old (n = 30) and 50–70 years old (n = 30). Empty and shaded circles represent the FRμNT90 values of the 20–30 and 50–70 age groups, respectively. c Assessment of the breadth of ZIKV neutralization among the four groups of donors dichotomized according to the presence (pos) or absence (neg) of protective or high neutralizing FRμNT90 titers against JEV and DENV, respectively. JEV-immune individuals with high (JEVposDENVhigh; n = 39) or low DENV immunity (JEVposDENVlow; n = 24) are represented by red filled or empty squares, respectively. JEV-negative donors with high (JEVnegDENVhigh ; n = 12) or low DENV (JEVnegDENVlow; n = 65) immunity are shown in blue filled or empty squares, respectively. d Effect of JEV and DENV immunity in DF individuals to ZIKV-neutralizing antibody titers. Fisher’s exact test was performed in n = 60 with a p-value of 0.000013. All data are representative of three independent experiments. In (a–c), the black solid horizontal bars represent the geometric mean titers (GMT) of samples with SD analyzed within each group. FRμNT90 titer <10 was represented with 5 for graphic display and statistical analysis. Significant differences were tested using one-way ANOVA followed by Tukey’s multiple comparisons post-test, with levels of significance defined by ***P < 0.0001; **P < 0.001. Each point shows the mean of data from two independent experiments. DENV dengue virus, JEV Japanese encephalitis virus, ZIKV Zika virus, FRμNT focus-reduction microneutralization test, VLP virus-like particle, ANOVA analysis of variance, SD standard deviation.CC BY · PubMed Central
Fig. 2 Functional characterization of anti-ZIKV human monoclonal antibodies (huMAbs) from donor KH1891. a Neutralizing antibody titers of donor KH1891 against the prototype strains of the DENV serotypes 1–4, JEV, and ZIKV at less than 1 month and 18 months post-infection plasma collection, representing the early and late convalescent stages, respectively. Significant differences were tested using two-way ANOVA followed by Bonferroni post-tests from three independent experiments. ****P < 0.001. b–e Characterization of purified huMAbs, expressed as IgG1, isolated from donor KH1891. b The binding reactivity against virus-like particles (VLPs) and d in vitro neutralization activities of 10 purified huMAbs against DENV-1 to -4, JEV, and ZIKV were measured by ELISA and FRμNT, respectively. Each VLP was properly titrated to obtain equimolar concentrations per well before use in antigen-capture ELISAs. Each point shows the mean ± SD of data from two and three independent experiments for binding ELISA and microneutralization assays, respectively. A nonlinear curve was generated for all antibody dilution series for all of the assays. The corresponding antibody identities are shown in the box. c, e Heatmap of VLP-binding profile (shades of blue) (c) and of neutralization profile (shades of red) (e) of the 11 huMAbs against recombinantly produced DENV-1 to -4, JEV, and ZIKV VLPs and prototype strains of DENVs, JEV, and ZIKV virus particles, respectively. The heatmaps were generated based on the minimal binding concentration (or endpoint titer values) against each VLP and the IC50 values for each virion; exact numerical values expressed in μg/mL are shown in each box. Each huMAb was tested in duplicates for each of the binding ELISA assays, while FRμNT data were representative of three independent experiments. A purified human IgG1 was used as an isotype control in all assays. Data are representative of three independent experiments. f, g FRμNT50 (μg/mL) values of K8b (f) and K5 (g) for neutralization of the prototype strains of the four DENV serotypes, JEV, and ZIKV. Each point shows the mean ± SD of data from three independent experiments. The geometric mean inhibitory concentration (μg/mL) at 50% (IC50) is indicated in parenthesis. DENV dengue virus, JEV Japanese encephalitis virus, ZIKV Zika virus, ELISA enzyme-linked immunosorbent assay, FRμNT focus-reduction microneutralization test, VLP virus-like particle, ANOVA analysis of variance, SD standard deviation.
Fig. 2 Functional characterization of anti-ZIKV human monoclonal antibodies (huMAbs) from donor KH1891. a Neutralizing antibody titers of donor KH1891 against the prototype strains of the DENV serotypes 1–4, JEV, and ZIKV at less than 1 month and 18 months post-infection plasma collection, representing the early and late convalescent stages, respectively. Significant differences were tested using two-way ANOVA followed by Bonferroni post-tests from three independent experiments. ****P < 0.001. b–e Characterization of purified huMAbs, expressed as IgG1, isolated from donor KH1891. b The binding reactivity against virus-like particles (VLPs) and d in vitro neutralization activities of 10 purified huMAbs against DENV-1 to -4, JEV, and ZIKV were measured by ELISA and FRμNT, respectively. Each VLP was properly titrated to obtain equimolar concentrations per well before use in antigen-capture ELISAs. Each point shows the mean ± SD of data from two and three independent experiments for binding ELISA and microneutralization assays, respectively. A nonlinear curve was generated for all antibody dilution series for all of the assays. The corresponding antibody identities are shown in the box. c, e Heatmap of VLP-binding profile (shades of blue) (c) and of neutralization profile (shades of red) (e) of the 11 huMAbs against recombinantly produced DENV-1 to -4, JEV, and ZIKV VLPs and prototype strains of DENVs, JEV, and ZIKV virus particles, respectively. The heatmaps were generated based on the minimal binding concentration (or endpoint titer values) against each VLP and the IC50 values for each virion; exact numerical values expressed in μg/mL are shown in each box. Each huMAb was tested in duplicates for each of the binding ELISA assays, while FRμNT data were representative of three independent experiments. A purified human IgG1 was used as an isotype control in all assays. Data are representative of three independent experiments. f, g FRμNT50 (μg/mL) values of K8b (f) and K5 (g) for neutralization of the prototype strains of the four DENV serotypes, JEV, and ZIKV. Each point shows the mean ± SD of data from three independent experiments. The geometric mean inhibitory concentration (μg/mL) at 50% (IC50) is indicated in parenthesis. DENV dengue virus, JEV Japanese encephalitis virus, ZIKV Zika virus, ELISA enzyme-linked immunosorbent assay, FRμNT focus-reduction microneutralization test, VLP virus-like particle, ANOVA analysis of variance, SD standard deviation.CC BY · PubMed Central
Fig. 3 Defining the human Ig genes and properties of broadly neutralizing ZIKV-CR huMAbs. a, c Pie plots showing immunoglobulin heavy (a) and kappa light chain (c) gene family utilization and distribution of ZIKV-CR neutralizing huMAbs isolated from KH1891. The proportion of IGHV and IGκV gene usage is reported beside the different color schemes. b, d Gene family assignments for the 10 purified IgG1 B cell clones from KH1891. IMGT was used to assign the germline reference sequence for IGHV (b) and IGκV (d) and the relative similarity with the germline clonotype expressed in %. e Relative percentage of somatic hypermutation (%SHM) in the paired IGHV and IGκV genes of ZIKV-CR huMAbs. f Comparison of the V-J genes and alleles of heavy and light chains of ZIKV-CR huMAbs. Values highlighted in red show increased SHM (>20%) relative to the germline gene family. ZIKV Zika virus.
Fig. 3 Defining the human Ig genes and properties of broadly neutralizing ZIKV-CR huMAbs. a, c Pie plots showing immunoglobulin heavy (a) and kappa light chain (c) gene family utilization and distribution of ZIKV-CR neutralizing huMAbs isolated from KH1891. The proportion of IGHV and IGκV gene usage is reported beside the different color schemes. b, d Gene family assignments for the 10 purified IgG1 B cell clones from KH1891. IMGT was used to assign the germline reference sequence for IGHV (b) and IGκV (d) and the relative similarity with the germline clonotype expressed in %. e Relative percentage of somatic hypermutation (%SHM) in the paired IGHV and IGκV genes of ZIKV-CR huMAbs. f Comparison of the V-J genes and alleles of heavy and light chains of ZIKV-CR huMAbs. Values highlighted in red show increased SHM (>20%) relative to the germline gene family. ZIKV Zika virus.CC BY · PubMed Central
Fig. 4 Mapping of target flavivirus epitopes of huMAb K8b. a Structure-based strategy and workflow for the epitope mapping of K8b using mature DENV-2 virus-like particles (mD2VLP). AlphaFold was used to predict the three-dimensional (3D) structure of huMAb K8b expressed as IgG1. K8b-IgG1 was utilized to form an immune complex with mD2VLPs and 3D reconstructed by cryo-EM. The derived structure was further improved by MODELLER to predict the key epitopes and build epitope models. The antibody and apparatus icons were sourced icons and created with biorender.com. b AlphaFold model structure for K8b-IgG1 with coloration based on model confidence. The complementarity determining regions (CDRs) were indicated. c The cryo-EM structure of K8b-IgG1 and mD2VLP complex revealed the arrangement of E glycoprotein at T = 1 icosahedral symmetry. In the configuration, 60 F(ab) molecules (depicted in blue) were bound to 60 envelope proteins on the VLP surface (shown in cyan), while the Fc region was not resolved in the structure. d Fitting the predicted structure of K8b-IgG1 into cryo-EM density maps is presented, with contour levels of 3.7 σ (left) and 1.2 σ (middle). Two perspectives are shown: side view (d, left and middle) and top view (d, right). The separation of variable domains from the constant domain was clearly defined in the density map at higher contour levels, as indicated by the arrows. The heavy and light chains of K8b-IgG1 were shown in purple and cyan, respectively. Heavy and light chain pairs of K8b-IgG1 bound to E proteins at the inter-dimer surfaces. Densities of F(ab) pairs of K8b-IgG1 were shaded in gray. The residues on the E protein interacting with the heavy and light chains of F(ab) were shown by purple and cyan spheres, respectively. Envelope domains, E-DI, E-DII, and E-DIII, are colored in red, yellow, and blue, respectively. The triangle represents an asymmetric unit. e A flexible refinement of immune complex structure into cryo-EM map showing ten conformers of K8b-IgG1 predicted by MODELLER. The heavy (H) and light (L) chain variable domains are represented by pink and cyan ribbons, respectively. The mD2VLP is shown as a yellow surface, and the EM map (0.1 σ) is represented as black meshes. The cross-correlation function (CCF) is 0.72. For (d) and (e), a scale bar of 5 nm is shown. f Interacting or contact residues between the E proteins on an mD2VLP and the heavy or light chain of K8b-IgG1, including the equivalent residues (and their AA positions) in other flaviviruses, predicted by MODELLER. g A representative amino acid sequence alignment of the E proteins among prototype flaviviruses including selected DENV serotypes, JEV, and ZIKV using Clustal Omega and visualized by JalView 2.11.1.4. Potential binding sites of K8b are highlighted in boxes: bc loop (magenta), ED I/II linker (cyan), hi loop (green), ED I/III linker (blue), and DE loop (flesh). Residues in bold and in red are surface-exposed (solvent-accessible surface area, QSASA > 0.30). Sequence identities were normalized by aligned length, while the positions of the amino acids in E were labeled according to ZIKV PRVABC59. The shaded residues show 100% conservancy among the selected flavivirus genera. Above the sequences, the arrows indicate β-strands, rectangles represent the helices, and lines show the spanning loops and strands in the E structure. The E domains I, II, and III are represented by arrows and lines in red, yellow, and blue, respectively. h, i Binding ELISA reactivity profile of purified K8b-IgG1 (h) and K5-IgG1 (i) at 10 and 1.0 μg/mL antibody concentrations against seven single-site mutants relative to the wild-type ZIKV VLP in ELISA. The consensus amino acid residues in the bc loop (ZIKV R73, T76, and D87), ED I/II linker (ZIKV R193), hi loop (ZIKV T231), ED I/III linker (ZIKV G302), and ED III DE loop (ZIKV T366) were mutated to alanine. For (h, i), the statistical differences were determined by two-way ANOVA with Tukey’s multiple comparisons test relative to the binding reactivity to the ZIKV WT VLP, and are defined as significant at: *P < 0.05, **P < 0.001, and ***P < 0.0001. The binding reactivity ELISA data shown in (h, i) are representative of four independent experiments, expressed as geometric means ± SD. DENV dengue virus, JEV Japanese encephalitis virus, ZIKV Zika virus, ELISA enzyme-linked immunosorbent assay, FRμNT focus-reduction microneutralization test, ANOVA analysis of variance, SD standard deviation.
Fig. 4 Mapping of target flavivirus epitopes of huMAb K8b. a Structure-based strategy and workflow for the epitope mapping of K8b using mature DENV-2 virus-like particles (mD2VLP). AlphaFold was used to predict the three-dimensional (3D) structure of huMAb K8b expressed as IgG1. K8b-IgG1 was utilized to form an immune complex with mD2VLPs and 3D reconstructed by cryo-EM. The derived structure was further improved by MODELLER to predict the key epitopes and build epitope models. The antibody and apparatus icons were sourced icons and created with biorender.com. b AlphaFold model structure for K8b-IgG1 with coloration based on model confidence. The complementarity determining regions (CDRs) were indicated. c The cryo-EM structure of K8b-IgG1 and mD2VLP complex revealed the arrangement of E glycoprotein at T = 1 icosahedral symmetry. In the configuration, 60 F(ab) molecules (depicted in blue) were bound to 60 envelope proteins on the VLP surface (shown in cyan), while the Fc region was not resolved in the structure. d Fitting the predicted structure of K8b-IgG1 into cryo-EM density maps is presented, with contour levels of 3.7 σ (left) and 1.2 σ (middle). Two perspectives are shown: side view (d, left and middle) and top view (d, right). The separation of variable domains from the constant domain was clearly defined in the density map at higher contour levels, as indicated by the arrows. The heavy and light chains of K8b-IgG1 were shown in purple and cyan, respectively. Heavy and light chain pairs of K8b-IgG1 bound to E proteins at the inter-dimer surfaces. Densities of F(ab) pairs of K8b-IgG1 were shaded in gray. The residues on the E protein interacting with the heavy and light chains of F(ab) were shown by purple and cyan spheres, respectively. Envelope domains, E-DI, E-DII, and E-DIII, are colored in red, yellow, and blue, respectively. The triangle represents an asymmetric unit. e A flexible refinement of immune complex structure into cryo-EM map showing ten conformers of K8b-IgG1 predicted by MODELLER. The heavy (H) and light (L) chain variable domains are represented by pink and cyan ribbons, respectively. The mD2VLP is shown as a yellow surface, and the EM map (0.1 σ) is represented as black meshes. The cross-correlation function (CCF) is 0.72. For (d) and (e), a scale bar of 5 nm is shown. f Interacting or contact residues between the E proteins on an mD2VLP and the heavy or light chain of K8b-IgG1, including the equivalent residues (and their AA positions) in other flaviviruses, predicted by MODELLER. g A representative amino acid sequence alignment of the E proteins among prototype flaviviruses including selected DENV serotypes, JEV, and ZIKV using Clustal Omega and visualized by JalView 2.11.1.4. Potential binding sites of K8b are highlighted in boxes: bc loop (magenta), ED I/II linker (cyan), hi loop (green), ED I/III linker (blue), and DE loop (flesh). Residues in bold and in red are surface-exposed (solvent-accessible surface area, QSASA > 0.30). Sequence identities were normalized by aligned length, while the positions of the amino acids in E were labeled according to ZIKV PRVABC59. The shaded residues show 100% conservancy among the selected flavivirus genera. Above the sequences, the arrows indicate β-strands, rectangles represent the helices, and lines show the spanning loops and strands in the E structure. The E domains I, II, and III are represented by arrows and lines in red, yellow, and blue, respectively. h, i Binding ELISA reactivity profile of purified K8b-IgG1 (h) and K5-IgG1 (i) at 10 and 1.0 μg/mL antibody concentrations against seven single-site mutants relative to the wild-type ZIKV VLP in ELISA. The consensus amino acid residues in the bc loop (ZIKV R73, T76, and D87), ED I/II linker (ZIKV R193), hi loop (ZIKV T231), ED I/III linker (ZIKV G302), and ED III DE loop (ZIKV T366) were mutated to alanine. For (h, i), the statistical differences were determined by two-way ANOVA with Tukey’s multiple comparisons test relative to the binding reactivity to the ZIKV WT VLP, and are defined as significant at: *P < 0.05, **P < 0.001, and ***P < 0.0001. The binding reactivity ELISA data shown in (h, i) are representative of four independent experiments, expressed as geometric means ± SD. DENV dengue virus, JEV Japanese encephalitis virus, ZIKV Zika virus, ELISA enzyme-linked immunosorbent assay, FRμNT focus-reduction microneutralization test, ANOVA analysis of variance, SD standard deviation.CC BY · PubMed Central
Fig. 5 Generation and functional characterization of purified K8b-IgG1, K8b-F(ab’)2, and K8b-F(ab). a Schematic representation of the designed gene constructs coding for full-length human IgG1, F(ab’)2, and F(ab) encoding the Ig variable heavy chain genes of K8b (K8b-VH), and hence termed as pVCHIg-hG1-K8b, pVCHIg-K8b-F(ab’)2-His and pVCHIg-K8b-F(ab)-His, respectively. The schematic diagram of the expression vector, pVCLIg-hκ, encoding the variable Ig kappa light chain domain of K8b (K8b-Vκ) is also shown in the upper right panel. Each of the molecules was expressed via co-transfection using the kappa light chain expressing plasmid, pVCLIg-hκ-K8b. Inset shows the detailed schematic map of the human IgƔ constant domains 1 (CH1) and 2 (CH2), including the IgG1 hinge regions and the actual location where cysteine (Cys) was introduced for increased thermal stability. CH1–3, human IgƔ constant domains 1–3; Cκ, human Igκ constant domain; 8x-His, poly-Histidine tag; SP, signal peptide; H, hinge; TGA, stop codon are highlighted. b SDS-PAGE of purified human monoclonal antibody, K8b, expressed as full-length IgG1, F(ab), and F(ab’)2 molecules visualized under nonreducing conditions. Red arrow marks show the dimeric species of K8b-IgG1 at 150 kDa, monovalent K8b-F(ab) at 48 kDa, and the bivalent K8b-F(ab’)2 at 110 kDa. The molecular mass of the protein marker is indicated on the left side, lane 1, for each gel panel. c Electrophoretic mobility of purified K8b-F(ab’)2 after size-exclusion chromatography (SEC) showing the separation of K8b-F(ab’)2 and K8b-F(ab) molecules from the pooled pre-SEC K8b-F(ab’)2 fractions in lane 1. Lanes 2 and 3 show the non-reduced form of K8b-F(ab’)2 post-SEC and lane 4 shows the K8b-F(ab) molecules post-SEC purity separation. The molecular mass of the protein marker is indicated on the right side, lane 5. d Cartoon representation of K8b IgG1, F(ab), and F(ab’)2 molecules. e Neutralization profile of K8b expressed as full-length IgG1, F(ab), and F(ab’)2. Prototype virus strains of DENV-1 to -4, JEV, and ZIKV were used for the FRμNT. f Binding profile of K8b expressed as full-length IgG1, F(ab), and F(ab’)2. Each VLP was properly titrated to obtain equimolar concentrations per well before use in antigen-capture ELISAs. In (e) and (f), each point shows the mean ± SD of data from two independent experiments. A nonlinear curve was generated for all antibody dilution series for all of the assays. DENV dengue virus, JEV Japanese encephalitis virus, ZIKV Zika virus, ELISA enzyme-linked immunosorbent assay, FRμNT focus-reduction microneutralization test, SD standard deviation, SASA solvent-accessible surface area.
Fig. 5 Generation and functional characterization of purified K8b-IgG1, K8b-F(ab’)2, and K8b-F(ab). a Schematic representation of the designed gene constructs coding for full-length human IgG1, F(ab’)2, and F(ab) encoding the Ig variable heavy chain genes of K8b (K8b-VH), and hence termed as pVCHIg-hG1-K8b, pVCHIg-K8b-F(ab’)2-His and pVCHIg-K8b-F(ab)-His, respectively. The schematic diagram of the expression vector, pVCLIg-hκ, encoding the variable Ig kappa light chain domain of K8b (K8b-Vκ) is also shown in the upper right panel. Each of the molecules was expressed via co-transfection using the kappa light chain expressing plasmid, pVCLIg-hκ-K8b. Inset shows the detailed schematic map of the human IgƔ constant domains 1 (CH1) and 2 (CH2), including the IgG1 hinge regions and the actual location where cysteine (Cys) was introduced for increased thermal stability. CH1–3, human IgƔ constant domains 1–3; Cκ, human Igκ constant domain; 8x-His, poly-Histidine tag; SP, signal peptide; H, hinge; TGA, stop codon are highlighted. b SDS-PAGE of purified human monoclonal antibody, K8b, expressed as full-length IgG1, F(ab), and F(ab’)2 molecules visualized under nonreducing conditions. Red arrow marks show the dimeric species of K8b-IgG1 at 150 kDa, monovalent K8b-F(ab) at 48 kDa, and the bivalent K8b-F(ab’)2 at 110 kDa. The molecular mass of the protein marker is indicated on the left side, lane 1, for each gel panel. c Electrophoretic mobility of purified K8b-F(ab’)2 after size-exclusion chromatography (SEC) showing the separation of K8b-F(ab’)2 and K8b-F(ab) molecules from the pooled pre-SEC K8b-F(ab’)2 fractions in lane 1. Lanes 2 and 3 show the non-reduced form of K8b-F(ab’)2 post-SEC and lane 4 shows the K8b-F(ab) molecules post-SEC purity separation. The molecular mass of the protein marker is indicated on the right side, lane 5. d Cartoon representation of K8b IgG1, F(ab), and F(ab’)2 molecules. e Neutralization profile of K8b expressed as full-length IgG1, F(ab), and F(ab’)2. Prototype virus strains of DENV-1 to -4, JEV, and ZIKV were used for the FRμNT. f Binding profile of K8b expressed as full-length IgG1, F(ab), and F(ab’)2. Each VLP was properly titrated to obtain equimolar concentrations per well before use in antigen-capture ELISAs. In (e) and (f), each point shows the mean ± SD of data from two independent experiments. A nonlinear curve was generated for all antibody dilution series for all of the assays. DENV dengue virus, JEV Japanese encephalitis virus, ZIKV Zika virus, ELISA enzyme-linked immunosorbent assay, FRμNT focus-reduction microneutralization test, SD standard deviation, SASA solvent-accessible surface area.CC BY · PubMed Central
Fig. 6 Neutralization antibody profile of murine sera and murine monoclonal antibodies (mAbs) generated from various DENV-2 and JEV VLP prime-boost immunizations. a BALB/c mice (n = 5) were primed and boosted at 0 and 28 days, respectively, with various combinations of mature DENV-2 VLP (mD2VLP) and JEV VLP antigens. Mice were bled at 84 days post-vaccination, and individual serum specimens were evaluated for 50% focus-reduction microneutralization (FRµNT50) activity against DENV, JEV, and ZIKV. The bar plots show GMT ± SD for each group. The p values (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) were determined on log-transformed data using one-way ANOVA with Tukey’s multiple comparisons post-test. b Schedule of boost and myeloma fusion experiments performed in representative mice from the homologous JEV VLP prime-boost immunization. c, d Representative mice (JEV FRµNT50 > 120) from JEV prime-boost immunization were further boosted for three consecutive times (112–114 days post-vaccination) with JEV VLP (c) or mD2VLP (d) and the splenocytes were harvested two days after the last boost and then individually fused with myeloma cells for hybridoma generation. Culture supernatants from selected polyclonal hybridomas were tested for positive VLP ELISA reactivity (P/N ratio ≥ 2) and further subcloned by limiting dilution to isolate monoclonal cells. c 71% of the 52 JEV-specific monoclones isolated from 3D2 polyclone secreted JEV-neutralizing mAbs with varying potencies at 50% inhibitory concentration (IC50, µg/mL) of unpurified supernatant. c Right: Pie plot showing murine monoclones selected from polyclonal hybridomas and the distribution of 52 type-specific 3D2 mAbs with varying breadth of neutralization. d A total of 36 monoclones were isolated from 5D5 polyclone. Percentages represent the proportions of secreted mAbs according to their breadth of cross-neutralization within the different serocomplexes. e Pie plot showing murine monoclones selected from polyclonal hybridomas and the distribution of 36 cross-neutralizing 5D5 monoclonal antibodies. The relative proportion of monoclones with varying breadth of neutralization is reported beside the different color schemes. f Heatmap of DENV, JEV, and ZIKV IC50 (µg/mL, unpurified supernatant) neutralization titers of the 36 cross-reactive mAbs. Data are representative of two independent experiments. g Distribution of immunoglobulin (IgG) isotypes identified from hybridoma screening. DENV dengue virus, JEV Japanese encephalitis virus, ZIKV Zika virus, VLP virus-like particle, ELISA enzyme-linked immunosorbent assay, GMT geometric mean titer, ANOVA analysis of variance, SD standard deviation.
Fig. 6 Neutralization antibody profile of murine sera and murine monoclonal antibodies (mAbs) generated from various DENV-2 and JEV VLP prime-boost immunizations. a BALB/c mice (n = 5) were primed and boosted at 0 and 28 days, respectively, with various combinations of mature DENV-2 VLP (mD2VLP) and JEV VLP antigens. Mice were bled at 84 days post-vaccination, and individual serum specimens were evaluated for 50% focus-reduction microneutralization (FRµNT50) activity against DENV, JEV, and ZIKV. The bar plots show GMT ± SD for each group. The p values (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) were determined on log-transformed data using one-way ANOVA with Tukey’s multiple comparisons post-test. b Schedule of boost and myeloma fusion experiments performed in representative mice from the homologous JEV VLP prime-boost immunization. c, d Representative mice (JEV FRµNT50 > 120) from JEV prime-boost immunization were further boosted for three consecutive times (112–114 days post-vaccination) with JEV VLP (c) or mD2VLP (d) and the splenocytes were harvested two days after the last boost and then individually fused with myeloma cells for hybridoma generation. Culture supernatants from selected polyclonal hybridomas were tested for positive VLP ELISA reactivity (P/N ratio ≥ 2) and further subcloned by limiting dilution to isolate monoclonal cells. c 71% of the 52 JEV-specific monoclones isolated from 3D2 polyclone secreted JEV-neutralizing mAbs with varying potencies at 50% inhibitory concentration (IC50, µg/mL) of unpurified supernatant. c Right: Pie plot showing murine monoclones selected from polyclonal hybridomas and the distribution of 52 type-specific 3D2 mAbs with varying breadth of neutralization. d A total of 36 monoclones were isolated from 5D5 polyclone. Percentages represent the proportions of secreted mAbs according to their breadth of cross-neutralization within the different serocomplexes. e Pie plot showing murine monoclones selected from polyclonal hybridomas and the distribution of 36 cross-neutralizing 5D5 monoclonal antibodies. The relative proportion of monoclones with varying breadth of neutralization is reported beside the different color schemes. f Heatmap of DENV, JEV, and ZIKV IC50 (µg/mL, unpurified supernatant) neutralization titers of the 36 cross-reactive mAbs. Data are representative of two independent experiments. g Distribution of immunoglobulin (IgG) isotypes identified from hybridoma screening. DENV dengue virus, JEV Japanese encephalitis virus, ZIKV Zika virus, VLP virus-like particle, ELISA enzyme-linked immunosorbent assay, GMT geometric mean titer, ANOVA analysis of variance, SD standard deviation.CC BY · PubMed Central

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Metadata

Authors
Salem, G., Galula, J., Wu, S.-R., Liu, J., Chen, Y.-H., Wang, W.-H., Wang, S.-F., Song, C.-S., Chen, F.-C., Wang, C.-Y., & Chao, D.-Y.
Affiliations
  • National Chung Hsing University (TW)
  • National Cheng Kung University (TW)
  • National Sun Yat-sen University (TW)
  • Kaohsiung Medical University (TW)
  • Agency for Science, Technology and Research
Corresponding author
Chao, Day-Yu
ORCID
Venue
Communications Biology
Publisher
Springer Science and Business Media LLC
Published
2023-06-07
Volume
7
Issue
1
Pages
15-15
ISSN
2399-3642 (ISSN-L)
DOI
10.21203/rs.3.rs-2969700/v1
PMC
PMC10808242
OpenAlex
W4391168722
Cited by
14
References
1
Citations by year
2026: 7 · 2025: 5 · 2024: 2
FWCI
5.77
Type
journal article
Language
en
Open access
Licence
Locations
  • Communications Biology (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Academia Sinica
  • Ministry of Science and Technology, Taiwan (MOST110-2313-B005-041)
  • National Cheng Kung University
Topics
  • Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Mosquito-borne diseases and control
  • Health Sciences › Medicine › Infectious Diseases › Viral Infections and Vectors
  • Health Sciences › Medicine › Infectious Diseases › SARS-CoV-2 and COVID-19 Research
Keywords
Zika virus, Japanese encephalitis virus, dengue virus, cross-reactive antibodies, monoclonal antibodies, humoral immunity, flavivirus cross-reactivity, somatic hypermutation, quaternary epitope, heterologous immunity
MeSH terms
Zika Virus Infection, Zika Virus, Animals, Antibodies, Monoclonal, Dengue, Encephalitis Virus, Japanese, Humans, Cryoelectron Microscopy, Mice

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