Fig 1.
Lead and F108A ZIKV VLP production and Transmission Electron Microscopy (TEM).
(A) Western blot of supernatant samples collected on days 2, 3, 4, and 5 days post-transfection (p.t.) from cells transfected with a plasmid containing either the lead or F108A ZIKV VLP cassette. Proteins were stained for E using a mouse ZIKV-specific mAb (MyBiosource, cat#mbs568050). (B) Transmission electron micrographs of purified lead (left panel) and F108A VLPs (right panel) negatively stained with uranyl acetate. The white scale bar depicted on images is 50nm long.
Fig 2.
Comparison of the maturation state of lead and F108A particles by prM/M protein detection and E epitope mapping.
(A) SDS-PAGE followed by Coomassie protein staining of 500ng and 1μg of purified lead and F108A ZIKV VLPs. Molecular weight ladders on this gel are the Novex Sharp (NS; Invitrogen, cat#57318, molecular weights for this ladder shown in kDa) and the Super Signal (SS; Thermo, cat#847868) (B) SDS-PAGE followed by immunoblotting analysis of 500ng and 1μg of purified lead and F108A ZIKV VLPs. An anti-prM polyclonal Ab against ZIKV (Genetex, cat#gtx133305) was used to detect VLP proteins. (C) Binding of each mAb to F108A VLP relative to lead VLP measured by Biolayer Interferometry. Binding was measured at 100 seconds (ZIKV-116, ZV67, IM-79), 250 seconds (ZIKV-117, ZV48, LM-081) or both 100 and 250 seconds (ZIKV-117). For ZIKV-117, only the highest F108A concentration tested fell on the standard curves for the 100 and 250-second measurements, so both are shown. For each Ab, 2 or 4 F108A VLP dilutions fell on the lead VLP standard curve. For those dilutions, the F108A VLP concentration was calculated by least squares fit from the 4PL curve, and the ratios of bound F108A VLP to bound lead VLP were calculated. Refer to S1 Fig for standard curves. Symbols represent the ratios from individual measurements, bars and numbers at the top of each group represent geometric mean ratios, and an equivalence line representing equal mAb binding to F108A and lead is shown at 1.
Fig 3.
Vaccination and induction of lead and F108A VLP binding Abs.
(A) Vaccination and bleeding schedule of AG129 mice are shown. Mice (5 per group) were immunized IM on day 0 and day 28 with doses of 0.1, 1, or 10μg VLP and alum adjuvant. Mice were subsequently SC challenged with ZIKV (Nica 2–16 strain) on day 52. Bleeding was performed on Days 21 and 49 of the study prior to viral challenge. Day 49 sera were tested by sandwich ELISA using captured lead (B) or F108A (C) VLPs, and endpoint titers were calculated. Each symbol represents the mean endpoint dilution of two independent measurements for an individual animal, and the geometric mean titer is depicted by a horizontal bar. The dotted line is the limit of detection (LOD) of the assay, and sera with undetectable binding were assigned a value of one-half LOD (25). Anti-E ZIKV mouse serum (Zoonogen, cat#pa10049) was used as a positive control and to demonstrate binding availability of E for the VLP types. For each capture antigen, endpoint titers of the VLP groups were compared by one-way ANOVA. The symbol † denotes a mouse that succumbed after viral challenge. (D) Relative Binding is the ratio of the lead VLP ELISA endpoint titer divided by the F108A VLP ELISA endpoint titer for each individual sample. Group geometric mean ratios are shown on top, and an equivalence line was placed at y = 1. One-way ANOVA followed by Sidak’s multiple comparison tests for each of the three doses were performed. (E) Comparison of Relative Binding Antibody (bAb) for combined lead vs combined F108A VLP groups is shown. Combined group geometric mean ratios and significance level by unpaired t test are shown on top. Dose group of F108A VLP-immunized mice that succumbed later to viral challenge is indicated. Comparison of the combined groups was by Mann-Whitney U test (ns, not significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).
Fig 4.
Lead and F108A VLP induction of nAb after booster immunization. The vaccination schedule is shown in Fig 3A.
(A) NT50 is defined as the reciprocal dilution of serum that neutralizes 50% of reporter virus luminescence calculated using linear regression analysis. Each symbol represents the NT50 for an individual animal, and † indicates a lethal ZIKV infection. Horizontal bars represent the geometric mean titer (GMT) of each group. The dotted line is the limit of detection (LOD) of the assay, and sera with undetectable neutralization activity were assigned a value of one-half LOD (5). Statistical comparisons of NT50 data were performed using Kruskal-Wallis test followed by Dunn’s multiple comparisons tests (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant). All VLP groups were compared to the Alum-only group. Additional pairwise comparisons were performed between titers of lead and F108A groups at each dose, with significance level for each comparison shown above each bar. (B) Comparison of NT50 titers for combined lead VLP groups compared to combined F108A VLP groups. GMT, individual NT50 values, assay LOD, † designation, and significance value by Mann-Whitney U test are as in (A). (C) Ab quality assessment by the ratio of NT50 to ELISA titer to lead VLP are shown for each dose of lead or F108A VLP. Group geometric mean titer ratios are shown above each group, individual ratios are shown by a symbol, group means by a horizontal bar, lethal infections by the symbol †, and statistical significance by Kruskal-Wallis followed by Dunn’s multiple comparison tests for each of the three doses as in (A). (D) Ab quality assessment by NT50 to ELISA titer to lead VLP ratios are shown for combined lead and combined F108A groups. Group GMT ratios, individual ratios, lethal infections, and statistical significance levels by Mann-Whitney U test are as in (A).
Fig 5.
Protection afforded by VLP immunization as measured by weight loss and survival. The vaccination schedule is shown in Fig 3A.
(A) Weight changes from day 4 post-challenge of F108A VLP-immunized (blue), lead VLP-immunized (red), and control [naïve challenged (naïve), alum-only immunized and challenged (alum-only), and naïve unchallenged (uninfected)] mice. Each symbol corresponds to the mean cumulative weight change on days 4 to 20 for each mouse group (n = 5), and error bars indicate standard error of the mean (SEM). Statistical comparisons of weight change per day were performed by one-way ANOVA followed by Dunnett’s multiple comparisons tests between all groups each day. Significance levels between control and VLP-immunized groups relative to the Alum-only group are indicated in S4 Fig. (B) Day 20 post-challenge weight changes were combined for F108A and lead VLP groups, respectively. Statistical significances were obtained by performing an unpaired t-test and means for each combined VLP dose groups are indicated on top. (C) Survival of mice was assessed up to 20 days post-challenge.
Fig 6.
Immunization with lead, but not F108A VLP, significantly reduces RNAemia, which inversely correlates with nAb responses.
Animals were challenged with 5,000 FFU of ZIKV strain (Nica 2–16) SC at Day 52 as depicted in Fig 3A. (A) Viral load in serum was measured on day 2 post-challenge by RT-qPCR. Each symbol represents the log10 GE/mL for an individual mouse, horizontal bars and values above each group show group means, significance values compared to the Alum-only group are just above group mean values, and the bars and significance levels on top were obtained by comparing lead and F108A groups. The dotted line is the limit of detection (LOD) of the assay set based on the lowest log GE/mL detected (0.59 GE/mL), and the symbol † denotes a mouse with a lethal infection. Measurements below the LOD were assigned a value of half the LOD. Statistical comparisons were performed using Kruskal-Wallis test followed by Dunn’s multiple comparisons tests (ns, not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001). (B) Combined lead and combined F108A VLP groups were compared to the Alum-only group. Individual viral load values, assay LOD, and the symbol † designation are as in (A). (C) The relationships between NT50 and viral load (both log10 transformed) for the combined lead VLP groups and the combined F108A VLP groups were assessed by Spearman correlation analysis with resulting r and P values shown.
Fig 7.
Ab-dependent Enhancement in vitro assay.
(A-C) Fold enhancement of infection is represented for each serum dilution tested, ranging from 10-fold up to 2,430-fold. Fold-enhancement reflects the ratio of the percentage of cells infected with diluted serum-treated virus to medium only-treated virus. The calculated cutoff for ADE activity, shown by dot and dashed line, was calculated as the mean fold-enhancement values of the negative control groups (Alum-only, naïve, and uninfected) + 3 standard deviations (SD). Each symbol indicates the geometric mean of a dose group (5 mice), and error bars represent geometric SD. The three dose groups, 0.1μg (A), 1μg (B) and 10μg (C), were separated into different graphs in which lead and F108A VLP-immunized mice are compared. The Alum-only group is shown in each panel for comparative purposes. Statistical comparisons of enhancement data were performed at each dose using one-way ANOVA test followed by pairwise Sidak’s multiple comparisons between lead and F0108A groups at each serum dilution (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant). (D) Fold-enhancement of infection of cells infected with the 10-fold serum dilution. Each symbol represents the geometric mean of two independent measurements for an individual animal. Horizontal bars represent the geometric mean titer of each group. The symbol † denotes a mouse that succumbed after challenge. Statistical comparisons were performed using ANOVA test followed by Dunnett’s multiple comparisons tests with significance levels denoted as in (A-C) above. All VLP groups were compared to the Alum-only group. Additional pairwise comparisons were performed between titers of lead and F108A groups at each dose with significance level for each comparison shown above each bar.