Preclinical Development of a Novel Zika Virus-like Particle Vaccine in Combination with Tetravalent Dengue Virus-like Particle Vaccines

Abstract

Declared as a Public Health Emergency in 2016 by the World Health Organization (WHO), the Zika virus (ZIKV) continues to cause outbreaks that are linked to increased neurological complications. Transmitted mainly by Aedes mosquitoes, the virus is spread mostly amongst several tropical regions with the potential of territorial expansion due to environmental and ecological changes. The ZIKV envelope protein's domain III, crucial for vaccine development due to its role in receptor binding and neutralizing antibody targeting, was integrated into sterically optimized AP205 VLPs to create an EDIII-based VLP vaccine. To increase the potential size of domains that can be accommodated by AP205, two AP205 monomers were fused into a dimer, resulting in 90 rather than 180 N-/C- termini amenable for fusion. EDIII displayed on AP205 VLPs has several immunological advantages, like a repetitive surface, a size of 20-200 nm (another PASP), and packaged bacterial RNA as adjuvants (a natural toll-like receptor 7/8 ligand). In this study, we evaluated a novel vaccine candidate for safety and immunogenicity in mice, demonstrating its ability to induce high-affinity, ZIKV-neutralizing antibodies without significant disease-enhancing properties. Due to the close genetical and structural characteristics, the same mosquito vectors, and the same ecological niche of the dengue virus and Zika virus, a vaccine covering all four Dengue viruses (DENV) serotypes as well as ZIKV would be of significant interest. We co-formulated the ZIKV vaccine with recently developed DENV vaccines based on the same AP205 VLP platform and tested the vaccine mix in a murine model. This combinatory vaccine effectively induced a strong humoral immune response and neutralized all five targeted viruses after two doses, with no significant antibody-dependent enhancement (ADE) observed. Overall, these findings highlight the potential of the AP205 VLP-based combinatory vaccine as a promising approach for providing broad protection against DENV and ZIKV infections. Further investigations and preclinical studies are required to advance this vaccine candidate toward potential use in human populations.

Keywords: Zika virus; dengue virus; vaccine; virus-like particles.

Figure 1

The envelope protein domain III from the Zika virus was successfully incorporated into AP205 dimer VLPs by genetic fusion. (a) A schematic illustration of the vaccine design. Amino acid (Aa) sequence 299–407 of the envelope protein from the Zika virus was C-terminally fused to the AP205 dimer (consisting of two AP205 monomers) and expressed in a bacterial expression system where the VLPs self-assembled. (b) Electron microscopy (EM) of AP205-ZV. Scale bar 100 nm. (c) Agarose gel analysis for visualization of packed RNA in the expressed VLPs and correlating protein staining with InstantBlue^TM Comassie. M. DNA Ladder, 1. AP205-ZV Batch 1, 2. AP205-ZV Batch 2, 3. AP205 dimer. Products indicated in the red circles. (d) 12% SDS-PAGE. M. Protein Marker, 1. ZIKV EDIII protein, 2. AP205-ZV, 3. AP205 dimer. Bands were visualized with InstantBlue^TM Comassie stain.

Figure 2

The AP205-ZV fusion product can be detected by α-AP205 and α-ZIKV EDIII monoclonal antibodies, indicating proper expression and folding of the protein. (a) A 12% SDS-PAGE analysis for AP205-ZV production. M. Protein Marker, 1. AP205 dimer, 2. AP205-ZV. Products indicated in the red circles. (b) A Western blot specific for AP205. M. Protein Marker, 1. AP205 dimer, 2. AP205-ZV. Products indicated in the red boxes. (c) Binding of α-ZIKV EDIII monoclonal antibody to the EDIII or AP205-ZV. EDIII and AP205-ZV coated with a concentration of 1 µg/mL. Control coated with PBS. Statistical analysis (mean ± SEM) using one-way ANOVA. One representative of two similar experiments is shown. A p-value < 0.05 was considered statistically significant (**** p < 0.0001).

Figure 3

Immunization with AP205-ZV vaccine elicits a strong and humoral immune response of high avidity. (a) Vaccination regimen (prime on day 0 and boost on day 28, 20 μg per mouse, subcutaneous injection), bleeding time points, and vaccination groups. Created with Biorender.com. (b) ZV-specific IgG titer on days 0, 7, 14, 21, 28, 35, 42, 49, and 56 from mice vaccinated with AP205-ZV. For the control, days 0, 28, and 56 are shown for the group injected with AP205. Data measured by ELISA; LOG10 OD50 shown. (c) ZV-specific IgG titer from days 28 and 56 from mice vaccinated with AP205-ZV. After serum incubation, one plate was treated with PBS + 0.05% Tween 20 and the other plate with 7M urea in PBS + 0.5% Tween 20. LOG10 OD50 shown. (d) Avidity index (AI) of ZV-specific IgG titer from days 28 and 56 of the group vaccinated with AP205-ZV. Statistical analysis (mean ± SEM) using one-way ANOVA for (b,c) and paired t-test for (d). Vaccine groups, n = 6. One representative of 3 similar experiments is shown. A p-value < 0.05 was considered statistically significant (ns non-significant, * p < 0.05, **** p < 0.0001).

Figure 4

Vaccination with AP205-ZV induces IgG2c- and IgG2b-dominant Zika virus envelope protein domain III-specific IgG subclass response. ZV-specific IgG subclass titer of day 42 from mice vaccinated with AP205-ZV measured by ELISA; LOG10 OD50 shown. Statistical analysis (mean ± SEM) using Student’s t-test. Vaccine group, n = 6. One representative of two similar experiments is shown. A p-value < 0.05 was considered statistically significant (ns non-significant, **** p < 0.0001).

Figure 5

Abs induced by vaccination with AP205-ZV can recognize DENV EDIII proteins but with low avidity. (a) DV-specific IgG titer of days 28 and 49 from mice vaccinated with AP205-ZV measured by ELISA; LOG10 OD50 shown. After serum incubation, one plate was treated with PBS + 0.05% Tween 20 and the other plate with 7M urea in PBS + 0.05% Tween 20. (b) Avidity index (AI) of DV-specific induced IgG titer of days 28 and 49 of the group vaccinated with AP205-ZV. Statistical analysis (mean ± SEM) using Student’s t-test. Vaccine groups, n = 6. One representative of two similar experiments is shown. A p-value < 0.05 was considered statistically significant (ns non-significant, * p < 0.05, *** p < 0.001, **** p < 0.0001).

Figure 6

Vaccination against ZIKV and DENV induces strong IgG responses against EDIII proteins. (a) Vaccination regimen (prime on day 0 and boost on day 28, 20 μg of total VLPs per mouse, subcutaneous injection), bleeding time points, and vaccination group. Figure created with Biorender.com. (b) DV1- (c) DV2- (d) DV3-, (e) DV4-, and (f) ZV-specific IgG titer on days 0, 7, 14, 21, 28, 35, 42, and 49 measured by ELISA; LOG10 OD50 shown. Statistical analysis (mean ± SEM) using Student’s t-test. Group 1, n = 6. One representative of two similar experiments is shown. A p-value < 0.05 was considered statistically significant, ** p < 0.01, **** p < 0.0001).

Figure 7

Vaccination against ZIKV and all four DENV serotypes induces high-avidity antibodies after two doses. (a) DV1-, (b) DV2-, (c) DV3-, (d) DV4-, and (e) ZV-specific IgG titer of days 28 and 49 from group 1 (see Figure 6a) measured by ELISA; LOG10 OD50 shown. After serum incubation, one plate was treated with PBS + 0.05% Tween 20 and the other plate with 7M urea in PBS + 0.05% Tween 20. (f) DV1-, (g) DV2-, (h) DV3-, (i) DV4-, and (j) ZV-specific avidity index from group 1 from day 28 and day 49. Statistical analysis (mean ± SEM) using Student’s t-test. Group 1, n = 6. One representative of two similar experiments is shown. A p-value < 0.05 was considered statistically significant (* p < 0.05, ** p < 0.01, **** p < 0.0001).

Figure 8

AP205-ZV efficiently induces ZIKV-neutralizing antibodies. (a) Foci reduction neutralization 50 (FRNT50) value from the negative control (NC), AP205-ZV-induced serum, and AP205 VLP-induced control serum. The serum was tested for ZIKV neutralization. (b) Foci reduction neutralization 50 (FRNT50) value from the negative control (NC), AP205-ZV-induced serum, and AP205 VLP-induced control serum. The serum was tested for DENV-2 neutralization. The serum used was from day 49. Naïve serum was used as the negative control. Statistical analysis (mean ± SEM) using one-way ANOVA. Vaccine groups, n = 6. One representative of two similar experiments is shown. A p-value < 0.05 was considered statistically significant (ns non-significant, * p < 0.05, *** p < 0.001).

Figure 9

Co-formulated administration of AP205-ZV together with DENV VLPs induces antibodies able to neutralize all five targeted viruses. (a–e) Reduction in (a) DENV-1 infection, (b) DENV-2, (c) DENV-3, (d) DENV-4, (e) ZIKV by vaccine (DV1-AP205/AP205~DV2/DV3-AP205/AP205-DV4/AP205-ZV)-induced serum (black), positive control (red), and negative control (blue). Serum dilution is shown in Log values. Serum used from day 49. Naïve serum used as negative control. ATCC serum used as positive control. Statistical analysis (mean ± SEM) using one-way ANOVA for (a–e). Vaccine groups, n = 6. One representative of two similar experiments is shown. A p-value < 0.05 was considered statistically significant (ns non-significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 10

Antibodies induced by vaccination with DV1-AP205/AP205-DV4/AP205-ZV do not enhance DENV-2, and antibodies induced by AP205-ZV do not enhance ZIKV infection. (a) Fold change in DENV-2 infection of the serum induced by vaccination with DV1-AP205/AP205-DV4/AP205-ZV. Day 28 shown in black; day 49 shown in grey. Negative control (naïve serum) shown in blue. The serum dilution shown in Log values. (b) Fold change in DENV-2 infection of mouse 4G2 antibody used as a positive control. Antibody concentration shown in Log values. (c) Fold change in ZIKV infection of the serum induced by vaccination with AP205-ZV. Serum from day 49. Negative control (naïve serum) shown in blue; positive control (monkey serum) shown in red. The serum dilution shown in Log values. Statistical analysis (mean ± SEM) using one-way ANOVA. Vaccine group, n = 6. One representative of two similar experiments is shown. A p-value < 0.05 was considered statistically significant (* p < 0.05, ** p < 0.01).