Induction of systemic and mucosal immune response against Zika virus by vaccination with non-infectious chimeric VLPs

Abstract

The Zika virus (ZIKV) causes acute febrile illness and can lead to complications such as Guillain-Barré syndrome and congenital disorders. As arbovirus outbreaks increase, vaccination becomes a crucial preventive strategy. Currently, no commercial vaccines are available for ZIKV, which is transmitted by mosquitoes and bodily fluids, underscoring the need for a safe vaccine that induces both systemic and mucosal immune responses. In this study, we present a ZIKV vaccine candidate utilizing virus-like particles (VLPs) technology combined with variant-specific surface proteins (VSP) from Giardia lamblia. Previous research demonstrated that these VSP act as effective adjuvants and are resistant to gastrointestinal degradation, expanding administration possibilities via orogastric routes in addition to the conventional subcutaneous route. To develop the immunogen, we engineered retrovirus-derived VLPs decorated with the ZIKV envelope glycoprotein (ZIKV-E) as the target antigen, incorporating VSPs on their surface. Immunocompetent Balb/c mice were immunized with VSP-VLPs ZIKV-E via oral and subcutaneous routes. Immune characterization revealed robust systemic and mucosal humoral responses, as well as a specific cellular activation. Moreover, a significant neutralizing capacity of serum antibodies was observed. These findings highlight the potential of the vaccine candidate to elicit a targeted immune response, achieved through different administration methods.

Keywords: Arbovirus; Mucosal response; Subunit vaccines; Zika virus.

Fig. 1

Schematic representation of the ZIKV immunogen and immunization protocol. (A) Organization of the genomic construct containing the signal peptide (SP), ectodomain Zika virus envelope (E), and the Vesicular Stomatitis Virus G glycoprotein transmembrane and cytosolic tail (VSVg TM-CT). (B) Overview of the experimental design in mice, including immunization, dosing and sampling schedules for oral and subcutaneous administration.

Fig. 2

Expression and assembly of VSP-VLPs ZIKV-E. (A) Fluorescence microscopy was conducted after transfecting VSP-expressing cells with the DNA constructs pZIKV-E and/or pGag. The expression of each specific protein is indicated at the top of each image, using the corresponding fluorescence color. Cells were analyzed for the expression of ZIKV-E and VSP proteins after incubating with the respective specific antibodies, followed by a fluorophore-conjugated secondary antibody. The expression of MLV-Gag was visualized by the fluorescence emitted from eYFP. In all fluorescence images, nuclei are labeled with DAPI (blue), and the yellow signal indicates co-expression. (B) Purified VSP-VLPs ZIKV-E were examined through transmission electron microscopy (TEM) and immunogold labeling, which confirmed the proper display of ZIKV-E (left), VSP (center) and MLV-Gag (right). Specific monoclonal antibodies were employed to identify the various components present in the particles. Immunogold-positive signals (black dots) corresponding to the specific markers are indicated by blue arrows.

Fig. 3

Detection of ZIKV-E from purified VSP-VLPs by Western blot. Purified VSP-VLPs ZIKV-E and VSP-VLPs without ZIKV-E (Ctrl) were resolved by SDS-PAGE, under reducing (5% β-ME) conditions, followed by immunodetection using anti-Zika envelope-specific antibody. The protein molecular weight marker (MW) is indicated on the left. The upper band (~ 58 kDa, red arrow) corresponds to the full-length ZIKV-E protein. A lower band (blue arrow), also recognised by the antibody, is marked as a putative truncated or degraded form of ZIKV-E.

Fig. 4

Induction of anti-ZIKV systemic humoral response in mice vaccinated with VSP-VLPs ZIKV-E. (A) Mice were vaccinated orally or subcutaneously with VSP-VLPs ZIKV-E and the specific anti-ZIKV IgG plus IgM were determined in sera 14 days after each dose. (B) Serum IgG plus IgM titer was evaluated at 42 days post immunization (d.p.i.) (C) Serum levels of IgG1 subclass were measured at 42 d.p.i. (D) Serum levels of IgG2a subclass were measured at 42 d.p.i. (E) Serum IgA levels were assessed at 42 d.p.i. n = 6 from two independent experiments for VSP-VLPs ZIKV-E vaccinated groups; n = 5 for mock group. Data are shown as means ± SEM. In the antibodies kinetic response (A) data were analyzed by two-way ANOVA and Bonferroni’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 respect to mock group at the same d.p.i.

Fig. 5

Specific anti-ZIKV mucosal response after VSP-VLPs ZIKV-E immunization. Mice were vaccinated orally or subcutaneously with VSP-VLPs ZIKV-E and the specific anti-ZIKV antibodies were determined in mucosal samples 14 days after the last dose (42 d.p.i.). (A) IgG + IgM levels in vaginal swabs were measured. (B) Specific IgA levels in vaginal swabs. (C) Specific IgA levels in faecal samples. n = 6 from two independent experiments for VSP-VLPs ZIKV-E vaccinated groups; n = 5 for mock group. Data are shown as means ± SEM. *p < 0.05, **p < 0.01.

Fig. 6

Induction of anti-ZIKV systemic cellular immune response in mice vaccinated with VSP-VLPs ZIKV-E. Balb/c mice were vaccinated orally or subcutaneously with VSP-VLPs ZIKV-E and the cellular immune response against ZIKV was evaluated 14 days after the last dose (42 d.p.i.) by the measurement of cytokines in splenocyte supernatants. n = 6 from two independent experiments for VSP-VLPs ZIKV-E vaccinated groups; n = 5 for mock group. Data are shown as means ± SEM. *p < 0.05, **p < 0.01.

Fig. 7

VSP-VLPs ZIKV-E immunization increases neutralizing antibodies. Balb/c mice were vaccinated orally or subcutaneously at days 0, 14 and 28 with VSP-VLPs ZIKV-E. Neutralizing antibody endpoint titers were determined in serum samples collected 14 days after the final dose using ZIKV-AR. The neutralization titer was defined as the serum dilution that reduces the cytopathic effect by 50% (MNT50) (A) or 80% (MNT80) (B). A total of n = 6 animals were analyzed from two independent experiments in the VSP-VLPs ZIKV-E vaccinated groups, while n = 5 animals were analyzed from the mock group. Data are shown as means ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001.