Construction of a recombinant avipoxvirus expressing the env gene of Zika virus as a novel putative preventive vaccine

Abstract

Background: Zika virus (ZIKV) has been declared a public health emergency that requires development of an effective vaccine, as it might represent an international threat.

Methods: Here, two novel DNA-based (pVAXzenv) and fowlpox-based (FPzenv) recombinant putative vaccine candidates were constructed that contained the cPrME genes of ZIKV. The env gene inserted into the fowlpox vector was verified for correct transgene expression by Western blotting and by immunofluorescence in different cell lines. The production of virus-like particles as a result of env gene expression was also demonstrated by electron microscopy. BALB/c mice were immunosuppressed with dexamethasone and immunized following a prime-boost strategy in a heterologous protocol where pVAXzenv was followed by FPzenv, to evaluate the immunogenicity of the Env protein. The mice underwent a challenge with an epidemic ZIKV after the last boost.

Results: These data show that the ZIKV Env protein was correctly expressed in both normal human lung fibroblasts (MRC-5 cells) and green monkey kidney (Vero) cells infected with FPzenv, and that the transgene expression lasted for more than 2 weeks. After mucosal administration of FPzenv, the immunized mice showed specific and significantly higher humoral responses compared to the control mice. However, virus neutralizing antibodies were not detected using plaque reduction assays.

Conclusions: Although BALB/c mice appear to be an adequate model for ZIKV infection, as it mimics the natural mild infection in human beings, inadequate immune suppression seemed to occur by dexamethasone and different immune suppression strategies should be applied before challenge to reveal any protection of the mice.

Keywords: Electron microscopy; Fowlpox virus; Immune response; Prime–boost vaccination; Recombinant vaccines; Zika virus.

Fig. 1

Plasmids construction and in-vivo recombination. Two plasmids were constructed: pFPzenv as the recombination plasmid for in-vivo recombination and pVAXzenv as the expression plasmid to prime the experimental mice. pFPzenv contains the cPrME gene sequence of ZIKV (zenv) obtained by retro-transcription of a 2015 Brazilian isolate. This sequence includes the genes that encode for the tail portion of the capsid protein (c), the membrane precursor (Pr), the membrane protein (M) and the whole envelope protein (E). Homologous in-vivo recombination occurred in specific pathogen-free primary chick embryo cells after infection of FPwt and transfection of the pFPzenv recombination plasmid. Recombinant plaques were identified by autoradiography after hybridization with the [³²P]-labelled zenv probe and different positive clones were subjected to multiple cycles of plaque purification until one clone (FPzenv) was selected for correct expression

Fig. 2

In vitro characterization of FPzenv-mediated transgene expression and VLP formation. a Expression of the Env protein of ZIKV by the FP recombinants in Vero cells. Vero cells were infected by the FP recombinants and examined using Western blotting, to determine the Env protein expression. The Env protein was always detected both when using the monoclonal or the polyclonal antibodies after infection with either ZIKV (Z, lanes 2) or FPzenv (zenv, lanes 4). Mock infected cells (m, lanes 1) and cells infected with FP wild-type (wt, lanes 3) were used as negative controls. b Heterologous protein expression by immunofluorescence in the CEFs and the Vero and MRC-5 cells. Immunofluorescence of the infected cells was performed to determine the subcellular localization of the Env protein expressed by FPzenv. The Env protein was expressed mainly in the cytoplasm (2a-2b-2c), and the intensity of the fluorescence signals was generally lower in cells infected with the recombinant than in the same cells infected with ZIKV (3b-3c). ZIKV did not infect the CEFs (3a). No immunofluorescence was detected in the FP-wild-type-infected cells used as negative controls (1a-1b-1c). c Expression of the env transcripts over time by FPzenv in replication-restrictive Vero cells. After infection of the Vero cells with FPzenv, the expression of the transgene was evaluated by RT-PCR every 3 days, over 27 days. The expression levels for FPzenv transcripts (661 bp) remained up to day 18 p.i.. Amplification of β-actin mRNA (518 bp) is shown. d Electron microscopy. Vero cells were infected with FPzenv to verify production of virus-like particles (VLPs). Left. Some empty VLPs were seen (white arrows), as well as clusters of FPzenv recombinants corresponding to the viral inoculum (black arrows) and DNA viral factories (V); bar, 0.2 µm. Inset, VLPs enlargement; bar, 50 nm. Right. ZIKV-infected cells (black arrows) were used as the positive control, and clusters of virus particles (50 nm, black arrows) were seen inside the cytoplasm; bar, 0.2 µm

Fig. 3

a Immunization protocols. Two different vaccination regimens (control, G1; experimental, G2) were followed using 7 mice per group. Two DNA recombinants were used for priming (pVAXgp, pVAXzenv, respectively), and two viral recombinants expressing the same genes were used for the boost (FPgp, FPzenv, respectively). The DNAgp and FPgp recombinants that contained the HIV-1 gag/pro genes were used as irrelevant immunogens. Each plasmid was administered in vivo by electroporation (10 + 50 µg/recombinant/mouse), and each virus was administered subcutaneously or intranasally (1 × 10⁶ PFU/recombinant/mouse). The challenge with ZIKV was administered subcutaneously at 1 × 10⁵ PFU/mouse. The mice were bled before each immunization, just before the ZIKV challenge (T6) and at further times after the challenge. b Analysis of the humoral immune response. The anti-Env antibody response was determined by ELISA, where Vero cells were infected with FPzenv and then lysed, as the plate-bound antigen. Serum was obtained from all of the mice at different times before each immunization, as well as before and after the ZIKV challenge. Each line represents an individual animal. Total IgG ELISA titres are shown. An anti-ZIKV Env-specific binding antibody response was seen soon after vaccination (G2, T4). It can be noted that at 10 weeks postvaccination, after boosting the animals by the intranasal route, the antibody titer was significantly higher as compared to the control mice (G2 vs G1, T5; AUC, p < 0.05). OD₄₅₀ is expressed after subtraction of the T0 values for each mouse. c Neutralizing activity using 1:50 serum dilution. Viral neutralization activity was determined using for each animal the pre-immune serum (T0) and sera from bleedings after the last immunization (T6). No inhibition of viral infectivity was found. Plaque numbers did not decrease when using hyper-immune or pre-immune sera (T6 vs T0) in the experimental vs the control animals (G2 vs G1)