Zanotto et al. Virol J (2021) 18:50 https://doi.org/10.1186/s12985-021-01519-x RESEARCH Open Access Construction of a recombinant avipoxvirus expressing the env gene of Zika virus as a novel putative preventive vaccine Carlo Zanotto1, Francesca Paolini2, Antonia Radaelli1*† and Carlo De Giuli Morghen3† Abstract Background: Zika virus (ZIKV) has been declared a public health emergency that requires development of an efec- tive vaccine, as it might represent an international threat. Methods: Here, two novel DNA-based (pVAXzenv) and fowlpox-based (FPzenv) recombinant putative vaccine can- didates were constructed that contained the cPrME genes of ZIKV. The env gene inserted into the fowlpox vector was verifed for correct transgene expression by Western blotting and by immunofuorescence in diferent 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 fbroblasts (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 specifc and signifcantly 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 diferent immune suppression strategies should be applied before challenge to reveal any protection of the mice. Keywords: Zika virus, Recombinant vaccines, Fowlpox virus, Prime–boost vaccination, Immune response, Electron microscopy Highlights 1. A recombinant avipoxvirus was constructed to express the env gene of Zika virus 2. Novel putative recombinant vaccines were used in a prime–boost immunization regimen *Correspondence: [email protected] 3. Mucosal immunization enhances the humoral †Antonia Radaelli and Carlo De Giuli Morghen: Joint last authors immune response 1 Laboratory of Molecular Virology and Recombinant Vaccine Development, Department of Medical Biotechnologies and Translational Medicine, University of Milan, Via Vanvitelli 32, 20129 Milan, Italy Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zanotto et al. Virol J (2021) 18:50 Page 2 of 14 Background protect against both homologous and heterologous Zika virus (ZIKV) was frst isolated in 1947 from rhesus virus strains [32]. In particular, avipox viruses have macaques in the Zika Forest Research Station of Uganda, taken on an important role in the development of novel and was then identifed in Aedes africanus mosquitoes recombinant immunogens, as they do not replicate in from the same forest [1]. ZIKV belongs to the Flavivi- most mammalian cells, although permissive for entry rus genus of the Flaviviridae family, which includes the and transgene expression [33, 34]. Moreover, avipox- Dengue, Yellow fever, West Nile, Japanese encephalitis, virus vectors do not cause the undesired side efects and Tick-borne encephalitis viruses, which have single- induced by vaccinia-based recombinants, and they are stranded, positive-sense RNA genomes of around 11 kb not neutralized in individuals who have already been [2]. In particular, ZIKV shows antibody cross-reactivity immunized against smallpox [35]. In particular, Fowl- to the four serotypes of Dengue virus [3]. Although ZIKV pox (FP)-based recombinants can express foreign anti- might also be sexually and vertically transmitted [4, 5], gens for long periods and induce protective immunity bites by Aedes aegypti and Aedes albopictus mosquitos in mammals [36–38]. represent the main route of ZIKV infection in humans Te structural proteins encoded by ZIKV after post- [6]. translational processing of the RNA genome include the Human infections were initially reported in Nigeria in capsid (c), the membrane precursor or pre-membrane 1954 [7], but the frst major outbreak occurred in 2007 (Pr), the membrane (M), and the envelope (E) proteins. on Yap Island, in the Federated States of Micronesia, In particular, the envelope proteins of faviviruses show where almost 75% of the population was shown to be very similar structures and functions, as they can mediate infected, and almost 20% developed symptomatic disease virus cell fusion [39] and elicit a cell-mediated response [8]. Large outbreaks also occurred in French Polynesia in [40]. Tey are therefore the main targets of neutralizing 2013 [9], and in South America [10]. ZIKV infections are antibodies, and can be related to ZIKV neurotropism mainly asymptomatic, but in spite of the generally mild [39]. self-limiting symptoms associated with maculopapular Here, we report on the construction of a novel DNA rash, headache, conjunctivitis, and musculoskeletal pain, recombinant (pVAXzenv) and a novel FP recombinant neurological complications can occur, such as micro- (FPzenv) putative vaccines that contain the cPrME genes cephaly in the developing fetus [4]. ZIKV has also been of ZIKV (Fig. 1). Tis sequence is related to cellular entry, associated with Guillain-Barré syndrome in adults, an and we evaluate the immunogenicity of the Env protein autoimmune neurological disease that is characterized by in a mouse model after challenge with an epidemic ZIKV muscle weakness, motor dysfunction, and in some cases, strain. Te fnal aim was to use the pVAXzenv recombi- paralysis [10, 11], as the virus can infect human neural nant as a prime and the FPzenv recombinant as a boost, progenitor cells [12]. administered also by the mucosal route. Tis novel FP Tus, since its introduction into Brazil in 2015, ZIKV construct was used to infect chick embryo fbroblasts has been declared a public health emergency of interna- (CEFs), normal human lung fbroblasts (MRC-5 cells), tional concern by the World Health Organization [13], and green monkey kidney (Vero) cells to assess transgene as it might represent an international threat [14]. Con- expression in vitro. Transcript expression in Vero cells sidering also its easy transmission from asymptomatic was tested to determine whether FPzenv induces long- patients, rapid development of a safe and efective vac- lasting responses. Te production of VLPs, as a result of cine is required to prevent further outbreaks. env gene expression, was also verifed by electron micros- Currently, there have been many attempts to develop copy. Mice immunization was performed by priming the candidate vaccines against ZIKV [15–17] that have animals with pVAXzenv by in-vivo electroporation (e.p.) included subunit and recombinant plasmid-based vac- and boosting them by subcutaneous (s.c.) and intranasal cines, inactivated or live-attenuated viral vaccines, (i.n.) administration of the FPzenv. Humoral responses recombinant vaccines [18, 19], and virus like particles were verifed before all bleeding times, and the virus (VLPs) [20, 21]. All these have shown diferent efca- neutralizing activity was tested before the challenge. Te cies in mice and nonhuman primate models [20, 22–27]. challenge with ZIKV was performed, after the last boost, Some of them have also been advanced to clinical evalua- on mice immunosuppressed with dexamethasone. Te tion, and are undergoing phase I and II clinical trials [12, experimentally immunized mice showed signifcantly 13, 28, 29]. higher antibody responses compared to the controls, Attenuated viral-vectored vaccines are among the especially after FPzenv administration by the mucosal most efective immunogens against infectious dis- route. Viral neutralizing activity could not be demon- eases [30, 31], as they are potent stimulators of anti- strated, as well as protection after the challenge with bodies and cell-mediated immunity, and they can ZIKV, as all of the mice survived. Zanotto et al. Virol J (2021) 18:50 Page 3 of 14 structural genes PrME signal non-structural genes c Pr M E RT-PCR cPrME (zenv) animal immunizaon pFPzenv pVAXzenv recombinaon plasmid expression