The latest advancements in Zika virus vaccine development

KEYWORDS:

• Flavivirus
• Zika virus
• vaccine
• advancement
• challenge

1. Introduction

Zika virus (ZIKV) was first discovered in Rhesus macaques in the Zika Forest of Uganda in 1947. It has caused major epidemics in the Americas and has spread to at least 84 countries and territories since 2015 (http://www.who.int/emergencies/zika-virus/en/), leading to significant public health concerns worldwide. ZIKV is linked to severe neurological diseases, such as Guillain–Barré Syndrome, transverse myelitis, encephalitis, meningoencephalitis, peripheral facial palsy, and thoraco-lumbosacral myelopathy in adults [1], as well as congenital Zika syndrome featured by microcephaly and other birth defects among children resulting from mothers with ZIKV infection during pregnancy [2]. Currently, no vaccines have been licensed against ZIKV infection, urgently calling for the development of safe and effective preventive strategies to stop the threat of ZIKV.

ZIKV is a single, positive-stranded RNA virus in the same Flaviviridae family as other mosquito-borne viruses, such as dengue virus (DENV), West Nile virus, and Japanese encephalitis (JEV). The ZIKV genome encodes a polyprotein consisting of capsid, precursor transmembrane (prM), and envelope (E) structural proteins, as well as NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 nonstructural proteins. The surface E protein mediates virus binding and membrane fusion, and it is thus a major target for the development of ZIKV vaccines.

2. Current status of ZIKV vaccine development

Currently developed ZIKV vaccines are categorized as those based on live-attenuated virus, inactivated virus, viral vectors, DNA, RNA, and recombinant subunit vaccines expressing ZIKV E protein. These vaccines, as described below, are tested for immunogenicity and efficacy in mice, nonhuman primates, and/or humans. While most ZIKV vaccines are in preclinical development, a few have been moved forward to clinical trials.

2.1. Live-attenuated ZIKV-based vaccines and infectious cDNA clones as a potential vaccine candidate

Live-attenuated vaccines are produced by weakening ZIKV to cause diseases, but preserving its ability to induce immune responses against viral infection. A ZIKV with a 10-nucletide deletion in the 3’-untranslated region of viral genome is attenuated, losing the ability to infect mosquitoes. This vaccine is immunogenic and protective against ZIKV (FSS13025, 10⁵ infectious focus unit: IFU; PRVABC59, 10⁶ IFU) infection in type I interferon receptor-deficient A129 mice with no detectable viremia [3]. Phase I clinical trials are scheduled in 2017 to identify the safety and immunogenicity of a live-attenuated monovalent ZIKV vaccine against ZIKV, and a multivalent vaccine against ZIKV and four types of DENV [4].

Using a reverse genetics system, several infectious cDNA clones of ZIKV have been generated and efficiently replicated in cultured cells. With their virulence attenuated, cDNA clones provide a genetics platform by which to develop alternative live-attenuated ZIKV vaccines [5].

2.2. Inactivated ZIKV-based vaccines

Inactivated ZIKV vaccines have been developed and tested in preclinical and clinical settings. A formalin-inactivated ZIKV vaccine, which was derived from the Puerto Rico PRVABC59 strain, provided protection of BALB/c mice against challenge with Brazil/ZKV2015 (10² plaque-forming unit: PFU), a strain that may cross the placenta and induce fetal microcephaly and intrauterine growth restriction in mice [6]. However, the complete protection, as measured by the absence of detectable viremia following challenge, was only revealed in the mice immunized by the intramuscular route, rather than the subcutaneous route [7]. It also elicited ZIKV-specific neutralizing antibodies, completely protecting immunized monkeys against ZIKV challenge from both Brazilian (Brazil/ZKV2015, 10³ PFU) and Puerto Rican (PRVABC59, 10³ PFU) strains with undetectable virus in blood, urine, CSF, colorectal and cervicovaginal secretions [8]. The inactivated ZIKV vaccine, named ZPIV, is being tested in a Phase I clinical trial [4]. This vaccine, formulated with Alum adjuvant, will be given intramuscularly to healthy adult volunteers and evaluated for safety and immunogenicity.

2.3. Viral-vectored ZIKV vaccines

Currently reported viral-vectored ZIKV vaccines are primarily based on adenovirus (Ad) and tested preclinically. A recombinant human Ad5-based vaccine expressing extracellular domain of E protein (Ad5.ZIKV-Efl) of ZIKV (BeH815744) completely protected 1-week-old pups, which were born to C57BL/6 mice immunized with this vaccine, against challenge with ZIKV (DAKAR41542, 10⁵ PFU). All mice survived from challenge with no weight loss or mild-to-no neurological signs [9]. Particularly, single-dose recombinant rhesus Ad52 (RhAd52)-vectored vaccine expressing ZIKV prM and E proteins induced ZIKV-specific neutralizing antibodies and protected monkeys against challenge with ZIKV (Brazil/ZKV2015, 10³ PFU), as shown by the absence of detectable viremia [8].

2.4. DNA-based ZIKV vaccines

ZIKV vaccines based on viral DNA have been developed and evaluated preclinically and clinically. A single-dose DNA vaccine expressing ZIKV prM and E (prM-Env of BeH815744 strain) afforded complete protection of BALB/c and C57BL/6 mice against challenge with ZIKV Brazil/ZKV2015 and PRVABC59 strains (10² PFU), and SJL mice against ZIKV Brazil/ZKV2015 challenge (10² PFU), as well as monkeys against challenge of ZIKV Brazil/ZKV2015 (10³ PFU), with no detectable viremia following challenge [7,8]. In addition, other prM-E-based DNA vaccines VRC5283 and VRC5288, in which the ZIKV (H/PF/2013 strain) prM signal sequence and/or E stem and transmembrane regions were replaced with the corresponding sequences of JEV, elicited ZIKV-specific neutralizing antibodies in mice and monkeys. Immunization with two doses of these vaccines provided 94% protection of monkeys against challenge with ZIKV (PRVABC59, 10³ focus-forming unit: FFU), showing the absence of detectable viremia [10]. Currently, ZIKV prM-E-encoding DNA vaccines, VRC-ZKADNA085-00-VP (NIAID) [4] and GLS-5700 (Inovio and GeneOne), are being tested in Phase I clinical trials. Healthy adult volunteers and/or DENV seropositive adults are injected intramuscularly and intradermally, respectively, with the specified vaccines, and evaluated for immunogenicity, safety, and tolerability. A Phase II trial of a DNA vaccine started in March 2017 to further evaluate its safety and immunogenicity [4].

2.5. RNA-based ZIKV vaccines

ZIKV vaccines are developed based on messenger RNA (mRNA) encapsulated with lipid nanoparticles (mRNA-LNPs), and evaluated for protective efficacy [11,12]. For example, two doses of LNP-encapsulated modified mRNA (IgE_sig-prM-E LNPs) encoding prM-E genes of ZIKV (Micronesia 2007 Asia strain) induced high titers of neutralizing antibodies in mice. It conferred complete survival to type I/II interferon receptor-deficient AG129 mice against challenge with ZIKV P6-740 (Malaysia 1966, 10⁴ PFU), as well as C57BL/6 mice treated with anti-IFNAR1 blocking antibody against challenge of mouse-adapted African ZIKV (Dakar 41519, 10⁵ FFU), with no weight loss and measurable viremia [11]. Similarly, a modified mRNA vaccine (JEV_sig-prM-E LNPs) containing a JEV leader sequence protected anti-IFNAR1 antibody-treated BALB/c mice against ZIKV (Dakar 41519, 10⁵ FFU) challenge, with undetectable viremia and viral RNA in the uterus or brain and reduced viral RNA in the spleen [11]. Furthermore, single-dose immunization of another nucleoside-modified mRNA-LNP vaccine encoding prM-E of ZIKV (H/PF/2013) induced potent and durable neutralizing antibody responses in mice and monkeys. While single dose of this vaccine protected BALB/c mice from ZIKV (PRVABC59, 10² PFU) challenge with undetectable viremia, it also conveyed sufficient protection of monkeys from detectable viremia after challenge with ZIKV (PRVABC59, 10⁴ 50% tissue culture infectious dose (TCID₅₀) [12]. One mRNA vaccine of ZIKV is being evaluated in Phase I trial [4].

2.6. Subunit ZIKV vaccines

ZIKV subunit vaccines can be developed based on recombinant viral proteins, particularly surface E protein. A subunit vaccine (ZIKV-rEfl) expressing E extracellular domain of ZIKV (BeH815744 strain) fused to the T4 fibritin foldon trimerization domain was prepared from mammalian cell 293T culture supernatant, and delivered through carboxymethyl cellulose microneedle array to adult C57BL/6 mice with prime-boost immunization regimen. It induced ZIKV-specific antibody responses and neutralizing antibodies in these mice, but only half of the 7-day-old pups born to immunized mice survived from lethal challenge of ZIKV (DAKAR41542, 10⁵ PFU), ~83% of which presenting neurological disease [9].

3. Challenge for development and application of ZIKV vaccines and potential strategies for improving their efficacy

Safety, stability, and immunogenicity are the major factors to be considered for vaccine development. While live-attenuated vaccines may provide continual antigenic stimulation to induce sufficient immune response and protection against viral infection, they might have safety and stability concerns due to the potential to revert to pathogenic forms and cause disease [13]. In contrast, inactivated vaccines are relatively safer and more stable than live-attenuated vaccines, but might require two to three doses to induce ideal immune response [14]. Vaccines based on viral vectors, such as Ad5, may present preexisting immunity, in which vector-specific antibodies may hinder the elicitation of vaccine antigen-specific immune response [15]. By comparison, DNA vaccines are safe and can be manufactured easily and rapidly. The biggest challenge for this vaccine type is the relatively poor immunogenicity in generating high-titer antigen-specific antibodies, calling for continual effort to improve the efficacy of human DNA vaccines in order for them to get success commercially. Similar to DNA vaccines, subunit vaccines, which do not contain any components of infectious virus, are safe and stable, with no risk of inducing disease and causing significant side effects to the injection sites. Nevertheless, some recombinant protein-based subunit vaccines may have relatively lower immunity than other vaccine types such as live-attenuated vaccines.

ZIKV vaccines can be primarily targeted to women of reproductive age, including pregnant women, and secondarily targeted to adolescent and adult males [16]. Due to the safety concerns and the potential of ZIKV to cross the placenta to infect fetus and cause microcephaly, it is more preferable for pregnant women to receive the replication-defective viral-vectored vaccines and nonreplicating vaccines (e.g. inactivated, DNA, or subunit vaccines) than the live-attenuated or replication competent viral vaccines. Inactivated or subunit vaccines with Alum, an adjuvant approved for human use, may be used as vaccine platform for pregnant women [16]. It is possible to extend the target population of vaccination to ZIKV-infected men if severe outcomes are identified from sexual transmission of ZIKV and there is sufficient vaccine supply [16].

It has been confirmed that ZIKV protective efficacy is mainly mediated by vaccine-induced ZIKV E-specific antibodies since adoptive transfer of purified IgG from prM-Env DNA-immunized mouse sera provided protection against ZIKV [7]. In addition, purified IgG from inactivated ZIKV-vaccinated monkeys also conferred passive protective efficacy in adoptively transferred rodents and primates [8]. Such protection against ZIKV infection is positively correlated with serum neutralizing activity [10]. There is an inverse correlation between neutralizing antibody titers and levels of ZIKV RNA, and a neutralizing antibody titer with a cutoff EC₅₀ (half-maximal inhibition of virus infection) value of ~1/10,000 is expected to completely prevent viremia and tissue dissemination in the vaccinated animals [11]. Therefore, defining of the neutralizing antibody titer required for protection is important for designing vaccine regimens to provide sustainable and long-term immunity. While most ZIKV vaccines, as described above, induce protective neutralizing antibody responses, some ZIKV vaccines may elicit CD8+ T cell responses, in protecting against ZIKV infection. It is shown that adoptive transfer of ZIKV-immune CD8+ T cells reduced viral loads and depletion of these cells resulted in high viral loads, and that CD8-deficient mice presented high mortality upon ZIKV infection [17].

ZIKV E protein is a key target for inducing protective neutralizing antibodies; consequently, several ZIKV vaccines, as noted above, are based on the full-length prM-E or E proteins. However, some antibodies to DENV E protein in DENV-infected people may cross-react with ZIKV E protein, but do not neutralize ZIKV, resulting in antibody-dependent enhancement (ADE) of ZIKV infection [18]. In addition, human monoclonal antibodies to linear epitopes of DENV E protein, including immunodominant fusion-loop epitopes, can bind, rather than neutralize, ZIKV, and thus enhanced ADE [18]. Antibodies specific to domain I and II of E protein (EDI/II) are cross-reactive and poorly neutralizing, enhancing ZIKV and DENV infection in vitro, while those specific to domain III of ZIKV E protein (EDIII) and quaternary epitopes on infectious virus have potent neutralizing activity against ZIKV infection [19]. A ZIKV vaccine was designed to mutate the conserved fusion-loop epitopes of E, leading to the increased protective immunity against ZIKV but reduced generation of antibodies that can enhance DENV in vitro and in vivo infection in animal models [11]. Although there are no data supporting the clinical significance of ZIKV ADE in humans, these studies will provide important guidance for designing safe and effective ZIKV vaccines that induce highly potent protective neutralizing, but not harmful, immune responses. Such vaccines will have particular application in regions where DENV and ZIKV (e.g. Brazil, Colombia, Puerto Rico, U.S. Virgin Islands, southwestern/southeastern U.S., etc.) or ZIKV (including areas in Africa, Caribbean, Central America, Pacific Islands, South America, and others) are pandemic [20,21], and for pregnant women infected with ZIKV [22].

A number of approaches may be applied to increase the efficacy of ZIKV vaccines. For example, special delivery technologies, such as electroporation or needleless pressure-based delivery, can be adopted for injection to improve the efficacy of DNA-based vaccines. The efficacy of subunit vaccines might be improved in formulation with appropriate adjuvants, selection of suitable injection routes, optimization of immunization regimens, or blocking of non-neutralizing epitopes while keeping neutralizing epitopes in the viral E protein. These strategies have been proven effective in developing subunit vaccines against other viral diseases [23], and could be employed to produce safer and more efficacious ZIKV vaccines for human use with the greatest potential to induce strong anti-ZIKV neutralizing antibodies. Other strategies, such as prime-boost vaccination, can be applied to promote vaccine components to elicit improved efficacy against ZIKV infection. A DNA vaccine prime-an inactivated ZIKV vaccine boost and an inactivated ZIKV vaccine prime-boost approaches are conducted in human clinical trials to evaluate vaccine’s safety and immunogenicity [24]. To overcome preexisting vector immunity and improve efficacy of viral-vectored (e.g. Ad) ZIKV vaccines, strategies such as modification of Ad5 genome, utilization of alternative-serotype Ad, and replacement of human Ad5 with chimpanzee-derived replication-defective Ad may be desirable, which have shown efficiency in protecting against other pathogens [25]. Overall, development of ZIKV vaccines with strong safety profile and protective efficacy is still urgently needed to prevent and reduce the threat of ZIKV.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Additional information

Funding

The manuscript was not funded.