Challenges of Vaccine Development for Zika Virus

Abstract

The emergence of outbreaks of Zika virus (ZIKV) in Brazil in 2015 was associated with devastating effects on fetal development and prompted a world health emergency and multiple efforts to generate an effective vaccine against infection. There are now more than 40 vaccine candidates in preclinical development and six in clinical trials. Despite similarities with other flaviviruses to which successful vaccines have been developed, such as yellow fever virus and Japanese Encephalitis virus, there are unique challenges to the development and clinical trials of a vaccine for ZIKV.

Introduction

Zika virus (ZIKV) is a mosquito-borne flavivirus, first identified in 1947. Until recently, the virus posed little concern, as infection is largely asymptomatic or associated with a mild rash and febrile illness (22), although it had occasionally been accompanied by the development of Guillain-Barre syndrome in adults (10). Public attention was drawn to an outbreak of ZIKV that emerged in Brazil in 2015 and that was associated with devastating fetal microcephaly and other neurological defects in the developing fetus as a consequence of infection during pregnancy (19,78). The WHO declared a state of public health emergency from February 1, 2016 to November 18, 2016 (95). It is now emerging that microcephaly may be the tip of the iceberg in terms of ZIKV damage to the developing fetus (16,17,68,101), as babies who appear normal at birth may manifest neurological defects later in development. ZIKV has been shown to target both neural progenitor cells and more mature neural cells (89), and can persist long-term in neurological sites (2). ZIKV has also been shown to persist in bodily fluids, including semen, consistent with its ability to be sexually transmitted, calling into question the potential for sustained transmission when mosquito vector populations may be low (64,67,97). This dramatic impact on human health and the rapid spread of the virus [now reported in at least 84 countries (95)] make the development of a vaccine against ZIKV an urgent public health priority.

Progress in ZIKV Vaccine Development

A global effort is underway to develop vaccines against ZIKV, and more than 40 candidates are currently under development, employing a wide variety of vaccine platforms. Here, we will review general approaches and challenges to ZIKV vaccine development, as detailed progress in the development of individual candidates and preclinical testing have been recently reviewed by others (5,6,26,59,90,91). An increasing number of Zika vaccine candidates are in phase I clinical trials, and some are progressing to phase II (6) (Table 1).

Table 1.

Zika Virus Vaccine Development and Ongoing Clinical Trials

Vaccine platform	Vaccine ID	Developer	Clinical phase	References
Whole virus inactivated	ZPIV (inactivated ZIKV) + Alum	WRAIR	Ph1	https://clinicaltrials.gov/ct2/show/NCT03008122 NLM identifier: NCT03008122
Nucleic acid (DNA and RNA)	mRNA-1325	Valera (Moderna)	Ph2	https://clinicaltrials.gov/ct2/show/NCT03014089 NLM identifier: NCT03014089
Nucleic acid (DNA and RNA)	pDNA (GLS-5700)	Inovio/GeneOne	Ph2	https://clinicaltrials.gov/ct2/show/NCT02809443 NLM identifier: NCT02809443
Recombinant or sub-unit	Synthetic Peptide- NIAID/SEEK AGS-v	NIAID	Ph1	Broad protection against mosquito-borne viruses, including DENV, ZIKV, and malaria, https://clinicaltrials.gov/ct2/show/NCT03055000 NLM identifier: NCT03055000
Recombinant or sub-unit	DNA (VREZKADNA085-00-VP)	VRC/NIAID	Ph2/2b	https://www.nih.gov/news-events/news-releases/phase-2-zika-vaccine-trial-begins-us-central-south-america NLM identifier: NCT03110770
Viral vector	MZ-ZIKA-101, Live attenuated Measles-Vectored rZIKV vx in a Theaxyn^® platform	Thermis BioScience (AT)	Ph1	https://clinicaltrials.gov/ct2/show/NCT02996890 NLM identifier: NCT02996890

DENV, dengue virus; ZIKV, Zika virus.

ZIKV vaccine candidate platforms include live attenuated virus, purified inactivated virus (PIV), purified virus-like particles (VLP), DNA vaccines, RNA vaccines, purified protein subunit vaccines, and vector-based vaccines (49). The development of PIV vaccines builds on the successful development of similar vaccines against other flaviviruses, including Japanese Encephalitis virus (JEV) (60) and tick-borne Encephalitis virus (TBEV) (53). PIV vaccines express multiple antigenic determinants and, thus, elicit a broad immune response, although important neutralizing epitopes may be destroyed by the inactivation procedure, as has been shown for JEV and TBEV; the overall impact is unknown considering their demonstration of clinical benefit in field trials (28,42). Inactivated vaccines may require multiple doses and administration of boosters that are remote from the primary vaccination series to ensure durable protection (30).

Live attenuated vaccines also elicit a broad response, but have the potential for increased reactogenicity compared with nonreplicating platforms and possess the theoretical risk of reversion to an under- or un-attenuated state. Thus, they are not recommended for vaccination during pregnancy or in the immunocompromised, although they may be appropriate for targeting populations of children before sexual activity (5).

Vector-based vaccines, VLP, chimeric vaccines, and RNA and DNA vaccine platforms are attractive because they can be formulated to express specific viral genes and, thus, direct antibody responses to particular viral structural components that preferentially elicit strong neutralizing activity rather than weaker, cross-reactive antibodies (see below). Although there are no currently licensed DNA vaccines for humans, preclinical testing in animals has shown such vaccines to be safe and protective after viral challenge (65). However, a concern with DNA vaccines is the potential for chromosomal integration by nonhomologous recombination, which can result in insertional mutagenesis and cell transformation. In this regard, RNA vaccines have an advantage over DNA vaccines, as they are translated by the host cellular machinery in the cytoplasm and, thus, pose limited risk of chromosomal integration. Recombinant viral vector-based vaccines are also being developed, employing virus backbones that are used successfully in previous vaccines.

Preclinical models of ZIKV infection in mice and nonhuman primates have been utilized for the initial characterization of vaccine safety and efficacy (1,7,8,24,35,51,72). Protection is believed to be associated with the development of neutralizing antibodies, consistent with accepted correlates of protection for other flavivirus vaccines (76). However, T cell immunity may also contribute to protection (54,59,94), and recent studies have raised the possibility that cellular immunity may be an essential component of long-lasting protection (36).

Cross-Reactivity with Dengue Virus and Antibody-Dependent Enhancement

Although there is only one ZIKV serotype (23), ZIKV has strong structural homology with dengue virus (DENV), which has four serotypes, and also other flaviviruses, including West Nile virus (WNV), yellow fever virus, and JEV. The cross-reactivity between ZIKV and DENV has been studied in detail, and it has been shown that antibodies generated by infection with DENV have high cross-reactivity and cross-neutralizing activity with ZIKV (3,21,77,87,88). This cross-neutralization activity has led to suggestions that the development of “universal vaccines” to protect against both DENV and ZIKV may be possible (3,21,88). However, the DENV-specific antibodies generated by infection that cross-neutralize ZIKV may not be sustained, and are rarely detected 6 months after DENV infection. With time, the neutralizing activity becomes less cross-reactive and more specific to DENV (15). The poorly neutralizing anti-ZIKV antibodies that remain introduce the possibility of antibody-dependent enhancement (ADE).

ADE, which has been extensively studied with regard to DENV, results from binding of poorly neutralizing antibodies to virus and the formation of immune complexes. Instead of neutralization, immune complexes bind to FcR on target cells, leading to enhanced infection [reviewed in Halstead (38)]. ADE is one hypothesis to explain the immunopathologic mechanisms leading to the increased disease severity sometimes observed after sequential DENV infections with different DENV serotypes. Although primary infection with one DENV strain appears to protect against re-infection and disease with the same strain for years to decades, sequential infection with a different strain of DENV can lead to an increased risk of severe disease, including hemorrhagic fever (9). Interestingly, however, a third or fourth DENV infection is not associated with enhanced frequency of DENV hemorrhagic fever (32).

Cross-reactive but poorly neutralizing antibodies between different DENV strains have been shown to mediate ADE in vitro, resulting in enhanced infection of FcR-bearing cells (38,39,63). In addition, it has been shown that passive transfer of antibodies in vivo mediates ADE (39,41). It has, however, been difficult to experimentally demonstrate an effect of ADE on disease severity in vivo (48,50). The specific in vivo immune evasion or immunopathologic mechanisms responsible for the rare, but severe and potentially fatal, phenotypes in human DENV disease, including DENV Hemorrhagic Fever, are incompletely understood, and both cellular and humoral immunologic mechanisms of ADE have been implicated (40,81,83).

Because of the cross-reactivity identified between DENV- and ZIKV-specific antibodies, many recent studies have examined, and successfully demonstrated, in vitro ADE activity between DENV- and ZIKV-specific antibodies (11,13,20,47,56,74). Also, it has been shown that administration of plasma from DENV- or WNV-convalescent patients resulted in enhanced viral titers and disease in mice (4). However, two independent studies in Rhesus macaques have failed to show an effect of ADE mediated by prior DENV infection on viral load or disease severity of ZIKV infection (61,71). In these experiments, Rhesus macaques infected more than 1 year earlier with DENV or other flaviviruses failed to show enhanced infection with ZIKV, despite the demonstration of enhancing activity in vitro. However, as the authors note, these studies do not rule out the possibility that mechanisms of ADE may enhance the transport of virus across anatomical barriers, such as the placenta via FcR-bearing Hofbauer cells. In addition, it is possible that ADE mediates enhanced infection of FcR-bearing neural cells, which is perhaps relevant for ZIKV-associated neurological complications (56). Further well-controlled in vivo experiments need to be carried out. Rather than just quantitation of overall viral burden, infection and pathology in specific anatomical sites need to be assessed.

Structural Basis for ZIKV Neutralization

To understand the mechanisms underlying the generation of antibody neutralization or antibody enhancement, the viral structural determinants responsible for eliciting strongly neutralizing or cross-reactive antibodies have been examined in detail. The flavivirus envelope glycoprotein (E) is essential for viral attachment and entry in the host (55,66). The E protein contains multiple immunodominant epitopes that elicit neutralizing antibodies after infection or vaccination. Antibodies elicited by the E protein bind to three distinct regions, termed DI, DII, and DIII (75). A recent longitudinal study after natural human ZIKV infection examined antibody specificity and antibody neutralizing/cross-reactivity (99). The data showed that titers of antibodies specific for ZIKV DI/DII peaked early, were weakly neutralizing and cross-reactive with DENV, but rapidly waned. In contrast, antibodies specific for ZIKV DIII increased in titer over time, were strongly neutralizing for ZIKV, and poorly cross-reactive with DENV. A potent monoclonal antibody (mAb) targeting DIII provided 100% protection against ZIKV infection of mice.

Panels of mAb have been developed and used to characterize neutralizing and cross-reactive antibodies to DI/II and DIII regions, respectively (87,100). Antibodies targeting an immunodominant epitope in the fusion loop in DII of the E protein have been shown to mediate ADE both in vitro (20,47,85,87) and in vivo in the mouse (87). In contrast, antibodies directed to DIII have been shown to be neutralizing (29,85,87,98 –100). As a consequence, some vaccine strategies have been directed toward eliminating responses to the fusion loop in the DII portion of the E protein, either by mutation (79) or by generating VLP-expressing E protein domain III (98), to elicit strong neutralizing activity with little cross-reactivity or potential ADE activity. However, it was recently shown that antibodies to DII or DIII alone can lead to viral escape, prompting the development of a bi-specific antibody directed toward both DII and DIII, which prevented viral escape and generated therapeutic and prophylactic protection in vivo (93). In other studies, it has been shown that antibodies specific for quaternary viral epitopes have cross-neutralization activities between ZIKV and the four stains of DENV, supporting the possibility of universal vaccines (3,29,46,82).

Influence of Pre-Existing Flavivirus Immunity on ZIKV Vaccines

Preclinical experiments with ZIKV vaccines have supported the progression to clinical testing in human trials. However, the preclinical vaccine studies were generally performed in flavivirus-naive mice or primates, whereas potential vaccines in ZIKV-endemic areas are likely to have had previous exposure to other co-circulating flaviviruses either through natural infection of immunization or will have an exposure in the future.

This raises the question of whether prior history of flavivirus exposure may adversely affect the safety, immunogenicity, or protective efficacy of a ZIKV vaccine, by ADE or some other mechanism (18,20,37,57). As previously stated, the physiological relevance of ADE for enhanced disease has not been established and it has proved difficult to demonstrate ADE in vivo. Thus, it is unknown whether administration of the ZIKV vaccine in DENV-endemic areas will lead to ADE or some other immunologic enhancement of ZIKV infection and disease. It is unclear whether these theoretical concerns will impact the approach of regulatory agencies or ethical review committees when evaluating ZIKV vaccine clinical development plans as they do for dengue vaccine development.

The only way to understand whether an association or causality exists between pre-existing ZIKV or flavivirus immunity and subsequent natural exposure is to conduct properly designed and randomized trials with aggressive sampling of blood to allow temporal associations between baseline immune profiles and outcomes of infection. Specifically linking, in a causal relationship, the immunologic mechanisms to human infections and severe clinical outcomes has been elusive.

Challenges for Development of the ZIKV Vaccine

There are important differences between the pathology induced by ZIKV compared with other flaviviruses. First, ZIKV infection during pregnancy can mediate devastating effects on fetal development, perhaps linked to the affinity of the virus for neuronal tissue (2,43,68,69,86). In addition to well-described fetal demise and microcephaly, there is growing evidence that neurological effects may be manifest later after birth in infants who appear normal at birth (16,17,101). These pregnancy-associated outcomes make it mandatory to develop a vaccine that will either protect a woman before or during pregnancy or protect the fetus from the harmful effects of maternal infection. Second, ZIKV can be harbored long-term in bodily fluids and can be sexually transmitted (31,64,97). This observation makes it important to test vaccines for their ability to prevent the establishment of even low levels of viral infection at specific anatomical sites.

Models for preclinical evaluation of vaccine candidates to explore these performance criteria are being developed. A variety of mouse pregnancy models have been used to study ZIKV infection. It has been shown that the NS5 protein of the ZIKV targets human STAT 2, which blocks type I interferon responses (34). However, ZIKV does not target mouse STAT 2, leaving type I interferon responses intact. To more closely mimic the human condition, different mouse models that lack a type I interferon or STAT2 response have been used for preclinical studies (52,62,79,92). These immunodeficient mouse models allow robust infection and profound pathogenesis, including fetal demise in pregnancy models (62). In other studies, immunocompetent mice have been used, to allow analysis of immunity to the viral infection (44,73).

Preclinical immunocompromised mouse pregnancy models have recently been used to test ZIKV vaccines. A modified RNA and a live attenuated vaccine have been tested for their ability to protect against fetal demise (80). Vaccinated mice that subsequently become pregnant were challenged at day six of gestation. Analysis of vaccinated mice showed reduced viral mRNA levels in maternal, fetal, and placental tissues. In addition, viable pups were born to vaccinated, but not unvaccinated, dams and the fetal heads had no measurable virus. These data are promising, however, sterilizing immunity was not achieved; it is unclear whether this is a requirement for protection during pregnancy. Also, increasing periods of time between vaccination and subsequent pregnancy and infection need to be tested.

In humans, active ZIKV infection can be asymptomatic and damage to the developing fetus can occur early in pregnancy, even before the woman is aware that she is pregnant. Therefore, rather than direct vaccination of pregnant women, a better approach may be to develop a vaccine that is capable of establishing potent and durable protective immunity that could be administered to children before sexual activity (58). Thus, preclinical efficacy studies will need to confirm that vaccination at various times before pregnancy will protect the developing fetus during subsequent pregnancy.

The finding that ZIKV infection can be sexually transmitted means that vaccines will need to prevent the establishment of persistent reservoirs of ZIKV in bodily fluids, including semen. Recently, the mouse model has been used to test the ability of vaccination to prevent virus-induced damage to the testis (35).

A final challenge is that, because of the theoretical potential for immunologic enhancement of disease, safety and efficacy must be assessed in both flavivirus-naive and flavivirus-experienced individuals. Also, the consequences of ZIKV vaccination in flavivirus-naive individuals over time, with potential exposure to DENV, need to be determined.

Establishing Clinical Benefit for a Zika Vaccine

The overall goal of ZIKV vaccination is to prevent infection and associated sequelae. There are several challenges to designing clinical trials to address this goal. A major challenge is the declining density of the infection. It appears the ZIKV epidemic is waning, as was the case earlier with WNV (14,58,70,91). A lower frequency of infection makes clinical trials more challenging, as vaccination cohorts need to be significantly larger.

A second challenge associated with clinical trials of ZIKV vaccines is establishing safety and efficacy in both flavivirus-naive and flavivirus-primed individuals (5). Randomized controlled clinical trials are preferred, as they address issues of regional and temporal variation in the incidence of ZIKV, and provide the best assessment of potential complications, including Guillain-Barre syndrome and immunologic enhancement (58). However, there are problems associated with identifying the required flavivirus-naive and flavivirus-primed populations. Not only is DENV endemic in many areas where ZIKV emerged, and DENV vaccination is currently being carried out, but there are now also high levels of ZIKV immunity. Thus, extensive prescreening is required to identify flavivirus-naive and flavivirus-primed individuals, and screening is complicated by cross-reactivity among the flaviviruses. In addition, in areas where ZIKV is now endemic, there may be insufficient numbers of ZIKV-naive individuals for trials. Restoration of an adequate ZIKV-susceptible population for vaccination trials may require waiting as long as 20 years.

A third challenge is the low incidence of symptoms during acute disease and the low frequency of sequelae. This makes the prevention of infection and associated sequelae difficult to confirm in clinical trials (26,58). For example, ZIKV infection is usually asymptomatic (as few as 18% of infected individuals having symptoms such as a rash) and is associated with a short period of viremia, although the virus can persist in the urine for up to 2 weeks (25,33). The two major sequelae are microcephaly or other manifestations of fetal demise, and Guillain-Barre syndrome, both of which are infrequent. Prevention of microcephaly is not a feasible readout of clinical trials, because the incidence is too rare, and it is estimated to range between 0.88% and 13.2% in the first trimester (45). Guillain-Barre syndrome is also rare—in one study, there were 0.24 cases of Guillain-Barre syndrome reported for every 100 cases of ZIKV (10).

A fourth challenge is establishing vaccine safety and efficacy in pregnant women, a population typically avoided during the development of vaccines. However, this population is a primary target for ZIKV vaccines, due to the devastating effects on the developing fetus. Ethics regarding maternal vaccination have been debated (96). Guidelines to ensure prioritization of the development of ZIKV vaccines that will be acceptable for use in pregnancy (27) and re-evaluation of risk-benefit analysis for immunizations during pregnancy (12) have recently come under consideration.

Finally, the challenges associated with clinical trials have led researchers to consider undertaking human challenge studies as an alternative. Human challenge studies for ZIKV are ethically complicated (26), because of the potential risks to third parties, including pregnant women and fetuses, and are complicated by reservoirs of viral persistence (43) and evidence for sexual transmission (64,67,97). Recent recommendations on ethical considerations for ZIKV human challenge trials were that human challenge studies were not, at this time, ethically justified (84). Future justification of human trials must take into consideration the high risk of the trial against the urgency to obtain knowledge because of lack of alternatives, for example, due to prohibitively difficult field trials and lack of suitable animal models.

Conclusions

There is an urgent need for a safe and effective ZIKV vaccine. ZIKV infection during pregnancy can mediate devastating effects on the developing fetus, and the potential for sexual transmission exposes risk to pregnant women even in countries outside of the endemic areas. The capacity of the virus to infect neurological tissues risks the development of congenital Zika syndrome, Guillain-Barre syndrome, and other, as yet poorly characterized neurological problems. These and other nuances of ZIKV transmission, virology, immunity, and potential for interaction with other flaviviruses increase the complexity of vaccine development.

Footnotes

Author Disclosure Statement

M.A.B., I.-J.K., and J.-S.L.: no competing financial interests exist. S.J.T.: Zika—inventor, U.S. Army ZPIV candidate vaccine; Dengue—consultant, advisory board or safety committee of Sanofi Pasteur, GSK Vaccines, Takeda, and Merck and Co.

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