What are the Implications of Silent Replication in Fetal and Newborn Monocytes for Zika Therapeutic development?

Zika virus (ZIKV) is an RNA virus belonging to the Flaviviridae family, which also harbors other pathogens of public health concern such as dengue virus (DENV) and yellow fever virus (YFV) [1]. Although identified in the 1950s in the Zika Forest of Uganda, ZIKV remained mostly ignored by medical and scientific communities, eventually being associated with outbreaks in restricted populations as the causative agent of a febrile, but mostly benign, illness [1]. The renewed interest in ZIKV studies was prompted by its arrival in the Americas, especially in Brazil, where the virus was associated with severe congenital malformations, most remarkably fetal microcephaly. While those manifestations – collectively named congenital Zika syndrome – may be compatible with life, they are nevertheless highly incapacitating and irreversible, with those children requiring lifetime healthcare support, which sometimes brings social stigma to the affected families [2].

Even though the most adverse effects of ZIKV infection occur during gestation, when the developing nervous system is highly susceptible to virus-induced cell damage [3], the impact of neonatal infection cannot be underestimated. Experimental evidence demonstrates that ZIKV infection can also promote some degree of neuroanatomical alterations and cognitive impairment in infant primates [4]. Considering transmission of ZIKV is mainly through the bite of an arthropod, usually mosquitoes from the Aedes genus, which are also the main vectors for DENV, the newborn can be easily infected in endemic areas. In addition, ZIKV can also be detected in breast milk [5], pointing to breastfeeding as an alternative infection route, although there is still no clear evidence supporting this concern.

Most knowledge about immunological response to ZIKV is derived from adult representative systems, which were key in uncovering essential roles played by innate immune components, such as the type I and type III interferon cascades, involved in viral restriction [6,7]. Yet, a great scientific gap exists in approaches focused on the neonatal immune system, since data regarding the fetal context were mostly surrounding the cellular mechanisms associated with the developmental abnormalities, with only brief immune insight. Essentially, the characteristic features of neonatal immunology reflect its ‘naivity’. Although we still do not fully understand the immune system at this stage of life; it is well acknowledged that neonates are biased toward T_H2/type 2 responses and do not present fully competent immunoregulatory networks [8]. While immune maturation is driven by constant challenges provided by environmental stimuli during the coming years, this neonatal window is a critical period of high susceptibility to infections that would not severely affect immunocompetent adults.

ZIKV spreads throughout host systems and tissues mainly by the blood but in addition to being delivered as free particles, the virus could be carried by blood cells. Among circulating leukocytes, monocytes were identified as preferential targets for ZIKV infection [9,10]. Recently, our group showed that newborn monocytes are also prone to ZIKV infection, supporting viral replication, but unable to produce a remarkable cytokine response [11]. While adult monocytes respond to viral infection, the dampened response of neonatal monocytes suggests that these cells could act as vessels for viral spread. This idea follows the Trojan horse hypothesis, which was well described for many viruses, as a strategy to achieve immune privileged sites residency (e.g., the nervous system) [12]. Considering monocytes can migrate to peripheral tissues, where they differentiate to replace the macrophage pool, they could efficiently help in ZIKV dissemination. Through endothelial barrier crossing, infection is favored in newborns. Thus, therapeutic targeting toward monocytes could be explored as an alternative for infection control.

Therapies directed toward modulating monocyte and macrophage activities are a clinical reality such as with the current use of clodronate in treating osteoporosis through its osteoclast functional modulation. With this said, monocyte depletion would be a harsh compromise in efforts to manage ZIKV infection. Our group has a track record for using innate immune agonists, including TLR ligands, as therapeutic coadjutants to promote neonatal immunity. For viral infections in particular, we demonstrated the potential for using TLR7/TLR8 agonists to restore the response of myeloid dendritic cells in HIV-exposed newborns [13]. Thus, while fetal monocytes may not present a robust antiviral response, adjuvant therapy could help to potentiate their activity. Ideally, a therapeutic strategy employing antiviral drugs, to interfere directly with viral replication, in association with immune-based approaches promoting host immunity, may be the most suitable way to achieve protection in a permissive host as the newborn.

Another remarkable point in ZIKV biology is its ability to be transmitted through sexual routes [14], overcoming the need for a vector and even prompting suggestions for ZIKV as a new sexually transmitted disease. The epidemiological relevance of this route is highlighted by the ability of ZIKV to persist in semen for months, far after symptoms cease and when the virus is no longer detectable in the blood [15]. The semen contains not only reproductive cells but also leukocytes; thus, in addition to shedding free viral particles, ZIKV could be carried by seminal monocytes as vehicles for effective transmission – an important infection strategy when the free virus will be unstable in acidic vaginal pH. Curiously, ZIKV can robustly replicate in the male reproductive tract and promote damage to those tissues, which can negatively affect individual fertility [16]. Since the testes are also an immune privilege site, silent ZIKV replicating monocytes can favor tissue invasion even before puberty and potentially cause reproductive problems.

A robust bulk of potential drugs for treatment have been uncovered in recent years; reviewed by Gorshkov et al. (2019) [17]. There are proposals for the use of natural products such as green tea extracts, classical anti-flaviviruses drugs such as sofosbuvir, and even unexpected drugs like the antibiotic azithromycin. However, even with the amount of data in the literature, there is still lacking in vivo validation, especially in human models – particularly in considering the outstanding differences in the pharmacokinetics and pharmacodynamics between adults and newborns, which adds another layer of complexity to drug design. Whether those drugs can efficiently act over silent replicating monocytes is a further open issue.

In parallel to drug screening, many groups seek the development of vaccines which could promote induction of cellular and humoral responses [18]. Considering some ZIKV and DENV antigens are structurally similar, the induction of neutralizing antibodies to their shared domains could sound a promising strategy for a vaccine design, particularly for areas where both viruses are endemic. A main concern for the applicability of broadly neutralizing antibodies, however, is the risk of antibody-dependent enhancement (ADE), which is well acknowledged during infection by different DENV serotypes. In ADE, antibodies raised against one DENV serotype can have a poor neutralizing ability against another, favoring viral infection mediated by the interaction between the coated viral particle and Fc receptors. However, the occurrence of ADE due to anti-ZIKV and anti-DENV antibodies cross-reactivity remains controversial [19]. In addition, it should be considered that the neonatal antibody response is not as robust as in adults, which may require differential vaccine formulations to achieve satisfactory protection. Nonetheless, if efficient anti-ZIKV neutralizing antibodies are indeed generated, they could still only be partially efficient where the virus is protected within monocytes and other reservoirs.

The development of an effective therapy requires considerable knowledge and understanding of disease pathogenesis. The full understanding of the neonatal ZIKV infection still has considerable gaps, which limits our power to identify critical steps in the interplay between the pathogen and the (young) host. The silent replication of ZIKV in fetal and newborn monocytes is one facet of the differential response at this life stage, but many other neonatal cells such as cytotoxic CD8⁺ T cell, NK cells or macrophages remain to be explored. Therefore, efforts devoted to uncovering infection pathogenesis during the neonatal window are needed in order to achieve successful development of efficient protective therapies.

Author contributions

FS Yamada Yoshikawa wrote the manuscript. MN Sato supervised the study and manuscript preparation.

Financial & competing interest’s disclosure

This work was supported by São Paulo Research Foundation (FAPESP) grant numbers 2016/16840-6 and 2016/09764-1. The authors have no other 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 apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.