The shedding of MERS-CoV was more extensive in the immunosuppressed animals, both in duration as in peak shedding. This suggests that the immune status has direct influence on virus shedding and subsequent potential of transmission. The epidemiological analyses of the 2015 MERS-CoV outbreak in South Korea clearly showed that only the level of MERS-CoV shedding was directly associated with transmission potential. Where spreaders had statistically lower Ct values compared to non-spreaders (25). The persistent MERS-CoV shedding in immunocompromised patients (23) could, therefore, contribute to enhanced nosocomial transmission.

As of yet, no specific treatment options have been identified for MERS-CoV infection. The results presented herein show that inflammatory responses contribute to the pathogenic process. This would suggest that treatment for patients with symptomatic infections would benefit from additional therapy that lessens the inflammatory response, especially in the lung, and not be based solely on therapies that are aimed at controlling virus replication.

# ETHICS STATEMENT

The use of study animals was approved by the Institutional Animal Care and Use Committee of the Rocky Mountain Laboratories and experiments were performed following the

# REFERENCES

guidelines of the Association for Assessment and Accreditation of the Laboratory Animal Care by certified staff in an approved facility. The guidelines and basic principles in the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals and the Guide for the Care and Use of Laboratory Animals were followed. All procedures were carried out under anesthesia using Ketamine by trained personnel under veterinarian supervision and efforts were made to provide for the welfare of animals and to minimize suffering. All animals were humanely euthanized at the endpoint of the study (6 days post-inoculation) by exsanguination under deep anesthesia. All standard operating procedures for MERS-CoV were approved by the Institutional Biosafety committee of the Rock Mountain Laboratories, and sample inactivation was carried out according to approved standard operating procedures prior to removal from high containment.

# AUTHOR CONTRIBUTIONS

JP, DF, EW, HF, and VM designed the study; JP, DS, HF, and VM analyzed the data; JP, HF, and VM wrote the manuscript; and JP, KH, FF, EH, VM, and DS performed the experiments and assays.

# ACKNOWLEDGMENTS

We would like to thank Doug Brining for veterinarian services; Dan Long, Tina Thomas, and Rebecca Rosenke for histology support; and Anita Mora for graphics. We would also like to thank Drs. Bart Haagmans and Ron Fouchier (Erasmus MC, Netherlands) for providing the MERS-CoV isolate.

# FUNDING

This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH).

marmoset. *PLoS Pathog* (2014) 10:e1004250. doi:10.1371/journal.ppat. 1004250

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Prescott, Falzarano, de Wit, Hardcastle, Feldmann, Haddock, Scott, Feldmann and Munster. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Flavivirus Receptors: Diversity, Identity, and Cell Entry

Mathilde Laureti 1,2†, Divya Narayanan1,2†, Julio Rodriguez-Andres 1,2†, John K. Fazakerley 1,2 and Lukasz Kedzierski 1,2 \*

*1 Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC, Australia, 2 Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Melbourne, VIC, Australia*

Flaviviruses are emerging and re-emerging arthropod-borne pathogens responsible for significant mortality and morbidity worldwide. The genus comprises more than seventy small, positive-sense, single-stranded RNA viruses, which are responsible for a spectrum of human and animal diseases ranging in symptoms from mild, influenza-like infection to fatal encephalitis and haemorrhagic fever. Despite genomic and structural similarities across the genus, infections by different flaviviruses result in disparate clinical presentations. This review focusses on two haemorrhagic flaviviruses, dengue virus and yellow fever virus, and two neurotropic flaviviruses, Japanese encephalitis virus and Zika virus. We review current knowledge on host-pathogen interactions, virus entry strategies and tropism.

#### Edited by:

*Alan Chen-Yu Hsu, University of Newcastle, Australia*

#### Reviewed by: *Alec Jay Hirsch,*

*Oregon Health & Science University, United States Namal P. M. Liyanage, The Ohio State University, United States*

\*Correspondence:

*Lukasz Kedzierski<EMAIL_ADDRESS>
*†These authors have contributed equally to this work*

#### Specialty section:

*This article was submitted to Viral Immunology, a section of the journal Frontiers in Immunology*

Received: *12 May 2018* Accepted: *04 September 2018* Published: *26 September 2018*

#### Citation:

*Laureti M, Narayanan D, Rodriguez-Andres J, Fazakerley JK and Kedzierski L (2018) Flavivirus Receptors: Diversity, Identity, and Cell Entry. Front. Immunol. 9:2180. doi: 10.3389/fimmu.2018.02180* Keywords: flaviviruses, Japanese encephalitis virus, Zika virus (ZIKV), dengue virus, yellow fever virus, entry receptor

# INTRODUCTION

The Flavivirus genus consists of more than 70 small, positive-sense, single-stranded RNA viruses transmitted by arthropods, in particular mosquitos and ticks.  These viruses are responsible for some of the most severe arbovirus infections affecting humans, pose a serious threat to global health and have the potential to cause severe outbreaks. These are exemplified by the global distribution of DENV (1), the recent ZIKV outbreak in South America (2), YFV outbreaks in Africa (3), and Brazil (4) and the spread of WNV across North America (5). Flavivirus infections range from asymptomatic, through mild fever and arthralgia to life threatening haemorrhagic or encephalitic diseases (6). Flaviviruses are also able to persist in patients and can be responsible for long-term morbidities (7). There are no antiviral treatments for flavivirus infection currently in clinical use, and despite licensed vaccines against several of the viruses including YFV, JEV, TBEV, or DENV, outbreaks still occur highlighting challenges in implementing effective vaccination programs (8).

The flaviviral genome of ∼11 kb contains a single open reading frame flanked by untranslated regions, and encodes 3 structural proteins (C, M, and E) and 7 non-structural proteins (NS). The mature virion features a surface densely covered with E glycoproteins and M proteins and a core consisting of capsid (C) protein and the RNA genome (9, 10). The entry into the target cell is dependent on E protein contact with its cognate receptor. E protein initially binds to attachment factors such as glycosaminoglycans. This effectively increases viral density on the cell surface, leading to high affinity receptor binding (11). The E protein ectodomain consists of three domains (E-DI, E-DII, E-DIII) of which E-DIII

**163**

is thought to interact with attachment factors and receptors (12). E-DIII domain's importance is highlighted by the fact that a vast majority of potent, neutralizing antibodies has been mapped to this region. Nevertheless, anti-DI and DII antibodies, although less potent, show broader cross-reactivity and form a major pool of anti-E protein specific immonoglobulins (13). Receptor binding is followed by clathrin-mediated endocytosis (14), which is considered to be a major mechanism of flavivirus cell entry, although there are exceptions described below. This leads to formation of endosomes and low pH dependent changes in the E glycoprotein with subsequent membrane fusion and release of nucleocapsid into the cytosol (15). In vitro, flaviviruses are able to infect a plethora of cell lines originating from rodents, non-human primates, humans and mosquitos. However in vivo, fewer cell types seem able to support flavivirus replication (16). A wide range of cell surface receptors has been implicated in flavivirus entry into different cells types (11). Amongst the entry receptors postulated to be involved in flavivirus entry, the best characterized to date include αvβ3 integrins (17, 18), C-type lectin receptors (CLR) (19–23), phosphatidylserine receptors TIM (Tcell immunoglobulin and mucin domain) and TYRO3, AXL and MER (TAM) (24). Recent studies indicate that flaviviruses can produce a range of structurally different virions. This structural heterogeneity may expand tissue tropism and ability to infect different cell types both in invertebrate and vertebrate hosts (25).

Flaviviruses are deposited into the skin epidermis by a mosquito bite where they encounter cells permissive to infection such as keratinocytes and skin dendritic cells (Langerhans cells) (26). Dendritic cells in particular appear to be a common initial target for flaviviruses. When infected, dendritic cells migrate to lymphoid organs where viral replication takes place allowing for flavivirus dissemination into circulation and internal organs (12). Viruses such DENV (27), JEV (28), ZIKV (29) have been shown to infect skin dendritic cells, and although there are no reports on YFV infecting Langerhans cells, it can nevertheless infect other types of dendritic cells (30). This interaction is mediated by DC-SIGN for JEV (28) and ZIKV (29), but appears to be DC-SIGN independent in case of DENV (31) and YFV (30).

Many flaviviruses are neuroinvasive and neurovirulent and cause central nervous system (CNS) damage (32). Neuroinvasive infections are observed with JEV, TBEV, and WNV (33, 34), and occasionally with haemorrhagic viruses including DENV (35). There is a paucity of knowledge regarding factors involved in CNS cell entry. While CLRs and TIMs are expressed by cells of the CNS (36–38), they are not expressed by neurons (39–41). However, members of the TAM family of receptors are expressed by different neuronal subtypes (42), though they are dispensable for ZIKV infection as ZIKV was able to infect and replicate in TAM receptor knockout mice (43).

As natural vectors, mosquitos and ticks are highly permissive to flavivirus infection. The virus can replicate in a range of arthropod tissues and cells (44, 45). Given that flaviviruses have only one glycoprotein, it seems likely that the mechanism of entry into vertebrate and invertebrate cells is evolutionarily conserved. A number of the cellular receptors implicated in flavivirus entry into mosquito cell lines overlaps with those identified for mammalian cells (46). Some flaviviruses are more selective regarding their arthropod host than others. For example, DENV is spread mainly by Aedes spp. mosquitos (6), WNV by Culex spp. (47), whereas JEV is transmitted by Aedes, Anopheles and Culex spp. (48). There appears to be a more restricted receptor repertoire used by flaviviruses for insect cell entry compared to mammalian cell entry. The range of clinical manifestations of flaviviral infection in the mammalian host suggests that these viruses may use a wide range of receptors. Mammalian tissues in general offer much greater range of receptors compared to invertebrates.

Identification of flavivirus entry receptors, particularly those involved in CNS infection, could lead to identification of novel therapeutic targets. For this review we will focus on four major flaviviruses of humans—DENV, JEV, ZIKV, and YFV, and discuss the differences and similarities in their mechanisms of entry into arthropod and mammalian cells.

# DENGUE VIRUS

DENV is one of the most common mosquito-borne viruses, mainly transmitted by Aedes aegypti mosquitoes, and occasionally by Ae. albopictus. Symptoms of DENV infection range from fever and muscle and joint pain (Dengue fever) to potentially life threatening haemorrhagic fever or shock syndrome. While DENV was endemic in <10 countries in the 1970's, it is presently a threat in over 128 countries and is responsible for almost 400 million human infections every year. About 24% of infections manifest in severe clinical symptoms. There is currently no treatment for DENV serotypes. There are four virus serotypes, and recovery from one serotype provides lifelong homologous immunity (49).

DENV is an icosahedral particle of 50 nm with a positive, single-stranded RNA genome of 10–11 kb (50). As in other flaviviruses, E protein is involved in receptor binding and fusion (51) and has the ability to bind to a wide range of cellular receptors to initiate DENV entry. The E-DIII domain has a role in cellular recognition (52) and has been suggested as a target for the development of a DENV vaccine (53).

Over the years, several cell membrane receptors involved in DENV entry have been identified. These include carbohydrate molecules (54–56), lectins (57, 58), and claudin-1 cell receptors (59). Carbohydrate molecules such as glycosaminoglycans (GAGs), sulphated polysaccharides, and glycosphingolipids (GSL) are widely expressed cell surface co-receptors for DENV entry and are believed to enhance viral entry efficiency. The highly sulphated form of GAGs, the heparan sulfates (HS) and heparan sulfates proteoglycans (HSPG), are essential for cellular adhesion to extracellular matrix and binding of polypeptide growth factors involved in intracellular signaling (56). It has been suggested that DENV first contacts HSPG, and that this weak interaction facilitates binding of virus to other receptors, which then results in virus internalization (55). Several studies have shown that pre-treatment with heparin can reduce or block DENV-2 infection (60, 61). However, the efficiency of inhibition of viral entry was dependent on numerous factors, such as the virus strains and the target cell (61). GSLs, a member of the same family of carbohydrate molecules as HS, are ubiquitous cellular components of eukaryotic plasma membranes that can also facilitate entry and binding of virus (54). However, GSLs are not required for DENV entry as the virus was able to enter GSL-deficient cells (62).

Cellular C-type lectin receptors (CLRs) are part of the host immune response to fungal, bacterial and viral infections (57). CLRs in mammalian cells include DC-SIGN/L-SIGN, mannose receptors (MR), and CLEC5A. DC-SIGN receptors are widely known because of their association with HIV. These receptors are also involved in DENV binding and internalization into dendritic cells (63). MR has been found to be the primary DENV cell receptor in macrophages. The CLEC5A receptor cooperates with DC-SIGN or MR to increase DENV binding and stability (58).

Other studies have suggested claudin-1 as a putative cell receptor for DENV entry through a direct interaction with the viral prM protein. Claudins are vital components of tight junction complexes and are essential for normal permeability of the epithelia (59, 64). DENV-2 entry was significantly reduced in claudin-1 deficient cells (59). Also, it has been demonstrated that caudin-1 is upregulated early in infection in order to facilitate entry and downregulated in late stage of infection (64).

Protein binding assays and mass spectrometry analysis have identified several additional potential flavivirus cellular receptors (65–67). Among them, the tubulin and tubulin-like proteins in C6/36 Ae. albopictus cell line (65). Heat shock proteins (HSPs) of ∼70 kDa and 80 kDa were also identified as cellular receptors for all four DENV serotypes in C6/36 cell line (66, 68). HSPs are chaperone proteins involved in the regulation of folding and unfolding of cellular, and upon infection, viral proteins (69). The 70 kDa protein, also known as heat shock cognate protein (HSC70) or HSPA8, acts as a chaperone protein during DENV entry (70, 71). Modulation of HSC70 expression was observed during DENV-2 infection, with an increase on the cell membrane during infection, suggesting that DENV-2 utilizes HSC70 for entry into mosquito cells (67). In addition to its role in viral entry, HSP70 is involved in virion biogenesis and RNA replication (71). It appears that all four DENV serotypes are dependent on this chaperone protein family, which makes HSP70 proteins an interesting target for the design of a tetravalent DENV therapy or vaccine (71).

HSP90, another heat shock protein, can also act as chaperone. This protein interacts with six DENV proteins (69). While the involvement of HSP70 and HSP90 in DENV binding to host cells has been reported (70–72), these proteins are not involved in internalization of virus into the host cell (73).

As mentioned before, TIM/TAM family receptors have been implicated in flavivirus entry. DENV express on its surface phosphatidylserine (PS) and phosphatidylethanolamine (PE) molecules. Both PS and PE are known to directly interact with TIM/TAM receptors and DENV is able to enhance its entry by exploiting these interactions (74).

After binding to cellular receptors, internalization of viral particles occurs. For DENV, internalization occurs via pHdependent endocytosis. Several endocytosis pathways are currently known, but clathrin-mediated endocytosis is the main pathway for DENV (75). The DENV use of clathrin-mediated endocytosis was demonstrated in C6/36 mosquito cells by biochemical inhibition of cell receptors (76), and in several human cell lines through siRNA silencing of genes associated with clathrin-mediated receptors (75, 77, 78). While this inhibition and specific gene silencing resulted in a decrease in viral load, a complete inhibition was not achieved, suggesting the existence of alternative entry pathways in mosquito and mammalian cells.

In addition to the exploitation of clathrin-mediated endocytosis, the host immune system can also promote viral entry (79). This phenomenon, known as antibody-dependent enhancement (ADE), was first described in 1964 by Hawkes (80) for WNV and JEV, and observed for DENV more than a decade later (81). Antibody-virus complexes are internalized by phagocytosis via Fc gamma receptors (FcγR) into macrophages, monocytes and dendritic cells (82) (**Figure 1**), thus facilitating virus entry (83). It has recently been shown that ADE increases membrane fusion activity, promoting DENV entry (79). Moreover, prM antibodies have the capacity to convert noninfectious, immature DENV particles into infectious particles and enhance their infectivity to levels comparable to wild-type

virus (50). ADE has been linked to the observation that one flavivirus infection can enhance another (84). However, a recent study showed that ADE is dependent on the level of neutralizing antibodies, particularly IgG and IgM (85); only patients with a low level of neutralizing antibodies showed enhancement of DENV infection (85). Antibodies, even at low concentration, against the EDIII domain were able to block viral entry of the four DENV serotypes without inducing antibody-dependent enhancement (86). However, high IgG titres were observed in patients with ADE following DENV infection, in particular IgG1 levels were the highest in patients with dengue fever or shock syndrome (87). ADE has been recently identified as consequence of sensitisation with Dengvaxia quadrivalent vaccine, leading to severe vaccine-enhanced disease resulting in hospitalization (88).

# JAPANESE ENCEPHALITIS VIRUS

Globally, Japanese encephalitis is the most clinically important arboviral encephalitis, with an estimated annual prevalence of up to 50,000 cases (89). As is the case with most arboviral encephalitic infections, humans are dead-end hosts unable to develop a sufficiently high viremia to transmit to feeding mosquitos. The majority of JEV infections are asymptomatic. Approximately a third of clinical cases are fatal and half of survivors have neurological or neuropsychiatric sequelae with symptoms resembling Parkinsonian movement disorders, poliomyelitis-like paralysis or impaired cognition (90). Disease is most common in children up to 14 years of age. JEV has been expanding its endemic areas in Asia (91) and poses an unpredictable and emerging global threat.

In humans, JEV has been found in different anatomical compartments and a variety of cell types is able to support its replication. These include endothelial cells, granulocytes, dendritic cells, macrophages and cells of the CNS including astrocytes, neurons and microglia (92). The virus spreads from dermal tissues (93) to lymphoid organs (94) and during the acute stage of infection can be found in blood (95). Although highly neuroinvasive, the mechanism of JEV entry into the CNS is unclear. Transport along the olfactory nerve and across the blood brain barrier have been implicated in JEV invasion of the CNS (96, 97). Studies in rodent models indicate that the blood brain barrier is disrupted following neuroinvasion (98), and might be a consequence of invasion rather than an entry route. Once in the brain, JEV can infect pericytes (99), astrocytes (100) and microglia (101), and has a predilection for developing neurons and neuronal progenitors (102, 103). As described below, a number of receptors mediate entry into different cells types. The distinctive neuronal tropism suggests the existence of JEV-specific receptors in the CNS, but their nature remains elusive (104).

In vitro studies on mouse neuroblastoma cells indicate heat shock protein (HSP) 70 as a putative entry receptor present on neuronal cells (105). This has not been corroborated by in vivo experiments, but in human hepatoma Huh7 cells, HSP70 is required for entry (106). Recently, a member of the HSP70 family, glucose-regulated protein (GRP) 78, has been implicated in JEV entry into Neuro2a and BHK-21 cells (107, 108). In addition to HSP70 and GRP78, HSP90β also interacts with E protein and may be used by JEV to enter mammalian cells (109). Another member of the HSP70 family is heat shock cognate (HSC) protein 70. HSC70 has been suggested to be a receptor for entry into mosquito cells (110). HSC70 isoform D is essential for clathrin-dependent endocytosis of JEV into C6/36 cells (111). Clathrin dependence seems to be critical for JEV entry into mammalian cells with the exception of neuronal cells (112), where JEV internalization into rodent neuroblastoma cell lines has been shown to be clathrin-independent (113, 114) and independent of HSP70 family proteins. JEV can enter human neuronal cells by caveolin-mediated endocytosis (115), a process that is receptor-independent (116). Interestingly, JEV has been shown to utilize the dopaminergic signal transduction pathway to increase neuronal susceptibility to infection (117). Infection of human dopaminergic neuroblastoma cells in vitro leads to increased levels of secreted dopamine and activation of the phospholipase C cascade. The latter enhances formation of structures known as focal adhesions on the cell surface and increases JEV binding and entry. One of the main components of focal adhesions is αvβ3 integrin that recruits vimentin to the cell surface (118), and is involved in JEV binding and infection of BHK-21 cells (18). Vimentin is a putative JEV receptor (119, 120). Thus, by signaling through dopamine D2 receptors and activating the phospholipase C cascade, JEV induces recruitment of surface molecules that enhance and propagate infection in adjacent cells. Enhanced infection of dopaminergic neurons also explains why JEV is predominantly found in brain areas rich in these cells including the thalamus and the midbrain (121, 122). Whereas JEV infection of neurons may be most directly relevant for disease, other cells types are also likely to have an important role in the disease process. Microglial cells may be a viral reservoir due to long-term, high level of virus production in these cells (123). CD4 has been identified as a major receptor for JEV entry into microglia (124). Presumably, CD4 can be used by JEV to enter other CD4 positive cells such as T cells, macrophages or dendritic cells. Published data are scarce, however, JEV productive infection of splenic macrophages and T cells has been reported in a mouse model of infection (125). T lymphocytes have also been reported as a reservoir of latent JEV in asymptomatic children following recovery from acute infection (126). The involvement of CD4 in microglial cell entry has not been reported for any other flavivirus, however CD4 is the main receptor for retroviral entry and is primarily localized in lipid rafts (127). As mentioned above, HSP70 in lipid rafts is involved in JEV entry into human cells and in general lipid rafts play a critical role in JEV entry (128, 129). Moreover, lipid rafts, as well as clathrin-coated pits and caveolae, contain sphingolipids such as sphingomyelin (SM) that is involved in JEV attachment and entry (130). Studies in SM synthase 1 deficient mice infected with JEV showed a reduction in disease, indicating a role for SM in JEV infection models (130).

Despite advances in identification of new receptors associated with JEV entry and its clear tropism for neuronal cells in the CNS, the identity of a specific neuronal receptor remains elusive. Notably, JEV has the ability to infect cells in the absence of above mentioned putative receptors although at a reduced rate (104). This suggests that the entry process involves multi protein interactions with high degree of redundancy and a single, specific entry receptor might not exist. Alternatively, the inability to identify such a receptor highlights the limitations of in vitro systems commonly used to investigate virus-cell interactions.

# ZIKA VIRUS

ZIKV is a mosquito-borne emerging pathogen that poses significant public health concerns due to recent rapidly expanding outbreaks. ZIKV was relatively unknown until 2007, when an outbreak occurred in Yap Island (Micronesia) (131). The virus was first isolated in the Zika Forest in Uganda from a rhesus monkey in 1947. In 1948 a second isolate from Ae. africanus mosquitoes was obtained from the same forest (132). Prior to the recent serious outbreak in French Polynesia, New Caledonia, the Cook Islands and Easter Island in 2013 and 2014 (133), Zika has not been reported to cause significant disease. Data from French Polynesia during the ZIKV epidemic documented the occurrence of Guillain-Barre syndrome and other neurological complications (134). The pathogenesis of ZIKV infection is poorly understood and involves a multifaceted interaction between viral and host factors. ZIKV has shown a significant tropism to the CNS and causes neurodegeneration, particularly of neural progenitor cells (135–137). ZIKV is also the only flavivirus known to have teratogenic effects in humans, including microcephaly, intracranial calcification and fetal death (138). As a result, the World Health Organization announced in 2016 that the ZIKV outbreak was a health emergency of international concern (139). Like other flaviviruses, ZIKV likely enters host cells through endocytosis instigated by an interaction of E glycoprotein with cell surface receptors. Identification of the entry receptor(s) for ZIKV is essential to understanding viral tropism and pathogenesis, and could lead to the development of novel therapeutics to treat the infection.

The first barrier for the virus to enter the host cell is the skin epidermis. ZIKV is transmitted by Aedes spp. mosquitoes, which deposit virus in the epidermis and dermis during the blood meal. Both dermal fibroblast and epidermal keratinocytes are permissive to ZIKV infection as are skin dendritic cells. Several entry receptors including the innate immune receptor DC-SIGN, transmembrane protein TIM-1 and TAM receptors (TYRO3, AXL, MER), have been shown to facilitate entry and enhance ZIKV infection (29). RNA silencing of TIM-1 and AXL in subsets of human skin cells showed a significant reduction in ZIKV titre in AXL knockdown, and in double AXL and TIM-1 knockdown, indicating that AXL is a major receptor for ZIKV entry at least in human skin cells. However, a recent study (43) investigating different infection routes of ZIKV, including subcutaneous, transplacental, vaginal, and intracranial infections in wild-type and TAM receptor null mice, showed no difference in viral titres. TAM receptors, at least in mice, are therefore not essential for ZIKV infection. Interestingly, WNV infection of neurons can be enhanced in mice lacking AXL and MER. This increase in infectivity was associated with changes in blood brain barrier permeability (140), suggesting that AXL and MER do not serve exclusively as receptors and might have other roles in WNV infection of the brain.

To reach the fetal brain, ZIKV must first be transported to the fetal circulation, and cross the placental barrier. The placental barrier is composed of placental barrier cells, trophoblasts and fetal endothelial cells, which separate the fetal blood in capillaries from maternal blood. ZIKV has been reported in the amniotic fluid of fetuses in Brazil (141). This observation strengthened the association of ZIKV with microcephaly in neonates. Moreover, it has been shown that microcephaly caused by maternal viral infection in mice could result from direct viral infection of the fetus via the trans-placental route as well as from a placental inflammatory response that affects fetal development (142). ZIKV can efficiently infect fetal endothelial cells, whereas WNV and DENV do not, highlighting ZIKV unique tropism among flaviviruses (143). These differences between flaviviruses are due to ZIKV ability to efficiently use AXL receptor to enter fetal endothelial cells (143).

TIM-1 was also observed to have an important role in placental entry of ZIKV (144). ZIKV was able to infect different human primary placental cell types and explants from chorionic villi. AXL, TYRO3, and TIM-1 were present in the primary placental cells and are found at the uterine-placental interface. Particularly high expression of TIM-1 has been observed in cells where maternal blood perfused placenta including basal decidua and neighboring chorionic villi. Expression of AXL and TYRO3 varied with explant donor, gestational age and cell type. Specific pharmacological inhibition of TIM-1 by duramycin (145) could inhibit ZIKV infection at the uterine-placental interface, indicating that TIM-1 is a putative receptor for ZIKV placental cell entry. However, the role of AXL, variation in the expression of AXL and TYRO3 in pregnant women and whether TIM-1 is the sole receptor for ZIKV infection of the placenta need further study.

Numerous studies on ZIKV infection in mice having defective interferon signaling, including IFNα/β knockout mice (146, 147), double knockout of IFNα/β and IFNγ (148), and triple knockout of IRF-3,-5,-7 (136, 149) showed viremia, microcephaly and death in young mice and viremia with recovery in adult mice. However, cell death and reduced proliferation was observed for adult neural stem cells (136) suggesting possible long term effects in adult brain followin ZIKV infection. ZIKV also infects other cell types, especially in the eye. ZIKV-inoculated mice develop ocular defects including conjunctivitis, pan uveitis, and infection of the optic nerve, cornea, iris, and ganglion and bipolar cells in the retina (150). AXL is expressed at high levels in retinal progenitor cells (151) suggesting a possible role in ZIKV infection of ocular cells. However, the ocular abnormalities were shown to be independent of AXL or MER, given that AXL−/−, MER−/−, and AXL−/− MER−/− double knockout mice sustained levels of infection similar to those of control animals. Nevertheless, AXL might have a role in ZIKV infection of glial cells via Gas6 mediated activation of AXL kinase (152).

In vitro and in vivo systems to study ZIKV infection of neural cells have been developed.  Microcephaly in these models is associated with inflammation, reduced proliferation of NPCs and neuronal cell death. ZIKV-BR can infect mouse fetuses and infection of pregnant mice also causes disease in embryos with intrauterine growth restriction, including signs of microcephaly (154). This study also demonstrated that ZIKV-BR infects human cortical progenitor cells, increasing the rate of cell death. ZIKV was found to directly infect human NPCs with high efficiency providing a plausible explanation for the observed developmental phenotypes and associated teratogenicity in the neonatal brains (137). Based on previous studies (29, 151, 152), AXL is a strong candidate receptor for the entry of ZIKV into cells of the developing brain. The potential role of AXL to facilitate ZIKV infection of the neonatal brain was explored by determining, at a single cell level, RNA expression profiles in the developing human cerebral cortex (151). The study revealed a higher expression of AXL in the radial cells and neural stem cells of the developing brain throughout neurogenesis and in capillaries and astrocytes. However, loss of AXL expression following CRISPR/Cas9 gene editing, did not affect ZIKV infectivity into hNPCs or cerebral organoids (155).

As is the case with JEV, the identity of the receptors involved in ZIKV receptor mediated endocytosis remains to be elucidated. There might be tissue specificity in receptor mediated viral entry, with variation in receptor repertoire in the skin, placenta, neurons, and other cell types. Alternatively, given ZIKV unique ability to cross the placenta and infect developing neurons in the fetal brain, there might be some as yet unidentified receptors facilitating this process.

# YELLOW FEVER VIRUS

YFV is the prototype and namesake virus of the Flavivirus genus; flavi means yellow in Latin. When infecting humans YFV replicates in liver, heart, kidneys, and lungs causing a broad spectrum of clinical symptoms. These vary from asymptomatic infection to renal and hepatic failures with severe haemorrhagic disease (156). A live attenuated YFV vaccine 17D was created over 70 years ago and has been used safely in over 500 million people. The parent strain of 17D is the virulent Asibi strain (157) isolated in Africa in 1927. 17D was passaged more than 230 times in mouse and chicken embryonic tissue. The adaptation of 17D to grow in tissue culture resulted in loss of viscerotropism, neurotropism, and mosquito tropism (156), making it an ideal candidate for a vaccine. The genome of both strains has been sequenced (158). The extensive passage history gave rise to 68 nucleotide mutations and 32 amino acid substitutions. Most of the genetic differences occur in the envelope (E) protein gene (157). Interestingly, the molecular determinants and mechanisms of this attenuation remain largely unknown. It has been suggested that the differences in the E protein and its involvement in cell entry are determinants of the difference in pathogenicity between the 17D and Asibi strains (159, 160). Mutations in the E gene have been suggested to allow the 17D strain to bind and enter hosts cells more efficiently.

YFV shares genome organization and entry by clarithinmediated endocytosis (CME) with most of the other flaviviruses. During YFV infection, the E protein binds to an unknown entry receptor that traffics the virion to endosomes. Similarly to other flaviviruses, increase in acidification of the endosome results in conformational changes in the E protein, membrane fusion and nucleocapsid release into the cytoplasm (156). The vaccine strain uses a clathrin- and caveolin-independent, but dynamin-2-dependent, pathway for infection (160). Dynamin-2 is a GTPase involved in cleaving off endocytic vesicles from the plasma membrane (161). The entry pathway of the 17D strain was further characterized as Rac1, Pak1, and cortactin independent (160). Clarithin-independent entry has been reported to mediate the internalization of a variety of viruses, such as rotavirus, human rhinovirus, influenza, and interestingly, JEV vaccine strain in neuronal cells (114, 162–164). Cells infected with 17D have been found to produce more viral RNA and INF-β, IL-29, ISG56, CCL5, and CXCL10 mRNA than those infected with the parental Asibi strain. In addition, 17D infected cells secrete INF-β, whereas cells infected with the Asibi strain do not. Virus entry through a clathrin-independent pathway allows for more efficient virion delivery into endosomes or protection from degradation, relative to entry via the classical clathrin-mediated route. This former entry route has been suggested to allow for a higher amount of viral RNA released into the cytoplasm (160). Viral RNA in the cytoplasm is detected by RIG-I, MDA5 and TLR7 (165), triggering strong innate immune responses. The Asibi strain on other hand, replicates at lower levels and inhibits the innate immune system. This difference in entry mechanism has been suggested to account for the differences in cytokine response between the two YFV strains, though further mutations in other proteins, such as NS2A could also be involved.

# CONCLUSIONS

Glycoprotein E is responsible for receptor-mediated attachment of flaviviruses to the host cell and membrane fusion. Although E protein of different flaviviruses share approximately 40% sequence identity (e.g., DENV and TBEV), their overall structural features are almost identical and this is assumed to apply to all flaviviruses (166). Cell entry is facilitated by a conserved peptide of 16 amino acids, located in E-DII region of the envelope glycoprotein (167). This conservation, coupled with highly organized conformational changes upon exposure to low pH (168), suggests evolutionary constraints allowing flaviviruses to enter both mammalian and arthropod cells. Yet, flavivirus receptors show diversity and significant cell type specificity. It is not unusual that a single molecule can bind to variety of targets as exemplified by immunoglobulins. However, their diversity and specificity are governed by V(D)J recombination, while the flaviviral glycoprotein E is conserved. The flavivirus infection is a consequence of multiple complex interactions between the virus and the target cell. It is clear that the flavivirus can exploit different endocytic routes that can be either clathrin or caveolae dependent or independent. The neurotropism of specific flaviviruses raises the question, is there a single specific neuronal receptor? What is the identity of this receptor and is the same receptor being used by all encephalitic flaviviruses? Another unresolved question is whether all flaviviruses share the same features of infection in the developing brain, or whether viruses such as microcephalycausing ZIKV, exhibit a different infection pattern. It is also relevant to note that the expression of entry receptors (e.g., CLRs or TAM) does not account for flavivirus tropism and cellular models lacking those receptors are still permissive to

# REFERENCES

infection. Identifying the relevant entry receptors is essential to deciphering the mechanisms of pathogenesis, tropism and viral biology. A better understanding of those processes will uncover new strategies for designing therapeutics and vaccines against flaviviruses.

# AUTHOR CONTRIBUTIONS

ML, DN, JR-A, and LK wrote sections of the manuscript, JF and LK edited and critically evaluated the manuscript.

for cellular attachment and infection. J Virol. (2006) 80:1290–301. doi: 10.1128/JVI.80.3.1290-1301.2006

regulation of actin reorganization. Future Microbiol. (2016) 1:1227–48. doi: 10.2217/fmb-2016-0002

alters proliferation. Cell Stem Cell (2016) 9:593–8. doi: 10.1016/j.stem.2016. 08.005

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Laureti, Narayanan, Rodriguez-Andres, Fazakerley and Kedzierski. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Cell-Mediated Immune Responses and Immunopathogenesis of Human Tick-Borne Encephalitis Virus-Infection

Kim Blom1 \*, Angelica Cuapio1 , J. Tyler Sandberg1 , Renata Varnaite1 , Jakob Michaëlsson1 , Niklas K. Björkström1 , Johan K. Sandberg1 , Jonas Klingström1 , Lars Lindquist 2,3, Sara Gredmark Russ 1,2 and Hans-Gustaf Ljunggren1 \*

#### Edited by:

Alan Chen-Yu Hsu, University of Newcastle, Australia

#### Reviewed by:

Alessandro Marcello, International Centre for Genetic Engineering and Biotechnology, Italy Manuela Zlamy, Innsbruck Medical University, Austria

#### \*Correspondence:

Kim Blom<EMAIL_ADDRESS>Hans-Gustaf Ljunggren<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology

Received: 27 March 2018 Accepted: 03 September 2018 Published: 26 September 2018

#### Citation:

Blom K, Cuapio A, Sandberg JT, Varnaite R, Michaëlsson J, Björkström NK, Sandberg JK, Klingström J, Lindquist L, Gredmark Russ S and Ljunggren H-G (2018) Cell-Mediated Immune Responses and Immunopathogenesis of Human Tick-Borne Encephalitis Virus-Infection. Front. Immunol. 9:2174. doi: 10.3389/fimmu.2018.02174 1 Department of Medicine Huddinge, Center for Infectious Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden, 2 Department of Infectious Diseases, Karolinska University Hospital, Stockholm, Sweden, 3 Unit of Infectious Diseases, Department of Medicine Huddinge, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden

Tick-borne encephalitis virus (TBEV) is a flavivirus that belongs to the Flaviviridae family. TBEV is transmitted to humans primarily from infected ticks. The virus causes tickborne encephalitis (TBE), an acute viral disease that affects the central nervous system (CNS). Infection can lead to acute neurological symptoms of significant severity due to meningitis or meningo(myelo)encephalitis. TBE can cause long-term suffering and has been recognized as an increasing public health problem. TBEV-affected areas currently include large parts of central and northern Europe as well as northern Asia. Infection with TBEV triggers a humoral as well as a cell-mediated immune response. In contrast to the well-characterized humoral antibody-mediated response, the cell-mediated immune responses elicited to natural TBEV-infection have been poorly characterized until recently. Here, we review recent progress in our understanding of the cell-mediated immune response to human TBEV-infection. A particular emphasis is devoted to studies of the response mediated by natural killer (NK) cells and CD8 T cells. The studies described include results revealing the temporal dynamics of the T cell- as well as NK cell-responses in relation to disease state and functional characterization of these cells. Additionally, we discuss specific immunopathological aspects of TBEV-infection in the CNS.

Keywords: cell-mediated immunity, flavivirus, NK cells, T cells, tick-borne encephalitis, tick-borne encephalitis virus, viral immunopathogenesis

# INTRODUCTION

Tick-borne encephalitis virus (TBEV) is a flavivirus that belongs to the Flaviviridae family. Flaviviruses comprise many human pathogens including the commonly known Dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), Yellow fever virus (YFV), and Zika virus (ZIKV) (1). With respect to TBEV, three subtypes of the virus exist: European (TBEV-Eu), Siberian (TBEV-Sib), and Far Eastern (TBEV-FE) (2).

**174**

TBEV is transmitted to humans primarily from infected ticks, mainly from the Ixodes family. The virus can also be transmitted from unpasteurized dairy products from infected livestock (3–5). Infection with TBEV causes tick-borne encephalitis (TBE), an acute viral infection that affects the central nervous system (CNS) with often severe long-term neurological consequences (3, 4, 6, 7). The first TBE-like disease was described as early as in the eighteenth century in Scandinavian church records (8). Traditionally, the disease is described as a syndrome with a biphasic course beginning with an influenza-like illness followed by a second neuroinvasive phase with neurological symptoms of variable severity, ranging from meningitis to severe meningoencephalitis with or without myelitis (3, 4, 6) (**Figure 1**). It shall be noted, however, that also monophasic patterns of disease development have been described (9). Upon infection, virus is detected in serum in the first phase of the disease but rarely in the second phase (10).

Due to increased geographic distribution of TBEV as well as a marked increase in morbidity in many areas, TBEV-infection has more recently caught attention as a public health problem. TBE is now observed in large parts of Europe as well as in northern Asia (3, 4). The main risk areas for TBE in Europe are primarily parts of central and eastern Europe as well as the Baltic and Nordic countries. With respect to central Europe, risk areas extend from Switzerland in the west into northern Italy and the Balkan countries (11). The incidence of TBEVinfection in endemic countries varies from year to year (12– 14), however, an overall upsurge has been reported in certain parts of Europe, including the borders between Austria, Slovenia, and Italy (15, 16). These changes have been related to climatic, ecological, environmental, and socioeconomic factors that all can lead to an increased risk of human exposure to infected ticks (17–20).

The total number of annual cases has been estimated to be up to 13,000, and as such the infection constitutes the most important tick-borne viral disease (4). More than 30% of patients with clinical symptoms from TBEV-infection develop prolonged sequelae, some of which may become life-long including neuropsychiatric symptoms, severe headaches, and a general decrease in quality of life (3, 4, 6, 7). The mortality rates differ between the strains. Infection with the Far Eastern strain (TBEV-FE) has a mortality rate of 5–35%, whereas the other two strains (TBEV-Eu and TBEV-Sib) have mortality rates of 1–3% (3, 4). There is no specific treatment (e.g., antivirals) for TBE; rather, symptomatic treatment is the only available option (3, 4, 9).

Of importance, TBE may be prevented by vaccination. There are in total four licensed vaccines to TBE. Two vaccines based on TBE-Eu subtype are licensed in Europe and two are licensed in Russia. Additionally, a TBEV-vaccine based on the Far Eastern subtype is produced and marketed in China. All vaccines are based on formalin-inactivated strains of TBEV (3, 4, 21, 22). In areas where the disease is highly endemic, WHO recommends that vaccination should be offered to all groups above 1 year of age (4, 23). Primary vaccination against TBE includes three doses of the vaccine within the first year, followed by revaccinations every third to fifth year to maintain immunity. Vaccination is generally considered effective and TBE incidence has decreased substantially in TBEV-endemic regions with successful vaccination-programs (24). Randomized controlled trials in large populations have shown high immunogenicity with often-strong antibody production and acceptable rates of adverse events following vaccination (25–28). Breakthrough TBE after vaccination is generally considered rare (4). However, over the last years, vaccine failures have been reported, in particular in middle-aged and elderly individuals, who have completed the primary vaccination (29–31).

Infection with TBEV triggers humoral and cell-mediated immune responses. A confirmed diagnosis of TBE is established by the detection of specific IgM and IgG in serum. IgM antibodies have been observed in sera very early in symptomatic TBE disease, whereas IgG antibodies peak in the convalescent phase of disease (32). IgG antibodies can persist over lifetime and prevent TBE (4, 33). Early after clinical disease onset, TBEV-specific antibodies can also be found in the cerebrospinal fluid (CSF) (32, 33). In contrast to the humoral immune response, the cellmediated immune responses elicited to natural infection have been rather poorly studied until recently. The latter responses may contribute both to host resistance against infection as well as to pathological reactions affecting the target organ of the virus, i.e., primarily the CNS.

Here, we review recent progress in studies of the cell-mediated immune response to human TBEV infection. A particular emphasis is devoted to natural killer (NK) cell- and T cellmediated responses. Responses to TBEV are discussed in context of cell-mediated immune responses toward other flavivirus infections. We also discuss some immunopathological aspects of TBE with a particular emphasis on cell-mediated immune reactions in the CNS. Cell-mediated immune reactions in the CNS may contribute to neural damage with severe consequences of brain function, and could in the worst cases lead to fatal outcome. First, however, some aspects of the TBEV itself are covered.

# TBEV AND OTHER FLAVIVIRUSES

All flaviviruses are enveloped and have a positive-sense single stranded RNA genome, which per se acts as messenger RNA upon entrance in the host cell. The RNA encodes for a polyprotein, which is co- and post-translationally cleaved by viral and cellular proteases into three structural proteins; capsid (C), precursor membrane (prM) and envelope glycoproteins (E), and seven non-structural proteins including NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (34). Flaviviruses enter the cell through clathrindependent endocytosis upon attachment of the E protein to a receptor. Heparan sulfate has been identified as such receptor for TBEV (35); however, there are most likely also other yet not identified receptors for the virus. Following cell entry, the flavivirus is delivered to endosomes (36), in which the low pH triggers the E protein to fuse with the endosomal membrane and the nucleocapsid is released into the cytosol. The assembly of immature flavivirus virions (36–38), including TBEV (39, 40), occurs in the ER, and the viral particles are transported to the

Golgi apparatus. The virion particles are immature until the envelope protein is rearranged and prM is cleaved by the host enzyme furin in the acidic environment in the Golgi apparatus. Immature particles are non-infectious and proteolytic cleavage of prM is a prerequisite for viral infectivity. However, studies have shown that complete cleavage of prM is not necessary for viral infectivity (41–43).

In general, species of flaviviruses have many similarities, but their preferred host cells differ. TBEV is shown to replicate 10,000-fold higher in human neuronal cells as compared with epithelial cells (44). A similar infection pattern has recently been shown for ZIKV (45).

# NK CELLS

NK cells are innate lymphocytes, though recent studies have revealed "adaptive" features of these cells (46, 47). They are perhaps best known for their ability to kill virus-infected and tumor cells. NK cell cytotoxicity is regulated by the expression of numerous activating and inhibitory receptors that sense ligands on neighboring healthy and altered cells. Several activating receptors recognize molecules that are up-regulated on cells during conditions of cellular stress, such as viral infection and transformation [reviewed in (48, 49)] whereas many inhibitory receptors, e.g., human killer cell Ig-like receptors (KIR) bind to HLA class I molecules. Additionally, NK cells have an important role in producing cytokines and chemokines, as well as by other means interacting with other immune and non-immune cells.

Human NK cells are classically defined as CD3− (T cell receptor negative), CD56+ cells and represent about 15% of peripheral blood lymphocytes. These cells have for long been divided into two main subsets; CD56bright and CD56dim cells (50). The CD56bright NK cells are thought to be less mature and are commonly known as primarily cytokine-producing cells with low cytotoxic ability, whereas CD56dim NK cells are best known for their potent cytotoxic activity upon target cell recognition (51). However, the latter are also ample cytokine producers upon interaction with target cells (51). Both "natural" and antibody-mediated NK cell cytotoxicity is mediated by exocytosis of cytoplasmic granules containing perforin and granzymes (52). Cytotoxic responses may also to various degrees involve TRAIL- and Fas-ligand-mediated induction of apoptosis (53, 54). CD56dim NK cells frequently express CD16 (FcγRIII), KIRs, and CD57, which regulate their function and define distinct stages of NK cell maturation (55), whereas CD56bright NK cells largely lack expression of these molecules.

# THE ROLE OF NK CELLS IN HUMAN TBEV INFECTION

Direct evidence for a protective role of NK cells has been found in experimental models of several viral infections, including cytomegalovirus and influenza, and a number of studies have indicated that they play a role also in protection against viral infections in humans. For example, NK cell-deficiencies in humans result in severe herpes virus infections in childhood and adolescence (56). NK cells may also have a protective role in human TBEV-infection. At the same time, responses mediated by these cells may be associated with development of symptoms in the course of TBEV-infection. Although there is only little known about NK cells in TBE, NK cells have been detected in CSF of patients with TBE (57), an observation that indicates transmigration through the blood brain barrier (BBB).

To gain a better understanding of the NK cell response to human TBEV-infection, we recently performed a longitudinal study providing an in-depth analysis of the human NK cell response to acute TBEV-infection in a well-defined cohort of TBE patients. The study had an emphasis on NK cell responses during the second stage of disease from which clinical samples were available. NK cell activation, as measured by expression of the proliferation marker Ki67, was apparent at the time of hospitalization (58) (illustrated in **Figure 2**). Concomitant with the increase in NK cell activation in the acute stage of disease, augmented levels of IL-12, IL-15, IL-18, IFN-γ, and TNF were detected in patient plasma. In parallel with high levels of activation, the activated NK cells expressed less perforin, granzyme B, and Bcl-2. By 3 weeks after hospitalization, the NK cell activation decreased to levels seen in healthy controls. This TBEV-induced NK cell activation was restricted predominantly to more differentiated CD57+CD56dim NK cells. Functionally, CD56dim NK cells responded poorly to target cells at the time of hospitalization, but they recovered functional capacity to healthy control levels during the convalescent phase. The poor functionality of NK cell responses was exclusive for target cell recognition, since NK cell responses induced by IL-18 and IL-12 remained unchanged throughout the disease (58).

# NK CELL RESPONSES TOWARD OTHER ACUTE FLAVIVIRUS INFECTIONS IN HUMANS

To be able to interpret the above-mentioned NK cell responses to acute TBEV infection, it is important to understand NK cell responses to other acute virus infections, including acute flavivirus infections. In this respect, NK cells have to various extent been studied ex vivo in other acute flavivirus infections, including DENV (60, 61) and WNV (62, 63) as well as hepatitis C virus (HCV), a distant relative within the Flaviviridae family (64, 65). NK cells have also been studied after vaccination with the live attenuated YFV 17D vaccine (66–68). They have been suggested to influence disease severity and outcome, and to contribute to viral control in these infections, even though underlying mechanisms are not well studied.

In this context, it was observed that the absolute number of NK cells in patients with a mild form of infection with DENV was higher as compared to patients with the more severe form of the infection, dengue hemorrhagic fever (DHF) (69). Reduced numbers of NK cells in the circulation may be indicative of migration toward peripheral target organs. Furthermore, a higher frequency of NK cells expressing CD69 early on during the infection in children developing severe DHF has been reported (70). In recent studies of DENV-infection, we found NK cells to be robustly activated during the first week after symptom debut. Here, the response seemed to be confined largely to the CD56bright subset of NK cells and less mature CD56dim NK cells (our own unpublished studies). Noteworthy in the context of acute TBEV infection, activation of NK cells may also occur very early, even before the onset of symptoms. This possibility is supported by the observation that the highest levels of NK cell activation in most TBEV infected patients were observed already at the time of hospitalization (58). This notion is corroborated in studies of YFV vaccinated individuals. An early response by NK cells was observed in study subjects vaccinated with YFV-17D, where expression of both Ki67 and CD69 was increased on NK cells as early as 1 week after vaccination (66). Accumulation of adaptive-like NK cells expressing the activating receptor CD94/NKG2C has been reported in some human viral infections (71–73); however, no expansion of NKG2C+ NK cells in blood has been observed in TBEV-infection or any other flavivirus infection (58). It can, however, not be excluded that this type of expansion could occur locally, e.g., at the site of infection.

In addition to the observed activation of NK cells in vivo in different flavivirus infections, a protective role of NK cells is also supported by in vitro data.  In addition, flavivirus-infected target cells have been reported to display virus-mediated up-regulation of MHC class I (74), and could thereby theoretically evade lysis from NK cells by engaging inhibitory receptors. The dampened NK cell responses to target cells in acute TBEV-infection further support this notion (58). On the other hand, increased MHC class I expression could result in enhanced T cell responses. In such a scenario, one may speculate that flaviviruses may have been driven more toward escape from innate immunity rather than from adaptive T cell immunity (75).

# T CELLS

In contrast to NK cells, CD8 and CD4 T cells recognize specific foreign peptide sequences presented by HLA class I and II molecules, respectively (76). Like NK cells, major functions of CD8 T cells are to kill infected cells through the release of perforin and granzymes, and to secrete cytokines such as IFN-γ, TNF, and IL-2. The cytotoxic T cell response to acute infection can typically be divided into three phases; priming and expansion, resolution and contraction, and memory formation. During the first phase, naïve CD8 T cells divide and differentiate into effector cells acquiring high cytotoxic ability (77). Following viral clearance, the effector T cell population contracts and the majority of the pathogen-specific T cells enter apoptosis. A small pool of pathogen specific T cells (5–10%) survives as memory cells in the third stage (78). Memory T cells are a principal component of immunity against intracellular pathogens such as viruses. They are distinguished by their capacity to survive long-term, and undergo rapid and robust proliferation and acquisition of effector function upon antigen re-exposure (78). Memory T cells can vary in their phenotype, localization, and function allowing them to protect the host against a broad array of potential insults.

Distinct stages of CD8 T cell differentiation are defined by the expression of specific surface markers such as the isoforms of CD45 and expression of the homing receptor CCR7. These stages of differentiation are useful in the characterization of responses to, e.g., anti-viral responses. The set stages define CD45RA+CCR7+ as naive (TN), CD45RA−CCR7+ as central memory (TCM), CD45RA−CCR7− as effector memory (TEM), and CD45RA+CCR7− as effector memory RA (TEMRA)

CD8 T cells (79, 80). TCM cells primarily reside in secondary lymphoid organs, possess the greatest proliferative potential among the memory T cell subsets and can rapidly expand and differentiate following re-challenge. TCM cells have higher sensitivity to antigenic stimulation, are less dependent on co-stimulation and provide better feedback to DCs and B cells compared to TN cells. TEM cells can migrate between tissues and secondary lymphoid organs and provide immune surveillance.

# THE ROLE OF T CELLS IN HUMAN TBEV INFECTION

Due to difficulties in identifying the acute phase of viral infection in humans, T cell responses to viral infections have to a large extent been addressed in studies of pathogens causing chronic infections such as HIV-1, EBV, HCV, and CMV (81–85). Such responses can be very robust, as exemplified by the massive clonal expansion of antigen-specific CD8 T cells seen in many infections (83, 85). Based on these studies, it has also become clear that the resulting populations of human CD8 T cells display striking phenotypic differences, as determined by the expression profiles of surface markers (79–81). In contrast to many other infections, including some flavivirus infections (86–89), there are only few studies of T cell responses to TBEV-infection in humans. This hold true for acute as well as cases with prolonged TBE disease. Noteworthy, however, one report has shown that TBEV-specific CD4 T cells from naturally infected patients show a higher level of polyfunctionality in response to antigen in the convalescent phase of disease, as compared to TBE-vaccine specific T cells (90).

The general lack of studies more systematically characterizing the human T cell response to TBEV-infection prompted us to study the primary T cell-mediated immune response in patients diagnosed with TBE with a particular emphasis of CD8 T cells (59). Similar to our study on NK cells (58), the T cell study focused on responses during the second stage of disease from which clinical samples were available. During this phase, CD8 T cells were strongly activated, as detected by increased expression of Ki67, within 1 week of hospitalization (illustrated in **Figure 2**). A large part of these CD8 T cells expressed high levels of perforin and granzyme B, and low levels of the anti-apoptotic protein Bcl-2. In contrast to CD8 T cells, CD4 T cells showed only low or at most moderate levels of activation. The TBEV-antigen specific CD8 T cells had a TEM PD-1+ phenotype throughout the course of disease. TBEVspecific CD8 T cells were predominantly Eomes+Ki67+T-bet+ in the acute stage of disease. This pattern was replaced by an Eomes−Ki67−T-bet+ profile in the convalescent phase of disease. TBEV-specific CD8 T cells were mainly monofunctional in the acute stage of disease, and tended to become more polyfunctional in the convalescent phase when clinical symptoms retracted (59).

# T CELL RESPONSES TOWARD OTHER ACUTE FLAVIVIRUS INFECTIONS IN HUMANS

To be able to better interpret the above-mentioned T cell responses to acute TBEV infection, we compared the present results with T cell responses to other acute virus infections, including infections by other flaviviruses. The live attenuated YFV-vaccine strain can replicate after vaccination leading to a detectable viral load similar to a mild infection. Thus, this vaccine can be utilized as a controlled model to study mild acute viral infection in humans. CD8 T cells become activated within 1–2 weeks after vaccination with the YFV vaccine (86, 91, 92). YFV antigen-specific CD8 T cells predominantly display a TEM PD-1 + phenotype, which transition into a TEMRA PD-1− memory phenotype (86). With respect to DENV-infection, a high level of functionality of DENV-specific T cells is associated with a better disease outcome (93). Similarly, patients hospitalized with (severe) TBE show a low level of T cell functionality in the acute stage of disease (59), indicating the importance of high function among virus-specific T cells for beneficial disease outcome. CD8 T cells have been shown primarily to respond with IFN-γ to JEV in asymptomatic JEV-exposed donors (87).

Activation of CD4 T cells with an optimal magnitude, specificity and kinetics may be a requirement for viral clearance and protective immunity. In the immune response induced by the YFV vaccine, activation of CD4 T cells (peak at 10 days after vaccination) precedes that of CD8 T cells, and this may be of importance to elicit strong immunological memory (86). Furthermore, CD4 T cell release of IFN-γ may have an impact on disease outcome since CD4 T cells, and not CD8 T cells, were shown to dominate the IFN-γ response in recovered Japanese encephalitis (JE) patients. In addition, a high quality polyfunctional CD4 T cell response can be associated with better disease outcome in JE patients (87). In murine models, a perforin-dependent mechanism by the CD8 T cells has been shown to clear WNV from infected neurons, thereby suggesting an immunopathological role of T cells in mice (88). In this context, it is of interest to note that TBEV-specific T cells have a high content of both perforin and granzyme B (59), but whether the same perforin-dependent mechanism is causing immunopathogenesis in acute infection with TBEV remains to be investigated.

# CROSS-REACTIVITY WITHIN THE FAMILY OF FLAVIVIRUSES

Immunological cross-reactivity between TBEV and other species within the flavivirus family may also contribute to disease (94, 95). Antibody-dependent enhancement (ADE) is a described phenomenon that can occur when non-neutralizing antibodies facilitate virus entry into host cells, leading to increased infectivity in the cells. ADE is commonly observed in vitro in cell culture-based models (96), but it is questioned as to which degree this phenomenon occurs in vivo. A recent study from non-human primates in vivo, did not observe increased ZIKV titers after prior infection with heterologous flaviviruses (97). Protective cross-reactivity of flaviviruses has been reported as well, opposing increased pathogenesis upon pre-exposure to other species of flaviviruses (98). TBEV has been suggested to cause both pathogenic and protective cross-reactivity. Polyclonal sera against members of the TBE serocomplex (including TBEV, Kyasanur Forest disease virus, Omsk hemorrhagic fever virus, and Langat virus) enhance viral replication of TBEV in vitro (96). However, it has recently been shown that antibodies generated from TBEV infection or from the TBEV vaccine can mediate cross neutralization against other, if not most, of the members of the TBE virus complex (99). Furthermore, sera from some individuals vaccinated against TBEV and JEV neutralized WNV, and the neutralization was enhanced by YFV vaccination in some recipients (95), altogether indicating that previous flavivirus exposure may sometimes provide a degree of protection to new flaviviruses.

Cell-mediated immunity and cross-reactivity caused by TBEV and other flaviviruses has been less well studied. Recently, a study demonstrated that JEV- and JE vaccine-specific T cells cross-react with DENV (87, 100). In line with this, it was also recently shown that vaccination with YFV vaccine could induce ZIKA-specific T cells, thereby suggesting cross-protection of flavivirus-specific T cells (101). The latter phenomena opened up a discussion as to the possibility of utilizing the YFV vaccine to protect against Zika virus infection (101). In the present context, it remains to be investigated whether YFV vaccination elicits protective cross-reactive immunity also toward TBEV.

# IMMUNOPATHOLOGICAL ASPECTS OF TBEV INFECTION IN THE CNS

In contrast to the significant interest in emerging infections such as the recent Zika pandemic [reviewed in (45, 102, 103)], studies of the immune response toward TBEV-infection as such, and TBEV-induced immunopathology in particular, have been rather limited.

Immune and none-immune mechanisms have been proposed contribute to the crossing of TBEV over the BBB and invasion of the CNS [reviewed in (104)]. Cytokines may facilitate this process. Cytokines such as TNF-a and IL-6 have an impact on endothelial cell permeability that may induce a BBB disruption (105, 106), leading to crossover of the virus into the CNS. A distinct mechanism by which the TBEV could possibly cross the BBB is the Trojan Horse mechanism (107), by which TBEV-infected immune cells such as dendritic cells, neutrophils, monocytes, macrophages, and T cells would migrate into the parenchymal compartment causing infection of neurons or other cells in the brain and the spinal cord. Yet, an alternative route is invasion via the olfactory epithelium (108, 109).

After the landmark discovery of the lymphatic system present in the meninges that connects the CNS to the peripheral blood (110, 111), the classical concept of the CNS as an immune privileged site has been replaced by a view of an immune regulated site. Hence, under normal conditions a continuous transmigration of lymphocytes, monocytes, DCs and macrophages occurs. They may serve to detect any kind of infection or injury in the brain [reviewed in (112)]. Similar to other CNS infections, increased frequencies of T cells have been reported in CSF of TBE patients (57). Hence, activated T cells are crossing the BBB in TBE; however, the role of T cells at this site not well understood (113, 114). On one hand they could contribute toward clearing viral infection but on the other hand they may mediate immunopathology within the CNS. Corroborating the latter speculations are findings in which granzyme B+ CD8 T cell infiltrates have been linked to cell-death in infected human neuronal tissue (113) and, in parallel, mice with CD8 T cell deficiency have been shown to have prolonged survival upon infection with TBEV compared to mice with adoptively transferred CD8 T cells to immuno-competent mice (114). Furthermore, studies of post-mortem tissue of TBE patients have shown a predominance of macrophages/microglia and CD3+ T cells (both CD4+ and CD8+) in brain parenchyma (113). As seen in other flavivirus infections, macrophages and microglia also play a role in tissue destruction in human TBE.

In relation to NK cells and their possible role in causing immunopathogenesis, it is of interest to note that also these cells have been detected in the cerebrospinal fluid (CSF), albeit in low numbers, in patients with severe TBE meningitis or encephalitis (57). Activated NK cells may be protective, but they may also, like T cells, take part in immunopathological reactions as they are known to participate in direct killing of infected cells, indirect killing through cytokines or chemokines, or by the recruitment of inflammatory cells into the tissues (115, 116). Although recent results support a role for NK cells in clinical TBEV-infection, more studies are needed to provide a better understanding of the role NK cells play in pathogenic processes of human TBE infection.

Knowledge and experience gained in the field of the immunopathogenesis of other diseases affecting the CNS and its immunological compartments could be helpful in understanding TBE-specific diseases patterns. For example, in multiple sclerosis (MS), an inflammatory disease with pathology affecting the CNS (117), the concentration of the Sphingosine-1-Phosphate (S1P) in CSF is elevated and S1P-signaling is altered. In MS, binding of S1P to S1P1-receptors expressed on lymphocytes leads to invasion of autoreactive T cells

## REFERENCES

into the CNS, the latter contributing to the hallmarks of the disease including demyelination and neurodegeneration (118). Interestingly, during the phase of acute infection in TBEV-infected patients, the levels of S1P in blood and CSF are highly elevated (119). This increase might promote a proinflammatory response. An increased production of extracellular S1P can be regulated by modulators of the S1P pathway, such as fingolimod, which is an immunomodulatory drug used in the treatment of MS (118). Therefore, therapeutic options used in other CNS diseases that share common immunopathogenic mechanisms with TBE could be used as models to aid in the development of new strategies for TBE treatment.

# CONCLUDING REMARKS

In the present review, we have focused our attention to recent insights into the cell-mediated immune response to human TBEV infection, with an emphasis on studies of NK cell and CD8 T cell mediated responses. Until recently, the latter have been poorly studied. As yet, however, much more needs to be learnt with respect to these responses and research in this area should be encouraged. We have also addressed some aspects of TBEV CNS pathogenesis, a process still far from understood in detail. Clearly, however, cell-mediated immune responses likely play an important role in this process. As TBE continues to be an increasing global health problem and challenge, much more research is needed into this emerging disease. Several areas of research of the TBEV itself, and the clinical disease TBE merit further studies. Not the least, the specific organ pathogenesis caused by TBEV and the immune response, including infiltrating immune cells, needs more investigation. Furthermore, the possibility of antiviral treatment and other possible treatment modalities needs much more thorough investigation to prevent disease development and the often severe sequeale following infection of humans with TBEV.

# AUTHOR CONTRIBUTIONS

KB, AC, and H-GL wrote the manuscript. All other authors provided valuable contributions and insights into the manuscript.

# FUNDING

The authors were founded by the Swedish Research Council, the Swedish Foundation for Strategic Research, Karolinska Institutet and the Stockholm County Council.

focus of infection. Eur J Epidemiol. (1996) 12:373–5. doi: 10.1007/BF00 145300

encephalitis vaccine FSME-IMMUN: results of two large phase 3 clinical studies. Vaccine (2006) 24:5256–3. doi: 10.1016/j.vaccine.2006.03.061

virus 17D as it matures from an effector- to a memory-type response. J Immunol. (2013) 190:2150–8. doi: 10.4049/jimmunol.1202234

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Blom, Cuapio, Sandberg, Varnaite, Michaëlsson, Björkström, Sandberg, Klingström, Lindquist, Gredmark Russ and Ljunggren. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Advances in Designing and Developing vaccines, Drugs, and Therapies to Counter ebola virus

*Kuldeep Dhama1 \*, Kumaragurubaran Karthik2 , Rekha Khandia3 , Sandip Chakraborty 4 , Ashok Munjal3 , Shyma K. Latheef 5 , Deepak Kumar 6 , Muthannan Andavar Ramakrishnan7 , Yashpal Singh Malik8 \*, Rajendra Singh1 , Satya Veer Singh Malik9 , Raj Kumar Singh10 and Wanpen Chaicumpa11*

*1Division of Pathology, ICAR-Indian Veterinary Research Institute, Bareilly, India, 2Central University Laboratory, Tamil Nadu Veterinary and Animal Sciences University, Chennai, India, 3Department of Biochemistry and Genetics, Barkatullah University, Bhopal, India, 4Department of Veterinary Microbiology, College of Veterinary Sciences and Animal Husbandry, Agartala, India, 5 Immunology Section, ICAR-Indian Veterinary Research Institute, Bareilly, India, 6Division of Veterinary Biotechnology, ICAR-Indian Veterinary Research Institute, Bareilly, India, 7Division of Virology, ICAR-Indian Veterinary Research Institute, Nainital, Uttarakhand, India, 8Division of Biological Standardization, ICAR-Indian Veterinary Research Institute, Bareilly, India, 9Division of Veterinary Public Health, ICAR-Indian Veterinary Research Institute, Bareilly, India, 10 ICAR-Indian Veterinary Research Institute, Bareilly, Uttar Pradesh, India, 11Center of Research Excellence on Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine SIriraj Hospital, Mahidol University, Bangkok, Thailand*

#### *Edited by:*

*Hiroyuki Oshiumi, Kumamoto University, Japan*

#### *Reviewed by:*

*Oscar Negrete, Sandia National Laboratories (SNL), United States Pei Xu, Sun Yat-sen University, China Yohei Kurosaki, Nagasaki University, Japan*

#### *\*Correspondence:*

*Kuldeep Dhama<EMAIL_ADDRESS>Yashpal Singh Malik<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology*

*Received: 01 April 2018 Accepted: 23 July 2018 Published: 10 August 2018*

#### *Citation:*

*Dhama K, Karthik K, Khandia R, Chakraborty S, Munjal A, Latheef SK, Kumar D, Ramakrishnan MA, Malik YS, Singh R, Malik SVS, Singh RK and Chaicumpa W (2018) Advances in Designing and Developing Vaccines, Drugs, and Therapies to Counter Ebola Virus. Front. Immunol. 9:1803. doi: 10.3389/fimmu.2018.01803*

Ebola virus (EBOV), a member of the family *Filoviridae*, is responsible for causing Ebola virus disease (EVD) (formerly named Ebola hemorrhagic fever). This is a severe, often fatal illness with mortality rates varying from 50 to 90% in humans. Although the virus and associated disease has been recognized since 1976, it was only when the recent outbreak of EBOV in 2014–2016 highlighted the danger and global impact of this virus, necessitating the need for coming up with the effective vaccines and drugs to counter its pandemic threat. Albeit no commercial vaccine is available so far against EBOV, a few vaccine candidates are under evaluation and clinical trials to assess their prophylactic efficacy. These include recombinant viral vector (recombinant vesicular stomatitis virus vector, chimpanzee adenovirus type 3-vector, and modified vaccinia Ankara virus), Ebola virus-like particles, virus-like replicon particles, DNA, and plant-based vaccines. Due to improvement in the field of genomics and proteomics, epitope-targeted vaccines have gained top priority. Correspondingly, several therapies have also been developed, including immunoglobulins against specific viral structures small cell-penetrating antibody fragments that target intracellular EBOV proteins. Small interfering RNAs and oligomer-mediated inhibition have also been verified for EVD treatment. Other treatment options include viral entry inhibitors, transfusion of convalescent blood/serum, neutralizing antibodies, and gene expression inhibitors. Repurposed drugs, which have proven safety profiles, can be adapted after high-throughput screening for efficacy and potency for EVD treatment. Herbal and other natural products are also being explored for EVD treatment. Further studies to better understand the pathogenesis and antigenic structures of the virus can help in developing an effective vaccine and identifying appropriate antiviral targets. This review presents the recent advances in designing and developing vaccines, drugs, and therapies to counter the EBOV threat.

Keywords: Ebola virus, Ebola virus disease, vaccines, prophylactics, drugs, therapeutics, treatment

# INTRODUCTION

Ebola virus (EBOV; *Zaire ebolavirus*) is the causative agent of a severe hemorrhagic fever disease, Ebola virus disease (EVD; formerly called Ebola hemorrhagic fever). It was first recognized in 1976 in northern Democratic Republic of Congo, at that time Zaire (1–3). Since then, EVD is endemic in Africa. Fruit bats are the best-known reservoirs of EBOV (4). EVD is a well-established zoonotic disease; the initial cases of the EVD outbreaks occur after contact with reservoir or materials contaminated with the virus and followed by human-to-human transmission (5). EBOV is not only a serious public health issue but now also designated as category A pathogen and considered as a potential bioterrorism agent (6, 7). EBOV causes high mortality rates of up to 88% in the infected humans (8); therefore, it is classified as a risk group 4 agent and handled under biosafety level-4 containment. The risk of mortality is relatively greater in the elderly and/or patients with high viral load and poor immune response at the initial stage of the infection (9).

The EBOV belongs to the *Filoviridae* family and has a unique thin filamentous structure that is 80-nm wide and up to 14-µm long. Its envelope is decorated with spikes of trimeric glycoprotein (GP1,2) which are responsible for mediating viral entry into target cells (function of GP1) (10) and release of viral ribonucleoprotein from endosome to cytoplasm for replication (function of GP2) (11, 12). EBOV infects primarily humans, simians, and bats; but other species such as mice, shrew, and duikers may also contact infection (3, 13). Of the five identified EBOV species, four species, *viz*., EBOV, Sudan virus (SUDV; *Sudan ebolavirus*), Tai Forest virus (TAFV; *Tai Forest ebolavirus*, formerly *Côte d'Ivoire ebolavirus*), and Bundibugyo virus (BDBV; *Bundibugyo ebolavirus*), are known to infect humans and cause disease, whereas Reston virus (RESTV; *Reston ebolavirus)* is non-human primate (NHP) pathogen.

After an initial incubation period of 3–21 days, the disease progresses quickly to fever, intense fatigue, diarrhea, anorexia, abdominal pain, hiccups, myalgia, vomiting, confusion, and conjunctivitis (14) which may lead to the loss of vision (15). EBOV can spread from males to females through semen (16) and from mother to fetus and infant during gestation and lactation, respectively (17). Of the note, in an EBOV-infected patient, higher concentration of Ebola viral RNA in semen was noticed during the recovery period than the viral concentration in the blood during peak time of infection, suggesting male genital organ as virus predilection site for replication (18). Usually the human immune system mounts a response against infectious pathogens by sensing the pathogen-associated molecular patterns *via* a variety of pathogen-recognition receptors. Nevertheless, in the case of EBOV, innate immunity is impaired by the immunosuppressive viral proteins including VP35 and VP24, and lymphocytes are depleted as a result of apoptosis caused by inappropriate dendritic cell (DC)–T-cell interaction (7, 19). A thorough understanding on the pathogenesis of this deadly virus is essential because of its severe health impacts (20).

The increased incidences and fast spread of EBOV paving into a pandemic flight has compelled more focus of research to develop strategies and remedial measures for mitigating the impact and consequential severity of the viral infection. Even before delineating the less studied Ebola viral genome fully, researchers throughout the globe and health industry were pressured to focus on the development of effective and safe Ebola vaccines and therapeutics (21, 22). As of now, no licensed vaccines and direct-acting anti-EBOV agents are available to protect against the lethal viral infection or to treat the disease. To minimize the suffering, EBOV-infected patients are only provided with symptomatic treatment and supportive care. Because of its high pathogenicity and mortality rate, preventive measures, prophylactics, and therapeutics are essential, and researchers worldwide are working to develop effective vaccines, drug, and therapeutics, including passive immunization and antibody-based treatments for EVD (23–26). Prior to the 2014–2016 EBOV outbreak in West Africa, which has been the deadliest EBOV outbreak to date, convalescent blood products from survivors of EVD represented the only recommended treatment option for newly infected persons. Administration of monoclonal antibody (mAb) cocktails (ZMapp, ZMAb, and MB-003) as post-exposure prophylactics have been found to reverse the advanced EVD in NHPs and/or effectively prevented morbidity and mortality in NHPs (27–30).

There is the need for an effective vaccine against EBOV, especially in high-risk areas, to prevent infections in physicians, nurses, and other health-care workers who come into contact with diseased patients (31). Regular monitoring and surveillance of EBOV is essential to control this disease. In the EBOV outbreak, novel surveillance approaches include contact tracing with coordination at the national level and "lockdown" periods, during which household door-to-door reviews are conducted to limit the spread of the virus. Swift identification and confirmation of the Ebola cases and immediate follow-up of appropriate prevention and control measures, including safe burial of dead persons, are crucial practices to counter EBOV (32).

After the onset of EVD, treatment is required, whereas, when EBOV is circulating in population dense areas before infection, prophylactic measures like vaccination are necessary. One of the main challenges in containing EBOV is its presence in remote areas that lack technology and equipment to limit the virus spread. Because of its lethality, EBOV can only be handled in laboratories with biosecurity level-4 containment; thus, only few laboratories in the world can conduct EBOV research and testing of the counter measures against the authentic virus. Recent efforts by several organizations have focused on identifying effective therapies and developing appropriate vaccination strategies (33). Several drugs and vaccines have been developed against EBOV, and the production of low-cost drugs and vaccines against EBOV is essential for everyone, including those in the high-risk areas of the world, to be protected (26, 34). As of the acquisition of better knowledge against the pathogen due to improvement in the field of genomics and proteomics, there has been expansion in the field of vaccine synthesis where epitope-based vaccines are gaining top priority (35–37).

The present review aims to discuss advances in designing and development of EBOV vaccines, drugs, antibody-based treatments, and therapeutics, and their clinical efficacy in limiting EVD, thereby providing protection against the disease and alleviating high public health concerns associated with EBOV.

# ADVANCES IN DEVELOPING VACCINES AGAINST EBOV

There is a clear need for an effective vaccine to prevent the rapid spread of EVD. An inactivated EBOV vaccine was first produced in 1980. This vaccine was tested for efficacy in guinea pigs (7). Since that time, several vaccines against EBOV have been developed, but no vaccine is licensed and available in the market (7). After the massive 2014–2016 outbreak of EBOV, several researchers have begun working to develop an effective vaccine (38). For an EBOV vaccine candidate, a long-lasting immune response is essential; as EBOV remains in the seminal fluid of EVD survivors as long as 401 days post-infection (39, 40). Keeping this window of virus persistence, a vaccine conferring immunity at least for 2 years is recommended by the Wellcome Trust-CIDRAP Ebola Vaccine Team B initiative (41). Vaccines like the chimpanzee adenovirus type 3 (ChAd3)-based non-replicating ChAd3-EBO vaccine, prime-boost recombinant adenovirus type 26 vector (Ad26.ZEBOV) followed by the modified vaccinia Ankara vector (MVA-BN-Filoa) vaccine, adenovirus 5-vectored EBOV vaccine, EBOV DNA vaccine, and recombinant vesicular stomatitis virus (rVSV) vector-based vaccine are undergoing clinical trials to evaluate their efficacy against EVD (38). The RNA-dependent RNA polymerase (L) epitope-based vaccine was designed using immunoinformatics. Various software have been used to analyze immunological parameters, and this epitope vaccine was found to be a good candidate for use against EVD (42). Two conserved peptides of EBOV, 79VPSATKRWGFRSGVPP94 from GP1 and 515LHYWTTQDEGAAIGLA530 from GP2, were identified as targets for the development of an epitope-based vaccine (43). Collection of the sequences of EBOV glycoproteins and examination for determining the proteins with greatest immunogenicity have been performed using *in silico* methods. The best corresponding B and T cell epitopes included peptide regions encompassing residues 186–220 and 154HKEGAFFLY162, respectively. Such predicted epitopes can confer the long-lasting immunity against EBOV with better ability of protection (36).

Ebola virus-GP fused with the Fc fragment of a human IgG1 subunit vaccine administrated with alum, QS-21, or polyinosinicpolycytidylic acid-poly-l-lysine carboxymethylcellulose adjuvant induced strong humoral immunity in guinea pigs (44). Effectiveness of a ring vaccine using rVSVΔG/EBOVGP in cases of simulated EBOV disease was studied and even this approach can be employed during an outbreak situation (45). Notably, the neutralizing antibodies play a major role in conferring protection against EBOV infections. Thus, an EBOV vaccine capable of effectively inducing a long-lasting neutralizing antibody response is desirable for developing appropriate prevention strategies in combating the infection. In this line, the mucin-like domain of EBOV envelope glycoprotein GP1 has been identified to be critical in induction of protective humoral immune response (46, 47). Filorab 1 vaccine revealed desirable immunogenicity without the side effects. The main advantage of this vaccine is its higher immune response induction in chimpanzees (captive) when given orally and also with a single dose [instead of multiple doses as is required by virus-like particle (VLP) vaccine] (48). Modified mRNA-based vaccine constructs, formulated with lipid nanoparticles (LNPs) to facilitate delivery, are being tested against EBOV challenge in guinea pigs. These mRNAs induced robust immune responses and conferred up to 100% protection from the infection (49).

It is important to note that compilation of data in relation to immune responses (both induced by vaccines and natural infection) and the records of community members showing IgG seropositivity should be kept systematically. Assimilation of such information will help to handle next outbreaks with more rigidity, thereby helping to check EVD-associated disasters at an early stage (50). *Vis-à-vis* public health workers should also be vaccinated and mass vaccination programs should be undertaken through standardized and coordinated efforts (51).

The following section describes the various types of vaccines and vaccine platforms which are being explored for the development of a successful EBOV vaccine.

# Inactivated Vaccines

Even though inactivated vaccines suffer with the problem of reversion to virulence due to inadequate viral inactivation, various strategies have been constantly explored in developing safe and potent non-replicating vaccine candidates for combating the EBOV infection (52). Both heat- and formalin-inactivated EBOV have been found protective against EBOV infection in a guinea pig model. Inclusion of inactivated vaccine with EBOV E-178 along with interferon (IFN) and immune plasma saved the life of a scientist working on EBOV (53). The protective efficacy of liposome-encapsulated irradiated EBOV, tested in a mouse model, was 100%. However, these viral particles failed to protect NHPs (54). This suggests that murine model is excellent for evaluating vaccine efficacy, but the level of protection might be different in different species and, hence, it is essential to test vaccines in NHPs before proceeding to clinical trials in humans. Heat-, formalin-, or gamma irradiation-killed EBOV vaccines have been found ineffective against EBOV disease; thus, the novel effective vaccine is essentially required (55).

# DNA Vaccines

In DNA vaccines, plasmids are used to express immunogenic antigens. This is an attractive vaccine approach because of the ease of production and simplicity. In addition, DNA vaccine induces both humoral and cellular immune responses. A threeplasmid DNA vaccine comprising the transmembrane-deleted GP sequences from EBOV species Zaire and SUDV-Gulu as well as nucleoprotein (NP) sequence from EBOV was tested in healthy adults. The vaccine was well tolerated, and both CD4+ and CD8+ T cell responses were elicited (56). An EBOV GP DNA vaccine designed on a consensus alignment of GPs (from strains obtained during 1976–2014), delivered intramuscularly and then electroporated, elicited a strong T cell response, and protected 100% of experimental mice from lethal challenge with EBOV (57). The DNA from three strains of EBOV was used to prime human volunteers and boosted with attenuated adenovirus, which acted as delivery vehicle for EBOV DNA into antigen-presenting cells, induced significant humoral- and cell-mediated immune (CMI) responses (58). Intramuscular inoculation of the DNA vaccine through electroporation with DNA plasmid containing codon-optimized GP genes of EBOV elicited high levels of IgG and a strong CMI response (measured by IFN-γ ELISpot assay) in cynomolgus macaques (59).

Though the preliminary trials using DNA constructs have provided the acceptable safety profiles, the development of low immune titer for a shorter window necessitates repeated vaccinations to overcome this problem. Thus, the use of a potent vaccination regimen based on DNA vaccine platforms does not appear logical for a large population (60, 61).

# Virus-Like Particles

Ebola VLPs (EBOV-VLPs or eVLPs) are generated from the expression of viral transmembrane glycoprotein (GP) and structural matrix protein (VP40) in mammalian cells, which undergo self-assembling and budding from host cells and display morphological similarity to infectious EBOV particles (47). Baculovirusderived eVLPs comprising GP, VP40, and NP of EBOV have been found to induce human myeloid DC maturation, suggesting their immunogenicity. Baculovirus-generated VLPs were able to elicit similar levels of protection as 293T cell-derived VLPs and showed protection against virus challenge in a dose-dependent manner (62). Nano-VLPs, produced by sonication of VLPs and filtering to have a mean diameter of approximately 230 nm, increased their thermostability. Unlike native VLPs where GP protein is denatured in a solution by heating, the nano-VLP maintained the conformational integrity of the GP protein at temperature up to 70°C and could confer protection in a mouse model (63). VLP containing only VP40 was sufficient to protect mice from EBOV infection. VLP injection leads to an enhanced number of natural killer (NK) cells, which play a crucial role in innate immune protection against lethal EBOV. NK cell protection is dependent on perforin, but not recombinant viral vector vaccines on IFN-gamma secretion (64).

Ebola virus VP40 and GP have been demonstrated to interact with the host protein, BST2, and are associated with viral infections by trapping the newly assembled enveloped virions at the plasma membrane in the infected cells, ultimately induce NF-κB activity. The effects of EBOV GP1,2, VP40, and BST2 converge on an intracellular signaling pathway leads to neddylation, resulting in the additive response with respect to the induction of NF-κB activity. Exploring the dynamics of this interaction could provide targets for vaccine developments and therapies that can modulate the inflammatory response during EVD (65).

Quantitation of EBOV antigenic particles using proteomic assays like liquid chromatography high resolution mass spectrometry method can be employed for determining the batch quality of vaccine constructs as well as in optimizing the dosages by assessing the amount of GP1 needed to confer effective protection (47).

It is to be noted that though anti-EBOV antibody can mediate effective protection, VLP-vaccinated murine models were shown to survive the EBOV challenge in the absence of detectable serum anti-EBOV antibodies (66). It could also be revealed that adjuvant signaling may circumvent the necessity for B-cell immunity in conferring protection against EBOV. These studies can be valuable for the future characterization, development, and optimization of effective EBOV vaccine candidates (66).

# Virus-Like Replicon Particles (VRPs)

The VRPs are the alternative to live-attenuated vaccines. The use of VRPs eliminates the risk of reversion to the original pathogenic form of live vaccine strains. To generate VRPs, generally filoviruses or alphaviruses are required. Here, while keeping the genes essential for replication, viral structural genes are deleted from full-length genomic cDNA clones. Viral structural genes are replaced with alternative gene(s) coding for an immunogen. Such replicons are able to replicate and transcribe upon transfection in competent cells. The resulting VRPs are able to infect cells only for one cycle. Because of the lack of structural genes, viral progeny are not formed. Viruses such as Venezuelan equine encephalitis virus (VEEV) can be used for production of EBOV antigen instead of structural proteins for the replicon vector. Thus, such vaccines are also quite safe (67). The gene inserted is typically GP, the main target of neutralizing antibodies. VRPs expressing EBOV VP24, VP30, VP35, and VP40 have been evaluated for their protective efficacy in a mouse model, but these were found not to be as protective as EBOV GP and NP antigens. VEEV replicons containing GPs from both EBOV and SUDV showed promising results in cynomolgus macaques after administration of a single dose. Here, two VRPs were constructed that contained the GP of EBOV or SUDV. The animals intramuscularly injected with both of the VRPs, survived viral challenge without exhibiting any clinical signs. The final results indicated that VRP-EBOV GP was able to confer cross-protection against SUDV, whereas VRP-SUDV GP was unable to provide complete protection against EBOV-Zaire challenge (68).

Recently, Ren et al. (69) constructed an alphavirus Semliki forest virus based recombinant replicon vector DREP for efficient and unchecked *ex vivo* co-expression of EBOV GP and VP40. Active immunization with recombinant DREP vectors possessing GP and VP40 induced cellular and humoral immune responses in murine model against EBOV antigens. This path breaking approach may provide key insights and strategies for designing further effective vaccines to contain EBOV permanently.

# Reverse Genetics System for EBOV Vaccine

A full-length recombinant EBOV infectious clone was constructed using cDNA. By employing reverse genetics method, viable but replication incompetent virus lacking entire VP30 ORF was constructed. The resultant EbolaΔVP30 is biologically contained and replication deficient, until VP30 is provided extraneously. Virus replication in cell culture was allowed by growing the virus in Vero cell line that stably expresses VP30, designated VeroVP30 (70). The safety of EbolaΔVP30 has been evaluated in mice and guinea pig model and was able to protect from lethal infection (71). The EbolaΔVP30 virus inactivated by using hydrogen peroxide protected NHPs after a single immunization. To avoid any incidence of potential recombination events that might result in regaining the replicative efficiency, the vaccine candidate was inactivated by hydrogen peroxide, that creates nicks and breakages in single- or double-stranded DNA or RNA and the virus is completely inactivated while retaining antigenic determinants unaffected (72).

# Recombinant Viral Vector Vaccines

Engineered viruses are gaining popularity because of their ability to efficiently induce CMI responses (a major part of adaptive immunity along with humoral response), as the antigen is expressed and processed in the cytoplasm. Replicationcompetent rVSV and chimpanzee adenovirus 3 (ChAd-3/cAd3) are the most efficient platforms for designing new vaccines (73). A recombinant vesiculovirus vector containing EBOV GP region (rVSVΔG/EBOVGP) was found to be highly effective after a single injection in NHPs (74, 75). The vaccine evaluated in pigs showed no disease development and no viral shedding. This indicated that the vaccine could be utilized for herd immunization and it also suggested the safety of the live-attenuated rVSVΔG/ EBOVGP vaccine (76). Recently, this rVSVΔG/EBOVGP vaccine was evaluated in a randomized double blinded placebo phase III trial in 1,197 humans. There were no adverse effects or death following vaccination, supporting its use as a vaccine (77). The vaccine protected immunocompromised rhesus macaques that had a high number of CD4+ T cells (78). The rVSVΔG/EBOVGP vaccine was also studied for its efficacy as a therapy in rhesus monkeys after exposure to EBOV-Makona. This vaccine showed minimal prophylactic efficacy after exposure (79). Efficacy trials initiated to test the rVSV-vectored EBOV vaccine showed greater efficiency at the time of EVD outbreak, if deployed following the strategy of ring vaccination (80).

Another recombinant vaccine (VSV based), i.e., rVSV-Zaire EBOV has been shown to provide substantial protection. From 10th day of vaccination with this vaccine, no report of any disease was documented, which proved efficacy and effectiveness of rVSV-vectored vaccine in preventing EVD (81). It is interesting to note that seroconversion has been noticed in recipients of recombinant VSV-EBOV (rVSV-EBOV) vaccine by the end of fourth week (i.e., by 28 days) against the Kikwit strain glycoprotein (82). Another recombinant vaccine *viz*., rVSV-EBOV vaccine was tested as a candidate vaccine. This particular vaccine is under trial in human (phase II/III). It provides protection against only EBOV and is clinically efficient in the clinical set up of ring vaccination format (38, 83, 84). EBOV and SUDV glycoproteins have been assimilated into a cAdVax vector (adenovirus-based vaccine). In mice, this vaccine has provided full protection (85, 86). During recent outbreak in Democratic Republic of the Congo (DRC), rVSVΔG-EBOV-GP is being used for ring vaccination in the affected area. Though the vaccine is yet not approved and still under investigation.

In Russia, clinical trial of a vaccine, GamEvac-Combi, has been performed and has been approved to enter in phase III clinical trial (87). The vaccine GamEvac-Combi contained two heterologous expression systems. One is live-attenuated rVSV and the second is a recombinant replication-defective adenovirus type-5 (Ad5). Both the vectors are expressing the same glycoprotein. The rationale to use a combination of two vectors expressing glycoprotein of EBOV is that widely present preexisting immunity to Ad5 limits the use of Ad5 and also a negative correlation between EBOV glycoprotein-specific immune response and preexisting antibodies to Ad5 has been reported (88). Hence, prime immunization with VSV vectored vaccine and then boosting with AD5 vectored vaccine might contribute in compensating negative impacts of preexisting immune response to Ad5. This heterologous vaccine evoked glycoprotein-specific immune response in 100% volunteers on day 28th. Also, the vaccine is well tolerated and did not significantly altered the body physiological parameters and vital organs. In Liberia, Sierra Leone, and Guinea; the VSV and ChAd3 vectored vaccine are in focus (89).

Another study in mice models has reported that the adoption of a heterologous prime-boost vaccine strategy can result in a durable EBOV-neutralizing antibody response. The chimpanzee serotype 7 adenovirus vectors expressing EBOV GP (AdC7-GP) was used for priming and a truncated version of EBOV GP1 protein (GP1t) was used for boosting. Vaccination response studies showed that AdC7-GP prime/GP1t boost strategy was more potent in generating a sustained and strong immune response as compared to using an individual vaccine construct (90).

Replication-defective recombinant chimpanzee adenovirus type 3-vectored EBOV vaccine (cAd3-EBO) elicited both cellmediated and humoral immunity in NHPs. A vaccine dose of 2 × 1011 particle units was found sufficient to induce protective immunity in the NHPs and to eliminate the effect of prior immunity to cAd3 (91). Recombinant VSV vaccine expressing EBOV GP and A/Hanoi/30408/2005 H5N1 hemagglutinin (VSVΔG-HA-ZGP) protected mice against challenge with both viruses and also cross-protected against H5N1 viruses (92).

The utility of adenovirus-vectored EBOV vaccines is limited with preexisting anti-adenoviral antibodies, which significantly lower the GP-specific humoral and T cell responses (88). Six mutations in the genome of MVA virus restrict its host specificity and make it unable to replicate in mammalian cells. A randomized study of a multivalent MVA vaccine encoding GPs from EBOV, SUDV, Marburg virus (MARV), and TAFV NP (MVA-BN-Filo) conducted in 87 participants resulted in no fever. The quadrivalent vaccine formulation has demonstrated the boosting up of both cellular and humoral immune responses against EBOV to several folds (93). Twenty-eight days after immunization, GP-specific IgG was detected with EBOV-specific T cell responses (94). EBOV GP and TAFV NP expressed in an MVA platform assembles into VLPs. Heterologous NPs enhanced VLP formation and offered GP-specific IgG1/IgG2a ratios comparable to those of MVA-BN-Filo (95).

Recombinant cytomegalovirus expressing EBOV GP was found to evoke protective immunity in rhesus monkeys challenged with EBOV (79). Baculovirus-expressed EBOV-Makona strain GP administered with Matrix-M (saponin adjuvant) showed better immunogenicity. Administration of Matrix M-adjuvanted vaccine resulted in increased IgG production and CD4+ and CD8+ T cell production (96). A human parainfluenza virus type 3-vectored vaccine expressing the GP of EBOV (HPIV3/EboGP) was developed as an aerosolized vaccine, and studies in Rhesus macaques showed 100% protection against challenge with EBOV (97).

Adenovirus 26 vectored glycoprotein/MVA-BN vaccine has recently passed the phase I trial (94). In the European countries including United Kingdom and United States, for the purpose of clinical trial, administration of ChAd-3 vectored vaccine has been adopted. This vaccine expresses the EBOV GP and is available in monovalent and divalent forms (91, 98).

Ebola vaccine potency trials employing replication defective adenoviral vectors (rAd) encoding EBOV GP have come up with promising results in NHP models. Based on such studies, multiple mutant glycoproteins were developed (such as glycoprotein with deleted transmembrane domain) which offers reduced *in vitro* cytopathogenicity but possessed reduced vaccine-mediated protection. In contrast to this, a point mutated glycoprotein has been reported to offer minimal cytopathogenicity and appropriate immune protection even with a two logs lower vaccine dose (99).

# Plant-Based Vaccines and Antibodies

Viral antigens, including GP, VP40, and NP, elicit protective immune responses. ZMapp, the cocktail of antibodies being used to treat EBOV, is a biopharmaceutical drug. To note, the component antibodies in ZMapp are manufactured in *Nicotiana benthamiana* using a rapid antibody manufacturing platform. Gene transfer is mediated by a viral vector, and the expression is transient. *N. benthamiana*-derived antibodies produced stronger antibody-dependent cellular cytotoxicity than the analogous anti-EBOV mAbs produced in a mammalian Chinese hamster kidney cell line (100). Phoolcharoen (101) expressed a GP1 chimera with the heavy chain of 6D8 mAb, forming an immune complex that was co-expressed with the light chain of the same mAb in leaves of tobacco plant. The ammonium sulfate-precipitated purified antibodies, along with poly(I:C) adjuvant, a synthetic analog of double-stranded RNA capable of interacting with toll-like receptor (TLR)-3, was found to elicit strong neutralizing anti-EBOV IgG. In addition, the immune complex along with poly(I:C) adjuvant was capable of stimulating a Th1/Th2 response. The experiment suggested the potential application of plant-produced Ebola immune complexes as vaccine candidates. EBOV VP40 was expressed in tobacco plants, and a mouse immunization study showed results that suggested this approach can be used to produce an EBOV vaccine (102).

The utility of plants as bioreactors for the bulk production of ZMapp could be considered to meet the required demand. The glycosylation pattern of mAbs may alter their efficiency and bioactivity, including their binding with the antigenic epitope. Several glycoforms of EBOV mAb13F6 have been prepared using a magnICON expression system. These glycoforms have humanlike biantennary N-glycans with terminal N-acetylglucosamine, resulting in a structure similar to that of human mAbs. Hence, these are beneficial for humans (103).

Both RNA and DNA viruses have been modified to serve as plant-based vectors for the expression of heterologous proteins. Bean yellow dwarf virus, a single stranded-DNA virus, can replicate inside the nucleus of plant cells using their cellular machinery. A vector containing deletions in the coat-encoding genes and gene for the desired antigen may be inserted to form an expression cassette. The delivery of vectors to plants is *Agrobacterium*mediated (23). mAbs against EBOV are produced by the process of agroinfiltration. In this context, it is noteworthy that lettuce acts as a very good host for the process of agroinfiltration. In lettuce cells, *Agrobacterium tumefaciens* has been used for delivering viral vectors (104). Neutralizing and protective mAb6D8 against EBOV has been expressed at a concentration of 0.5 mg/g of leaf mass. This quantity is similar to that generated in magnICON expression system (105). The plant-derived approach to vaccine development is attractive because of the large amount of transient proteins that can be expressed, with the potential for use during high demand for therapeutics and prophylactics (106). Advances in the field of vector expression like plant transient expression system and associated host cell engineering and manufacturing processes paved way for developing biopharmaceutical proteins and therapeutics in commercial basis. The great potentials of such novel approaches have been exploited for evolving therapeutics to counter emerging pandemics of EBOV and influenza that is evidenced from the production of experimental ZMapp antibodies (107).

An overview of various types of vaccines for countering EVD is presented in **Table 1** and depicted in **Figure 1**.

# ADVANCES IN DEVELOPING DRUGS AND THERAPIES AGAINST EBOV

Momentous leap has been witnessed toward the designing of efficacious EBOV drugs and therapeutics during the short span of only few years, even though the efficacy of several biologicals and vaccines were evaluated during the recent West African outbreak, it remained elusive to ratify a licensed EBOV disease treatment regimen (123).

Management of suspected or confirmed EVD patients includes quarantine, symptomatic, and supportive treatments, including fluid replacement, electrolyte imbalance correction, treating complicated infections, and preventing shock (124). For mitigation of the huge fluid loss and resultant hypovolemia, oral rehydration solutions should be provided adequately and if required anti-diarrheal and anti-emetic drugs need to be administered (125). Brincidofovir, a drug used to treat dsDNA viruses such as adenovirus, herpesviruses, orthopoxviruses, papillomavirus, and polyomaviruses, was approved for emergency treatment of two patients with EBOV infection; however, the clinical efficacy of the drug is unknown (126). Many drugs are being tested to identify specific antiviral drugs to treat EBOV, and new drug candidates are being developed by researchers worldwide (26, 127, 128).

Favipiravir (T-705), an antiviral drug found useful in treating influenza, has been studied and found effective against EBOV (129–131). Insertional mutagenesis, a high-throughput method to identify genes responsible for virus replication, can be used to develop drug candidates (132). Molecular docking experiments with EBOV GPs can be used for drug designing and the development of therapeutics (133, 134). Novel flexible nucleosides called fleximers were found to be effective against recombinant EBOV in Huh7 cells (135). Ribavirin antiviral can be recommended for the treatment of EBOV, since in mouse and monkey models, treatment with ribavirin delayed the death and increased survival rate (136). However, adverse effects associated with its use may limit ribavirin use (137). Lamivudine, an anti-retroviral drug, has been tested by Liberian doctor on 15 EBOV patients with survival of 13 patients (138). However, study by Cong et al. (139) found no survival benefits in Guinea pig model. Similar results were obtained by Hensley et al. (140), with no significant antiviral Table 1 | Vaccines for treating Ebola virus disease.

(*Continued*)

EBOV Vaccines, Drugs, and Therapies

#### TABLE 1 | Continued

EBOV Vaccines, Drugs, and Therapies

activity of lamivudine against EBOV in Vero E6 cells. Hence, the use of lamivudine may not be advocated.

The docking of VP40, VP35, VP30, and VP24 has been achieved using small molecules belonging to the class of flavonoids and derivatives. Gossypetin and Taxifolin (the two flavonoids of top ranks) showed higher docking scores for every EBOV receptor (141). A virtual analysis of more phytochemicals could help to identify plant-derived products with comparatively higher efficacy and lower toxicity. Adenosine nucleoside analogs such as BCX4430 have been found effective against EBOV in a mouse model. GS-5734, also a nucleoside analog, was found effective against EBOV in a NHP model (142). Using molecular dynamics simulations, graphene sheets are found to associate strongly with VP40 (matrix) protein of EBOV and disrupt VP40 hexamer–hexamer association, crucial to form virus matrix, thereby graphene and similar nanopolymers may be used as therapy or at least disinfectant to reduce the risk of transmission at time of epidemic (143).

The potential of retro-type drugs (molecules that block the retrograde trafficking of bacterial and plant toxins within mammalian cells) must be explored for designing novel therapy against filovirus (144). Retro-2 along with its other two derivatives, Retro-2.1 and compound 25 could effectively block EBOV and MARV progression *in vitro*. The derivatives were shown to be more potent inhibitors of filoviral penetration, replication, and progression when compared with their parent compound, as evidenced by pseudo-typed virus assays (144).

# EBOV Entry and Inhibitors

The cell entry of EBOV involves virus binding to the cell surface receptors followed by internalization through macropinocytosis, processing by endosomal proteases, and transport to Niemann– Pick C1 (NPC1; an internal receptor for EBOV) containing endolysosomes. Phosphatidylinositol-3-phosphate 5-kinase is essential for maturation of endosome, a critical step to the EBOV infection (145). *In vitro* studies using apilimod, an antagonist of phosphatidylinositol-3-phosphate 5-kinase, showed inhibition of EBOV by blocking the viral particle trafficking to NPC1 containing endolysosomes (146). IFNs are natural antivirals, and type I (IFN-α/β), particularly, is being widely used for the treatment of viral diseases. Type I IFN-α2b has been evaluated for the treatment of EBOV; however, IFN-α2b was not successful, as only delayed death but could not prevent mortality in EBOV-exposed monkeys. IFN-γ reduced the mortality rate in mice when administered either before or after EBOV infection (147), suggesting its promise as a prophylactic and/or therapeutic drug for use in EBOV infections. As IFN-γ has already been used to treat certain chronic medical conditions and has been approved by the FDA, it can be readily adapted for use against EBOV infections.

Silvestrol extracted from *Aglaia foveolata* was found to inhibit replication of EBOV and has been suggested as a therapeutic drug to treat acute EBOV infection (148). Supportive treatments like oral rehydration therapy are recommended for children under 5 years of age (149). Web-based identification of therapeutic agents indicated that a single siRNA can inhibit mRNA transcription of three species of EBOV, whereas 75 siRNAs can inhibit at least two species of EBOV. The web server Ebola VCR has been developed, with details available for the development of suitable therapeutic agents (150). Numerous treatment options for EVD are discussed below.

Ebola virus possesses only one surface protein and is responsible for both the receptor binding and fusion of virus-to-host cell endosomal membrane. EBOV glycoprotein binds with lectin receptor DC-SIGN (151). The infection initiated with the binding of EBOV glycoprotein to lectin receptors and internalization of virus majorly through macropinocytosis and as alternative mechanism through clathrin-dependent endocytosis (152). In the low pH of endosome, cysteine proteases including cathepsin B and L proteolytically cleave GP. Possibly this proteolytic cleavage exposes the putative receptor-binding region that interacts with NPC1, a receptor facilitating the filovirus entry (153). TIM-1 receptors directly interact with phosphatidylserine on the viral envelope, suggestive of GP independent virus attachment onto the cells. In poorly permissive cells, EBOV infection enhanced by exogenously expressed TIM-1 by 10- to 30-folds (154). Other phosphatidylserine interacting proteins like TIM-4 and Axl (a receptor tyrosine kinase) also have been demonstrated to enhance the infection of several enveloped virus. A benzylpiperazine adamantane diamide-derived compound obtained after screening of a library of small molecules, targets endosomal NPC1, and inhibit infection by VSV particles (VSV) pseudotyped with EBOV GP (155).

Tetrandrin is a potent drug that inhibits the EBOV entry into the cells (156). Two estrogen receptor drugs, clomiphene and toremifene, have been reported to hinder EBOV infection in mice by blocking cell entry and fusion with host cells (157). Amiodarone, an ion channel blocker, has been found to inhibit EBOV entry into cells (158, 159). Dendrimers and fullerene C60 have unique symmetrical properties and were recently found effective in inhibiting EBOV entry *in vitro* (160). Clarithromycin, an antibiotic, inhibits the release of calcium (stimulated by nicotinic acid adenine dinucleotide phosphate) from lysosome and exhibit anti-EBOV activity. Alike clarithromycin, posaconazole, an anti-fungal agent also shows similar anti-EBOV activity. In addition, it also inhibits the functions of NPC1 protein and acid sphingomyelinase activity. Both drugs, i.e., clarithromycin and posaconazole, ultimately inhibit the entry of EBOV into the host cell (161). The drug 5-(*N*-ethyl-*N*-isopropyl) amiloride inhibits the process of macropinocytosis (a process required for uptake of large filamentous virions, like EBOV) and thus interferes with the viral entry into the cell. Compounds like MLS000394177 and MLS000733230 also inhibit the viral entry into cells (162).

*Prunella vulgaris,* a Chinese herb, was found to inhibit EBOV entry into cells, using an EBOV-GP-pseudotyped-human immunodeficiency virus (HIV)-1-based vector system (163). Pseudovirions containing EBOV-GP were used for screening of the Prestwick chemical library, which contains 1,200 FDA approved drugs. The assay was based on cell entry of HIV-1-based surrogate in 384-well format. Twenty chemicals were found to inhibit more than 80% entry and 16 out of them were identified as G protein-coupled receptor (GPCR) antagonists, which target a range of GPCRs including adrenergic receptors, 5-HT (serotonin) receptors, histamine receptors, muscarinic and acetylcholine receptors. The time-of-addition studies suggested that EBOV entry is stopped at level of initial attachment prior to fusion of virus and cell membrane (164). Quercetin 3-β-*O*-d-glucoside (Q3G), a flavonoid derivate, was found to protect mice against EBOV challenge by targeting viral entry (165).

During replication of EBOV, surface GPs undergo proteolytic cleavage in the endosome by several proteases, including cathepsin B (CatB) (166). Thus, proteases may be a good target for the inhibition of EBOV replication. One study showed that, using a synthetic serine protease inhibitor, nafamostat mesilate (NM), caused a reduction in CatB release in rat pancreases. NM was also found to have anti-coagulant properties, which would also be useful in EBOV infections, as EBOV causes disseminated intravascular coagulation. Thus, this drug should be examined in clinical trials to be approved for the treatment of EVD (167). Chemically modified human serum albumin with 3-hydroxyphthalic anhydride (HP-HSA) has been demonstrated with the potential of a therapeutic candidate in resisting the EBOV infection (168).

## Transfusion of Convalescent Blood/Serum

Convalescent serum by definition contains immunoglobulins IgM and IgG but is devoid of red blood cells and clotting factors. Transfusing convalescent whole blood and convalescent plasma from disease survivors has been found to neutralize EBOV and reduce its load; thereafter, the immune response of the patient can provide protection against EBOV (169, 170). The use of whole blood and convalescent serum was approved by the World Health Organization (WHO) during critical EBOV conditions (171, 172). Screening of plasma is needed to rule out the presence of residual EBOV RNA and other blood-transmitted pathogens such as HIV, hepatitis B virus, and hepatitis C virus. Protection is conferred in NHPs through antibody therapy (post-exposure). In humans, this has ultimately paved the way for filovirus therapy by the use of polyclonal/mAb (approved by Food and Drug Administration) (173).

Valuable emergency therapeutics for the treatment of EBOVinfected persons include passive immunization with neutralizing antibodies by the transfer of sera from individuals recovering from EVD (174, 175), although it is not considered to render 100% protection especially after exposure (3 days post-exposure) to EBOV (e.g., Zaire Ebolavirus Makona) (176). Precise immunoglobulins retrieved from equine serum against EBOV were found safe and effective as prophylactic therapy in non-allergic patients (177). Recently developed mAb-based treatments for EVD include mAb114 and MB-003, ZMAb, ZMapp, and MIL-77E cocktails (25). ZMAb, consisting of three murine mAbs (1H3, 2G4, and 4G7), administered at a dose of 25 mg/kg three times, completely protected cynomolgus macaques against EVD. Administration of ZMAb with adenovirus-vectored IFN-α resulted in 75 and 100% survival of cynomolgus and rhesus macaques, respectively (29). mAbs that bind to the base of GP (4G7 and 2G4) are neutralizing antibodies, whereas mAbs that bind to the glycan cap (mAb114, 1H3, and 13C6) are nonneutralizing antibodies. The chimeric human mAbs 13C6, 6D8, and 13F6 possess the variable region from mice and Fc region of human; mAbs 13C6 and 6D8 neutralize EBOV in the presence of complement proteins (24). By repeated immunization of mice with glycoproteins of filovirus, generation of pan-EBOV-specific (as well as pan-filovirus) mAbs have been obtained. These pan-EBOV mAbs have shown reaction with RESTV, SUDV, and other viruses (178).

The components of ZMapp are mAbs (chimeric), *viz*., c13C6 from MB-003 (already known cocktail of antibody) and c2G4 as well as c4G7 from ZMab (different cocktail of antibody). This drug reversed clinical signs in 100% of rhesus macaques, even when administered as late as 5 days after EBOV exposure (30). Use of ZMapp, humanized-mouse antibodies, as a therapeutic agent has shown promise in NHPs (30, 84, 179) and it is a WHOapproved treatment regimen for EVD. Recently, a baby born to an EBOV-infected mother was found positive for EBOV on the first day of life. The baby was treated with ZMapp and the broad spectrum antiviral GS-5734, and, on day 20, the baby was found negative for EBOV (180). MB-003 is another mAb cocktail which is found to be effective in NHPs against variants of EBOV that are resistant to ZMapp (181).

The mechanism of action of mAbs is that they identify the inter-protomer epitope of the GP fusion loop, which is essential for viral membrane fusion, and also neutralize the entry of virus (182). Although several mAbs are available that can neutralize EBOV, there are few mAbs that can neutralize GPs from different EBOV species. In a study by Duehr et al. (183), a panel of eight murine mAbs derived from animals immunized with *Zaire ebolavirus* was evaluated. The mAbs were tested for binding breadth using a set of recombinant surface GPs from RESTV, TAFV, BDBV, EBOV, SUDV, and MARV. Of the eight, two mAbs (KL-2E5 and KL-2H7) showed binding ability. These two mAbs did not neutralize EBOV; however, they protected mice from infection with a VSV expressing the *Zaire ebolavirus* GP. Duehr et al. (183) also suggested that Fc-FcR interactions are responsible for the protection of mice in the absence of neutralization. Although ZMapp was found to be effective against Zaire EBOV, it has not shown cross-protection against other species of EBOV. FVM04 (a mAb) has shown cross neutralizing activity against SUDV. So, it can be used to replace one of the components of ZMapp, thereby increasing the range of protection against SUDV, ultimately leading to generation of cross-protective mAbs cocktail (184).