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Profiling the human immune response to Ebola virus infection: mechanisms of immune evasion, response signaling, and potential

therapeutic strategies.

Alden Parker, BS 752 – Immunobiology, University of the Sciences in Philadelphia, 4/22/2015

Introduction : Ebola virus (EBOV) is a negative-sense, single-stranded RNA virus from the

filoviridae family that often induces viral hemorrhagic fever (VHF) upon infection in humans and

non-human primates. The EBOV genome encodes seven proteins: viral protein (VP) 24, VP30,

VP35, VP40, glycoprotein (GP), nucleoprotein (NP), and an RNA – dependent RNA polymerase

(Han et al., 2003). VP24 and VP40 are matrix proteins that are involved in nucleocapsid and

virion assembly (Nanbo et al., 2013). VP30 binds RNA and is involved in viral genome

transcription (Martinez et al., 2008). VP35 inhibits interferon (IFN) and is a transcription factor

for viral genome synthesis (Luthra et al., 2015). GP is the only protein expressed on the virion

surface and is the main ligand that mediates attachment and membrane fusion to host cells

(Lee et al., 2009). Finally, NP is involved in viral genome replication and virion assembly

(Watanabe et al., 2006).

Though hypothesized to be rodents or bats, the natural reservoir for EBOV has not been

confirmed (Feldmann et al., 2012), but it is transmitted primarily via close contact with body

fluids of infected patients, especially affecting caretakers of infected patients (Beeching et al.,

2014). Recently, EBOV has garnered significant attention due to international outbreaks and the

high mortality rate associated with its induced VHF (Camacho et al., 2014). Different EBOV

strains have different mortality rates, with Zaire EBOV having up to 90% (Kuhn et al., 2010), and

the immune response induced by particular strains appears to be a highly significant factor in

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patient outcome (Sobarzo et al., 2015). Thus, profiling the human immune response to EBOV

infection has great potential to elucidate optimal treatment and prevention methods and, in

turn, to increase the survival rate of EBOV-infected patients. Moreover, it will provide insight

into the morphology and pathogenicity of EBOV itself, potentially providing an increase in

therapeutic targets after strain characterization and comparison. Defining commonalities for

both immune response and infection-essential EBOV features is crucial to the development of

optimal therapeutic and preventative strategies. A major challenge in vaccine development is

the fact that there are different EBOV strains that induce individual immune responses,

rendering an umbrella treatment for all strains elusive. Furthermore, via mutation against

selective pressures, EBOV strains have evolved many mechanisms of immune evasion including:

fusion of viral and host plasma membranes to avoid non-self recognition by T-cell receptors

(TCR) or major histocompatibility complex (MHC) (Cook et al., 2013), utilizing host mannose-

binding lectin (MBL) to enhance infectivity (Brudner et al., 2013; Mason et al., 2015), blocking

dendritic cell (DC) maturation (Lubaki et al., 2013; Yen et al., 2014), inhibiting apoptosis in

infected cells (Zinzula et al., 2013; Noyori et al., 2013), reducing epitope content and mimicking

host TCR structures (He et al., 2014), suppression of both innate and adaptive immune

responses (Misasi et al., 2014), and mechanisms involving both immune activation for infection

enhancement (Escudero-Pérez et al., 2014) and inhibition of host immune signaling (Garcia-Dorical et

al., 2014; Lemaître et al., 2014). Thus, the evasive tactics of EBOV are dynamic, complex, and

largely specific to particular strains, much like the individual immune responses elicited by each

strain. Therein lies the major obstacle in developing a ubiquitously applicable therapeutic

strategy: the lack of strain and induced immune response commonality. Thus, until common,

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combatable EBOV features are identified, analyzed, and neutralized, there will likely continue to

be major EBOV outbreaks. Furthermore, the fact that the natural reservoir has not been

confirmed exacerbates this problem in terms of outbreak preventative strategies. As more and

more outbreaks occur, it will provide EBOV with more opportunities for genetic drift, which

could result in more virulent strains with higher mortality rates. Hence, it is of utmost

importance to minimize the impact of EBOV infections before a hypervirulent strain causing a

pandemic EBOV outbreak develops. Moreover, minimizing the impact of EBOV infection on a

patient level will decrease pathogenicity, limiting EBOV spread as well as reducing the EBOV

mortality rate from induced VHF.

Principle Approaches: One of the few positive aspects of EBOV is that there are

survivors from virtually every major outbreak, and their immune profiles can be determined

during and after EBOV infection via serum analysis. Fatal EBOV cases can only be analyzed if

their sera was collected and properly stored during infection, but this is also extremely useful

information, as it can be compared to the sera of survivor cases (Sobarzo et al., 2013; Sobarzo

et al., 2015) in the search for common immunological trends among survivors and their

differences compared to the immune responses of fatalities. Particularly interesting is the

variation in immune responses between different strains of EBOV. Comparison between groups

has primarily been achieved with immune profiling via enzyme-linked immunosorbent assays

(ELISA) and EBOV-infected cell culture followed by plaque neutralization assays to determine

response efficacy of survivor versus fatality sera (Shedlock et al., 2013; Sobarzo et al., 2013;

Sobarzo et al., 2015). Most often, uninfected patients from the same region of the given EBOV

outbreak being studied are used as controls to ensure the obtained results are not because of

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endemic immune expression profiles. These collection and analysis methods generate reliable

baseline immunological requirements for survival of EBOV infection against particular EBOV

strains, and when compared to survivors of other EBOV strains, survivor immune response

commonalities can be determined. They can also potentially elucidate the what, in particular,

fatal cases lacked in their immune response or determine the possible mechanism of a patients’

death, such as a cytokine storm (Tisoncik et al., 2012). However, this approach does not

account for a variety of other factors that contribute to variability in patient outcome,

including: genetics, drug availability, general health before infection, care during infection, age,

sex, race, environmental factors, route of infection, time after infection of sample collection,

sample collection site, sample collection method, and sample storage. Thus, normalization

should be considered in collection practices across different studies, and the other factors need

to be carefully considered when drawing conclusions for any immune responses against a given

EBOV strain and for a particular patient. In the case of the plaque neutralization assay, it is a

purely in vitro assay, so it does not fully address the actual in vivo EBOV infection scenario.

Moreover, the infected cells used in such assays are often vastly different from normal human

cells, so they do not necessarily illicit the same immune responses normal human cells would

because their presentation machinery (i.e. MHC) is likely not identical. Hence, the TCRs in

human experimental sera may not react as they would in vivo. Finally, a drawback of plaque

assays is that they take days to weeks, whereas ELISA can be performed in minutes to hours.

Because EBOV is so dangerous to handle, there are only a handful of laboratories in the world

capable of safely conducting research on live virus. Therefore, a major technique in studying

EBOV is using recombinant DNA technology to generate virus – like particles (VLPs) with EBOV –

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specific proteins (Brudner et al., 2013; Ayithan et al., 2014; Yen et al., 2014). Upon challenge

with recombinant EBOV protein-expressing VLPs, immune cell maturation and response type is

commonly determined by looking at particular immunological markers by ELISA, quantitative

reverse transcriptase polymerase chain reaction (qRT-PCR), or by looking for surface proteins by

FACS (Yen et al., 2014). This approach allows for the identification of the function and

mechanism of particular EBOV proteins through induced-mutation in the proteins followed by

functionality assays and, importantly, how the proteins contribute to whole virion infection or

immune evasion (Yen et al., 2014). It also provides information about the human immune

response to individual, antigenic EBOV proteins. Moreover, it provides a safer environment

than working with live virus because VLPs are not infectious, as they do not contain any viral

genetic material other than the antigenic protein they code for. The limitation of this approach

is that it is unable to discern whether or not these proteins have the same function and

mechanism in the presence of other EBOV proteins or the virus as a whole. Thus, VLPs give a

hypothesized in vitro account of the function and mechanism of a given protein, but they do

not allow for definitive, comprehensive in vivo conclusions to be made.

These issues for both plaque neutralization assays and VLPs would be partially alleviated by the

use of live virus, but the risk is simply too high to allow for that except under highly controlled

environments. Thus, plaque neutralization and VLPs followed by FACS and ELISA represent the

best-suited technologies to date in terms of studying human immune responses to EBOV

infection, as well as mechanisms of EBOV immune evasion in humans.

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Other methods of studying EBOV and the human immune response to EBOV infection include

cryo-electron tomography (Tran et al., 2014) or X-ray crystallographic characterization (Leung et

al., 2010) of EBOV proteins or human immune structures like retinoic acid-inducible gene I (RIG-

1) (Kwok et al., 2014), which is a pattern recognition receptor (PRR) for most RNA viruses

(Tinoco et al., 2010; Siu et al., 2014), including EBOV. This provides insight into the architecture

of functional domains of viral proteins and corresponding host receptors or inhibitory

molecules and also provides insight into new potential drug targets. The main limitation of this

method is that it requires an ultra-pure crystal of the target protein, which is often extremely

difficult to obtain.

Present Knowledge: The immune profile of EBOV survivors is strongly correlated with a

strong humoral immune response upon VLP challenge of sera (Figure 1: Sobarzo et al., 2015).

Both TH1 and TH2 responses are significantly elevated in Gulu EBOV survivor sera due to the

elevated interferon (IFN) γ and interleukin (IL) – 5, respectively. Kibaale EBOV survivor sera only

have an elevated TH2 response. Uninfected patient sera are significantly impaired in their

immune response upon EBOV VLP challenge compared to survivor sera of both survivor groups.

Interestingly, the Kibaale EBOV strain induces extremely high levels of IL-10, which is highly

inflammatory. This implies that even though the patients survived EBOV infection, Kibaale EBOV

is highly virulent in humans. This can help explain the high mortality rate from the VHF when

other contributing factors are considered. For instance, low drug availability could certainly

Figure 1:______________________________________________________________________________

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Figure 1 Legend:

= Kibaale survivor group (2012)

= Uninfected, healthy group = Gulu survivor group (2001) IL = Interleukin, IFN = Interferon, TNF = Tumor Necrosis Factor

Blood from survivors of two different EBOV outbreaks was analyzed for cytokine signaling using chemiluminescent ELISA (Sobarzo et al., 2015). Both survivor groups had enhanced cytokine signaling compared to the uninfected group, but the profiles had significant differences. Upon EBOV immune stimulation, the Gulu group had 3 to 500 fold increased signaling compared to uninfected patients for all measured cytokines except for IL-10, indicating that Gulu survivors have a strong humoral response to EBOV infection but that their response may be highly inflammatory given the low level of IL-10. Upon EBOV immune stimulation, the Kibaale group had 2 to 500 fold increased cytokine signaling compared to uninfected patients for all measured cytokines except for IFNγ and TNFα, both of which were about equal. The high level of IL-10 suggests that the mode of Kibaale survivor immunity may be largely anti-inflammatory. Both survivor groups showed increases in T cell-associated cytokines, implying that both groups had immunological memory from their previous EBOV infection. Uninfected patients displayed increased IL-2, IL-5, and TNFα expression compared to resting cultures. For the survivor groups, levels are relative to the uninfected group, and for the uninfected group, levels are relative to resting cultures (+++ : >3 fold greater levels,

++ : 2-3 fold greater levels, + : 1 fold greater levels, = : no significant change). Ebola virion images adapted from Ian M. Mackay, PhD: http://virologydownunder.blogspot.com.au (visited on 3/27/15)_______________________________________________________________

contribute to enhanced inflammation, and as a result, an increased death rate. Wide-ranging T

cell responses are also correlated with EBOV survival in mammal models (Shedlock et al., 2013).

Further supporting the strong humoral response hypothesis, a significant TH1 response occurs in

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guinea pigs and mice upon EBOV challenge. Moreover, antibody-mediated responses are

essential for survival against challenge with Zaire EBOV VLPs in non-human primates (Marzi et

al., 2013) and bats (Olival et al., 2013). Importantly, animal study findings do not directly apply

to human cases, as they lack full genetic homology. Nevertheless, survivor sera samples in both

human and animal studies have enhanced plaque reduction in plaque assays against the same

EBOV strains, so there are clearly some immunological commonalities contributing to EBOV

resistance in mammals (Marzi et al., 2013; Sobarzo et al., 2013; Shedlock et al., 2013; Sobarzo

et al., 2015).

Lending more credence to the involvement of a strong humoral immune response, survival of

EBOV infection is correlated with immunoreactivity to NP, VP30, and GP (Figure 2: Sobarzo et

al., 2013). Sera from survivors neutralize recombinant EBOV VLP – infected human cells

expressing these proteins while sera from uninfected patients usually do not. This is not always

the case, as some survivor serum does not react to these proteins, highlighting genetic

differences in patients and the fact that other factors need to be accounted for when

considering treatment options against particular EBOV strains.

With studies like these in mind, should an EBOV strain for a particular outbreak be identified to

have high similarity to a strain from which survivor sera have been analyzed, the appropriate

immune response for survival can be postulated, yielding an optimized treatment regimen for

infected patients.

Figure 2:_____________________________________________________________________________

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Figure 2 Legend: NP = Ebola virus nucleoprotein, VP30 = viral protein 30, GP= Glycoprotein.

Survival of Ebola virus infection has been shown to often depend on a humoral immune response (Sobarzo et al., 2013). In general, serum from survivors has been shown to be immunoreactive against Ebola viral proteins NP, VP30, and GP, while serum from fatalities has shown little or no immunoreactivity to them. It is important to note that a humoral response does not guarantee survival and that a lack of one does not guarantee death. Rather, these are generalized outcomes from the majority of data using recombinant viral proteins for immune stimulation in analyses. Thus, the chances of the given outcomes are correspondent to the thickness of the arrows, with GREEN arrows being survival and RED arrows being death. In either case, Ebola infection is a veritable war for the human immune system due to its evasive tactics and the inflammation often caused by infection. The toxic symbol in the lower left symbolizes death and was adapted from http://centurionsigns.co.uk/shop/product-tag/chemical-hazard-signs/page/2/ (visited 3/27/15). The symbol in the upper right of the figure symbolizes survival, as it is the Adrinka (West African) symbol of survival, adapted from http://www.adinkra.org/htmls/adinkra/aya.htm (visited 3/27/15). The balance was adapted from http://www.clipartpanda.com/categories/balance-20clipart (visited 3/27/15).________

One of the major challenges in EBOV treatment and prevention is the lack of treatments that

effectively counteract its mechanisms of immune evasion. EBOV VLPs inhibit human DC

maturation with Wild Type (WT) VP35 by blocking RIG-1 signaling (Figure 3: Yen et al., 2014),

resulting in a diminished immune response due to reduced T cell activation. This reduces

nuclear factor kappa-light-chain-enhancer of activated B cells (NF-кB) and interferon regulatory

Figure 3:______________________________________________________________________________

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Figure 3 Legend: Ag = antigen, VP35 = Ebola Viral Protein 35, RIG-1 = retinoic acid-inducible gene 1, MDA5 = Melanoma Differentiation-Associated Protein 5, TLR = Toll-Like Receptor, TRIF = TIR-domain-containing adapter-inducing interferon-β, TRAM = TRIF-related adapter molecule,

TRAF = tumor necrosis factor receptor associated factor

= TLR – mediated DC activation pathway (partially blocked by VP35) = RIG-I – like DC activation pathway (fully blocked by VP35)

Ebola VP35 has been shown to inhibit dendritic cell maturation in human cells by blocking RIG-I and MDA5 pathways, which results in less activation of NF-кB and IRF-3, resulting in impaired inflammatory signaling. VP35 inhibits but does not block TLR3 and TLR4 signaling. Therefore, DCs are able to be activated and mature through this pathway. It is hypothesized that VP35 affects some downstream part of TLR signaling, such as acting as a decoy substrate, which would support the partial inhibition (Yen et al., 2014). However, there is not a full blockage of TLR signaling, so DCs are still able to be activated via this pathway in the presence of EBOV VP35. Thus, antiviral strategies could be developed to further activate this pathway or to inhibit VP35 from interacting with RIG-1 or MDA5 in order to enhance overall DC activation for a better chance of immune clearance of EBOV.___________________________________________________________________

factor (IRF) – 3 signaling and gives the virus free reign to replicate upon host cell infection,

enhancing pathogenesis and virulence. Mutated VP35 – expressing cells effectively activate T

cells, resulting in enhanced inflammatory cytokine signaling but reduced viral protein

production compared to WT-VP35. Thus, a structural basis for the VP-35 – mediated

mechanism of immune evasion and EBOV pathogenesis has been identified as a target for

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therapeutic treatments. Both WT and mutant VP35 inhibit toll-like receptor (TLR) signaling but

do not fully prevent it, so there is also an alternative pathway for DC activation that could

potentially compensate for blocked RIG-1 signaling. Hence, there is yet another potential

therapeutic target for enhancing the human immune response against EBOV by agonizing TLR

signaling pathways. Studies like this highlight the intracellular mechanisms of EBOV immune

evasion, and they provide insight into therapeutic targets that could be used to reduce

virulence and pathogenesis, in turn, reducing mortality rate in infected patients.

EBOV GP inhibits Fas – mediated apoptosis by sterically shielding the Fas receptor (Fas) on

infected cells from binding the Fas ligand (FasL) expressed by cytotoxic T cells (Figure 4: Noyori

et al., 2013). Thus, GP acts similarly to Decoy receptor 3 (DcR3), which competitvely binds FasL

(Ge et al., 2011), except GP acts on Fas itself. Expression of GP in the host cell membrane is

necessary for this action, and it displays yet another mechanism of immune evasion:

incorporation of viral epitopes onto host cells so that they do not get recognized by host cell

MHCs. The effect of GP on Fas may be due to co-localization in lipid rafts, and GPs are clearly

involved in shielding the Fas/FasL interaction, resulting in reduced apoptosis in EBOV-infected

cells. In this scenario, T cells have been activated, so this is an evasive tactic that is downstream

from blocking RIG-1 and inhibiting TLR signaling. Thus, EBOV immune evasion occurs at a

number of cellular defense levels, which is a large contributor to its severe pathogenicity.

However, because it is known to block FasL binding, EBOV GP can be utilized as a therapeutic

target.

Figure 4:______________________________________________________________________________

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Figure 4 Legend: T(x) = transcription, T(L) = translation, GP = Ebola Glycoprotein, Fas = Fas receptor,

FasL = Fas Ligand, FAD = Fas-Associated protein with Death Domain

= Ebola Glycoprotein pathway = Fas-mediated apoptosis pathway

Ebola Glycoprotein has been shown to inhibit Fas-mediated apoptosis by sterically shielding Fas receptors on GP-expressing (i.e. infected) human cells. Here, negative strand RNA coding for Ebola GP has been transcribed into mRNA in the cytoplasm, translated into GP in the cytoplasm, and incorporated into the cell membrane. There, it inhibits Fas-mediated apoptosis (extrinsic pathway) by sterically hindering Fas receptors from binding Fas Ligands of other cells. This displays a powerful immune evasion mechanism used by Ebola and other filoviruses because it keeps cells alive allowing for the production of more virions. The degree of steric hindrance has been linked to the severity of the pathogenesis for certain strains (Noyori et al., 2013)._________________________________________________________________________

Interestingly, EBOV GP is shed from infected cells via cleavage by a host TNF-α converting

enzyme (TACE), and cleaved GP exerts a couple of effects: shed GP binds to and inhibits

neutralizing antibodies against surface GPs on infected cells allowing for enhanced replication

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and virion assembly in infected cells, and shed GP actually activates human DCs and

macrophages (Escudero-Pérez et al., 2014), which is somewhat unexpected. Upon binding to

DCs and macrophages, shed GP induces both pro- and anti-inflammatory cytokine transcription,

ultimately resulting in increased endothelial permeability. Essentially, GP disrupts cytokine

signaling to the point that it is no longer regulated, and the balance between inflammation and

anti-inflammation is disrupted, resulting in massive recruitment of non-infected antigen-

presenting cells, increased inflammation, and possible organ failure. Increased endothelial

permeability allows newly produced virions and shed GP to enter into new tissues once the

replication machinery at the initial infection site has been expended. Thus, EBOV evades normal

human immune responses by blocking neutralizing antibodies and increasing inflammation to

the point of tissue damage in order to spread into uninfected tissue for further replication.

Moreover, EBOV utilizes human immune machinery and signaling for its own evasion. In this

manner, EBOV has multiple mechanisms of molecular mimicry in order to get its own proteins

processed and to activate host antigen presenting cells (APCs) for infection enhancement.

Recombinant human mannose – binding lectin (rhMBL) enhances EBOV VLPs expressing GP

(Figure 5: Brudner et al., 2013). Utilizing a host receptor meant for complement recognition of

other antigenic epitopes is a disturbing action of EBOV because it seems as if the cell actively

transports the virus across the plasma membrane, where it can replicate as well as go

undetected by other immune cells until it has replicated into many virions. However, this

highlights a therapeutic target in that MBL could be inhibited in EBOV-infected patients in order

to decrease infectivity and hopefully pathogenicity overall. Host cathepsin protease is necessary

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Figure 5: ______________________________________________________________________

Figure 5 Legend: MBL = mannose-binding lectin. T(X) = transcription, T(L) = translation, VP35 = EBOV

VP35, = EBOV nucleocapsid (genome, NP, matrix proteins)

EBOV utilizes MBL to enhance attachment and penetration into human cells (Brudner et al., 2013). Mutated MBL allelic variants have been identified in areas where EBOV infection is prevalent, and these mutations lead to infection inhibition in vitro. This potentially highlights a therapeutic target to reduce infectivity and virulence. Once EBOV GP binds to MBL, the virion penetrates host cells by micropinocytosis, where it fuses its glycosylated membrane to the vesicle. At this point, host cathepsin activates EBOV VP35, which along with the cholesterol transporter Niemann –Pick C1 facilitates lysosome recruitment for the proteolytic fragmentation of the vesicle membrane and nucleocapsid penetration into the cytoplasm (Carette et al., 2011). There the nucleocapsid and accessory protiens are able to use more host cell machinery to replicate the EBOV genome and produce more virions. Thus, this form of immune evasion is the utilization of a host receptor to gain access to cells before antigenic ligands can be recognized in the extracellular matrix. Once inside the cell, EBOV cannot be found by the immune system until antigenic peptides are expressed on the cell surface, and that also presents its own set of problems like steric shielding of apoptotic signaling (Noyori et al., 2013)._ Ebola virion images adapted from Ian M. Mackay, PhD: http://virologydownunder.blogspot.com.au (visited on 3/27/15) _______________________________________________________________

for VP35 activation upon micropinocytosis, and the host cholesterol transporter Neimann-Pick

C1 facilitates lysosomes to break down the vesicle membrane, allowing the EBOV nucleocapsid

into the cytoplasm (Carrette et al., 2011). Knockdown of lectin receptors results in reduced

infection, resulting in identification of two novel EBOV attachment mediators that play a role in

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MBL-dependent infection enhancement, and rhMBL was shown to significantly increase

infectivity for an array of wild type glycosylated viruses, indicating that a common theme may

exist. Hence, EBOV not only utilizes host cell immune response molecules like MBL, which is a

major player in complement – mediated responses, but it also uses intracellular proteases and

signaling mechanisms that are commonly used in many signaling cascades in order to facilitate

and enhance infection.

Clearly, EBOV has evolved many mechanisms of immune evasion against selective pressures in

the human immune response, and as outbreaks continue to happen, it will continually have the

opportunity to further develop these evasive tactics. On a positive note, it appears that human

genetics have counter-evolved against some of these tactics, as is evidenced by the higher

presence of MBL allelic variants in areas where EBOV is endemic (Brudner et al., 2013).

Analysis of the genetics and immune profiles from both survivors and fatalities will be an

important aspect of EBOV management in all future outbreaks. Moreover, determining what

EBOV strain is causing a particular outbreak will also help medical professionals optimally craft

treatments for EBOV-infected patients.

Future Directions: Obviously, one of the main goals in researching EBOV is the

development of prophylaxis or a vaccine that is cross-reactive to all EBOV strains and effective

for patients of all genetic backgrounds. To this end, multiple studies have shown adenoviral-

vector vaccines to confer long-term protection against lethal EBOV infection in mice (O’Brien et

al., 2014) and non-human primates (Choi et al., 2014; Stanley et al., 2014; Ye et al., 2015).

Moreover, human clinical trials using a similar vaccine have shown significant promise in

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conferring both adaptive and innate immune responses, though sometimes at the expense of

adverse effects (Zhu et al., 2015). Clinical trials in infected patients for EBOV vaccines are quite

controversial because many believe that using a placebo-controlled group when analyzing a

virus with such a high mortality rate is unethical (Cohen et al., 2014; Cooper et al., 2015). Thus,

some organizations have proposed that if the vaccine does not produce adverse effects in

recipients and does initiate the desired immune response that it bypass phase I and phase II

trials to proceed to phase III efficacy trials in areas where EBOV is endemic. This would

potentially decrease the spread of EBOV, minimizing the potential global impact as well as

minimizing the number of opportunities for EBOV to evolve into a hypervirulent strain as it

encounters novel selective pressures in new genetic populations. Contributing to vaccine

design, genetic analysis of EBOV codon usage sheds light on mechanisms of viral evolution,

regulation of viral genome replication and transcription, and localization requirements for viral

protein expression in order to induce innate and adaptive immune responses (Cristina et al.,

2015).

Interestingly, complement has been shown to inhibit EBOV infection (Brudner et al., 2013), but

the lectin-dependent pathway of complement activation enhances EBOV infection. Thus, there

must be an important response mechanism involving either the classical or alternative

complement pathway. Positive identification of this complement response pathway would

significantly contribute to supplementing the immune response of infected patients.

Designing antiviral drugs against identified EBOV protein targets or the host machinery EBOV

utilizes for infection is another method of supplementing the immune response. Monoclonal

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antibody treatments have proven effective in non-human primates (Qui et al., 2013). As for

targeting specific EBOV viral proteins, inhibition of GP would decrease EBOV attachment

efficiency (Lee et al., 2009) and minimize steric shielding of apoptotic signaling (Noyori et al.,

2013). Inhibition of VP35 would decrease viral genome replication as well as cytoplasmic

penetration of the nucleocapsid (Carrette et al., 2011; Brudner et al., 2013). Most previous

studies have focused on only one EBOV protein product. This is useful in determining function

and mechanism, but it does not give a clear indication of how the particular protein may work

in the presence of the other viral proteins. Thus, studies where VLPs express multiple viral

proteins should be examined. This will allow for a clearer representation of the in vivo function

of EBOV proteins, as they are all present together in the cells of EBOV-infected patients.

Immunosuppressing the MBL pathway could potentially reduce attachment and penetration in

infected patients (Brudner et al., 2013). Thus, there are multiple levels of drug targets that will

be important to investigate in the future. Until effective compounds are developed, there

should be normalization in the collection and storage of sera from EBOV-infected patients, and

patients should be monitored post infection. Moreover, particular EBOV strains that cause

outbreaks should be analyzed to determine if they have high homology to previously analyzed

strains. This will allow for cross comparison of survivor sera from different outbreaks, which will

contribute to an optimal immune response scenario for successful survival of EBOV infection.

Conclusions: It is relatively easy to see why EBOV is virtually incurable and extremely

difficult to treat and prevent. The fact that EBOV outbreaks occur most often in impoverished

African countries where natives do not always trust the local healthcare system based on

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previous negative experiences also exacerbates issues of treatment and prevention. Moreover,

as world travel becomes more and more common and efficient, the potential for massive

spreading of highly infectious and pathogenic diseases only increases. Conversely, as more

outbreaks have occurred, more information about EBOV and other diseases have been

elucidated, and as a result, potential for curative and preventative therapies are extant or are in

development. For EBOV, monitoring strains, virus spread, outbreak demographics, patient

genetics, and immune profiles will be extremely important moving forward in treatment,

prevention, and research. These considerations together have the potential to prevent EBOV

with the opportunity to evolve into a hypervirulent strain that may not be treatable and could

even be more contagious should it evolve into utilizing other transmission routes such as

aerosol. Curative and preventative treatments will limit EBOV spread, limiting the selective

pressures put on EBOV evolution, resulting in more treatable mechanisms of immune evasion

and symptoms as a whole. This will ultimately yield a more manageable and treatable disease,

reducing pathogenicity, virulence, and most importantly, mortality rate.

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