Review

Differential Immune Responses to Hemorrhagic

Fever-Causing Arenaviruses
Emily Mantlo 1,2 , Slobodan Paessler 1 and Cheng Huang 1, *
1 Department of Pathology, University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA;
ekmantlo@utmb.edu (E.M.); slpaessl@utmb.edu (S.P.)
2 Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston,
TX 77555, USA
* Correspondence: chhuang@utmb.edu




Received: 30 August 2019; Accepted: 30 September 2019; Published: 2 October 2019


Abstract: The family Arenaviridae contains several pathogens of major clinical importance. The Old
World (OW) arenavirus Lassa virus is endemic in West Africa and is estimated to cause up to 300,000
infections each year. The New World (NW) arenaviruses Junín and Machupo periodically cause
hemorrhagic fever outbreaks in South America. While these arenaviruses are highly pathogenic
in humans, recent evidence indicates that pathogenic OW and NW arenaviruses interact with the
host immune system differently, which may have differential impacts on viral pathogenesis. Severe
Lassa fever cases are characterized by profound immunosuppression. In contrast, pathogenic NW
arenavirus infections are accompanied by elevated levels of Type I interferon and pro-inflammatory
cytokines. This review aims to summarize recent findings about interactions of these pathogenic
arenaviruses with the innate immune machinery and the subsequent effects on adaptive immunity,
which may inform the development of vaccines and therapeutics against arenavirus infections.

Keywords: arenavirus; hemorrhagic fever; immunity; interferon; innate sensing; Lassa virus;
Junín virus; Machupo virus

1. Introduction
Arenaviruses are enveloped, negative-sense, single-stranded RNA (ssRNA) viruses [1]. The family
Arenaviridae currently consists of four genera, Mammarenavirus, Reptarenavirus, Hartmanivirus,
and Antennavirus [2,3]. With the exception of the trisegmented Antennavirus genus, arenavirus
genomes are bi-segmented, with one large (L) segment of around 7.2 kb and one small (S) segment of
around 3.4 kb. Each segment contains two open reading frames (ORFs) encoding two gene products
in opposite orientation, allowing the virus to assume an ambisense coding strategy. The two ORFs
are separated by a highly structured intergenic region (IGR) that functions to terminate viral RNA
transcription [4]. The conserved termini regions of each genomic segment form pan-handle structures
and mediate viral RNA replication and transcription [5,6]. The S segment encodes the viral glycoprotein
(GP) precursor, which is post-translationally cleaved into stable signal peptide (SSP) and mature
GP1 and GP2 [7–9]. All three of these cleaved products form the glycoprotein complex and are
incorporated into virions, with GP1 and GP2 forming the spikes on the surface of virions that bind to
host receptors and mediate cell entry [10]. The S segment also encodes the nucleoprotein (NP), which is
the most abundant viral protein produced during infection and the major structural component of
the nucleocapsid [1]. The L segment encodes the RNA-dependent RNA polymerase L protein and a
small, zinc finger protein (Z), which acts as the arenavirus matrix protein that drives the assembly and
budding of virus particles [11–13].

Vaccines 2019, 7, 138; doi:10.3390/vaccines7040138 www.mdpi.com/journal/vaccines

Vaccines 2019, 7, 138 2 of 16

Within the family Arenaviridae, all human pathogens are members of the Mammarenavirus genus [2].
Mammarenaviruses are further separated into two groups based on geography and phylogeny: the
Old World (OW) arenaviruses and the New World (NW) arenaviruses [14]. Lassa virus (LASV) is
endemic in West Africa and is therefore classified as an OW arenavirus. The prototypic lymphocytic
choriomeningitis virus (LCMV) is also classified as an OW arenavirus based on similar phylogeny [15].
Meanwhile, NW arenaviruses are endemic to South America and can be further divided into four
clades (A–D). Clade B contains all the pathogenic NW arenaviruses, including Junín (JUNV) and
Machupo viruses (MACV), the causative agents of Argentine hemorrhagic fever (AHF) and Bolivian
hemorrhagic fever (BHF), respectively. Notably, clade A contains the prototypic Pichinde virus that,
while non-pathogenic to humans, causes hemorrhagic disease in rodents that is similar to Lassa fever
(LF) in humans [16,17].
Mammarenavirus (with the exception of Tacaribe virus) are rodent-borne viruses, which usually
infect specific rodent species. Therefore, the geographic distribution of each arenavirus is defined
by the range of the habitat of its host rodent species. Mastomys natalensis, the reservoir for LASV, is
found across much of Africa, though most LASV infections occur in M. natalensis monophylogenetic
group A–I in West Africa [18,19]. NW arenaviruses likewise each primarily infect a single species of
rodent in the Americas. Arenaviruses often persistently infect their natural hosts without overt disease
signs and are shed via excreta from infected animals. The transmission of pathogenic arenaviruses
to humans occurs largely through aerosol exposure to rodent excreta or consumption of rodent
meat [1,20]. Most infections occur in a rural setting, often during cyclical outbreaks. However,
nosocomial transmission of LASV, JUNV, and MACV has been reported [1,21,22].
Within endemic areas, both OW and NW arenaviruses are responsible for significant human
disease. Among the highly pathogenic arenaviruses, LASV is the most prevalent and clinically
important, with an estimated 100,000–300,000 infections and 5000 deaths in West Africa each year [23].
While most LASV infections are asymptomatic, severe LF can have case fatality rates ranging from
9.3–18% among hospitalized patients [24]. For pathogenic NW arenaviruses (JUNV and MACV),
the case fatality rates can be as high as 15–35% [25,26].
In addition to the severe acute disease and high mortality rates in humans, long-term sequelae are
common but often neglected among survivors. Patients recovering from AHF and BHF often experience
a protracted convalescence period, with hair loss and neurological symptoms such as dizziness and
headaches lasting up to several months after the acute infection [1,25,26]. Neurological sequelae have
also been reported in LF cases [27]. Recently, the prevalence and impact of LASV-induced hearing loss
is becoming increasingly recognized as a significant social and economic burden in affected areas [28].
Approximately 33% of LF survivors develop unilateral or bilateral sudden-onset sensorineural deafness
that may be permanent [29]. The exact mechanisms behind the development of long-term sequelae after
infection by highly pathogenic arenaviruses remain to be determined, but cell-mediated immunity may
be involved. Currently, vaccines and treatments are very limited for these hemorrhagic fever-causing
arenaviruses. The World Health Organization has listed LF in the Blueprint list of priority diseases for
which there is an urgent need for accelerated research and development.

2. Immune Response to Hemorrhagic Fever-Causing Arenaviruses

While highly pathogenic OW and NW arenaviruses cause severe diseases in humans, the innate
immune responses are remarkably different (Figure 1). Despite high unchecked levels of viremia,
severe LF cases are generally characterized by profound immunosuppression, including a weak or
delayed Type I interferon (IFN-I) and pro-inflammatory cytokine response particularly in the early
stage of illness [30,31]. In contrast, pathogenic NW arenavirus infection stimulates a robust IFN-I
response in patients. In AHF cases, serum IFN-α levels have been reported to peak at 64,000 IU/mL,
one of the highest circulating interferon levels recorded for any viral disease [32]. Elevated levels of
IL-6, IL-8, IL-10, and TNF-α are also observed in the serum of AHF patients. High IFN-I and cytokine
Vaccines 2019, 7, 138 3 of 16

levels are correlated with the severity and outcome of AHF disease [32,33]. Also, there is evidence that
theVaccines
IFN response
2019, 7, 138 may contribute to thrombocytopenia in AHF patients [34]. 3 of 15

Figure
Figure 1. 1.AAschematic
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Pathogenic OW and NW arenavirus infections also induce differential adaptive immune

responses, which could be linked to the profound difference in the innate response (Figure 1). The
host innate immune response not only acts directly to counteract virus infection, but also motivates
Vaccines 2019, 7, 138 4 of 16

Pathogenic OW and NW arenavirus infections also induce differential adaptive immune responses,
which could be linked to the profound difference in the innate response (Figure 1). The host innate
immune response not only acts directly to counteract virus infection, but also motivates and regulates
virus-specific, adaptive immune responses that provide long-term protection. Dendritic cells (DC)
and macrophages, which present viral antigens to T cells, are essential components of both the innate
immune response and the adaptive immune response. These cells are also the early targets in arenavirus
infection. LASV efficiently infects monocyte-derived dendritic cells (moDC) and macrophages without
stimulating IFN and cytokine responses. Consequently, LASV-infected moDCs and macrophages fail
to activate T cells, which explains the delayed cellular immune response [30,35,36]. The nonpathogenic
OW Mopeia virus (MOPV) is genetically closely related to LASV and has been used as a control to
study the pathogenesis of LASV. In contrast to LASV, MOPV infection induces strong IFN and cytokine
responses in moDCs and macrophages [37], leading to T cell activation. In the host, myeloid dendritic
cells (mDCs) are specialized in antigen presentation and the induction of T cell responses. A recent
study has identified that LASV and MOPV infection of mDCs ex vivo are abortive [36] due to IFN
and cytokine responses. Blocking IFN enables productive infection for both LASV and MOPV in
mDCs. Interestingly, in a mDC and autologous T cell coculture model, mDC activation and the IFN
response are remarkably reduced and delayed in LASV infection, while mDC activation and the IFN-I
response in MOPV infection are increased and prolonged [36]. Consistently, LASV-infected mDCs do
not activate T cells, while MOPV-infected mDCs efficiently activate T cells. The mechanism underlying
the apparent negative regulation of mDC activation and IFN response by T cells in LASV infection
remains unknown. It is also unclear if other cell types, e.g., NK cells, might be present in the coculture
and contribute to the phenotype. Plasmacytoid dendritic cells (pDCs) are an important IFN producer
in vivo. LASV and MOPV infection of pDCs are both abortive, though LASV infection produces a
less sustained and lower level of IFN-I response than MOPV [38]. Nevertheless, it has been found
that LF survivors develop virus-specific CD4+ and CD8+ T cell responses during the acute stage
of infection [39,40], which is associated with LASV clearance and recovery. It is generally believed
that the humoral response is not important in the clearance of LASV as even LF survivors do not
develop neutralizing antibodies until months after the acute infection has resolved [41]. In contrast,
AHF patients develop neutralizing antibodies to JUNV during the acute phase of disease [42], which is
essential for virus clearance. Furthermore, both AHF and BHF can be successfully treated using
convalescent immune sera from survivors [25,43].
Research using animal models for pathogenic arenavirus infections has recapitulated many of
the clinical observations. NHPs infected with LASV typically develop lymphopenia, though survival
is correlated with increased numbers of CD4+ and CD8+ T cells [44,45]. Survival of NHPs from
LASV infection has also been correlated with an early IFN-I response, while fatal NHP infections
are characterized by a lack of type I IFN production [44,45]. A LF chimeric mouse model, in which
wild-type bone marrow cells are transplanted into irradiated type I interferon receptor knockout
mice, has been developed recently and reproduces the main symptoms of LF [46]. In this model,
T cell-mediated immunopathology apparently plays an essential role in LF disease, highlighting the
dual activity of the T-cell response during LF. More studies are required to determine whether an early
T-cell response aids in LASV clearance, or whether a late and dysregulated T-cell response may actually
contribute to the severity of LF. On the other hand, JUNV infection elicits a robust IFN-I response
in both NHPs and guinea pigs [47,48]. As observed in LF patients, neutralizing antibodies against
LASV do not form in either surviving NHPs or guinea pigs during the acute phase of infection [45,49].
In contrast, neutralizing antibodies that develop early in infection are critical for clearance of JUNV
infection of guinea pigs [50]. MACV infection also induces a potent IFN response in NHPs [51] and
guinea pigs [52]. In addition, as demonstrated during human cases of AHF, immune plasma is an
effective treatment for AHF in NHPs [53]. These data demonstrate that hemorrhagic fever-causing
OW and NW arenaviruses trigger a vastly different immune response, which has implications in viral
pathogenesis as well as the development of vaccines and therapeutics.
 Vaccines 2019, 7, 138 5 of 15

RNA (dsRNA) molecules might form as byproducts of viral RNA replication (Figure 2). Host pattern
recognition receptors (PRRs) can efficiently recognize these pathogen-associated molecular patterns
Vaccines
(PAMPs) 7, 138
2019, as danger signals of virus infection and initiate downstream signaling cascades5 ofthat 16
ultimately result in an antiviral response. Among these PRRs, the Toll-like receptors TLR7 and TLR8
are known to recognize ssRNA in endosomes, but their role during pathogenic arenavirus infection
3. Arenavirus Interactions with Pattern Recognition Receptors
has not been determined. The majority of research into PRR recognition of arenaviruses has focused
Viral that
on PRRs RNAs often contain
primarily unique
recognize featuressuch
dsRNA, suchas 50 -triphosphate
asretinoic groups gene
acid-inducible and double-stranded RNA
I (RIG-I) and protein
structures that are distinguishable
kinase R (PKR) (Figure 2). from host RNAs. In addition, viral-derived double-stranded RNA
(dsRNA) moleculesgenomes
Arenavirus might form as byproducts
contain non-self ofRNAviralpatterns
RNA replication
that might(Figure
act as2).PAMPs;
Host pattern
both recognition
ends of the
receptors
genome form (PRRs) 5′ can efficiently recognize
ppp-containing panhandle these pathogen-associated
structures, molecularregions
while the intergenic patterns (PAMPs)
naturally as
form
danger signals of virus infection and initiate downstream signaling cascades that ultimately
hairpin loops to facilitate transcription termination [1]. In line with the JUNV-induced IFN response, result in an
antiviral
a recent response.
imaging study Among hasthese PRRs,dsRNA
revealed the Toll-like receptorsduring
accumulation TLR7 and JUNVTLR8 are known
infection [54].to recognize
The dsRNA
ssRNA in endosomes, but their role during pathogenic arenavirus infection
signals co-localize with the PRRs RIG-I and PKR as well as melanoma differentiation-associated has not been determined.
The majority
protein of researchstrongly
5 (MDA-5), into PRRsuggesting
recognitionthat
of arenaviruses
these dsRNAs has focused on PRRs that
are recognized by primarily recognize
and activate PRRs
dsRNA, such as retinoic
during arenavirus acid-inducible
infection [54]. gene I (RIG-I) and protein kinase R (PKR) (Figure 2).

Figure2.2. Schematic
Figure Schematic diagram
diagram of of interactions
interactions between
between arenavirus
arenavirus proteins
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andhost
hostinnate
innateimmune
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arenavirus infection,
infection, viral-derived
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double-stranded RNA RNA (dsRNA)
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during viral
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RNAsynthesis
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andrecognized
recognized by byhost
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Thismay
maytrigger
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responseand andPKRPKRresponse,
response,respectively.
respectively.The
Thenucleoprotein
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exonuclease
(ExoN)
(ExoN)efficaciously
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of
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arenaviruses also block the the IFN
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Arenavirus genomes
RIG-I has been contain non-self
demonstrated RNA patterns
to mediate the IFNthat might act
response to as PAMPs; both
pathogenic NWends of the
arenavirus
genome form 5 0 ppp-containing panhandle structures, while the intergenic regions naturally form
infection, while pathogenic OW LASV has seemingly evolved the ability to evade the RIG-I response.
hairpin loops to facilitate
RIG-I primarily transcription
recognizes termination
5′ triphosphate ssRNA [1]. In line with
or dsRNA [55], the
thenJUNV-induced IFN response,
undergoes a conformational
ashift
recent
andimaging study has
ubiquitination, revealed
and further dsRNA
interactsaccumulation
and activates during JUNV infection
mitochondrial anti-viral[54]. The dsRNA
signaling protein
signals co-localize with the PRRs RIG-I and PKR as well as melanoma differentiation-associated
protein 5 (MDA-5), strongly suggesting that these dsRNAs are recognized by and activate PRRs during
arenavirus infection [54].
Vaccines 2019, 7, 138 6 of 16

RIG-I has been demonstrated to mediate the IFN response to pathogenic NW arenavirus infection,
while pathogenic OW LASV has seemingly evolved the ability to evade the RIG-I response. RIG-I
primarily recognizes 50 triphosphate ssRNA or dsRNA [55], then undergoes a conformational shift
and ubiquitination, and further interacts and activates mitochondrial anti-viral signaling protein
(MAVS) [56,57] (Figure 2). MAVS activation leads to downstream signaling cascades that induce
the activation and nuclear translocation of interferon regulatory factors 3 (IRF3) and 7 (IRF7) as
well as nuclear factor kappa B (NF-κB), which ultimately upregulate gene expression of IFN and
pro-inflammatory cytokines. While studies have shown that transfected LASV RNA can activate the
IFN-β promoter in a RIG-I-dependent manner [58], IFN-β expression is not upregulated in the context
of LASV infection [59], indicating that LASV efficiently evades or inhibits the IFN response in the
context of virus infection. In contrast, IFN and interferon-stimulated genes (ISGs) are upregulated
in both JUNV and MACV infection [59,60]. Knockdown of RIG-I using siRNA substantially reduced
IFN-β and ISG production in JUNV-infected cells, indicating that the IFN pathway is activated in a
RIG-I dependent manner during JUNV infection. A recent screen of genes upregulated during NW
arenavirus infection identified elevated IFN and ISG gene expression (including RIG-I) during both
non-pathogenic NW Tacaribe virus (TCRV) and the Candid#1 vaccine strain of JUNV [61] infections.
JUNV NP is found co-localized with RIG-I in infected cells in imaging studies [54,62], though the
influence of NP on RIG-I function is still unknown. The Candid#1 strain of JUNV has also been shown
to induce nuclear translocation of IRF3, indicating that the RIG-I-mediated IFN response is functional
during JUNV infection [60].
Another cytosolic RIG-I-like receptor (RLR), MDA-5, has also been shown to play a role during
arenavirus infection. MDA-5 binds preferentially to long dsRNA ligands and activates the IFN pathway
similarly to RIG-I [55]. In JUNV infection, MDA-5 has been visualized co-localizing with dsRNA
along with NP, suggesting that MDA-5 is recognizing dsRNA during JUNV infection [54]. Unlike
RIG-I, MDA-5 is not required for stimulation of IFN-β upon transfection of LASV RNA [58]. A recent
study with myeloid dendritic cells revealed that the non-pathogenic MOPV, but not LASV, induces
upregulated MDA-5 gene expression [36]. Since MDA-5 is an ISG, the upregulation of MDA-5 is very
likely mediated by the IFN response. Upregulation of MDA-5 is also observed during non-pathogenic
TCRV infection as well as during Candid#1 infection [61].
Apart from the RLRs, the dsRNA-activated protein kinase R (PKR) also becomes activated
following JUNV and MACV, but not LASV infection (Figure 2). PKR is ubiquitously expressed in
cells and can be further transcriptionally induced by IFN [63,64]. Upon dsRNA recognition, PKR
undergoes autophosphorylation and becomes enzymatically activated. PKR then phosphorylates the
eukaryotic translation initiation factor 2 (eIF2α), leading to translational shutoff (Figure 2). LASV
infection does not activate PKR [65], nor does it prevent PKR activation in the presence of the dsRNA
analog poly (I:C), implying that LASV evades PKR detection rather than directly interfering with its
kinase activity [65]. In contrast, PKR is activated during JUNV (pathogenic Romero strain) and MACV
infections, concomitant with eIF2α phosphorylation and translation inhibition [65]. Expression of
IFN and ISGs (e.g., OAS1 and ISG15) is augmented in PKR-deficient cells as compared with that in
wild-type cells during JUNV and MACV infections. The pathogenic Romero strain of JUNV and MACV
replicate slightly more efficiently in wild-type cells than in PKR-deficient cells. These results suggest
that pathogenic NW arenaviruses exploit PKR activation to facilitate virus infection by attenuating
ISG expression. The vaccine Candid#1 strain of JUNV also activates PKR. Interestingly, Candid#1
JUNV infection neither results in eIF2α phosphorylation nor translation inhibition [61,66], unlike
the pathogenic Romero strain JUNV. Knockout of PKR does not affect Candid#1 JUNV replication.
Candid#1 infection blocks poly (I:C)-induced eIF2α phosphorylation. It is worth noting that NP
interacts with PKR in Candid#1 infected cells [54,66]. Whether and how JUNV NP affects the PKR
pathway needs to be addressed in future studies. The non-pathogenic TCRV also induces PKR
activation and phosphorylation of eIF2α [61]. Knocking out PKR facilitates TCRV infection, indicating
that TCRV is susceptible to the PKR-mediated antiviral response.
Vaccines 2019, 7, 138 7 of 16

4. Molecular Mechanisms for Arenavirus Antagonism of the IFN Response

Arenaviral NP has been identified as an IFN antagonist targeting different steps in the IFN pathway
(Figure 2). Examination of the crystal structure of LASV NP has revealed a DEDDh exonuclease (ExoN)
motif that preferentially targets dsRNA [67–69]. Purified NPs of LASV, TCRV, MOPV, and PICV have
been shown to degrade dsRNA in biochemistry assays [69–71]. The ExoN motif is highly conserved
among all arenaviruses [67,68] and has been proposed to degrade viral dsRNA produced during
viral replication, preventing recognition by non-self RNA sensors such as RIG-I and MDA-5 that
otherwise will trigger an innate immune response (Figure 2). However, dsRNA can be readily detected
in JUNV-infected cells and co-localizes with PRRs (RIG-I and PKR) [54], which is consistent with
the robust IFN/PKR responses observed in JUNV infected cells. Therefore, the role of NP ExoN in
the context of virus infection remains to be examined in future studies. It is possible that the NP
ExoN activity or its regulation is different among arenaviruses. It is also possible that the highly
conserved NP ExoN may have an additional function(s) that is critical for arenaviruses. In addition to
the ExoN activity, NPs of pathogenic arenavirus have been demonstrated to block IRF3 and NF-κB
activation [72,73]. However, the IRF3 pathway does not appear to be completely blocked during
NW arenavirus infection [60]. One possibility is that the IFN antagonist activities of the NPs of
pathogenic NW arenaviruses (JUNV and MACV) are weaker than that of LASV NP during virus
infection, explaining why the RIG-I/IRF3 signaling pathway is active in JUNV and MACV but not in
LASV infection.
The NPs of non-pathogenic TCRV and MOPV degrade dsRNA in biochemistry assays. Expressed
TCRV NP inhibits IFN reporter gene expression similarly to LASV NP [69]. Hence it seems that
non-pathogenic and pathogenic arenaviruses sometimes have similar anti-IFN activities. It is worth
noting that these data are based on assays with purified or plasmid-expressed NP. Nevertheless,
TCRV or MOPV infection induces an IFN response [36–38,61]. During infection, the kinetics and the
level of NP expression, as well as the distribution of NP are different from that in plasmid-expressed
cells. Additionally, NP also engages in viral RNA replication, RNP formation, and virion formation.
Therefore, the interaction of NP with the IFN pathway may be different in infected cells compared to
that in plasmid-expressing cells, which may reconcile the difference sometimes observed in expression
and infection studies.
Differences in interplay with PKR are also observed among arenavirus NPs. Unlike Candid#1
JUNV NP, neither LASV nor LCMV NP bind to PKR in co-expressed cells [66]. Co-localization of PKR
with NW arenavirus NP can also be visualized in Candid#1 or TCRV infected cells [54,61]. Though more
research is needed, the NPs of arenaviruses interact with the RIG-I and PKR pathways differentially,
which may contribute to the differences in innate immune responses observed in clinical cases.
Recent research has also revealed that arenaviral NP inhibits the PACT-enhanced RIG-I-mediated
IFN response [74]. PACT is a 313-amino-acid cellular protein that binds directly to both dsRNA
and RIG-I [75] (Figure 2). EBOV VP35, IAV NS1, HSV-1 Us11, and MERS-CoV 4a and N, have been
demonstrated to block PACT enhancement of the RIG-I-mediated IFN response by disrupting PACT and
RIG-I interaction [76–79]. NPs of various arenaviruses (LASV, JUNV, MACV, TCRV, and PICV) inhibit
PACT-enhanced, RIG-I-mediated IFN reporter gene expression [74]. This inhibition is not through
blocking of PACT-RIG-I binding [74], but rather depends on the NP exonuclease activity, as both LASV
and PICV NP mutants with defective exonuclease activity are unable to inhibit PACT-potentiated
IFN-β production [74]. Thus, arenavirus NP is speculated to degrade dsRNA and thereby prevent
PACT from binding to dsRNA. PACT is known to activate PKR [80], though the role of arenaviral NPs
on PACT-mediated PKR activation remains unknown.
A recent large-scale protein interaction screen revealed that both OW and NW arenavirus NPs
interact with the host DEAD-box ATP-dependent RNA helicase, DDX3 [81]. DDX3 exhibits a proviral
role for arenaviruses, as replication of LCMV, LASV, and JUNV was substantially impaired in DDX3
knockout cells [81]. Two different mechanisms have been proposed for the proviral effect of DDX3.
Early in infection, DDX3 assists in viral RNA replication, as supported by the reduced arenavirus
Vaccines 2019, 7, 138 8 of 16

minigenome replication in DDX3 knockout cells [81]. Mutation in the DDX3 ATPase and helicase
domains also leads to reduced minigenome replication, indicating that these domains are essential
for the proviral function of DDX3 [81]. Late in infection, DDX3 may act to suppress IFN production
and facilitate LCMV infection [81]. By contrast, DDX3 does not affect IFN-β transcription in JUNV
infection [81].
In addition to NP, arenavirus Z protein has also been implicated as a suppressor of the host innate
immune response. Z has been shown to directly interact with RIG-I and prevent its association with
MAVS, thus blocking IFN signaling [82–84] (Figure 2). Originally, differential roles for Z protein in OW
and NW arenaviruses were assumed, as Z proteins of NW arenaviruses seemed uniquely capable of
inhibiting RIG-I-mediated signaling [83]. However, more recent research has revealed that Z proteins
of pathogenic arenaviruses, but not from nonpathogenic arenaviruses, are able to inhibit RIG-I and
MDA-5 signaling [82]. The inhibition is mediated by interaction of the N-terminal domain on Z protein
with the CARD domain on RLRs that disrupts RLR and MAVS interactions. The IFN antagonist activity
of Z protein is found more prominently in macrophages, raising the possibility that Z proteins might
inhibit the IFN response mainly in macrophages, while NPs act mainly in DCs. Further work will be
needed to study the roles of Z protein in modulating the host immune response.

5. Arenavirus Subversion of Other Host Antiviral Defenses

The autophagy pathway is an important component of host antiviral defense, functioning to
degrade pathogens and trigger both innate and adaptive immunity. While many viruses have evolved
to suppress the autophagy pathway, others hijack it to assist in viral replication [85]. The role of
autophagy during arenavirus infection is largely unknown, but recent research has provided evidence
of a proviral role for autophagy. Two groups observed that both virulent and attenuated strains of
JUNV induce autophagy early in infection [86,87]. Induction of autophagy prior to infection results
in increased NP expression [87]. The OW MOPV also transiently induces autophagy during the first
two days of infection, though LASV does not [88]. Nevertheless, depleting the essential autophagic
vesicle protein ATG5 impairs MOPV, LASV, and JUNV replication [86–88], indicating that both OW
and NW arenaviruses may utilize components of the autophagy pathway to aid in replication. While
ATG5 is the key component in autophagy, there is increasing evidence that ATG5 also plays roles
in non-autophagy processes, including negative regulation of RIG-I or MDA5 [89–91]. Thus, future
studies are required to define if ATG5 is directly involved in arenavirus replication.
The mechanism behind autophagy induction in arenavirus infection is unclear. One group
determined that, unlike replication-competent JUNV, UV-inactivated JUNV did not induce autophagy
up to 9 hours post-exposure [86]. However, another group observed autophagy induction 24 hours
after exposure to UV-inactivated JUNV [87], though it is unclear whether this was virus-driven.
Autophagy likely plays a role late in arenavirus infection, as depleting ATG5 reduced extracellular,
but not intracellular, LASV and MOPV RNA levels [88]. Several viral proteins have been demonstrated
to interact directly with key components of the autophagy pathway. MOPV Z, and to a lesser extent
LASV Z, interact with the sequestosome 1-like receptors NDP52 and TAX1BP1 [88]. JUNV NP has
also been visualized co-localizing with LC3 [87]. However, the biological significance of viral protein
interaction with components of autophagy remains unknown.
Another means by which pathogenic arenaviruses may subvert the host immune response may
be through preventing or avoiding the activation of natural killer (NK) cells. NK cell activation is
dependent on a mixture of activating and inhibiting signals presented by APCs [92]. It has been shown
that LASV-activated macrophages are able to activate NK cells, but the activation is incomplete and
ultimately does not suppress viral replication [93]. NP-mediated immunosuppression likely plays a
role in preventing NK cell activation, as mutants lacking NP exonuclease function are able to fully
activate NK cells in coculture with APCs [94]. Furthermore, it has been hypothesized that LASV may
utilize NK inhibitory receptors to escape detection. LASV-activated NK cells are able to lyse K562 cells
which lack HLA class-1 expression, but they cannot lyse LASV-infected DCs that are constitutively
Vaccines 2019, 7, 138 9 of 16

expressing HLA class-1 [93]. A recent study revealed that certain LASV epitopes bound to HLA-C1
molecules activate the KIR2DL2 inhibitory NK cell receptor, thus preventing NK cell activation [95].
Patients who succumb to LF are more likely to carry the KIR2DL2 gene than survivors [95]. The specific
interactions between LASV bound to HLA molecules and the variety of inhibitory NK cell receptors
may explain some of the clinical variability seen in LF cases.

6. Adaptive Immune Responses Following Successful Arenavirus Vaccination in Animal Models

For arenaviruses, the attenuated Candid#1 JUNV is the only vaccine that is approved for use in
humans in Argentina. The vaccine elicits strong and protective neutralizing antibody responses in
those vaccinated and has been successful in controlling AHF within endemic regions [96]. A chimeric
MACV expressing the GPC from Candid#1 JUNV is highly attenuated in a mouse model and protects
animals from lethal MACV challenge [97]. This protection correlates with high titers of MACV-specific
neutralizing antibody before challenge.
Several LASV vaccine candidates currently in the preclinical stage are very promising based on
their safety, immunogenicity, and efficacy in animal models. ML29 is a LASV/MOPV reassortant virus
that contains the S segment of LASV and the L segment of MOPV. This vaccine platform expresses
the main LASV antigens (GPC and NP). ML29 vaccination efficaciously protects NHPs and guinea
pigs from LASV challenge through cell-mediated immunity; meanwhile the neutralizing antibody
is below the detection level [98,99]. A recombinant vesicular stomatitis virus vector expressing
the LASV GPC (VSV∆G/LVGPC) efficaciously protects NHP from lethal LASV challenge [100].
After VSV∆G/LVGPC immunization, a low level of neutralizing antibody is detected in vaccinated
animals, meanwhile the T-cell response is measurable in one of four animals. Nevertheless, all vaccinated
NHPs survived challenge and developed potent humoral and cellular immune responses. Finally,
vaccination of NHPs with a vaccinia virus vector expressing LASV G1 and G2 confers protection
from lethal LASV challenge [101]. The antibody response is low in survivors. Protection is very
likely through cell-mediated immunity as vaccinia virus is known to induce the cellular immune
response predominantly.

7. Impacts of the Adaptive Cellular Immune Response on Post-Infection Sequelae

Survivors of pathogenic arenavirus infection often develop post-infection sequelae in the months
following the acute stage of illness. Neurological symptoms are common during NW arenavirus
infection. This correlates with robust viral replication in neurological tissues as evidenced by high
viral titers in the brains of infected animals [52,102]. MACV antigen can also be preferentially detected
within neurons [52]. Interestingly, treatment of AHF and BHF with convalescent serum increases the
likelihood of developing a long-term neurologic syndrome [43,103]. Whether neurological damage is
directly caused by the virus infection or through an immune-mediated mechanism is still unknown.
In LASV infection, accumulating evidence has raised the possibility that the neurological sequelae
are likely caused by virus-induced immunological injury. A recent study using NHPs as a model for
LASV infection demonstrated that NHP survivors developed pathological findings consistent with
autoimmune-associated vasculitis [104]. Two out of three NHP survivors also developed sensorineural
hearing loss similar to that observed in human cases. Histopathological examination of the inner ear
revealed inflammation of vessels and perivascular tissue at 45 days post-infection, long after the acute
infection had subsided. Furthermore, serological analysis revealed that survivors developed elevated
C-reactive protein and antineutrophil cytoplasmic antibodies, which are indicators of autoimmune
disease [104]. These findings in NHPs indicate that hearing loss acquired after LF may be due to
chronic inflammation.
Studies using a rodent model of LASV-induced hearing loss have provided further evidence
that hearing loss may be caused by a cell-mediated immune response rather than through direct
viral damage [105]. STAT1 knockout mice infected by clinical LASV isolates develop sensorineural
hearing loss, though IFNαβ/γ receptor knockout mice do not. Interestingly, while LASV antigen can
Vaccines 2019, 7, 138 10 of 16

be detected in the inner ears of both types of mice, tissue damage was only observed in the STAT1
knockout mice concomitant with profound CD3-positive lymphocytic infiltration [105]. It would be
interesting to determine if depletion of T cells could prevent hearing loss in this model.
One study using guinea pigs as a model for LASV infection determined that anterior uveitis was
common during both fatal and nonfatal LASV infections [106]. This ocular inflammation was largely
T-cell-mediated. However, low levels of LASV RNA were detected in the eyes of all guinea pigs who
succumbed to infection as well as 3 of the 7 survivors. While viral antigen was not detected in the eye
during this study [106], immunohistochemical staining has revealed the persistent presence of LASV
in the smooth muscle of arteries in both a guinea pig and NHP model of infection, likely contributing
to the long-lasting vasculitis [104,107]. Thus, it is currently hypothesized that persistently low levels of
LASV replication trigger a chronic activation of the adaptive cellular immune response that leads to
long-lasting inflammation.

8. Conclusions
Arenaviruses represent a continuing emerging threat as humans increasingly come into contact
with their rodent reservoirs. Data from both clinical and animal model studies demonstrate that
pathogenic OW and NW arenaviruses elicit vastly different immune responses, which have implications
in viral pathogenesis. LASV infection is characterized by weak or delayed IFN-I/ cytokine induction
and T-cell responses, while pathogenic NW arenaviruses (JUNV and MACV) trigger a robust IFN-I
and pro-inflammatory cytokine response. Though the mechanisms behind these differences remain
poorly defined, several observations have been noted. JUNV and MACV infections activate a variety
of PRRs likely through dsRNA accumulation, while LASV seems to evade PRR detection. For all
arenaviruses tested so far, arenaviral NP and Z proteins are capable of interfering with PRR activation
and blocking innate immune signaling in expression studies. Nevertheless, their activity during viral
infection remains to be determined. Differences in the innate immune response likely account for the
differences seen in the adaptive response to hemorrhagic fever-causing arenaviruses. Overall, LASV
clearance is associated with an early and strong cellular immune response, while recent findings have
implicated the cellular immune response as a key contributor to the chronic inflammation and sequelae
seen in LF survivors. This has profound implications in LASV vaccine development, particularly for
those LASV vaccine candidates that are based on a T-cell response. Protection and recovery from
pathogenic NW arenavirus infection are mediated by the humoral response. However, pathogenic
NW arenaviruses that invade the immune-privileged central nervous system may evade clearance,
which potentially causes neurological sequelae. This knowledge may inform the development of a
neutralizing antibody-based therapy to treat AHF and BHF patients. Appreciation of the differential
immune response to highly pathogenic NW and OW arenaviruses should facilitate the rational design
of targeted therapeutics and vaccines.

Author Contributions: E.M., S.P. and C.H. compiled and wrote the paper.
Funding: C.H. was supported by UTMB Commitment Fund P84373 and UTMB IHII grant P84501. S.P. was
supported by Public Health Service grant RO1AI093445 and RO1AI129198.
Acknowledgments: C.H. would like to acknowledge Galveston National Laboratory (supported by the UC7
award 5UC7AI094660) for support of research activity.
Conflicts of Interest: The authors declare no conflict of interest.

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