Immunity: Gaining a Winning Position in Lightning Chess Aurélie Ploquin, Yan Zhou and Nancy J. Sullivan This information is current as J Immunol 2018; 201:833-842; ; of September 23, 2021. doi: 10.4049/jimmunol.1700827 http://www.jimmunol.org/content/201/3/833 Downloaded from References This article cites 151 articles, 42 of which you can access for free at: http://www.jimmunol.org/content/201/3/833.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Ebola Immunity: Gaining a Winning Position in Lightning Chess Aure´lie Ploquin, Yan Zhou, and Nancy J. Sullivan Zaire (EBOV), one of five in the ge- infected secretions or fluids (4–6). The assumption that EBOV nus Ebolavirus, is the causative agent of the hemor- persists sublethally in its natural reservoir but causes rapid disease rhagic fever disease epidemic that claimed more than progression and death in highlights the role of host 11,000 lives from 2014 to 2016 in West Africa. The immune system in determining infection outcomes. combination of EBOV’s ability to disseminate broadly and rapidly within the host and its high pathogenicity Ebola infection pose unique challenges to the immune system Insights into the natural history of EBOV infection in humans postinfection. Potential transmission from apparently have been obtained through contact tracing and exposure Downloaded from healthy EBOV survivors reported in the recent epi- history and have established the EBOV incubation period to be demic raises questions about EBOV persistence and from 2 to 21 d after initial contact with the (5, 7). The immune surveillance mechanisms. Clinical, virological, delay between self-reporting of symptoms (fever, cough, rash, and immunological data collected since the West Africa abdominal pain) and urgent care admission hinders the col- epidemic have greatly enhanced our knowledge of lection of immediate postexposure samples, so the majority of http://www.jimmunol.org/ host–virus interactions. However, critical knowledge human biological samples are not obtained until days after the gaps remain in our understanding of what is necessary onset of symptoms (on average 6 d postonset) (Fig. 1) (8–14), for an effective host immune response for protection resulting in a dearth of data on early infection events and against, or for clearance of, EBOV infection. This re- initial host immune responses. During the next phase of the view provides an overview of immune responses against disease, infected individuals display extreme fatigue, head- ache, vomiting, diarrhea, dyspnea, hypovolemic shock, and EBOV and discusses those associated with the success organ failure, with death generally occurring from 7 to 14 d or failure to control EBOV infection. The Journal of after the onset of initial symptoms (9, 11, 12, 14, 15). Sur-

Immunology, 2018, 201: 833–842. vival is broadly associated with a lower viral load at the time by guest on September 23, 2021 of admission (16) and, although previously underappreciated, n 1976, the first documented case of Ebola infection was postrecovery, long-term sequelae have been identified in follow-up reported in sub-Saharan Africa (1), caused by the pro- studies in West African survivor cohorts (17, 18). I totypical Zaire ebolavirus (EBOV), whose most recent Although there are limited historical case reports suggesting that EBOV replicates in sites of immunological privilege, the emergence in 2014 caused a global emergency 2014–2016 epidemic provided reproducible case data on with more than 28,000 cases and 11,000 deaths (2). From EBOV in the CNS, eye, and gonads (4, 19–26). These ob- 1976 to 2014, the recognized outbreaks of EBOV had a cu- ∼ servations highlighted the fact that EBOV can persist in the mulative reported case count of 1500 people and an average infected host long after viral clearance from the circulation fatality rate of 78% (2). Most of these EBOV outbreaks were (plasma and urine) in convalescent individuals (Fig. 1). This rapidly contained because of the small, isolated populations theory is further supported by case reports of possible sexual involved, the limited population movement outside of those transmission events from survivors to their partners long after areas, and the effective implementation of contact tracing and symptom resolution (6) and recurrence of viremia potentially quarantine, but the West African epidemic illustrated the seeded from immunologically privileged sites (22). The failure potential for uncontrolled spread. Analysis of index cases from of the immune system to prevent the establishment of viral outbreaks suggests that and other can serve as persistence and reinitiation of infection is a newly recognized intermediate hosts, but the natural reservoir for EBOV may immunological challenge for the clearance of EBOV, adding be (3). Following the initial jump into humans, trans- to the spectrum of events from very early infection to long- mission occurs from person to person via close contact with term sequelae that require elucidation.

Vaccine Research Center, National Institute of and Infectious Diseases, National Health, 40 Convent Drive, Building 40, Room 2509, Bethesda, MD 20892. E-mail Institutes of Health, Bethesda, MD 20892 address: [email protected] Received for publication June 7, 2017. Accepted for publication May 5, 2018. Abbreviations used in this article: ADCC, Ab-dependent cellular cytotoxicity; ADCP, Ab-dependent cellular phagocytosis; ADE, Ab-dependent enhancement; DC, dendritic This work was supported by the Intramural Research Program of the Research cell; EBOV, Zaire ebolavirus; ISG, IFN-stimulated gene; MDA5, melanoma differenti- Center, the National Institute of Allergy and Infectious Disease, and the National ation–associated 5; mDC, monocyte-derived DC; MDM, monocyte-derived Institutes of Health. macrophage; NP, ; NPC1, Niemann–Pick C1; PD-1, programmed Address correspondence and reprint requests to Dr. Nancy J. Sullivan, Vaccine Research death–1; RBD, receptor binding domain; RIG-I, retinoic acid–inducible gene I. Center, National Institute of Allergy and Infectious Diseases, National Institutes of www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700827 834 BRIEF REVIEWS: EBOLA VIRUS IMMUNITY Downloaded from

FIGURE 1. Schematic representation of EBOV infection and immune-response time course in human survivors. EBOV replicates locally and triggers innate immune responses (yellow). The increase of viremia in the systemic circulation (blue) corresponds to the self-reported onset of symptoms leading to the hos- http://www.jimmunol.org/ pitalization of an infected person a few days after symptoms onset (fever, abdominal pain). During clinical onset, IgM (light green), IgG (dark green), and cellular responses (orange) are detected. During the recovery phase, the virus can persist at immune-privileged sites in the absence of viremia. The average time period for nonsurvivors is indicated by a shaded blue box as reference. Interrogation points represent estimations for curve shape because of lack of reported human data.

Because of the difficulty of early case identification and about vaccine or therapeutic efficacy from these models are sampling during outbreaks, most data from infected humans often not transferrable to large animal models (40, 41). are limited to the symptomatic phase of the disease. As a result, Ebola disease in macaques shares many of the same clinical early events that preceded the onset of symptoms, in which features as human infection, but time to death is accelerated, and by guest on September 23, 2021 virus–host dynamics are key to determining infection out- the mortality rate (100%) greatly exceeds that in humans, likely come, remain a critical gap in knowledge. If EBOV behaves due to the high dose infectious challenge (more than 100 3 similarly to what has been observed for other RNA LD99) and i.m. route of exposure in experimental infections. (hepatitis C virus, HIV, influenza virus), transmission will However, similar to human infection, EBOV elicits disease occur by a tiny fraction of selected variants from the exposing characterized by fever, rash, anorexia, multiple organ failure, and virus swarm that can successfully establish infection in a new coagulation dysfunctions in macaques (42). Although the chal- host (27–29). After the founding event, host immune selective lenge route may result in Ag recognition by different populations pressure and mutations introduced by the viral polymerase of immune cells than would be present at mucosal or skin sur- shape a new virus swarm that replicates to high circulating faces exposed during natural human infection, similarities such titers (27, 30, 31). Although extensive sequencing has been as the kinetics of immune responses relative to phase of infection, performed to characterize circulating EBOV after systemic lymphopenia, and a late-stage cytokine storm suggest this model spreading of the infection, little is known about the trans- can yield insights into the mechanisms of immunologic control mitted founder virus, which may be the most relevant for of EBOV. In light of the similarities between the macaque vaccine targeting and evaluation in animal models (32–34). model and human disease, this review will focus primarily on studies that characterize the immune responses to EBOV in- Animal models fectioninthesetwohosts. Mouse EBOV models require species adaption of the virus The game: host response to EBOV infection away from the human strain to overcome the murine I IFN response and generate lethal infection (35). Collaborative EBOV replicates in the spleen and lymph nodes prior to Cross inbred mice lethally infected with mouse-adapted virus systematic dissemination, at which point circulating virus can develop some signs of hemorrhage, a hallmark of human be detected (43). Early infection of monocytes, macrophages, EBOV pathogenesis (36). However, mouse-adapted EBOV in and dendritic cells (DCs) is the gateway for virus dissemina- most other mouse models does not result in an infection that tion, and represents the initial assault on the host immune fully recapitulates the fever, rash, or hemorrhagic clinical system by viral (Fig. 2). Thus, the contest between manifestations observed in humans (37), nor the immune host and virus starts within these immune cells, which not responses, such as TNF-a production (38). Early insights into only constitute the first line of immune defense, but are also protective immunity are derived primarily from vaccination in pivotal for initiating the adaptive immune response. small animal models (39). Although mouse models are still The opening move: type I IFN response. When EBOV infects cells used to screen preliminary vaccine candidates, observations in vitro, viral dsRNA formed in the during the The Journal of Immunology 835

FIGURE 2. Model of immune re- sponse against EBOV. Left, Innate im- mune responses. EBOV initially targets macrophages and DCs. Recognition of viral dsRNA by RIG-I and MDA5 in- duce type I IFN responses and the pro- duction of proinflammatory cytokines andchemokines,whichservetoinhibit viral replication and recruit and activate other leukocytes. EBOV blocks multiple aspects of the type I IFN response and interferes with T cell activation by in- ducing aberrant DC maturation. Right, Adaptive immune responses. Abs control viral infection by preventing EBOV entry into target cells. Ab-mediated effector functions, such as complement-dependent cytotoxicity (CDC), ADCC, or

ADCP, could also contribute to viral Downloaded from clearance. However, ADE could oc- cur when Abs are at suboptimal con- centrations. CD8+ T cells may play a major role in clearing infection.

- http://www.jimmunol.org/ replication cycle is recognized by the pattern recognition driven by immune activation and the upregulation of inhib- receptors, the retinoic acid–inducible gene I (RIG-I), and itory receptors (59, 60). In EBOV infection, initial viral melanoma differentiation–associated protein 5 (MDA5), which replication takes place in the spleen and lymph nodes, days leads to induction of type I IFN responses and the expression of before the virus spreads through the circulation and the rise of IFN-stimulated genes (ISGs) that inhibit viral replication (44, systematic type I IFN responses (43). It is possible that local 45). In vivo, plasma IFN-a and upregulation of ISGs become type I IFN responses at the tissue level prior to the onset of detectable after the onset of symptoms in both EBOV patients viremia are critical to determining survival outcomes. by guest on September 23, 2021 and infected macaques (43, 46–49).However,insteadofthe The strength of tissue-level type I IFN responses during the transient peak that is observed in nonlethal viral infections earliest phase of virus replication is likely determined by DCs (50–52), plasma IFN-a remains high throughout the and macrophages, as they are the first cell types associated with remainder of the EBOV disease course in macaques (43, EBOV RNA in lymph nodes (43). Monocyte-derived DCs 53). Despite the large amount of circulating IFN-a, EBOV (mDCs) do not secrete IFN-a, nor do they undergo normal viral load in macaques continues to rise to .106 PFUs/ml, maturation when infected by EBOV in vitro (61, 62). This is and the outcome is uniformly fatal (43). largely due to suppression of the type I IFN response by VP24 There are several possible explanations for the failure of (63). VP35 is another that interferes with the observed type I IFN responses to promote protective immu- activation of RIG-I, prevents the recognition of viral dsRNA nity. First, there is evidence that type I IFN responses could be by RIG-I/MDA5, and disrupts signaling events triggered by blunted by EBOV antagonism at steps downstream of IFN RIG-I/MAD5 engagement (45). In monocyte-derived mac- production. For example, EBOV protein VP24 blocks the IFN rophages (MDMs), in vitro EBOV infection induces IFN-a signaling pathway by preventing nuclear localization of a production between 9 and 72 h (46, 64). However, the level transcription factor STAT1, which is essential for ISG ex- of IFN-a induction is limited by viral IFN antagonists, as pression (54). As another example, EBOV glycoprotein an- shown by reduced IFN-a production by EBOV-infected tagonizes the action of tetherin, one of the ISGs that prevents MDMs in response to a synthetic IFN-a stimulant (46). virus budding (54). Second, the timing and magnitude of the The ability of EBOV proteins to impair IFN-a production by type I IFN responses affect its antiviral impacts. Early type I MDMs upon viral infection is further illustrated with a IFN responses induced by the administration of exogenous recombinant Newcastle disease virus encoding VP35, which type I IFN protect animals against lymphocytic choriomen- induces 8000 times lower IFN production in infected MDMs ingitis virus, severe acute respiratory syndrome coronavirus, as compared with a Newcastle disease virus encoding no VP35 and SIV infection (55–57). In contrast, delayed type I IFN (65). Impairment of the local type I IFN response by EBOV responses, due to either the ablation of early type I IFN before the onset of viremia could profoundly affect subse- producing cells or viral antagonism, are associated with rapid quent viral–host interactions through inefficient containment virus propagation (57, 58), reduced frequencies of virus- of local viral replication, failure to activate innate immune specific CD8+ T cells (55, 58), and pathogenic inflamma- cells such as NK cells, and poor priming of T cell responses in tion (55). Further, excessive type I IFN responses can be the absence of type I IFN responses (Fig. 2). This hypothesis detrimental, as observed in chronic lymphocytic choriomen- is indirectly supported by experiments in which treatment ingitis virus infection, in which CD8+ T cell exhaustion is of macaques on the day of infection with IFN-a2b extends 836 BRIEF REVIEWS: EBOLA VIRUS IMMUNITY survival time by 2 d (66), and early treatment with IFN-b subsets in primates can be explored to directly test their im- extends mean survival time from 8 to 13 d (67). portance in protecting the host against EBOV (83). The setup: inflammation and immune cell recruitment. Systemic in- The capture: Ab responses. In EBOV-infected humans and flammation is a key feature of EBOV infection. Proinflammatory vaccinated macaques, high titers of plasma IgM and IgG are cytokinessuchasIL-6andTNF-a, as well as anti-inflammatory observed in survivors (84, 85). In humans, Ag-specific IgM chemokines such as IL-10, are detected in the circulation levels increase coincidentally with viral load decreases, and Ag- following the onset of symptoms in humans and macaques specific IgG first appears 1 wk after the appearance of IgM (43, 48, 68–72). In survivors of EBOV infection, the level of in the blood (Fig. 1) (8, 26, 47, 84). Although an early circulating cytokines peaks briefly and subsides as infection is study administering convalescent whole blood from EBOV controlled. However, in nonsurvivors, cytokine levels continue to survivors to patients suggested a potential therapeutic benefit increase despite the presence of high levels of IL-10 (68, 70), of Abs, subsequent studies administering either convalescent resulting in uncontrolled systemic inflammation analogous to whole blood or plasma to EBOV-infected humans did not that seen in bacterial and fungal sepsis (73). demonstrate a survival advantage in recipients (10, 86–90). In contrast to impaired type I IFN responses, inflammatory In most cases, the amount of Ag-specific IgG in the donor responses are effectively triggered by EBOV infection in early samples was not quantified prior to infusion, so negative results target cells. MIP-1a and MIP-1b chemokines are secreted by could be explained by insufficient quantities of Ag-specific Ab both mDCs and macrophages upon EBOV infection in vitro transferred to recipients. Similar studies in macaques, delivering (62, 64). In addition, macrophages infected in vitro produce EBOV-convalescent whole blood to recipients, did not provide Downloaded from inflammatory cytokines such as TNF-a, reactive oxygen protection against challenge; however, as in the human studies, species, and reactive nitrogen species (61, 64, 74). These data this could be due to the quantity of Ag-specific Ab present, as suggest that EBOV is capable of inducing local inflammation evidenced by the low IgG titers measured a few days after infusion early in infection (Fig. 2). The fact that the type I IFN re- (91). Follow-up studies used IgG purification and concentration sponse and inflammatory response are differentially regulated to maximize levels in the recipient but still failed to protect, http://www.jimmunol.org/ by EBOV within the same target cells is reminiscent of innate possibly owing to species mismatch between the donor and immune modulation by 1918 influenza virus infection (75). recipient, resulting in rapid clearance of Abs postinjection or In contrast to a seasonal influenza virus that induces strong inferior Fc-mediated effector functions (66, 92). Indeed, type I IFN responses and transient inflammation in the in other experiments, species-matched IgG, transferred from EBOV-convalescent to naive macaques, remained elevated in bronchi of infected macaques, pandemic 1918 influenza virus the recipients for several weeks and did provide a survival suppresses the type I IFN response but induces strong benefit (93). However, the failure of high-titer, species-matched, and sustained inflammation in the lung (75). High levels of vaccine-generated polyclonal IgG to protect in the more stringent chemokines and cytokines result in infiltration of inflamma- cynomolgus macaque model illustrates that the requirements for by guest on September 23, 2021 tory cells, eventually leading to severe hemorrhage (76). Ab-mediated protection are multifactorial (94). The requirements Dysregulation of innate responses at an early stage of infection for successful passive immunotherapy with Ig must be defined by may be a common characteristic shared between EBOV and macaque studies in which the Ag history of the donor 1918 influenza virus, contributing to the high virulence in (vaccination and challenge survival versus vaccination alone), both cases. species, regimen, formulation, and importantly, Ab quality, an Following EBOV triggering of inflammatory responses, aspect not addressed by these studies but likely impacting on prominent neutrophilia is observed prior to the onset of viremia in protection, are all controlled. The mixed success of Ig transfer macaques (69), indicating widespread recruitment to sites of is not surprising because EBOV has evolved a morphology infection. This increase in peripheral neutrophils is detectable in and replication strategy that present special challenges for either relative or absolute counts, with the latter approaching recognition and inhibition by the humoral immune system. what is observed with bacterial infections (77, 78). Neutrophils EBOV particles are in some cases more than 10 mminlength are activated by EBOV infection in vitro, resulting in the se- with a high density of glycoproteins stably anchored to the virion cretion of proinflammatory cytokines, which potentially con- surface, suggesting that a high local concentration of Ab may be tribute to the overall proinflammatory milieu (79). Through required to efficiently coat the virion and block infection (85), these proinflammatory contributions, it is possible that neutro- with polymorphic virion shapes potentially impeding Ab access to phils play a role in EBOV immunopathogenesis. some glyoprotein trimers (95). Perhaps more problematic is the Peripheral NK cells have been reported to transiently decline promiscuous use of multiple factors to mediate initial attachment upon EBOV infection in humans and macaques, suggesting to cells, ensuring that no single Ab specificity will successfully that either cell death or redistribution into infected tissues is block this critical first step in entry (Fig. 3). This nonspecific occurring (47, 80). Although NK cells are enriched in liver attachment is followed by rapid uptake into the cell by and spleen, which are major replication sites in early infection macropinocytosis (96–101). Once in the endosome (102), Ab (43), their contribution to the control of EBOV infection access to the virion is limited (unless Abs have bound prior to remains to be demonstrated. Genetic analyses among EBOV- virion uptake), and high affinity interactions will be necessary to infected patients could provide more information regarding remain associated under conditions of endosomal acidification the role of NK cells in vivo, as specific allelic combination of (103). Throughout the initial entry steps, the EBOV receptor killer cell Ig-like receptors and HLA loci has been associated binding domain (RBD), a primary target for neutralization, is with slow disease progression in HIV infection (81) or virus largely protected from Ab recognition because of its placement clearance in hepatitis C virus (82). Furthermore, adoptive recessed within the glycoprotein core (Fig. 3). Exposure of the transfer of in vitro expanded NK cells or depletion of NK cell RBD occurs only after cathepsins cleave off ∼130 kDa of the The Journal of Immunology 837 Downloaded from http://www.jimmunol.org/

FIGURE 3. EBOV life cycle and . EBOV attaches to the cell surface through nonspecific attachment factors, which induce virion internalization via by guest on September 23, 2021 macropinocytosis. Once EBOV is in the endosomal compartment, acidification activates cysteine proteases cathepsin B and L (CatB/L), which cleave glyco- proteins, exposing the receptor binding site (RBD). Cleaved gycoprotein then engages with the cellular endosomal receptor NPC1, allowing membrane fusion and entry of the EBOV ribonucleoprotein complex into the cytoplasm. EBOV has a negative-sense, ssRNA genome composed of seven genes: NP, VP35, the matrix protein (VP40), the glycoprotein (GP), VP30, VP24, and the RNA-dependent RNA polymerase (L). VP30/VP35/L protein complex transcribe the NP-coated genome (green) into mRNA (144–150). The major mRNA product from the GP gene codes for a secreted dimeric form of glycoprotein (sGP). The membrane-bound trimeric form of glycoprotein found in mature virions is expressed from mRNA produced through stuttering by the polymerase L that results in a frameshift owing to the addition of a nontemplated adenosine (151). Replication of the viral genome is mediated by a VP35/L protein complex (152, 153). Viral proteins and NP-coated genome assemble to form new viral particles, which bud from the infected cell (148, 154).

150 kDa trimer, leaving only a subset of the Ab binding emergency outbreak conditions (114). Ideally, mAbs can be epitopes that were present on the native trimer. This cleavage identified to target specific sensitivities in virus entry or on exposes the glycoprotein RBD for interaction with the glycoproteins to reduce the number of mAbs required for intracellular receptor, Niemann–Pick C1 (NPC1), which potent protection. Even in the case of mixtures, formulations triggers membrane fusion and ribonucleoprotein complex may be simplified and potency may be enhanced by elimi- liberation into the cytoplasm (Fig. 3) (104–108). The success nating mAbs that compete for glycoprotein binding and de- of some polyclonal IgG preparations to protect macaques fining mAb mechanisms of action (115, 116). For example, supports a potential strategy of using mAb mixtures targeting Abs that target epitopes in the portion of glycoproteins re- multiple sites on the glycoprotein, for mAbs that do not moved by cleavage, and which demonstrate potent neutrali- completely protect when given individually (109). Preclinical zation in vitro, failed to protect animals against challenge studies in macaques have demonstrated that postexposure (109). An Ab targeting the glycoprotein base that is retained transfer of mAb mixtures improves survival outcomes (110, after cleavage, and which shows near complete neutralization 111), and in some studies, complete protection was achieved by in vitro, still failed to control infection in vivo, potentially infusion of mAb mixtures administered 3–5 d after infectious owing to a lack of stable binding at low pH (117). The most challenge (109, 112). Moreover, a human clinical trial suggested intuitive mechanistic Ab target is inhibition of the critical a modest trend toward greater survival when administration of glycoprotein–NPC1 receptor binding interaction. However, mAb mixtures in combination with standard of care was Abs targeting the glycoprotein RBD must gain access to the compared with standard of care alone (113). recessed RBD prior to virion uptake, tolerate acidic condi- A potential caveat of mAb mixtures is that the approach can tions in the endosome, and retain high affinity binding after be compromised if manufacturing multiple mAbs impedes cathepsin cleavage (Fig. 2). Although such Abs appear to be production and regulatory approval for use, especially under rare, one mAb to date fulfills these criteria (103). Indeed, 838 BRIEF REVIEWS: EBOLA VIRUS IMMUNITY when delivered up to 5 d after lethal EBOV challenge, this death–1 (PD-1) and CTLA-4 (72, 87). CD8+ T cell activation monotherapy mediates uniform protection of macaques with phenotype was first reported among EBOV patients who no sign of illness (112). received advanced care and treatments in the United States A vexing challenge in the search for efficacious EBOV mAbs is and Europe, which could have potentially impacted immune the limitation of in vitro neutralization assays and small animal responses (87, 129). However, a similar observation was also models to predict Ab efficacy in the macaque challenge model made among patients with limited access to advanced care in (109, 117, 118). Viewed as an opportunity, those limitations West African clinics (72), suggesting that the activation of CD8+ provide an impetus to define the relative importance in vivo T cells is not associated with treatments, but rather an intrinsic of all potential Ab effector functions, including complement- character of the immune response against EBOV. dependent cytotoxicity, Ab-dependent cellular phagocytosis The kinetics of CD8+ T cell activation could shed light on (ADCP), or Ab-dependent cellular cytotoxicity (ADCC) (Fig. 2) the possible role of CD8+ T cells in controlling EBOV in- (119). For other viruses, the cooperative interactions between fection. A previous study reported CD8+ T cell activation Abs and components of cell-mediated immunity have been during the recovery phase in survivors (84). However, in more demonstrated in vivo by abrogating the FcR-binding functions recent studies, CD8+ T cell activation was detected during the of neutralizing mAbs and showing a loss of protective capacity symptomatic phase (72, 129) and coincided with the decline in, for example, simian/human immunodeficiency chimeric virus of viral load (84, 87). The discrepancy in detection of CD8+ infected macaques or influenza-infected mice (120, 121). T cell activation during the symptomatic phase could result

However, interpretation of studies using Fc-mutated mAbs from the difference in sample time points and sets of markers Downloaded from should take into account the fact that Fc modification will analyzed, or the difference in detection methods used. In one affect multiple non-ADCC related functions such as mAb study, CD8+ T cell activation was inferred from RT-PCR distribution in tissues, and Ag uptake for subsequent processing analysis of bulk immune cells (84), whereas the more recent and presentation to the cellular immune system. Because NK studies monitored CD8+ T cell activation by flow cytometric cells are enriched in spleen and liver, which are major sites of analysis of specific marker-based subpopulations (72, 84, 87, + EBOV replication, ADCC may play a role in controlling viral 129). More importantly, CD8 T cells recognizing MHC http://www.jimmunol.org/ replication in these organs. Abs can mediate ADCC against class I epitopes in EBOV nucleoprotein (NP) are detectable in EBOV in vitro (112), but its importance in vivo in controlling four survivors early in the symptomatic phase of infection EBOV replication remains to be defined in primates. (72). Although this is a small sample size, the temporal as- Well-defined EBOV and monotherapeutic mAbs sociation between the accumulation of virus-specific CD8+ provide indispensable model systems to identify the role of NK T cells and a decline in viremia suggests CD8+ T cells con- cells and Fc-mediated Ab effector functions in vivo (112, 122), tribute to the control of EBOV replication in humans (72). and to determine if there are negative consequences to the However, to identify the role of the CD8+ T cell response in

host of the FcR–Ab interaction. The rapid dissemination of controlling EBOV infection, the functionality of EBOV- by guest on September 23, 2021 EBOV in infected individuals and failure of some Ab thera- specific CD8+ T cells in the symptomatic phase should be pies to protect even when they exhibit in vitro neutralization defined. raises the question of whether EBOV entry is being aided by A similar level of CD8+ T cell activation is also observed in mechanisms such as Ab-dependent enhancement (ADE) nonsurvivors, although the quality of these CD8+ T cells may (Fig. 2). ADE has been observed for dengue virus and re- be inferior to those in survivors. For example, a larger pro- spiratory syncytial virus infection in humans in which the portion of CD8+ T cells express the inhibitory receptors PD-1 presence of low levels of Ag-specific Abs contribute to disease and CTLA-4 in nonsurvivors than in survivors (72). Fur- severity (123–125). ADE is thought to occur when Ab–virus thermore, although NP-specific CD8+ T cells are detected in complexes interact with FcR present on immune cells (e.g., nonsurvivors, they fail to increase in frequency over time, monocytes, macrophages, and neutrophils), or with compo- suggesting their prolonged presence in survivors may impact nents of the complement system, resulting in enhanced favorably on clinical outcomes (72). Higher viral loads and proximity of the virus to cellular membranes, thus facilitating excessive inflammation found in fatal EBOV cases may drive infection (126). In vitro, the use of plasma derived from the increase in PD-1/CTLA-4 expression and impair CD8+ macaques and human survivors demonstrated enhancement in T cell function and proliferation (72). It is also possible that EBOV infectivity of an immortalized cell line (127, 128); failure of the immune system to overcome viral IFN antag- however, more in vitro research and in particular in vivo onism early in infection and aberrant DC maturation may set studies are required to fully understand what role, if any, ADE the stage for subsequent defects in T cell responses. As a re- plays in the course of natural EBOV infection. sult, the virus replicates without control, leading to leukope- The checkmate: the role of T cell immunity. The longstanding nia and T cell death by intravascular apoptosis during the last belief that immune suppression by EBOV thwarts an effective 5 d of a fatal disease course (84, 132). T lymphocyte response bears reconsideration in light of recent In contrast to limited data on human T cell responses against observations that activated CD8+ T cells are effectively induced EBOV infection, animal models have provided useful insights during the symptomatic phase of EBOV infection. Single-cell for the role of CD8+ T cells in controlling EBOV infection, as immunophenotyping revealed that at peak response, 10–60% CD8+ T cell responses can be either enhanced through of CD8+ T cells display activation phenotype (72, 87, 129), vaccination or eliminated by immunodepletion. In recombi- similartothelevelofCD8+ T cell activation induced by nant adenovirus–vaccinated macaques, protection is associated other virus infections or vaccination (130, 131). CD8+ T cells with robust CD8+ T cell responses that include IFN-g and expressed markers associated with cell proliferation (87, 129), TNF-a dual-expressing, cytotoxic CD8+ Tcells,insomecases cytotoxicity (87), and immunoinhibitory receptors programmed in the absence of a humoral response (133–135). Furthermore, The Journal of Immunology 839 treatment of recombinant adenovirus–vaccinated macaques Although passive transfer of optimized mAb preparations with a CD8-a–depleting Ab immediately before challenge provides a survival benefit in macaque studies, host-generated abolished vaccine-mediated protection (94), suggesting CD8+ Ab responses in natural infection may not mature to suffi- T cells, and possibly CD8+ NK cells, are necessary for virus cient affinity and potency to provide protection, thus perhaps clearance. A different result was obtained in recombinant ve- explaining the inability of some subjects to resolve infection. The sicular stomatitis virus–vaccinated macaques, in which CD8- aggressive nature of EBOV infection may not allow adequate a–depletion during the vaccination phase did not affect time for host-generated Abs to acquire desired qualities such as vaccine-mediated protection (136). The interpretation of the the capacity to block interaction with the EBOV receptor (103). result, however, is complicated owing to the detection of CD8+ On the other hand, even a nonoptimal Ab response, alone or T cells at a time shown in other studies to immediately precede combined with innate immunity, may contribute to the con- rapid rebound to near predepletion CD8+ Tcelllevels(137– tainment of viral replication and allow time for T cell responses 139). Nevertheless, because immune correlates and mecha- to develop. Ab-mediated effector functions such as ADCC and nisms of protection will likely vary across different vaccine ADCP have been studied in vitro, and their role in vivo in platforms, the relative importance of cellular and humoral re- the clearance of EBOV infection of primates remains to be sponses for protection against EBOV should be determined demonstrated. empirically for each vaccine strategy (140). Experimental data support a role of CD8+ T cells in immune CD8+ T cells may play an important role beyond viremia protection against EBOV in macaques. Samples from the clearance in the control of residual replication following re- West African epidemic demonstrated for the first time, to Downloaded from covery and in the prevention of the recurrent infection by our knowledge, the early activation and development of EBOV that has persisted in the body. Infectious EBOV has EBOV-specific CD8+ T cells, which associate with virus been recovered from breast milk, urine, ocular aqueous hu- clearance from the circulation. CD8+ T cells maintained mor, and semen of recovering patients (141). In EBOV pa- effector phenotype for more than a month after viremia tients treated at intensive care units who experienced severe becomes undetectable, suggesting that CD8+ T cells may be + disease and high viral load, CD8 T cells remained activated activated by persistent viral Ag and could contribute to http://www.jimmunol.org/ for up to 48 d following the resolution of viremia, suggesting elimination of residual EBOV in tissues. a role for CD8+ T cells in clearing residual noncirculating Finally, the persistence of the virus in immune privileged lo- viral Ag (87, 129, 142). More importantly, the majority of cations is a particularly alarming observation confirmed by the these CD8+ T cells store significant quantities of intracellular 2014–2016 epidemic. Immune analysis of survivors will help granzyme B (129, 142) and produce cytokines upon EBOV to clarify whether the persistence of EBOV raises the risk Ag stimulation (87, 142), suggesting that they are fully of long-term transmission, and in turn, if that has the functional effector cells and capable of killing in- capability to increase a worldwide re-emergence of EBOV.

fected cells upon a future resurgence of viral replication. Understanding these immunological mechanisms of virus by guest on September 23, 2021 However, treatment such as immunotherapy and antiviral clearance will inform the development of vaccines and ther- drugs received by these patients could have separately im- apeutics in preparation for future EBOV outbreaks. pacted immune responses, including memory CD8+ T cell development (87, 129, 142). Therefore, monitoring EBOV- specific cellular responses in recovered survivors who did not Acknowledgments We thank Ken Abe, Kendra Leigh, and John Misasi for comments and critical received advanced treatment will shed light on the potential + reading of the manuscript. We apologize to those in the field whose important role of CD8 T cells in clearing residual EBOV infection. work was not included in this Brief Review because of space limitations. Conclusions Disclosures Despite the high mortality rate of EBOV infection, survival in the The authors have no financial conflicts of interest. absence of therapeutics demonstratesthatsomehostsareableto mount effective immune responses. Although intense efforts are References focused on developing preventative vaccines and therapeutic 1. International-commission. 1978. Ebola haemorrhagic fever in Zaire, 1976. Bull. measures, there is increasing interest in understanding the basic World Health Organ. 56: 271–293. immunological mechanisms mediating survival. In addition, 2. Centers for Disease Control and Prevention. 2015. Outbreaks chronology: Ebola virus disease. Atlanta, GA: Centers for Disease Control and Prevention. http:// models for predicting survival based on EBOV viral loads or www.cdc.gov/vhf/ebola/outbreaks/history/chronology.html. Accessed: November peripheral immune cell transcription profiles of infected patients 11, 2015. 3. Leroy, E. M., B. Kumulungui, X. Pourrut, P. Rouquet, A. Hassanin, P. Yaba, are being developed (16, 49, 143). Before the onset of viremia, A. De´licat, J. T. Paweska, J. P. Gonzalez, and R. Swanepoel. 2005. Fruit bats as interactions with the innate immune system are likely playing an reservoirs of Ebola virus. Nature 438: 575–576. important role in determining outcome. Hosts that can over- 4. Bausch, D. G., J. S. Towner, S. F. Dowell, F. Kaducu, M. Lukwiya, A. Sanchez, S. T. Nichol, T. G. Ksiazek, and P. E. Rollin. 2007. Assessment of the risk of comethetypeIIFNantagonismofEBOVwillbeabletoin- Ebola virus transmission from bodily fluids and fomites. J. Infect. Dis. 196(Suppl. hibit viral replication, delay the spread of infection, and mount 2): S142–S147. 5. Dowell, S. F., R. Mukunu, T. G. Ksiazek, A. S. Khan, P. E. Rollin, and an adaptive immune response. An aggressive inflammatory re- C. J. Peters. 1999. Transmission of Ebola hemorrhagic fever: a study of risk factors in sponse initiated by infected macrophages and mDCs must be members, Kikwit, Democratic , 1995. Commission de tempered to avoid recruitment of additional inflammatory im- Lutte contre les Epide´mies a` Kikwit. J. Infect. Dis. 179(Suppl. 1): S87–S91. 6. Mate, S. E., J. R. Kugelman, T. G. Nyenswah, J. T. Ladner, M. R. Wiley, mune cells (e.g., neutrophils) and damage caused by excessive T. Cordier-Lassalle, A. Christie, G. P. Schroth, S. M. Gross, G. J. Davies-Wayne, inflammation. Future animal studies focusing on tissue-based et al. 2015. Molecular evidence of sexual transmission of Ebola virus. N. Engl. J. Med. 373: 2448–2454. responses and early time points in infection will provide a bet- 7. Centers for Disease Control and Prevention. 2015. 2014 Ebola outbreaks in West ter understanding of the innate responses against EBOV. Africa: case counts. Atlanta, GA: Centers for Disease Control and Prevention. 840 BRIEF REVIEWS: EBOLA VIRUS IMMUNITY

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