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The Journal of Immunology, 2005, 174: 4198-4202.
Copyright © 2005 by The American Association of Immunologists

CD8-Mediated Protection against Ebola Virus Infection Is Perforin Dependent

Manisha Gupta1,*, Patricia Greer{dagger}, Siddhartha Mahanty2,*, Wun-Ju Shieh{dagger}, Sherif R. Zaki{dagger}, Rafi Ahmed{ddagger} and Pierre E. Rollin*

* Special Pathogens Branch and {dagger} Infectious Diseases Pathology Activity, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333; and {ddagger} Emory Vaccine Research Center and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD8 T cells have been shown to play an important role in the clearance and protection against fatal Ebola virus infection. In this study, we examined the mechanisms by which CD8 T cells mediate this protection. Our data demonstrate that all normal mice infected s.c. with a mouse-adapted Ebola virus survived the infection, as did 100% of mice deficient in Fas and 90% of those deficient in IFN-{gamma}. In contrast, perforin-deficient mice uniformly died after s.c. challenge. Perforin-deficient mice failed to clear viral infection even though they developed normal levels of neutralizing anti-Ebola Abs and 5- to 10-fold higher levels of IFN-{gamma} than control mice. Using MHC class I tetramers, we have also shown that perforin-deficient mice have 2- to 4-fold higher numbers of Ebola-specific CD8s than control mice. These findings suggest that the clearance of Ebola virus is perforin-dependent and provide an additional example showing that this basic immunologic mechanism is not limited to the clearance of noncytopathic viruses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD8 T cells and NK cells play an important role in viral clearance. There are three mechanisms by which they perform this function. The first involves the secretion of cytokines, such as IFN-{gamma}, TNF-{alpha}, and chemokines that either interfere with viral attachment, entry, or replication or induce programmed cell death of the infected cells (1). The second method involves the exocytosis of a pore-forming protein called perforin, accompanied by the release of serine proteases and activating caspases that trigger programmed cell death in the target cells. The third method involves the induction of programmed cell death by a Fas-Fas ligand (FasL)3-mediated interaction that also activates caspase-dependent pathways in the infected cell. Previous reports suggested that IFN-{gamma} plays an important role in the clearance both of cytopathic viruses, such as vaccinia and Sindbis virus, and of noncytopathic viruses, such as lymphocytic choriomeningitis virus (LCMV) (2, 3, 4). However, perforin, which induces viral clearance by causing cell lysis, has traditionally been associated only with the clearance of noncytopathic viruses (5, 6).

Ebola virus replicates in a variety of cell types, including hepatocytes, Kupffer’s cells, macrophages, and endothelial cells, causing cell lysis. Many of the features of Ebola hemorrhagic fever are caused by host responses to infection, including the release of proinflammatory cytokines and chemokines from infected cells and the initiation of coagulopathy through synthesis of tissue factor (7). The factors responsible for severe tissue damage that can result in a septic shock are still not very clear.

CD8 T cells have been shown to play important role in protection against Ebola virus infection (8, 9). In this study using a mouse model of Ebola virus infection, we show that IFN-{gamma} plays a role in controlling viral replication, but is not sufficient to protect against lethal infection. We found that protection against Ebola virus infection is independent of Fas-FasL-mediated killing and requires the presence of perforin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Virus and mice and infection protocol

A mouse-adapted strain of Ebola virus was derived from Ebola Zaire 1976 as previously described (10). All infected samples and animals were handled under maximum containment in the biosafety level (BSL)-4 laboratory at the Centers for Disease Control and Prevention (Atlanta, GA). All blood and tissue samples from the BSL-4 laboratory were gamma-irradiated (5 x 106 rad) before further processing under BSL-2 and -3 conditions.

Perforin–/– (Pfn–/–; Pfptm1Sdz), Fas–/– (Tnfrf6lpr), IFN-{gamma}–/– (Ifngtm1Ts), and wild-type (C57BL/6 background) mice, matched for sex and age (6–8 wk), were obtained from a commercial supplier (The Jackson Laboratory). All mice were maintained under pathogen-free conditions and allowed to acclimate to the BSL-4 laboratory for 3–4 days before use in our experiments.

Mice were infected by a single s.c. inoculation of 100 PFU of mouse-adapted Ebola virus (in 0.2 ml of PBS). In some experiments, mice were infected by the i.p. inoculation of 10 PFU of mouse-adapted Ebola virus.

Viral Ag and Ab titers

Levels of circulating Ebola viral Ags in sera and tissue homogenates and anti-Ebola Igs were measured by a capture ELISA, as previously described (11).

T cell depletion

Mice were depleted of CD8 T cells by injecting them with protein-A purified anti-CD8 Abs (clone L2.43). The Abs (300–400 µg/mouse) were given i.p. one day before s.c. virus challenge and weekly thereafter for 3 wk.

Purification of Ig from immune serum

Mice infected s.c. with 100 PFU of mouse-adapted Ebola virus were euthanized between 16 and 21 days after infection. Sera were collected, pooled, gamma-irradiated (5 x 106 rad), and titrated for anti-Ebola Igs by ELISA. Igs were purified from pooled immune serum by an ammonium sulfate precipitation method. Partially purified Igs were dialyzed against PBS (pH 7.2) and filter-sterilized for injection and titers were determined by ELISA as described previously (11).

Surface and intracellular staining

IFN-{gamma} was induced in vitro in the presence of peptides as previously described (12). Splenocytes (2 x 106) were cultured in the presence of either NP44–52 (YQVNNLEEI), NP388–396 (FQQTNAMVT), or NP297–305 (ARLLNLSGV) peptide at a 20-µg/ml concentration in the presence of 20 ng/ml human IL-2 (BD Pharmingen) and 1 µl/ml brefeldin A (Golgi plug; BD Pharmingen) for 4 h at 37°C. Cells were stained with IFN-{gamma}-allophycocyanin and CD8-FITC (BD Pharmingen) using a Cytofix/Cytoperm kit according to the manufacturer’s instructions.

To quantitate Ebola Ag-specific CD8s, splenocytes were stained with MHC class I tetramers specific for NP44–52 (National Institutes of Health tetramer core facility), NP388–96 (National Institutes of Health tetramer core facility), and NP297–305 (Beckman Coulter) coupled to PE. Cells were costained with CD8-FITC. All staining was performed in a BSL-4 facility. Specificity of each tetramer was determined by using splenocytes from LCMV infected day 8 mice and the frequency of CD8+tetramer+ cells were below 0.05%. Cells were acquired on FACSCalibur inside a BSL-4 laboratory and analyzed on FlowJo software (BD Biosciences).

IFN-{gamma} assay

IFN-{gamma} levels in tissue homogenates and serum were determined using a commercial ELISA (R&D Systems) according to the manufacturer’s instructions. Values were calculated and plotted as nanograms per milliliter of homogenized sample.

Histopathology and immunohistochemistry

Viral Ag was detected by immunohistochemistry assays performed using a streptavidin-biotin labeled technique for the detection of Ebola virus Ags in formalin-fixed tissues (13).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD8 T cell-mediated protection against Ebola virus infection require perforin

To confirm the role of CD8 T cells in protection against Ebola infection, +/+ mice were depleted of CD8 T cells one day before s.c. challenge and observed for illness, and tissue viral titers were determined. Mice depleted of CD8 T cells had high viral Ag titers (≥105) on days 10 and 14 postinfection (data not shown), and most of them (6 of 8) died by day 15 (Fig. 1A), indicating that CD8 T cells are required for protection against Ebola virus infection.



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  		FIGURE 1. CD8 T cell-mediated protection against Ebola virus is perforin dependent. A, Survival in +/+ (n = 10) and CD8 T cell-depleted +/+ (n = 8). Mice were challenged s.c. with 100 PFU of Ebola virus and observed for survival. B, Survival in +/+, Pfn–/–, IFN-{gamma}–/–, and Fas–/– mice challenged s.c. with 100 PFU of Ebola virus. One hundred percent of +/+ (n = 30) and Fas–/– (n = 8) and 90% of IFN-{gamma}–/– (n = 29) survived infection, In contrast, all Pfn–/– (n = 29) mice died between 13 and 33 days after infection. C, Viral Ag titers in +/+, Pfn–/–, Fas–/–, and IFN-{gamma}–/– mice following Ebola virus challenge. Mice were challenged s.c. with 100 PFU of Ebola virus and assayed for viral Ag in tissue homogenates and serum (three mice per time point per group). Data are plotted as mean ± SE. Detection limit of the assay is shown as dotted lines. Viral Ag levels were below the limit of detection in control mice and in both IFN-{gamma}–/– and Fas–/– mice by 14 days postinfection, whereas Pfn–/– mice had viral titers of 103–105 in the tissues tested. Experiment was performed three times. D and E, Immunohistochemistry of liver section for Ebola Ag. D, (x25 magnification) Pfn–/– mice were challenged s.c. with 100 PFU of Ebola virus and liver section was stained for Ebola Ag (red stain). Arrows indicate virus grows in multiple foci. E, (x150 magnification) Arrows indicate viral Ag (red stain) in hepatocytes, while arrowheads indicate viral Ag in histiocytes (Kupffer’s cells). Staining was repeated two times.

 
We next examined the mechanisms of this protection by testing the resistance of mice deficient in perforin, IFN-{gamma}, or Fas to s.c. viral challenge. Mice deficient in Fas (Fas–/–; n = 8), perforin (Pfn–/–; n = 29), and IFN-{gamma} (IFN-{gamma}–/–; n = 29) were challenged s.c. with 100 PFU of Ebola virus and observed for survival, and viral titers in tissues were measured. All control mice survived infection without becoming ill, as did all Fas–/– mice. However, all of the Pfn–/– mice became ill and eventually died between 13 and 33 days after infection (Fig. 1B). A few (10%, n = 3/29) of the IFN-{gamma}–/– mice also died between days 8 and 10, but the remainder did not show any signs of illness during the 50-day observation period (Fig. 1B).

Viral Ag levels in Fas–/– mice were comparable to levels in control mice (Fig. 1C) and 1000-fold lower than the levels (titer ≥105) found in CD8 T cell-deficient mice (data not shown). The kinetics of viral infection in IFN-{gamma}–/– mice was similar to that in the control group: titers peaked at day 8 and were below the level of detection by day 15 postchallenge. However, viral Ag levels in these mice were 5- to 10-fold higher than in controls on days 8 and 10 after infection, suggesting that IFN-{gamma} plays a role in controlling viral replication (Fig. 1C).

Pfn–/– mice showed a biphasic pattern of viral kinetics, with the first peak in the viral Ag load seen on day 8 and a second peak on day 16 after challenge (Fig. 1C). These mice had tissue viral Ag levels 10- to 100-fold lower than the levels in CD8 T cell-depleted mice (data not shown) and 5- to 10-fold higher than levels in +/+ control mice on day 8 postinfection (Fig. 1C). Pfn–/– mice were unable to clear infection from tissues, and died with high viral Ag titers in the spleen (1:102,400), liver (1:409,600), and kidneys (1:6,400). They also displayed splenomegaly and liver hyperplasia on days 10, 15, and 20 after infection. The livers of Pfn–/– mice weighed, on average, twice as much as those of normal controls on day 20 after infection (2.69 vs 1.33 g, respectively). The liver lobes contained white patches indicative of necrosis, and immunohistochemistry showed small foci of virus-infected cells surrounded by lymphocytic infiltrates (Fig. 1D), with viral Ag localized primarily to hepatocytes and histiocytes (Fig. 1E).

High levels of IFN-{gamma} correlate with viral Ag levels in Pfn–/– mice

We determined IFN-{gamma} levels in tissue homogenates and serum from Pfn–/– mice at various time points after infection, and found that IFN-{gamma} levels peaked at 8 days after s.c. challenge both in Pfn–/– and control mice (Fig. 2). IFN-{gamma} then disappeared in control mice, but remained elevated in the Pfn–/– mice, declining by day 10, but then increasing over the remainder of the course of infection (Fig. 2). The high levels of IFN-{gamma} correlated with elevated viral Ag levels (Fig. 1C) and were maintained until death. Early in infection, IFN-{gamma} levels were 5- to 10-fold higher in Pfn–/– than in control mice, correlating with higher viral replication in Pfn–/– mice. These data suggest that high levels of IFN-{gamma} produced in response to viral infection are insufficient to clear the virus in the absence of perforin.



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  		FIGURE 2. IFN-{gamma} levels in Pfn–/– mice. Normal and Pfn–/– mice were challenged s.c. with 100 PFU of Ebola virus. IFN-{gamma} levels were determined in tissue homogenates and serum on days 4–26 postinfection by ELISA. IFN-{gamma} levels were plotted as mean ± SE (three mice per time point per group). IFN-{gamma} levels in Pfn–/– mice were 5- to 10-fold higher than in +/+ mice, correlating with their viral Ag levels. Levels were estimated three times in separate experiments.

 
Perforin-deficient mice have high numbers of Ebola-specific CD8 T cells

Previous reports suggest that perforin plays an important role in the down-regulation of T cell responses (5). In the absence of perforin, CD8 T cells undergo enhanced expansion and maintain high CD8 T cell numbers for a long period and produce high levels of IFN-{gamma} (5). We next estimated numbers of Ag-specific CD8 T cells and IFN-{gamma}-producing CD8s in perforin-deficient mice. We used three different tetramers to estimate Ag-specific CD8s. Our results indicate that tetramer-positive CD8s peaks on day 8 and stay high until day 10 in both the groups (Fig. 3). Perforin-deficient mice have 2- to 4- fold higher numbers of NP44-, NP388-, and NP297-specific cells (Fig. 3). Similarly IFN-{gamma}-producing CD8s following peptide restimulation is 3- to 5-fold more than control groups (Fig. 3). These data indicate that despite generating large Ag-specific CD8 response and IFN-{gamma} levels, mice are unable to clear infection in the absence of perforin.



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  		FIGURE 3. Ag-specific CD8 T cells in Pfn–/– mice. Splenocytes from Pfn–/– (n = 3 mice/time point) and control mice (n = 3 mice/time point) were stained with MHC-class I tetramers specific for NP44, NP388, and NP297 on days 6, 8, 10, and 13 postinfection. Cells were gated on CD8 and tetramer-positive cells. Tetramer-positive cells were calculated based on total numbers of CD8s in spleen. CD8 T cell numbers were calculated based on the following formula: (percentage of CD8-positive cells/100) x total number of splenocytes. Splenocytes were also restimulated in the presence of different peptides at a 20-µg/ml concentration. Cells were gated on IFN-{gamma}-producing CD8 T cells. IFN-{gamma}-producing cells were calculated using this formula: ([percentage of IFN-{gamma} positive after restimulation – percentage of IFN-{gamma}-positive without restimulation]/100) x CD8 T cell numbers. Frequency of IFN-{gamma}-positive CD8 T cells before restimulation was in the range of 0.05–2.0%. Data is plotted as mean ± SE. Experiment was repeated two times.

 
Anti-Ebola Abs produced by Pfn–/– mice protect naive mice against lethal infection

It has been shown previously that anti-Ebola Abs can protect mice against lethal Ebola virus infection (11). To confirm that Pfn–/– mice produce levels of Ab comparable to controls and that the Abs are protective, we measured anti-Ebola Ig levels in the serum following s.c. challenge. Pfn–/– mice showed kinetics of Ab production and equivalent levels of anti-Ebola Ig similar to those of control mice (Fig. 4A). The levels of purified anti-Ebola Ab (titer of 20,000–24,000) were similar in control and Pfn–/– mice. We next determined whether these Abs could protect mice against lethal infection, as shown previously in a passive transfer study (11). Immune serum was collected from Pfn–/– and from normal control mice between 16 and 21 days after s.c. challenge, purified for Igs, titrated, and administered i.p. (1 ml) to naive mice 1 day before lethal challenge with mouse-adapted virus. Serum from uninfected normal mice was used as negative control. Circulating titers of immune serum in recipients were similar (1:6,400) in both groups of mice except in one mouse (1:1,600). All six mice that received immune serum from Pfn–/– mice and four of five that received immune serum from +/+ mice survived the infection, whereas none of the mice that received normal mouse serum survived (Fig. 4B). These data indicate that the Abs produced by Pfn–/– mice in response to Ebola viral infection are protective and can neutralize the virus in vivo. We also performed in vitro neutralization assay and did not find any difference in the titers between the two groups (data not shown).



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  		FIGURE 4. Pfn–/– mice have normal levels of anti-Ebola Abs and are protective in vivo. A, Levels of anti-Ebola Abs in Pfn–/– mice. +/+ and Pfn–/– mice were challenged s.c. with 100 PFU of Ebola virus and assayed for anti-Ebola Abs on indicated days by capture ELISA (three mice per time point per group). Data is plotted as mean ± SE. B, This experiment was performed four times. Protective efficacy of immune serum from Pfn–/– mice. Naive mice were inoculated i.p. with either purified immune serum (IS) from +/+ (n = 5) or from Pfn–/– (n = 6) mice or with serum from normal mice (n = 5; NMS) 1 day before i.p. challenge (10 PFU) with Ebola virus, and were then observed for survival. This experiment was done twice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Previous studies show that IFN-{gamma} plays an important role both in the initial and/or the long-term control of cytopathic viruses such as Sindbis, vaccinia, and MHV-68 (mouse herpes virus), and of noncytopathic viruses such as LCMV (2, 3, 4, 14, 15). IFN-{gamma} is involved in the site-specific clearance of virus (3, 16), and the absence of IFN-{gamma} may result in persistent infection (4). Our study of Ebola virus infection suggests that IFN-{gamma} plays an important role in restricting viral replication. Some IFN-{gamma}-deficient mice are unable to control infection and develop lethal illness, suggesting that IFN-{gamma} plays an important role in suppressing initial viral replication. However, IFN-{gamma}-deficient mice that did not die in the early phase of infection did not show any virus (viral RNA and plaque assay) in tissues up to 150 days after infection (data not shown) and were protected against Ebola rechallenge (data not shown), suggesting that IFN-{gamma} is not required for long-term protection. Our data demonstrate that the large amounts of IFN-{gamma} produced by Pfn–/– mice were unable to clear the infection, suggesting that IFN-{gamma} is not sufficient for protection against Ebola virus infection.

Perforin is a pore-forming molecule that delivers granzymes into cells, activating caspase pathways that result in DNA fragmentation. A variety of cytopathic viruses, such as vaccinia, Semliki forest virus, Sindbis, rotavirus, and MHV-68, have previously been shown to be suppressed by CD8 T cells in a process that does not require Fas or perforin (1, 6, 17, 18). In contrast, perforin plays a crucial role in the clearance of noncytopathic viruses such as LCMV and mouse CMV (5, 19). These observations have led to a general hypothesis that protection against cytopathic viruses depends on IFN-{gamma} and Abs, while perforin is effective in the clearance of noncytopathic viruses. However, some exceptions to this rule have been identified, because both Theiler’s virus and mousepox virus (ectromelia) are highly sensitive to perforin-mediated cell lysis (20, 21). Some other cytopathic viruses, such as respiratory syncytial virus and influenza, up-regulate Fas expression on target cells. Clearance of these viruses is more sensitive to Fas-FasL-mediated cell lysis than to perforin (22, 23).

Our results provide evidence that perforin-mediated protection and viral clearance are not limited to noncytopathic viruses. In Pfn–/– mice, virus levels peaked on day 8, declined, and then peaked again at day 16, suggesting that an early control mechanism restricts viral replication, facilitated by IFN-{gamma} and anti-Ebola Abs. In an attempt to clear infection, CD8s kept expanding in Pfn–/– mice and produce IFN-{gamma}. We found 2- to 3-fold increase in total (data not shown) and Ag-specific CD8s on day 8, 10, and 13 after infection. The fact that virus was not cleared from hepatocytes and histiocytes in the absence of perforin suggest that perforin is crucial for viral clearance. Of interest, our study also demonstrates that protection against acute Ebola disease, unlike other cytopathic viruses such as vesicular stomatitis virus, vaccinia, and Semliki forest virus, requires CD8 T cells, and is not dependent on Abs (2, 24).

Our data suggest that cell-mediated killing can play an important role in the clearance of infection by a cytolytic virus. Two mechanisms may explain how this could occur. First, cell-mediated immune mechanisms could potentially kill an infected cell before the virus can use it for amplification. Second, the disruption of virus-infected cells releases free virus particles that are easily accessible to Abs, and also releases viral proteins that can activate APCs.

Pfn–/– mice had much higher levels of viral replication than normal mice, but their production of anti-Ebola Abs was similar to that of controls. Abs produced by Pfn–/– mice were protective following transfer to naive mice, but could not clear infection in Pfn–/– mice. There are two possible explanations for this outcome: either the level of Abs was not sufficient to clear infection in Pfn–/– mice, or else the Abs were not effective in clearing cell-associated virus, requiring perforin for virus release. It is possible that anti-Ebola Abs protect naive mice against Ebola infection by neutralizing free virus and delaying viral replication before the host immune response develops, as shown previously (11). How effective these Abs are in clearing cell-associated virus is yet to be studied.

Our findings highlight novel mechanisms for understanding the biology and protective mechanisms used by the immune system against cytolytic viruses. Our data provide evidence that perforin plays an important role in protection against cytolytic viruses and implicates the importance of CD8 T cells in protection against Ebola virus infection.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Acknowledgments
 
We thank Dr. Mike Bray for providing mouse-adapted Ebola virus and for helpful suggestions, Dr. Thomas Ksiazek for providing Ebola reagents, and Gary Reynolds for assistance in animal maintenance. We acknowledge National Institutes of Health Tetramer core facility for providing MHC class I tetramers specific for NP44–52 and NP388–396. We acknowledge Jon Towner for his help in determining Ebola RNA levels and Claudia Chesley for editorial assistance.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Manisha Gupta, Special Pathogens Branch, G-14, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333. E-mail address: mgupta{at}cdc.gov Back

2 Current address: Malaria Vaccine Development Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852. Back

3 Abbreviations used in this paper: FasL, Fas ligand; LCMV, lymphocytic choriomeningitis virus; Pfn–/–, Perforin–/–; BSL, biosafety level. Back

Received for publication October 8, 2004. Accepted for publication January 12, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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