This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Olinger, G. G.
Right arrow Articles by Hart, M. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Olinger, G. G.
Right arrow Articles by Hart, M. K.

 Previous Article  |  Next Article 

Journal of Virology, November 2005, p. 14189-14196, Vol. 79, No. 22
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.22.14189-14196.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Protective Cytotoxic T-Cell Responses Induced by Venezuelan Equine Encephalitis Virus Replicons Expressing Ebola Virus Proteins

Gene G. Olinger, Michael A. Bailey, John M. Dye, Russell Bakken, Ana Kuehne, John Kondig, Julie Wilson, Robert J. Hogan, and Mary Kate Hart*

United States Army Medical Research Institute of Infectious Diseases, Division of Virology, 1425 Porter Street, Frederick, Maryland

Received 11 January 2005/ Accepted 15 August 2005


arrow
ABSTRACT
 
Infection with Ebola virus causes a severe disease accompanied by high mortality rates, and there are no licensed vaccines or therapies available for human use. Filovirus vaccine research efforts still need to determine the roles of humoral and cell-mediated immune responses in protection from Ebola virus infection. Previous studies indicated that exposure to Ebola virus proteins expressed from packaged Venezuelan equine encephalitis virus replicons elicited protective immunity in mice and that antibody-mediated protection could only be demonstrated after vaccination against the glycoprotein. In this study, the murine CD8+ T-cell responses to six Ebola virus proteins were examined. CD8+ T cells specific for Ebola virus glycoprotein, nucleoprotein, and viral proteins (VP24, VP30, VP35, and VP40) were identified by intracellular cytokine assays using splenocytes from vaccinated mice. The cells were expanded by restimulation with peptides and demonstrated cytolytic activity. Adoptive transfer of the CD8+ cytotoxic T cells protected filovirus naïve mice from challenge with Ebola virus. These data support a role for CD8+ cytotoxic T cells as part of a protective mechanism induced by vaccination against six Ebola virus proteins and provide additional evidence that cytotoxic T-cell responses can contribute to protection from filovirus infections.


arrow
INTRODUCTION
 
Ebola viruses (EBOV) cause a severe hemorrhagic fever disease characterized by a high mortality rate. No licensed vaccines are currently available for human use. Various vaccine approaches for filoviruses, including DNA vaccination, vaccination with virus-like particles, liposome encapsulation of irradiated virus, and expression of filovirus proteins from viral vectors, are being evaluated (10-12, 14, 20-22, 26, 28, 30, 31a, 34, 35, 38, 39). Nonhuman primates were protected from challenge after vaccination against Ebola virus glycoprotein (GP) and nucleoprotein (NP) either by a series of DNA inoculations followed by administration of an adenovirus-vectored booster or with a high dose of the adenovirus-vectored product alone (27, 28). More recently, protection against Ebola and Marburg viruses has been achieved using a chimeric virus in which the vesicular stomatitis virus glycoprotein was replaced with either the EBOV glycoprotein or Marburg virus glycoprotein (14).

A mouse model of Ebola virus Zaire (ZEBOV) infection is available for evaluating the immune responses to potential vaccines and to the virulent virus. The mouse-adapted virus is lethal to immunocompetent mice when administered intraperitoneally (i.p.) (5). However, it is not lethal when given subcutaneously (s.c.), unless the mice lack {alpha}/ß interferon (IFN-{alpha}/ß) receptors or are treated with anti-{alpha}/ß-IFN polyclonal antiserum, indicating a role for innate immune responses in survival (4). Subsequent studies indicated that the virus administered s.c. induces an attenuated inflammatory response, early production of antiviral cytokines, and reduced rates of viral replication compared to i.p. administration (16). Recently, Warfield et al. demonstrated a potential role for NK cells in mice vaccinated with virus-like particles composed of GP and VP40 (31). In that study, vaccinated mice that lacked NK cell function were unable to survive subsequent challenge with the mouse-adapted virus.

EBOV vaccine research efforts have emphasized the potential for generating protective adaptive immune responses to the surface GP and, in some cases, the NP. Protective Ebola virus GP-specific monoclonal antibodies (37) and polyclonal sera (8, 35) have been described previously, and to date, the GP is the only ZEBOV protein for which protective B-cell responses have been identified. Cytotoxic T cells (CTLs) to NP or GP were observed after vaccination with plasmids with or without a booster (24, 28, 30, 38), vaccination with liposomes (22, 23), or vaccination with packaged Venezuelan equine encephalitis virus replicons (VRPs) (36). Our previous study demonstrated that cells with in vitro lytic activity to the NP 43-53 sequence protected filovirus-naïve mice after adoptive transfer (36). We also demonstrated that vaccination with other ZEBOV proteins (VP24, VP30, VP35, and VP40) expressed from VRPs induces immunity to viral infection in mice (35). Vaccination with VRPs expressing one of these proteins reduced but did not prevent viremia in challenged mice, and passive transfer of polyclonal sera to the VP proteins did not protect recipient mice from subsequent challenge. The immune mechanism mediating protection has not been identified in mice vaccinated with the various Ebola virus VP proteins.

Six Ebola virus proteins were examined in this study. The GP forms the specific receptor fusion domain required for entry into the host cell. The NP, VP30, VP35, and polymerase proteins combine with the viral genomic RNA to form the ribonucleoprotein complex (6). VP30 also functions as an EBOV-specific transcription activation factor that recognizes a secondary stem-loop structure during transcription (18, 32). VP35 has been suggested to act as an interferon antagonist (2) by blocking interferon regulatory factor 3 activation (1). The exact role of VP24 remains unclear, but it has been demonstrated to participate in the spontaneous formation of the nucleoprotein complex (13). VP40 functions as a matrix protein, which is essential for viral assembly and budding (3, 9, 15, 17, 19, 29).

This study evaluated the induction of CD8+ T cells after vaccination with VRPs expressing ZEBOV GP, NP, VP40, VP35, VP30, or VP24 and their role in protection against infection. We used overlapping peptides of the Ebola virus proteins to identify epitopes that induce CD8+ T cells to ZEBOV in two mouse strains and to assess the lytic function and protective capacity of these CD8+ T cells.


arrow
MATERIALS AND METHODS
 
Virus, animals, and infections. Mouse-adapted Ebola virus was obtained from Mike Bray (5). A comparison of the protein sequences of GP, NP, and the VP proteins of Zaire 1976 virus (GenBank accession number AF086833) and the mouse-adapted Ebola viruses (GenBank accession number AF499101) is shown in Table 1.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Comparison of the protein sequences of GP, NP, and VP proteins of Zaire 1976 and mouse-adapted Ebola viruses

The amino acid changes in the glycoprotein are also in the Ebola virus from which the mouse-adapted virus was derived and are not the result of adaptation (5). Female C57BL/6 or BALB/c mice (5 to 8 weeks old) were obtained from the National Cancer Institute (Frederick, Md.) and housed under specific-pathogen-free conditions. Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adhered to principles stated in the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). The facility where this research was conducted is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International. For infection, mice were inoculated i.p. with 1,000 PFU (30,000 x the 50% lethal dose) of mouse-adapted Ebola virus in a biosafety level 4 laboratory. Mice were observed for 28 days after challenge by study personnel and by an impartial third party. Daily observations of mice included evaluation of mice for clinical symptoms such as reduced grooming, ruffled fur, hunched posture, subdued response to stimulation, nasal discharge, or bleeding.

Peptides. Overlapping peptides for each protein were purchased as pin-synthesized PepSets from Mimotopes Inc. (Clayton, Victoria, Australia). Each peptide was 15 amino acids long and overlapped the previous sequence by 5 amino acids. Once a positive sequence was determined, individual peptides were synthesized by Mimotopes, New England Peptide (Fitchburg, Mass.), or Sigma Genosys (Woodlands, Tex.). The purity of individual peptides, evaluated by using reverse-phase high-performance liquid chromatography analysis, was >70%.

VRPs and vaccinations. The production of VRPs expressing ZEBOV proteins was performed as previously described (20, 21, 35, 36). The estimation of replicon titer was performed using previously described methods (35) and using a flow cytometry-based assay. Tenfold serial dilutions of VRPs were incubated on 1 x 105 Vero or BHK cells for 12 to 16 h in a 48-well culture plate. Adherent cells were resuspended with 1 mM EDTA (phosphate-buffered saline [PBS]), washed, fixed, and made permeable with a 0.5% saponin solution. They were stained first with mouse monoclonal antibodies (MAbs) (VP40 MAb MHD06-A10A, VP35 MAb HMCO1-AF06-AF11, VP24 MAb ZAC1-BG11-01, GP MAbs 6D3-1-1 and 13C6) or with antigen-specific pooled serum (mouse anti-VP30 or anti-NP serum or primate anti-Lassa NP serum) and then with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse or goat anti-primate antibody (Kirkegaard & Perry Laboratories, Gaithersburg, Md.). The titer was derived from the dilutions that resulted in antigen expression in more than 20% and less than 80% of the cells. The titer, expressed in infectious units (IU), was calculated by multiplying the percentage of cells expressing protein by the appropriate VRP dilution factor(s). Titers were usually a log lower than by immunofluorescence assay such that mice received an overall larger dose of replicons in this study than in previous studies by Wilson et al. (35). Mice were injected s.c. in the dorsal neck region with 2 x 106 IU of VRPs encoding the ZEBOV protein. On days 0 and 30, or as stated, mice received booster vaccinations.

In vitro restimulation of splenocytes. Single-cell suspensions, mixed-lymphocyte reactions (MLRs), and restimulation of cells were performed as previously described (35). Briefly, splenocytes were isolated from mice and cultured for 5 h with peptides and stained for intracellular expression of IFN-{gamma}. Alternatively, cells were restimulated for 5 to 7 days with peptides and expanded following the addition of human recombinant interleukin (IL)-2 (10 U/ml; National Cancer Institute) and 10% concanavalin A added on day 2 during restimulation. Control mice received PBS or restimulated splenocytes that were directed towards the previously described Lassa N epitope RPLSAGVYMGNLSSQ (33).

Intracellular IFN-{gamma} staining. Splenocytes were isolated and cultured at 37°C for 5 h in the presence of 1 to 5 µg of peptide(s) or phorbol myristate acetate (PMA; 25 ng/ml) and ionomycin (1.25 µg/ml) in 100 µl of RPMI-Eagle’s Ham amino acid medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 10 µg of gentamicin per ml, 5 mM HEPES, and 0.05 mM ß-mercaptoethanol medium and containing 10 µg/ml of brefeldin A (Epicenter Technologies, Madison, Wis.). Cells were blocked with MAb to FcRIII/II receptor and stained with anti-CD44 FITC and either anti-CD8 or anti-CD4 Cy-Chrome (Pharmingen, San Diego, Calif.) in staining wash buffer (PBS, 2% FBS, 0.01% sodium azide; Sigma, St. Louis, Mo.), with brefeldin A (10 µg/ml). The cells were fixed in 1% formaldehyde (Ted Pella, Redding, Calif.), made permeable with staining wash buffer containing 0.5% saponin (Sigma, St. Louis, Mo.), and stained with anti-IFN-{gamma} phycoerythrin (PE) (Pharmingen, San Diego, Calif.). The data were acquired by using a FACSCalibur flow cytometer and analyzed with CELLQuest software (Becton-Dickinson Immunocytometry Systems, San Jose, Calif.). Samples were considered positive if the percentage of CD8+, CD44+, IFN-{gamma}-positive cells was greater than twofold above background. Background was determined by adding an irrelevant peptide from Lassa N or no peptide in a solution that contained an equivalent amount of the dimethyl sulfoxide used to dilute peptides.

IFN-{gamma} ELISPOT assay. Millipore Immobilon-P plates were coated with 5 µg/ml of primary antibody (clone AN18; Mabtech, Cincinnati, Ohio) diluted in PBS overnight at 4°C. The plates were then washed three times with PBS and blocked using complete medium (RPMI, 10% FBS, 2% L-glutamine, 2% HEPES buffer, 1% penicillin-streptomycin, 0.02 mM 2ME) for 2 h at 37°C. One hundred thousand cells per well were added along with 2 µg/ml of peptide(s) or PMA (25 ng/ml) and ionomycin (1.25 µg/ml) diluted in complete medium. Plates were incubated for 18 to 24 h at 37°C and then washed eight times with PBS plus 0.05% Tween-20. Secondary antibody (clone R4-6A2; Mabtech) was added in buffer containing PBS plus 0.05% bovine serum albumin at a concentration of 1 µg/ml and incubated for 2 h at 37°C. Plates were then washed six times with PBS, and 3-amino-9-ethylcarbazole conjugate (Vector Labs, Burlingame, Calif.) was added and incubated for 1 h at room temperature. Plates were washed six times with PBS and developed with amino-9-ethylcarbazole (AEC) substrate (Vector Labs) for 4 min. Development was stopped by flushing with tap water. Plates were allowed to dry completely, and spots were visualized using the Autoimmun Diagnostika GmbH enzyme-linked immunospot (ELISPOT) reader (Strasberg, Germany).

51Cr release assays. Cytotoxicity assays were performed as described previously (36). Briefly, target cells (EL4, L5178Y) were labeled with 51Cr (Na2CrO4; New England Nuclear, Boston, Mass.) and pulsed with 1 µg/ml of peptide for 1.5 h. Unpulsed target cells were used as negative controls. Various numbers of effector cells were incubated with 2,500 target cells for 4 h. Percent specific release was calculated by using the following formula: % specific release = (experimental release – spontaneous release)/(maximum release – spontaneous release) x 100. Spontaneous release values were obtained by incubating target cells in medium alone and were routinely <15% of maximum release. Maximum release values were obtained by adding 100 µl of 1% Triton X-100. Experiments were repeated a minimum of two times.

Adoptive transfer experiments. Adoptive transfers were performed, and the mice were challenged 4 h later with 1,000 PFU of mouse-adapted virus as previously described (36). The mean time to death of unvaccinated mice was 6 to 8 days. Mice that survived 28 days (four times the mean time to death) were considered protected. Experiments were performed a minimum of two times.

Cell separation and analysis. Effector cell cultures were separated using the Dynabeads mouse CD4 (L3T4) and mouse CD8 (Lyt 2) system (Dynal) according to the manufacturer's protocol as previously described (36). The phenotype of the cells was determined by staining with rat anti-mouse CD8a (Ly-2, clone 53-6.7), rat anti-mouse CD4 (L3T4, clone GK1.5), rat anti-mouse CD3 (molecular complex, clone 17A2), and CD45/B220 (clone RA3-6B2). All antibodies were produced by Pharmingen. Cells were washed in staining wash buffer, fixed with 1.0% paraformaldehyde, and analyzed on a FACSCalibur.


arrow
RESULTS
 
Increased protective efficacy of VP vaccination in mice. We previously reported that BALB/c (H-2d) mice were easier to protect with vaccination to Ebola virus VP24, VP30, and VP40, with fewer than half surviving challenge after vaccination with VRPs expressing VP35. In contrast, C57BL/6 (H-2b) mice were easier to protect using VRP expressing VP35, with fewer than half surviving challenge after vaccination with VP30 or VP40 and no mice surviving challenge after vaccination with VP24. Increasing the dose of replicons (~10-fold based on flow titer estimation) improved the protective efficacy of most VRP-VP vaccines to 80% or better (Table 2). However, C57BL/6 mice vaccinated with VRP expressing VP24 were not protected, despite changes in dose or increased numbers of booster vaccinations. As in our earlier study, no protection was observed with sera derived from VP-vaccinated mice.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Protection induced by Ebola VRP vaccinationa

Induction of CD8+ T cells specific for epitopes within ZEBOV GP, NP, VP24, VP30, VP35, and VP40. The improved efficacy of the VRPs permitted analysis of the role of CD8+ T-cell responses in mediating protection. We identified CD8+ T-cell responses for each protein using a combination of assays and stimulation with peptides. The first analysis was conducted using cells ex vivo after a brief exposure to peptides. Overlapping peptide pools for each protein were generated using a matrix in which each peptide was present in two different pools (Fig. 1A). The peptide pools were used to induce IFN-{gamma} production and secretion following 5 h of incubation with freshly isolated splenocytes from vaccinated mice (Fig. 1A). Individual peptides were selected for further testing based on their presence in two positive pools (e.g., peptide 43 in Fig. 1A). The utility of this approach for enabling rapid selection of potential epitope sequences was confirmed by testing each peptide from the VP24 protein individually (Fig. 1B and C). In vitro restimulation with peptides was used next to expand responsive cells. Splenocytes from vaccinated mice were restimulated in vitro with peptides for 5 to 7 days and further characterized using ELISPOT, intracellular cytokine staining (ICC), and chromium release assays (Table 3; Fig. 2).



View larger version (40K):
[in this window]
[in a new window]
  		FIG. 1. Screening of CD8+ cells' specific responses to VP24 peptides after vaccination with VRPs expressing Ebola virus VP24. BALB/c mice were vaccinated twice at 30-day intervals. Seven days after the second vaccination, pooled splenocytes from five mice were restimulated in vitro for 5 h with or without the indicated VP24 peptide matrix pools (A) or individual overlapping peptide sequences (B and C) and subjected to surface and intracellular staining with anti-CD44 FITC and anti-IFN-{gamma} PE and anti-CD8 Cy-Chrome. (C) The percentage of CD44+, IFN-{gamma}+ cells among CD8+ lymphocytes is indicated in the upper right quadrant of each plot with background for each assay. These data are representative of results from two independent experiments.


View this table:
[in this window]
[in a new window]
  		TABLE 3. Ebola virus protein sequences recognized by murine CD8+ T cellsa



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 2. Restimulation and lytic function of BALB/c CD8+ T-cell responses to VP24 peptides after vaccination with VRPs expressing Ebola virus VP24. BALB/c mice were vaccinated twice at 30-day intervals. Seven days after the second vaccination, pooled splenocytes from five mice were restimulated in vitro with the VP24159, VP24171, or VP24214 peptides, and on day 2, 10 U/ml of IL-2 and concanavalin A supernatants (10%) were added. On day 7, after being washed, cells were assayed in vitro for 5 h with PMA/ionomycin, without peptide, or with the VP24 specific peptides. The cells were subsequently surface stained with anti-CD8 Cy-Chrome or anti-CD4 Cy-Chrome and intracellularly stained with anti-IFN-{gamma} PE. The percentage of CD44+, IFN-{gamma}+ cells among CD8+ lymphocytes is indicated in the upper right quadrant of each plot with background for each assay. The ability of the epitope-specific cells to lyse peptide-pulsed target cells was determined. The filled squares represent the MHC-matched target cells pulsed with the specific peptide, while the open circles represent the lysis of MHC-matched target cells incubated with no peptide. The data represent results from at least two experiments for each peptide sequence. In all experiments, the percentage of lysis observed for either spontaneous or mismatched controls did not exceed 15%.

We examined the CD8+ T-cell responses induced by VRPs in BALB/c (H-2d) and C57BL/6 (H-2b) mice. BALB/c mice had detectable CD8+ T-cell responses to all six of the tested ZEBOV proteins (Table 3). The responses were directed toward one to three peptide sequences per protein. With our standard vaccine dose, we observed no CD8+ T-cell responses to GP; however, when the dose was increased 10-fold, a response was detected towards VSTGTGPGAGDFAFHK (GP141). VRP-NP vaccination induced a CD8+ response to a previously identified (24) NP peptide sequence, SFKAALSSL (NP279). Three CD8+ T-cell responses were identified in BALB/c mice vaccinated with VRP expressing Ebola virus VP24: KFINKLDALH (VP24159), NYNGLLSSI (VP24171), and PGPAKFSLL (VP24214). Identifying BALB/c CD8+ T-cell responses to VP30, VP35, and VP40 required three booster vaccinations. Responses were directed toward two sequences each in VP30 and VP35: KFSKSQLSLLCETHLR (VP30181), DLQSLIMFITAFLNI (VP30231), TVPQSVREAFNNL (VP35190), and PGFGTAFHQLVQVICK (VP35233). Three VP40 sequences stimulated IFN-{gamma} production by H-2d CD8+ T cells: AFLQEFVLPPVQLPQ (VP40160), YFTFDLTALK (VP40171), and TESPEKIQAI (VP40232). In BALB/c mice, no CD4+ cell responses to the sequences that stimulated IFN-{gamma} in CD8+ T cells were detected.

Vaccinated C57BL/6 (H-2b) mice had a detectable CD8+ T-cell response to a single peptide sequence in Ebola virus GP: WIPYFGPAAEGIYTE (GP531). This peptide also induced an IFN-{gamma} response by CD4+ T cells (not shown). NP vaccination induced a response to a previously described epitope, VYQ VNNLEEIC (NP44) (36), and to two other peptides: GQF LSFASL (NP148) and DAVLYYHMM (NP663). Freshly isolated splenocytes from vaccinated C57BL/6 (H-2b) mice did not respond to any VP24 peptides regardless of the dose or number of vaccinations administered. VP30 vaccination induced CD8+ T-cell responses to the VP30181 and VP30231 sequences also recognized by BALB/c mice. C57BL/6 mice responded to three sequences in VP35—CDIENNPGL (VP3545), MVAKYDHL (VP35138), and RNIMYDHL (VP35225)—and to one peptide from VP40—LRIGNQAFLQEFVLPP (VP40150).

Cytolytic function after in vitro restimulation and adoptive transfer of CTLs. Cytolytic activity was tested using restimulated splenocytes from vaccinated mice. A representative chromium release assay (Fig. 2) indicates marked lysis of peptide-pulsed, but not unpulsed, H-2-matched target cells using effector cells from VP24-vaccinated BALB/c mice. Similar results were observed for all but one of the newly identified CD8+ T-cell epitopes (Table 3). Lysis was not observed using the GP531 peptide recognized by C57BL/6 CD8+ T cells, although the CD8+ and CD4+ T cells expanded after in vitro restimulation with the GP531 peptide (from 0.21% ex vivo to 1.54% for CD8+ T cells and from 1.4% ex vivo to 2.89% for CD4+ cells). Purifying the CD8+ T cells from the CD4+ T cells to reduce competition for the peptide did not result in lysis of labeled cells.

CD8+ T cells were adoptively transferred into filovirus-naïve mice approximately 4 h before challenge, and cytotoxic activity was confirmed. Adoptive transfer of 5 x 106 cells with ZEBOV-specific lytic function was protective with no morbidity noted in survivors, whereas the transfer of cells from control MLR cultures or restimulated Lassa N-specific CTLs failed toprotect or significantly change the mean time to death (Table 3). The CD8+ T cells specific for the GP531 sequence that did not exhibit lytic activity also did not protect mice from challenge with mouse-adapted Ebola virus.

Cellular responses in convalescent mice. To determine whether T-cell responses to the newly identified epitopes are induced during infection rather than by vaccination, some convalescent mice that were vaccinated against one protein, or that were pretreated with MAbs to Ebola virus GP, were evaluated for the de novo induction of cellular responses to other proteins after challenge (Table 4). To date, we have identified cellular responses to 15 of the epitopes in convalescent mice that were not exposed to those sequences before challenge. For example, C57BL/6 mice vaccinated with VRP expressing Ebola virus GP had T-cell responses to Ebola virus NP44 and NP663 that were detected 30 days after challenge. Similarly, CD8+ T-cell responses to NP279, VP24171, and VP24214 were observed after challenge in BALB/c mice vaccinated with Ebola virus GP VRP. Furthermore, mice that were treated 1day before challenge with a mixture of 100 µg each of three protective monoclonal antibodies (13F6, 13C6, and 6D8) (37) also had CTL responses detected after challenge.


View this table:
[in this window]
[in a new window]
  		TABLE 4. De novo CTL responses detected in convalescent micea


arrow
DISCUSSION
 
Vaccine research efforts for filoviruses have been hampered by a lack of knowledge regarding the adaptive immune mechanisms needed for protection and by the difficulty of assessing cellular responses in animal models, such as the guinea pig and nonhuman primate. This study identified CD8+ CTLs as a protective immune mechanism induced by vaccination of mice against each of the VP proteins. It also extended our earlier findings and other reports of in vitro cytolytic activity (23, 24, 30, 38) by demonstrating protective CTL responses in adoptive transfer studies. This analysis of the CD8+ cellular responses to these six ZEBOV proteins offers insights on the specificity and breadth of responses that may be important to vaccine research efforts. BALB/c and C57BL/6 mice were protected after vaccination with VRPs expressing NP, VP30, VP35, and VP40; for each of these combinations, we identified or confirmed at least one protective CTL response. Twice we observed that an individual peptide induced a protective CTL response in both mouse strains (see VP30 in Table 3), but not surprisingly, the peptide specificity of the majority of the responses was not the same, as the mouse strains express major histocompatibility complex (MHC) class I molecules with different peptide-binding motifs. The mice also differed in the number of CTL responses generated towards each protein. For example, C57BL/6 mice had CTL responses to one peptide derived from the Ebola virus VP40, whereas three protective VP40-specific CTL responses were identified in BALB/c mice. However, the number of VRP-induced responses to each protein was low (≤3) in both mouse strains.

The murine responses to VP24 were also interesting, in that the presence or absence of detectable CTL activity corresponded with protection. Vaccination to VP24 induced three CD8 T cell responses in BALB/c mice and protected them from challenge, while similarly vaccinated C57BL/6 mice failed to produce any detectable CD8+ T-cell response to VP24, and none survived challenge, even after modification of the vaccine regimen. The lack of detectable CTL responses to VP24 suggests that cells of the H-2b haplotype do not present peptides from VP24 that induce lytic activity. Analysis of the VP24 sequence using predicted binding motifs for Kb and Db class I molecules indicates at least five peptide sequences with the potential to be presented by these molecules; however, studies comparing motif peptides and CTL responses for other viruses demonstrated that not all motif peptides actually induce CTLs (25). The same observation may apply to C57BL/6 mice and VP24 predicted peptide sequences.

Alternatively, the failure to induce protection in VRP-VP24-vaccinated C57BL/6 mice could indicate a suboptimal vaccine regimen. Both mouse strains had equivalently low, but detectable, antibody responses to VP24 after vaccination. There may be differences in the levels of VP24 protein expressed in the mouse strains, or in the threshold needed to activate their CTL, such that expression of VP24 from another vector would be better able to induce protective CTLs to VP24 in these mice. However, this is not supported by our inability to detect VP24-specific T-cell responses in convalescent C57BL/6 mice after challenge.

We had some difficulty identifying protective CD8+ T-cell responses to GP, despite reports of in vitro lytic activity against target cells expressing GP with cells from mice vaccinated with plasmid vaccines (30, 38) or liposome-encapsulated virus (22). The plasmid vaccine platforms induced solid lytic activity to cells expressing GP but did not identify the specific epitope(s) recognized. The study using liposome-encapsulated virus identified two CTL epitope sequences (GP 161 to 169 and GP 231 to 239). We did not observe any response to those peptides after vaccination with VRPs expressing GP; however, we did detect T-cell responses to the peptide at positions 231 to 239 after challenge (Table 4). The reported lytic activity to those sequences was modest, even with the encapsulated virus, generally being less than 25% at E:T ratios as high as 100:1, and the different vaccine regimens used in the two studies likely contributed to the different observations. Alternatively, the protective antibody responses known to be induced in both mouse strains following vaccination with VRPs expressing GP (35) may have contributed to clearance of antigen-expressing cells before these cytolytic T-cell responses were firmly established in these mice. The response to GP531 is interesting because it contains the putative fusion domain (7) and because both CD4+ and CD8+ cells responded to it. Within this sequence there is some variation at amino acid position 544 (T to I) in both Ebola virus Zaire and Sudan isolates. The GP in the VRP has an I at amino acid 544, but the mouse-adapted challenge virus has a T. Preliminary data suggested that both sequences induced IFN expression by the CD4 and CD8 responses in C57BL/6 mice vaccinated with VRP (data not shown). Our expansion strategy used the peptide sequence containing the threonine residue since it is found in the mouse-adapted challenge virus.

Three assays were used to try to identify cellular immune responses and were examined for their ability to predict protection accurately. The ELISPOT assay was the least sensitive of the three, often detecting only the most dominant responses. The ICC assay was more sensitive, identified more epitopes, and was generally predictive, with the single exception of the GP531 response in C57BL/6 mice. The 51Cr assay was the most reliable indicator of protection, as it accurately predicted protection when cells were lysed and predicted the lack of protection by the CD8+ T cells specific for the GP531 epitope. The lack of cytolytic activity could be from competition between the CD4+ and CD8+ T cells for the peptide; however, we could not identify any cytolytic activity even when the CD8+ cells were separated from the CD4+ cells during restimulation. This may be another example of a peptide that is presented but does not activate a cytolytic response. The inability of these CD8+ Tcells to protect mice, despite inducing IFN-{gamma}, lends further support to the role of CTL effectors in protection, although wecould not confirm that the cells continued to secrete IFN-{gamma} in vivo.

Our demonstration of protection by CTLs specific for 20 different epitopes in six proteins provides evidence that cellular immunity has a role in resolving Ebola virus infection in mice. This study, together with prior reports (8, 22-24, 30, 34-38), indicates a growing consensus that, in the mouse model of Ebola virus infection, both humoral and cellular responses contribute to protection. This is supported by our prior studies identifying protective MAbs and CTLs, our observation that Ab responses to GP arise after challenge in mice provided CTLs, and the observation that CTLs arise after challenge of mice vaccinated with VRP-GP or pretreated with protective monoclonal antibodies. Prior lack of information regarding T-cell epitopes recognized by mice has limited the ability to examine de novo responses occurring in animals surviving challenge, including in our prior antibody study (37). We conducted preliminary studies to further examine the contributions of both antibodies and CTL responses in protection. We found that mice vaccinated with one protein, or passively administered a mixture of antibodies, generated new CTL responses after challenge, suggesting that both antibody and cellular responses are working together to provide protection. Either response alone may be able to limit virus replication until both arms of the immune response are present to clear infection.

The induction of both antibody and cellular responses was also reported in the studies that successfully protected nonhuman primates against filovirus challenge with adenovirus vaccination against GP and NP (27, 28). T-cell responses, assessed according to the production of tumor necrosis factor alpha, were demonstrated in five of eight macaques successfully protected by vaccination (27) or with chimeric viruses (14). If CTL responses contribute to protection, it is likely that the epitope specificity of the responses will vary among species, and ultimately among humans, as it does among mouse strains. It is not feasible to test every recipient of an eventual human use vaccine to evaluate CTL responses; therefore, producing a vaccine that includes multiple proteins increases the chances of inducing CTL responses in the majority of recipients. This study indicates that there are at least six Ebola virus proteins capable of inducing protective CTL responses. The GP elicits protective humoral immunity in addition to cellular immunity, but inclusion of the other ZEBOV proteins may be required in an eventual human use vaccine formulation to induce sufficient CTL responses in humans.


arrow
ACKNOWLEDGMENTS
 
We thank Connie Schmaljohn for providing some peptides used in this study, Mehrl Gibson for technical support, and the National Cancer Institute Biological Resources Branch (BRB) Preclinical Repository for supplying the recombinant human IL-2.

The research described herein was sponsored by the U.S. Army, Project No. 024-7J-098. G. G. Olinger was a recipient of the National Research Council Fellowship.

Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: United States Army Medical Research Institute of Infectious Diseases, Division of Virology, 1425 Porter Street, Frederick, MD 21702-5011. Phone: (301) 619-4917. Fax: (301) 619-2290. E-mail: marykate.hart{at}amedd.army.mil. Back


arrow
REFERENCES
 
    1
  1. Basler, C. F., A. Mikulasova, L. Martinez-Sobrido, J. Paragas, E. Muhlberger, M. Bray, H. D. Klenk, P. Palese, and A. Garcia-Sastre. 2003. The Ebola virus VP35 protein inhibits activation of interferon regulatory factor 3. J. Virol. 77::7945-7956.[Abstract/Free Full Text]
    2. 2
  2. Basler, C. F., X. Wang, E. Muhlberger, V. Volchkov, J. Paragas, H. D. Klenk, A. Garcia-Sastre, and P. Palese. 2000. The Ebola virus VP35 protein functions as a type I IFN antagonist. Proc. Natl. Acad. Sci. USA 97:12289-12294.[Abstract/Free Full Text]
    3. 3
  3. Bavari, S., C. M. Bosio, E. Wiegand, G. Ruthel, A. B. Will, T. W. Geisbert, M. Hevey, C. Schmaljohn, A. Schmaljohn, and M. J. Aman. 2002. Lipid raft microdomains: a gateway for compartmentalized trafficking of Ebola and Marburg viruses. J. Exp. Med. 195:593-602.[Abstract/Free Full Text]
    4. 4
  4. Bray, M. 2001. The role of the Type I interferon response in the resistance of mice to filovirus infection. J. Gen. Virol. 82:1365-1373.[Abstract/Free Full Text]
    5. 5
  5. Bray, M., K. Davis, T. Geisbert, C. Schmaljohn, and J. Huggins. 1998. A mouse model for evaluation of prophylaxis and therapy of Ebola hemorrhagic fever. J. Infect. Dis. 178:651-661.[Medline]
    6. 6
  6. Elliott, L. H., M. P. Kiley, and J. B. McCormick. 1985. Descriptive analysis of Ebola virus proteins. Virology 147:169-176.[CrossRef][Medline]
    7. 7
  7. Gomara, M. J., P. Mora, I. Mingarro, and J. L. Nieva. 2004. Roles of a conserved proline in the internal fusion peptide of Ebola glycoprotein. FEBS Lett. 569:261-266.[CrossRef][Medline]
    8. 8
  8. Gupta, M., S. Mahanty, M. Bray, R. Ahmed, and P. E. Rollin. 2001. Passive transfer of antibodies protects immunocompetent and immunodeficient mice against lethal Ebola virus infection without complete inhibition of viral replication. J. Virol. 75:4649-4654.[Abstract/Free Full Text]
    9. 9
  9. Harty, R. N., M. E. Brown, G. Wang, J. Huibregtse, and F. P. Hayes. 2000. A PPxY motif within the VP40 protein of Ebola virus interacts physically and functionally with a ubiquitin ligase: implications for filovirus budding. Proc. Natl. Acad. Sci. USA 97:13871-13876.[Abstract/Free Full Text]
    10. 10
  10. Hevey, M., D. Negley, J. Geisbert, P. Jahrling, and A. Schmaljohn. 1997. Antigenicity and vaccine potential of Marburg virus glycoprotein expressed by baculovirus recombinants. Virology 239:206-216.[CrossRef][Medline]
    11. 11
  11. Hevey, M., D. Negley, P. Pushko, J. Smith, and A. Schmaljohn. 1998. Marburg virus vaccines based upon alphavirus replicons protect guinea pigs and nonhuman primates. Virology 251:28-37.[CrossRef][Medline]
    12. 12
  12. Hevey, M., D. Negley, L. VanderZanden, R. F. Tammariello, J. Geisbert, C. Schmaljohn, J. F. Smith, P. B. Jahrling, and A. L. Schmaljohn. 2001. Marburg virus vaccines: comparing classical and new approaches. Vaccine 20:586-593.[CrossRef][Medline]
    13. 13
  13. Huang, Y., L. Xu, Y. Sun, and G. J. Nabel. 2002. The assembly of Ebola virus nucleocapsid requires virion-associated proteins 35 and 24 and posttranslational modification of nucleoprotein. Mol. Cell 10:307-316.[CrossRef][Medline]
    14. 14
  14. Jones, S. M., H. Feldmann, U. Stroher, J. B. Geisbert, L. Fernando, A. Grolla, H. D. Klenk, N. J. Sullivan, V. E. Volchkov, E. A. Fritz, K. M. Daddario, L. E. Hensley, P. B. Jahrling, and T. W. Geisbert. 2005. Live attenuated recombinant vaccine protects nonhuman primates against Ebola and Marburg viruses. Nat. Med. 11:786-790.[CrossRef][Medline]
    15. 15
  15. Licata, J. M., R. F. Johnson, Z. Han, and R. N. Harty. 2004. Contribution of Ebola virus glycoprotein, nucleoprotein, and VP24 to budding of VP40 virus-like particles. J. Virol. 78:7344-7351.[Abstract/Free Full Text]
    16. 16
  16. Mahanty, S., M. Gupta, J. Paragas, M. Bray, R. Ahmed, and P. E. Rollin. 2003. Protection from lethal infection is determined by innate immune responses in a mouse model of Ebola virus infection. Virology 312:415-424.[CrossRef][Medline]
    17. 17
  17. Martin-Serrano, J., T. Zang, and P. D. Bieniasz. 2001. HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress. Nat. Med. 7:1313-1319.[CrossRef][Medline]
    18. 18
  18. Modrof, J., E. Muhlberger, H. D. Klenk, and S. Becker. 2002. Phosphorylation of VP30 impairs Ebola virus transcription. J. Biol. Chem. 277:33099-33104.[Abstract/Free Full Text]
    19. 19
  19. Noda, T., H. Sagara, E. Suzuki, A. Takada, H. Kida, and Y. Kawaoka. 2002. Ebola virus VP40 drives the formation of virus-like filamentous particles along with GP. J. Virol. 76:4855-4865.[Abstract/Free Full Text]
    20. 20
  20. Pushko, P., M. Bray, G. V. Ludwig, M. Parker, A. Schmaljohn, A. Sanchez, P. B. Jahrling, and J. F. Smith. 2000. Recombinant RNA replicons derived from attenuated Venezuelan equine encephalitis virus protect guinea pigs and mice from Ebola hemorrhagic fever virus. Vaccine 19:142-153.[CrossRef][Medline]
    21. 21
  21. Pushko, P., J. Geisbert, M. Parker, P. Jahrling, and J. Smith. 2001. Individual and bivalent vaccines based on alphavirus replicons protect guinea pigs against infection with Lassa and Ebola viruses. J. Virol. 75:11677-11685.[Abstract/Free Full Text]
    22. 22
  22. Rao, M., M. Bray, C. R. Alving, P. Jahrling, and G. R. Matyas. 2002. Induction of immune responses in mice and monkeys to Ebola virus after immunization with liposome-encapsulated irradiated Ebola virus: protection in mice requires CD4+ T cells. J. Virol. 76:9176-9185.[Abstract/Free Full Text]
    23. 23
  23. Rao, M., G. R. Matyas, F. Grieder, K. Anderson, P. B. Jahrling, and C. R. Alving. 1999. Cytotoxic T lymphocytes to Ebola Zaire virus are induced in mice by immunization with liposomes containing lipid A. Vaccine 17:2991-2998.[CrossRef][Medline]
    24. 24
  24. Simmons, G., A. Lee, A. J. Rennekamp, X. Fan, P. Bates, and H. Shen. 2004. Identification of murine T-cell epitopes in Ebola virus nucleoprotein. Virology 318:224-230.[CrossRef][Medline]
    25. 25
  25. Stevanovic, S., and H. Schild. 1999. Quantitative aspects of T cell activation—peptide generation and editing by MHC class I molecules. Semin. Immunol. 11:375-384.[CrossRef][Medline]
    26. 26
  26. Sullivan, N., Z. Y. Yang, and G. J. Nabel. 2003. Ebola virus pathogenesis: implications for vaccines and therapies. J. Virol. 77:9733-9737.[Free Full Text]
    27. 27
  27. Sullivan, N. J., T. W. Geisbert, J. B. Geisbert, L. Xu, Z. Y. Yang, M. Roederer, R. A. Koup, P. B. Jahrling, and G. J. Nabel. 2003. Accelerated vaccination for Ebola virus haemorrhagic fever in non-human primates. Nature 424:681-684.[CrossRef][Medline]
    28. 28
  28. Sullivan, N. J., A. Sanchez, P. E. Rollin, Z. Y. Yang, and G. J. Nabel. 2000. Development of a preventive vaccine for Ebola virus infection in primates. Nature 408:605-609.[CrossRef][Medline]
    29. 29
  29. Timmins, J., S. Scianimanico, G. Schoehn, and W. Weissenhorn. 2001. Vesicular release of Ebola virus matrix protein VP40. Virology 283:1-6.[CrossRef][Medline]
    30. 30
  30. Vanderzanden, L., M. Bray, D. Fuller, T. Roberts, D. Custer, K. Spik, P. Jahrling, J. Huggins, A. Schmaljohn, and C. Schmaljohn. 1998. DNA vaccines expressing either the GP or NP genes of Ebola virus protect mice from lethal challenge. Virology 246:134-144.[CrossRef][Medline]
    31. 31
  31. Warfield, K. L., J. G. Perkins, D. L. Swenson, E. M. Deal, C. M. Bosio, M. J. Aman, W. M. Yokoyama, H. A. Young, and S. Bavari. 2004. Role of natural killer cells in innate protection against lethal Ebola virus infection. J. Exp. Med. 200:169-179.[Abstract/Free Full Text]
    32. 31
  32. Warfield, K. L., C. M. Bosio, B. C. Welcher, E. M. Deal, M. Mohamadzadeh, A. Schmaljohn, M. J. Aman, and S. Bavari. 2003. Ebola virus-like particles protect from lethal Ebola virus infection. Proc. Natl. Acad. Sci. USA 100:15889-15894.
    33. 32
  33. Weik, M., J. Modrof, H. D. Klenk, S. Becker, and E. Muhlberger. 2002. Ebola virus VP30-mediated transcription is regulated by RNA secondary structure formation. J. Virol. 76:8532-8539.[Abstract/Free Full Text]
    34. 33
  34. Whitton, J. L., A. Tishon, H. Lewicki, J. Gebhard, T. Cook, M. Salvato, E. Joly, and M. B. Oldstone. 1989. Molecular analyses of a five-amino-acid cytotoxic T-lymphocyte (CTL) epitope: an immunodominant region which induces nonreciprocal CTL cross-reactivity. J. Virol. 63:4303-4310.[Abstract/Free Full Text]
    35. 34
  35. Wilson, J. A., C. M. Bosio, and M. K. Hart. 2001. Ebola virus: the search for vaccines and treatments. Cell. Mol. Life Sci. 58:1826-1841.[CrossRef][Medline]
    36. 35
  36. Wilson, J. A., M. Bray, R. Bakken, and M. K. Hart. 2001. Vaccine potential of Ebola virus VP24, VP30, VP35, and VP40 proteins. Virology 286:384-390.[CrossRef][Medline]
    37. 36
  37. Wilson, J. A., and M. K. Hart. 2001. Protection from Ebola virus mediated by cytotoxic T lymphocytes specific for the viral nucleoprotein. J. Virol. 75:2660-2664.[Abstract/Free Full Text]
    38. 37
  38. Wilson, J. A., M. Hevey, R. Bakken, S. Guest, M. Bray, A. L. Schmaljohn, and M. K. Hart. 2000. Epitopes involved in antibody-mediated protection from Ebola virus. Science 287:1664-1666.[Abstract/Free Full Text]
    39. 38
  39. Xu, L., A. Sanchez, Z. Yang, S. R. Zaki, E. G. Nabel, S. T. Nichol, and G. J. Nabel. 1998. Immunization for Ebola virus infection. Nat. Med. 4:37-42.[CrossRef][Medline]
    40. 39
  40. Yang, Z. Y., L. S. Wyatt, W. P. Kong, Z. Moodie, B. Moss, and G. J. Nabel. 2003. Overcoming immunity to a viral vaccine by DNA priming before vector boosting. J. Virol. 77:799-803.

Journal of Virology, November 2005, p. 14189-14196, Vol. 79, No. 22
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.22.14189-14196.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Halfmann, P., Ebihara, H., Marzi, A., Hatta, Y., Watanabe, S., Suresh, M., Neumann, G., Feldmann, H., Kawaoka, Y. (2009). Replication-Deficient Ebolavirus as a Vaccine Candidate. J. Virol. 83: 3810-3815 [Abstract] [Full Text]  
  • Bradfute, S. B., Warfield, K. L., Bavari, S. (2008). Functional CD8+ T Cell Responses in Lethal Ebola Virus Infection. J. Immunol. 180: 4058-4066 [Abstract] [Full Text]  
  • Dowling, W., Thompson, E., Badger, C., Mellquist, J. L., Garrison, A. R., Smith, J. M., Paragas, J., Hogan, R. J., Schmaljohn, C. (2007). Influences of Glycosylation on Antigenicity, Immunogenicity, and Protective Efficacy of Ebola Virus GP DNA Vaccines. J. Virol. 81: 1821-1837 [Abstract] [Full Text]  
  • Martin, J. E., Sullivan, N. J., Enama, M. E., Gordon, I. J., Roederer, M., Koup, R. A., Bailer, R. T., Chakrabarti, B. K., Bailey, M. A., Gomez, P. L., Andrews, C. A., Moodie, Z., Gu, L., Stein, J. A., Nabel, G. J., Graham, B. S., the VRC 204 Study Team, (2006). A DNA Vaccine for Ebola Virus Is Safe and Immunogenic in a Phase I Clinical Trial. CVI 13: 1267-1277 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Olinger, G. G.
Right arrow Articles by Hart, M. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Olinger, G. G.
Right arrow Articles by Hart, M. K.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»