The development of a mouse-adapted strain of Ebola virus (3
) has facilitated the ability to examine the role of antibodies and CTLs in mediating protection from Ebola virus. The mouse-adapted virus is uniformly lethal to immunocompetent mice when delivered intraperitoneally (3
). This virus is also lethal to guinea pigs and causes severe illness that can be lethal to rhesus monkeys (M. Bray, personal communication). The ability to transfer immune effectors into naive, genetically identical mice prior to challenge provides the opportunity to examine the in vivo contribution of CTLs in protection, thereby aiding in the identification of protective immune effectors critical for Ebola virus vaccine development.
Vaccination with Ebola virus NP expressed from a VEE virus replicon has been shown to induce protection in BALB/c mice against lethal challenge with mouse-adapted Ebola virus (12
). Our studies extended this observation to C57BL/6 mice, which differ from BALB/c mice at the MHC. Two or three injections of 106
focus-forming units of VEE virus replicons expressing the Ebola virus NP induced protection from lethal Ebola challenge in 75 to 80% of the mice (Table ). Ebola virus was detected in the sera of vaccinated animals after challenge (Table ), indicating that mice vaccinated with the Ebola virus NP replicons were not resistant to infection. However, the viral load in Ebola virus NP-vaccinated mice was markedly reduced compared with that in control animals vaccinated with VEE virus replicons expressing the Lassa virus N gene (Table ).
Protective efficacy of Ebola virus NP replicons in C57BL/6 micea
The vaccinated mice had mean end point antibody titers to the Ebola virus NP of 3 to 3.5 logs in ELISA prior to challenge. These antibodies did not inhibit plaque formation by mouse-adapted Ebola virus at a 1:20 dilution. To determine if antibodies to the Ebola virus NP were capable of protecting against lethal Ebola virus challenge, 1 ml of immune serum pooled from mice vaccinated three times with Ebola virus NP replicons was passively transferred to unvaccinated mice 24 h before lethal challenge with mouse-adapted Ebola Zaire virus. The recipient mice had serum antibodies to the Ebola virus NP (end point titers of 2.5 to 3 logs in ELISA) 5 h prior to challenge. However, as was observed with BALB/c mice (12
), passive transfer of immune serum to the Ebola virus NP did not protect unvaccinated C57BL/6 mice from lethal disease (Table ) or extend the time to death (data not shown).
CD8+ T lymphocytes protect against lethal Ebola virus challenge
To determine if NP-specific CTLs were involved in protection from lethal Ebola virus challenge, we used a computer algorithm (11
) and predicted binding motifs (15
) to examine the Ebola virus NP sequence for possible H-2b
class I-restricted epitopes. Both methods predicted that an amino acid sequence contained within VYQVNNLEEIC would be bound by H-2Db
class I molecules. This peptide was chemically synthesized and used to restimulate immune splenocytes in vitro before cells were examined for functional activity.
Spleen cells from mice vaccinated with the Ebola virus NP expressed from VEE virus replicons were restimulated in vitro with the NP peptide (VYQVNNLEEIC) and tested for lysis in a chromium release assay. These cells had high levels of cytolytic activity against syngeneic target cells (EL4) coated with the Ebola virus NP peptide (Fig. ). These CTLs failed to lyse either untreated EL4 target cells, EL4 target cells coated with a peptide recognized by Lassa virus-specific CTLs, or H-2-incompatible cells (L5178Y, H-2d) coated with the Ebola virus NP peptide (Fig. ). In vivo priming of the CTL response was required, as no lysis of Ebola virus NP peptide-coated target cells was observed after in vitro Ebola virus NP peptide stimulation of spleen cells from unvaccinated C57BL/6 mice or C57BL/6 mice vaccinated with the control Lassa virus N replicon (data not shown).
FIG. 1 Lysis of target cells by cytotoxic T cells from mice vaccinated with the VEE replicon expressing the Ebola virus NP. Syngeneic EL4 cells coated with the Ebola virus NP peptide were tested for lysis by unfractionated (closed squares), CD8-enriched (closed (more ...)
FACScan analysis indicated that the cultures obtained in different experiments contained 6 to 33% CD4+
cells and 54 to 90% CD8+
cells. These cell populations were separated by magnetic beads coated with antibodies to CD4 or CD8 to determine the phenotype of the cells responsible for CTL activity. In separate experiments, FACScan analysis indicated that the cell separations yielded CD4-enriched cultures containing 95 to 98% CD4+
cells and 2 to 3% CD8+
cells and CD8-enriched cultures with 94 to 96% CD8+
cells and <3% CD4+
cells. Chromium release assays performed using the fractionated cell populations as effector cells demonstrated that the CD8+
cells mediated the CTL activity (Fig. ). To confirm that the CTLs were restricted to the NP peptide bound by Db
class I molecules, as predicted by the binding motif (11
), we tested for lysis of concanavalin A-stimulated spleen cells obtained from inbred mice with different K or D class I molecules. At an effector/target cell ratio of 50:1, 50% specific release was observed against Ebola virus NP peptide-coated H-2 Kk
target cells from B10.A(2R) mice, but only 16% specific release was observed using peptide-coated H-2Kb
target cells from B10.A(5R) mice.
To determine if the CTLs were efficacious in vivo, either the unfractionated, the CD8-enriched, or the CD4-enriched cells were injected i.p. into unvaccinated C57BL/6 mice 4 h before a lethal challenge with mouse-adapted Ebola Zaire virus. Mice were protected from Ebola virus challenge if they received either the unfractionated or the CD8-enriched T cells (Table ). The adoptive transfer of as few as 1 million cells protected at least 80% of the mice from death after challenge with up to 3,000 times the Ebola virus LD50 (Table ). This was similar to the efficacy achieved with vaccination. The survival of all mice receiving eightfold more CTLs suggests that the vaccination strategy may be optimized to achieve 100% efficacy.
In contrast to what was observed with the adoptive transfer of the unfractionated and CD8-enriched T cells, the CD4-enriched T-cell population did not protect mice from lethal disease (Table ). Similarly, no protection was afforded by the transfer of either control CTLs specific for allogeneic BALB/c cells (approximately 80% CD8+) or cells that were obtained from unvaccinated C57BL/6 mice and stimulated in vitro with the Ebola virus NP peptide (Table ). These data demonstrate that vaccination with replicons expressing the Ebola virus NP induces CTLs that are protective in vivo against Ebola virus challenge.
We chose to examine the efficacy of the CTLs by transferring cells into naive recipients rather than by in vivo depletion of CD4+
cells prior to vaccination or challenge. This strategy enabled us to directly examine the effect of specific T cells with defined lytic capability in the absence of any Ebola virus-specific antibodies that would be present as a result of vaccination or increased levels of cytokines and chemokines resulting from the in vivo lysis and clearance of cells. The latter point is relevant to Ebola virus challenge, as several reports have suggested that innate immunity or the production of cytokines and chemokines may affect the outcome of human disease progression (1
Examination of sera drawn from surviving mice 28 days after challenge demonstrated the presence of antibodies to the Ebola virus. This was not unexpected, as the lysis of virus-infected cells by CTLs exposes the humoral immune system to viral antigens. Therefore, it is not known whether the CTLs were entirely responsible for eliminating the virus or if they worked in conjunction with antibodies that were induced to other viral proteins, such as the Ebola virus GP, in response to viral replication in infected cells. However, the CTLs specific for the Ebola virus NP controlled infection sufficiently to enable survival of the mice, which would have otherwise succumbed to lethal infection within a week after challenge. The finding that control CD8+ CTLs neither protected mice nor extended the time to death after challenge suggests that the ability of the Ebola virus NP-specific CTLs to lyse infected cells was essential for protection from lethal Ebola challenge and that protection was not simply due to nonspecific mediators or cytokines being secreted by the transferred cells.
This study represents the first demonstration of CTL-mediated protection against a lethal Ebola virus challenge. This is important in that it formally demonstrates that CTLs contribute to protection from Ebola virus and stresses the need to closely examine the ability of all viral proteins to induce protective cellular immune responses for the development of effective vaccine strategies for Ebola hemorrhagic fever. Taking these results together with our previous study that identified protective monoclonal antibodies to the Ebola virus GP, it is likely that an optimal Ebola virus vaccine will need to induce protective humoral as well as protective cellular responses to efficiently clear both free virus and virus-infected cells. Due to the MHC -restriction of CTL responses and the limited number of CTL epitopes on viral proteins (17
), the inclusion of several viral proteins capable of eliciting protective CTLs may be necessary to ensure the induction of adequate protection by an eventual human-use vaccine.