Infection with many emerging viruses, such as the hemorrhagic fever disease caused by the filoviruses, Marburg (MARV), and Ebola virus (EBOV), leaves the host with a short timeframe in which to mouse a protective immune response. In lethal cases, uncontrolled viral replication and virus-induced immune dysregulation are too severe to overcome, and mortality is generally associated with a lack of notable immune responses. Vaccination studies in animals have demonstrated an association of IgG and neutralizing antibody responses against the protective glycoprotein antigen with survival from lethal challenge. More recently, studies in animal models of filovirus hemorrhagic fever have established that induction of a strong filovirus-specific cytotoxic T lymphocyte (CTL) response can facilitate complete viral clearance. In this review, we describe assays used to discover CTL responses after vaccination or live filovirus infection in both animal models and human clinical trials. Unfortunately, little data regarding CTL responses have been collected from infected human survivors, primarily due to the low frequency of disease and the inability to perform these studies in the field. Advancements in assays and technologies may allow these studies to occur during future outbreaks.
The
Filoviruses are enveloped, nonsegmented, negative-stranded RNA viruses. The virion comprises a core ribonucleocapsid complex surrounded by a lipid envelope which is derived from the host cell plasma membrane. The ~19 kb noninfectious genome encodes seven structural proteins with the following gene order: 3′ leader, a nucleocapsid protein (NP), structural virion protein (VP) 35 (VP35), a matrix protein VP40, glycoprotein (GP), two additional structural proteins VP30, VP24, and the RNA-dependent RNA polymerase L protein, and 5′ trailer [
The filoviruses cause severe acute hemorrhagic fever in humans, with a high mortality rates. Disease onset is sudden, beginning with fever, malaise, chills, loss of appetite, muscle aches, and headache. These may be followed by abdominal pain, nausea, vomiting, cough, sore throat, arthralgia, diarrhea, and hemorrhage, with death occurring from shock. A maculopapular rash often develops 5 to 7 days into the illness. The mortality observed in outbreaks has ranged from 25% to 90% [
Outbreaks of filovirus infection cannot be predicted despite growing evidence that bats are among, and perhaps principle among, the natural reservoirs and/or vector(s) [
There are several promising vaccine candidates that have demonstrated immunogenicity and efficacy in animal models of disease. These platforms include the Venezuelan equine encephalitis (VEE) virus-like replicon (VRP), adenovirus 5 (Ad5), vesicular stomatitis virus-(VSV-) based vaccines, and virus-like particles (VLPs) [
Currently, there are no medical interventions approved for the treatment of filovirus infections in humans and current standard of care is supportive medical treatment (e.g., fluid replacement, transfusions, antibiotics for prevention of secondary infections) [
In this review, we have summarized the findings of recent investigations on cellular responses against EBOV and their importance in survival from lethal filovirus challenge. Understanding the protective immune responses and immunopathology induced by these viruses may be critical to the advancement of the filovirus vaccine platforms and potentially to postexposure treatment strategies for filovirus-infected individuals.
The innate immune system is the cornerstone for recognizing and effectively eliminating viral infections; rapid detection of the microbe and subsequent activation of the host innate immune response is key for developing effective adaptive immunity to invading pathogens. Antigen-presenting cells including monocytes, macrophages, and dendritic cells (DCs) are central in both activation of innate immunity and initiation of adaptive immunity. Antigen-presenting cells drive immune responses by inducing cytokines and chemokines; antigen presentation; interactions with B, T, and NK cells; and direct cytotoxic activity against target cells [
First, survivors of filovirus infection have an early and short-lived rise in serum chemokines, indicative of innate immune system induction [
Second, filoviruses evade the immune system by preventing the maturation of DC, the cornerstone of innate and adaptive immunity [
Third, activation and maintenance of natural killer (NK) cells appears to be vital to protection against lethal filovirus infection [
Subversion of innate immunity combined with a lag in activation of adaptive immune responses likely results in uncontrolled, disseminated, filovirus infection [
Infected individuals who succumb to filovirus infection fail to mount a substantial cellular or humoral immune response. In non-survivors, activation of immune cells and secretion of cytokines and chemokines are detected early in infection; however, these early cellular responses appear to be attenuated and are not detectable at the time of death while the levels of proinflammatory cytokines and chemokines reach enormous levels before a fatal outcome. Fatal cases of filovirus hemorrhagic fever are associated by a marked lack of detectable adaptive immunity. After onset of symptoms, a massive loss of CD4+ and CD8+ T cells occurs. In fatal cases of disease, gross numbers of CD4+ and CD8+ T cells are greatly reduced in non-survivors as compared to survivor (6–10% versus 20–40%, resp. [
In EBOV survivors, the early and apparently regulated inflammatory response is quickly followed by a detectable T cell response with an increase in markers suggesting the activation of cytotoxic T cells (CTL) [
Mechanistic studies regarding the role of B and T cells are difficult in nonhuman primate models; therefore, a number of studies examining the role of B and T cells in protection from lethal filovirus disease have been conducted using a mouse model of EBOV (requiring a mouse-adapted strain for lethal disease [
Humoral responses have long been deemed important for protective immunity against viral infections and, indeed, all those vaccines shown to provide 100% protection from lethal challenge in NHP have demonstrated the ability to drive filovirus-specific IgG [
A great deal of effort has focused on the passive transfer of antibodies to achieve protection and demonstrate a definitive requirement for antibodies in mediating protection from filovirus infection. Passive transfer of serum containing antibodies or purified IgG specific to EBOV or MARV can provide protection in rodent models of disease [
After successful protection after immunotherapy,
A critical role in vaccine-induced protection was identified for T cells by VLP vaccination of
ZEBOV protein sequences recognized by murine CD8+ T cells. The table is adapted and expanded from [
ZEBOV protein | Epitope sequence | Amino acid position | Restriction | Protectivea | Reference |
---|---|---|---|---|---|
Glycoprotein | VSTGTGPGAGDFAFHK | 141–155 | H-2d | Yes | [ |
LYDRLASTVI | 161–169 | H-2d | NT | [ | |
EYLFEVDNL | 231–239 | H-2d | NT | [ | |
WIPYFGPAAEGIYTE | 531–545 | H-2b | No | [ | |
TELRTFSI | 577–584 | H-2k | NT | [ | |
Nucleoprotein | VYQVNNLEEIC | 44–52 | H-2b | Yes | [ |
GQFLFASL | 148–156 | H-2b | Yes | [ | |
FLSFASLFL | 150–159 | HLA-A2.1 | NT | [ | |
RLMRTNFLI | 202–210 | HLA-A2.1 | NT | [ | |
SFKAALSSLA | 279–287 | H-2d | Yes | [ | |
FQQTNAMVT | 388–396 | H-2b | NT | [ | |
KLTEAITAA | 404–412 | HLA-A2.1 | NT | [ | |
DAVLYYHMM | 663–671 | H-2b | Yes | [ | |
VP24 | KFINKLDALH | 159–168 | H-2d | Yes | |
NYNGLLSSI | 171–179 | H-2d | Yes | [ | |
PGPAKFSLL | 214–222 | H-2d | Yes | ||
VP30 | KFSKSQLSLLCETHLR | 181–196 | H-2d | Yes | |
H-2b | Yes | ||||
DLQSLIMFITAFLNI | 231–245 | H-2d | Yes | [ | |
H-2b | Yes | ||||
VP35 | CDIENNPGL | 45–53 | H-2b | Yes | |
MVAKYDHL | 138–145 | H-2b | Yes | ||
TVPQSVREAFNNL | 190–202 | H-2d | Yes | [ | |
RNIMYDHL | 225–323 | H-2b | Yes | ||
PGFGTAFHQLVQVICK | 233–248 | H-2d | Yes | ||
VP40 | LRIGNQAFLQEFVLPP | 150–165 | H-2b | Yes | [ |
AFLQEFVLPPVQLPQ | 160–175 | H-2d | Yes | [ | |
YFTFDLTALK | 171–180 | H-2d | Yes | [ | |
TESPEKIQAI | 232–241 | H-2d | Yes | [ |
aProtection from lethal challenge demonstrated by either peptide vaccination or by adoptive transfer experiments (Yes, >50% protection observed).
NT: Not tested.
MARV protein sequences recognized by murine H-2d CD8+ T cells. Summary of data is from [
MARV protein | 15-mer peptide sequence | Minimal peptidea | Amino acid position | Adoptive transfer protectionb |
---|---|---|---|---|
Glycoprotein | FLISLILIQGTKNLP | ILIQGTKNL | 11–19 | 50 |
ILIQGTKNLPILEIA | QGTKNLPIL | 14–22 | 20 | |
TCYNISVTDPSGKSL | VTDPSGKSL | 97–105 | NT | |
SGKSLLLDPPTNIRD | LLLDPPTNI | 105–113 | 0 | |
SPPPTPSSTAQHLVY | TPSSTAQHL | 420–428 | 0 | |
GILLLLSIAVLIALS | LLLSIAVLI | 659–667 | 100 | |
LSIAVLIALSCICRI | LSIAVLIAL | 661–669 | 20 | |
LIALSCICRIFTKYI | IALSCICRI | 667–675 | 40 | |
Nucleoprotein | AINSGIDLGDLLEGG | NSGIDLGDL | 43–51 | 80 |
KFNTSPVAKYLRDAG | NTSPVAKYL | 73–81 | 20 | |
EPHYSPLILALKTLE | HYSPLILAL | 108–116 | 10 | |
VP40 | QHKNPNNGPLLAISG | KNPNNGPLL | 218–226 | 40 |
a9 mer peptide sequence derived from HLA binding predictions.
bPercentage of animals protected from lethal challenge with mouse-adapted Marburg virus after adoptive transfer of CD8 lymphocytes specific for the 9-mer peptide sequences.
NT: not tested.
Studies have also demonstrated a less important role for CD4+ T cells in protection by filovirus vaccines [
The discovery and identification of virus-specific CTL epitopes have primarily been based on computer algorithms or the use of overlapping synthetic peptides of the viral proteins. The protective capacity of the CTL virus-specific epitopes has been demonstrated in passive transfer studies, vaccination, and in the analysis of postchallenge immune responses in mice [
To date, the use of tetramer and similar technologies offers the ability to analyze the CD8+ T cell virus epitope-specific frequency, phenotypes, and functional abilities without
Nonlethal infection or vaccination generates complex responses in the host, which include innate and adaptive arms of the immune system. For filovirus vaccines, successful vaccination has generated a protective immune response protecting animals from lethal virus challenges. While both antibody responses and cellular responses have been monitored in past studies, the analysis of these responses was secondary to the objective of assessing the vaccine candidate’s efficacy in the animal model. While we are unsure of the contribution of B and T cell responses during vaccination, the collective data suggest that both are necessary for viral clearance [
After successful vaccination, adaptive responses to the glycoprotein encompass both humoral and cellular immune responses [
Without assessing ELISA antibody titers, examining T cell responses after vaccination has been less predictive when used in out-bred populations, specifically macaques. This is likely due to the complexity of measuring cellular immunity following vaccination. Measuring cellular immune responses is difficult since the assays are laborious, tedious, difficult to replicate, and expensive. Likewise, immunogen quality (peptides or protein) used in the assays is paramount and currently poorly understood for filoviruses. Unlike most humoral assays, cellular assays are typically performed
To date, LFA and chromium release assays have been limited to evaluating immune response in rodents and primarily in mice. LFAs are unique in that they allow the assessment of both CD4+ and CD8+ or when fractionated CD4+ or CD8+ T cell proliferation. In the presence of radioactive (i.e., thymidine) or nonradioactive molecules, which are incorporated into DNA during proliferation, a stimulation index can be determined for a protein or peptide antigen. Similarly, the chromium release assay is a means to measure specific lysis of target cells expressing antigen by CD8+ T cells; however the assay has been primarily limited to studies with clearly defined MHC systems and primarily for analyzing T cell responses of vaccinated in-bred mice.
The two primary assays utilized with macaques have been ELISpot- and ICC-based assays. Both assays provide semi-quantitative analysis of the total T lymphocyte responses or analysis of indicators of T cell activation such as cytokine production in
The ELISPOT assay is considered by many to be a gold standard for monitoring specific cellular immune responses, especially in humans. The assay can detect single cells secreting molecules primarily cytokines following exposure to a specific antigen [
ICC methods have been the primary method to assess cellular responses in macaques. To date, these assays have relied on three- to four-color flow cytometry. In these assays, irradiated virus, recombinant proteins, or overlapping peptides have been used to stimulate rodent or macaque PBMCs
Newer, more-sensitive methods for analysis of cellular immunity offer promise for more-detailed assessment of T cell phenotype and function. Another emerging approach to measure cellular responses is the use of multiparameter flow cytometry. The aforementioned techniques used for filoviruses reveal a cellular reactivity to virus protein(s); however the data lack critical information that can discriminate between “good” or nonprotective immunity. Thus, the issue is the relative values of the easily obtained data (magnitude of the response to antigen) and the more difficult assays which ascertain the sensitivity/specificity of the immune response. The later aspect has been difficult to assess in the past and can be costly to obtain. Assays for both magnitude and quality of the response have been best described by Roederer et al. for the development of multicolor flow cytometry methods to assess T cell frequency and, more importantly, T cell quality in memory and effector cells [
Unfortunately, our understanding of these various functional cell subsets is not clear. Therefore, a “good” vaccine cellular response can only be determined empirically using animal model(s) and then translated to human-use. The assays developed by Roederer et al. have defined a method that allows sampling of heterogeneous populations of antigen-specific cells and their relative functional capacities. Multiparametric flow cytometry methods allow for simultaneous T analysis of several parameters (~four to five parameters and growing [
In initial work by Sullivan et al., they suggest that similar profiles exist for protective responses to filoviruses [
Due to the nature of filovirus hemorrhagic fever, efficacy trials in humans are not ethical. Primary objectives of clinical testing efforts for filovirus vaccines will be the safety and immunogenicity of different dosage levels of the candidate filovirus vaccine [
In the first clinical trial of a filovirus vaccine, 27 subjects were vaccinated in a dose-escalation study having four tiers of 0, 2, 4, or 8 mg of DNA given three times at >21 day intervals [
In the second clinical trial, a replication-defective adenovirus serotype 5 (Ad5) vaccine expressing the ZEBOV and SEBOV GPs was tested in 31 human volunteers with 23 receiving the Ad5 vaccine [
The reports for the first two clinical trials of filovirus vaccines show the ability to successful induce filovirus-specific humoral and cellular responses [
Because of their lethality and other key properties that characterize the filoviruses as a bioweapon threat, a focused effort to develop medical countermeasures has been directed against EBOV and MARV infections [
The authors thank Sabrina Stronsky for reference assistance and support. Dr. Olinger is supported by the U.S. Army and the Defense Threat Reduction Agency funding to develop assays for assessing protective responses following virus infection. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army.