Genus Ebolavirus, family Filoviridae has five viral species [180] of which four species Zaire Ebolavirus (ZEBOV), Sudan Ebolavirus (SEBOV), Taï Forest Ebolavirus formerly Côte d’Ivoire Ebolavirus (TAFV) and Bundibugyo Ebolavirus (BEBOV) cause the Ebola hemor- rhagic fever in humans and other primates. In large outbreaks due to ZEBOV, SEBOV and BEBOV, case fatalities have ranged from 32 − 90% [101, 51, 107, 167, 111, 120, 365, 215]. The fifth species Reston Ebola virus (REBOV) causes severe disease in non-human primates [239, 136, 110] and can infect swine [20]. Figure 2.9 shows the number of fatalities caused by Ebola Zaire, Sudan and Bundibugyo between 1976 and 2014. As we can see from the figure, the number of fatalities in the latest outbreak (2014) has been by far the largest in the last four decades. And we can also see that Zaire Ebolavirus strains were the causative agents in majority of the outbreaks.
Figure 2.9: Number of death by classification (in order they are Ebola Zaire, Sudan, Bundibu- gyo) by year and ordered from most impacted to least impacted year. Due to the severity of the latest outbreak in 2014, it has been shown separately. Source: http://www.who.int/ csr/disease/Ebola/situation-reports/archive/en/.
Results of earlier attempts to develop protective EBOV vaccines in guinea pigs and non- human primates have been inconsistent [212, 237]. No human disease has been associated with REBOV species even though human infection with the species has been documented [20, 240, 109]. Even though the severe pathogenic disease caused by the different species in humans and non-human primates are very similar, there is 30 − 45% nucleotide differences in their genomic sequences [347, 155]. Figure 2.10 shows a neighbour joining tree of 60 Zaire Ebolavirusgenomes. We can see from the figure that the viral stains that caused outbreaks in certain periods of time are genetically quite different from those that caused an outbreak at an- other time. This again implies that the strains accumulate mutations and/or gather genes from other strains before they cause an outbreak again. Due to these extreme (and not yet well under- stood) genetic variations and the socio-economic impact of Ebola, it is another good candidate for in silico vaccine prediction.
The Ebola virus (EBOV) contains a linear, single-stranded, negative-sense RNA genome. It encodes seven structural proteins and a non-structural protein. Structural proteins are nucleo-
Figure 2.10: A neighbour joining tree of 60 Zaire Ebolavirus genomes. The tree shows a model rooted by a clade of the 1976 outbreaks. The asterisk identifies a clade of 1994-1996 isolates from Gabon. All sequences were obtained from NCBI http://www.ncbi.nlm. nih.gov/gquery/gquery.fcgifor multiple alignments and the tree was generated by the ClustalW algorithm in Mega version 7.0. The bootstrap values for 1000 replicates are given at each node.
protein (NP), polymerase cofactor (VP35), (VP40), glycoprotein (GP), transcription activator (VP30), VP24, and RNA polymerase (L). It is an enveloped virus with a lipid bilayer and with anchored GP forming the viral spikes on the surface [190]. Figure 2.11 shows the arrangement of the seven structural proteins in the Ebola genome.
Figure 2.11: Arrangement of the seven structural proteins in the Ebola genome. Structural Proteins Involved In Viral Antigenicity:
Most studies of EBOV epitopes have been on mice [370]. Despite the encouraging results of the these studies aimed at developing candidate EBOV vaccines, none of the strategies that protected mice or guinea pigs from lethal infection with this virus have been shown to provide similar protection in nonhuman primates [118]. Sullivan et al. [333] reported the development of an accelerated vaccine for Ebola where a single immunization with adenoviral (ADV) vectors encoding the Ebola GP and NP was able to induce highly effective protection against lethal challenge in non-human primates. Warfield et al. [370] described the production of virus-like particles (VLPs) of Ebola by coexpressing GP and VP40 proteins. Mice vaccinated with Ebola VLPs developed virus-specific antibodies including neutralizing antibodies and these mice were 100% protected against lethal field virus strain. While these investigations provide insights into the genetic markers of EBOV that stimulate murine effector T cell activation in the context of specific MHC4 haplotypes, the information derived from such studies may not be applicable to human MHC determinants. Sundar, et al. used a combination of computational prediction methods together with in vitro and in vivo assays to identify three human HLA-A2.1-restricted CTL (Cytotoxic T lymphocytes)5 epitopes for the nucleoprotein of EBOV. They were able to predict five MHC class I-binding NP peptides within the structural proteins of EBOV, namely, FLS, RLM, KLT, AMN and YQG (first three letters of the peptides that were nine amino acids long) [337]. These EBOV-NP peptides induced CTL responses in HLA-A2-transgenic mice and hence these could be useful in the development of protective immunogens for this hemorrhagic virus. In the absence of any confirmed epitopes for vaccine production in Ebola, we look at these five epitopes, to check for any correlates with antigenicity.
4The major histocompatibility complex (MHC) is a set of cell surface proteins essential for acquired immune
system to recognize foreign molecules in vertebrates, which in turn determines histocompatibility. The main function of MHC molecules is to bind to peptide fragments derived from pathogens and display them on the cell surface for recognition by the appropriate T-cells. [150]
5Cytotoxic T lymphocytes are lymphocytes that kill other “target” cells. Targets may be virus-infected cells,
cells infected with intracellular bacterial or protozoal parasites, allografts such as transplanted kidney, heart, lungs, etc. and cancer cells.