To  What  Extent  Is  It  Possible  To  Develop  A  Prophylactic  for  Ebola?  James  Baxter  

ABSTRACT  Ebola  is  a  virus  with  the  capability  of  inducing  a  lethal  viral  haemorrhagic  fever,  causing  massive  internal  bleeding  in  humans   and   non-­‐human   primates.   It   was   first   encountered   in   1976   in   the   Democratic   Republic   of   the   Congo  (formerly  Zaire)  in  which  the  fatality  rate  was  88%.  Further  outbreaks  have  occurred  since  with  fatality  rates  ranging  from  25%-­‐100%,  yet   there   is  currently  no  approved  course  of   treatment  nor   licensed  vaccine.  The  report  contains  detail   on   the   pathogenesis   of   Ebola   (EBOV)   before   pursuing  ways   in  which   it   could   be   prevented.   Using   research  carried   out   by   the   London   School   of   Hygiene   and   Tropical   Medicine,   the   American   military   and   Médecins   Sans  Frontières,  it  was  found  that  whilst  several  prototype  vaccines  are  being  researched,  it  is  not  expected  that  any  will  be  ready  for  large  scale  administration  within  the  next  5  years.        

The  first  recorded  outbreaks  of  Ebola  occurred  simultaneously  in  Zaire,  now  the  Democratic  Republic  of  the  Congo,  and  Sudan,  now  South  Sudan,  in  1976.  The  emergence  in  the  towns  of  Yambuku,  Zaire  and  Nzara,  Sudan  killed  280  of  318  and  151  of  284  cases  respectively.  Named  after  a  river  near  Yambuku,  Ebola  returned  again  the  following  year,  killing  one,  and  again  to  the  Democratic  Republic  of  the  Congo  in  1995  in  Kikwit  –  a  town  of  population  in  excess  of  500,000   with   a   mortality   rate   equally   as   shocking   the   first   outbreaks   (Fleck,   2011).   More   recently   it   has   been  reported  that  there  is  currently  an  on-­‐going  outbreak  in  Guinea,  West  Africa  (BBC,  2014).  It  was  the  persistent  90%  death  rate  of  the  major  outbreaks  of  Ebola  (WHO,  2012)  that  brought  about  the  initial  interest  in  Ebola,  primarily  to  look  at  how  the  condition  was  treated  and,  as  a  keen  traveller  and  a  prospective  medical  student,  whether  vaccination  was   a   viable   method   of   protection   against   the   virus.   Upon   discovering   that   there   was   at   present   no   vaccination  programme  for  any  strain  of  Ebola,  it  was  decided  to  research  more  about  the  pathogenesis  of  the  virus  and  examine  the   current   efforts  made  by   international  medical   organisations   to   develop   effective   treatments   and   a   vaccine.     In  order  to  establish  a  common  ground  of  knowledge,  the  report  opens  with  an  introduction  to  fundamental  virology.  

   

An  Introduction  to  Viruses      Viruses  are  unlike  any  other  organism  on  earth:  their  origins  are  unknown  and  there  is  still  significant  debate  as  to  whether  they  are  even   ‘alive.’  Yet  these  less  than  microscopic  organisms  pose  some  of  the  biggest  health  threats  to  humans   including:  Human   Immunodeficiency  Virus   (HIV),   SARS   coronavirus   and  Rabies.   The   latter   stimulated   the  discovery  of  viruses  when  Louis  Pasteur  was  unable  to  find  a  pathogen  responsible  for  the  onset  of  the  disease  and  proposed   that   there  may  be  an   instigator   too  small   to  be  viewed  under  a   light  microscope  (Wikipedia,  2013).  At  a  similar  time,  the  Russian  Botanist  Dmitri  Ivanovsky  showed  that  after  passing  an  infectious  sample  of  Tobacco  plants,  suffering  from  Mosaic  Disease,  through  a  filter  with  pores  able  to  prevent  the  flow  of  bacteria,  the  solution  retained  its   potential   to   infect   (Soper,   1997a).   Dutch  microbiologist,  Martinus   Beijerinck   repeated   this   experiment,   using   a  diluted  sample  that  regained  its  original  concentration  after  the  infection  of  a  second  plant,  demonstrating  that  the  infectious  agent  could  reproduce,  defining  it  as  a  living  organism.  In  1903  the  director  of  the  Pasteur  Institute,  Pierre  Roux,  defined  the  characteristics  of  ‘filterable  viruses,’  into  3  properties:    

• Filterable:  Organisms  small  enough  to  pass  through  a  Chamerberland  Filter  • Invisible:  Organisms  not  possible  to  be  viewed  under  a  light  microscope  • Non  –  cultivable:  Organisms  that  cannot  be  grown  on  a  bacterial  culture  plate.  

 Throughout   the   early   20th   Century,   more   was   discovered   about   the   morphology   of   viruses,   but   very   little   was  appreciated  about  their  mechanisms.  In  an  attempt  to  understand  this,  chicken  eggs  were  infected  with  a  pathogen,  after  which  a  plaque  upon  the  embryo  membrane  developed.  It  was  first  observed  on  the  inoculated  membranes  of  chick  embryos  that  viruses  multiplied  inside  the  cells,  often  killing  the  infected  cell  (Crawford,  2000a).    The  Invention  of  the  electron  microscope  in  1938  revealed  the  identities  of  such  pathogens:    

Figure  1:  Electron  Micrograph  Images  of  Some  Viruses  (Stannard,  1995)  

   Influenza  Virus   Rotavirus  (common  cause  of  diarrhoea)  

   

Hepatitis  B  Virus   Adenovirus  (causes  conjunctivitis)    

Whilst  viruses  could  now  be  seen,  many  questions  still  remained  over  their  construction.  Experiments  whereby  the  Tobacco  Mosaic  Virus  was  crystalized  suggested  that  it  consisted  of  pure  protein.  However  as  understanding  of  the  biological   significance   of   chromosomes   and   DNA   grew,   it   became   apparent   that   viruses   also   contained   genetic  material.   This   discovery   distinguished   the   differences   between   bacteria   and   viruses   in   a   way   that   was   finally  unequivocal  (Crawford,  2000b).      

Figure  2:  Comparing  the  size  of  a  virus  (Openstax,  2013)  

   Virus  Anatomy    Viruses  are  the  simplest  known  organisms  in  existence,  consisting  of  solely  a  protein  shell  and  containing  a  genetic  code.  Virions  are  mature  viruses  and  range  from  20nm  –  400nm  in  size,  a  minor  fraction  the  size  of  any  cell  (Martin  &  Hine,   2000a).   As   there   are   no   provisions   for   respiration,   viruses   cannot   perform   any  metabolic   reactions   such   as  protein  synthesis  or  reproduction,  which  leads  to  significant  debate  as  to  whether  they  are  deemed  to  be  truly  living  (Crawford,  2000b).  The  protein  ‘capsid’  that  encases  the  inner  core  of  nucleic  acid  consists  of  many  identical  protein  subunits  arranged  into  an  isometric  3-­‐D  shape.  This  is  typically  a  20-­‐sided  icosahedron  or  a  thin  spiral  tube.  In  some  cases  a  second  capsid  called  the  viral  envelope,  is  also  present  on  the  exterior  of  the  capsid.  This  is  formed  from  the  surface  membrane  of  the  host  cell  and  can  be  used  to  evade  the  host  organism’s  immune  response  (Smith,  1995a).        The   genetic   information   in   the   interior   of   the   virus  may   be   stored   either   as   deoxyribonucleic   acid   (DNA),   where  typically   a   double   helix   structure   consisting   of   two   polynucleotide   strands   is   present,   or   ribonucleic   acid   (RNA),  which  is  a  single-­‐stranded  polynucleotide  (Smith  1995a).  In  viruses,  single  stranded  DNA  (ssDNA)  is  more  common  than  its  double-­‐stranded  (dsDNA)  counterpart,  as  found  in  more  complex  organisms.  Double-­‐stranded  RNA  (dsRNA)  viruses  are  also  an  anomaly  to  the  standard  form  of  single  stranded  RNA  (ssRNA).    The  nucleic  acids  can  be  further  categorised  by  a  characteristic  known  as  ‘sense.’  This  is  defined  as  ‘the  concept  used  to  compare  the  polarity  of  nucleic  molecules,  such  as  DNA  or  RNA,  to  other  nucleic  molecules’  (Wikipedia,  2014).  In  DNA  in   general,   there   are   two   antiparallel   strands   of   nucleotides   that   are   bonded   together   by   inter-­‐molecular   forces  between  the  complementary  nitrogenous  bases.  One  of  these  strands,  which  has  the  same  base  code  as  the  messenger  RNA  (mRNA)  involved  in  protein  synthesis,  is  labelled  as  ‘positive  sense,’  whilst  the  complimentary  strand  of  DNA  is  called  ‘negative  sense.’  The  gene  on  the  positive  sense  strand  is  the  same  permutation  that  is  seen  on  mRNA  during  the   translation  phase   of   protein   synthesis,   but   is   not   actually   used  during   transcription.  As   the  mRNA  must   be   an  exact  copy  of  the  coding  DNA  positive  sense  strand,  the  complimentary  pairs  of  the  DNA  negative  sense  strand  (also  called  the  transcribed  strand)  are  used  in  transcription  so  that  an  identical  mRNA  strand  of  the  DNA  positive  sense  strand   is   formed.   The   same  principle   exists   for  RNA,  whereby   the   negative   strand   is   complimentary   to   the  mRNA  whereas  the  positive  strand  is  a  copy.      This  can  be  used  to  classify  viruses,  because  if  a  positive  sense  nucleic  acid  is  present,  once  it  enters  the  host  cell  it  may   undergo   direct   replication   and   then   translation,   utilising   the   host   cell’s   ribosomes.   For   nucleic   acid   negative  sense  strands  however,  RNA-­‐dependent  RNA  polymerase  or  DNA-­‐dependent  DNA  polymerase  enzymes  are  required  to   replicate   the   respective   nucleic   acid   and   convert   the   produced   genetic  material   into   the   positive   sense   strand.  Ambisense  viruses  also  exist   in  which  the  nucleic  acid  has  a  double  strand  intermediate  preceding  transcription.  In  this  instance,  transcription  could  occur  on  either  strand  (Wikipedia  2014).  Retroviruses  have  a  different  way  again  of  storing   their  genome  and  store  all   the  genetic   information   for   the  virus  as  mRNA.  Upon  entering   the  host   cell,   the  

mRNA   is   turned   into   either  RNA  or  DNA  using   the   virus’   reverse   transcriptase   enzyme   (Martin  &  Hine,   2000b).  A  useful  method  of  classifying  viruses  thus  transpires:  (Baltimore,  1971):    

I   Double  strand  DNA  viruses  II   Single  strand  DNA  viruses  III   Double  strand  RNA  viruses  IV   Positive  sense  single  strand  RNA  viruses  V   Negative  sense  single  strand  RNA  viruses  VI   RNA  retroviruses  VII   DNA  retroviruses  

   The  Virus  Life  Cycle    The  virus  life  cycle  is  split  into  two  distinct  stages:  a  dormant  stage  in  which  the  mature  virion  is  effectively  inert  and  a  post-­‐infection  stage  in  which  reproduction  occurs  within  the  host  cell.  There  are  two  types  of  virus  lifecycle,  which  greatly  affect  the  nature  of  the  virus.  In  lytic  cycles,  the  virus  infects  the  host  cell  with  its  nucleic  acid,  which  is  used  by   the   cellular   machinery   present   in   the   cytoplasm   to   synthesise   proteins   and   more   nucleic   acid   to   create   new,  progeny  viruses  that   then  burst   from  the  cell  during  cell   lysis.   In  a   lysogenic  cycle,   the  nucleic  acid   is   incorporated  into  the  host  cell’s  DNA  and  is  therefore  copied  with  the  host  DNA  during  cell  division.   It  will  remain  dormant   in  a  state  known  as  prophage  after  which  it  may  be  activated  and  cause  cell  death  with  the  same  mechanism  as  in  the  lytic  stage  (Soper,  1997b).  A  cell  is  said  to  have  been  activated  when  it  host  to  a  viral  pathogen.      This  type  of  cell  death  is  known  as  necrosis,  whereby  death  is  brought  about  by  disease,  physical  or  chemical  injury,  or  a  reduction  of  the  blood  supply  (Martin,  2002a).  This  is  contrary  to  programmed  natural  cell  death  where  cells  die  as   part   of   normal   development,   maintenance   and   renewal   of   cells,   tissues   and   organs.   This   process   is   coined  apoptosis  (Martin,  2002b)    

Figure  3:  The  virus  lifecycle  (as  modelled  by  the  Influenza  virus)  (OpenStax,  2013)  

   

 Ebola  

 Ebloa  Virus  (EBOV)  is  a  threadlike,  filamentous  organism  belonging  to  the  Filoviridae  family  of  viruses  (Roddy  et  al.,  2012).  The  nucleic  acid  within  the  tubular  protein  capsid  is  negative  sense,  single  stranded  RNA  (Baltimore  class  V)  and  consists  of  a  genome  of  near  19,000  nucleotides  (Rutgers,  2011).  By  comparison,  the  human  genome  is  believed  to  contain  in  the  region  of  3,200,000,000  nucleotides  (Brown,  2002).  There  are  currently  5  known  Ebola  species,  each  of  which  has  it’s  own  abbreviation  (ICTV,  2012):    

• Zaire  ebolavirus  (ZEBOV)  • Sudan  ebolavirus  (SEBOV)  • Reston  ebolavirus  (REBOV)  • Côte  d’Ivoire  ebolavirus  (CIEBOV)  • Bundibugyo  ebolavirus  (BEBOV)  

 The   ZEBOV,   SEBOV   and   BEBOV   strains   of   Ebola   are   known   to   induce   a   viral   haemorrhagic   fever   (VHF),   which   is  similar   to   the   other   virus   of   the   filoviridae   family,  Marburg   virus   (WHO,   2012).   Both   have   very   similar   attributes  whereby   they   cause   their   respective   VHFs   –   Ebola   haemorrhagic   fever   (EHF)   and   Marburg   haemorrhagic   fever  (MHF),  and  both  originate  from  sub-­‐Saharan  Africa.  There  have  been  24  recorded  outbreaks  of  EHF  in  humans,  but  only  11  in  MHF  (Roddy  et  al.,  2012),  although  given  the  remote  location  and  relatively  primordial  medical  resources,  it  is  likely  that  both  these  figures  are  underestimates  of  the  actual  number  more  recent  outbreaks.  Both  EHF  and  MHF  are  described  as  ‘severe  acute  viral  illness[es]  often  with  the  loss  of  blood,’  by  World  Health  Organisation  (Fleck,  2011).    Transmission    Ebola  Virus  is  considered  to  be  zoonotic  because  it  must  be  transmitted  from  a  non-­‐human  vertebrate  to  a  human  in  order  to  start  an  epidemic.  A  zoonotic  virus  is  said  to  be  capable  of  infecting  both  non-­‐human  vertebrates  as  well  as  humans.  Once  a  member  of  the  human  population  has  been  infected,  the  virus  is  quickly  spread  through  person-­‐to-­‐person   contact,   which   in   the   case   of   Ebola   Virus   involves   the   contact   of   bodily   secretions   and   fluids.   This   is  exacerbated   in   a   healthcare   environment  where   used   needles   and   unsterilized   equipment   can   harbour   pathogens  (NIAID,   2010).   It   has   also   been   noted   that   cultural   burials   have   been   linked   to   the   transmission   of   Ebola,   where  mourners  have  direct  contact  with  the  deceased  (WHO,  2012).      Pathogenesis    Once  EBOV  has  entered  the  body,  it  is  swift  to  act  and  death  is  normally  brought  about  in  an  incubation  period  of  less  than  21  days  (Fleck,  2011).  The  early  symptoms  of  the  haemorrhagic  fever  include  weakness,  headache,  vomiting  and  rash,  all  of  which  could  lead  to  differential  diagnosis  (WHO,  2012).  This  is  where  several  diseases  share  the  same  or  very  similar  symptoms  (Martin,  2002c)  and  as  such  several  diagnoses  could  be  made.  This  could  mean  that  it  is  not  beyond  reasonable  doubt  that  the  small  minority  of  survivors  of  earlier  Ebola  outbreaks  were  in  fact  misdiagnosed.  The  way  in  which  EBOV  brings  about  EHF  is  not  entirely  understood  by  the  scientific  community  but  the  consensus  is  that  death  is  a  result  of  hypovolemic  shock  as  a  result  of  mass  haemorrhaging  or/and  organ  failure  (Rutgers,  2011).    Whereas  most  viruses  target  a  specific  type  of  cell  for  infection,  there  is  evidence  that  in  the  case  of  EBOV,  the  initial  site   of   viral   replication   is   within   dendritic   cells   and   macrophages,   before   then   proceeding   to   endothelial   cells  (Geisbert  et  al.,  2003).  The  ability  to  infect  a  wide  range  of  primate  cells  is  considered  to  be  a  result  of  the  way  two  identified   glycoproteins   on   the   virus   capsid   interact   with   target   molecules   on   the   cell   surface,   in   particular   the  cholesterol  transporter  Niemann-­‐Pick  C1  (Carette  et  al.,  2011)(Bray  &  Geisbert,  2004).    The  importance  of  the  infection  of  dendritic  cells  should  not  be  underestimated,  as  they  are  essential  to  the  immune  response  of   the  mammalian  body.  Located  within   the   skin,   lungs  and  gastrointestinal   tract,   these   cells   react   in   the  presence  of   foreign  antigens,   transferring  them  to   lymph  nodes  where   lymphocytes  are   located.  These  white  blood  cells  produce  antibodies,  which  are  essential  in  the  immune  response  to  infection  (Banchereau  &  Steinman,  1998).      The  death  of  dendritic  cells  due  to  necrosis  prevents  the  passing  of  viral  antigens  to  the  lymphocytes,  which  results  in  the  failure  to  produce  antibodies  against  the  virus.  Likewise  Natural  Killer  Cells,  which  engulf  activated  cells,  do  not  receive  the  antigen  signal  and  therefore  do  not  act  upon  the  viral   infection  and  soon  become  depleted  (Baize  et  al.,  1999).  Whilst  the  virus  does  not  infect  lymphocytes  themselves,  they  are  lost  in  high  concentrations  during  the  illness  through  apoptosis,  inducing  a  condition  known  as  lymphopenia,  which  is  synonymous  with  symptoms  of  sepsis  and  septic  shock  (Bray  &  Mahanty,  2003).  The  second  site  of  viral  infection  is  in  macrophages,  which  are  white  blood  cells  (leucocytes)  that  remove  foreign  pathogens  through  phagocytosis  whereby  the  cell  engulfs  the  target.  As  these  cells  are  not  fixed  to  lymph  nodes,  they  become  the  vectors  for  the  spread  of  the  virus  throughout  the  body.  The  infected  macrophages   release   proinflammatory   cytokines,   chemokines   and   nitric   oxide   (NO)  which   all   act   as   inter-­‐cellular  communicators  (Bausch  et  al.,  2008).  It  is  thought  that  this  response  is  brought  about  by  the  binding  of  viral  products  to   pattern   recognition  molecules  within   the  macrophage.  The   released  proteins   attract  macrophages   to   the   site   of  infection  and  at  the  same  time  mobilise  immature  neutrophils  from  the  bone  marrow  and  blood  vessel  walls  (Bray  &  Geisbert,  2005).      Neutrophils  are  a  specific  type  of  white  blood  cell  called  granulocytes  and  are  capable  of  digesting  foreign  pathogens  (Martin,   2002d),   although   it   is   assumed   that   these   are   summoned   to   ensure   the   maximum   dispersal   of   the   viral  infection   around   the   body   is   achieved.   The   chemokines   and  NO   also   remove   inflammatory   cells   by   increasing   the  permeability  of  the  endothelium  through  a  process  known  as  vasodilatation.  In  effect,  the  virus  hijacks  a  process  that  on  a  small  scale  is  therapeutic,  and  implements  it  simultaneously  throughout  the  patient  as  the  activated  leucocytes  disperse,  causing  severe   internal  haemorrhaging  (Bray  &  Geisbert,  2005).  Another  common  observation  during  the  

autopsy  of  non-­‐human  primate  models  of  EHF  was  that  there  was  ‘widespread  deposition  of  fibrin  in  the  circulation,’  (Geisbert  et  al.,  2003).  This  has  since  been  attributed  to  disseminated  intravascular  coagulation  (DIC)  and  is  linked  to  the  interaction  of  EBOV  with  macrophages.      

Figure  4:  The  Pathogenesis  of  EBOV  (Feldmann  &  Geisbert,  2011)  

   When  infected  these  cells  synthesise  cell-­‐surface  tissue  factor  (TF)  which  interferes  with  clotting  factors  VIIa  and  X,  resulting   in   the   deposition   of   fibrin   on   the   surface   if   infected   cells   (Bray   &   Geisbert,   2005).   Factors   are   naturally  occurring  proteins,  which  are  essential   in  the  coagulation  of  blood.   In  addition  to  the   increased  permeability  of  the  endothelium  as  a  result  of  the  emission  of  chemokines  and  NO,  EBOV  also  targets  endothelial  cells  in  the  later  stages  of   infection.   Once   activated,   these   cells   have   a   limited   future   before   they   undergo   cytolysis.   This   is   the   process  through   which   a   cell   loses   its   structural   integrity   as   a   result   of   the   virus   leaving   the   cell   through   the   surface  membrane.   This   destroys   the   endothelium   of   blood   vessels   and   the   gastrointestinal   tract,   causing   massive  haemorrhage  and  systemic  organ  failure  (Bausch  et  al.  2008).      

 Prognosis    If   a   positive   diagnosis   of   EBOV   is  made,   then   the   outlook   for   a   patient   is   relatively   bleak   as   there   is   currently   no  specific   therapeutic   treatment.   The   standard   practice   is   known   as   supportive   treatment   in   which   the   patient   is  administered  drugs,  both  intravenously  and  orally,  that  rehydrate  and  alleviate  symptoms  or  pain  caused  as  a  result  of  the  infection  (Roddy  et  al.,  2011).  Whilst  it  is  little  comfort  to  neither  patients  nor  their  relatives,  each  outbreak  of  EBOV   provides   significant   advances   both   in   terms   of   pharmaceutical   treatments   and   the   quality   of   treatment  delivered.        

Existing  Post-­‐Exposure  Treatments    

Post  exposure  treatments  are  under  intensive  research  and  several  lines  of  investigation  are  being  made,  looking  at  potential   weaknesses   in   the   pathogenesis   of   the   virus   and   counteracting   some   of   the   effects.   One   of   the   main  problems  of  developing  both  prophylactic  and  post  exposure  treatments  is  that  clinical  investigations  are  limited  to  in  vitro  and  in  vivo  in  non  human  primates.  In  some  cases,  the  first  in  vivo  testing  is  done  on  mice,  for  which  EBOV  must  be  altered  to  achieve  a  successful  infection.  The  result  is  that  while  a  treatment  may  be  successful  for  this  particular  strain  of  EBOV,   it  may  not  be  as  effective,   if  at  all,   for  human  EBOV  (Rutgers,  2011).  As  such  there  are  currently  no  licensed  antiviral  drugs  for  EBOV  (Bausch  et  al.,  2008).    One  direction  of  current  research  is  centred  about  the  two  glycoproteins  on  the  exterior  of  the  EBOV  capsid.  Together  these  proteins  enable  the  infection  of  cells,  binding  to  Niemann  –  Pick  C1  (NPC1)  Cholesterol  Transporters,  which  are  embedded  into  the  phospholipid  bilayer  of  the  cell  membrane.  This  is  confirmed  by  means  that  cells  which  lack  this  molecule,   such   as   those   in   NPC1   Disease   patients,   are   resistant   to   the   infection   of   EBOV   (Carrette   et   al.,   2011).  Glycoprotein   1   (GP1)   attaches   the   virion   to   the   host   cell  membrane  whereas   Glycoprotein   2   (GP2)   fuses   the   host  membrane   to   the  viral   envelope,  permitting   the  virus’  RNA   to  enter   the   cell   through  endocytosis.  Essentially,   each  glycoprotein  has  two  conformations  –  pre  and  post  attachment  (Rutgers,  2011).  This  fact  is  of  particular  importance  when  it  is  considered  that  EBOV  antibodies,  found  in  a  survivor  of  the  1995  ZEBOV  outbreak,  in  Kikwit,  Democratic  Republic  of  The  Congo  act  upon  the  tertiary  structure  of  these  proteins.  KZ52  prevents  the  change  of  conformation  from  pre  –attachment  to  post  –attachment,  therefore  removing  the  virion’s  ability  to  infect  a  cell  (Jeffrey  et  al.,  2008).      During  the  same  outbreak,  this  was  used  as  a  form  of  treatment  in  which  8  patients,  recently  diagnosed  with  EBOV,  were  administered  antibody-­‐containing  blood  transfusions  from  ‘5  convalescent  patients.’  This  was  a  success  because  out  of  the  sample,  the  fatality  rate  was  only  12.5%  (1  fatality),  which  is  significantly  lower  than  the  80%  fatality  rate  of   the   ZEBOV   Kikwit   epidemic   (Mupapa   et   al.,   1999).   Despite   this   initial   success,   there   was   significant   failure   in  laboratory   testing   succeeding   the  outbreak  on  macaques,  which   lead   to   the  dissolution  of   this   course  of   treatment  (Oswald  et  al.,  2007).      A   successor   to   this   is   the   administration   of   monoclonal   KZ52   antibodies.   These   are   clones   of   KZ52,   which   were  isolated   from   recovering   patients   of   the   Kikwit   outbreak.   Clinical   trials   of   this   treatment   in   which   3   doses   are  administered  3  days  apart,  starting  24  hours  post-­‐infection  have  shown  100%  success  rates  in  Non-­‐human  primates,  although  this  diminishes  to  50%  if   the  treatment  starts  at  48  hours  post   infection  (Qiu  et  al.,  2012).  An  alternative  pathway  for  treating  EBOV  could  be  based  on  anti-­‐apoptotic  therapies  designed  to  reduce  the  number  of  lymphocytes  lost  throughout  the  course  of  the  infection.  As  B-­‐lymphocytes  produce  antibodies  specific  to  each  pathogen,  it  would  help  to  reinstate  the  otherwise  defective  immune  system  brought  about  by  the  Ebola  virus  (Parrino  et  al.,  2007).  This  would  appear  to  be  another  promising  solution  because  the  virus  first  infects  the  immune  system  before  inducing  any  visible  effect  on  any  other  tissue  as  shown  in  the  table  below.  This  means  that  given  the  therapeutic  help,  the  immune  system  would  retain  sufficient  strength  to  counteract  the  virus,  before  the  activation  of  endothelial  cells  posed  a  life-­‐threatening  risk.      

 Figure  5:  A  table  showing  the  spread  of  Ebola  throughout  the  body  in  non-­‐human  primates  (Geisbert  et  al.,  2003)  

Animal  Number   Days  P.I.   HEV   Sin   Spl   Lam  prop  

Ren  cap  

Alv  cap  

Heart   Brach  Plex  

Brain  

CQ9877   1   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐  CQ9890   1   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐  0717CQ   1   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐  CQ9846   2   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐  0331CQ   2   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐  CQ9028   2   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐  32Q   3   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐  CQ8667   3   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐  CQ9093   3   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐  CQ8681   3   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐  28-­‐427   4   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐  CQ9887   4   -­‐   +   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐  CQ9108   4   -­‐   +   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐  CQ9095   4   +   +   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐   -­‐  28-­‐221   5   ++   ++   +   +   +   -­‐   -­‐   +   -­‐  0359CQ   5   +   ++   +   +   +   -­‐   -­‐   -­‐   -­‐  0323CQ   5   ++   ++   +   +   +   +     +   -­‐  48-­‐143   5   ++   ++   +   +   +   +   +   +   +  CQ9878   6   ++   ++   +   +   +   +   +   +   +  28-­‐332   6   ++   ++   +   ++   +   +   ++   +   ++  CQ9112   6   ++   ++   +   ++   +   +   ++   +   ++  +,  EBOV  RNA-­‐  and  antigen-­‐positive  cells  were  rarely  detected;  ++,  EBOV-­‐positive  cells  were  occasionally  detected;  +++,  EBOV  positive  cells  were  frequently  detected;  and  -­‐,  no  EBOV-­‐positive  cells  were  detected.    P.I.,  post   infection;  HEV,  high  endothelial  venules  of   lymphoid  tissues;  Sin,  hepatic  and  adrenal  cortical  sinusoids;  Spl,  endothelial  cells  in  spleen;  Lam  prop,  lamina  propria  of  tongue,  nares,  lip  larynx  and  intestines;  Ren  cap,  renal  capillaries;  Alv  cap,  alveolar  capillaries;  Heart,  endothelial  cells  in  the  heart;  Brach  Plex,  endothelial  cells  of  brachial  plexus;  Brain,  endothelial  cells  of  the  capillaries  and  venules  of  the  brain.      

Prophylaxis    

 By  definition,  prophylaxis  is  ‘any  means  taken  to  prevent  disease’  (Martin  2002e)  and  of  all  courses  of  treatment,  it  is  the  most  preferred  since  it  takes  a  proactive  approach  to  combat  a  disease.  An  essential  step  in  the  development  of  prophylactic   care  however,   is   to  determine   the  natural   reservoir  of   the  pathogen.  This   is  an  organism,  or  group  of  organisms  that  harbour  the  virus  and  provide  a  sustainable  vector  to  ensure  its  long-­‐term  survival.  For  example  the  reservoir  of  the  Lassa  Virus,  which  also  induces  it’s  own  haemorrhagic  fever,  is  Mastomys  natalensis;  more  commonly  known   as   the   Natal   Multimammate  Mouse.   The   natural   reservoir   of   EBOV   is   as   yet   not   known   although   there   is  substantial  evidence,  which  suggests  that  three  species  of  fruit  bat,  Hypsignathus  monstrosus,  Epomops  franqueti,  and  Myonycteris  torquata,  are  likely  vectors  (Leroy  et  al.,  2005).  For  example,  the  natural  population  of  the  Pteropodidae  family  to  which  all  of  the  named  species  belong  forms  boundaries  similar  to  the  geographical  extent  of  reported  non-­‐imported  cases  of  EBOV  (fig.  2).  The  named  species  were  shown  to  be  asymptomatic  to  EBOV  after  a  comprehensive  study  on  small  vertebrates  that  were  collected  during  ZEBOV  outbreaks  in  the  Republic  of  the  Congo  (adjacent  to  the  Democratic  Republic  of  the  Congo)  between  2001  and  2003  (Leroy  et  al.,  2005).  There  is  however  a  lack  of  evidence  as   to   how   these   bats   facilitate   the   infection   of   primates,  which   is   essential   knowledge   for   public   health   education  (Feldmann   et   al.,   2004).  More   recently,   in   an   on-­‐going   outbreak   in   Guinea,  West   Africa,   there   is   a   theory   that   the  introduction  of  EBOV  to  humans  may  have  occurred  through  the  consumption  of  bats,  which  locally  are  considered  a  delicacy  (BBC,  2014).  Overall,  public  health  education  is  an  effective  form  of  prophylaxis  in  two  means,  because  not  only  is  it  possible  to  reduce  the  likelihood  of  an  epidemic  occurring;  it  raises  the  chances  of  quick  isolation  if  it  does.  Furthermore,  it  is  highly  cost  effective,  not  requiring  significant  scientific  research,  nor  heavy  international  presence  in  its  implementation.    Education  however  should  not  only  be  directed  at   the   lay  population,  but  at   local  medical  professionals,   informing  them  of  the  necessity  of  infection-­‐control  care  aimed  to  prevent  nosocomial  infection.  Particular  reference  is  made  to  the  wearing  of  Personal  Protective  Equipment  and  the  disinfection  of  gloves  after  contact  with  each  patient  (Borchert  et  al.,  2011).  Despite  efforts  made,  it  is  difficult  to  maintain  widespread  access  to  such  specialised  equipment  in  rural  communities   in   developing   countries.   It   is   therefore   often   non-­‐governmental   organisations   such   as  Médecins   sans  frontières  who  implement  the  first  effective  isolation  wards  under  outbreak  conditions.      

Figure  6:  A  map  plotting  the  known  geographical  distribution  of  Pteropodidae  family  bats  against  recorded  outbreaks  of  EBOV  (WHO,  2009)  

 

A  more  absolute  prophylactic  is  vaccination,  which  is  believed  to  predate  the  16th  Century  with  its  origins  in  eastern  Asia   (Lombard   et   al.,   2007),   before   Edward   Jenner’s   breakthrough   with   the   development   of   a   vaccination   for  Smallpox  in  1796.  The  term  vaccination  was  coined  by  Louis  Pasteur  in  honour  of  Jenner,  who  used  Cow  Pox  virus,  Vaccina  virus,   to  prevent   the   infection  of  Smallpox  –  Valoria  major/minor.  The   latter  was  certified   to  be  eradicated  from  the  globe  by  WHO  in  1979  (WHO,  2014).      Vaccines  function  by  providing  antigens  of  a  pathogen,  triggering  an  immune  response.  In  doing  so,  the  body  creates  antibodies   that  can   then  be  readily  called  upon   to  prevent  an   infection  becoming  serious.  An  antigen   is  a  chemical  found   on   the   exterior   surface   of   cells,  which   is   used   to   recognise,  whether   a   cell   is   foreign.   If   a   foreign   antigen   is  detected,   an   immune   response,  whereby   a   unique   antibody   to   the   antigen   is   synthesised   in  massive   quantities,   is  triggered.  These  antibodies  bind  to  the  antigens  and  to  other  antibodies,  isolating  a  number  of  pathogens,  therefore  enabling  phagocytosis  –  the  consumption  of  a  pathogen  by  certain  types  of  white  blood  cell.      It  is  important  to  note  that  vaccination  is  significantly  different  to  inoculation,  which  is  the  practice  in  which  the  real  pathogen  is  administered  in  doses  not  considered  to  be  life  threatening.  It   is  now  however,  thought  to  be  unethical  because  there  were  a  high  percentage  of  cases  where  the  pathogen  could  take  a  hold  on  the  body  and  as  a  result,  it  is  a  discontinued  form  of  prophylaxis.    

Figure  7:  A  table  displaying  generic  types  of  vaccine  (NIAID,  2013)  Vaccine  Category   Description  Inactivated  Vaccine:   A  pathogen  is  grown  in  a  culture  and  then  killed  using  

heat  or  formaldehyde.  Whilst  the  dead  pathogens  cannot  infect  nor  harm,  the  viral  capsid  or  bacterial  walls  remain  intact  to  enable  recognition  by  the  immune  system.  

Attenuated  Vaccine:   Live  pathogens  are  administered,  but  only  those  with  low  or  localised  replication.    

Virus-­‐like  Particle  Vaccine:   The  viral  proteins  of  the  capsid  will  form  to  create  ‘empty’  viruses  that  do  not  contain  a  nucleic  acid.  

Subunit  Vaccine:   Presents  an  antigen  to  the  immune  system  without  introducing  the  pathogen  itself.  This  effectively  tricks  the  immune  system  into  believing  there  is  a  minor  infection,  creating  antibodies  in  response.  

DNA  Vaccine:   The  genetic  material  of  the  vaccine  is  converted  to  dsDNA,  which  is  then  isolated  and  prepared  in  solution  to  create  a  vaccine  that  provokes  an  immune  response  upon  administration.    

Recombinant  Vector  Vaccine:   Similar  to  DNA  Vaccine,  but  a  virus  or  bacteria  acts  as  a  carrier  molecule  or  vector  to  the  nucleic  acid.  

 Although  there  are  currently  no  licensed  vaccines  for  any  strain  of  EBOV,  there  is  significant  international  interest  in  their  development.  There  are  two  main  categories  of  vaccine:  replicating  competent  and  non-­‐replicating.  The  former  has  significant  advantages  over  the  latter  in  that  the  vaccine  is  more  durable,  typically  requires  fewer  immunisations  and   provides   longer-­‐lasting   immunity.   On   the   other   hand,   replicating-­‐competent   vaccines   are   live   and   have   the  potential  to  become  unsafe  in  the  presence  of  another  pathogen.  This  creates  a  dilemma  over  whether  it   is  morally  better   to   administer   a  more   effective,   yet   potentially   dangerous   vaccine   or   one   that   is   safer   but   also   necessitates  stronger  doses,  more  immunisations  and  leads  to  a  shorter  period  of  immunisation  (Falzarano  et  al.,  2012).    Replication-­‐Competent  Vaccines    

Recombinant  Vesicular  Stomatitis  Virus  (rVSV)  This  virus  has  proven  to  be  a  useful  attenuated  and  replication-­‐competent  vector  for  vaccines  acting  against  a  number   of   pathogens   including   influenza,   hepatitis   B   and   HPV   amongst   several   others.   It   is   genetically  engineered   so   that   the   VSV   glycoprotein   is   replaced   by   the   GP   of   EBOV   to   create   viral   particles.   These   are  structurally  similar  to  VSV,  but   instead  contain  EBOV  GP  rather  than  VSV  GP  (Jones  et  al.  2005).  Whilst   initial  clinical  trials  were  successful,  subsequent  trials  using  a  combination  of  SEBOV,  ZEBOV  and  the  other  virus  of  the  filoviridae  family,  Marburg  (MARV)  were  less  so.  It  was  found  that  whilst  rVSV-­‐MARV  GP  adequately  immunised  against   the   different   strains   of   MARV,   rVSV-­‐ZEBOV   GP   did   not   provide   any   immunity   to   SEBOV,   which   is   a  different  species.  The   lack  of  cross-­‐protection   is  a  significant  downfall,  however   it   is   feasible  that  an   injection  may  contain  several  vaccines  that  offer  protection  for  filoviridae  as  an  entity.  It  is  considered  that  the  fact  rVSV  vaccines   can   be   administered   orally,   nasally   and   intramuscularly   would   be   an   advantage   in   the   extra-­‐rural  community  (Falzarano  et  al.,  2012).    Recombinant  Human  Parinfluenza  Virus  3  (rHPIV3)    HPIV3   is   a   common   paediatric   respiratory   virus,   which   can   be   genetically   modified   to   express   ZEBOV   GP  forming   a   rHPIV3-­‐ZEBOV   GP   complex,   in   the   same  manner   as   rVSV-­‐ZEBOV   GP.   It   is   known   to   only   provide  

immunity   for  ZEBOV,  but   studies  using  non-­‐human  primates  have  shown   that  2  doses  are   required   to  obtain  100%  survival  (Bukreyev  et  al.,  2007).  The  major  disadvantage  with  rHPIV3  is  that  nearly  all  adults  have  gained  immunity  to  it  through  childhood,  which  may  restrict  the  function  of  rHPIV3  as  a  vaccine  platform  (Falzarano  et  al.   2012).  The   fact   that   rHPIV3   can  be  given   in   intranasal  does  however,   is   an  advantage  as   it   enables   easier  administration  in  rural  areas  (Falzarano  et  al.,  2012).    

Non-­‐replicating  Vaccines    

DNA    Whereas  the  first  recombinant  vaccines  have  an  EBOV  GP  within  the  fabric  of  their  own  protein  capsid,  the  DNA  vaccine  contains  3  plasmids  of  DNA  that  code  for  the  SEBOV  GP  and  nucleoprotein  (NP)  (Martin  et  al.,  2006).  Whilst   DNA   vaccines   bring   many   benefits   including   the   fact   that   they   can   be   adapted   quickly   as   pathogens  evolve,   the   level   of   immunity   provided   has   been   less   than   satisfactory.   Therefore   it   is   highly   unlikely   that   a  stand-­‐a-­‐lone  DNA  vaccine  will   be   pursued   in   future   research,   but   rather   in   combination  with   another   vector  (Falzarano  et  al.,  2012)    Recombinant  Adenovirus  5  (rAd5)  In   many   ways,   this   vaccine   platform   is   better   than   the   combined   DNA/rAd5   approach   due   to   the   far   more  compact   vaccination   course.   This  means   that   in   the   event   of   an   outbreak   or   bioterrorism   event,   the   vaccine  could  be  administered  swiftly,  providing  100%  survival  4  weeks  post  vaccination  in  non-­‐human  primates.  In  an  identical  manner   to  other   recombinant  vaccine  platforms,   rAd5   is  genetically  modified  so   that   it   contains   the  ZEBOV  GP.  Similarly,  the  immunity  offered  by  rAd5-­‐ZEBOV  GP  is  thought  to  be  species  specific  although  a  pan-­‐filovirus   adenovirus   platform   has   been   developed   containing   SEBOV   GP,   EBOV   GO   and   the   GPs   of   different  strains   of   MARV.   A   further   disadvantage   of   rAd5-­‐ZEBOV   GP   is   that   it   requires   a   single   large   dose   of   1010  particles  in  order  to  ensure  survival  (Falzarano  et  al.,  2012).    Combined  DNA  and  rAd5  The   combination   platform   of   DNA/rAd5   has   received   significant   scientific   interest,   as   it   was   the   first   to  demonstrate   100%   immunogenicity   in   non-­‐human   primates.   The   significant   benefit   of   combining   the   two  vectors  is  that  the  immune  response  is  significantly  increased  in  comparison  when  they  are  applied  separately.  Within   the  combination,   there  was  a   ratio  of  3:1  between   the  DNA  vaccine,   coding   for  ZEBOV  GP,  SEBOV  GP,  CIEBOV  GP  and  ZEBOV  NP;  and  the  rAd5-­‐ZEBOV  GP  vaccine.  When  presented  with  ZEBOV,  SEBOV  or  CIEBOV,  there  was  no  development  of  disease.   In  addition  to  the  species  mentioned,  despite  a  significant  difference   in  the  primary  structure  of  its  GP,  BEBOV  is  also  protected  against,  contrary  to  logical  assumptions  (Hensley  et  al.,  2010).  The  principle  disadvantage  with  this  vaccine  is  that  the  course  of  vaccinations  can  take  up  to  18  months,  which  in  filovirus  endemic  regions  is  verging  on  logistical  impossibility  (Falzarano  et  al.,  2012).    

 Virus  Like  Particle  There  are  several  key  advantages  that  VLP  has  over  a  traditional  inactivated  vaccine.  These  include  a  high  level  of   safety   because   VLPs   are   non-­‐replicating   and   an   ability   to   engage   the   wider   immune   response.   A   VLP  containing   GP,   NP   and   VP40   derived   from   EBOV   will   assume   the   approximate   morphology   of   the   original  pathogen.  In  clinical  trials,  eVLP  protected  100%  of  the  nonhuman  primate  cases,  which  failed  to  display  any  of  the  characteristic  symptoms  of  an  EBOV  infection  (Warfield  et  al.,  2007).  In  all  the  laboratory  testing,  eVLP  was  produced  using  mammalian  cells.  This  is  not  however  cost  effective  and  alternative  production  methods  such  as  using  insect  cells  are  currently  being  studied  (Falzarano  et  al.,  2012).    ∆VP30  ZEBOV  In  order  to  obtain  the  vaccine  platform,  the  gene  coding  for  VP30  within  the  EBOV  genome  is  replaced  with  a  reporter  gene  called  neomycin.  A  reporter  gene  acts  a  marker  and  can  help  determine  the  dispersal  of  a  gene  throughout   an  organism.  The   result   of   this   replacement   is   the   generation  of   Ebola  ∆VP30-­‐neo,  which   is   then  encouraged  to  replicate.  When  a  vaccine  consisting  of  Ebola  ∆VP30-­‐neo  was  administered  to  mice  at  a  quantity  of  106  particles,  all  the  specimens  were  resistant  to  the  modified  ZEBOV  strain.  Although  this  is  a  success  in  the  development   of   this   vaccine   platform,   it   is   unlikely   to   be   progressed   due   to   the   fact   that   85%  of   the   ZEBOV  genome   remains   intact.   This   could   potentially   give   rise   to   infection,   which   is   considered   to   be   a   risk   that  outweighs  the  potential  cross-­‐protection  benefits  (Falzarano  et  al.,  2012).    

   

Figure  8:  A  comparison  of  the  properties,  benefits  and  limitations  of  current  prototype  vaccines  for  EBOV  (Falzarano  et  al.,  2012)  Platform   Protection   Cross-­‐protection   Benefits   Limitations  DNA   Prophylactic   No   Non-­‐replicating  

Production  Multiple  immunisations  Limited  immunity    

DNA/rAd5   Prophylactic   Blended   Multivalent  Vaccine  

Non-­‐replicating  Cross-­‐protection  

Large  dose  (Ad)  Multiple  immunisations  Two  components  

rAd5   Prophylactic   Blended   Multivalent  Vaccine  

Non-­‐replicating  Multiroute  delivery§  

Pre-­‐existing  immunity  Large  Dose  

VLPs   Prophylactic   Blended   Multivalent  Vaccine§  

Non-­‐replicating  Successful   platform  (HPV,  HBV)  

Multiple  Immunisations  Production  

∆VP30  ZEBOV§  

Prophylactic   Unknown   Non-­‐replicating  Potential   for   cross-­‐protection  

Reintegration  of  VP30  Limited  to  ZEBOV  

rVSV   Prophylactic   &   Post  Exposure  

Blended   Multivalent  Vaccine  

Single  immunisation  Multiroute  delivery  

Replication-­‐competent  (attenuated)  

rHPIV3   Prophylactic   Unknown   Single   immunisation  potential  Mucosal  delivery  

Replication-­‐competent  (attenuated)  Pre-­‐existing  immunity  Limited  to  ZEBOV  

§  Not  tested  in  nonhuman  primates  HBV:  Hepatitis  B  virus;  HPV:  Human  papillomavirus  

(Falzarano  et  al.,  2012)      

Challenges  of  the  Implementation  of  Vaccines  Beyond  the  Laboratory    There  are  a  number  of  significant  factors  that  may  impede  both  the  progress  of  the  development  of  a  vaccine  and  the  consequential   administration.   Primarily   there   is   little   evidence   that   favours   a   prophylactic   vaccine   over   a   post-­‐exposure  vaccine  and  vice  versa.  Whilst   in  an  ideal  world  long-­‐term  prophylactic  vaccination  would  be  the  ultimate  goal,   it   would   be   exceedingly   difficult   to   achieve.   The   argument   centres   around   the   demographic   that   requires  immunisation  the  most  –  Sub-­‐Saharan  Africa.  Within  this  broad  group,  the  sectors  that  are  more  at  risk  from  infection  can   be   found   in   rural   areas,  where   there   is  more   likely   to   be   interaction   between   the   human   population   and   the  suspected   fruit   bat   reservoir.   In   logistical   terms   this  makes   a   comprehensive  prophylactic   vaccination  programme  practically  impossible  due  to  the  degree  of  remoteness  of  these  communities.  It  is  known  that  from  other  vaccination  programmes,  such  as  polio  in  Uganda,  that  obtaining  a  high  level  of  coverage  is  extremely  difficult,  with  just  59%  of  1yr   olds   receiving   their   vaccinations   (WHO).   Despite   this,   it   is   possible   to   prevent   an   epidemic   by   vaccinating   a  significant  proportion  of  the  population  –  it  is  only  necessary  to  vaccinate  the  entire  population  in  order  to  ensure  the  pathogen  is  eradicated.  The  justification  for  even  attempting  to  administer  countless  prophylactic  vaccines  for  a  virus  that  has  had  2,300   reported   cases   throughout   its   entire  known  history   is   again  nearly   impossible   to  make.  This   is  particularly  the  case  when  it  is  considered  how  localised  the  epidemics  are.      Not  only  do  vaccines  present  a  logistical  problem,  but  a  political  one.  When  it  is  considered  that  EBOV  is  most  greatly  concentrated   in   central,   Sub-­‐Saharan   Africa,   where   political   instability   is   rife,   the   feasibility   of   any   vaccination  programme   is   questionable.   Similarly,   non-­‐governmental   organisations   such   as   Médecins   Sans   Frontières   (MSF),  which   may   be   expected   to   aid   in   such   a   programme   are   fully   occupied   in   countries   such   as   the   Central   African  Republic  where  this  currently  a  civil  war.  A  further  political  factor  is  that  Ebola  is  unlikely  to  ever  be  a  serious  threat  to  any  western  population,  due   to   the   fact   the  virus  has  only  affected  a  small  minority.  This   is   in  contrast   to  other  international   vaccination  efforts   such  as   in   the   case  of  Polio  or  Smallpox  where   the   illness  was   truly  global.  These  combined  reasons  create  a  void  of  political  will  to  fund  the  development  of  a  vaccine,  which  is  likely  to  ultimately  lead  to  more  outbreaks  and  consequently  more  fatalities.      An  alternative  form  of  prophylactic  vaccine  could  be  a  localised  campaign  whereby  the  immediate  population  to  an  outbreak  are  immunised.  Whilst  this  is  probably  the  most  efficient  form  of  vaccination  for  EBOV,  certain  issues  still  remain.   The   principal   problem   is   that  whilst   the   local   population   is   significantly   smaller   than   the   entirety   of   sub-­‐Saharan  Africa,  there  is  still  a  significant  enough  population  to  well  exceed  the  capacity  for  a  comprehensive  vaccine.  Furthermore,  if  any  attempt  was  made  to  enact  such  a  programme,  it  would  draw  a  vast  quantity  of  manpower  from  the  infection  control  wards,  where  foreign  aid  is  required  the  most.  An  additional  problem  could  be  that  if  a  vaccine  is  administered,   and   then   that   patient   subsequently   develops   EHF   (or   indeed   any   illness),   the   foreign   medical  organisations   would   lose   the   confidence   of   the   local   population.   This   would   have   drastic   effects   on   the   entire  operation,   especially   if   cooperation   is   lost.   In   that   scenario,   an   EBOV   epidemic   could   potentially   be   out   of   control  (MacNeil  &  Rollin,  2012).    

Although  vaccines   for  any  species  of  EBOV  are  unlikely  to  be  approved   in  the  nest  5  years  (Falzarano  et  al.,  2012),  scientific   advances  will   likely   create   an   effective   vaccine.   Some   of   the   prototype   vaccines   already   show   significant  promise   and   could   be   highly   effective  when   administered,   but   in   the   case   of   Ebola,   the   necessary   clinical   trials   to  licence  a  vaccine  are  considered  to  be  unethical  due  to  the  high  probability  of  death  if  the  vaccine  fails.  The  main  issue  however  is  that  research  is  costly  and  there  is  not  sufficient  political  desire  amongst  developed  countries  to  fund  the  research  of  the  vaccine.  That  is  however  of  little  relevance  given  the  practical  difficulties  of  the  implementation  of  any  vaccine   for   Ebola.   It   is   highly   likely   therefore   that   any   approved   vaccine   will   be   administered   only   to   medical  professionals  who  work  in  close  quarters  with  EBOV.      As   is   the  way  with  the  human  race,   it   is  probable  that  efforts  will  concentrate  more  on  the  solution,  or   in  this  case  post-­‐exposure  treatment,  rather  than  preventing  it.  Despite  this,  it  is  imperative  that  the  natural  reservoir  for  all  the  species   of   EBOV   is   known   so   that   at   least   a   certain   degree   of   public   health   education   can   occur.   For   the   human  population  of   sub-­‐Saharan  Africa,   that   is   likely   the  best  method  of  prophylaxis   they  will   receive   in   the   foreseeable  future.          

 References:    Baize   S,   Leroy  E,  Georges-­‐Courbot  M,   Capron  M,  Lansoud-­‐Soukate   J,   et   al.   (1999)   ‘Defective  humoral   responses   and   extensive   intravascular  apoptosis   are   associated   with   fatal   outcome   in  Ebola  virus-­‐infected  patients.’  Natural  Medicine  5  (4)  pp423-­‐6    Baltimore  D   (1971)   ‘Expression  of  Animal  Virus  Genomes.’  Bacteriological  Reviews,  35  (3)  pp235-­‐238    Banchereau  J,  Steinman  R  (1998)  ‘Dendritic  cells  and  the  control  of  immunity.’  Nature,  392  pp245-­‐252    Bausch   D,   Sprecher   A,   Jeffs   B,   Boumandouki   P  (2008)   ‘Treatment   of   Marburg   and   Ebola  haemorrhagic   fevers:   A   strategy   fir   testing   new  drugs   and   vaccines   under   outbreak   conditions.’  Antiviral  Research,  78  pp150-­‐161    BBC  (2014)  ‘West  Africa  on  Ebola  virus  high  alert’  Available  at  http://www.bbc.co.uk/news/health-­‐26740686  [Access  date  25/3/14]    Borchert   M,   Mutyaba   I,   Van   Kerkhove   M,  Lutwama   J,   Luwaga   H   et   al.   (2011)   ‘Ebola  haemorrhagic   fever   outbreakin   Msindi   District,  Uganda;   outbreak   description   and   lessons  learned.’  BMC  Infectious  Diseases   11  Available   at  http://www.biomedcentral.com/content/pdf/1471-­‐2334-­‐11-­‐357.pdf  [Access  date:  30/9/13]    Bray  M,  Geisbert  T  (2005)   ‘Ebola  virus:  The  role  of   macrophages   and   dendritic   cells   in   the  pathogenesis  of  Ebola  h[a]emorrhagic  fever.’  The  International   Journal   of   Biochemistry   and   Cell  Biology,  37  pp1560-­‐1566    Bray  M,  Mahanty  S  (2003)  ‘Ebola  H[a]emorrhagic  Fever  and  Septic  Shock.’  The  Journal  of  Infectious  Diseases  188  pp1613-­‐1617    Brown  TA  (2002)  Genomes.  2nd  edition.  Oxford:  Wiley-­‐Liss.   Chapter   1,   The   Human   Genome.  Available   from:  https://www.ncbi.nlm.nih.gov/books/NBK21134/  [Access  date  03/02/14]    Bukreyev  A,  Rollin  P,  Tate  M,  Yang  L,  Zaki  S  et  al.  (2007)   ‘Successful   topical   respiratory   tact  immunisation   of   primates   against   Ebola   Virus.’  Journal  of  Virology  81  (12)    Carette   J,   Raaben   M,   Wong   A,   Herbert   S,  Obernosterer   G   et   al.   (2011)   ‘Ebola   virus   entry  requires   the   cholesterol   transporter   Neimann-­‐Pick  C1.’  Nature  477  (7364)  pp340-­‐343    Crawford   DH   (2000a)  The   Invisible   Enemy:   A  Natural   History   of   Viruses   Oxford   University  Press,  Oxford  pp13-­‐14  

 Crawford   DH   (2000b)  The   Invisible   Enemy:   A  Natural   History   of   Viruses   Oxford   University  Press,  Oxford  pp14-­‐15    Falzarano   D,   Geisbert   T,   Feldman   H   (2012)  ‘Progress   in   filovirus   vaccine   development:  evaluating   the   potential   for   clinical   use.’   Expert  Revue  Vaccines  10  (1)  pp63-­‐77    Feldmann   H,   Geisbert   T   (2011)   ‘Ebola  Haemorrhagic  Fever.’  Lancet  377  (9768)  pp849-­‐862      Feldmann   H,   Wahl-­‐Jensen   V,   Jones   S,   Ströher   U  (2004)   ‘Ebola   virus   ecology:   a   continuing  mystery.’  Trends  in  Microbiology   12   (10)  pp433-­‐437    Fleck   F   (ed.)   (2011)   Bugs,   drugs   and   smoke  World  Health  Organisation  pp121-­‐122    Geisbert  T,  Young  H,  Jharling  P,  Davis  K,  Larsen  T,  et   al.   (2003)   ‘Pathogenesis   of   Ebola  H[a]emorrhagic   Fever   in   Primate   Models.’  American   Journal   of   Pathology   163   (6)   pp2371-­‐2382    Hensley  L,  Mulangu  S,  Asiedu  C,  Johnson  J,  Honko  A  et  al.  (2010)  ‘Deomstration  of  cross-­‐protectivie  vaccine   immunity   against   an   emerging  pathogenic  Ebolavirus  Species.’  PLoS  Pathogens  6  (5)   Available   at  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2873919/pdf/ppat.1000904.pdf   [Access   date  24/02/14]    International  Committee  on  Taxonomy  of  Viruses  (ICTV)   (2012),   Virus   Taxonomy,   Available   at  http://www.ictvonline.org/virusTaxonomy.asp  [Access  date:  04/02/2014]      Jeffrey  E,   Fusco  M,  Hessell   A,  Oswald  W,  Burton  D,   Ollmann   Saphire   E   (2008)   ‘Structure   of   the  Ebola   virus   glycoprotein   bound   to   an   antibody  from  a  human  survivor.’  Nature  454  pp177-­‐182      Jones   S,   Feldmann   H,   Ströher   U,   Geisbert   J,  Fernando   L   et   al.   (2005)   ‘Live   attenuated  ecombinant   vaccine   protects   non   human  primates   against   Ebola   and   Marburg   viruses.’  Natural  Medicine  11(7)  pp786-­‐790    Leroy   E,   Kumulungui   B,   Pourrut   X,   Rouquet   P,  Hassanin  A  et  al.  (2005)  ‘Fruit  Bats  as  reservoirs  of  Ebola  Virus.’  Nature  438  pp575-­‐576    Lombard  M,  Pastoret  P,  Moulin  A  (2007)  ‘A  Brief  History   of   Vaccines   and   Vaccination.’   Revue  Scientifique  et  technique  26  (1)  pp29-­‐48    MacNeil   A,   Rollin   P   (2012)   ‘Ebola   and   Marburg  H[a]emorrhagic   Fevers:   Neglected   Tropical  Diseases?’  PLoS  Neglected  Tropical  Diseases  6  (6)  Available   at  

http://www.plosntds.org/article/fetchObject.action?uri=info%3Adoi%2F10.1371%2Fjournal.pntd.0001546&representation=PDF   [Access   date  24/02/14]    Martin  E  (ed.)  (2002a)  Concise   Colour  Medical  Dictionary   Oxford   University   Press,   Oxford  “necrosis”  p459    Martin  E  (ed.)  (2002b)  Concise  Colour  Medical  Dictionary   Oxford   University   Press,   Oxford  “apoptosis”  pp44-­‐45    Martin  E  (ed.)   (2002c)  Concise   Colour  Medical  Dictionary   Oxford   University   Press,   Oxford  “differential  diagnosis”  p195    Martin  E  (ed.)  (2002d)  Concise  Colour  Medical  Dictionary   Oxford   University   Press,   Oxford  “neutrophil”  p469    Martin  E  (ed.)  (2002e)  Concise   Colour  Medical  Dictionary   Oxford   University   Press,   Oxford  “prophylaxis”  p469    Martin  E,  Hine  RS  (2000a)  Dictionary  of  Biology  Oxford   University   Press,   Oxford   “Virus”   pp620-­‐621    Martin  E,  Hine  RS  (2000b)  Dictionary  of  Biology  Oxford   University   Press,   Oxford   “Retrovirus”  p519    Martin   J,   Sullivan   N,   Enama   M,   Gordon   I,  Roederer   M   et   al.   (2006)   ‘A   DNA   Vaccine   for  Ebola  Virus  Is  Safe  and  Immunogenic  in  a  Phase  1  Clinical   Trial.’   Clinical   and   Vaccine   Immunology  13  (11)      Mupapa   K,   Massamba   M,   Kibadi   K,   Kuvula   K,  Bwaka   A   et   al.   (1999)   ‘Treatment   of   Ebola  H[a]emorrhagic   Fever   with   Blood   Transfusions  from   Convalescent   Patients.’   The   Journal   of  Infectious  Diseases  179  (suppl  1)  ppS18-­‐S23    National   Institute   of   Allegry   and   Infectious  Diseases   (NIAID)   (2010)   Transmission   of  Ebola/Marburg   Viruses,   Available   at  http://www.niaid.nih.gov/topics/ebolaMarburg/understanding/Pages/transmission.aspx  [Access  date:  16/02/14]      National   Institute   of   Allegry   and   Infectious  Diseases   (NIAID)   (2013)   Types   of   Vaccine  http://www.niaid.nih.gov/topics/vaccines/understanding/pages/typesvaccines.aspx   [Access  date:  22/02/14]      OpenStax   College   (2013)   ‘Viruses.’   Available   at  http://cnx.org/content/m45541/latest/#fig-­‐ch17_01_02  [Access  date:  22/03/14]    Oswald   W,   Geisbert   T,   Davus   K,   Geisbert   J,  Sullivan   N   et   al.   (2007)   ‘Neutralising   Antibody  Fails   to   Impact   the   Course   of   Ebola   Virus  Infection   in   Monkeys.’   PLoS   Pathogens   3(1)  

Available   at  http://www.plospathogens.org/article/fetchObject.action?uri=info%3Adoi%2F10.1371%2Fjournal.ppat.0030009&representation=PDF   [Access  date:  23/02/14]    Parrino   J,   Hotchkiss   R,   Bray  M   (2007)   ‘Immune  Cell   Apoptosis   Prevention   as   Potentential  Therapy   for   Severe   Infections.’   Emerging  Infectious  Diseases   13   (Internet   Serial)   Available  at:   http://wwwnc.cdc.gov/eid/article/13/2/06-­‐0963.htm  [Access  date:  22/02/14]    Qui   X,   Audet   J,   Wong   G,   Pillet   S,   Bello   A   et   al.  (2012)   ‘Successful   treatment   of   ebola   virus-­‐infected  cynomolgus  macaques  with  monoclonal  antibodies.’   Science   Translational   Medicine   4  (138)  pp138-­‐181    Roddy   P,   Colebunders   R,   Jeffs   B,   Palma   P,   Van  Herp   M,   Borchert   M   (2011)   ‘Filovirus  H[a]emorrhagic   Fever   Outbreak   Case  Management:   A   Review   of   Current   and   Future  Treatment   Options.’   The   Journal   of   Infectious  Diseases  204  (suppl  3)  pp791-­‐795    Roddy  P,  Howard  N,  Van  Kerkhove  MD,  Lutwama  J,  Wamala  J,  et  al.  (2012)   ‘Clinical  manifestations  and   case   management   of   Ebola   Haemorrhagic  Fever   caused   by   a   newly   identified   virus   strain,  Bundibugyo,   Uganda   2007-­‐2008.’   PLoS   ONE,  7(12)   Available   at  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3532309/pdf/pone.0052986.pdf   [Access   date  20/08/13]    Rutgers   (2011)   Molecular   Anatomy   Project,  Ebola   Virus,   Available   at  http://maptest.rutgers.edu/drupal/?q=node/453  [Access  date  03/02/14]    Smith  T  (ed.)   (1995a)  Complete   Family  Health  Encyclopaedia   The   British  Medical   Association,  Dorling  Kindersley,  London  “Viruses”  p1055    Soper  R  (ed.)  (1997a)  Biological   Science  1  &  2  Cambridge  University  Press,  Cambridge  2.41    Soper  R  (ed.)  (1997b)  Biological   Science  1  &  2  Cambridge  University  Press,  Cambridge  2.43    Stannard   LM   (1995)   Division   of   Medicial  Virology,   University   of   Cape   Town.   Available   at  http://web.uct.ac.za/depts/mmi/stannard/emimages.html  [Access  date  03/11/13]    Wikipedia   (2013)   Virus.   Available   at  http://en.wikipedia.org/wiki/Virus   [Access   date  03/11/13]    Wikipedia   (2014)   Sense   (Molecular   Biology).  Available   at  https://en.wikipedia.org/wiki/Sense_(molecular_biology)  [Access  date  05/01/14]    

World   Health   Organisation   (2009)   ‘Geographic  distribution   of   Ebola   haemorrhagic   fever  outbreaks  and  fruit  bats  of  Pteropodidae  Family.’  Available   at  http://www.who.int/csr/disease/ebola/Global_EbolaOutbreakRisk_20090510.png?ua=1   [Access  date:  29/9/13]    World   Health   Organisation   (2012).   Ebola  Haemorrhagic  Fever.  Fact  sheet  103  Available  at  http://www.who.int/mediacentre/factsheets/fs103/en/    [Access  date  21/08/13]