development of an oral vaccine against candidiasis using

Seiji SHIBASAKI1, Mitsuyoshi UEDA2

1Hyogo University of Health Sciences, Kobe, Japan, 2Kyoto University, Kyoto, Japan

Introduction       Candidiasis   is   one   of   serious   infec.ous   disease   caused   by   the   virulent   fungus  Candida   albicans   and   other   Candida   species   [1].   Pharmacotherapy   of   candidiasis  o=en   involves   the   administra.on  of   amphotericin  B,   caspofungin   and  micafungin.  Unfortunately,   several   mutants   of   Candida   with   reduced   suscep.bility   to   these  drugs   have   emerged   [2].   Preven.on   of   infec.on   by   vaccina.on   against   Candida  species  is  important  strategy  in  efforts  to  control  candidiasis.           Recently,   we   have   developed   the   molecular   display   technology,   which   uses  gene.cally  engineered  microorganisms  to  produce  foreign  proteins  on  their  surface  [3].  The  use  of  molecular  display   to  prepare  target  proteins   is  well  established   for  use   with   the   yeast   Saccharomyces   cerevisiae.   We   present   that   the   an.gens  displayed  on  yeast  generated  using  molecular  display  technology  offer  a  novel  type  of  oral  vaccine  against  candidiasis.  The  enolase  1  protein  (Eno1p)  from  C.  albicans  was   selected  as   the  model  an.genic  protein   to  be  displayed  on   the   surfaces  of  S.  cerevisiae  cells  [4,  5].  

(1)  Molecular  display  of  Eno1p  on  yeast  cell  surface  The  fragment  of  the  gene  encoding  ENO1  was  inserted  into  pULD1  plasmid  that  had  been  digested  with  NotI  and  XhoI.  Eno1p  coding  sequence  was  fused  to  5’-­‐end  of  the  cell  wall  anchoring  protein,    α-­‐agglu.nin,  coding  sequence  in  the  plasmid.  

(2)  Oral  administra.on  of  yeast  displaying  Eno1p  Female  C57BL/6  (7  w)  mice  were  used  for  oral  immuniza.on  experiments.  A  suspension  of  S.  cerevisiae  cells  displaying  Eno1p  on  the  surfaces  (1.6  ×  109  cells/400  ml)  were  administered  to  ten  mice  per  dose  at  weeks  0,  1,  and  3  and  at  week  7.  The  host  strain  of    S.  cerevisiae  was  used  as  a  control.  All  inoculums  were  suspended  in  PBS  (400  μL)  and  administered  via  an  intragastric  tube  a=er  2  h  of  fas.ng,  once  per  day  for  5  days  per  week.  Blood  samples  were  taken  at  week  9  to  determine  the  .ter  of  serum  IgG.  For  survival  studies,  mice  were  infected  with  1.1  ×  105  cells  of  C.  albicans  resuspended  in  100  μl  PBS  by  tail-­‐vein  injec.on  2  weeks  a=er  the  last  immuniza.on.  

Materials and Methods

Results

Conclusions

    A   vaccine   produced   using   molecular   display  technology   avoids   the   need   for   protein  purifica.on.   This   oral   vaccine   offers   a   promising  alterna.ve   to   the   use   of   conven.onal   and  injectable   vaccines   for   preven.ng   a   range   of  infec.ous  diseases.  

References [1]  Nucci  M  &  Marr  KA  (2005)  Clin.  Infect.  Dis.  41:  521–526.  [2]  Rodloff  C,  Koch  D  &  Schaumann  R  (2011)  Eur.  J.  Med.  Res.  16:  187–195. [3]  Shibasaki  S  &  Ueda  M  (2010)  Recent  Pat.  Biotechnol.  4:  198-­‐213.  [4]  Shibasaki  S,  Aoki  W,  Nomura  T,  Miyoshi  A,  Tafuku  S,  Sewaki  T  &  Ueda  M  (2013)  Pathog.  Dis.  69:  262-­‐268.  [5]  Shibasaki  S,  Aoki  W  &  Ueda  M  (2013)  Yakugaku  Zasshi.  133:  1145-­‐1151.  

Development of an oral vaccine against candidiasis using molecular display technology

10μm  

B

C

A

D

Fig.  1.  Fluorescence  micrographs

(1)  Construc.on  of  Eno1p-­‐displayed  yeast  cell  

Fig.  2.  Evalua.on  of  surface  display  of  Eno1p  on  yeast  cells

Fig.  4.  Survival  ra.o  of  vaccinated  mice  a=er  challenge  of  C.  albicans

Fig.  3.  An.body  response  in  administra.on  of  yeast  cells

(2)  Oral  administra.on  of  yeast

     This  study  shows  that  the  only  availability  of  a  DNA  sequence-­‐encoding  the  an.genic  protein  is  needed  to  produce  oral  vaccine  based  on  molecular  display  technology.  

Eno1p-­‐displaying  yeast

Administra.on +  C.  albicans Examina.on  of  survival  rate

+  pULD1 Yeast  cell

C.  albicans  genome  DNA

PCR

   Gene.cally  engineered  cells  were  stained  with  IgG  labeled  with  Alexa-­‐488  (Fig.1).                  Fluorescent  stained  cells  were  analyzed  to  evaluate  surface  display  quan.ta.vely  (Fig.2).

   An  an.body  response  in  oral  administra.on  was  measured  by  ELISA  (Fig.3).        Survival  rates  were  evaluated  for  vaccinated  and  control  mice  a=er  challenge  of  C.  albicans  (Fig.  4).