Next  Genera*on  Synthe*c  DNA  Vaccines  in  Prime  Boost  Studies  

David  B.  Weiner,  Ph.D.  Professor,  Department  of  Pathology  &  Laboratory  Medicine  

Chair  Gene  Therapy  &  Vaccine  Graduate  Program  CAMB  

University  of  Pennsylvania  

Disclosure:  DBW  notes   several   commercial   rela3onships   -‐     These   include  Consul3ng,  Board  Service,  Stock,  SRA,  Speaking,  others  etc.  with  -‐  Novar3s,     Inovio,  VGXi,  BMS,  Medarex,  Pfizer,  Virxsys,   Ichor,  Merck,  Althea,  among  others  which  are  managed  through  Penn  COI  commiNee.    

WHO/NIH Workshop: Heterologous Prime-Boost Vaccine Strategies for

HIV, Malaria and TB

April 2012

Comparison  of  Immune  Response  by  Vaccine  PlaKorms    

These  data  relegated  DNA  to  second  class  status    

CMI  responses  at  highest  feasible  dose  in  rhesus  macaques    

10  

100  

1000  

Individual  Macaque  Geom.  Mean  

Gag-‐spe

cific  T  cells/  

1e6  PB

MC  

           Poxviruses  

(-‐)                DNA          MVA          ALVAC      NYVAC      VEE        Ad5  

First  Focus  -‐  Crossing  the  Line  

in  the  Sand  

Improve  the  T  cell  responses  to  equal  or  surpass  the  best  viral  vector  plaKorm  –  Ad5  

Focus  improvement  in  NHP  studies  and  validated  quan*ta*ve    T  cell  assays  –  Improving  T  cells  will  improve  Ab  Responses      

Focus  on  Three  Important  Steps  

Original  DNA  Vaccines  

1  

2  

3  Enhanced  DNA  Vaccines  

1-‐  Increase  per  cell  an*gen  produc*on  by  gene  engineering  of  plasmid  &  an*gens  improve  formula*ons  inserts  are  synthe*c  sequences  

2-‐  Increase  the  number  of  cells  transfected  invivo  –EP/ES  Delivery  

3-‐  Make  the  an*gens  delivered  excite  and  direct  the  immune  system  -‐  Gene*c  Adjuvants-‐IL12,etc.  

Mul*ple  DNA  Synthe*c  Op*miza*on  Approaches  are  U*lized  

Op3miza3on  per  gene  Computer  designed  immunogens  based  on  Con  hypothesis  using  early  

transmiNer  phenotype  framework  

Changes:  Synthe3c  high  AT  low  GC  leader  which  is  highly  efficient  at  targe3ng  the  an3gens  to  the  cell  membrane  

Species  specific  codon  usage  targeted  Modify  GC  content  to  increased  usage  in  the  body  of  the  an3gen  

Remove  cryp3c  splice  sites,  RNA  instability  sequences  Ubiqui3n  targeted  regions,  secondary  start  signals,  addi3onal  splice  sites,  

NLS,  consider  novel  glycosyla3on  sites,  avoid  fusion  an3gens,  modify  regions  of  high  secondary  structure,  destory  RNA  repeat  regions,  eliminate  excess  

RES,  delete  internal  TATA  boxes,    use  synthe3c  furin  cleavage  sites  to  preserve  proteasome  cleavage.  

For  novel  plasmids  promoter  design,  spacing,  ORI  op3miza3on,  polyA  track  stability,  others.  New  high  Concentra3on  Formula3ons!  

U3lize  Synthe3c  Consensus  Designed    Immunogens  to  focus  immune  responses  ,  delivery  of  many  many  genes  corrdinately    

Synthe*c  DNA  Vaccine  Delivery  by  EP/ES  •  Originally  used  to  enhance  chemotherapy  delivery  to  

solid  tumors  •  Original  devices  very  crude  and  painful  •  Refocused  on  tranfec*on  effeciency  not  adjuvant  

effects  of  EP.    New  arrays  focused  delivery  and  parameters  /devices  

•  With  injec*on  of  plasmid  electrode  needles  which  surround  the  injec*on  site  are  ac*vated  with  controlled  pulsing  and  pulse  pabern    

•  Electric  pulses  cause  a  temporary  increase  in    membrane  permeability  

•  Electric  current  drives  macromolecules  direc*onally  through  invivo  electrophoresis  together  these  ac*ons  increase  transfec*on    efficiency  

•  Allows  for  unprecedented  control  over  invivo  transfec*on  condi*ons.      

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Elispot  Responses  

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Cellectra  EP  

AD5  vs  E-‐DNA  Macaque    Collabora*ve  Study  Merck/Penn/Inovio  

Ad5  is  established  as  the  most  potent  T  cell  driving  platform  in  humans  &  NHP    We  sought  to  compare  DNA  induced  responses  with  Recombinant  Ad5  vaccine  in  Macaques  

 Ad5  SIV  Vaccine  (gag/pol/nef)  1010  VP/Dose/vector  

Vac-  Wk  0,  4,  24  

       

SIV  DNA  +  EP  (CELLECTRA-‐IM)  Concentrated  formula*on  Vaccine  SIV  (gag/pol/env)  1mg/construct/dose  –  Wk  0,4,  8,  24  

Note  -‐  for  Assays  pol  and  gag  pep*des  pools  were  matched  to  Ad5  insert  to  prevent  bias  Prolifera3on  

Ref:    Hirao  et  al.  Mol  Therapy  2010  

 DNA  +  EP            Ad5                                    DNA  +  EP              Ad5  

IFN-‐γ  Elispot  

DNA  +  EP    Ad5  

Weak CTL Potent CTL

CTL Scale T-Helper Response Scale Antibody Response

CD86 C3d IL-5 IL-10 IL-4 CD80 Fas IL-12

Mut. Caspase 3 Flt-3L IL-1β IL-2 IL-8 IgG1 FC TNF-α

HSP70 RelA GM-CSF IL-15 Rantes Calreticulin IL-18 ICAM-1 FrC TT

increase

increase

Some of the DNA Vaccine adjuvants studied

PENNVAX  B  Clinical  Trial    

Vaccine  An*gens  

Consensus  Immunogens  to  Focus  CTL  Responses  

pGag,  pPol,  pEnv,  

1mg/construct  x3  immuniza*ons  

Imm  @  0,  1    3  months  

080  with  EP  &  Il-‐12  

Placebo  10/2  randomiza*on  

HVTN  Studies  

Protocol  080  (EP)    -‐  Spyros  Kalams  PI    

48  subjects  -‐  Opened  in  2009  

Sponsors  &  Collaborators  

U.  Penn  NIAID-‐DAIDS,  HVTN,  Wyeth/Profectus,  Inovio  

What  was  observed  in  the  clinic  this  *me  around?  

HVTN  080  Summary  of  Results  

•  CD4  T  cell  responses  were  induced  in  80%  of  volunteers  by  1  month  post  the  3d  immuniza3on.      

•  CD8  T  cell  responses  were  induced  in  52%  of  volunteers  by  1  month  post  the  3d  immuniza3on.  Overall  90  percent  of  persons  vaccinated  produced  either  a  CD4  or  CD8  or  both  responses.    

•  Memory  T  cells  persist  in  80%  of  responders  for  more  than  6  months.      

•  T  cell  responses  were  comparable  to  prime  boost  data  generated  in  ac3ve  HVTN  studies  (DNA  +pox  &  DNA+adeno,  or  Ad  +  Ad  protocols)  in  ½  the  3me  with  less  immuniza3ons.      

Trimble  C  L  et  al.  Clin  Cancer  Res  2009;15:361-‐367  

IM  injec*on:  5/15  pa*ents  showed  E7-‐specific  responses,  2/15  pa*ents  showed  E6-‐specific  responses,  the  T  cell  responses  required  culture  to  observe.  Vaccina*on  did  not  induce  Abs  

Improving  Immune  potency  important  

HPV  Clinical  Reports    -‐  Many  Previous  Studies  provide  a  rich  background  of  data:    o  MVA  –  based  HPV  E6/E7  vaccines  from  Transgene:  Phase  IIa  (n  =  21)  completed;  Phase  IIb  (n  =  200)  near  

comple*on  o  Ad5,  SFV  (Semliki  Forest  virus)-‐based  HPV  E6/E7  vaccines  o  Protein/Pep*de-‐based  vaccines    o  Listeria-‐based  vaccines      o  DNA-‐based  vaccines  (TC  Wu)  -‐  Use  gene  gun  techniques  to  introduce  HPV  DNA  vaccines  directly  into  APCs  However  in  these  prior  studies…  o  Low  immune  responses  observed  -‐  cultured  ELISpots  needed  to  see  cellular  responses  

-‐  no  an*body  responses  induced  

Transla*on  to  a  second  T  cell  relevant  target  -‐  HPV  Therapy    Remains  Second  largest  Cancer  Killer  of  women  world  wide  -‐  250K  deaths  per  year  

Elispot Responses at Peak vs Memory Time points

HPV:001 T cells induced determined by ELISPOT Assay

Trial Design

Results: T cell responses were induced in 90% of volunteers as measured in standard Elispot. T cell responses persist in 90% of responders 6 months post the final immunization. T cell killing function is induced by the vaccine in all dose groups tested (9/11) individuals evaluated display this activity. Antibody responses are induced in WB and in Elisa in 14/15 evaluable patients.

These responses persist for more than 9 months.

HIV  DNA  Vaccine  Induced  An*bodies:  The  Search  for  the  Holy  Grail  

The DNA platform has been a poor inducer of antibody responses in the absence of a boost technology.

1- Can we improve the ability of DNA to generate/ prime for antibody responses – including functional antibody responses?

Original Clade B DNA was T cell focused – Developed new constructs with focus on T cells + improved antibody responses, larger loops included, surface

expression.

2- Can we use DNA technology to improve the induction of Mucosal Immunity? Ultimate focus is on NHP for translation to the clinic

Consensus  A,C  gp160  E-‐DNA  enhances  HIV  an*body  priming  in  Rabbits  

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Reciprocal titer

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Antibody  &  Nab  titer  

HIV-‐1Env  (DNA  prime/100ug)    

Weeks   1   2  0   3   4    5   6  

HIV-‐1  rEnv-‐(100ug)  

K. Muthmani et al. pV

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DNA  Improvements  translate  to  improved  Ab  responses  in  NHP  

15 NHP were vaccinated with clade C env at weeks 0, 4, & 12

Results: -Clade C binding titers > 103 -Clade B and C Tier 1 Neuts with DNA alone

MID delivery - Nabs assay rabbits - TZM-bl cells Average of 5 animals/group responses

post 4 immunizations using high concentration & ID delivery

PENN-Lab

Base Line

Tier 1

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100

1000

10

100

1000

Tier 1 Tier 2

DM-Lab

10

100

1000

Small footprint Lower voltage

Higher DNA concentrations

Improved AB responses in NHP

Redirec*ng  Immune  Responses  Invivo  Mucosal  B-‐cell  Chemokines  

•  CTACK  (CCL27)  –  Expressed  by  skin  

epithelium  –  Binds  CCR10  

•  MEC  (CCL28)  –  Expressed  by  epithelia  

including  small  and  large  intes3ne  

–  Binds  CCR10  

Kunkel et. Al. Nature Reviews Immunology. 2003

 CCR10 commonly expressed on IgA secreting plasmablasts which functions to recruit them to mucosal tissues

Co-immunization with Mucosal Chemokines in NHP Enhances Serum and Vaginal Antibody Responses

Data from the laboratory of J. Mestecky

IgA IgG

Vagi

nal

Seru

m

Pilot  Study  to  Evaluate  protec*ve  immunogenicity  by  an  Intravaginal  repeat  E660  

challenge  

•  Animals  intravaginally  challenged  bi-‐weekly  4  3mes  with  500  TCID50  SIVsmE660  (TRPC)  

•  E660  viral  stock  obtained  from  the  AIDS  Research  and  Reference  Reagent  Program,  Division  of  AIDS,  NIAID,  NIH:  SIVsmE660  from  Dr.  Vanessa  Hirsch  and  Dr.  Philip  Johnson.  

•  Viral  load  measured  by  bDNA  with  a  limit  of  detec3on  of  165  vp/ml  

•  Animals  were  studied  for  Trim5a  genotype  but  it  did  not  appear  to  play  a  clear  role  in  this  study.    

Rhesus  Macaque  Challenge  Study  

Naïve  (6)   DNA  (5)   DNA  +  CCR10L  (9)  

Uninfected   1   2   2  

Abor*ve  infec*on   0   0   6  

Progressive  Infec*on   5/6   3/5   1/9  

Percent  Protec*on   16%   40%   89%  

Naive DNA Only

CCL10L Adjuvant

*note 11/14 vaccinated animals abort infection

P

E-‐DNA  relevance  to  Prime  Boost    

•  Mul3ple  improvements  to  the  DNA  planorm  (synthe3c  design,  controlled  EP  delivery,  gene  adjuvants)  make  it  a    stronger  cellular  driving  planorm  in  NHP  &    the  clinic  (HVTN  080,  HPV-‐001)  

•  Adjuvant  Plasmids  are  an  important  part  of  the  potency  of  this  planorm  •  Improved  formula3ons  and  beNer  an3gens  result  in  improved  an3body  

responses  driven  by  E-‐DNA  alone  •  Boos3ng  with  Protein  an3gen  further  drives  the  magnitude  of  responses,  

Breath  is  enhanced  by  the  E-‐DNA  prime.    •  New  EP  and  concentrated  formula3ons  delivered  to  the  skin  appear  to  

improve  an3body  responses  •  E-‐DNA  combined  with  viral  vectors  extend  their  u3lity  in  both  prime  boost  

as  well  as  boost  prime  protocols.  •  Many  unknown  factors-‐  intrinsic  immunogenicty,  3ming  in  protocols  

phenotype  of  unique  prime  boost  combina3ons  etc.    

Prior DNA Enhanced -Synthetic DNA

NIH/DAIDS Mike Pensiero Chris Butler Jeff Pulen

Jim Bradac Nancy Miller Jon Warren

Carl Dieffenbach

Mark Connors

HVTN Julie McElrath, Larry Corey, Jim Kublin &

Spyros Kalams, Nicole Fram

& the entire 070 and 080 Teams as well as the study volunteers

Merck John Shiver

Danny Cassimiro Adam Finnefrock

Andrew Bett

Duke David Montefiori Celia LeBranch

Guido Ferrari Justin Pollara

Penn Mike Betts

Morgan Reuter Sally Yuan

George Makedonas Pablo Tebas

BMS Maria Jure Kunkel

Pfizer (Wyeth) Vaccine Discovery

Kathrin Jansen Emilio Emini

Inovio Jian Yan

Amir Khan Kate Broderick

Matt Morrow Feng Lin

Xuefei Shen Steven Kemmerer

Jessica Lee Mary Giffear

Niranjan Sardesai Mark Bagarazzi

J.J. Kim

VGXi Eddie Park Rob Juba

Moonsup Jeong Young Park

USF Ken Ugen

Tulane Preston Marx Bapi Pahar

Meredith Hunter Tessa Williams

UAB Jiri Mestecky

Zina Moldoveanu

Penn Clinical Jean Boyer

Ann Lan Dai Mina Naji

Lindsey Phillipson-Weiner

Rita Tamburrino

DBW Lab Natalie Hutnick

Muthumani Karuppiah Panyupa Pankhong Bernadette Ferraro

Devon Shedlock Devin Myles

Kendra Talbott Veronica Scott Danny Choo Billie Bian

Obie Nyamekye • Dan Villarreal • Colleen Lucke

Drexel Michele Kutzler Noshin Kathuria

Al Sylvester Jeff Jacobsen

Novartis Susan Barnet

Jeff Ulmer

NYU Susan Zoller Pazner

Disclosure:  DBW  notes   several   commercial   rela3onships   -‐     These   include  Consul3ng,  Board  Service,  Stock,  SRA,  Speaking,  others  etc.  with  -‐  Novar3s,    Inovio,  VGXi,  BMS,  Medarex,  Pfizer,  Virxsys,  Merck,  Althea,  among  others  which  are  managed  through  the  Penn  COI  commiNee.    

Related  Funding  P01AI071739-‐01A1  

U19  AI078675      P01  AI082282    

MVI  research  award