nucleic acid vaccine technologies: dna and mrna · nucleic acid as a vaccine platform technology 6...
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Nucleic Acid Vaccine Technologies:
DNA and mRNA
Barney S. Graham, MD, PhD
Deputy Director
Vaccine Research Center, NIAID, NIH
Nucleic Acid as a Vaccine Platform Technology
6 18 12
12-21-2006 2-11-2006
8-27-2009 4-27-2009
4 m
12-13-2004 4-14-2003 20 m
11 m
H5 Indonesia/06
H1 California/09
SARS Gene-based delivery including mRNA, DNA, and vectored approaches
Self-assembling nanoparticles and VLPs
X
Nucleic Acid Vaccines – DNA vs. mRNA
Lymph node
Antibodies produced by B cells
Protection
Muscle cells
Needle-free Delivery Devices for DNA Vaccines
Biojector®
Gene Gun
Pharmajet®
Influenza Vaccine Clinical Trials
Prime-boost interval matters: A randomized phase I study to identify the minimum interval to observe the H5 DNA influenza vaccine priming effect. Ledgerwood JE, et al. and VRC 310 study team JID 2013; 208:418-422. DNA priming prior to H5N1 inactivated influenza vaccination expands the antibody epitope repertoire and increases affinity maturation in a boost-interval-dependent manner in adults. Khurana S, et al. and VRC 310 study team JID 2013; 208:413-17.
DNA Priming and Influenza Vaccine Immunogenicity: Two Phase 1 Open Label Randomised Clinical Trials
Ledgerwood JE, et al. the VRC 306 and 310 Study Teams.
Lancet Infect Dis. 2011 ; 11:916-924.
West Nile Virus VRC DNA Vaccine
Ledgerwood et al. J Infect Dis. 2011;203:1396-1404
Ab (EC50) by RVP neutralization assay responses
A West Nile Virus DNA Vaccine Utilizing a
Modified Promoter Induces Neutralizing
Antibody in Younger and Older Healthy
Adults in a Phase I Clinical Trial.
Ledgerwood JE, the VRC 303 Study Team,
et al.
May 15, 2011
6
Major mRNA Platforms
• Conventional GC-rich nucleotides with protamine or lipid formulation
– CureVac (Tübingen, Germany)
• Self-amplifying mRNA
– GSK (from Novartis)
• In vitro synthesized alternative nucleoside mRNA
– Drew Weissman, UPenn
– Moderna/Valera
Company Innate Immunity Modulation Formulation Translation
Curevac “GC-Enrichment” algorithm • Protamine
• LNP
• 5’&3’ UTR engineering
GSK Induces large response • Cationic Nano-emulsions
• LNP
• SAM
Moderna Naturally-occurring modified
nucleotides
• LNP • Codon Optimization
• 5’&3’ UTR engineering
Fig 3. Protective capacity of mRNA vaccine against lethal intracerebal (i.c.) rabies challenge infection.
Schnee M, Vogel AB, Voss D, Petsch B, Baumhof P, et al. (2016) An mRNA Vaccine Encoding Rabies Virus Glycoprotein Induces Protection against
Lethal Infection in Mice and Correlates of Protection in Adult and Newborn Pigs. PLOS Neglected Tropical Diseases 10(6): e0004746.
doi:10.1371/journal.pntd.0004746
http://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0004746
Curevac Rabies mRNA Vaccine Shows Protection
Against a Rabies Challenge in Mice
GSK Self-Amplifying mRNA (SAM) Platform
Alphavirus-based SAM platform amplifies genome >100 fold
greater than other viral vectors
Slide courtesy of Rino Rappuoli
Self-Amplifying mRNA (SAM)
Replication Machinery
Innate Immune Sensing of SAM
Deering RP, et al.Expert Opinion on Drug discovery. 2014
• Self-contained RNA-dependent RNA polymerase • Amplicon/replicon - High level of transcription/translation • “Self-adjuvanted” -Triggers innate responses
Geall AJ, et al. Nonviral delivery of self-amplifying RNA vaccines.
PNAS U S A 2012; 109(36):14604-9.
GSK SAM formulation: Cationic Nanoemulsion (CNE)
13 Brito LA, et al. Advances in Genetics.2015 McCullough KC, et al. Vaccines. 2014
none
RNA
LNP/R
NA
VRP
F/alum
10 1
10 2
10 3
10 4
10 5
10 6
10 7
RS
V p
fu/g
lung
none
RNA
LNP/R
NA
VRP
F/alum
10 0
10 1
10 2
10 3
10 4
10 5
p<0.05
ns
p<0.05
60
% R
SV
ne
utr
aliz
atio
n t
ite
r
Neutralizing antibodies Viral load
Geall et al. Proc. Natl. Acad. Sci., 2012
CD8+ T cells CD4+ T cells
Self-amplifying mRNA Elicits Potent Humoral and
Cellular Protective Immunity
RSV F in mice
adapted from Rino Rappuoli
In vitro Synthesized Alternative Nucleoside mRNA
15
Immune Activation
Unmodified mRNA
Poly(I:C)
No mRNA
Pseudouridine mRNA
Level of Translation
Unmodified mRNA
Pseudouridine mRNA
Less immune activation=greater and longer translation
Kariko, K et al. Molecular Therapy. 2008 Anderson BA et al. Nucleic Acids Res. 2011 Nov.
mRNA synthesized in vitro using alternative nucleosides (pseudouridine, 5-methylcytidine or 1-methylpseudouridine)
Uridine Pseudouridine
5-Methylcytidine 1-Methylpsuedouridine
mRNA Encapsulated in Lipid Nanoparticles
16
White - PEGylated lipids Green – lipids Yellow - mRNA
Acuitas
Advantages of LNPs • Protects mRNA from degradation • Enhances uptake into endosomes • Delivers mRNA into cytoplasm • Can potentially incorporate immunomodulators
or targeting molecules Cationic lipids Phosphatidylcholine Cholesterol
Slide 17 Confidential and Proprietary · © 2017 Valera Confidential and Proprietary · © 2017 Valera
• RNA Protection
• Cellular uptake and delivery
through the ER
• Cellular targeting
• Immune stimulation ?
• Translation efficiency • Modulation of indiscriminate Innate Immunity
recognition
• Ribosomal initiation and processivity
• mRNA half-life
• Protein sequence and activity
• Cellular-specific Expression modulation
Moderna H7 Influenza mRNA Vaccine
Bahl K et al. Mol Ther. 2017
NHP
Human
June 2016
Drug Product
Manufacturing
19
ZIKV DNA Vaccine Development
May 23, 2016
IND
Submission
IRB Review
2013
Oct 14, 2016
First vaccination
VRC 319
(Phase 1)
Zika virus outbreak
French Polynesia
Alerted to Zika
problem (doenca
misteriosa) by
Brazilian physician
Dec 2015
2016 2014 2015
2013 - 2014
Zika virus
spread to over
20 countries
in Western
hemisphere
Feb 2016
Product
Release
July 2016 July 21, 2016
Projected Date
Feb 10, 2016
Pre-IND Apr 24, 2016
Zika Virus Outbreak ?
Mar 30, 2017
First vaccination
In Phase 2a/2b
DNA Vaccine
Sequence
Selected
2017
May 2015
July 2015
1st reports of
Zika infection
in Brazil
Initial ZIKV
constructs
received
Jan 2016
Reagents
designed to
explore potential
for Zika vaccine
development
Aug 2, 2016
Preclinical
data published
Dec 12, 2016
First vaccination
VRC 320
(Phase 1)
ZIKV prM-E Constructs
Devika Sirohi et al. Science 2016;science.aaf5316
Top view Side view
Numbering based on H/PF/2013 (GenBank# AHZ13508.1)
21
VRC5283 4mg X2
VRC5283 1mg X2
VRC5288 4mg X2
VRC5288 1mg X1
VRC8400 control
Viral Load NT Antibody
Days Post-Challenge Weeks Post-Vaccination Weeks Post-Vaccination VRC/NIAID/NIH Confidential
Post-Challenge Fold-Change in NT
1 2 3 4 5 6 71
2
3
4
5
6
Lo
g10 G
en
om
e c
op
ies/m
l VRC8400, 4mg x2
1 2 3 4 5 6 71
2
3
4
5
6
Lo
g10 G
en
om
e c
op
ies/m
l VRC5283, 4mg x2
1 2 3 4 5 6 71
2
3
4
5
6
Lo
g10 G
en
om
e c
op
ies/m
l VRC5283, 1mg x2
1 2 3 4 5 6 71
2
3
4
5
6
Lo
g10 G
en
om
e c
op
ies/m
l VRC5288, 4mg x2
1 2 3 4 5 6 71
2
3
4
5
6
Lo
g10 G
en
om
e c
op
ies/m
l VRC5288, 1mg x1
0 1 2 3 4 5 6 7 81
2
3
4
5
Lo
g10 E
C50 D
ilu
tio
n-1
VRC8400, 4mg x2
0 1 2 3 4 5 6 7 81
2
3
4
5
Lo
g10 E
C50 D
ilu
tio
n-1
VRC5283, 4mg x2
0 1 2 3 4 5 6 7 81
2
3
4
5
Lo
g10 E
C50 D
ilu
tio
n-1
VRC5283, 1mg x2
0 1 2 3 4 5 6 7 81
2
3
4
5
Lo
g10 E
C50 D
ilu
tio
n-1
VRC5288, 4mg x2
0 1 2 3 4 5 6 7 81
2
3
4
5
Lo
g10 E
C50 D
ilu
tio
n-1
VRC5288, 1mg x1
8 10 12 140
10
20
30
Neu
t T
iter
Fo
ld C
han
ge
8 10 12 140
10
20
30
Neu
t T
iter
Fo
ld C
han
ge
8 10 12 140
10
20
30
Neu
t T
iter
Fo
ld C
han
ge
8 10 12 140
100
200
300
Neu
t T
iter
Fo
ld C
han
ge
8 10 12 140
100
200
300
400
500
Neu
t T
iter
Fo
ld-C
han
ge
DNA Vaccine-Induced Antibody and Protection
VRC 319: Phase I Zika DNA
Group Subjects* Day 0 Week 4 Week 8 Week 12 Week 20
1 20 X X
2 20 X X
3 20 X X X
4 20 X X X
Total 80 All injections are ZIKV DNA vaccine, 4 mg/mL
Principal Investigators:
Julie Ledgerwood (NIH)
Srilatha Edupuganti (Emory)
Monica McArthur (UMD Baltimore)
Enrolled: Aug 2– Sept 30, 2016
Sites: NIH, UMD, Emory
1st Candidate: Phase I Clinical Trial, VRC 319
(plasmid 5288)
300μg X2
100μg X2
1mg X2
VRC5283 VRC5288
Viral Load Post-Challenge
Fold Change in NT Viral Load Post-Challenge
Fold Change in NT
VRC/NIAID/NIH Confidential
1 2 3 4 5 6 71
2
3
4
5
6
Lo
g10 G
en
om
e c
op
ies/m
l
1 2 3 4 5 6 71
2
3
4
5
6
Lo
g10 G
en
om
e c
op
ies/m
l
1 2 3 4 5 6 71
2
3
4
5
6
Lo
g10 G
en
om
e c
op
ies/m
l
8 10 120
20
40
60
Neu
t T
iter
Fo
ld-C
han
ge
8 10 120
20
40
60
Neu
t T
iter
Fo
ld-C
han
ge
8 10 120
20
40
60
Neu
t T
iter
Fo
ld-C
han
ge
1 2 3 4 5 6 71
2
3
4
5
6
Lo
g10 G
en
om
e c
op
ies/m
l
1 2 3 4 5 6 71
2
3
4
5
6
Lo
g10 G
en
om
e c
op
ies/m
l
1 2 3 4 5 6 71
2
3
4
5
6
Lo
g10 G
en
om
e c
op
ies/m
l
8 10 120
20
40
60
Neu
t T
iter
Fo
ld-C
han
ge
8 10 120
20
40
60
Neu
t T
iter
Fo
ld-C
han
ge
8 10 120
20
40
60
Neu
t T
iter
Fo
ld-C
han
ge
1mg 0.3mg 0.1mg 1mg 0.3mg 0.1mg1
2
3
4
Log
10 E
C50 D
ilution
-1
Neutralizing Antibody Titers
Week 8
VRC5283 VRC5288
p=0.0068
Red=Infected
Blue=Bump in NT
5283-Induced Antibody is Qualitatively Better than 5288
A Phase I, Randomized Clinical Trial to Evaluate the Safety and
Immunogenicity of a Zika Virus DNA Vaccine Administered via Needle and
Syringe or Needle-free Injector, PharmaJet, in Healthy Adults
VRC 320 Study Schema
Group Administration
Method Subjects
Administration Schedule
Day 0 Week 4 Week 8
1 Needle & Syringe 15 1 injection of 1 mL 1 injection of 1 mL 1 injection of 1 mL
2 Needle & Syringe 15 2 injections of 0.5 mL 2 injections of 0.5 mL 2 injections of 0.5 mL
3 PharmaJet 15 2 injections of 0.5 mL 2 injections of 0.5 mL 2 injections of 0.5 mL
Total 45
PI: Grace Chen (NIH)
Enrolled: Dec 12, 2016 – April 19, 2017 at NIH
All injections are ZIKV DNA vaccine, 4 mg/mL
2nd Candidate: Phase I Clinical Trial, VRC 320
(plasmid 5283)
A Phase 2b, Randomized Trial
to Evaluate the Safety and
Immunogenicity of a Zika Virus
DNA Vaccine Healthy Volunteers Ages 15-35
>20 sites in the US,
Caribbean, Central
and South America
Protocol Chairs: Julie Ledgerwood and Grace Chen
IND Sponsor: VRC/NIAID
Part B proceeds if Phase 1 and Part A results promising
Blinded evaluation of case rates to increase sample size as needed
Accelerated Planning: Phase 2/2b
Zika DNA WT Vaccine Candidate (plasmid 5283)
Enrollment started March 30, 2017
Search for Sites and Modeling Projections
NIH / CDC Modeling Collaboration • Estimate areas of higher attack rates
• Using 3 different modeling approaches to identify areas with high likelihood of ZIKV transmission of >5% incidence in 2017.
• Models use weather, demographic, transportation, temperature, Dengue & CHIKV rates, mosquito data and ZIKV surveillance data.
• 158 Site Evaluation Questionnaires emailed to investigators in 23
countries
• 140 Questionnaires received and evaluated
• from 20 countries
• 21 Site Evaluation Visits (SEVs) conducted
– Southern Continental USA (5)
– Puerto Rico (4)
– Brazil (5)
– Costa Rica (2)
– Mexico (1)
– Panama (1)
– Ecuador (2)
– Colombia (1)
Drew Weissman at UPENN: A single dose of alternative
nucleoside mRNA protects against ZIKV Challenge
Pardi N, et al. Nature 2017 Feb 2. doi: 10.1038/nature21428.
Differences from Moderna ZIKV Vaccine • Intradermal delivery • MHC class II signal peptide • Variations in 5’ cap, poly-A tail, GC content
Neutralizing antibodies
Viral Load
Rhesus Macaques
• DNA and mRNA vaccines are immunogenic and appear to be safe
• Evidence of efficacy in animal models
• Multiple formulations and delivery approaches
? Efficacy in adequately powered studies in humans
? Duration of immunity
? Long term safety
? Scalability of manufacturing and formulation at commercial scale
? Long term stability data and cold chain compatibility
Summary and Outstanding Questions
Viral Pathogenesis Laboratory in NIAID VRC
Top row, left-to-right: Man Chen, Masaru Kanekiyo Truck bed, back: Tracy Ruckwardt, Erez Bar-Haim, April Killikelly, Jie Liu Truck bed, front: Rebecca Gillespie, Seyhan Boyoglu-Barnum, Kizzmekia Corbett, Assanatou Bamogo, Michelle Crank Standing: Syed Moin, Brian Fisher, Azad Kumar, Joan Ngwuta, Deepika Nair, La Che Wiggins, Kaitlyn Morabito, Adrian Creanga, Monique Young Not Pictured: Leda Castilho, Emily Phung, Erez Bar-Haim, Julia Lederhofer, Rebecca Loomis, Geoffrey Hutchinson
NIAID Ted Pierson Anthony Fauci Hilary Marston
NIAID Vaccine Research Center John Mascola Mario Roederer Richard Koup Daniel Douek Jason Gall Robert Seder Peter Kwong Nancy Sullivan Adrian McDermott Judy Stein Abe Mittelman Marybeth Daucher Julie Ledgerwood & Clinical Trial Program Diane Scorpio & Animal Care Program Richard Schwartz &Vaccine Production Program David Lindsay & Vaccine Clinical Material Program
Other VRC Wing Pui Kong Sung-Youl Ko Wei Shi
Companies Moderna - Giuseppe Ciaramella Curevac –Susanne Rauch GSK – Dong Yu, Rino Rappuoli
Production of Zika Virus DNA Vaccines
Moderna Preclinical Data
Model: C57Bl/6 Mice with IFN blockage before challenge
Richner J et al. Cell 2017