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