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

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20 m 4-14-2003 12-13-2004

SARS Gene-based delivery including mRNA, DNA, and vectored approaches

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H5 Indonesia/06

Self-assembling nanoparticles and VLPs 4 m 4-27-2009 8-27-2009

H1 California/09 Nucleic Acid Vaccines – DNA vs. mRNA

Protection

Muscle cells Antibodies produced by B cells

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Lymph node Needle-free Delivery Devices for DNA Vaccines

Gene Gun

Biojector® Pharmajet® Influenza Vaccine Clinical Trials

DNA Priming and Influenza Vaccine Immunogenicity: Two Phase 1 Open Label Randomised Clinical Trials Prime-boost interval matters: A randomized phase I study to Ledgerwood JE, et al. the VRC 306 and 310 Study identify the minimum interval to observe the H5 DNA influenza Teams. vaccine priming effect. Lancet Infect Dis. 2011 ; 11:916-924. 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. West Nile Virus VRC DNA Vaccine

Ab (EC50) by RVP neutralization assay responses

May 15, 2011

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.

Ledgerwood et al. J Infect Dis. 2011;203:1396-1404 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 • 5’&3’ UTR engineering • LNP GSK Induces large response • Cationic Nano-emulsions • SAM • LNP Moderna Naturally-occurring modified • LNP • Codon Optimization nucleotides • 5’&3’ UTR engineering

Curevac Rabies mRNA Vaccine Shows Protection Against a Rabies Challenge in Mice

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 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)

Innate Immune Sensing of SAM

Replication Machinery

• 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.

Deering RP, et al.Expert Opinion on Drug discovery. 2014 GSK SAM formulation: Cationic Nanoemulsion (CNE)

Brito LA, et al. Advances in Genetics.2015 McCullough KC, et al. Vaccines.13 2014 Self-amplifying mRNA Elicits Potent Humoral and Cellular Protective Immunity

RSV F in mice

Neutralizing antibodies Viral load CD4+ T cells CD8+ T cells

p<0.05

10 5 ns 10 7

10 4 p<0.05 10 6 10 5 10 3 10 4 10 2 10 3 1 10 lung pfu/g RSV 10 2

10 0 10 1 60% neutralization RSV titer

VRP none RNA none RNA VRP F/alum F/alum LNP/RNA LNP/RNA

Geall et al. Proc. Natl. Acad. Sci., 2012 adapted from Rino Rappuoli In vitro Synthesized Alternative Nucleoside mRNA mRNA synthesized in vitro using alternative nucleosides Immune Activation (pseudouridine, 5-methylcytidine or 1-methylpseudouridine) Poly(I:C)

Uridine Pseudouridine Unmodified mRNA

No mRNA

Pseudouridine mRNA

Level of Translation 5-Methylcytidine 1-Methylpsuedouridine

Pseudouridine mRNA

Unmodified mRNA Less immune activation=greater and longer translation

Kariko, K et al. Molecular Therapy. 2008 15 Anderson BA et al. Nucleic Acids Res. 2011 Nov. mRNA Encapsulated in Lipid Nanoparticles

Acuitas

Advantages of LNPs • Protects mRNA from degradation • Enhances uptake into endosomes • Delivers mRNA into cytoplasm White - PEGylated lipids • Can potentially incorporate immunomodulators Green – lipids Cationic lipids Yellow - mRNA Phosphatidylcholine or targeting molecules Cholesterol 16

• RNA Protection • Translation efficiency • Cellular uptake and delivery • Modulation of indiscriminate Innate Immunity through the ER recognition • Cellular targeting • Ribosomal initiation and processivity • Immune stimulation ? • mRNA half-life • Protein sequence and activity • Cellular-specific Expression modulation

Slide 17 Confidential and Proprietary · © 2017 Valera Moderna H7 Influenza mRNA Vaccine

Human NHP

Bahl K et al. Mol Ther. 2017 ZIKV DNA Vaccine Development

2013 2014 2015 2016 2017 Zika Virus Outbreak ?

2013 - 2014 May 2015 Feb 2016 Feb 10, 2016 July 2016 July 21, 2016 Aug 2, 2016 Dec 12, 2016

Zika virus outbreak 1st reports of Zika virus Pre-IND Product IND First vaccination First vaccination Apr 24, 2016 French Polynesia Zika infection spread to over Release Submission VRC 319 VRC 320 in Brazil 20 countries (Phase 1) (Phase 1) in Western DNA Vaccine hemisphere Sequence Selected Mar 30, 2017 July 2015 Jan 2016 Oct 14, 2016 May 23, 2016 June 2016 Preclinical Dec 2015 First vaccination data published Alerted to Zika Initial ZIKV In Phase 2a/2b IRB Review problem (doenca constructs misteriosa) by received Drug Product Brazilian physician Manufacturing Reagents designed to explore potential for Zika vaccine development

Projected Date

19 ZIKV prM-E Constructs

Side view Top view

Numbering based on H/PF/2013 (GenBank# AHZ13508.1) Devika Sirohi et al. Science 2016;science.aaf5316 DNA Vaccine-Induced Antibody and Protection

NT Antibody Viral Load Post-Challenge Fold-Change in NT 5 6

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Weeks Post-Vaccination Days Post-Challenge Weeks Post-Vaccination 21 VRC/NIAID/NIH Confidential 1st Candidate: Phase I Clinical Trial, VRC 319 (plasmid 5288)

Enrolled: Aug 2– Sept 30, 2016 Sites: NIH, UMD, Emory

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 Principal Investigators: Julie Ledgerwood (NIH) 3 20 X X X Srilatha Edupuganti (Emory) Monica McArthur (UMD Baltimore) 4 20 X X X Total 80 All injections are ZIKV DNA vaccine, 4 mg/mL 5283-Induced Antibody is Qualitatively Better than 5288 VRC5283 VRC5288 Neutralizing Antibody Titers Post-Challenge Viral Load Post-Challenge Viral Load Week 8 Fold Change in NT Fold Change in NT 4 p=0.0068

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VRC/NIAID/NIH Confidential 2nd Candidate: Phase I Clinical Trial, VRC 320 (plasmid 5283)

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 Administration Administration Schedule Group Subjects Method 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 All injections are ZIKV DNA vaccine, 4 mg/mL

Enrolled: Dec 12, 2016 – April 19, 2017 at NIH

PI: Grace Chen (NIH) Accelerated Planning: Phase 2/2b Zika DNA WT Vaccine Candidate (plasmid 5283)

A Phase 2b, Randomized Trial to Evaluate the Safety and Immunogenicity of a Zika Virus DNA Vaccine Healthy Volunteers Ages 15-35

Part B proceeds if Phase 1 and Part A results promising

>20 sites in the US, Caribbean, Central and South America

Blinded evaluation of case rates to increase sample size as needed

Enrollment started March 30, 2017 Protocol Chairs: Julie Ledgerwood and Grace Chen IND Sponsor: VRC/NIAID 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 Neutralizing antibodies

Differences from Moderna ZIKV Vaccine • Intradermal delivery • MHC class II signal peptide Viral Load • Variations in 5’ cap, poly-A tail, GC content

Rhesus Macaques

Pardi N, et al. Nature 2017 Feb 2. doi: 10.1038/nature21428. Summary and Outstanding Questions

• 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 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 Vaccine Research Center Companies John Mascola Mario Roederer Moderna - Giuseppe Ciaramella Richard Koup Daniel Douek Curevac –Susanne Rauch Jason Gall Robert Seder GSK – Dong Yu, Rino Rappuoli Peter Kwong Nancy Sullivan NIAID Other VRC Adrian McDermott Judy Stein Ted Pierson Wing Pui Kong Abe Mittelman Marybeth Daucher Anthony Fauci Sung-Youl Ko Julie Ledgerwood & Clinical Trial Program Hilary Marston Wei Shi Diane Scorpio & Animal Care Program Richard Schwartz &Vaccine Production Program David Lindsay & Vaccine Clinical Material Program Production of Zika Virus DNA Vaccines Moderna Preclinical Data

Model: C57Bl/6 Mice with IFN blockage before challenge

Richner J et al. Cell 2017