Alphavirus-Vectored Vaccines:
From VRP to SAM® Vaccines
Christian Mandl, PhD, MD
Novartis Vaccines and Diagnostics, Cambridge, MA USA
Munich, May 30th, 2013
Viral Vectors
Why a viral vector vaccine ?
 Viral vectors provide a number of advantages as vaccine
candidates
• In vivo expression
• Comprehensive immune response (durability similar to live vaccine?)
• Platform for multiple and complex antigens
 Virus-like replicon particles (VRP) derived from
Alphaviruses are a single-cycle infection system
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Production of alphavirus replicon particles (VRPs)
m7G 5’ nsProteins
Replicase
G.O.I.
3’ A (n)
Amplification
m7G
Glyco (E1,E2)
m7G
Capsid (C)
G.O.I. 3’ A (n)
m7G 5’ nsProteins
(G.O.I.= Gene of Interest)
(= Vaccine antigen)
GOI
Production of large amounts of
particles encoding antigen
A (n)
A (n)
C, E1, E2
GOI
GOI
GOI
| Alphavirus-Vectored
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SAM vaccine
Review
| A. Geall
Sep
2012
| |SAM
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CMV Phase I Clinical Trial Design
AlphaVax, Inc.
 CMV vaccine candidate: bivalent virus-like replicon particle
(VRP) vaccine (gB VRP & pp65/IE1 VRP)
 Randomized, double-blind, placebo control
 CMV seronegative men and women 18-45 y/o (40 subjects)
 Dose / Route / Immunization
• Two dosage levels [1 x 107 or 1 x 108 Infectious Units (IU)]
• Two routes of administration (intramuscular and subcutaneous)
• One VRP given per arm (gB and pp65/IE1)
• Three injections (weeks 0, 8 and 24)
Bernstein et al., 2010. Vaccine 28, 484-493
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Abbreviations: SC, subcutaneous; IM, intramuscular; GMT, geometric mean titer
Bernstein et al., 2010. Vaccine 28, 484-493
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pp65-Specific IFN- ELISPOT- LD vs. HD
SFCs/10^6 PBMCs: Mean and SE
800
*
Placebo
Low Dose
High Dose
*
600
*
*
*
400
*
**
*
*
200
*
*
4
9
*
*
*
*
*
0
0
1°
1
2°
week 8
* Denotes statistical significance at the 0.05 level
compared to Week 0 time point. p values: Wilcoxon
matched-pairs signed-rank test.
12
24
25
28
39
52
3°
Study Week
Bernstein et al., 2010. Vaccine 28, 484-493
Alphavirus-Vectored
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Non-viral delivery of self-amplifying mRNA vaccines
The vision
• Reinvent the gene vaccine
• Develop a synthetic vaccine with in vivo properties of a viral vectored vaccine
• Without the complications of cell culture production and anti-vector immunity
• That is safe, scalable and widely applicable (platform)
SAM™ vaccine
Viral Delivery
Lipid nanoparticle (LNP)
Conversion to a
synthetic, safer
delivery system
RNA
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| Munich GVN
How do SAM® vaccines work?
Synthetic vaccine without the complications of a packaging cell line,
contamination with replication competent virus and anti-vector immunity
RNA is enzymatically
synthesized in vitro
from a DNA template
RNA is delivered, e.g. in
a liposomal formulation,
to cytoplasm of host cells
RNA self-amplifies in the
vaccinee and expresses
vaccine antigen (or
therapeutic molecule)
Amplification
+ self-adjuvant
Expression
Potent immune
response
8 | Alphavirus-Vectored Vaccines | Christian Mandl | May 30th, 2013 | Munich GVN meeting |
Mechanism of non-viral delivery of SAM vaccine
Like other viral vectors, SAM should “fool” immune system by hitting PRRs
RNA + Delivery System
Endocytosis
- Non-structural
proteins
Direct fusion
3’
Genomic (+) RNA
5’
AAAAn
GOI
m7G-
Translation of nsp’s & synthesis of
complementary RNA
Endosomal release/
decomplexation
RDRP
3’
5’
Replication
3’
5’
m7G-
UUUUn
GOI
Genomic (-) RNA
GOI
Nucleus
Innate immune response
(via MDA-5, RIG-I, PKR, etc.)
RDRP
AAAAn
m7G-
m7G7
m Gm7Gm7G7
m G-
Protein of interest
9 | Alphavirus-Vectored Vaccines | Christian Mandl | May 30th, 2013 | Munich GVN meeting |
5’
Transcription
3’
AAAAn
GOI
AAAAn
GOI
AAAAn
GOI
AAAAn
GOI
AAAAn
GOI
AAAAn
GOI
Translation
RNA synthesis and purification
RNA synthesis
Enzymatic transcription reaction from a DNA template
T7 RNA polymerase
3’
3’
5’
RNA transcript
DNA template
Base
P-P-P
kit-independent IVT
5’
Ambion MegaScript
Promoter region
NTP
 In vitro transcription reactions extensively described in the literature
 Commercial kits available for small scale production (≈ 1 mg)
9 kb
 GMP production of smaller (≈ 2 kb) mRNA already established
 While production of larger (≈ 9 kb) RNA will add additional
challenges, these are not insurmountable
11 | Alphavirus-Vectored Vaccines | Christian Mandl | May 30th, 2013 | Munich GVN meeting |
yield [mg/ml] 5.7 5.0
Producing RNA at a commercial scale
Does not require cell culture, complex purification, or novel equipment
1) Cell-free
synthesis
RNA is produced from a
DNA template using
an enzymatic
transcription reaction
2) Purification
3) Sterile
filtration
Enzymes and DNA
fragments are removed
RNA drug
substance
0.2 µm filter
300 µg/ml
 Composition of the transcription reaction is well defined and a simple
proprietary purification process yields >99.5% pure RNA
 Process is generic and not construct specific
 Research scale process (2 ml transcription reaction) yields 10+ mg of RNA
drug substance, equivalent to 100+ human doses (100 µg RNA)
12 | Alphavirus-Vectored Vaccines | Christian Mandl | May 30th, 2013 | Munich GVN meeting |
Producing RNA at a commercial scale
Does not require cell culture, complex purification, or novel equipment
RNA production compared to protein subunit
RNA (100 µg human dose, 90% yield)
4000 ml
 4 ml (5 mg/ml RNA) = 180 doses
 40 ml (5 mg/ml RNA) = 1,800 doses
Protein subunit (50 µg human dose, 20% yield)
 4000 ml (0.05 - 0.15 mg/ml protein) = 800 – 2,400
doses
13 | Alphavirus-Vectored Vaccines | Christian Mandl | May 30th, 2013 | Munich GVN meeting |
4 ml
Non-viral delivery
Non-viral delivery with Lipid nanoparticles (LNPs)
Encapsulation technology based on scaled-down industrial process
Component
D=141 nm
Function
Neutral Lipid
Particle base
Cationic Lipid
RNA Loading
Cholesterol
Particle stabilization
PEG - Lipid
Particle stabilization
Charge shielding
Self-replicating
RNA
15 | Alphavirus-Vectored Vaccines | Christian Mandl | May 30th, 2013 | Munich GVN meeting |
Antigen expression
Delivery system protects RNA from RNases
1
9 kb RNA
2
3
4
5
Self-amplifying RNA
protected from Rnase
degradation
(1)RNA ladder
(2)Self-amplifying RNA
(3)Self-amplifying RNA after exposure to RNase A
(4)Phenol-chloroform extraction of self-amplifying RNA from an LNP
(5)Phenol-chloroform extraction of self-amplifying RNA from an LNP
after exposure to RNase A
16 | Alphavirus-Vectored Vaccines | Christian Mandl | May 30th, 2013 | Munich GVN meeting |
In vivo data
LNP delivery enhances gene expression
Secreted embryonic alkaline phosphatase (SEAP)
In vivo SEAP expression 6 days after i.m. injection of 1 µg self-amplifying RNA in phosphate buffered saline (RNA),
0.1 µg self-amplifying RNA encapsulated in an LNP (LNP/RNA), and 0.1 µg self-amplifying RNA mixed (not
encapsulated) with LNP (LNP + RNA).
18 | Alphavirus-Vectored Vaccines | Christian Mandl | May 30th, 2013 | Munich GVN meeting |
RSV
Fusion
protein (F)
 Member of the family Paramyxoviridae
 Enveloped virion
 Single stranded, negative polarity RNA
genome encoding 11 viral proteins
 Primary target in host is respiratory
epithelial cells
Attachment
glycoprotein (G)
 Antibodies generated to viral surface proteins – F and G
• These antibodies neutralize the virus and inhibit infection
 CD4 and CD8 T cells respond to several viral proteins
• Neither subset is required for protection, but either can afford protection
19 | Alphavirus-Vectored Vaccines | Christian Mandl | May 30th, 2013 | Munich GVN meeting |
RSV-SAM®
vaccinate vaccinate
(i.m.)
(i.m.)
Potency of
vaccine
comparable to viral delivery technology Day: 0
21
*
35
49
RSV F-specific IgG titers
Mouse immunogenicity study of a LNP/RNA vaccine candidate encoding RSV F. 8 mice/group (except LNP/DNA, 4 mice/
group) were vaccinated on days 0 and 21, and sera were collected on day 35. F-specific IgG titers were determined by
ELISA. Data are from individual mice (depicted as dots), and the geometric mean titers are depicted as solid lines.
20 | Alphavirus-Vectored Vaccines | Christian Mandl | May 30th, 2013 | Munich GVN meeting |
Seroconversion after a single dose
of RSV-SAM® vaccine
(a) RSV F-specific IgG titers 2wp1
*
vaccinate vaccinate
(i.m.)
(i.m.)
Day: 0
*
14 21
*
35
49
(b) RSV F-specific IgG titers 2wp2
*
Mouse immunogenicity studies of a LNP/RNA candidate vaccine encoding RSV F. 8 mice/group were vaccinated on days
0 and 21, and serum was collected on days 14 and 35. F-specific IgG titers were determined by ELISA for (a) 2wp1 and
(b) 2wp2. Dots depict measurements from individual mice and solid bars depict the geometric mean titers
21 | Alphavirus-Vectored Vaccines | Christian Mandl | May 30th, 2013 | Munich GVN meeting |
vaccinate vaccinate RSV challenge
(i.m.)
(i.m.)
(i.n.)
*
RSV-SAM®
vaccine protects cotton
rats from viral challenge
(b) IgG titers 2wp2
*
p<0.05
10 3
10
2
10 1
10 0
49 54
10 6
10 5
ns
p<0.05
10 4
10 3
10 2
10 1
10 0
10 7
10 6
10 5
10 4
10 3
10 2
10 1
no
ne
LN RN
P/ A
R
N
A
VR
F/ P
al
um
ns
no
ne
LN RN
P/ A
R
N
A
VR
F/ P
al
um
10 4
35
p<0.05
RSV F-specific IgG titer
10 5
no
ne
LN RN
P/ A
R
N
A
VR
F/ P
al
um
60% RSV neutralization titer
p<0.05
21
(c) Lung viral load
*
RSV pfu/g lung
(a) Neut titers 2wp2
Day: 0
Cotton rat data demonstrating potency and efficacy of a RNA vaccines encoding RSV F. Groups of 8 rats were
vaccinated i.m. on days 0 and 21 with naked RNA (1 μg), LNP/RNA (1 μg), VRP (5 x 106 IU ), alum-formulated RSV F
protein (10 μg), or were not vaccinated. All animals were challenged intranasally with 1 x 10 5 pfu RSV on day 49. (a)
Serum RSV neutralization titers and (b) serum F-specific IgG titers 2 weeks after the second vaccination (day 35). (c)
Lung viral load 5 days after the RSV challenge (day 54).
22 | Alphavirus-Vectored Vaccines | Christian Mandl | May 30th, 2013 | Munich GVN meeting |
HIV
Envelope surface glycoprotein
(gp120)
Viral Polymerase
(Pol)
 Lentivirus, member of the family Retroviridae
 Enveloped virion
Envelope transmembrane protein
(gp41)
 2 copies of positive-stranded RNA
genome encoding 9 viral proteins
 Primary target cells in host are CD4+ T lymphocytes,
macrophages, dendritic cells
 Antibodies generated to viral surface protein (gp120) and
transmembrane protein (gp41)
• These antibodies neutralize the virus and inhibit infection
 CD4+ and CD8+ T cells respond to several viral proteins
23 | Alphavirus-Vectored Vaccines | Christian Mandl | May 30th, 2013 | Munich GVN meeting |
Gag proteins
(p17 and p24)
HIV SAM vaccine expressing HIV-1 gp140 is potent in NHPs
6/6 rhesus macaques seroconverted after the 2nd vaccination
9.00
 Regimen simulates the
VRP (1x108 IU)
7.00
prime-boost used in RV144
log10 Env-specific IgG titer
5.00
3.00
1.00
9.00
7.00
Neutralization titers (week 38)*
HIV SAM vaccine (50 μg)
5.00
3.00
1.00
9.00
7.00
Env/MF59 (100 μg)
5.00
3.00
SAM, VRP, or protein prime
1.00
0 4 8 12162024283236404448
*
protein boost
Bogers et al., and Geall et al., AIDS Vaccine 2012, Boston, MA
24 | Alphavirus-Vectored Vaccines | Christian Mandl | May 30th, 2013 | Munich GVN meeting |
HIV SAM vaccine expressing HIV-1 gp140 is potent in NHPs
Macaques demonstrated a stronger T cell response after the 2nd vaccination
VRP
HIV SAM
ENV/MF59
 Median data from IFNgamma Elispots on
peptide pool-pulsed
PBMCs document the
T-cell potency of HIV
SAM, which in this
study was superior to
VRPs
SAM, VRP or protein prime
protein boost
Bogers et al., and Geall et al., AIDS Vaccine 2012, Boston, MA
25 | Alphavirus-Vectored Vaccines | Christian Mandl | May 30th, 2013 | Munich GVN meeting |
CMV
 Perinatal transmission rate to
infants in utero in CMV
seronegative women exposed
during pregnancy is ~40%
• ~ 1% (86,000) newborn infections
each year in EU & US; resulting in
~17,500
serious permanent outcomes
including death
• Leading viral cause of birth
defects in US (mental retardation,
hearing loss, other)
 Reactivation of CMV due to immunosuppression in transplant and
HIV/AIDS patients is a major cause of mortality
26 | Alphavirus-Vectored Vaccines | Christian Mandl | May 30th, 2013 | Munich GVN meeting |
SAM technology can also be used to deliver complex antigens
LNP-formulated replicons that encode more than one antigen are potent in mice
 Mice vaccinated with gH/gL SAM vaccine
produced antibodies that neutralized the
infection of hCMV clinical isolates (VR1814
and TB40) on human epithelial cells
(ARPE-19)
 Neutralizing titers in sera from mice
immunized with gH/gL SAM are
substantially higher than those from mice
immunized with gB SAM
 Combinations of gH/gL SAM with gB SAM
did not detectably increase neutralization
Loomis et al., 2013, Vaccine 31:919
27 | Alphavirus-Vectored Vaccines | Christian Mandl | May 30th, 2013 | Munich GVN meeting |
gB SAM
gH/gL
SAM
gB + gH/gL gB/gH/gL
SAM
SAM
Summary
 Proof of concept achieved for the SAM® vaccines
 Potential as a platform technology to address multiple disease targets
 Immune responses are comparable to a viral vectored vaccine
 HIV-SAM® vaccine is immunogenic in NHPs at a 50 μg RNA dose
 Next steps
 Establish GMP production process and test in human clinical trials
 Explore additional disease targets
 Develop next generation vaccines
 Improved vectors
 Optimized and new delivery systems
28 | Alphavirus-Vectored Vaccines | Christian Mandl | May 30th, 2013 | Munich GVN meeting |
Acknowledgements
Formulations
• Luis Brito
• Michelle Chan
• Siddhartha Jain
• Sushma Kommareddy
• Madhura Shidhore
• Ayush Verma
• Yen Cu
• Amanda Scampini
Immunology
• Jason Debasitis
• Christine Dong Lee
• Lamine Mbow
• Pu Zhang
• Katrin Ramsauer
• Mary Schaefer
• Christy Shaw
• Tina Scalzo
• Alberto Visintin
• Kaustuv Banerjee
• Carlo Iavarone
• Flaviana Mosca
• Igor Leykin
• Anne-Marie Pulichino
MMB
• Jake Archer
• Kara Balabanis
• Armin Hekele
• Mithra Rothfeder
• Melissa Sackal
• Avi Nandi
• Anders Lilja
• Becky Loomis
• Scilla Buccato (Siena)
Protein Biochemistry
• Yingxia Wen
• Kyoko Uehara
• Ethan Settembre
• Kurt Swanson
Serology
• John Donnelly
• Giuseppe Palladino
• Jim Monroe
• Kristian Friedrich
RNA Core Team
• Andy Geall (RNA
platform leader)
• Christian Mandl
• Jeff Ulmer
• Derek O’Hagan
• Manmohan Singh
• George Santos
• Peter Mason
• Clayton Beard
• Nick Valiante
• Gib Otten
• Drew Natenshon
• Phil Dormitzer
• Andrea Carfi
Others in NV&D
• Susan Barnett
• Hetal Patel
• Karen O’Brien
• Gene Palmer
29 | Alphavirus-Vectored Vaccines | Christian Mandl | May 30th, 2013 | Munich GVN meeting |
BPRC, Netherlands
• Willy Bogers
• Herman Oostermeijer
• Petra Mooij
• Gerrit Koopman
• Ernst Verschoor
• David Davis
Duke University
• David Monfefiori
• Celia LaBranche
Funding
• NIH P01AI066287,
Susan Barnett, PI
Backup slides
Kinetics of gene expression
In vivo bioluminescence imaging (RNA/DNA encoding luciferase)
VRP (day 7)
LNP (day 7)
DNA + EP (day 7)
Average radiance over 63 days after administration of self-amplifying RNA (1 μg) encapsulated in LNP, electroporated
pDNA (10 μg) or of VRPs (1 x 106 IU). The limit of detection is 1.0 x 103, as indicated by the dashed line.
31 | Alphavirus-Vectored Vaccines | Christian Mandl | May 30th, 2013 | Munich GVN meeting |
SAM® vaccine research is being published/reviewed
in high profile journals: PNAS & Nature Reviews
32 | Alphavirus-Vectored Vaccines | Christian Mandl | May 30th, 2013 | Munich GVN meeting |