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1
Recombinant, Insect Cell-Derived RSV Nanoparticle Vaccine
Gregory Glenn Chief Medical Officer MVADS-Copenhagen 4 July 2012
2 2
Agenda for RSV Discussion
• Overview of Insect Cell Technology
• Respiratory Syncytial Virus Burden of Disease and Pathophysiology
• The RSV Nanoparticle Vaccine
• Preclinical Evaluation
• Clinical Evaluation
• Overview of Future Development
3 3
Insect Cell Vaccine Technology for Manufacture of Recombinant Nanoparticle Vaccines
• Select genetic sequences encoding vaccine antigens • Genes cloned into baculovirus • Baculovirus infects insect (Sf9) cells • Antigens expressed and purified as multimeric nanoparticles • Immunogenic particle with antigens in native configuration
8/1/12 Confidential to Novavax
Genes coding for antigens cloned into baculovirus
rBaculovirus
Baculovirus-infected insect(Sf9) cells Proteins form
nanoparticles
4 4
Clinical Applications for and RSV Vaccine
• Major new target for vaccine developers
• Infects all children by age 2 years
• Is the leading cause of bronchiolitis and pneumonia in infants <1 year of age ‒ 75,000 to 125,000 hospitalizations
annually (in US)
• A leading cause of pneumonia in older adults ‒ 14,000 deaths and 175,000
hospitalizations annually
• No vaccine currently available
5 5
Respiratory Syncytial Virus (RSV)
• Fusion (F) protein – Virus entry and syncytia formation – Type I trimeric glycoprotein – F protein conserved across RSV-
A/B strains – Target of palivizumab (Synagis®) – Ideal vaccine target
• Attachment (G) protein – Cell attachment – Type II tetrameric glycoprotein – Ga and Gb subgroups, accounts
for year to year strain variation – More challenging vaccine target
6 6
Recombinant Nanoparticle Vaccines: RSV F Nanoparticle Antigen
8/1/12
Trimers Form RSV F Nanoparticles
Trimeric F Protein
F Protein expressed
BV/SF9 System
EM of Insect cell-derived RSV F nanoparticles
F Protein Gene
Infect Sf9
Insect Cells
Insert into Baculovirus
7 7
Recombinant RSV F Nanoparticle (F2+F1): Modifications in Fusion Peptide and Cleavage Site
sp FP F2 F1
137 - 154 FD
CS RSV F wild type
tm NH - - COOH
S-S
KKRKRR
sp FP F2 F1 CS
KKQKQQ furan cleavage site mutation
RSV F Nanoparticle BV683
tm NH - - COOH
S-S
RSV F nanoparticles present antigen in its native configuration, which is important for induction of functional antibodies
8 8
Electron Microscopy: Purified RSV F Forms Trimer-Based Nanoparticles
9
Preclinical Summary
10 10
Cotton Rat Immunogenicity, Neutralizing Antibodies against RSV/A
• RSV F Nanoparticles were administered with and without Adjuphos at day 0 and 21.
• Immune evaluations were perfomred after the second dose at day 49.
• RSV F with AdjuPhos resulted in higher titers compared to no alum groups (p <0.0001)
• RSV-Infection groups had similar neutralizing titers to vaccine groups
• FI-RSV vaccine did not induce neutralizing antibody response consistent with the observation made by Murphy et al J.Clin Microbiol 1986 Aug;24(2):197-202.
11 11
Cotton Rat Immunogenicity – Anti-RSV F IgG
Keys PBS = phosphate buffered saline, PBS+Chal = PBS RSV+Chal = prior infection with RSV FI-RSV = Formalin-inactivated RSV vaccine RSV F = RSV F nanoparticle vaccine
• Robust RSV F specific IgG response in vaccine groups
• FI-RSV group did not induce antibodies against RSV F nanoparticle
• AdjuPhos Adjuvant groups had higher responses compared to unadjuvanted groups (p >0.0001)
• RSV Infection group log2 titer is significantly lower compared to the vaccine groups (p>0.0001)
12 12
Cotton Rat Immunogenicity – Palivizumab Competitive ELISA
3
4
5
6
7
8
9
10
11
12
PB
S
PB
S+
Cha
l
RS
V+C
hal
FI-R
SV
RS
V F
1!g
RS
V F
1!g
+ A
lum
RS
V F
6!g
RS
V F
6!g
+ A
lum
RS
V F
30!
g
RS
V F
30!
g +
Alu
m
Pal
iviz
umab
com
petit
ive
IgG
Tite
r Lo
g 2
• Palivizumab competitve ELISA using pooled sera
• FI-RSV and RSV IN challenge did not induce palivizumab competitive antibodies
• RSV F vaccine induced high levels of palivizumab competing antibodies
13 13
Challenge Outcomes • RSV/A virus was detected only in the
PBS control and FI-RSV groups after (IN) challenge with RSV/A Long virus at 106 pfu/0.1mL
Cotton Rat– Lung Viral Titers/Histopathology
* No virus detected at the lowest dilution tested.
Histopathology: • Adjuphos groups and placebo had lowest
histopathology scores • No evidence of disease exacerbation • With upper doses of RSV F/Adjuphos, the
vaccine antigen shows protection similar to prior RSV infection
14
Clinical Data from Phase 1 Trial
15 15
Study Design
• Randomized, observer-blind, placebo-controlled design • 150 healthy adults
‒ Male and female ‒ 18 to 49 years of age inclusive
• Two-dose series at Days 1 and 30, IM administration • Dose escalation under SMC supervision, with placebo recipients
embedded in each cohort ‒ 5, 15, 30, and 60 µg of F-protein with adjuphos (n= 20 per group) ‒ 30 and 60 µg of F-protein without adjuvant (n= 20 per group) ‒ Placebo (cumulative n= 30)
• 7-day reactogenicity diary + pre/post clinical safety labs + standard AE follow-up for 6 months
• Serologic sampling at Days 1, 30, and 60
16 16
Safety Summary
• Overall vaccine was well tolerated • Majority of AEs were local pain and tenderness and the majority
were mild – Local AEs were higher in vaccine group compared to placebo – No dose effect or AE trend based on dose
• No systemic signal in the vaccine groups • No vaccine-related serious adverse advents (SAEs)
17 17
F Protein ELISA: Assay Methodology
Binding palivizumab to F protein nanoparticle
x axis
10 100 1000 10000 100000 1e60
0.5
1
1.5
2
2.5
Synagis
4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A B C D R^2Plot#2 (S#06: Dilution vs Values) 2.45 1.23 9.16e+03 0.115 0.999Plot#1 (S#05: Dilution vs Values) 0.121 20.6 2.47e+03 0.0415 0.692
__________Weighting: Fixed
OD
at 4
50nm
Palivizumab Conc. (serial dilution)
Binding of Palivizumab to Recombinant RSV F nanoparticle. ELISA plates were coated with 2ug/ml RSV F nanoparticle antigen. Palivizumab at 10 ug/ml concentration serially diluted four fold and reacted to RSV F on the plate. Anti-human HRP reaction was used to determine the Palivizumab binding to RSV F nanoparticles. An unweighted four parameter logistic regression curve is presented for both assays.
18 18
F Protein ELISA: Anti-F Specific IgG Geometric Mean Titers
!"
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)*+,-.+/-$#*0123$
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4*5"%!"
19 19
Placebo 5 µg+ 15 µg+ 30 µg+ 60 µg+ Day 30 1.2 4.4 4.7 8 14
Day 60 1.2 7.1 7.6 11.1 19.1
0
2
4
6
8
10
12
14
16
18
20
Fold Rise
F Protein ELISA: Fold-Rise Dose Response for Adjuphos Groups Only
20 20
Relevance of Palivizumab
• Palivizumab is a monoclonal antibody that binds to a key antigenic site on the F protein
• Palivizumab has been shown to prevent RSV disease in infants in multiple studies
• Palivizumab has provided an important window into the functionality/efficacy of the immune responses to the RSV F nanoparticle vaccine
Antigenic site II: Palivizumab epitope Amino acids 254-278
NSELLSLINDMPITNDQKKLMSNNV * McLellan, et al. JV Aug. 2011, p. 7788-96.
21 21
Peptide ELISA: Assay Methodology
Binding Palivizumab to Palivizumab peptide
Dilution
10 100 1000 10000 100000 1e60
1
2
3Syangis
4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A B C D R^2Plot#2 (S#04: Dilution vs Values) 2.88 1.37 591 0.123 0.998Plot#1 (S#05: Dilution vs Values) 0.0395 65.4 864 0.0599 0.871
__________Weighting: Fixed
OD
at 4
50nm
Palivizumab Conc. (serial dilution)
Binding of palivizumab mAb to palivizumab epitope peptide. ELISA plates were coated with streptavidin at 5ug/ml. Palivizumab peptide at 1ug/ml was bound on to Streptavidin. Palivizumab at 10ug/ml was serially diluted four fold and incubated to the peptide on the plate. Palivizumab binding was detected using anti-human HRP reaction.
22 22
0
500
1000
1500
2000
2500
3000
1 2 3 4 5 6 7
ELISA
Unit
Treatment Group
Day 1
Day 30
Day 60
Peptide ELISA: Anti-Palivizumab Peptide IgG
23 23
Palivizumab-Competitive ELISA: Assay Methodology
24 24
Competitive ELISA with anti-RSV F Vaccinated Mouse Serum and Palivizumab. ELISA plates were coated with RSV F nanoparticle at 2 ug/ml. Pre immune and a pool of day 28 serum from mice immunized on day 0 with 30 ug RSV F nanoparticle with aluminum phosphate were mixed with 50 ng/ml biotin-Palivizumab then serially diluted and incubated with purified RSV F coated ELISA plate. Streptavidin was used to determine Palivizumab bound to the plate. An unweighted four parameter logistic regression curve is presented.
Dilution
10 100 1000 10000 100000 1e60
1
2
3mouse RSV serum+ synagis
4-P Fit: y = (A - D)/( 1 + (x/C)^B ) + D: A B C D R^2S#01 (S#01: Dilution vs Values) -6.99 0.00492 4.6e-72 5.88 0.369S#02 (S#02: Dilution vs Values) 0.0394 1.85 2.25e+03 2.01 0.999
__________Weighting: Fixed
OD
at 4
50nm
Mouse pre immune sera
Mouse post immune sera
Mouse anti-RSV F and Palivizumab (50ng/m) competitive binding
anti-RSV F immune serum (1/dilution)
Palivizumab-Competitive ELISA: RSV F Induced Antibodies vs Palivizumab in Mice
25 25
Palivizumab-Competitive ELISA: Fold-Rise of Palivizumab-like Antibodies Induced by Vaccine
Vaccine-induced antibodies competing with palivizumab for palivizumab epitope on F Protein: Fold rise of the 50% inhibitory titer
26 26
Placebo
5 µg + AlP04
15 µg + AlP04
30 µg + AlP04
30 µg
60 µg + AlP04
60 µg
Study Day (N=26) (N=17) (N=16) (N=18) (N=17) (N=13) (N=18)
Study Day 1 N 26 17 16 18 17 13 18 GMT 11 12 14 10 14 14 20 95% CI 10, 12 10, 16 11, 20 NA , NA 21, 20 10, 20 13, 31
Study Day 30 N 26 17 16 18 17 13 18 GMT 13 70 81 105 112 227 174 95% CI 11, 17 41, 120 61, 107 80, 137 69, 180 167, 308 117, 260
Study Day 60 N 26 17 16 18 17 13 18 GMT 14 111 128 142 100 337 157 95% CI 11, 18 75, 162 103, 161 114, 175 69, 145 254, 448 108, 230
Palivizumab-Competitive ELISA: Translating Palivizumab-like Antibody Concentration (µg/mL)
• Palivizumab-like activity (µg/ml) determined using conversion factor of 2.1 µg/ml per 50% inhibition unit
• In cotton rats, levels of ~30 µg/ml provided a 100-fold reduction in viral load • MedImmune used a trough level of 40 µg/ml to guide efficacy studies for palivizumab
27 27
Concordance between Palivizumab-Competitive and Anti-F IgG ELISA’s
As anti-F antibodies rise in response to immunization, functional Palivizumab-like antibodies also rise in concordant manner
28 28
Neutralizing Titers
Virus + Serum (1:10)
Plaque
Virus +Hep-2
RSV Virus
F
Y Y
Y
Y
Y Y
Y
Y
Y Y Y
Y Serum+Virus +Hep-2
Plaque Positive
F
F
Virus Neutralized
Neutralizing Antibody Assay: Methodology Performed by Baylor College of Medicine
29 29
Neutralization Antibody Assay: RSV-A Microneutralization Titers
• Minimum protective titer in MN assay is log2 6 (Piedra et al, Vaccine)
• Pre-existing neutralizing antibodies above the protective titer, as expected
• Post-immunization neutralizing antibodies increased by ~2 fold
30 30
Neutralizing Antibody Assay: Reverse Cumulative Distribution of RSV-A Microneutralization Titers
31 31
Neutralizing Antibody Assay: Reverse Cumulative Distribution of RSV-A Microneutralization Titers
32 32
MN Fold Rise by Starting Titer
Baseline MN Titer Level (log2)
Geo
. Mea
n Fo
ld R
ise
(95%
CI)
<=6 <=7 <=8 <=10 <=120
1
2
3
4
5
6
7
8
9
10 (n=11)
(n=33)
(n=94) (n=99)(n=61)
33 33
Study Conclusions
• The vaccine was well-tolerated, with no dose related toxicity • The vaccine was immunogenic as measured by:
‒ Anti-F IgG ‒ Palivizumab epitope binding antibodies ‒ Rise in neutralization titers detected against both RSV-A and RSV-B
strains • Concordance seen with anti-F IgG and palivizumab epitope binding
antibodies • Palivizumab-like antibodies are functional and may be predictive for
vaccine efficacy ‒ Quantitation indicate antibodies above protective levels (>40ug/ml)
• Neutralization titers above protective levels ‒ Minimal protective titer log2 6 ‒ No low titer subjects after immunization
34 34
Conclusion
• Novavax RSV F recombinant nanoparticle vaccine is a conserved, immunogenic antigen, in its native confirmation
– Induces functional, high affinity immunity, critical for an RSV vaccine ‒ Baculovirus/insect cell system appears ideal to produce natively
configured antigen
– Recombinant RSV F nanoparticle made via viral infection in eukaryotic cell
• Clinical immunogenicity data may be predictive of protection and de-risks development program
– MN compare well with naturally induced, protective levels
• Protective in both elderly and pediatric sero-epidemiology studies – Palivizumab-like antibodies compare well with mAb protective levels
– Effective levels established RSV infant prophylaxis
8/1/12 Confidential to Novavax
Clinical data indicates RSV F nanoparticle vaccine warrants further development