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Novel vaccines targeting all age groups
Mariagrazia PizzaCEREHA kickoff meetingNovember 28, 2012
20th Century VaccinesInitiated by Jenner, Developed by Pasteur
Vaccination remains the medical intervention with highest impact on health
-75%
- 67%
- 61%
- 41%
Rheumatic fever and rheumatic heart disease
Hypertensive heart disease
Ulcer of stomach and duodenum
Ischemic heart disease
Drop in death rate for diseases prevented or treated with innovative medicines (pharmaceuticals)1965 – 1999
- >97%Infectious Diseases
(polio, measles, Hib, HVB, Hib etc)
VACCINATION
THERAPEUTICS
Source: EFPIA 1999 – 2002
Key questions
In the 20th century most childhood diseases were eliminated by vaccines based on old technologies
Which is the role of vaccines in the 21st century and which tecnologies?
During the last 30 years, several new technologies made possible vaccines that were previously impossible
Poverty
New vaccines to address the major health challenges of the 21st
century are needed
21st century society, “aging society“
3
1
Emerging infections
2
people live longer
Which factors influenced this change?
Crimmins et al. Attribute the Increase of Life Expectancy to the Conquest of Infectious Diseases
Less Infectious Diseases
Reduced infant mortality
Increased Life
Expectancy
Less inflammation
Reduced Mortality in the
Elderly
Life Expectancy is outpacing PredictionWith an aging society, we need a new model for health care
R.Rappuoli, C. Mandl, S: Black , E. De GregorioNature Reviews Immunology | November 2011; doi:10.1038/nri3085
Vaccines for every age
R.Rappuoli, C. Mandl, S: Black , E. De GregorioNature Reviews Immunology | November 2011; doi:10.1038/nri3085
During the last 30 years, several new technologies made possible vaccines that were previously impossible
Conjugate vaccines
Many bacteria have a capsular polysaccharide
Glyco-conjugation improves the immunogenicity of polysaccharides
Non immunogenic polysaccharide
immunogenic conjugate
MenC Conjugate Vaccine (red) Induced high level of Bactericidal Antibodies in Infants. Plain Polysaccharide (blue) was a poor
Immunogen
Conjugate vaccines for Meningococcus Celiminated the disease in the UK
Laboratory Confirmed Cases of Serogroup C Meningococcal Disease (England & Wales)
Week No. (totals from mid-year)
VaccineSince the introduction of the UK MenC vaccine in 1999
>12,000 cases prevented> 1,200 deaths prevented>2,400 permanent sequelae prevented
A Antigen C Antigen W Antigen Y Antigen
Conjugated capsular polysaccharides induce protection in all ages against serogroups A, C, W, Y
Meningococcus B capsule polysaccharide resembles a self antigen and cannot be used for vaccination
During the last 30 years, several new technologies made possible vaccines that were previously impossible
Reverse Vaccinology
expressionand
purification
purified proteins
~350 proteins successfully expressed in E.coli, purified, and used to immunize mice
Based on the genome sequence of MC58, 600 ORFs that potentially encoded novel surface exposed or exported proteins were identified
100,000
200,000
300,000
400,000
500,000
600,000
700,000
800,000
900,000
1,000,0001,100,0001,200,000
1,300,000
1,400,000
1,500,000
1,600,000
1,700,000
1,800,000
1,900,000
2,000,000
2,100,000
2,200,000IHT-A
IHT-B
IHT-C
1
Reverse Vaccinology Allowed the Identification of Novel MenB Antigens
immunizations
Pizza M. et al. Science 2000
NadA NHBAfHbp variant 1
Testing for bactericidal
activity
4CMenB Vaccine Composition� Three protein antigens (two fusion proteins and one single polypeptide)
� Outer Membrane Vesicle (OMV) component (NZ PorA is P1.4)
Dose NHBA-GNA1030
fHbp-GNA2091
NadA OMV Al 3+
0.5ml 50 µg 50 µg 50 µg 25 µg 0.5 mg
� 4CMenB is a suspension for injection
NHBA GNA1030
GNA2091 fHbp
NadA
N
N
N C
C
C
Class 5
PorA
Class 4
PorB
LPS
OMV
Clinical Development Program
� 4CMenB has been evaluated in 12 studies with 7,190 subjects (from 2 months of age) who received at least one dose of 4CMenB• 5,395 infants and toddlers from 2 months to 2 years of age
- 1,630 received a booster dose in the second year of life
• 169 children 2 to 10 years of age
• 1,712 adolescents and adults ≥11 years of age
� Geographically diverse (EU, North and South America)• 4CMenB has been studied in the Northern and Southern Hemispheres
The 4CMenB vaccine has 4 major antigenic components: fHbp, PorA, NadA, and NHBA.
Serum sample
Reference Men B Strains Used to Measure Antigen-Specific SBA Responses in Vaccinees
5–99
H44/76SL
NZ98/254
M10713
Indicator MenB Strain
Test bactericidal response against MenB “indicator” strains, each
one primarily susceptible to killing by bactericidal antibodies against
one of the major vaccine antigens.
hSBA titers ≥1:4 is the protective level of antibody(titer of ≥1:5 gives 95% confidence)
4CMenB Immunogenicity in InfantsPercentage of infants with bactericidal titers ≥1:5 and immune response to a booster dose
Baseline Post-primary* Pre-booster Post-booster†
Vesikari T, et al. Presented at the 17th International Pathogenic Neisseria Conference (IPNC); 11-16 September 2010; Banff, Canada; Poster #180; Vesikari T, et al. Presented at the 29th Annual Meeting of the European Society for Paediatric Infectious Disease (ESPID); 7-10 June 2011; The Hague, The Netherlands; Poster #749.
Phase III in Infants Study V72P13 and V72P13E1 in EU Countries
3 4 1
33
100 100
8484
81
99
20
61
100 10095 98
0
20
40
60
80
100
% S
ubje
cts
with
ba
cter
icid
al ti
ters
≥1:
5
44/76-SL fHbp
5/99 NadA
NZ98/254 PorA P1.4
StrainAntigen
M10713 NHBA‡
*Blood drawn at 7 months, N=1149–1152.†Blood drawn at 13 months, N=421–424. ‡N=100.
0
20
40
60
80
100
44/76-SL 5/99 NZ98/254
4CMenB Immunogenicity in AdolescentsPercentage of adolescents with bactericidal titers ≥1:4 1, 2 and 6 month schedules
Indicator Strain:
Vaccine Antigen: fHbp NadA PorA P1.4
% S
ubje
cts
with
hS
BA
titer
s ≥1:
4
53184 212 238 GMTs69 466 713 579
4391 122 121
4.4
2.6
3.6
Baseline 4CMenB 1 dose
4CMenB 2 doses (1 month apart)
4CMenB 2 doses (2 months apart)
4CMenB 2 doses(6 months apart)
Santolaya ME, et al. Lancet. 2012;379(9816):617–624.
Baseline: n=1355; 4CMenB 1 dose: n=223; 4CMenB 2 doses (1 month apart): n=222; 4CMenB 2 doses (2 months apart): n=215; 4CMenB 2 doses (6 months apart): n=86.Data shown is 1 month post the last dose in the series.
Phase IIb/III in AdolescentsStudy V72P10 in Chile
Conclusions on immunogenicity
� Immunogenicity– 4CMenB was shown to induce functional bactericidal antibodies in infants,
toddlers, adolescents and adults– Adequate immune responses for routine vaccines concomitantly
administered with 4CMenB – In infants, robust booster responses at 12 months of age following
2-4-6 month schedule– Evidence of persisting bactericidal antibodies to at least 40 months of age
in infants and 18 months after adolescent series
25
4CMenB antigen characterizationBackground and latest results
NHBA is a surface exposed lipoproteinNeisseria Heparin Binding Antigen
� NHBA is present in all strains
• Classified in >20 peptides
• Variably expressed in different strains
� NMR structure has been solved
� NHBA is naturally cleaved by hLF and meningococcal protease NalP
� NHBA binds heparin & heparansulfatePGs via the Arg-rich region
• Enhanced serum resistance
• Adhesion to hepithelial cellsAnti-NHBA
Anti-HS
Nm encounters different temperatures during colonization and disease
27
Rouadi P et al., 1999; Cole O. 1953; Herbowski et al., 2011
naso-oropharynx
30-32°C
cerebrospinalfluid
34-36°C
blood37°C
Nm growth in the lab37°C
NHBA expression is maximal at ~30°C, a temperature detected in the upper respiratory tract
28
MC58ST32p0003
8047ST11p0020
M11719ST162p0020
40ºC 37°C 35ºC 30ºC 28ºC
NHBA
NHBA
NHBA
T° testedstrains
FACS8047 37°C
∆287 30°C
8047 30°C
NadA is a surface-exposed trimeric autotransporterNeisseria Adhesin A
� NadA promotes bacterial adhesion to and invasion of human epithelial cells• Anti-NadA antibodies decrease adherence to
host epithelial cells
� NadA antibodies are present in convalescent sera
� NadA distribution and genotype varies across MenB strains • nadA gene is not present in all Nm strains
• Classified into 3 main variants (with cross-protective activity) + 2 rare variants
X-ray structure of NadA has recently been solved
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NadA expression is variable among strains and influences hSBA results
31
NadA
NadR
NadA
∆NadR
NadA expression is induced by HPA derivatives, physiologically relevant molecules
NadR
Derivatives of Hydroxy Phenylacetic Acid (HPA) are secreted in saliva or
produced during inflammation
NadA
- + - 4HPA
NGP165
- - + 3Cl-4-HPA
NadR
תורשפא ןיא הנומת גיצהל.תעכ וז
fHbp is a surface exposed lipoproteinFactor H binding protein
Masignani V et al., JEM 2003; Cantini F, et al. JBC 2009; Giuliani MM et al., Vaccine 2010; Lucidarme et al Clin Vacc Immun 2011
� fHbp gene is present in most meningococcal strains
� Expression of fHbp is highly variable among strains
� fHbp exists in 3 different genetic and immunogenic variants (v1, v2, v3)
� NMR structure has been solved
SCR7
SCR6
fHbp binds the human complement regulatory protein factor H (fH)
Madico G et al., JI 2006; Schneider et al, 2009; Seib KL et al., I&I 2009
fHbp
hfH
� Binding to fH enables bacterial survival in the bloodstream
� Antibodies against fHbp can block the binding of fH and increase the susceptibility to killing by the complement alternative pathway
� Mechanism of fH binding has been fully elucidated
� Binding site is spread along the fHbp sequence
� All variants & subvariants bind fH
� Several mAbs were generated against fHbp
fHbp Is an Important Virulence Factor
� fHbp deletion mutant is killed in human blood
� Antibodies directed against fHbp can block the binding of fH and increase the susceptibility to killing by the complement alternative pathway
� However: • there are a minority of strains that
do not expressing fHbp
• fHbp is not the only N. meningitidis factor involved in fH binding and complement evasion
Time (min)
Madico G et al., JI 2006; Granoff et al., 2009; Seib KL I&I et al., I&I 2009;; Lewis LA et al., Plos Pathogens 2010
Invasion
Complement mediated killing
Bacteria Are Recognized as Non-self by the Alternative Pathway of Complement, C3 Binds and Complement Kills Them
Neisseria meningitidis Coated by Factor H Is Not Recognized as Non-self by C3, Survives and Multiplies in Human Blood
Invasion
Survival and growth
fHbp binds only human factor H, this explains the species specificity of meningococcus and the absence of an animal model for this bacterium
A novel functional role for fHbp ?
� fHbp binds enterobactin in vitro
� fHbp–enterobactininteraction site was identified by NMR
� Enterobactin binding site was distinct from the site involved in binding to human factor H
� The biological significance of this interaction remains to be investigated.
| Presentation Title | Presenter Name | Date | Subject | Business Use Only38
Enterobactin binding site
180°
fH binding site
180°
Antigenic Components of the 4CMenB:Important for Meningococcal Survival, Function, or Virulence
� NadA: neisserial adhesin A• Promotes adherence to and invasion of
human epithelial cells1-3
• Antibodies could interfere in colonization
� fHbp: factor H binding protein• Binds factor H, which enables bacterial
survival5,6 in the blood
• Binds the bacterial siderophoreenterobactin (in vitro)4
� NHBA: neisserial heparin-binding antigen
• Present in virtually all strains
• Binds heparin, which may increase the serum resistance of bacteria7-9
� NZ PorA 1.4: porin A• Major outer membrane vesicles protein –
induces strain specific bactericidal response
1. Comanducci M, et al. J Exp Med. 2002;195:1445-1454; 2. Capecchi B, et al. Mol Microbiol. 2005;55:687-698; 3. Mazzon C, et al. J Immunol. 2007;179:3904-3916; 4. Veggi D, et al. Presented at IPNC. Banff, Canada. September 11-16, 2010; 5. Madico G, et al. J Immunol. 2006;177:501-510; 6. Schneider MC, et al. J Immunol. 2006;176:7566-7575; 7. Serruto D, et al. Proc Natl Acad Sci U S A. 2010;107:3770-3775; 8. Welsch JA, et al. J Infect Dis. 2003;188:1730-1740; 9. Plested, et al. Clin Vaccine Immunol. 2008;15:799-804.
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Towards a meningitis free worldCan we eliminate meningococcal meningitis?
Reverse vaccinology allowed us to target manypathogens that were difficult or impossible beforeIncluding SUPERBUGS
Group B StreptococcusGroup A Streptococcus
Gonococcus
Pneumococcus
Chlamydiatrachomatis and Pneumoniae
Yersinia pestis
Porphyromonas gingivalis
Malaria
Tuberculosis
43 | Presentation Title | Presenter Name | Date | Subject | Business Use Only
ExPEC antigen selection of antigens by multiple genome anal ysis
� Neonatal meningitis-associated strains (NMEC)
- IHE3034 (Novartis with TIGR)
- RS218
� Uropathogenic strains (UPEC)
- CFT073
- 536
� Non pathogenic strains
- MG1655
- DH10B
267 200 9
Protection
ExPEC - The most protective candidates selected in t he mouse model of sepsis
P value* calculated by Fisher’s exact text: statist ically significant if < 0.05
P.E.: protection efficacy calculated as = % dead co ntrol - % dead immune% dead control
Genome sequence told us that there is no genetic segregation between intestinal and extraintestinal E. coli
they share common antigens
Group B Streptococcus (GBS)invasive neonatal infections
� The major cause of invasive neonatal infections (pneumonia, septicemia and
meningitis) in the industrialized world
� Over 80% of neonatal infections occur in the first 7 days of life (early-onset
diseases) acquired before or at the birth by direct mother to baby transmission
� Main reservoir is lower gastrointestinal- genital tract, around 30% of women
colonized by GBS
� An increasingly important cause of invasive infections in the elderly and in
patients with underlying diseases
� Beta-haemolytic Gram positive bacterium
� 10 known capsule serotypes : Ia, Ib, II, III, IV, V, VI, VII, VIII, IX
� non-typeable (NT) GBS account for up to 8-14% of all strains isolated
Group B Streptococcus (GBS)invasive neonatal infections
Group B streptococcus, a pangenome analysis
Multiple genome analysis allowed the development of a vaccine covering all serotypes
Group B Streptococcus: bacterial challenge and OPA on 356 antigens
Passive maternal Immunization/ neonatal challenge : Immunize female mice and test protection from GBS challenge in pups.
Vaccine
GBS challenge
OPK: Bacterial killing by human phagocytic cell line in the presence of a sample serum and complement.
Y
Y
YC’
HL60C’
At least four antigens identified as protective in the animal model
GBS 322, GBS 80, GBS 104, GBS 67
Their combination protect against 92% of the tested strains
Two of the protective antigens were found to becomponents of pili structures
• Pili may play an important role in the virulence of Gram-positive bacteria
• The GBS program allowed discovery of pili also in GAS (group A streptococcus) and pneumococcus.
Staphylococcus, 4 antigens combo gives the best protection in the mouse lethality model
0
10
20
30
40
50
60
70
80
90
100
110
D-0 D-1 D-2 D-3 D-4 D-5 D-6 D-7 D-8 D-9 D-10 D-11 D-12 D13 D14
Sur
viva
l (%
)
Time after challenge (Days)
Sur
viva
l (%
) at
diff
eren
t tim
e po
nits
(da
ys)
alum
Combo 2
IsdB
Combo 4
Reverse vaccinology :
� By searching the complete genomes of pathogens, it has increased of several order of magnitude the number of antigens available for vaccine development. This technology has delivered and continues to deliver novel vaccine candidates to previously difficult or impossible targets, for all age groups.
� The new protective antigens may have a role in virulence and pathogenesis. The study of their function can help unravelling important mechanisms of pathogenesis and host defence.
During the last 30 years, several new technologies made possible vaccines that were previously impossible
Structural Vaccinology
From reverse vaccinology to structural vaccinology
54
100,000
200,000
300,000
400,000
500,000
600,000
700,000
800,000
900,000
1,000,0001,100,0001,200,000
1,300,000
1,400,000
1,500,000
1,600,000
1,700,000
1,800,000
1,900,000
2,000,000
2,100,000
2,200,000 1
Analysis of Sequence diversity
3D structureEpitope mapping
Antigen identification
Rational optimitazion
Epitopes are usually larger than described
55
Standard approaches of epitope mapping provide an incomplete picture
56
N-termC-term
Standard approaches of epitope mapping provide an incomplete picture
57
FL C-term N-term
Dot-Blot N-termC-term
Standard approaches of epitope mapping provide an incomplete picture
58fHbp wt Lys302 ����Ala Neg ctrl
Site-directed mutants
N-termC-term
Standard approaches of epitope mapping provide an incomplete picture
59
PepScan
N-termC-term
Standard approaches of epitope mapping provide an incomplete picture
60
M13 Phage DisplayN-termC-term
Standard approaches of epitope mapping provide an incomplete picture
61
HDX-MS analysisPeptide 104-119
0.1 1 10 1000
2
4
6Control12C1/D7
Deuteration Time (Min)
# D
eute
rons in
corp
ora
ted
Peptide 233-245
0.1 1 10 1000
1
2
3
Deuteration Time (Min)
# D
eute
rons
inco
rpor
ated
N-termC-term
X-ray crystallography of the fHbp-12C1Fab complex identifies a much larger epitope
62
N-termC-term
1.8Å resolution X-ray structure of fHbp + 12C1 Co-crystal structure reveals canonical folds burying 1000Å2 on fHbpsurface
| Presentation Title | Presenter Name | Date | Subject | Business Use Only63
C N
VH VL
CL
C N
VH VL
CH1 CL CH1
Xray structure of fHbp-Jar5 complex has also beensolved
| Presentation Title | Presenter Name | Date | Subject | Business Use Only64
Epitope mapping: aa contributing to immunogenicity of thedifferent variants are located in non-overlapping regions
Cantini, F. et al. J Biol Chem 281, 7220-7227 (2006)Mascioni, A. et al. J Biol Chem 284, 8738-8746 (2009)Cantini, F. et al. J Biol Chem 284, 9022-9026 (2009)
C-terminal domain sub-divided into 11 different par tially overlapping areas ranging from 900 to 2000 Å 2 * 54 mutants designed from multiple sequence alignment of all sub-variants, expressed, purified and used for mouse immunization
*approximate size of conformational epitopesRubinstein, N.D. et al. Computational characterization of B-cell epitopes. Molecular Immunology 45, 3477-3489 (2008)Chakrabarti, P. Dissecting protein-protein recognition sites. Proteins Structure Function and Genetics (2002)Davies, D.R. & Cohen, G.H. Interactions of protein antigens with antibodies. Proc Natl Acad Sci USA 93, 7-12 (1996)
var.1 var.2 var.3
Screening for induced protection of the 54 mutantsSerum bactericidal activities of the eight most promising candidates
Scarselli, M. et al., Sci Transl Med., In press (2011)
fHbp-G1 mutant induces protective immunity against different antigenic variants
var.1 var.2 var.3
Screening for induced protection of the 54 mutantsSerum bactericidal activities of the eight most promising candidates
Scarselli, M. et al., Sci Transl Med., In press (2011)
Crystal structure of the G1 mutant
The 23 substitutions changed the exposed surface, without inducing any local disorder
Mutations do not interfere with fH binding
Backbone structures of G1 and fHbp-WT are virtually identical
Structure-based design of novel antigens is a powerful approach to generate novel antigens with optimal and broadly protective immune response.
The knowledge of the 3D structure of the antigen is a necessary prerequisite to design the epitope grafting
This approach can be in principle applied in all cases where variability hampers the use of otherwise effective immunogens
Conclusions
During the last 30 years, several new technologies made possible vaccines that were previously impossible
Adjuvants
MF59: An established adjuvant in aEuropean-licensed seasonal trivalent vaccine
� Oil-in-water emulsion adjuvant licensed for use in seasonal influenza vaccine FLUAD* since 1997
• More than 100 million commercial doses distributed
� Adjuvanted vaccine provides heterologous responses to drifted strains
� >120 Clinical studies, >200,000 subjects
• No safety signals in either pharmacovigilance database or meta-analysis of clinical trial database with6 month subject follow-up (filed with CBER)
� Pediatric studies and efficacy trial in3,000 subjects
MF59 adjuvant emulsion
SPAN 85 TWEEN 80Antigens
160nm
*FLUAD is a registered trademark of Novartis. FLUAD is not licensed in the Unites States. FLUAD is recommended for active prophylaxis of influenza in the elderly
oil
MF59. more CD4+ T cells, more neutralizing (MN) antibodies
Non-Adj-15
MF59-7.5
MF59-15
10
100
1000
1 22 130 202 223 38243
A/V
N/1
1194
/04
MN
-GM
T
*
*
*
*
*
1:40
Galli et al, Proc Natl Acad Sci USA 2009
H5-
CD
4+(in
10
6to
t CD
4)
days
*
0
250
500
750
1000
1 22 130 202 223 38243
* *
*CD4+
MN
Fluad
TIV–0.6
–0.4
–0.2
0.0
0.2
0.4
0.6
0.8
1.0
Vac
cine
effi
cacy
vs. n
on-in
fluen
za c
ontr
ol
0 20 40 60 80 100 120 140 160 180 200 220
Days post-second dose
Vesikari T, et al. NEJM. In press.
Efficacy Study (n=4707)Efficacy was robust, observed early, and persisted
Vaccine also showed satisfactory safety profile:• Increased local reactogenicity• No increase in serious adverse
experiences vs. control
Vaccines for every age are possible with the newtechnologies
| RNA Platform slides for public disclosure | C. Mandl / A. Geall | From April 2012 | Self-amplifying RNA vaccines | Business Use Only
76
Introduce detecting antibodyIntroduce free antigens to
each coated well, incubate, and wash away unbound antigen
Lyse cells with detergent
to free antigens
Collect and grow bacterial strain (test strain) Create suspension
with standardized concentration of bacteria
Coat wells with capture antibody
for individual antigens
Compare quantities of vaccines antigens expressed in test strains to those in reference
strains to determine relative potency
Detergent
� MATS – Meningococcal Antigen Typing System
A MATS ELISA developed in houseto evaluate level of expression and antigenic reactivity
MATS for each antigen
78
Is MATS correlating with sensitivity of strains to bacterial killing?PBT defined as threshold MATS value to predict killing
Killed in SBA: infant serum (post4) pool titer ≥1:8Not killed in SBA79
MATS Standardization 5 European Countries and US
� National reference labs qualified for an interlab study for assay standardization
Eight meningococcal reference laboratories have been qualified for MATS analysisTwo laboratories under qualification
| Presentation Title | Presenter Name | Date | Subject | Business Use Only81
1. Health Protection Agency (HPA), Manchester, UK2. University of Würzburg, Germany3. Institut Pasteur, France4. National Center for Microbiology, Institute of He alth Carlos III, Spain5. Norwegian Institute of Public Health, Norway6. Queensland Paediatric Infectious Diseases Lab. (Q PID), Australia7. Istituto Superiore di Sanità, Italy8. Center of Disease Control, US
• 2012-2013: National Microbiology Laboratory - Public Health Agency of Canada• 2012-2013: Institute Adolfo Lutz, Brazil
Predicted capsular group B strain coverage by 4CMenB vaccine by country is high
| Presentation Title | Presenter Name | Date | Subject | Business Use Only82
4CMenB-immunized sera protect infant rats from infection with strain NGP165
83
i.p. inoculation 105CFU NGP165
(mismatched for the other antigens)
CFU count
3hrs 18hrs
pre-immune serum
4CMenB pooled infant
serum (post 4)
Num
ber
of C
FU
s n=5 n=4**
2000-
1500-
1000-
500-
0-
bacteremia
Broadly neutralizing influenza antibodies
Structural Vaccinologyengineering a stable F protein for RSV
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