Mariagrazia Pizza CEREHA November 28,

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

| Presentation Title | Presenter Name | Date | Subject | Business Use Only30

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.

| Presentation Title | Presenter Name | Date | Subject | Business Use Only40

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