www.pei.de The Use of Novel Adjuvants to Enhance and Broaden the Immune Response Elicited by Human

Ralf Wagner Section for Viral Vaccines Paul-Ehrlich-Institut Langen / Germany

Section Viral Vaccines

 CONTENT AND STRUCTURE OF PRESENTATION

• WHY IS THERE A NEED FOR ADJUVANTS Open issues and challenging aspects where optimisation is needed

• BRIEF HISTORY OF ADJUVANTs and ADJUVANTED VACCINES

• MECHANISMS/MODES OF ACTION OF SELECTED NOVEL ADJUVANTS Selection of those ADJUVANTS recently licensed with novel vaccines

• BRIEF OVERVIEW OF REGULATORY REQUIREMENTS FOR NOVEL ADJUVANTS

• ADJUVANT EFFECTS IN RECENTLY LICENSED NOVEL VACCINES Efficacy and Safety profiles of selected vaccines Special Issue: Narcolepsy associated with AS03-adjuvanted pandemic vaccine

• FUTURE PERSPECTIVES

Section Viral Vaccines  WHY is there a NEED for (NOVEL) ADJUVANTS??

Section Viral Vaccines (NOVEL) ADJUVANTS HOLD HUGE POTENTIAL FOR…

• Overall Increase in immune response against pathogen (number of doses needed) • enhanced immune response to poorly immunogenic antigens subunit vaccines, expressed highly purified proteins (no good PAMP motif) • promoting specific arms of the immune response (eg humoral, CMI, memory…) • induction of a broader immune response resulting in better cross-protection to antigenically diverse/variable infectious agents (“antigenic drift”)

• specific immune response in specific populations • efficient priming in naïve subjects • enhancement of immune response in otherwise poorly responsive population groups (elderly, immune-compromised)

• antigen sparing to ensure vaccine supply in cases where production capacity is limited or huge amounts of doses are needed within a very short time frame (pandemic)

Section Viral Vaccines Hence, there a numerous good reasons for the development and integration of novel adjuvants into today´s and tomorrow´s vaccines!!

Section Viral Vaccines  BRIEF HISTORY OF ADJUVANT DEVELOPMENT

Section Viral Vaccines A CENTURY OF ADJUVANT DEVELOPMENT

Only during the last 30 years adjuvanted vaccines are more rapidly emerging

Licensed

Under (pre)clinical Development

From: De Souza Apostolico et al., 2016 • Positive opinion by EMA in Art. 58 procedure for „Mosquirix“ • Currently under EMA evalation in CP for “Shingrix” Zoster Vaccine

Section Viral Vaccines THE NUMBER OF ADJUVANTED VACCINES IS CONSTANTLY INCREASING

From: Di Pasquale et al., Vaccines 2015

Section Viral Vaccines OVERVIEW TO ADJUVANTS IN LICENSED VACCINES To be discussed in Presentation

MF59

MPL

QS21

AS01

AS03 AS04

Section Viral Vaccines  MODE/MECHANISMS OF ACTION OF SELECTED ADJUVANTS  What is known  What is proposed  Immune-potentiating effects Derived mainly from in-vitro and animal studies

 Potential immunological targets for adjuvants

Section Viral Vaccines OVERVIEW TO IMMUNOLOGICAL EFFECTS/FUNCTIONS OF ADJUVANTS

1) Depot formation 2) Secretion of cytokines “Innate“ 3) Immune cell recruitment

4) Antigen uptake

APC 5) Maturation / Activation of immune cells 6) Antigen processing presentation on MHC

7) Activated APCs traffick to draining lymph node

Lymph node

“Adaptive“ 8) Immunmodulation

Adapted from: Awate et al., 2013

Section Viral Vaccines  EXAMPLES OF ADJUVANTS AND THEIR MODE OF ACTION

Section Viral Vaccines OIL-IN-WATER ADJUVANTS: COMPOSITION OF AS03 AND MF59

Structural representation of MF59 – similar to AS03

Quantitative Composition (mg/dose) MF59 AS03 Squalen 9,75 10,69 DL-α-Tocopherol --- 11,86 Tween 80 1,175 4,86 Sorbitan Trioleate 1,175 --- (Span 85) Manufacturing Microfluidization Particle size ≈ 150 nm 120-200 nm

Section Viral Vaccines MODE OF ACTION OF OIL-IN-WATER ADJUVANTS: AS03 AND MF59 Mostly affect innate immune responses to create a “pro-inflammatory environment”  as the basis for an efficient/enhanced adaptive immune response

Activation / Modulation of “adjuvant core response genes“: • Cytokines, such as - Chemokines, IFN, IL • Innate immune receptors • Adhesion molecules Recruitment/differentiation of immune cells to/at site: • Monocytes • Neutrophils, eosinophils • Macrophages Trafficking to draining • Dendritic cells, APC lymph node: for induction of adaptive immune response  Enhanced AB titers EFFECTS ON ADAPTIVE  AB cross-reactivity IMMUNE RESPONSE (higher B-cell diversity)

 Expanded TH cells activity Section Viral Vaccines ADJUVANT EFFECT ON IMMUNOGENICITY – STUDY DESIGN

Study in seronegative ferrets Vaccine Formulations applied: A - Plain (non-adjuvanted) pandH1N1 antigen (split), 10µg/dose B - pandH1N1 antigen with MF59 (oil-in-water) adjuvant (Focetria®) C - pandH1N1 antigen with AS03 (oil-in-water) adjuvant (®) D - pandH1N1 antigen with Diluvac® (well established veterinary adjuvant)

Immunization schedule

1st dose 2nd dose Challenge

0 8 21 42 days 4 months

       Blood draws for serological examination

Section Viral Vaccines ADJUVANTS EFFECTS ON IMMUNOGENICITY – RESULTS

HI-titers against A/HH/05/2009

VN-titers against A/HH/05/2009 immunisation

immunisation Schmidt et al., 2016

Section Viral Vaccines AS04 ADJUVANT COMPLEX: MPL ADSORBED TO AIOH3 Composition: MPL (50µg) adsorbed to AlOH3 (500µg) – per dose of “” vaccine (3-O-desacyl-monophosphoryl lipid A)

• Lipid A is a component of the LPS complex of bacterial cell walls with pronounced immune-stimulatory effect • Lipid A shows high toxicity, pyrogenicity

 MPL is detoxified version of Lipid A that retains the immune-stimulatory activity  Detoxification of Lipid A by removal of phosphate group and fatty acid

Lipid A MPL

Detoxification

Due to the production/detoxification procedure MPL is a mixture of congeners with different numbers of fatty acid side chains (4-7) Section Viral Vaccines AS04 ADJUVANT COMPLEX: A POTENT TRL-4 AGONIST

Like LPS, MPL exhibits its adjuvanting function via the TLR-4 signalling pathway TLR-4 LPS Lipid A MPL Other agonists: - dsRNA: TLR-3 - ssRNA: TLR-7 - CpG: TLR-9

Adapted from: APC De Souza Apostolico et al., 2016

Pro-inflammatory state Upon Binding of MPL to TLR-4 and MD2 Increase in MyD88  IL,TNFα  activation of intracellular pathways Adaptive AB TRIF  Interferon response (TH1)

Section Viral Vaccines AS01 ADJUVANT COMPLEX: MPL PLUS QS-21

AS01: liposome-based complex containing two immune stimulants and cholesterol - MPL - QS-21: molecule extracted from the bark of Quillaja saponaria (fraction 21) water-soluble triterpene glycoside with amphiphillic character hemolytic activity – eliminated in cholesterol-containing liposomes (as in AS01) exact mechanism of adjuvanting function currently not fully understood

Chemokine release attracts granulocytes and monocytes Activated APC Trafficking to draining lymph node Induction of IFN-pathway Proinflammatory cytokines Cellular and cytokine Antigen-specific reponses peak at day 1 - CD4+ T-cells resolved by day 7 - CD8+ T-cells Adapted from: Didierlaurent et al., 2017 AIM: ENHANCED CELLULAR AND HUMORAL IMMUNE RESPONSES

Section Viral Vaccines  REGULATORY ISSUES Brief overview to Existing Guidance and pivotal Licensing Requirements

Section Viral Vaccines LICENSING REQUIREMENTS FOR NOVEL ADJUVANTED VACCINES

Regulation/licensure of adjuvanted vaccines is quite challenging: - extremely diverse nature of substances (origin, composition, manufacture, antigens…) - extremely diverse functions/modes of action (multiple factors and mechanisms) - extremely diverse safety profile ⇒ Difficult/Impossible to conclusively predict all potential safety risks

Selected Available regulatory guidance/recommendations: - Guideline On adjuvants In Vaccines For Human Use (EMEA/CHMP/VEG/134716/2004) - Guidelines on the non-clinical evaluation of vaccine adjuvants and adjuvanted vaccines (WHO, 2013) - Guideline on clinical evaluation of new vaccines (EMEA/CHMP/VWP/164653/2005) - Guidelines on clinical evaluation of vaccines: regulatory expectations (WHO, 2017)

 Basic framework of concepts rather than specified instructions for individual substances

Section Viral Vaccines LICENSING REQUIREMENTS FOR NOVEL ADJUVANTED VACCINES

Guiding principle: Evaluate Adjuvant alone AND adjuvant in combination with specific antigen!! Inclusion of adjuvants to be justified ⇨ Clear emphasis on safety over efficacy for healthy recipients Brief outline of requirements (not exhaustive): Quality: Comprehensive data package required

description, source materials, manufacturing process, in-process controls, testing programme and specifications, physico-chemical identification, impurities, association with antigen, stability,

Preclinical: Comprehensive data package required

Mode of action: immunological effects, Suitability of animal models (species specificty of immunity), protection vs immunogenicity, dose-response, repeated doeses

Safety: local tolerance, systemic toxicity, reprotoxicity (women pregnant or child-bearing age), hypersensitivity, pyrogenicity, distribution (case by case)

Clinical: Comprehensive data package required

Efficacy: immune response (AB, humoral, CMI,…), specific population/age groups (start with healthy adults), adhere to requirements for “first in man” trials, dose-finding studies

Safety: adverse events, local and systemic effects, monitoring of clinical parameters, adhere to requirements for “first in man” trials

Section Viral Vaccines …no turning to real life data….

 ADJUVANT EFFECTS IN SELECTED VACCINES Efficacy and Safety Profiles

Section Viral Vaccines CERVARIX: AS04 ADJUVANTED HUMAN PAPILLOMAVIRUS VACCINE

Types contained in the vaccine: HPV-16/18, VLP expressed in insect cells

 Facts on Immunogenicity:

- High GMTs after (≈ 12-fold higher than after natural ) - Almost all vaccinees negative at baseline seroconverted post vaccination (p.v.) - GMTs peak at months 7 p. v., reach plateau after 18-24 months p. v. and then slightly decline – but still ≈ 10-fold higher than after natural infection after 9 years - Functional neutralising AB - Robust immune response in all tested age groups (10-14 yoa, 15-25 yoa, > 25 yoa) - Serum AB correlated with AB detected in cervico-vaginal secretions (mucosal AB)

• Head-to-head comparison with Al-Phosphate adjuvanted vaccine: - Higher GMTs for Cervarix in all age strata - 2,7-fold higher antigen-specific B-cells for Cervarix Cave: Clinical relevance of these findings currently unknown (different antigens!) Could contribute to long-term persistency of AB

Section Viral Vaccines CERVARIX: AS04 ADJUVANTED HUMAN PAPILLOMAVIRUS VACCINE

Overview to selected vaccine efficacy (VE) data from clinical trials against HPV16/18:

VE against virological endpoints in ATP cohort (acc.to protocol, negative at baseline, 15-25 yoa)

VE against high-grade cervical lesions associated with HPV-16/18 in ATP cohort (as above)

Data extracted from SmPC

 Very high vaccine efficacy in subjects negative at baseline  Efficacy is lower in subjects with baseline immunity (data not shown)

Section Viral Vaccines CERVARIX: AS04 ADJUVANTED HUMAN PAPILLOMAVIRUS VACCINE

Overview to selected vaccine efficacy (VE) data from clinical trials: CROSS-PROTECTION

VE against high-grade cervical lesions irrespective of HPV DNA type in lesions

 In naive subjects very high efficacy against CIN3+ irrespective of HPV type!!  Protection also against non-vaccines HPV types

Cross-protection data for non-vaccines types available: - Against HPV types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 - 6-months persistent infection: 0 – 77% efficacy - CIN2+ : 26.8 – 87.5% efficacy  There is good cross-protection to certain non-vaccine HPV-types

Section Viral Vaccines CERVARIX-AS04: CLINICAL SAFETY EVALUATION

Safety profile of “Cervarix” Pooled analysis of data available from more than 30.000 girls and women (≈ 46.000 doses) Higher rates in vaccinees for solicited local events (injection site) – but mild and short duration Summary of main findings: Serious adverse events 2.8% Cervarix vs 3.1% control pregnancies outcomes no differences medically significant conditions 19.4% vs 21.4% new onset of chronic diseases 1.7% vs 1.7% new onset of autoimmune diseases 0.4% vs 0.3 %  Overall acceptable and favourable safety profile

No increase in safety signals after “Cervarix” vaccination

• Unsolicited adverse events • Medically significant conditions • Serious adverse events • Potentially immune-mediated diseases From: Angelo et al., 2014

Section Viral Vaccines OIL-IN-WATER ADJUVANTS FOR INFLUENZA VACCINES: AS03

AS03 strongly increases adapative immune response in H5N1 pandemic (same as for MF59-adjuvanted vaccine)

350 HI-Titer 300 250 200 150 100 50 0

450 Neutralization-Titer 400 350 300 250 200 150 Non-adjuvanted 100 AS03 adjuvanted 50 0 3.8 7.5 15 30 3.8 7.5 15 30 µg antigen after dose 1 after dose 2 From: Garcon et al., 2012 Section Viral Vaccines OIL-IN-WATER ADJUVANTS FOR INFLUENZA VACCINES: AS03

AS03 effect on Seroconversation rates after H5N1 pandemic Influenza vaccination

Against vaccine-homologous Influenza strain (A/Vietnam/1194)

Indonesia/5/05 Turkey/Turkey/1/05 Anhui/1/05 Clade 2.1.3 Clade 2.2 Clade 2.3.4 Against vaccine-heterologous Influenza A strains as indicated

3.8µg 3.8µg 3.8µg 3.8µg 3.8µg 3.8µg AS03 AS03 AS03 From: Leroux-Roels, Lancet 2007 Section Viral Vaccines OIL-IN-WATER ADJUVANTS FOR INFLUENZA VACCINES: MF59

Binding capacity of serum IgG to HA1 protein (by surface plasmon resonance) • MF59 strongly increases binding to HA1 • Most prominent effect in toddlers (naïve)

IgG avidity to HA protein MF59 strongly increases avidity Again, most prominent effect in toddlers (naïve)

From: Khurana et al, 2011

Section Viral Vaccines OIL-IN-WATER ADJUVANTS: OVERALL SAFETY PROFILE

Safety profile of vaccines adjuvanted with AS03 or MF59 Large data base available from clinical trials – children and adults/elderly Almost 200 Mio doses of MF59-vaccine administered (“Fluad” for elderly!! Licensed for 20 years) “Focetria” (MF59) and “Pandemrix” (AS03) used in “Swineflu pandemic” mass vaccination campaign

Summary of main findings:

In children: - slightly higher rates of mild-to-moderate local reactions at injection site (such as pain, redness, swelling, tenderness) - Slightly higher rates of solicited systemic reactions (myalgia, headache, fatigue, malaise) - For AS03: increased frequency of fever after second dose (in < 5 yoa) In adults: No significant differences as compared to non-adjuvanted influenza vaccines No specific safety signals in Pharmacovigilance databases (1997 – 2006)

Overall, “Focetria” with slightly better tolerability than “Pandemrix” (→ higher oil and tocopherol content)

Both vaccines with acceptable and favourable overall safety profile

However: Pandemrix  Association with Narcolepsy

Section Viral Vaccines ASSOCIATION OF AS03 ADJUVANTED PANDEMIC VACCINE WITH NARCOLEPSY

Narcolepsy • rare chronic sleep disorder with excessive daytime sleepiness that can be associated with cataplexy. • Incident rate is about 0.74 – 1.37 per 100.000 person-years. • Strong association with HLA DQB1*06:02 genotype • Caused by selective loss of hypocretin-producing cells in the hypothalamus and resulting low hypocretin levels in CSF, probably as consequence of an autoimmune disorder

What happened in the aftermath of the “Swineflu vaccination campaign”….

In 2009: Mass vaccination campaign in EU against “Swineflu“ A/H1N1 influenza pandemic: - AS03 adjuvanted vaccine “Pandemrix”: more than 30 Mio doses administered in EU - MF59 adjuvanted vaccine “Focetria”: several Mio doses (much less that “Pandemrix”)

In 2010: first reports from scandinavian countries Finland and Sweden - Increase in narcolepsy – more than 10-fold as compared to background incidence - Potential Association with AS03-adjuvanted vaccines (Pandemrix and Arepanrix)

 Later also detected in other EU countries – but mosly to much less extent

 In total, so far more than 1300 cases of vaccine-associated (excess) cases of Narcolepsy registered by the European Medicines Agency (EMA)

Section Viral Vaccines ASSOCIATION OF AS03 ADJUVANTED PANDEMIC VACCINE WITH NARCOLEPSY

Data from Finland: Incidence peak follows vaccination peak Number of cases per age and reporting year

Annual incidence by age group and year of diagnosis

 Clear increase in Narcolepsy cases in children and adolescents (less than 20 yoa) following “Pandemrix” vaccination  A clear signal of association!  Regulatory Measures imposed by EMA  Investigations into potential mechanisms

Section Viral Vaccines ASSOCIATION OF AS03 ADJUVANTED PANDEMIC VACCINE WITH NARCOLEPSY

Potential proposed mechanisms for Pandemrix / AS03 to cause Narcolepsy:

Issue still very much under debate Some putative modes of action / mechanisms have been described:

 AB induced against the NP component of “Pandemrix” cross-react with hypocretin receptor type 2 and lead to disruption of hypocretin signalling in the brain

 Molecular similarity between influenza HA-Epitop and hypocretin causes HA-directed AB to react with and inactivate hypocretin

 Alpha-tocopherol in AS03 (not present in MF59) triggers a pathway that finally leads to a degradation of hypocretin producing neurons

None of these has been finally proven and/or confirmed. Currently no confirmed causative role for “Pandemrix”/AS03 in the emergence of Narcolepsy YET: temporal association of narcolepsy with “Pandemrix” vaccination is evident!!

Section Viral Vaccines ADJUVANT AS01 IN MALARIA/ “MOSQUIRIX”

Vaccine Composition (per dose): 25 µg of RTS,S: Portion of P. falciparum circumsporozoite protein fused with hepatitis B surface antigen (RTS), and combined with hepatitis B surface antigen (S) AS01 adjuvant: Quillaja saponaria Molina, fraction 21 (QS-21) (25 µg) and MPL (25 µg)

Clinical evaluation: Large Phase III trial in seven sub-Saharan African countries Almost 9.000 young children (5-17 months) and 6500 infants (6 – 12 weeks) included 3 doses to be given at monthly intervals, at 18 months after first dose recommended

Vaccine showed moderate efficacy (VE) against malaria clinical manifestations:

VE against: Clinical Malaria Severe Malaria Hospitalisation

infants

young children

VE quite rapidly decreasing – can be slightly improved by the fourth recommended dose

Section Viral Vaccines ADJUVANT AS01 IN HERPES ZOSTER () VACCINE “SHINGRIX” Currently under evaluation for licensure in the EU

Vaccine Composition (per dose): 50 µg of gE antigen: (VZV) glycoprotein E (gE) produced by recombinant DNA technology in Chinese Hamster Ovarian (CHO) cells AS01 adjuvant: Quillaja saponaria Molina, fraction 21 (QS-21) (50 µg) and MPL (50 µg)

Indication: For prevention of herpes zoster (HZ) and HZ-related complications, such as post-herpetic neuralgia (PHN) in adults 50 years of age or older

2 doses to be administered

Clinical evaluation: Two large Phase III trials ≥ 50 yoa: > 15.000 subjects ≥ 70 yoa: ≈ 14.000 subjects

Evaluation for efficacy (HZ and PHN) and safety

Section Viral Vaccines ADJUVANT AS01 IN HERPES ZOSTER (SHINGLES) VACCINE “SHINGRIX”

 Vaccine efficacy against Shingles and PHN

Shingrix Non-adjuvanted Zoster vaccine control

against HZ

against PHN

• VE of Shingrix much higher than that of the non-adjuvanted control • Strong adjuvanting effect for AS01 in combination with the VZV antigen

Section Viral Vaccines ADJUVANT AS01 IN HERPES ZOSTER (SHINGLES) VACCINE “SHINGRIX”

 In humans AS01 strongly enhances the frequency of VZV gE-specific CD4+ T-cells

Section Viral Vaccines SAFETY PROFILE OF ADJUVANT AS01

In children: data from “Mosquirix” vaccine trials (more than 10.000 children included for safety) Overall, acceptable safety profile Similar numbers of severe adverse events as compared to control vaccine groups Certain systemic symptoms (fever, drowsiness, irritability) slightly higher in “Mosquirix” recipients

Increased number of Meningitis in Mosquirix group. But relatedness to vaccination is questionable and currently unclear: Most likely a “chance finding”

In adults and elderly: Data from “Shingrix” trials (Lal et al., NEJM 2015) Overall, acceptable safety profile Rate of serious adverse events not increased by “Shingrix” vaccination (within 30 day p. v.) No difference in long-term follow up (3,5 years) between Vaccine and placebo recipients

Higher rate of solicited and unsolicited adverse symptoms mostly mild-to-moderate, transient (1-3 days) • injection-site reactions (81,5% for Shingrix vs 11,9% for placebo) (such as pain) • systemic reactions 66,1% vs 29,5%) (such as myalgia)

Section Viral Vaccines  FUTURE PERSPECTIVES

Huge Battery of novel adjuvants currently under preclinical and/or clinical investigation - dsRNA analogues, SSDNA: poly I:C, CpG motifs - Lipid A analogues: GLA, RC529,… - Flagellin - - ISCOMS - Cationic liposomes; CAF - Polysaccharides: Inulin

There are currently several important Unknowns (Mechanisms, Safetey) Major Questions and Issues to be addressed/answered in the future • Are these adjuvants applicable to be used for human vaccines? • Detailed knowledge on the mode of action • Do they allow for deliberate shaping of the vaccine immune response induced? (eg for efficient priming, immune senescence,…) • Can we better understand antigen-specific effects of individual antigens? • (How) can we prevent/minimise very rare serious adverse events?

Section Viral Vaccines THANK YOU VERY MUCH FOR YOUR ATTENTION!!!

Any Questions??

Section Viral Vaccines Section Viral Vaccines