1 Adjuvants (understanding modern vaccines)Leonard Friedland, MD Vice President, Scientific Affairs and Public Health GSK Vaccines 12th Statewide Immunization Ohio Conference November 16, 2016

2 Vaccines are Complex Biological Mixtures of Several ComponentsTrace components Adjuvant Diluents Antigens Stabilizers The vaccines we give to our patients are complex biological mixtures of several components. Vaccines are formulations of antigens, and may also include adjuvants, diluents, trace components and stabilizers. This presentation will review the role of adjuvants in vaccines. CDC. Ingredients of vaccines – Fact sheet. Accessed Feb 5, 2016.

3 From Latin, adiuvare: to aid Adjuvant From Latin, adiuvare: to aid Pharmacological/immunological agent that modifies the effect of other agents Substances that enhance or shape the immune response Immunological adjuvants added to vaccines stimulate the host immune system’s response to target antigen, but do not themselves confer immunity Old technology, made new The word adjuvant comes from the Latin root “to aid.” Adjuvants are immunological agents that modify, enhance and shape the immune system’s response to the target antigen. Adjuvant technology is old technology, dating back to the 1920s. This old technology is now being made new as the result of recent advances in our understanding of biology, chemistry, immunology and bioengineering. Garçon N, et al. Understanding modern vaccines, Perspectives in vaccinology, Vol 1, Amsterdam: Elsevier; 2011; Chapter 4: p

4 Major discoveries: adjuvantsUse of aluminium salts as vaccine adjuvants by Alexander Glenny Discovery of adjuvants by Gaston Ramon The adjuvant story starts in the 1920s, with the discovery by the French veterinarian Gaston Ramon and the British immunologuist Alexander Glenny in that certain substances could increase yields of immune sera containing antitoxins. This was a major contribution to vaccine development, and started the use of adjuvants in vaccines. Aluminum was first used as an adjuvant by Glenny in 1926, and became the only human licensed adjuvant for the following 75 years. Copyright, Wellcome Library, London Copyright, Wellcome Library, London Bonanni et al. Understanding modern vaccines, Perspectives in vaccinology, Vol 1, Amsterdam: Elsevier; 2011; Chapter 1: p1–24

5 Antigens May Need Help: Role for AdjuvantsCURRENT CHALLENGES FOR VACCINES1 Challenging populations due to impaired immune system (e.g. elderly, children, immunocompromised) Need for booster vaccinations Recombinant antigens Generally less immunogenic than live or attenuated organism vaccine2 Pathogens that requires broad and complex immune response Need for antigen sparing Potential supply problems (e.g. pandemic flu) Reduce the amount of antigen needed (dose-sparing) Increase the level of the immune response Prolong the duration of the immune response, improve immune memory, and protection Overcome a weakened immunogenicity Induce the generation of a high and broad immune response The history of vaccine development over the past 2 centuries has been driven by the continuous increased knowledge of vaccine science, innovative research, refinements, new technologies and a mission to improve public health. The development of today’s vaccines presents challenges in elderly immunoscenescent and immunocompromised patients, issues with duration of vaccine protection, obstacles with poorly immunogenic recombinant antigens, difficulties with complex pathogens such as malaria, and the need for antigen sparing in the context for example of pandemic flu. Adjuvants can help to overcome many of these challenges by improving the strength and durability of the immune response, often with reduced levels of antigen. Let us take a few minutes to discuss how adjuvants work. Garçon N, et al. Understanding modern vaccines, Perspectives in vaccinology, Vol 1, Amsterdam: Elsevier; 2011; Chapter 4: Petrovsky N, et al. Immunology and Cell Biology (2004) 82, 488–496; doi: /j

6 Adjuvants Activate Innate Immunity1Adaptive immunity Min 　　Hours Days Months–Years Antigen-specific B- and T-cell responses ‘Inflammatory responses’ Antigen(s) MHC-peptide TCR Antigen- presenting Cell (APC) T-cell Vaccine = tolerance/ignorance = immune response Co-stimulation Cytokines Defense triggers or adjuvants An adjuvant can activate the innate immune system by acting like pathogen-associated molecular patterns and thus can enhance or restore the ability of the immune system to identify a vaccine antigen as a pathogen with subsequent activation/maturation of APCs and activation of the adaptive immune system.2 This illustration shows the interplay between innate and adaptive immune responses. The key cell bridging the innate and adaptive arms of immunity is the antigen presenting cell. Some definitions: Innate immunity is the part of the immune system that acts rapidly and has no capacity for immune memory. Adaptive immunity is the part of the immune system that is the antigen-specific line of defence, and has immune memory. Antigen presenting calls INTEGRATE INFORMATION FROM FOREIGN STIMULI, for example components of pathogens, or vaccines, or adjuvants in vaccine formulations, and ORCHESTRATE THESE SIGNALS into adaptive immune responses, with immune memory. Adjuvants work by activating innate immunity, directing and stimulating the adaptive immune system. Adapted with permissions from Ishii et al. Curr Pharm Des 2006;12(32):4135–42. Reed SG, et al. Nature Medicine. 2013:19; MHC = major histocompatibility complex; TCR = T-cell receptor.

7 Stimulate/direct immune responseMicrobial Structures1,2,3 r contain: Antigens Defence triggers (danger signals), e.g. PAMPs act as intrinsic immune-triggers Defense triggers Antigens Pathogen Alert immune system Stimulate/direct immune response Specific immune response To understand how adjuvants work it is useful to first look at a pathogen, shown here in blue. The pathogen contains antigens and intrinsic triggers of immune defence, referred to as PAMPS, pathogen associated molecular patterns, also known as danger signals. The danger signals sound an alert that is recognised by cells of the innate immune system, directing and stimulating a robust adaptive immune response, with immune memory. 1. Dougan G & Hormaeche C. Vaccine 2006;24(S2):S13–S19 2. O’Hagan DT & Valiente NM. Nat Rev Drug Discov 2003;2:727–35 3. Garçon N, et al. Understanding modern vaccines, Perspectives in vaccinology, Vol 1, Amsterdam: Elsevier; 2011; Chapter 4:89-113 PAMPs = pathogen-associated molecular patterns

8 Adjuvants act as substitutes for natural immune-defense triggersSelecting From Nature: Right Antigens & Well Characterized Adjuvants1,2 Vaccine antigens alone may exhibit insufficient immunogenicity Some selected adjuvants act as substitutes for natural immune-defense signals, enhancing and directing the immune response Defense triggers Antigens Vaccine Adjuvants act as substitutes for natural immune-defense triggers Specific immune response On this slide we have a vaccine, with its antigen on the right. During the vaccine inactivation or purification process the vaccine can loose its innate defence triggers, its danger signals, and in this case the antigens alone may not be able to elicit a sufficient immune response. The discovery of the intrinsic immune defence triggers and the recognition of the link between innate and adaptive immunity, has facilitated the development of a series of innovative adjuvants. [CLICK] Some specifically selected and well characterized adjuvants can be formulated with the vaccine, such as MPL a TLR4 agonist, or CPG a TLR9 agonist. The adjuvant can act as a substitute for the natural defense triggers, and by rational design enhance, direct and shape the antigen-specific immune response. In general, adjuvants act in a similar way to the immune-defence triggers present in pathogens by interacting with APCs and promoting appropriate immune responses. 1. Dougan G & Hormaeche C. Vaccine 2006;24(S2):S13–S19 2. O’Hagan DT & Valiente NM. Nat Rev Drug Discov 2003;2:727–35

9 Antigens May Need Help: Why and When? The Role of Adjuvants1,2High Adjuvants Purified antigens/ (single adjuvant, combination adjuvants) Native virus Replicating (live attenuated pathogen) Immunogenicity Non-replicating (whole inactivated pathogen) Subunit (toxoids, split virus, fragments of pathogens) Purified antigens (various antigens, recombinant proteins) Low High Tolerability On this slide we have various types of vaccines, with live attenuated vaccines on the left and recombinant antigen vaccines on the right. The x-axis is patient tolerability to the vaccine from low to high, and the y-axis is immune response to the vaccine from low to high. Here we see that live attenuated vaccines and whole cell vaccines tend to be highly immunogenic . Subunit and recombinant protein vaccines tend to have high tolerability by patients, yet during the vaccine inactivation or purification process the subunit and recombinant protein vaccines can loose their innate defence triggers and in this case the antigens alone may not be able to exhibit sufficient immunogenicity. [CLICK] The addition of adjuvants to the vaccine formulation can enhance and modulate vaccine immunogenicity to the target antigens, while tending to increase reactogenicity. Illustrative figure based on concepts from: 1. Pasquale AD, Preiss S, Da Silva FT et al. Vaccine adjuvants: from and beyond. Vaccines : 2. Garçon N, et al. Understanding modern vaccines, Perspectives in vaccinology, Vol 1, Amsterdam: Elsevier; 2011; Chapter 4: 9

10 Expected Impact of Adjuvants on Vaccine Immune ResponseAdjuvanted formulation Stronger/broader immune response Immune response Longer-term immune response Non-adjuvanted formulation Earlier immune response Time Compared with the non-adjuvanted formulation, the expected advantages of vaccines formulated with adjuvants are: An earlier immune response. A stronger/broader immune response. A sustained or long term immune response. These effects do not necessarily appear all together, it depends on the vaccine formulation (antigen−adjuvant combination) and the specific target population. Illustrative figure based on concepts from: Garçon N, et al. Understanding modern vaccines, Perspectives in vaccinology, Vol 1, Amsterdam: Elsevier; 2011; Chapter 4:89-113

11 Adjuvanted vaccine design principleAntigen(s) Intrinsic immune defense triggers or adjuvants Specificity of the immune response Can enhance and modulate the immune response to vaccine antigen Adapted from: Garçon et al. Expert Rev Vaccines 2007;6:723–39. Garçon N, et al. Understanding modern vaccines, Perspectives in vaccinology, Vol 1, Amsterdam: Elsevier; 2011; chapter 4: p

12 General Adjuvant Mode of Action

13 Adjuvant: Expected Impact on Vaccine Immune ResponseMuscle/Injection site Draining lymph node Periphery/ Site of infection Antibody Macrophage APC CD4+ T cell Without adjuvant B cell (memory) Plasma cell B-cell (naїve) Increased number of activated APCs migrating to the DLNs Blood vessel Monocyte Enhancement and modulation of the adaptive response Increased recruitment of innate cells at the site of injection Plasma cell Antibody CD4+ T cell B cell (memory) B-cell (naїve) With adjuvant Increased uptake of antigen by APCs Antibody Monocyte Macrophage Plasma cell CD4+ T cell (diversity impacted) Blood vessel APC B-cell (naїve) Antibody (wider profile) APC = Antigen presenting cells; DLN = Draining lymph node Granulocyte I show on this slide in the top panel a vaccine without an adjuvant being administered into a muscle, and on the bottom panel a vaccine with an adjuvant. Each adjuvant has its own specific mechanism of action. Recent basic science discoveries have revealed some basic mechanistic principles. When comparing a vaccine with an adjuvant to a vaccine without an adjuvant, [CLICK] the adjuvanted vaccine increases recruitment of innate cells (monocytes, macrophages and dendritic cells) at the site of injection; [CLICK] this then leads to increased uptake of antigen by the antigen presenting cells and immune mediator/cytokine release, [CLICK] then leading to increased numbers of antigen presenting cells migrating to the draining lymph nodes, [CLICK] ultimately resulting in enhancement and modulation of the adaptive immune response. Cytokines Adjuvant Antigen Cytokines (improved pattern) Major histocompatibility complex Illustrative figure based on concepts from: Pasquale AD, Preiss S, Da Silva FT et al. Vaccine adjuvants: from and beyond. Vaccines : Garçon N, et al. Understanding modern vaccines, Perspectives in vaccinology, Vol 1, Amsterdam: Elsevier; 2011; Chapter 4:89-113

14 Role of Innate and Adaptive Immune Response in Adjuvant ResponseInnate Response (0 – 72h) Adaptive Response (Day 1 to Weeks) T APC B Granulocyte Macrophage Monocyte Cytokines Antigen Site of infection/injection Lymph node Blood Stimulation of the local innate system Local cytokine response Recruitment of innate immune cells Adaptive immune response Antigen presenting cells integrate the messages from innate immunity to T and B cells Garçon N, et al. Understanding Modern Vaccines, Perspectives in Vaccinology, Vol 1, Amsterdam: Elsevier; 2011; chapter 4: p89-113

15 Safety considerations for the development of vaccines with novel adjuvants

16 The Safety Refers to the Vaccine as a Whole and Each Vaccine is DifferentAntigen X Other components Adjuvant Y Vaccine X Antigen A Other components Adjuvant Z Vaccine A As mentioned at the beginning of this presentation, vaccines are complex mixtures of antigens, adjuvants and other components. Each vaccine formulation is distinct, and thus the safety of the vaccine refers to the whole vaccine received by the patient, not its components. WHO. Vaccine safety basics learning manual. . Accessed Feb 5, 2016.

17 Continuous Safety Assessment and MonitoringSafety is of Primary Importance From the Start of Development and Throughout the Entire Life of a Vaccine Preclinical Clinical Post-Licensure 5–15 years 5–15 years For Entire Life-cycle Continuous Safety Assessment and Monitoring Vaccines are carefully evaluated under tight process controls and overseen by regulatory authorities Preclinical safety profile is established through toxicology studies in animals Each vaccine is a separate entity and requires independent safety evaluation Safety monitoring is designed to rapidly identify rare and/or serious adverse events temporally linked to vaccination Adverse events of ‘special interest’, including potential immune mediated diseases, are a focus for safety evaluation Follow safety for a long period – typically 12 months after vaccination Safety is important for any vaccine development program, and is continuously assessed at every step of vaccine development: preclinical, clinical and after licensure. Safety monitoring from the start of development and throughout the entire life cycle is designed to rapidly identify rare and/or serious adverse events temporarily linked to vaccination. Leroux-Roels et al. in: Understanding Modern Vaccines: Perspectives in Vaccinology, Vol 1. Amsterdam: Elsevier; 2011 Chapter 5:115–150

18 Adjuvanted Vaccines: General Reactogenicity and Safety ProfileAdjuvanted vaccines often have increased reactogenicity, especially at the injection site Local symptoms are usually mild/moderate, short-lasting and do not impact compliance The safety profile of aluminium salt adjuvants has been well established through the use of billions of doses, in different populations, over more than 80 years Licensed, adjuvanted vaccines have clinically acceptable benefit-risk ratios Garçon N et al. Understanding Modern Vaccines: Perspectives in Vaccinology, Vol 1. Amsterdam: Elsevier; 2011 (Chapter 4: p89–113)

19 Potential Immune-Mediated Diseases: Example of List included in ProtocolsNeuroinflammatory disorders Musculoskeletal disorders Skin disorders Cranial nerve disorders, including paralyses/paresis (e.g. Bell’s palsy) Optic neuritis Multiple sclerosis Transverse myelitis Guillain-Barré syndrome, including Miller Fisher syndrome and other variants Acute disseminated encephalomyelitis, including site specific variants: e.g. non-infectious encephalitis, encephalomyelitis, myelitis, myeloradiculoneuritis Myasthenia gravis, including Lambert-Eaton myasthenic syndrome Immune-mediated peripheral neuropathies and plexopathies, (including chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy and polyneuropathies associated with monoclonal gammopathy). Narcolepsy Systemic lupus erythematosus and associated conditions Systemic Scleroderma (Systemic sclerosis), including diffuse systemic form and CREST syndrome Idiopathic inflammatory myopathies, including Dermatomyositis, Polymyositis, Antisynthetase syndrome Rheumatoid arthritis and associated conditions including Juvenile chronic arthritis and Still’s disease) Polymyalgia rheumatica Spondyloarthritis, including ankylosing spondylitis, reactive arthritis (Reiter's Syndrome) and undifferentiated spondyloarthritis Psoriatic arthropathy Relapsing polychondritis Mixed connective tissue disorder Psoriasis Vitiligo Erythema nodosum Autoimmune bullous skin diseases (including pemphigus, pemphigoid and dermatitis herpetiformis) Alopecia areata Lichen planus Sweet’s syndrome Localised Scleroderma (Morphoea) Liver disorders Gastrointestinal disorders Endocrine disorders Autoimmune hepatitis Primary biliary cirrhosis Primary sclerosing cholangitis Autoimmune cholangitis. Inflammatory Bowel disease, including Crohn’s disease, ulcerative colitis, microscopic colitis, ulcerative proctitis Celiac disease Autoimmune pancreatitis Autoimmune thyroiditis (including Hashimoto thyroiditis) Grave's or Basedow’s disease Diabetes mellitus type I Addison’s disease Polyglandular autoimmune syndrome Autoimmune hypophysitis Vasculitides Blood disorders Others Large vessels vasculitis including: giant cell arteritis such as Takayasu's arteritis and temporal arteritis. Medium sized and/or small vessels vasculitis including: polyarteritis nodosa, Kawasaki's disease, microscopic polyangiitis, Wegener's granulomatosis, Churg–Strauss syndrome (allergic granulomatous angiitis), Buerger’s disease (thromboangiitis obliterans), necrotizing vasculitis and anti-neutrophil cytoplasmic antibody (ANCA) positive vasculitis (type unspecified), Henoch-Schonlein purpura, Behcet's syndrome, leukocytoclastic vasculitis. Autoimmune hemolytic anemia Autoimmune thrombocytopenia Antiphospholipid syndrome Pernicious anemia Autoimmune aplastic anaemia Autoimmune neutropenia Autoimmune pancytopenia Autoimmune glomerulonephritis (including IgA nephropathy, glomerulonephritis rapidly progressive, membranous glomerulonephritis, membranoproliferative glomerulonephritis, and mesangioproliferative glomerulonephritis) Ocular autoimmune diseases (including autoimmune uveitis and autoimmune retinopathy) Autoimmune myocarditis/cardiomyopathy Sarcoidosis Stevens-Johnson syndrome Sjögren’s syndrome Idiopathic pulmonary fibrosis Goodpasture syndrome Raynaud’s phenomenon With regard to adjuvanted vaccines, some considerations for safety monitoring should be taken into account: Adverse events of special interest need to be identified. There should be a special focus on autoimmune or autoinflammatory diseases, such as neuroinflammatory disorders, musculoskeletal and connective tissue diseases and GI disorders. Throughout the entire study period, enhanced case reporting for potential autoimmune adverse events should be established. A longer safety follow-up period should be considered. References: 1. Adapted from Marion F Gruber, PhD, Acting Director, OVRR/CBER/, Vaccine Forum, Washington Available at: (Accessed November 2014). 2. Leroux-Roels. Vaccine 2010;28S:C25–36. 3. Verstraeten et al. Vaccine 2008;26:6630–6638. Data on File. Study

20 Safety evaluation: Key PointsSafety evaluation of a vaccine is ultimately based on the evaluation of the final vaccine (antigen and adjuvant) Preclinical and early clinical testing should provide justification for the use of the adjuvant in the final vaccine Clinical programme methodology should be standardised to allow for comparison and pooling of data (early and late trials) Case reporting should be enhanced throughout entire study period for characterising potentially immune-mediated diseases Regulatory pathways supporting development and approval of vaccines formulated with novel adjuvants are the same as for unadjuvanted vaccines Garçon N et al. Vaccine 2011;29:4453–9

21 Among all the Possibilities, How is an Adjuvant Selected?1,2Identify need for adjuvant Compatibility with antigen? Understand: Host-pathogen interaction Antigen selection and production Optimized immunological tools In case classical aluminium not sufficient, consider: Stability over time? Formulation: scalable, robust, reproducible? Immune response? Safety and reactogenicity? When aluminium salts do not sufficiently induce the relevant immune response, alternative adjuvants are evaluated. Understanding of the host pathogen interaction, selection and production of protective antigens as well as the availability of the adequate immunological tools to is necessary to select the most appropriated adjuvant. Immune assays need to be developed in order to assess the impact of the adjuvant on the immune response. To be a candidate for the vaccine, the adjuvant needs to be compatible with the antigen, be stable over time, induce the immune response deemed necessary for protection, and have a safety and reactogenicity profile that is acceptable for the target population. Leroux-Roels et al., in: Understanding Modern Vaccines, Perspectives in Vaccinology, Vol 1, Amsterdam, Elsevier, 2011, Chapter 5:115–150. Leroux-Roels. Unmet needs in modern vaccinology adjuvants to improve the immune response. Vaccine S:C25-C36.

22 Adjuvant Development Fluad® (influenza) (AS04) Cervarix® (human papilloma virus) Prepandrix® (pandemic H5N1 influenza) AS01 Mosquirix® (malaria) Many potent adjuvants failed, often due to unacceptable reactogenicity MF59 and MPL + Alum (AS04) were first new adjuvants in ~75 years Additional adjuvants are now emerging Cervarix, Prepandrix and Mosquirix are trademarks of the GlaxoSmithKline group of companies; Fluad is trademark of Sequirus, Inc Garçon N, et al. Understanding modern vaccines, Perspectives in vaccinology, Vol 1, Amsterdam: Elsevier; 2011; Chapter 4: FDA. Accessed June 23, 2016. EMA. Cervarix. June 23, 2016. EMA. Prepandrix. Accessed June 23, 2016. GSK. Accessed June 23, 2016. The brilliant science of Ramon and Glenny in the 1920s led to aluminum being used in human vaccines starting in the 1930s. Scientists looked to develop newer and novel adjuvants throughout the 20th century; however these experimental adjuvants failed due to high reactogenicity and a lack of understanding of mechanism of action. The last 25 years have brought increased knowledge of immunology, biology, chemistry, and bioengineering, resulting in the development of novel adjuvants, and advances in understanding their mechanism of action. We are now entering a golden age of new adjuvant development, licensure and use in human vaccines; beginning with the inclusion of MF59 and AS04 adjuvants in human vaccines in the late 90s and early 2000s, the first new adjuvants in 75 years.

23 Adjuvants in Clinical Trials/Licensed VaccinesAdjuvant name Mechanism or receptor Clinical phase or licensed product Aluminum salts (for example, aluminum oxyhydroxide, aluminum phosphate) Nalp3, ITAM, Ag delivery Numerous license products (e.g. Infanrix®, Engerix-B®, Havrix®) Lipid A analogues (for example, MPL, RC529, GLA, E6020) TLR4 Cervarix® AS04 (MPL, aluminum salt) Cervarix Emulsions (for example, MF59, AS03) Immune cell recruitment, ASC, Ag uptake Fluad®, Pandemrix ® Imidazoquinolines (for example, Imiquimod, R848) TLR7 and TLR8 Aldara® CpG ODN TLR9 Phase 3 Saponins (for example, QS21) Unknown AS01 (MPL,QS21, liposomes) AS02 (MPL,QS21, emulsion) AS15 (MPL, QS21, CpG, liposomes) TLR4 and TLR9 ISCOMs (saponin, phospholipid) Phase 2 dsRNA analogues (for example, poly(I:C)) TLR3 Phase 1 Flagellin TLR5 C-type lectin ligands (for example, TDB ) Mincle, Nalp3 CD1d ligands (for example, α- galactosylceramide) CD1d GLA-SE (GLA, emulsion) IC31 (CpG, cationic peptide) CAF01 (TDB, cationic liposomes) Mincle, Ag delivery Ag = antigen; ASC= apoptosis-associated speck-like protein containing caspase recruitment domain; dsRNA = double-stranded RNA; ITAM = immunoreceptor tyrosine-based activation motif; TDB = trehalose dibehenate. Some particulate formulations (such as aluminum salts and emulsions) also generate immunomodulatory activity. GSK has been one of the leaders in adjuvant science, and as shown in this table the use of vaccine adjuvants is an extremely active field of research. Most of the adjuvants are natural substances. For example, MF59 is an oil-water emulsion based on squalene oil that is naturally derived from shark liver; QS21 is a natural saponin molecule purified from the bark of a tree; MPL is purified, non-toxic endotoxin derivative prepared from the lipopolysaccharide of a Salmonella strain. As we understand more about adjuvant science, the research is now exploring synthetic adjuvants. For example, CpG oligodeoxynucleotides. Infanrix, Engerix-B, Havrix, Cervarix and Pandemrix are trademarks of the GlaxoSmithKline group of companies; Fluad is trademark of Sequirus, Inc.; Aldara is trademark of Valeant Pharmaceuticals International, Inc. Adapted from Reed SG et al, Nature Med 19: , 2014

24 GSK Adjuvant System (AS) Families

25 GSK’s adjuvanted vaccine design principleAntigen Adjuvant System Specificity of the immune response Designed to enhance and modulate the immune response by combining the effect of two or more adjuvants Goal is to select the antigen/Adjuvant System combination which can guide the immune response, delivering enhanced and sustained protection Garçon N et al. Expert Rev Vaccines 2007;6:723–39; Leroux-Roels G. Vaccine 2010;28S:C25-C36.

26 GSK Adjuvant System: Nomenclature PrinciplesAS 01 AS or Adjuvant System written in capital letters Adjuvant System Family number Variant letter Components + Quantity Variant refers to the absolute amount of the immunostimulant /immunoenhancer(s) and liposome or emulsion. Exception for AS04 where the variant refers to the aluminum salt used (phosphate or hydroxide). GSK follows specific nomenclature for our adjuvant systems as shown here. The numbering is based on the order these adjuvant systems were created, however, the first one to be licensed was AS04 as part of HPV vaccine, Cervarix.

27 Creation of Rationally Designed Adjuvant SystemsType of formulation Hepatitis B virus (in pre-haemodialysis- Fendrix®) AS04 Aluminum salts Human papilloma virus (Cervarix®) Oil/water emulsions Alum/TLR7 Phase I (POC) Liposome Immuno-enhancers AS03 Pandemic Influenza MPL (TLR4) QS-21 SMIP (TLR7) Plasmodium (Malaria) VZV (Zoster) Tocopherol AS01 CpG (TLR9) MPL = 3-O-desacyl-4’-monophosphoryl lipid A; QS = Quillaja saponaria (Antigenics Inc., a wholly owned subsidiary of Agenus Inc., Lexington, MA Cervarix and Fendrix are trade marks of the GlaxoSmithKline group of companies.

28 Leroux-Roels et al. Clinical Immunology 2016:169;16-27One size does not fit all - The ‘adaptive signature’ of different AS in humans (naive adults, HBs as model antigen) Seropositivity Cut-off Alum MPL+ QS21 (lip) MPL+ alum Specific CD4+ T cells (CD40L+ per 1.10e6 cells) Technical Cut-off Anti-HBs Antibodies (mIU/mL) study shows that different adjuvants have a profound impact on the kinetics and the magnitude of T and B cell responses to a given protein vaccine in humans. Leroux-Roels et al. Clinical Immunology 2016:169;16-27

29 GSK Adjuvant System: an historical perspectiveIn-licensing of MPL, QS21 and CpG Cohen J et al. Human Vaccines 2010; 6(1):90-96. Garçon et al. Expert Rev Vaccines 2007;6:723–39. Garçon et al. Expert Rev Vaccines 2011;10(4): RTS,S Clinical Trial Partnership. NEJM 2012; 367: Lal H, et al. NEJM 2015;372: Novel adjuvant development has been a GSK Vaccines R&D core platform for the past 3 decades. We started work with the technology platform in the 1980s with in-licensing of the adjuvants MPL, QS21 and CpG,. Our early work with adjuvants was through empirical, trial and error design. Increased knowledge of immune-pathogen interactions, and the discovery of new biology, immunology, chemistry, and “omics” knowledge and tools in the 2000’s has opened the door to increased understanding of the mechanism of action of adjuvants. This has led to the rational design of new vaccines formulated with novel adjuvants. We are now entering a golden age of adjuvanted vaccines, starting with the licensure in the US of AS04-adjuvanted HPV vaccine, and today GSK has AS01-adjuvanted vaccines in development for malaria and shingles. Zoster and Malaria phase III data

30 Experience with investigational malaria vaccine

31 The Scientific Approach Behind Malaria Vaccine Candidate1,2Antibodies to block infection T cells to kill intra-hepatic parasites and eliminate infected hepatocytes Protection against Infection Impact on Incidence and Severity of Disease Vekemans J et al. Vaccine 2009: 27:G67-G71 Vekemans J et al Expert Review Vaccines 2008: 7(2): To induce strong and persisting immunity against the early stage of the malaria parasite life cycle in human

32 The Malaria Candidate Vaccine Composition1,2Specificity of the immune response Enhances the immune response to vaccine antigen Antigen RTS,S Adjuvant System AS01 Vaccine MPL from microbial membrane LPS MPL Saponin QS-21* Liposome Illustration by Franz Eugen Köhler Repeat T epitope, S antigen (RTS,S) *Antigenics Inc., a wholly owned subsidiary of Agenus Inc., Lexington, MA Vekemans J et al. Vaccine 2009: 27:G67-G71 Vekemans J et al Expert Review Vaccines 2008: 7(2):

33 First Proof of Concept for Efficacy of the RTS,S Candidate Vaccine Against P. falciparum Infection1,2 Human challenge model at the Walter Reed Army Institute of Research (WRAIR) Conducted at WRAIR 3 doses of adjuvanted RTS,S candidate vaccine (0, 4, 24 weeks) 3 weeks post-dose 3: challenge with infectious mosquitoes Follow up: parasites in blood smears Group Protected/ Challenged Prepatent Period (days)* 1) RTS,S + AS04 1/8 12.6 2) RTS,S + AS03 2/7 15.6 3) RTS,S + AS02 6/7 >16 4) Controls 0/6 12 * Prepatent period = interval between challenge and the detection of parasitemia Safety: After 1st dose – mild discomfort at the injection site After 2nd dose – more reactions in group 2 and 3. Three subjects (two from Group 2 and one from Group 3) experienced pain, malaise, feverishness, headache and myalgias within 24 hours. Two subjects with the most symptoms did not get 3rd dose, one each from group 2 and 3 The 3rd dose was reduced all in group 2 and 3. All of which were well tolerated. The standard dose of vaccine 1 was 1 ml, and that of vaccines 2 and 3 was 0.5 ml; each dose delivered 50 mg of RTS,S antigen. The third dose of vaccines 2 and 3 was reduced to 0.1 ml in response to adverse reactions after the second dose. Stoute J A et al. NEJM 1997, 336:86-91 Garçon et al. Expert Rev: Vaccines 2003, 2:

34 Antibody Response Does Not Correlate with Protection in Challenge ModelRTS,S/AS /7 RTS,S/AS /8 RTS,S/AS /7 Protected IgG CS Repeats 100 P<0.02 P<0.02 10 lgG Antibody (g/ml) Different RTS,S vaccine formulations with different Adjuvant Systems Antibody responses against CS tandem-repeat epitopes (ELISA with recombinant R32LR) 1 30 44 150 194 220 0.1 14 Day of Study CS = circumsporazoite; ELISA = enzyme-linked immunosorbent assay; R32LR = recombinant protein composed of the repeat region of P. falciparum CS protein Stoute J A et al. NEJM 336(2),86–91, 1997 34

35 Cell Mediated Immune ResponsesInterferon (IFN)- Responses in Volunteers Immunized with Different Formulations of RTS,S and Association with Protective Efficacy AS04 AS03 AS02 Number of ELISPOTs/106 cells Post dose 3 Pre-immune Subjects Not protected Protected 600 400 200 Safety information was not collected in this study ELISPOT = enzyme-linked immunospot Sun et al J of Immunol: 171 (2), (2003)

36 Can We Improve Vaccine Efficacy Through Alternative FormulationCan We Improve Vaccine Efficacy Through Alternative Formulation? AS01 Compared to AS02 α γ AS01 = Liposomes/ MPL/QS-21*; AS02 = Oil-in-water emulsion/MPL/QS-21; DOC = day of challenge; M1 = 1 month post-vaccination; M2 = 2 months post-vaccination; Post II = 1.5 months post-vaccination *QS21, Antigenics Inc., a wholly owned subsidiary of Agenus Inc., Lexington, MA Kester KE et al. J Infect Dis 2009; 200:

37 RTS, S/AS01 was introduced into the clinical development planCan We Improve Vaccine Efficacy (VE) Through Alternative Formulation? AS01 Compared to AS02 Protected Infected VE* 1) RTS,S/AS02 14 30 32% (95% CI, 18%-48%) 2) RTS,S/AS01 18 50% (95% CI, 33%-67%) AS02 MPL QS-21† Emulsion based AS01 MPL QS-21 † Liposome based Safety Pain was the most common solicited adverse event (82% for Group 1 & 81% for Group 2); others included swelling & redness Fatigue was the most common solicited systemic adverse event (44.1% for Group 1 and 35.9% for Group 2); trend others included headache, malaise, myalgia, gastrointestinal, arthralgia and fever No increase solicited adverse events with subsequent administration of the vaccines in either groups No serious adverse events were associated with study vaccines RTS, S/AS01 was introduced into the clinical development plan VE = vaccine efficacy * P = 0.11 for comparison between AS01 and AS02 † Antigenics Inc., a wholly owned subsidiary of Agenus Inc., Lexington, MA Kester K E et al. J Infect Dis 2009; 200:

38 Multi-Center RTS,S Malaria Phase III Vaccine Efficacy TrialKintampo, Ghana Nanoro, Burkina Faso Agogo, Lambaréné, Gabon Manhiça, Mozambique Lilongwe, Malawi Bagamoyo, Tanzania Korogwe, Kilifi, Kenya Siaya & Kombewa, Study sites Unstable risk Pf Malaria free Country boundary Water bodies 11 centers, 7 African countries, a range of different malaria transmission intensities Designed in collaboration with Clinical Trials Partnership Committee, with feedback of World Health Organization, African National Regulatory Authorities, Food and Drug Administration and European Medical Agency 15,460 children enrolled RTS,S Clinical Trial Partnership, NEJM 2011; 365: ; NEJM 2012; 367: ; Adapted from Hay et al, PLoS Med 2009;6:e ClinicalTrials.gov. NCT

39 Strategies to Address Remaining Challenges in Vaccine DevelopmentStrategies to overcome challenges New antigens New antigen presentation (DNA/RNA) New delivery strategies (Live Vectors) New Adjuvants Pathogens or diseases: HIV, Tuberculosis, CMV, ... Special populations: infants, elderly, immunocompromised individuals, ... Before we conclude our discussion, let’s go back to challenges vaccine developers encounter today; including complex pathogens and special populations such as the elderly and immunocompromised. While new adjuvants are a promising and proven tool to overcome today’s vaccine challenges, there are other strategies that vaccine scientists are utilizing. These include the discovery of new antigens, new ways to present antigens through RNA and DNA vaccines, and new delivery methods, such as vector vaccines as being used with the 2 most advanced investigational Ebola vaccines. HIV = human immunodeficiency virus; CMV= cytomegalovirus Stanberry et al. Chapter 6 in: Garçon et al. Understanding modern vaccines, Perspectives in vaccinology, Vol 1, Amsterdam. Elsevier 2011;p151–99

40 “Vaccinology” Vaccinology Vaccine Safety New and old vaccinesadjuvants Vaccinology Vaccine Safety New and old vaccines Adjuvanted-Vaccines Effective communication about adjuvants ties together vaccinology, vaccine history, vaccine safety and adjuvanted vaccine approaches. Experts, educators communicate the need for new or improved vaccines, how they work to enhance the quality and quantity of the immune response and how they meet the highest quality and safety standards Make the science of vaccines with its heritage and modern insights (including adjuvants) accessible and relevant to the healthcare provider community

41 Vaccine science: two centuries of continuous research, improvements and achievementsThe history of vaccine development over the past 2 centuries has been driven by the continuous increased knowledge of vaccine science, innovative research, some serendipity, refinements, new technologies and a mission to improve public health. After clean water, vaccines are judged to be the second most significant contribution to the control and eradication of infectious diseases. ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Hillemacher (French painter): Edward Jenner Vaccinating a Boy Family in modest home, back of cow sharing the premises is visible behind the crib. Parents lean over trustingly-family’s future is safe. Jenner is pioneering public health’s crowing achievement, immunization. “Edward Jenner Vaccinating a Boy” EE Hillemacher, Courtesy of Wellcome Trust, Wellcome Library, London

42 Vaccines, today an explosion of new technologies2015 Looking at today, our increased understanding of many disciplines including biology, immunology, chemistry, genetics and informatics makes possible an explosion of new technologies. These next generation technologies, such as synthetic biology, structural vaccinology, and adjuvants are being employed in the discovery and development of new vaccines. Front Immunol. 2014; 5: 12.  doi: /fimmu

43 Vaccines for every age Rappuoli R Editorial / Vaccine 33S (2015) B1–B2 Fig. 1

44 Vaccines for today’s societyRappuoli R Editorial / Vaccine 33S (2015) B1–B2 Fig. 1

45 Q&A

46 Experience with investigational zoster vaccine

47 Latent VZV infection and reactivationVZV-specific memory T-cells decline with age or in specific immune impairment conditions VZV immunity may be boosted periodically by exposure to varicella or silent reactivation from latency VZV primary infection induces VZV T-cell immune memory VZV T-cells The decline below a threshold correlates with an increased risk of HZ disease HZ threshold Varicella HZ Age → Immunity to HZ correlates with VZV T-cell levels HZ, herpes zoster; VZV, varicella zoster virus Kimberlin and Whitley. N Engl J Med 2007; 356: CONFIDENTIAL

48 Scientific approach behind zoster vaccine candidateelicit strong cellular & humoral immune responses against VZV in older adults (≥50 yr) & immunocompromised adults ≥18 yr Vaccine antigen: Varicella-zoster virus (VZV) glycoprotein E (gE) Abundantly expressed in the virion envelope and membranes of VZV-infected cells Prominent target of VZV-specific cellular and humoral immune responses Vaccine adjuvant: Adjuvant System 01B (AS01B) AS01 adjuvant system: liposome based adjuvant, contains 2 immunostimulants: QS-21* and MPL Enhance both cellular and humoral immune responses to subunit antigens Induce robust gE-specific CD4+ T cell and humoral immune responses in mice * Antigenics Inc., a wholly owned subsidiary of Agenus Inc., Lexington, MA

49 zoster candidate vaccine composition (HZ/su)Antigen gE Adjuvant System AS01 Specificity of the immune response Enhances the immune response to vaccine antigen MPL from microbial membrane LPS MPL Saponin QS-21* Liposome gE Illustration by Franz Eugen Köhler *Antigenics Inc., a wholly owned subsidiary of Agenus Inc., Lexington, MA Dendouga N et al., Vaccine 30 (2012), 3126:3135

50 Specific gE antibody response only raised when gE antigen is adjuvanted in micePriming with live attenuated GSK OKA virus vaccine, followed a month later by specified formulation (D0,28) IM Dendouga N et al., Vaccine 2012; 30:3126:3135

51 All components of AS01B are required to induce an adequate cellular response - miceSpleen cells from VZV-primed mice (OKA live-attenuated vaccine), 30 days post dose 2, Intracellular Cytokine Staining (ICS) OKA gE AS01B> gE + Liposome + QS-21 or gE + Liposome + MPL at 30 post dose 2 ( fold, p<0.05) gE AS01B> gE alone or gE + Liposome (15 fold, p<0.05) L= Liposome; MPL = 3-O-desacyl-4’-monophosphoryl lipid A; QS-21 = Quillaja saponaria Molina, fraction 21; Dendouga N et all, Vaccine 30 (2012), 3126:3135

52 HZ/su PoC study: cellular immune responsesOpen-label, randomized; N=155 gE/AS01B and/or VZV live attenuated (OKA) vaccine* administered separately or concomitantly 2 doses, Months 0, 2 gE/AS01B alone 2-doses gE/AS01B + OKA* (co-admin) 2-doses OKA* alone 2-doses response to natural zoster** months Cytokine-expressing cells/106 (median) Healthy older adults (50-70 years; n=135; 45x3 group) Young adults (18-30 years; n=20; 10x2 group) gE-specific CD4 T cell responses- A CMI response was defined as the frequency of cytokine-positive CD4 T cell producing at least 2 different cytokines among CD40L, IFN-γ, IL-2 or TNF-α. A CMI response was defined as the frequency of cytokine-positive CD4 T cell producing at least 2 different cytokines among CD40L, IFN-γ, IL-2 or TNF-α. * Minimum release titer = pfu/dose; actual titer of VZV used in this study: 104 pfu/dose ** Data from a GSK Vaccines HZ natural history study in which cellular and humoral immune responses were measured 1 month after natural HZ in immunocompetent adults ≥60 years. Mols J et al J Virol Method 2013; 188: Leroux-Roels G, et al., J Infect Dis 2012; 206:

53 HZ/su Phase III efficacy studies in healthy older adultsFinland Sweden Estonia UK Germany CZ Rep France Italy Spain Canada US Japan Taiwan Hong Kong Australia S. Korea Mexico Brazil Almost 31,000 subjects in 18 countries Lal et. al. NEJM 2015:372; Cunningham, et al. NEJM 2016:375;