Adjuvants. Giuseppe Del Giudice, MD. Global Head Translational Medicine, Novartis Vaccines. Siena, 23 September 2009

Transcription

1 Adjuvants Giuseppe Del Giudice, MD Global Head Translational Medicine, Novartis Vaccines Siena, 23 September 29

2 Gaston Ramon ( ) 2 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

3 Adjuvants included in licensed vaccines Fluad (Flu) Fendrix (HBV) Cervarix (HPV) Prepandrix (pan.flu) Many potent vaccine adjuvants in pre-clinical models have failed in clinical trials due to an unacceptable safety profile 3 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

4 Vaccine adjuvants and elderly Very little work done so far Most of the work done with influenza vaccine Most of the work done with the oil-in-water MF59 adjuvant 4 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

5 MF59: a successful o/w emulsion adjuvant Appearance: milky white oil in water (o/w emulsion) H 2 O H 2 O H 2 O H 2 O H 2 O OIL H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O OIL H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O H 2 O Composition:.5% Polysorbate 8.5% Sorbitan Triolate 4.3 % Squalene Water for injection 1mM Na-citrate buffer Density: Size: Viscosity:.9963 g/ml 16nm close to water, easy to inject 5 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

6 In mice, MF59 proved to be the strongest adjuvant Serum HI titers after one and two doses in naïve mice 3 A/H3N2 8 A/H1N1 3 B MF59 Serum HI Titers Alum CAP PLG Non-adjuvanted Postdose 1 Postdose 2 Postdose 1 Postdose 2 Postdose 1 Postdose 2 Alum masked by CAP. CAP masked by non-adjuvanted. CAP and PLG masked by non-adjuvanted. Also in humans, alum is a poor adjuvant for influenza vaccines (both seasonal and pandemic) Alum = aluminum hydroxide; CAP = calcium phosphate; PLG = polylactide coglycolide. Wack et al Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

7 MF59 induces an immunostimulatory environment at the injection site MF59 was the most potent activator of mouse transcriptome at injection site MF59 induced transcription of chemokines and cytokines with the potential to recruit and activate APCs 7 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only Mosca et al, Proc Natl Acad Sci USA 28

8 MF59 induced a rapid recruitment of CD11b+ cells Blue: Utrophin Red: PI Green: αcd11b 8 Presentation Adjuvants G. Title Del Giudice Presenter Siena, Name 23 Date September Subject 29 Business Business Use Use Only Only Mosca et al, Proc Natl Acad Sci USA 28

9 MF59 induces the release of chemokines but not inflammatory cytokines into the blood 9 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only Mosca et al, Proc Natl Acad Sci USA 28

10 MF59: dual adjuvant function at injection site Ag MF59 2 Indirect Immune potentiator Muscle fibers JunB, Ptx3 3h 1 DC Antigen Delivery IL1β TNFα Activation of resident APCs Local immunocompetent environment TNFα, IL1β, CCLs Circulating APC recruitment and activation 6h 1d-4d Monocytes DC precursors Migration to lymph node CD4 T cell activation 1 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only How these activities of MF59 on Ag delivery and of immunomodulation translate into clinical experience?

11 MF59 (oil-in-water)-adjuvanted vaccines Children CMV: Toddlers HIV: Newborns (1-3 days) Seasonal Flu (Fluad ): 6-36 months old H5N1: 6-36 months old Adults HSV, HIV, HPV, HCV, HBV, UPEC, CMV Immunocompromised patients Interpandemic Flu vaccine (Fluad ) in - HIV+ subjects - Pharmacological treatment (e.g. chronic corticosteroids, transplantations, etc) >45 million doses Elderly Licensed for use in > 65 years-olds in Fluad trivalent, seasonal vaccine in Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

12 MF59 Safety Database Completed and ongoing clinical studies (June 29) Influenza Studies Non-influenza Studies Combined Total >219, subjects in studies Age Group (+)MF59 (-)MF59 (+)MF59 (-)MF59 (+)MF59 (-)MF59 Children/Adolescents a Non-Elderly Adults (18-64 yrs) b Elderly Adults 65 yrs Elderly Adults 65 yrs from LIVE study (only) b N/A N/A Subjects with underlying medical conditions Overall (excluding LIVE study) b N/A 5482 c N/A c Overall (including LIVE study) b a for flu studies includes subjects from 6 months to <18 years of age; for non-influenza studies 15 subjects aged 1-2 years and 4 aged less than 18 years in the (+)MF59 group; b observational study; 12 c Adjuvants three subjects G. (two Del in Giudice study V42P2 Siena, and 23 one September in V5P1) had 29 no age Business documented Use Only and have not been included in the age cohort analysis, but only in the analysis of all age groups combined.

13 MF59: magnitude of immune responses Fluad Immunogenicity in elderly Meta-analysis of 2 trials 1st vaccination 2nd vaccination 3rd vaccination Post-vaccination GMR CI GMR MF59-adjuvanted vaccine better B A/H3N2 A/H1N1 control better 13 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only Podda A. Vaccine 21;19:

14 MF59: magnitude of immune responses Fluad Immunogenicity in elderly at risk Chronic diseases MF59-adjuvanted vaccine, N=175 Non-adjuvanted subunit vaccine, N=171 P<.1 Subjects with underlying chronic moderate to severe cardiovascular, respiratory or metabolic diseases 14 Geometric Mean Ratios P<.1 P<.1 A/H3N2 A/H1N1 B Baldo V, et 14 al. Adjuvants Data presented G. Del at Giudice OPTIONS Siena, VI. 23 Toronto, September Canada, 29 June Business 17-23, Use 27. Only

15 FLUAD has been shown to reduce hospitalization rates for pneumonia, cardiovascular disease and cerebrovascular disease in the elderly compared with unvaccinated individuals Community-dwelling elderly (> 64 years of age) FLUAD recipients were compared with unvaccinated controls with respect to rates of emergency hospitalization for pneumonia (PNA), acute coronary syndrome (ACS), and cerebrovascular (CVA) disease 1 Vaccination with FLUAD reduced the probability of hospitalization due PNA, ACS and CVA by 69%, 87% and 93%, respectively, during periods of peak influenza virus circulation 1 Hospitalization Rate per 1 Subjects ACS CVA PNA Disease FLUAD Unvaccinated 1. Puig-Barberà et al Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

16 MF59-adjuvanted clade 1 H5N1 vaccine is seroprotective in adult and elderly individuals SRH antibody response against a homologous H5N1 Vietnam strain at day 42 Seroprotection Rate Seroprotection (%) Seroconversion Rate Seroconversion (%) Geometric Mean Ratio Geometric Mean Ratio (D42/D) Adults (18 65 years; n=149) Elderly (>65 years; n=84) CHMP criteria for vaccine protective immunity in adults and the elderly Note: CHMP, Committee for Medicinal Products for Human Use; CI, confidence interval; GMR, geometric mean ratio; GMT, geometric mean titer. Randomized, controlled, observer-blind, multi-center, Phase II study in adults (n=149) and elderly (n=84) Vaccine schedule: Two doses at day and 21; AFLUNOV Banzhoff et al, PLoS ONE Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

17 WHO monitors strain drifts and recommends strains for vaccine inclusion Drift has led to 15 changes in recommended strains since A/H1N1 A/H3N2 B Bayern Beijing New Caledonia Solomon Islands Brisbane Wuhan Sydney Moscow Fujian California Wisconsin Brisbane Beijing Sichuan Hong Kong Shanghai Malaysia Florida Influenza Season (year) 1. WHO 27a; 2. WHO 27b; 3. WHO 27c; 4. Wood JM 2; 5. WHO Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

18 Despite strain updates, vaccine-virus mismatches still occur Vaccine-virus mismatch for A/H3N2 in Canada over 13 influenza seasons ( ) Similarity of Isolates (%) * *Vaccine strain vs. laboratory-confirmed influenza infections. 18 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only Influenza Season (year) 65% average match (all seasons combined) Ellis et al

19 Vaccine efficacy is impacted by drift, H3N2 vaccine component High mismatch Moderate mismatch Exact match Vaccine Efficacy From Literature (%) Influenza Season Carrat and Flahault Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

20 MF59-adjuvanted vaccine: greater seroprotection than nonadjuvanted vaccines against drifted strains Elderly subjects ( 61 years of age) were vaccinated with FLUAD or nonadjuvanted subunit or split vaccines (all containing A/Panama (H3N2) 1 Seroprotection (HI titer 4) was assessed postvaccination day 21 against the drifted A/Fujian-like (H3N2) strain (A/Wyoming/3/23) Seroprotection Rate: Subjects With HI Titer 4 (%) Prevaccination * Postvaccination Fluad (n = 6) Nonadjuvanted subunit vaccine (n = 29) Nonadjuvanted split vaccine (n = 3) *P =.1 vs. nonadjuvanted subunit and split vaccines. 1. Del Giudice et al., 26 2 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

21 MF59-adjuvanted vaccine: higher levels of crossreactive antibodies against drifted H3N2 strains Elderly subjects ( 65 years of age) were vaccinated with FLUAD or a nonadjuvanted subunit vaccine, each containing A/Wyoming/3 (H3N2) Antibody titers (GMT) were assessed on day 21 against consecutive drifted A/H3N2 strains Corrected Postvaccination Titer (GMT) ** ** FLUAD (n = 25) Nonadjuvanted vaccine (n = 25) HI assay ** * NT assay ** * Wisconsin67/5 Wellington1/4 California7/4 Wyoming3/3 Panama27/99 Sydney5/97 Pan/99 Wyo/3 Cal/4 Wisc/5 Pan/99 Wyo/3 Cal/4 Wisc/5 **P <.1, *P <.5, FLUAD vs. nonadjuvanted subunit vaccine; error bars = standard deviation. 1. Ansaldi et al Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

22 MF59-adjuvanted clade 1 H5N1 vaccine induces seroprotection also against clade 2 virus Elderly Age groups Adults (< 65 year-old) Seroprotection rate post Seroprotection rate post Banzhoff et al, PLoS ONE Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

23 Potential targets of adjuvants to counteract effects of agin on immune responses Subclinical ubiquitous inflammatory processes increase the threshold for danger signal induced by vaccine + adjuvant SITE OF INJECTION Macrophage Dendritic cell Naïve T? Naïve T Thymus Functional defects of APC (impaired uptake & presentation) Thymic involution progressive loss of naïve T cells: reduced chance to respond to primary immunizations LYMPH NODE Memory B Naïve B Naïve CD4 Naïve CD8 Antibody secreting B Reduced B cell production, defects in isotype switching & somatic hypermutation weak & low affinity antibody responses BLOOD BONE MARROW YYYYYY YYYYYY Memory & Effector CD4 23 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only Long-lived plasma cell Memory CD4 Memory & Effector CD8 Memory CD8 Increased number of effector T cells restricted diversity of the T-cell repertoire reduced efficacy of vaccination Increased number of effector T cells restricted diversity of the T-cell repertoire reduced efficacy of vaccination Adapted from Weinberger et al, CID 28

24 H5-specific CD4+ T cells are already induced after one dose of H5N1 + MF59 and strongly expanded after boosting H5 - CD4 + H5-CD4 + (in 1 6 tot CD4) days Non-Adj-15 MF MF59-15 Galli et al, Proc Natl Acad Sci USA 16 (1): , Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

25 Model for Th1 effector/memory differentiation Naive Tcm TNFα + IL-2 +? TNFα + IL-2 + IFN-γ + TNFα + IL-2 + Tem IFN-γ + IFN-γ + IFN-γ + TNFα + IL-2 + Effector Memory +/- - +/ MF59-H5N1 Adapted from Bob Seder 25 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

26 Frequency of H5N1- memory B cells significantly increase after heterologous boost only in people previously primed with MF59-adjuvanted H5N3 vaccine Unprimed H5N3 primed MF59-H5N3 primed Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only Galli et al, Proc Natl Acad Sci USA 16 (19): , 29

27 Pre-pandemic influenza vaccination is feasible if MF59 is used in the vaccine Priming with H5N3 and boosting with H5N1-derived vaccines 1 By day 7 post-boost most of subjects have already protective neutralizing antibody titers against all virus strains Homologous H5N1 Clade 1 1 Heterologous H5N1 Clade 2.2 Protective titer (1:4) 1 1 With MF59 Heterologous H5N1 Clade 2.3 Heterologous H5N1 Clade w/o MF years Homologous H5N1 Clade 1 No MF59 Priming with H5N3 Days Boost with H5N1 clade 1 with MF59 Months Nicholson et al, Lancet 357: , 21 Galli et al, Proc Natl Acad Sci USA 16 (19): , Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

28 MF59 increases and changes the quality of protective antibodies against H5N1 This region is recognized by protective monoclonal antibodies and by sera from people who recovered from H5N1 infection This region contains the enzymatically active region of the neuraminidase Unadjuvanted vaccine mainly induces antibodies against nonfunctional regions MF59 allows the production of antibodies directed against the protective sites of HA and NA Collaboration with Hana Golding, FDA 28 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

29 MF59 increases the antibody titers and drives the response towards the protective regions H5N1 plain or with Alum Y Neutralizing (= Protective) sites Y Y H5N1 plus MF59 Y Y Y Y Y Y YY Y Y Y Y Y Y Y Y Y Y Y Y YY Y Y The majority of antibodies are directed against non-rpotective sites of HA 29 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only MF59 not only increases the quantity of anti-ha antibodies, but also changes the quality of these antibodies which are now directed against the neutralizing (= protective) regions

30 In summary MF59 enhances the immune response, quantitatively and qualitatively, to vaccines in people at all ages, including elderly Some of these effects (e.g. differentiation of IL-2-producing CD4+ cells, induction of memory B cells, etc) may contribute to counteract the effect of aging on poor responsiveness to vaccines (e.g. influenza) Most of the studies on the mechanism of action of MF59 (and of other adjuvants) have been carried our in fully immunocompetent, adult individuals (mice & humans) need to expand these mechanistic studies at the elder age Opportunities abound in the field of vaccine adjuvants and elderly! 3 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

31 Backups 31 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

32 The growth rate of elderly population is very high in developing countries 32 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

33 The increase of elderly is mirrored by the decrease of young 33 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

34 Reduced efficacy of vaccination in the elderly Disease/bacteria Influenza A/H1N1 Influenza A/H3N2 Influenza B Hepatitis A Hepatitis A Hepatitis B Herpes Zoster Pneumococci Tickborne encephalitis Vaccine Inactivated Inactivated Inactivated Inactivated Virosome Subunit Live attenuated Polysaccharides Inactivated Efficacy in elderly people, % 55 (32) 58 (46) 41 (29) (97)* (18) * Seroprotection after priming (and after boosting). In parentheses efficacy in people aged over 8 years Adapted from Weinberger et al, CID Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

35 Several approaches are being pursued for better vaccines for elderly Increased dosage of vaccine Intradermal immunization Cytokines Mucosal immunization Use of strong adjuvants 35 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

36 The public health and economic impact of seasonal influenza is considerable 36, Deaths* Annual estimates for US 1 294, Hospitalizations* Million Hospitalization days Million Cases of Influenza Million MD Visits 3 44 Million Lost Productivity Days billion dollars of direct medical costs billion dollars of lost earnings billion dollars of total economic burden 3 Highest morbidity and mortality in the elderly: 44, excess deaths (33, with underlying cardiorespiratory causes) in those aged 65 years 1 *Influenza-associated respiratory/circulatory causes, all ages. 1. Thompson et al. 23; 2. Thompson et al. 24; 3. Molinari et al Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

37 Influenza can significantly impact specific subpopulations 1,2 The young Infants Children and adolescents The elderly Nursing home residents Persons with chronic illness Healthy working adults Healthcare workers Travelers 1. Bridges et al. 28; 2. CDC Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

38 Adjuvants for influenza vaccines, an old solution Hennessy A,V, and Davenport F,M; Pub. Health Repts 76, , 1961 Polyvalent vaccines Aqueous; aqueous Test strain and antibody response Swine PR8 PR31 Before second dose After second dose Adjuvant; adjuvant Before second dose After second dose 1,229 1,741 1,818 Mineral oil adjuvant vaccine is remarkably effective for stimulating high, broad, uniform, and persistent antibody levels against prototype strains of influenza A A phenomenal economy can be affected in the requirement of antigen The adjuvant was not licensed because mineral oil caused sterile abscesses 38 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

39 Influenza-related hospitalizations are high among young children Influenza-related Hospitalization Rate per 1 Children Aged -5 months Aged months Aged 6-23 months Aged -59 months Poehling et al Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

40 The elderly are at greatest risk for influenza-associated hospitalization Hospitalizations/1, Person-Years Age-specific annual rates of influenza-associated cardiorespiratory hospitalizations, United States Age (years) Thompson et al Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

41 Influenza mortality is highest in the elderly Deaths per 1, Person-Years Estimated average annual influenza-related cardiopulmonary and all-cause mortality rates, United States, Cardiopulmonary All-cause deaths < > 65 Age (years) 9% of deaths (32,651/36,155) were in persons 65 years old Thompson et al Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

42 Complications are more frequent in adults with chronic disease Serious complications include 1 Exacerbation of underlying chronic pulmonary and cardiovascular diseases, such as: - Congestive heart failure - Chronic obstructive pulmonary disease - Asthma Viral and bacterial pneumonia Complication rate higher for those subjects who have chronic heart disease (46.9%) vs. those without (22.8%) 2 Pneumonia is frequent in hematopoietic stem-cell transplant recipients (63% of patients) 3 1. Bridges et al. 28: 2. Irwin et al. 21; 3. Whitley and Monto Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

43 Influenza vaccination may reduce hospitalization and death in the elderly Adjusted Odds Ratio and 95% CI y y All 85 y Hospitalization for pneumonia or influenza y y All 85 y Hospitalization for cardiac causes y y All 85 y Hospitalization for cerebrovascular causes y y All 85 y Hospitalization for any cause N = 14, y y Death All 85 y Nichol et al Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

44 Influenza vaccination is effective in reducing hospitalization and death in community-dwelling elderly Adjusted Odds Ratio % effective N = 713,872 person-seasons 48% effective Hospitalization Death Nichol et al Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

45 Antigenic drift can reduce vaccine efficacy season 1 (poor vaccine-virus match) season 1 (good vaccine-virus match) season interim estimate 2 Vaccine efficacy: 5% P =.33 vs. placebo Vaccine efficacy: increased significantly to 86% Vaccine efficacy: 44% (moderate vaccine-virus match) 1. Bridges et al. 2; 2. CDC Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

46 Influenza-related complications are high among young children Hospitalization rates are high in children /1, population (aged < 5 years) vs. 8.8/1, for adults (aged years) Most common complication of influenza in children is acute otitis media 2 2%-6% of cases, depending on age and other risk factors Acute exacerbations of asthma, in children with asthma, are common 3 Increased frequency of influenza-associated encephalopathy 4 and febrile seizures 1. Glezen et al. 1987; 2. Heikkinen Neuzil et al. 2; 4. Togashi et al. 24; 5. Whitley and Monto Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

47 Influenza impacts working adults and their employers An estimated 15 million working days are lost in the United States each year due to influenza 1 Influenza related illnesses greatly impact the employer Cost to the employer depends on wages, severity of influenza season and underlying health of employee Average number of productivity days lost:.5-15 days 2 Value of lost productive day: $145 (23) 1 Presenteeism costs due to 3%-7% reduced productivity 1. Molinari et al. 27; 2. Prosser et al Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

48 Influenza in healthcare workers Healthcare workers (HCW) have increased risk of acquiring influenza during outbreaks community as well as hospitalized population exposure Reported HCW attack rates vary from 11%-59% during nosocomial outbreaks 1 A serological study in the UK found that up to 23% of HCW contracted clinical or subclinical influenza during community outbreaks 2 Up to 75% of healthcare workers with influenza-like illness worked while ill 1. Bellei 27; 2. Salgado Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

49 Healthcare workers can transmit influenza to patients and each other Influenza can be transmitted between patients and healthcare workers in the hospital setting One nosocomial outbreak of influenza occurred from February 1 to February 26, 21 in a hospital ward The patient population was generally immunocompromised: 45% had AIDS, while 55% had other infectious diseases 29 of 57 healthcare workers (51%) and 8 of 23 hospitalized patients (34%) had symptoms of influenza Epidemiological analysis showed that the index case of this outbreak was an unvaccinated nurse s aid Horcajada et al Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

50 Influenza in travelers Seasonal influenza second most frequent vaccine-preventable illness in travelers Guidelines on influenza vaccination for international travel are scarce On the basis of some national recommendations, influenza vaccine should be used in the elderly (aged > 5-65 years), infants (aged 6-23 months), and those with comorbidities Close contact with high transmission has been documented in cruise ships and air travel Travelers crossing to the opposite hemisphere during influenza-peak season may need protection. Those visiting the tropics are at moderate risk of infection and illness during the entire year Franziska et al Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

51 Vaccination may reduce the incidence of influenzarelated complications, European Union Vaccination strategies target those considered most at risk of complications In the European Union 25 member states, the at-risk population is million people Currently, only 35.4% of the at-risk population is being vaccinated This analysis suggests that if this proportion were higher, benefits may be increased Reduction in Cases, Compared With no Vaccination (%) % Influenza 5% 49% 17% 35.4% vaccinated 1% vaccinated PCP visits for treatment 12% 35% Hospital admissions 22% 62% Excess deaths Ryan et al Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

52 Antigenic drift can reduce vaccine efficacy season 1 (poor vaccine-virus match) Vaccine efficacy: 5% P =.33 vs. placebo season 1 (good vaccine-virus match) Vaccine efficacy: increased significantly to 86% season interim estimate 2 (moderate vaccine-virus match) Vaccine efficacy: 44% 1. Bridges et al. 2; 2. CDC Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

53 MF59 is the most active TLR-independent adjuvant Singh et al Vaccines 25 nil Alum MF59 3 H3N2 8 H1N1 35 B 25 3 Serum HI antibody titers post-1 post-2 post-1 post-2 post-1 post-2 53 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only Wack et al Vaccines 28

54 MF59 enhanced Ag uptake at the injection site and LN FITC-Ag Ag + MF59 im Dil-MF59 FITC-Ag Dil-MF59 Ag alone FITC-Ag MF59 increased uptake of coadministered HSV gd2 antigen at the injection site. (Dupuis et al. 1998) CD11b+ cells in the muscle double positive for MF59 and antigen were recovered from muscle and LN 48h after injection (Dupuis et al. 21) MF59 did not activate DC in vitro MF59 was described as an antigen delivery system 48h 54 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

55 Chemokines secretion induced by MF59 in human monocyte-derived macrophages 5 MCP-1 MIP-1α 4 9 MIP-1β CCL2 (MCP-1) CCL3 (MIP-1b) CCL4 (MIP-1a) [pg/ml] [pg/ml] 6 3 MF59 4.5h 9h 24h 48h 4.5h 9h 24h 48h IL-8 IL h 9 h 2 4 h 4 8 h 4.5h 9h 24h 48h 4.5h 9h 24h 48h 4.5h 9h 24h 48h IL-1ra IL-1ra [pg/ml] [pg/ml] MF59 4.5h 9h 24h 48h 4.5h 9h 24h 48h 4.5h 9h 24h 48h 4.5h 9h 24h 48h 55 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only Seubert et al, J Immunol 28

56 MF59 adjuvant improves survival to lethal influenza challenge in vaccinated mice Mice were vaccinated with A/Texas H1N1, A/Beijing H3N2 and B/Panama (with or without MF59) 1,2 Survival assessed after intranasal challenge with heterologous influenza virus (A/Taiwan H1N1; challenge dose 5 LD 5 or 9 LD 5 on days 56 and 126 respectively) 1 MF59-adjuvanted vaccine Nonadjuvanted vaccine Animals Surviving 14 Days Post Challenge (%) Day 56 Day 126 Influenza Vaccine Dose (µg) Influenza Vaccine Dose (µg) 1. Cataldo and Van Nest 1997; 2. Higgins et al. 1996;. 56 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

57 FLUAD: greater antibody response vs. nonadjuvanted vaccines on repeat vaccination Comparison of FLUAD with nonadjuvanted subunit or split vaccine following consecutive annual vaccination of elderly subjects ( 65 years of age) 1 Year 1 2 n, FLUAD n, Comparator Postvaccination GMT Ratio GMT ratio > 1 FLUAD more immunogenic GMT ratio < 1 nonadjuvanted vaccine more immunogenic Year A/H3N2 A/H1N1 B 1. Podda, 21. Copyright 21 with permission from Elsevier. Error bars denote the 95% confidence interval (CI). 57 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

58 MF59: magnitude of immune responses Fluad Immunogenicity in adults at risk - HIV MF59-adjuvanted vaccine, N=44 Non-adjuvanted subunit vaccine, N=4 HIV-1 seropositive subjects under HAART 1 Seroprotection Rates 1 Seroconversion Rates 9 9 * * A/H3N2 A/H1N1 B A/H3N2 A/H1N1 B Randomized study in HIV-1-seropositive subjects under highly active antiretroviral therapy: subjects were randomized to receive a single.5 ml IM dose of the MF59-adjuvanted or conventional subunit influenza vaccine. * EU Committee for Medicinal Products for Human Use (CHMP) immunogenicity threshold for healthy adults Iorio AM, 58 et al. Adjuvants Vaccine 23;21: G. Del Giudice Siena, 23 September 29 Business Use Only

59 Safety and tolerability of MF59: an extensive clinical database was pooled for a safety analysis 59 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

60 MF59 Safety Database Completed Trials (as of July 28) Influenza Studies Non-influenza Studies Combined Age Group (+)MF59 (-)MF59 (+)MF59 (-)MF59 (+)MF59 (-)MF59 Overall Children/Adolescents a Non-Elderly Adults(18-64 yrs) b Elderly Adults 65 yrs Subjects with underlying medical conditions N/A N/A Studies included since DMF: V87P5; V87P6, V7P7S, V11P1, V14P1 6 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

61 MF59 Safety Database Ongoing Trials Estimated numbers from ongoing* trials since July 28 Age Group (+)MF59 Influenza Studies (-)MF59 Overall Children/Adolescents A Non-Elderly Adults(18-64 yrs) B Elderly Adults 65 yrs C LIVE STUDY (C7P1) Elderly Adults 65 yrs YEAR 1 YEAR 2 (+)MF (-)MF YEAR 3 (Estimated enrolled) YEAR 4 (PLANNED) Overall * Ongoing trials: studies not yet completed (CSR not available) A Estimated numbers from blinded studies V7P5 (1 and 2 year) and V14P2 B from studies V89P1, V87P11, V87P12, V87P13 C from studies V87P11, V87P13, V14P3 61 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

62 The CD4+ T cells induced by H5N1+MF59 are Th1-effector / memory cells CK+ CD4 + (in 1 6 tot CD4 + ) weeks post-boost Day Non Adj-15 MF MF59-15 Non Adj-15 MF MF * IL IL-2 + IFN-γ - IL-2 + IFN-γ + IL-2 - IFN-γ H5-CD4 + H5N1-CD4 + Galli et al, Proc Natl Acad Sci USA 16 (1): , Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

63 In summary MF59-adjuvanted vaccines are very well tolerated at all ages MF59-adjuvanted influenza vaccines confer strong, longlasting, and heterotypic immunity in those people who more need seasonal vaccination against flu: kids, elder people, people with chronic disorders MF59-adjuvanted (pre)-pandemic vaccines allow dosesparing, induce strong and long lasting immunological memory (T s and B s), and broad heterotypic immunity 63 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

64 Vaccination against Alzheimer s disease (mouse) 25 µg CAD16, subcutaneously Age of mice (months) Cortex Control -VLP) (Qβ) End of Experiment CAD16 (Qβ-Aβ1-6) Hippocampus Thalamus Caudate putamen Plaque area (%), Cortex Cortical plaque area ** Brain Aβ42 content PBS Qβ CAD16A Courtesy of A. Graf and the AD Team, Novartis 64 Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only Amyloid plaque area ~8 % lower than in control mice

65 Coverage rates are below targets set for influenza vaccination of the elderly Vaccination Rate (%) Influenza vaccination coverage rate in the elderly (aged 65 years) during 26/27 CDC target by 21 2 WHO target by Germany 1 1 Italy 1 1 UK 1 1 Spain 1 1 France 1 1 US 2 2 Country 1. Blank 28; 2. CDC 28; 3. WHO Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

66 FLUAD: increased seroprotection rates vs. nonadjuvanted vaccine in the elderly 75 years Immunogenicity analysis included elderly ( 65 years of age) subjects vaccinated with FLUAD (n = 595) or a nonadjuvanted split vaccine (n = 591) 1 Seroprotection (HI titer 4) was assessed postvaccination day 21; graph indicates results for the subset of subjects 75 years of age Seroprotection: Subjects With HI Titer 4 (%) FLUAD Vaxigrip A/H3N2 A/H1N1 B Squarcione et al Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

67 MF59-adjuvanted vaccine: broader immune response against drifted strains vs. nonadjuvanted vaccines Elderly subjects ( 65 years of age) were vaccinated with MF59-adjuvanted vaccine (Fluad) (n = 15) or a nonadjuvanted subunit vaccine (n = 16) 1 Antibody titers (GMT) against a homologous (matched) and a heterovariant (drifted) A/H3N2 virus strain were assessed postvaccination day 28 GMT ** * FLUAD/Homologous (A/Beijing/32/92) FLUAD/Heterovariant (A/Shangdong/9/93) Nonadjuvanted subunit vaccine/ Homologous (A/Beijing/32/92) Nonadjuvanted subunit vaccine/ Heterovariant (A/Shangdong/9/93) 1 Day Day 28 *P =.11; **P =.2 vs. nonadjuvanted vaccine. 1. De Donato et al Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

68 Estimated annual deaths and mortality rates by RSV and Influenza in the USA, / Thompson et al, JAMA 289: 179, Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only

69 Reduced efficacy of influenza vaccines in the elderly Goodwin et al, Vaccine Adjuvants G. Del Giudice Siena, 23 September 29 Business Use Only