Clinical trials of an oral inactivated ETEC vaccine (ETVAX) in - PowerPoint PPT Presentation

Clinical trials of an oral inactivated ETEC vaccine (ETVAX) in children in developing countries and in travelers Ann ‐ Mari Svennerholm Department of Microbiology and Immunology The Sahlgrenska Academy University of Gothenburg (UG) ECMIS , Ghent June 5, 2019 .

Enterotoxin ‐ producing E. coli (ETEC) – important global diarrheal pathogen in humans ETEC is one of the most common causes of diarrhea in ……… children ≤ 5 years in developing countries • ETEC causes several hundred million cases and >50.000 diarrheal deaths annually • Repeated ETEC diarrheas may cause undernutrition, retarded growth and decreased cognitive functions ETEC is also the most common cause of diarrhea in travellers ‐ 80 M 20 ‐ 50 % of all diarrheas in travellers 28 M are caused by ETEC 12.6 M

Why is no human ETEC vaccine available yet? Large heterogeneity of diarrheagenic ETEC and lack of robust immunological markers of protection makes vaccine development complicated Enterotoxins: LT (~40%), ST (STh and STp; ~30%), LT+ST (~30%) Colonization factors (CFs): > 25 CFs; C FA/I and CS1 ‐ CS7, CS14 and CS17 on 50 ‐ 80% of all ETEC strains ( Gaastra &Svennerholm 1996) O ‐ antigens : >70 in ETEC Putative protective surface antigens: e.g. EtpA, EatA, flagellin, TibA (Fleckenstein et al)

The ETEC vaccine, ETVAX, developed at UG in collaboration with Scandinavian Biopharma, Stockholm Multivalent ETEC vaccine (ETVAX) dmLT adjuvant CFA/I ± CS3 + LCTB A CS5 CS6 10 11 recombinant bacteria +1mg toxoid 10 µg dmLT overexpressing CFs (Lebens , Holmgren et al) ( J Clements et al) (Tobias & Svennerholm)

Evaluation of ETVAX in Phase I double ‐ blind placebo ‐ controlled studies in adult Swedes Four groups with altogether 129 subjects receiving: A. Placebo (bicarbonat buffer only); n=34 B. ETVAX alone; n=35 C. ETVAX + 10 µg of dmLT; n=30 D. ETVAX + 25 µg of dmLT; n= 30 Boosting groups A ‐ C with single dose ETVAX 1 ‐ 2 years later Is ETVAX safe ?  No significant differences in side ‐ effects between vaccinees and placebos Can ETVAX induce?:  Mucosal immunse responses : SIgA antibody responses fecal extracts and …intestine ‐ derived circulating antibody secreting cell (ASC/ALS IgA) responses  Serum antibody responses against the primary vaccine antigens  Cross ‐ reactive immune responses against other CFs than those in the vaccine  Immunologcal memory

Mucosal ALS IgA and/or fecal SIgA immune responses against ETVAX antigens in adult Swedes Frequencies of responders to two oral doses of ETVAX against the five primary . vaccine antigens (CFA/I, CS3, CS5, CS6 and LTB) Subjects Placebo ETVAX ETVAX + 10 ETVAX + 25 All Vaccinees responding to alone µg dmLT µg dmLT n=95 ≥ 4 antigens 0% 91% 87% 85% 87% 5 antigens 0% 74% 83% 75% 77% A Lundgren et al, Vaccine 2014

Induction of cross ‐ reactive mucosal immune responses against CFs not included in the vaccine in adult Swedes ALS IgA Fecal SIgA Non-vaccine Vaccine Vaccine Non-vaccine CFs CFs CFs CFs CFA/I CS14 CS17 CS1 CFA/I CS14 CS17 CS1 100 100 CFA/I Group % % 50 50 21/21 15/21 15/21 15/21 9/14 9/14 11/14 14/14 0 0 CS5 CS7 CS5 CS7 100 100 CS5 Group % % 50 50 17/17 15/17 14/14 10/14 0 0 S Leach et al, 2017

Immunological memory responses : Higher and earlier appearing ALS responses were induced by a single dose of ETVAX in previously (1 ‐ 2 years earlier) vaccinated ( Gr II and III) compared to in naive (Gr I) Swedish adults CFA/I CS3 P=0.006 P=0.03 P=0.004 30 P=0.06 ) M 100 ) M E E S S + + M M (G 30 (G 10 its its n n u u 10 A A IS IS L L E E 3 3 Day: Day: 0 4/5 7 0 4/5 7 0 4/5 7 0 4/5 7 0 4/5 7 0 4/5 7 Group I Group II Group III Group I Group II Group III RF: 13% 63% P=0.003 59% P=0.004 RF: 26% 69% P=0.019 88% P<0.001 CS5 CS6 P=0.03 P=0.006 P=0.07 P=0.01 ) M ) M 10 E E 10 S S + + M M (G (G its its n n u 5 u 5 A A IS IS L L E E 3 3 Day: Day: 0 4/5 7 0 4/5 7 0 4/5 7 0 4/5 7 0 4/5 7 0 4/5 7 Group I Group II Group III Group I Group II Group III RF: 13% 69% P<0.001 59% P=0.004 RF: 13% 56% P=0.009 53% P=0.01 Lundgren, Jertborn & Svennerholm, 2016

Phase I/II study of ETVAX in descending age groups in Bangladesh Main Objective : Safety: To identify the highest safe and immunogenic dose of ETVAX +/ ‐ dmLT adjuvant Secondary objective: Immunogenicity: To determine immune responses against the …………. five primary ETVAX antigens (CFA/I, CS3, CS5, CS6 and LTB) Mucosal immune responses ‐ IgA antibodies in ALS specimens ‐ SIgA /total SIgA antibodies in fecal extracts Systemic immune responses ‐ IgA and IgG antibodies in plasma Can dmLT adjuvant enhance immune responses ?

Phase 1/II trial of ETVAX in Bangladesh (495 subjects ) Cohort ETVAX dmLT N Cohort ETVAX dmLT N ¼ dose ----- 15 PART A: C1 Full dose ----- 15 18-45 years, Placebo ----- 10 A1 Full dose 10 ug 15 PART C: inclusive ½ dose ----- 15 Placebo 15 C2 12-23 Placebo ----- 10 Total 45 months, ½ dose 2.5 µg 15 inclusive C3 Placebo ----- 10 ½ dose 5 µg 15 Cohort ETVAX dmLT N C4 Placebo ----- 10 ¼ dose ----- 15 B1 Total 100 Placebo ----- 10 ½ dose ----- 15 B2 Placebo ----- 10 Cohort ETVAX dmLT N 1/8 dose ----- 15 PART B: Full dose ----- 15 D1 B3 Placebo ----- 10 24-59 Placebo ----- 10 months, ¼ dose ----- 15 D2 ½ dose 2.5 µg 15 PART D: 6- Placebo ----- 10 inclusive B4 Placebo ----- 10 11 months, ½ dose ----- 15 D3 15 ½ dose 5 µg inclusive Placebo ----- 10 B5 ¼ dose 2.5 µg 15 Placebo ----- 10 D4 Placebo ----- 10 ½ dose 10 µg 15 B6 ¼ dose 5 µg 15 Placebo ----- 10 D5 Placebo ----- 10 Total 150 Total 200 F Qadri, A ‐ M Svennerholm et al

Introduction of a new approach to assess ALS responses by an electrochemiluminescence assay Meso Scale Discovery (MSD) Sulfo ‐ tag Light Vaccine specific antibody Carbon based plate with electrodes Main benefits of ECL assays compared to ELISA:  High sensitivity – small sample volumes required (5 µl/analysis), e.g for use in young children  Wide dynamic range – no sample titration required Lundgren & Svennerholm

Excellent correlation between ALS IgA responses by ELISA and ECL MSD assay (Immune responses to CFs and LTB in adult Bangaldeshis given ETVAX d ± d mLT or placebo) CFA/I (day 19) CS3 (day 19) 1000 1000 MSD (fold rises) r=0.90 MSD (fold rises) r=0.91 P<0.0001 P<0.0001 100 100 10 10 1 1 0.1 0.1 0.1 1 10 100 1000 0.1 1 10 100 1000 ELISA (fold rises) ELISA (fold rises) ELISA (fold rises) ELISA (fold rises) CS6 (day 19) LTB (day 19) r= 0.95 r= 0.91 MSD (fold rises) P< 0.0001 P< 0.0001 MSD (fold rises) MSD (fold rises) MSD (fold rises) ELISA (fold rises) ELISA (fold rises) Akhtar, Lundgren et al Vaccine 2018

Strong and specific ALS IgA responses against CFs and LTB in adult Bangladeshis given two doses of ETVAX ± dmLT or placebo (determined by the ECL MSD assay) CS3 CFA/I Responder freq: 7% 100% 100% Responder freq: 0% 100% 100% CS6 LTB Responder freq: 7% 100% 100% Responder freq: 0% 100% 100% Also strong responses to CS5 Akhtar et al 2018

Frequent IgA responses in plasma against CFs and LTB in adult Bangladeshis: given two doses of ETVAX +/ ‐ dmLT or placebo day 0 and 14 CFA/ I CS6 1000 IgA (ELISA titer) 100 10 Pre Day 7 Day 19 Pre Day 7 Day 19 Pre Day 7 Day 19 Vaccine + Vaccine Placebo dmLT LTB 10000 1000 IgA (ELISA titer) 100 10 Pre Day 7 Day 19 Pre Day 7 Day 19 Pre Day 7 Day 19 Akhtar et al 2018 Placebo Vaccine Vaccine + dmLT

A phase 2b trial of ETVAX in Finnish travelers to Benin (PI Anu Kantele, University of Helsinki) A double – blind, placebo ‐ controled study in 800 volunteers to evaluate: • Safety • Immunogenicity (serum antibody responses ) Protection against mild ‐ moderate ETEC diarrhea with ETEC as • only pathogen Volunteers were given 2 doses of ETVAX or placebo (buffer only) 2 weeks apart in Finland 1 ‐ 2 weeks before departure Surveillenc for diarrhea was conducted during stay in Benin for 12 days + one week after return to Finland

Thank you! L Bourgeois, R Walker, N Bauer et al Stiftelsen för Strategisk forskning GU, Institute of Biomedicine, B Sjöstrand & Sahlgrenska N Carlin Academy ETEC group icddrb; Jan Holmgren F Qadri et al ETEC group GU