Development of a VP6 subunit rotavirus vaccine A dual role of VP6 as a vaccine antigen and an adjuvant

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1 30 August 2018, Minsk 13TH INTERNATIONAL ROTAVIRUS SYMPOSIUM Development of a VP6 subunit rotavirus vaccine A dual role of VP6 as a vaccine antigen and an adjuvant Dr. Vesna Blazevic Head of Laboratory Vaccine Research Center University of Tampere 1

2 Rotavirus (RV) VP6 RV VP6 Triple-layered RV particle: 1. Inner capsid VP2 2. Intermediate capsid VP6 Highly conserved protein Groups (A-H) Subgroups (SGI, II, I+II, non I/II) 3. Outer capsid VP7 with VP4 spikes -> a dual role of VP6 as a subunit rotavirus vaccine and an adjuvant in a combination norovirus (NoV) + RV vaccine candidate developed by our laboratory 2

3 Rationales for a non-live subunit RV vaccine No adverse issues of live RV vaccines No competition of uptake with enteric viruses in the gut Live RV vaccines (RV1 and RV5) show lower efficacy in Africa and Asia Live RV vaccines show decline of efficacy in the 2nd year in developing countries Genetic background of the population not critical (e.g. secretors/nonsecretors) 3

4 Combination NoV+RV vaccine: recombinant NoV virus-like particles (VLPs) + RV VP6 Non-live subunit vaccine against childhood gastroenteritis All vaccine antigens are oligomeric proteins produced in insect cells by recombinant baculovirus expression vector system (BEVS) NoV VP1 RV VP6 VP2 roteins/rota%20cut-away.gif ~100 nm capsid protein VP1 BEVS capsid protein VP6 + capsid VP2 or or 100 nm 100 nm NoV VLPs ~30-40 nm VP6 nanotubes (VP6T) ~75 nm, µm long (ph 7) VP6 nanospheres (VP6S) ~75 nm (ph 4) Double-layered 2/6-VLPs ~65 nm M. Estes et al., J Virology 1987 (rvp6 produced in BV expression system) 4

5 Trivalent combination vaccine formulations -extensively studied in our laboratory NoV GII.4 VLPs + GI.3 VLPs + RV VP6 oligomers (VP6T or VP6S or dlvlps) GII.4 VLPs GII.4 VLPs GI.3 VLPs GI.3 VLPs RV rvp6 VP6 T dlvlps a representative of GI and GII NoVs no external adjuvants! 5

6 The combination vaccine induces strong NoV and RV type-specific and cross-reactive immunity PLOS One Jul 26;8(7):e VP6-specific serum IgG VP6-specific mucosal IgG VP6-specific mucosal IgA OD 490nm 2.5 Trivalent (GII-4+GI-3+VP6) OD 490nm VP6 IM VP6 IN Control IM Control IN VP6 IM VP6 IN Control IM Control IN Sample dilution Sample dilution Strong cross-reactive serum antibodies to heterologous RV strains Low levels of IgA in feces of IM immunized mice Rapid increase after live RV challenge 6

7 VP6 protects mice from live RV EDIM challenge RV antigen shedding OD 450nm VP6 IN VP6 IM Control 66 % reduction 65 % reduction Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Days post-challenge Protection correlated with VP6- specific serum IgA levels >50 % reduction in RV antigen shedding in the stool considered significant protection from virus challenge 7

8 Mechanisms of VP6 protection No traditional neutralizing antibodies, instead non-serotype specific protective immunity VP6-specific cytotoxic T lymphocytes (CD8 + T cells) (H Greenberg s group) VP6-specific CD4 + T cells (R Ward s group) Direct cytotoxic mechanism and anti-viral cytokines 50 polymeric/secretory IgA antibodies (H Greenberg, J Crowe) Intracellular neutralization by VP6-specific IgA antibodies following transcytosis VP6-specific piga inhibits RV replication at the transcription level by blocking channels on dlrv particles and preventing RV mrna release (Aiyegbo et al. 2013, 2014) 8

9 Correlates of protection in children VP6 RV-specific serum IgA antibody levels were shown to correlate to RV1 and RV5 vaccines efficacy (Patel et al. 2013) VP6-specific serum IgA in children years Serum IgA against rvp r=0.683 (p<0.0001) Serum IgA against Wa (Lappalainen et al. 2016) Early live bovine RV vaccine RIT4237 induced the strongest response against VP6 (Svensson et al. 1987) Could VP6 induced immunity be responsible for most of the protection against severe RV AGE? 9

10 VP6 adjuvant effect on NoV immunity Study design VLP IM T µg NoV VLP + VP6 VLP VP6T/S + co-delivery (mix) NoV GII.4-specific serum IgG NoV GII.4 -specific T cell responses OD 490nm µg GII-4 10µg GII-4 0.3µg GII µg VP6 1:100 1:200 1:400 1:800 1:1600 1:3200 1:6400 1: : : : : : : Serum Dilution IFN-γ SFC / 10 6 splenocytes (Blazevic et al. 2015, Malm et al. 2017) 0 GII.4 peptide pool OVA peptide 0.3µg VLP 0.3µg VLP + 10µg VP6T 0.3µg VLP + 10µg VP6S Experimental Group Ctrl 10

11 RV VP6 acts as a delivery vehicle for NoV VLPs 0.3 µg GII.4 VLP 10 µg VP6 T 3.0 NoV GII.4 -specific IgG titers co-delivery 1h between injections contralateral sites OD 490 nm T Serum dilution IM (Malm et al. 2017) 11

12 VP6 is internalized by antigen presenting cells (APC) RAW ibmdc RV VP6 CM Anti-VP6 FITC Anti-VP6-FITC Intracellular staining with anti-vp6-fitc ab shows that VP6 is internalized by macrophages and dendritic cells (DC) 12

13 RV VP6 activates macrophages and DC CM VP6 TNF-a (pg/ml) TNF-α production by RAW cells CM BSA 50 ug/ml VP6 100 ug/ml 5000 Mean fluorescence intensity (MFI) CM BSA VP6 VP6 % increase MHC II CD IL-6 (pg/ml) 0 IL-6 production by JAWSII cells 3000 CM BSA 50 ug/ml 2000 VP6 100 ug/ml 1000 increased cell surface expression of antigen presentation and co-stimulatory molecules 0 24h (Malm et al. 2016) Induced cytokine production by macrophages and DC 13

14 Conclusions VP6 VP6 T VP6 S RV VP6-specific immune response protects from heterologous RV challenge in mice Importance of non-serotype-specific immunity induced by VP6 in protection from RV infection RV VP6 acts as an adjuvant for norovirus VLPs RV VP6 activates antigen presenting cells (APC) RV VP6 is an excellent subunit vaccine candidate against RV 14

15 Acknowledgements Vaccine Research Center University of Tampere, Finland Suvi Heinimäki Maria Malm Kirsi Tamminen Eeva Jokela Sanna Kavén Nina Koivisto Timo Vesikari Instituto de Biotecnología, Universidad Nacional Autónoma de México, Morelos, México Ana Ruth Pastor Laura A. Palomares Facultad de Medicina, Universidad Autónoma del Estado de Morelos, Morelos, México Vanessa López-Guerrero Fernando Esquivel-Guadarrama Thank you 15