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Aerosolized Ebola vaccine protects primates and elicits lung-resident T cell responses Michelle Meyer, Tania Garron, Ndongala M. Lubaki, Chad E. Mire, Karla A. Fenton, Curtis Klages, Gene G. Olinger, Thomas W. Geisbert,Peter L. Collins and Alexander Bukreyev Alexandra Scalvini & Émilien Gimaret Ebola virus (EBOV) Mononegavirales Filoviridae Ebolavirus RNA (-) 19 kB 970 nm long 80 nm diameter Target : human and NHP (non human primate) Severe hemorrhagic fever. Fatality ranges from 50% to 90%. Last outbreak (2014-2015 : 28601 cases, 11300 deaths WHO) Transmition : Aerosol, through mucosal surfaces, biological fluid droplets, fomites. Background • Vaccine strategy with aerosolized EBOV has never been tested. • Aerosolized vaccines have many advantages : needle free, do not require trained medical personal. • Aerosolized vaccines create a different immune response that is poorly studied in human. Could aerosolized vaccine be protective against EBOV? The aerosolized vaccine • Human Parainfluenza virus type 3-vectored vaccine that expresses the glycoprotein (GP) of EBOV (HPIV3/EboGP) • Delivered through nebulizer to the respiratory tract (site of replication of HPIV3) HPIV3 Nebulizer Study 1 Immune responses induced by the vaccination, in NHP Experiment * *BAL = Broncho-Alveolar Lavage • • 2 injections days 0 and 28 Lungs, blood and spleen samplings Aims: - Study of both mucosal and systemic antibody responses and cellmediated responses induced by aerosolized vaccine. - Comparison to both liquid HPIV3/EbovGP and VRP vaccines. Serum IgG, IgA and neutralizing antibody responses in NHP Dose 1 Dose 2 Dose 1 Dose 2 Method: EBOV-specific serum IgG and IgA analysed by ELISA HIPV3/EboGP aerosol HIPV3/EboGP IN/IT HPIV3 VRP Serum IgG, IgA and neutralizing antibody responses in NHP Dose 1 Dose 2 Dose 1 Dose 2 Both aerosolized and liquid vaccines induce EBOVspecific serum IgG and IgA responses. HIPV3/EboGP aerosol HIPV3/EboGP IN/IT HPIV3 VRP Serum IgG, IgA and neutralizing antibody responses in NHP Ebola virus Method: Serum-neutralizing antibody responses against EBOV determined by plaque-reduction assays. HIPV3/EboGP aerosol HIPV3/EboGP IN/IT HPIV3 VRP Serum IgG, IgA and neutralizing antibody responses in NHP Ebola virus Induction of a systemic antibody responses comparable to those with liquid vaccine. HIPV3/EboGP aerosol HIPV3/EboGP IN/IT HPIV3 VRP Ability to cross-neutralize EBOV Bundibugyo (BDBV) and Sudan (SUDV) Ebola virus Bundibugyo virus Sudan virus Method: Serum-neutralizing antibody responses against EBOV, BDBV and SUDV determined by plaque-reduction assays. HIPV3/EboGP aerosol HIPV3/EboGP IN/IT HPIV3 VRP Ability to cross-neutralize EBOV Bundibugyo (BDBV) and Sudan (SUDV) Ebola virus Bundibugyo virus Sudan virus Antibody cross-neutralization is stronger after the second dose vaccine. Considerable animal-to-animal variability. HIPV3/EboGP aerosol HIPV3/EboGP IN/IT HPIV3 VRP Mucosal antibody-responses in the respiratory tract Method: - EBOV-specific serum IgG and IgA analysed by ELISA - Serum-neutralizing antibody responses against EBOV determined by plaque-reduction assays. HIPV3/EboGP aerosol HIPV3/EboGP IN/IT HPIV3 VRP Mucosal antibody-responses in the respiratory tract Both aerosolized and liquid vaccines induce strong mucosal antibody responses in the respiratory tract HIPV3/EboGP aerosol HIPV3/EboGP IN/IT HPIV3 VRP Cell-mediated response Non-mucosal CD8 CD4 % of cells Mucosal CD8 MFI Method: - Flow cytometry HIPV3/EboGP aerosol HIPV3/EboGP IN/IT HPIV3 VRP Cell-mediated response Mucosal CD8 Non-mucosal CD8 CD4 % of cells Strong T cell responses predominantly in the respiratory tract. Limited, but detectable, systemic spread. MFI Mainly IFNγ- and TNFα-secreting T cells. HIPV3/EboGP aerosol HIPV3/EboGP IN/IT HPIV3 VRP Polyfunctional CD8 and CD4 T cell responses Rhesus macaque 1 marker (IFN-γ or TNF-α or IL-2 or CD107a) 2 markers (e.g. 25% of the cells expresse one marker and 75% expresse two markers). 3 markers 4 markers Polyfunctional CD8 and CD4 T cell responses 1 marker 2 markers CD8 3 markers 4 markers CD4 - Polyfunctional response higher in the lungs Mainly polyfunctional for 3 or 4 markers - Polyfunctional response higher in the lungs Mainly polyfunctional for 1 or 2 markers HIPV3/EboGP aerosol HIPV3/EboGP IN/IT HPIV3 VRP Polyfunctional CD8 and CD4 T cell responses 1 marker 2 markers CD8 3 markers 4 markers CD4 - Polyfunctional response higher in the lungs Mainly polyfunctional for 3 or 4 markers - Polyfunctional response higher in the lungs Mainly polyfunctional for 1 or 2 markers HIPV3/EboGP aerosol HIPV3/EboGP IN/IT HPIV3 VRP Polyfunctional CD8 and CD4 T cell responses 1 marker 2 markers CD8 3 markers 4 markers CD4 Both aerosolized and liquid vaccines induce a greater activation of lung CD8 and CD4 T cells. Stronger activation of the CD8 T cells. HIPV3/EboGP aerosol HIPV3/EboGP IN/IT HPIV3 VRP IFNγ expression in polyfunctional CD8 and CD4 T cells CD8 Greater level of activation of CD8 T cells is accompanied by a greated level of IFNγ secretion. CD4 Same trend for CD4 T cells? 4markers 3markers HIPV3/EboGP aerosol 2markers HIPV3/EboGP IN/IT 1marker HPIV3 VRP STUDY 1: Conclusions Aerosolized vaccine induce strong systemic and mucosal antibody responses, comparable to those obtained with liquid vaccine. Induction of neutralizing antibodies able to crossneutralize different types of EBOV. Greater activation of T cells in the lungs, by aerosolized vaccine, may be the consequence of a more efficient delivery to the pulmonary bronchiole. Study 2 Protection conferred by aerosolized vaccine Experiment • • • • 2 injections days 0 and 28 Or 1 injection at day 28 (purple) EBOV infection at day 55 Blood sampling Aims: - Determine the protection that aerosolized vaccine could confer against EBOV infection. Serum and mucosal antibody responses Results similar to the ones obtain in the study 1. Lower responses with only 1 injection HIPV3/EboGP aerosol (2D) HIPV3/EboGP aerosol (1D) HIPV3/EboGP IN/IT HPIV3 Markers of EBOV infection, clinical sickness scores, survival and viremia during EBOV infection Liver, hemolysis, kidney, A single administration of aerosolized vaccine completely protects against EBOV infection. HIPV3/EboGP aerosol (2D) HIPV3/EboGP aerosol (1D) HIPV3/EboGP IN/IT HPIV3 Take home messages Aerosolized vaccine induces strong systemic (high antibody and T cell responses) and mucosal responses that can be accelerated with a second dose. Aerosolized vaccine confered cross-protection and can protect against viral hemorrhagic fever. Aerosolized vaccine can be easly implemented in case of an outbreak, or during bioterrorism and warfare scenarios. Perspectives: • Application to other heamorrhagic fevers. • Improve the cross-proctection efficiency. • Phase I clinical study. Thank you for your attention ! Micrograph from F. A. Murphy, University of Texas Medical Branch, Galveston, Texas.