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Influenza virus Membrane-enveloped enveloped virus Major envelope glycoprotein: Hemaglutinin HA Neuraminidase NA Virosome Virus envelope

Virosome for Influenza Vaccines Virosome encapsulating model antigen, ovalbumin (OVA) Fusion-inactive virosome with OVA Activate MHC class II Fusion-active virosome with OVA Activate MNC class I and II

Generation of Virosomes Preparation analysis Buffer Free indocarbocyanin (Cy5)-labeled sirna 10% sucrose Virosome band 50% sucrose Encapsulated sirna More than 35% (36.7 14.2%) of the sirna could be recovered when 34% DODAC was included in the preparation.

Characterization of virosome composition Equilibrium density analysis sirna protein Virosomes contained 2.75 pmol sirna/ug protein, 60-70 sirna molecules/virosome

Characterization of virosome morphology Electron Microscopy 60nm 100nm Rosette-like particle Virosomes with encapsulated sirna Native influenza virus

Delivery of sirna to cultured cells Toxicity test on BHK21 and A2780 cells 80-100% cell viability in sirna virosomes infection up to 72h. Microscopical analysis of sirna (labeled with FAM) delivery Binding of virosomes to cellular receptors Internalization via endosomes

Drug targeting using virosomes Cytotoxic drug Antibody fragment targeting rneu Tumor antigen Targeting HER-2/neu with Antirat Neu Virosomes for Cancer Therapy Waelti E, Wegmann N, Schwaninger R, Wetterwald A, Wingenfeld C,Rothen-Rutishauser B, Gimmi CD, 2002:, Cancer Res 62, 437-444

Summary Virosome Synthetic delivery systems - well defined - safe Desired properties - active translocation of sirna into cytoplasm - targeting to broad cell types In vivo delivery of sirna - via respiratory epithelium - targeting cells with Fc receptors

virus-like particles (VLPs) formed by the self-assembly of envelope and/or capsid proteins from many viruses have found a number of biomedical applications e.g. vaccines and novel delivery systems for nucleic acids and small molecules

VLPs: promising vaccine candidates against virus infection General feature: non-infectious highly immunogenic size range of viruses (22 150 nm) easily scalable and safer to produce than those based on attenuated viruses

Natural occurring VLPs In the blood of hepatitis B virus (HBV) infected patient, subviral particles (SVPs( SVPs) ) are far more than the virion itself. Notably, these plasma- derived SVPs provided the first-generation HBV vaccines. Expression of the small envelope protein of HBV in yeast or mammalian cells leads to the formation of 22 nm VLPs that are essentially identical to the SVPs,, which take over as the current HBV vaccine.

Natural occurring VLPs Human papillomavirus (HPV) replication also release natural empty particles contain the L1 and L2 proteins. Using mammalian cells, baculovirus,, yeast expression systems, expression of the HPV L1, the major capsid protein, leads to the assembly of VLPs that are similar to the empty virus particles formed during HPV replication. The HPV-VLPs VLPs consisting of L1 protein is current HPV vaccine for prevention and therapy.

Virus Particle composition Type/expression system Size Vaccine status HBV Small envelope protein (HBsAg) HBsAg PreS1+2 and HBsAg HBsAg rec VLP (yeast) (Recombivax( Recombivax-HB; Engerix-B) rec VLP (potato) rec VLP (CHO cells) (Sci( Sci-B-Vac; BioHepB) Native SVP (plasma) 22 nm 17 nm 22 nm 22 nm Licensed Preclinical Licensed Licensed (3rd world) HPV L1, major capsid protein recvlp (mammalian cells;baculovirus; ; yeast) 45-50 50 nm Preclinical Gardasil,Cervarix HEV Truncated major capsid rec VLP (baculovirus( baculovirus) 23.7 nm protein (ORF2) Influenza HA, NA, matrix recvlp (baculovirus) 80-120 nm Preclinical HCV Core, E1, E2 recvlp (baculovirus) 40-60 nm Preclinical Poliovirus Capsid (VP0,1,3) recvlp (baculovirus) 27 nm Preclinical HIV Pr55gag, envelope recvlp (mammalian cells;baculovirus; ; yeast) 10-120 120 nm Preclinical Ebola; Marburg Glycoprotein (GP) and matrix (VP40) recvlp (mammalian cells) Filovirus- like particles Preclinical Norwalk capsid recvlp (baculovirus;; transgenic potatoes) 38 nm Preclinical Rotavirus VP2,VP6,VP7 recvlp (baculovirus) 70-75 75 nm Preclinical SARS-CoV S, E and M recvlp (baculovirus) 100 nm Preclinical

Building blocks of VLPs One or multiple capsid proteins of viral core particle : non-enveloped virus e.g. HPV; enveloped virus, e.g. HBcAg. One or more envelop proteins of viral envelop: only applied to enveloped virus

DC preferentially ingest smaller (viral size range) particles, while MC take up more of the larger, (bacterial size) particles Immunology and Cell Biology (2004) 82, 506 516

Recombinant VLPs of influenza A virus closely mimic the native virus particle Flu-VLPs have a similar size to the native influenza virus particle (~120 nm), and provide a useful alternative to egg-derived or cell-culture culture derived vaccines based on inactivated virus. The egg-based technology may not be suitable to respond to a pandemic crisis. The H5 avian influenza virus strains responsible for recent epizoonotic outbreaks in Asia are lethal to chicken eggs. The current methods of production are fragile in ensuring an adequate and timely supply of flu vaccine. Certain viral strains replicate slowly in chicken embryo and cell culture.

Purification of influenza VLPs and electron microscopy examination influenza virus A/PR8 HA (H1N1) 0% >20% 20-30% 30-60% Suc.% 100 nm trypsin

Influenza virus-specific specific total serum IgG antibody responses OD 450 OD 450 OD 450

Neutralization activity. against A/PR8 against A/WSN

Protection of mice from lethal PR8 and WSN challenge PR8 challenge WSN challenge

Virus titers in lungs on day 4 postchallenge with a lethal dose of PR8 or WSN a, P < 0.01; b, P < 0.05

Mucosal antibody responses following challenge infection IgG IgG IgG IgA

Cytokine-secreting splenocytes following challenge infection ELISPOT assays

Proinflammatory cytokines in the lungs after virus challenge

VLP immunization maintains long- lasting protective immune responses Postimmunization 5 months antibody-secreting cells in the bone marrow lethal challenge

Long-lasting protective immune responses of immune sera Group Clinical sign(s) Protection (%) Naïve + PR8 Normal sera + PR8 Immune sera + PR8 Naïve + WSN Normal sera + WSN Immune sera + WSN Sick, loss of wt, dead Sick, loss of wt, dead Healthy Sick, loss of wt, dead Sick, loss of wt, dead Healthy 0 0 100 0 0 100

Reference readings Flu-VLPs vaccine consisting of HA and M1 without any additional adjuvant are protective (J. (J. Virol.. 81: 3514-3524, 3524, 2007) Virus-like particles: Passport to immune recognition (Methods 40: 60-65,2006) 65,2006)

利 SARS VLPs gene transfer S M GFP Inducible plasmid E InduceVLP expression and secretion VLPs 類粒 Purify for application

Transmission Electron Microscopy of SARS VLPs 50 nm 100 nm VLP (fraction 9-15) SARS-CoV

Protocol for SARS VLPs vaccination in mice by subcutaneous injection 0 week 2 weeks 3 weeks 4 weeks 6 weeks Primary immunization G1 PBS PBS G2 20 g 0 g Booster immunization 1.Cellular immune response analysis by ELISPOT assay 2.Determine the IgG titer and subtype in serum 3. SARS-specific plasma cells in BM G3 20 g G4 20 g 5 g 20 g

Humeral immune response of SARS VLPs vaccination in mice 1 week 2 weeks O.D. 3 2 after boost PBS 20-0 20-5 20-20 O.D. 3 2 after boost PBS 20-0 20-5 20-20 1 1 0 1:50 1:250 1:1250 1:6250 0 1:50 1:250 1:1250 1:6250 O.D. 3 2 4 weeks after boost PBS 20-0 20-5 20-20 O.D. 3 2 1 PBS 20-0 20-5 20-20 1 0 1:50 1:250 1:1250 1:6250 0 2 3 4 5 6 7 weeks after first immunization (1:1250)

Cellular immune responses of SARS VLPs vaccination in mice 1 week 2 weeks Spots/3x10 5 splenocytes 150 100 50 0 INF-γ after boost IL-4 PBS 20-0 20-5 20-20 Spots/3x10 5 splenocytes 300 200 100 0 INF after boost IL4 PBS 20-0 20-5 20-20

Application of VLP: vaccine antigen of SARS virus VLP SARS gene transfer S M GFP E VLPs 類粒 Safe!? infection replication Killed or attenuated virus Therapeutic vaccine 療

2.0 Immunization with Killed SARS CoV on 0,2, 7 49 week and with VLP on 82 week Immunization with SARS VLP on 82 week 405 nm λ O.D. 1.5 1.0 SARS-CoV Coating Plate VLP Coating Plate 0.5 0.0 0 10 20 30 40 50 60 70 80 90 Weeks

Application of VLP: diagnosis of SARS infection VLP SARS VLPs were coated in the ELISA plates. The Serum Sample 1: ELISA 2: The Serum Sample VLP-secreting cells were fixed and coated in the glass. Confocal microscopy

Application of VLP in drug screening VLP VLP (Assay for VLPs binding to Vero E6 cells). 1 Fluorescent 2 nd Ab Flowcytometry : 讀數量 Confocal microscopy : Quantitative 量 Qualitative VLPs Anti-VLP Ab Vero E6 cells 2(ELISA) HRP Quantitative 量