Lichenase: a versatile, stable carrier molecule for vaccine and reagent development R. Mark Jones Center for Molecular Biotechnology, Fraunhofer USA New Cells, New Vaccines VII, From Protein to Product Wilmington DE 17 March 20 March 2013
Vaccinations: 27 Vaccine-Preventable Diseases Anthrax Cervical Cancer Diphtheria Hepatitis B Haemophilus influenzae type b (Hib) Human Papillomavirus (HPV) H1N1 Flu (Swine Flu) Influenza (Seasonal Flu) Mumps Measles Meningococcal Monkeypox Rabies Typhoid Fever Pertussis (Whooping Cough) Pneumococcal Smallpox Poliomyelitis (Polio) Rotavirus Yellow Fever Rubella (German Measles) Shingles (Herpes Zoster) Tuberculosis Tetanus (Lockjaw) Varicella (Chickenpox) Hepatitis A Japanese Encephalitis (JE) Classes: Live attenuated Inactivated Toxoid Conjugate / Subunit FDA approved 3 recombinant vaccines: Hepatitis B surface antigen Human papillomavirus major capsid protein Influenza Hemagglutinin
Compromises in Vaccine Development: Recombinants are generally considered to have better safety profiles but the trade-off for safety can be efficacy (strength). The cost of precision antigen targeting can lead to reduced immunogenicity and potential for incorrect immunology memory?
Lichenase Hydrolysis site of -glycans Structure of the Lichenase 27 a.a. 224 a.a. 18 a.a. 65 a.a. SP catalytic domain Pro- Thr dockerin domain
Predicted 3D structure of Lichenase Target proteins can be engineered as N (4) or C (3) terminal fusions or in the internal loop (2) A B Circular permutation B A (4) N-terminal fusions (2) Loop fusions (3) C-terminal fusions
Expression System Diversity for LicKM E.coli Mammalian Plant Public health image library. CDC.gov Histology-world.com Fraunhofer-CMB M 1 2 3 4 1 2 4 1 2 3 4 M Magic marker 1 Control 2 LicKM 3 LicKM-PAD4 4 LicKM-LFD1 E.coli Mammalian Plant
Expressed Antigen Fusion Diversity for LicKM Plant Bacillus anthracis antigens Yersinia pestis antigens Trypanosoma ssp. antigens Plasmodium ssp. Antigens Various growth factor reagents Influenza virus antigens Yellow Fever virus antigens Respiratory Syncytial Virus antigens Human Papilloma Virus antigens Filoviral antigens
Expressed Antigen Fusion Diversity for LicKM HPV Proven in therapeutic and prophylaxis test against TC-1 tumor model in mouse. LicKM showed increased efficacy/protection compared to non- LicKM fused antigen. tmw: 38 kda Anthrax Proven efficacy against lethal challenge studies in mice. Used as bi-valent vaccine (mixture of LicKM-PAD4 & LicKM- LFD1) showed 100% protection from challenge. Plague Double antigen fusion to LickM showed 100% protection in NHP challenge. Mixture of individual components showed gave protection to 7/8 subjects. Filovirus Fusions show potential for reagents: positive responses in Western blot and Elisa with neutralizing and non-neutralizng antibodies tmw: 95 kda MALS: 130 kda
FhCMB Malaria Transmission Blocking Vaccine Program Funding through the Bill & Melinda Gates Foundation TB Vaccine program targets sexual stage antigens and proteins. Pfs/Pvs 25, Pfs48/45, Pfs230. Proteins expressed transiently in N. benthamiana. Pfs25 LicKM Pfs25
Mechanism of Pfs25 TBV
FhCMB Pfs25 Candidates: ± LicKM Fusion Pfs25 LicKM Pfs25 60 50 40 PAGE WB PAGE WB 60 50 40 30 30 25 20 20 25F 25F-LickM
Mouse Study Experimental Design 5, 1, 0.2 µg of antigen administered s.c. with Alhydrogel Primary Boost DAY -1 0 28 56 112 168 Pre Bleed Bleed Bleed Bleed
Mouse Study Experimental Design 5, 1, 0.2 µg of antigen administered s.c. with Alhydrogel Primary Boost DAY -1 0 28 56 112 168 Pre Bleed Bleed Bleed Bleed
25F versus 25F-LicKM: Standard Membrane Feeding Assay Vaccine Candidate Adjuvant Dose (µg) SMFA Total # Oocysts Day 56 Day 112 Day 168 25F Alhydrogel 5 0 1 4 25F Alhydrogel 1 249 nd 25F Alhydrogel 0.2 340 nd 25F-LicKM Alhydrogel 5 3 4 0 25F-LicKM Alhydrogel 1 9 0 2 25F-LicKM Alhydrogel 0.2 162 nd LicKM Alhydrogel 5 575 333 393 nd= not determined based on negative results of the serum sample in an immunofluorescence assay 25F-LicKM elicited transmission reduction activity at lower doses and over a longer period compared to non-lickm fusion candidate. 25F-LicKM was selected for further modification and testing.
Second generation of Pfs25-LicKM (MM) LicKM Pfs25 Point mutations: M M M M N-linked glycosylation site removed from LicKM Free cysteine removed from LicKM N-linked glycosylation sites removed from Pfs25 Animal study design, single dose with immunizations at days 0 & 21. SMFA performed on sera drawn at days 42 and 168.
Pfs25-LicKM (MM) Saline/Alhydrogel Formulation 10 mg dose at Days 0 & 21 Pfs25-Subunit SMFA Day 42 Group # Storage Time SMFA 4 C 37 C #mosq #mosq Total # Oocysts per feeder infected dissected Oocysts Mean Lowest Highest % TB* 1 Month 38 48 298 6.1 0 24 84.8 ± 3.8 3 Months 1 50 1 0 0 1 99.8 ± 0.2 1 Month 0 50 0 0 0 0 100 ± 0 3 Months 9 49 18 0.4 0 7 99.1 ± 0.2 Saline N/A 129 144 3412 20 0 157 0 Serum Only N/A 132 147 2989 23.4 0 111 0 MRA39 (rabbit pab) N/A 116 150 550 5.6 0 44 69.2 ± 21 *Data represented as average ± STD of 2 SMFA Performed by Sanaria
Pfs25-LicKM (MM) Saline/Alhydrogel Formulation 10 mg dose at Days 0 & 21 Pfs25-Subunit SMFA Day 168 Group # Storage Time SMFA 4 C 37 C #mosq #mosq Total # Oocysts per feeder infected dissected Oocysts Mean Lowest Highest % TB* 1 Month 48 50 1376 27.5 0 66 54.6 3 Months 8 40 21 0.5 0 7 99.3 1 Month 16 49 42 0.9 0 6 99.2 3 Months 37 50 230 4.6 0 19 92.4 Saline N/A 143 144 11280 79.1 0 245 0 Serum Only N/A 137 139 14978 107.4 0 309 0 MRA39 (rabbit pab) N/A 138 149 4729 32 0 140 71 *Data represented as average ± STD of 2 SMFA Performed by Sanaria
Conclusions: Our engineered Lichenase can add stability, solubility and increase expression of targets across multiple expression systems. The fusion positions options; N-terminus, C-terminus or surface loop, offers increased opportunity for successful expression of full size proteins or individual domains. Lichenase fusions create potent, stable molecule that have been shown to generate functional immune responses that protect against Anthrax, Plague, HPV cancer model, and Malaria. Added stability of Lichenase offers the potential for stable reagent production platform.
Acknowledgments Fraunhofer R&D, Project Management, and Administration Staff The Bill & Melinda Gates Foundation U.S. Department of Defense (DARPA, DTRA) ibio, Inc. (IBIO:AMEX) Departments of Medical Microbiology, Nijmegen Center for Molecular Life Sciences, Nijmegen, The Netherlands NIH/NIAID; Rockville, MD USA Division of Cell and Molecular Biology; Imperial College London; London, UK ENEA, Italian National Agency for New Technologies, Energy and the Environment, BAS BIOTEC GEN, C.R. Casaccia, Via Anguillarese 301, 00123 Rome, Italy