© 2016 Instituto Butantan – reproduction without permission is prohibited.
Zika vaccine development at Butantan Institute
Outubro de 2016
Paulo Lee Ho ([email protected])
Laboratório Especial de Inovação e Desenvolvimento Industrial
Instituto Butantan
Alphavirus: Chikungunya, encefalite equinas da Venezuela, Oriental e Ocidental
Flavivirus: Febre amarela, dengue, zika, encefalite japonesa, west do nilo
DENGUE, CHIKUNGUNYA and ZIKA are arboviruses
1) RNA fita positiva é análogo ao mRNA: a tradução de proteínas virais é feita a partir desta fita.
2) Possui uma RNA polimerase RNA dependente: síntese da fita de RNA fita negativa que servirá como
molde para a replicação da fita genômica positiva de RNA viral.
Dengue, Chikungunya e zika:
Zika virus is transmitted by Aedes aegypti mosquito that also transmittes
dengue, yellow fever, west nile flavivirus as well as
chikungunya alphavirus
Agosto
Dengue epidemiology
What about Zika? The same trend is expected...
Em 2016, foram registrados 1.182.250 casos prováveis de dengue no País até a Semana Epidemiológica
(SE) 18 (3/1/2016 a 7/5/2016)
Zika virus from Brazil is more related to Asian and Micronesia than Africa strains
Zika virus is more related to Japanese Encephalomielitis Virus (JEV)
Dengue, Chikungunya and Zika: Many challenges
and questions
1) Diagnostics
2) Therapies
3) Enhancement
4) Cross-protection
5) Microcephaly
6) Other disorders
7) Guillain-Barré Syndrome
8) Effectiveness of vaccines
9) Safety of vaccines
10) Other issues
Butantan´s vaccine developments:
a) inactivated zika virus;
b) live attenuated dengue-zika chimera (NIH);
c) live attenuated zika virus;
d) other approaches (depend on opportunities)
Live attenuated tetravalent dengue vaccine (Butantan/NIH):
Phase 3 trial
Dengue virus genome
Three are structural proteins: the capsid (C), envelope (E), and membrane (prM) proteins. Seven
are nonstructural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. These nonstructural
proteins play roles in viral replication and assembly. During virus maturation, prM is cleaved by
cellular furin protease to M mature protein.
-GCA G CAACAA C A A A G A G-C G G C-G A A G-C G*U G-C A-U G-C C-G G-CC-GA C G C-GACUA C-GA A A A U C-GG A-U C-G C-G A-U G-C G-C G-C G A A U A G G C U
DEN1 DEN2
The D30 mutation removes a homologous structure (TL-2)
in each dengue virus serotype
-AGCAA C ACAAAA- A A A A G A U A G G G-C A A G-C G*U G-C A-U G-C C-G G-CC-GA C G C-GACUA C-GA A A A G G*UG G-C U-A G-C A-U G-C A-U U*G G A A U A G G C U
DEN4
-AGC A ACAAAA- A C A A G A A A G G G-C A A G-C G*U G-C A-U G-C C-G G-CC-GA C G C-GACUA C-GA G A A G A G G*U C-G C-G A-U G-C G-C A-U G-C G A A U A G G C U
DEN3
-GCA G ACAAAA- C A A A G A G-C G T C-G A A G-C G*U G-C A-U G-C C-G G-CC-GA C G C-GACUA C-G G A A A G-CA C-G U U C U U C G-C A-U G-C G-C G A A U A G G C U
Homology
with other
DEN types
100%
38 - 66%
66 - 90%
2 new approaches for development of DEN3 vaccine candidates:
1) Additional deletions in the 3’ UTR
CTG G T A A A C AGA A C A GCC A G G G ||| A G-C A CGG A T-A C A A-T G-C C-G T*G C-G C-G T-A A-T T-A A G A-T A AT C-G C-G T*G G-C A-T T-A C-G C-GACTA G-C | G A-T G-CC-GC C-G G-C C-G T T A-T A-T C T AAC G-C G*T C-G C A G-C A A T-AAT GGTTCT--3’ G-C G G C || ||| A G --GTAAAACCT-A G A TGTCGTCCTAGAGACC A T |||||| |||| GACACAAC AAACAGCA CTGG G G A TATTGACG G A C A A T G A C A A A G A G-C G G C-G A A G-C G*T G-C A-T G-C C-G G-CC-GA C G C-GACTA C-G G A A A G-CA C-G T T C T T C G-C A-T G-C G-C G A A T A G G C T
D30
CTG G T A A A C AGA A C A GCC A G G G ||| A G-C A CGG A T-A C A A-T G-C C-G T*G C-G C-G T-A A-T T-A A G A-T A AT C-G C-G T*G G-C A-T T-A C-G C-GACTA G-C | G A-T G-CC-GC C-G G-C C-G T T A-T A-T C T AAC G-C G*T C-G C A G-C A A T-AAT GGTTCT--3’ G-C G G C || ||| A G --GTAAAACCT-A G A TGTCGTCCTAGAGACC A T |||||| |||| GACACAAC AAACAGCA CTGG G G A TATTGACG G A C A A T G A C A A A G A G-C G G C-G A A G-C G*T G-C A-T G-C C-G G-CC-GA C G C-GACTA C-G G A A A G-CA C-G T T C T T C G-C A-T G-C G-C G A A T A G G C T
D86
CTG G T A A A C AGA A C A GCC A G G G ||| A G-C A CGG A T-A C A A-T G-C C-G T*G C-G C-G T-A A-T T-A A G A-T A AT C-G C-G T*G G-C A-T T-A C-G C-GACTA G-C | G A-T G-CC-GC C-G G-C C-G T T A-T A-T C T AAC G-C G*T C-G C A G-C A A T-AAT GGTTCT--3’ G-C G G C || ||| A G --GTAAAACCT-A G A TGTCGTCCTAGAGACC A T |||||| |||| GACACAAC AAACAGCA CTGG G G A TATTGACG G A C A A T G A C A A A G A G-C G G C-G A A G-C G*T G-C A-T G-C C-G G-CC-GA C G C-GACTA C-G G A A A G-CA C-G T T C T T C G-C A-T G-C G-C G A A T A G G C T
D30D31
Newly constructed vaccine candidates: rDEN3D86
rDEN3D30/31 (currently used in dengue
vaccine formulation)
D31
Zika Vaccine developments at Butantan
1) Dengue - Zika chimera based on the NIH platform (NIH-Butantan)
D31
Zika Vaccine developments at Butantan
2) Live attenuated zika virus
ZIKV
Zika Vaccine developments at Butantan
3) Inactivated Zika virus
Zika virus is more related to Japanese Encephalomielitis Virus (JEV)
Inactivated Vero cell derived
JEV vaccine
PIV - Purified Inactivated Virus
DNA or Purified Inactivated Virus vaccines protect against Zika virus challenge in mice model
DNA or Purified Inactivated Virus or recombinant adenovirus 52 vaccines protect Rhesus against
Zika virus challenge
Zika Vaccine developments at Butantan
3) Inactivated Zika virus
Target Product Profile • Product description: Zika virus inactivated vaccine – composed by whole
zika virus chemically inactivated adjuvanted with alum
• Mechanism of action: Induction of humoral immune response although some cellular immune response can be observed. Production of neutralizing antibodies by primed B-cells, induction of memory immune response.
• Immunity Duration: Based on immune response kinetic information
• Indication for use: All the population in general (main target, adolescents and adult population). For not pregnant women that are planning to be pregnant. Pregnant women (?)
• Primary efficacy endpoints: Protection against Zika infection in more than 80% of vaccinees.
• Secondary efficacy endpoints: Antibody titers, neutralizing antibody titers in PRNT assay above threshold (threshold to be determined) in 80% of vaccinees.
Target Product Profile
• Expected safety outcome: As usual. No enhancement of diseases caused by other arboviruses; no increase in GBS; no neurological disorders due to maternal immune activation.
• Dosage and administration: 10E3 to 10E6 inactivated virus/administration; 2-3 administration, (0.5-2 months between first and second dose; 0.5-6 months between the second and third dose), subcutaneous or intramuscular, 0,5 ml/dose
• Contraindications: Autoimmune diseases, neurological diseases
• How supplied: Multidoses (5 doses) in vials.
• Aonde estamos?
• Perspectiva?
Vero cells (requeriment for use in human vaccine production)
27
Vero cell: ATCC CCL- 81.4 (p. 123)
QUALIFICATION
Identity
Karyotyping
Tumorinicity/
Oncogenicity
Electron
Microscopy
Assays for adventicious agents
PCR
Símio: retrovírus 1, 2, 3; SIV;
SRV; SFV; CMV; SV40
Human:
Retrovíruse
HTLVs
HIV 1 E 2
Hepatitis A, B e C
Cytomegalovirus
Epstein Bar virus
Mycoplasma
Porcine Circovírus (PCV) Chicken embryonated
eggs
sterility and mycoplasma
(culture)
Cellular assays
Vírus: 3 cell lines
Porcine - 8 cell lines
Bovine - 8 cell lines
Animal assays:
guinea pig, rabbit
mice
28
1 criotube Vero (ATCC)
Amplification (subcultures 1:3 a 1:5)
•Sterility
• Identity
•Karyotyping
•Micoplasma
•Adventicious agents
•Hemadsorbent viruses
VERO cells: Master cell bank and working cell
bank
Adaptation on SFM
Amplification (subcultures 1:3 a 1:5)
Working cell bank Master cell bank
Certified Banks
Storage in liquid nitrogen
Zika virus working Bank
Harvest
One passage on Vero cells grown in serum free media
Virus production Zika virus bank Certified
Storage in liquid nitrogen
ZIKA Virus dengue: Production of Master and
working virus bank
• Sterility
• Identity
• Micoplasma
• Virus titer
• Adventicious agents
• Hemadsorbent virus
Zika virus Master bank
Virus titration by plaque forming units
Sample vírus 2- T1-IB ZIKA Vero
• Source - Derived from infection on T3 C6/36 (ICBUSP) 1012 pfu/mL
250 µL MOI 7.000
• 6 T25 Vero: 2x media A, 2x media B e 2x DMEM(10% SFB, piruvate and essential aas)
• Plaque forming in 2 days
• Harvest in 2 days- 20mL for each medium
• Viral titers: A - 4.106pfu/mL
B - 8,5.105pfu/mL
DMEM- 2,25.106pfu/mL
Control A Zika - VA
Control B Zika - B Control 10%FCS +DMEM Zika - 10%FCS +DMEM
MEDIA TEST, 10% Fetal calf serum in DMEM, serum free media (A and B)
Lot - vírus 3- T1-IB ZIKA Vero
• Source - Infection on T3 C6/36 (ICBUSP)
MOI 0,05/ 0,08/ 0,1
• 3 T25 Vero
• Plaque forming in 4-5 days
• Harvest in 5 days- 10mL for each MOI
• Viral titers: MOI 0,05- 9,25.105 pfu/mL
MOI 0,08- 1,0.105 pfu/mL
MOI 0,1 - 6,0.105 pfu/mL
MOI 0,05
MOI 0,08 MOI 0,1
3 days 5 days 3 days 5 days
3 days 5 days
CONTROLE
Multiplicity of Infection (MOI) studies
VERO cells infected with Zika virus
Harvest
Ultrafiltration
Ultracentrifuge
Sucrose gradient
Clarification – filtration 0.22 m
Ultrafiltration
Inactivation
Chromatography
Diafiltration and concentration
Virus titration for yield determination
Diafiltration
50 kDa or 100 kDa or 300 kDa cut off
formaldehyde or
b-propiolactone
affinity
ion-exchange (2 different producers)
captocore
Zika virus Purification on ion exchange support
Fraction 1B1 Fraction 1C1
SDS-PAGE of samples from the anion exchange colunm
1ª) Cultura antes da passagem na coluna – 10 ul 1b) cultura antes da passagem -2 x 5 ul
7) fração 1B4 - 15 ul (3x5 ul)
2) flow through – 15 ul (3x5 ul) 8) fração 1B5 - 15 ul (3x5 ul)
3) fração 1A5 -15 ul (3x5 ul) 9) fração 1C1 - 15 ul (3x5 ul)
4) fração 1B1 - 15 ul (3x5 ul) 10) pool frações B - 15 ul (3x5 ul)
5) fração 1B2 - 15 ul (3x5 ul)
6) fração 1B3 - 15 ul (3x5 ul)
1.
Fraction 1B1
Tests also performed with samples from serum free medium: Zika virus binds to ion exchange resin
Inactivation protocol underway
Inactivation protocol: formaldehyde
or b-propiolactone Immunogenicity in mice model: underway
Drug Substance definitions depend on the process developed
Special thanks to:
Instituto Butantan Instituto Evandro Chagas
Jorge Kalil Pedro Vasconcelos
Cosue Miyaki
Neuza Maria Gallina Frazatti
Renato Astray BARDA
Rosana Paoli
Soraia Jorge Chuong Huynh
Viviane Botosso Rick Bright
Alessandra Schanoski Thomas Warf
Cláudia Sossai Soares
Universidade de São Paulo
Benedito Fonseca (FMRP)
Danillo Esposito (FMRP)
Edson Durigon (ICB)
Luís Carlos Ferreira (ICB)
Paolo Zanoto (ICB)
Butantan Zika Vaccine Plan
2016 Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr
Tox Start
Jun 1
Pilot Scale Established
Mar 15
Research Virus Seed Produced
Oct 15
GLP lot mfg
Apr - Sep
Preclinical Testing
Oct - Jun
Engineering runs and scale-up
Oct - Sep
GLP/GMP Preparations
Oct - May
Process and Analytical Dev.
Jun - May
2017 Jul May Jun Aug Sep
Phase 1 Start
Sep 30