zika vaccine development at butantan institute 3... · one passage on vero cells grown in serum...

© 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


D86


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


2) Live attenuated zika virus

ZIKV


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


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)



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

zika vaccine development at butantan institute 3... · one passage on vero cells grown in serum...

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