Jon Heinrichs, Ph.D.

Associate Vice President & Segment Head

Early and Pre-Development Projects

Zika Vaccine Project Leader

The scope of the Zika pandemic and implications for vaccine development

Emergence of Zika virus

Draft for Review | 2

NY Times, April 5, 2016

Newsweek.com

Isolation of Zika virus

Draft for Review | 3

www.who.int

jme.oxfordjournals.org

Worldwide spread of Zika

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Zika virus outbreak to date

● ZIKV virus first isolated in Zika Valley, Uganda in 1947 from a febrile Rhesus monkey

● Before 2007 caused only a few mild human cases in Africa and Asia

● 2007 outbreak in Micronesia: the first Zika outbreak outside Africa and Asia

● in Yap Island, 919 residents (18%) with mild symptoms

● 2013 outbreak in French Polynesia

● ~ 8,750 suspected cases between October 2013 and April 2014

● 42 cases of Guillain-Barre syndrome (GBS) after the initial Zika infection (encephalitis,

meningitis, paraesthesia, facial paralysis and myelitis)

● In May 2015, Brazil reported first case of Zika and disease has subsequently spread to

other parts of Brazil and other Latin American countries

● In November 2015 the Brazilian Ministry of Health declared a public health emergency in

relation to an unusual increase in the number of children born with microcephaly (MC)

● > 10-fold increase in the incidence of microcephaly

● By Jan 2016, an estimated 1.3M cases and ~ 3530 MC cases including 46 deaths in 20 states

● On February 1, 2016 WHO declared Zika a Public Health Emergency of International

Concern

● April 13 CDC concluded Zika is definitive cause of microcephaly

● April 29- First reported death from Zika in the U.S. (Puerto Rico)

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Areas with Zika active transmission

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CDC Website, As of April 29, 2016

Status of Zika in Brazil

Malone RW, et al. (2016) Zika Virus: Medical Countermeasure Development Challenges. PLoS Negl Trop Dis 10(3): e0004530.

doi:10.1371/journal.pntd.0004530

States in Brazil investigating

microcephaly cases for

association with Zika virus

infection

States in Brazil with

confirmed circulation of

Zika virus

Zika virus background

● ZIKV is a single-stranded, positive-sense RNA virus● Member of the genus Flavivirus (family Flaviviridae), which also includes yellow fever

virus (YFV), Japanese Encephalitis virus (JEV), West Nile virus (WNV), and the four

Dengue viruses (DENV-1 to -4)

● ZIKV is mostly transmitted by Aedes spp. mosquitoes, and in the case of the

Americas more specifically by Aedes aegypti● Other transmission routes include sexual transmission, as well as a potential for

transmission via blood transfusion

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Image courtesy of Oloo and Oomen Devika Sirohi et al. Science

2016;science.aaf5316

3’SP genes NS genes

Genomic RNA (positive-polarity, ~ 11 kb)

5’

• E protein main immunogen eliciting neutralizing

antibodies (the generally accepted correlate of protective

immunity)

• T cell epitopes predominantly in E and NS3 proteins

Flavivirus genome organization

?C prM E NS1 2a 2b NS3 4a 4b NS5

Furin

M

Signalase

Viral

protease

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Phylogeny of members of the genus Flavivirusand ZIKV complete coding region sequences

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Nuno Rodrigues Faria et al. Science 2016;science.aaf5036

Yap 2007Cambodia 2010

Thailand 2013

Cook Islands 2014

FP + Americas

Viruses 2015, 7(1), 219-238; doi:10.3390/v7010219

Flavivirus nucleotide sequence alignment

● Nucleotide sequences of a

representative strain of most

flaviviruses of importance for

public health relevance were

selected

● The full genomes were aligned

together and an identity table

was generated, showing the

percentage of pairwise identity of

all aligned sequences

● There is homology between ZIKV

vs DENVs of ~60% (slightly

higher than YFV vs DENVs)

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The most distant sequences are indicated in green whereas closest sequences are shown in red

Flavivirus Sequence Alignment Created by Yves Girerd-Gambaz, provided by Bruno Guy

Zika virus impairs growth in human neurospheres and brain organoids

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Patricia P. Garcez et al. Science 2016;science.aaf6116

ZIKV infects human neural stem cells

alters morphology and

halts the growth of

human neurospheres

induces death in human

neurospheres

reduces the growth rate of human

brain organoids

Clinical aspects of Zika disease

● Zika virus usually produces a mild clinical syndrome:

● conjunctival hyperemia or bilateral non-purulent conjunctivitis

● maculo-papular rash

● low-grade fever (<38.5°C)

● transient arthritis/arthralgia with possible joint swelling

● general non-specific symptoms including myalgia, asthenia and

headaches

● Incubation period ranging between 3 and 12 days. Disease symptoms

usually mild and short lasting (2–7 days)

● Zika-associated Guillain-Barré syndrome (GBS) first described during

the outbreak in the French Polynesia during 2013-14

● Zika-associated microcephaly first described in the context of the current

outbreak in Brazil (2015-16)

The link between Zika and GBS has yet to be confirmed

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Zika and microcephaly

● Primary microcephaly is a severe brain malformation characterized by the reduction

of the head circumference

● Patients display a heterogeneous range of brain impairments, compromising motor,

visual, hearing and cognitive functions

● Microcephaly is associated with decreased neuronal production as a consequence

of proliferative defects and death of cortical progenitor cells

● During pregnancy, the primary etiology of microcephaly varies from genetic

mutations to external insults

● The so-called TORCHS factors (Toxoplasmosis, Rubella, Cytomegalovirus, Herpes

virus, Syphilis) are the main congenital infections that compromise brain

development in utero

● The increase in the rate of microcephaly in Brazil has been associated with the

recent outbreak of ZIKV. So far, ZIKV has been described in:

● the placenta and amniotic fluid of microcephalic fetuses

● in the blood of microcephalic newborns

● the brain of a microcephalic fetus

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Zika and microcephaly

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Petersen, et al. 2016. NEJM www.bbc.com

Microcephaly cases in Brazil: significant increase in 2015

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- 150 - 200 per year between 2010 and 2014

- In 2015, as of 28 November, 1248 suspected cases, of which 509 cases

reported between 21 and 28 November 2015.

Guillain-Barré syndrome

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• 0.8 -1.9 cases per 100,000

people per year in US & EU

• Observed after Campylobacter

jejuni, CMV, Epstein-Barr

virus, flu A, Mycoplasma

pneumoniae, Haemophilus

influenzae infections, and

after rabies and Flu

vaccination, etc

• Ag mimicry believed to be

involved but interplay with host

factors not understood

• Autoimmunity does not arise

in most individuals (>99%)

exposed to stimulus

Willison et al. 2016 Lancet Feb 29. pii: S0140-6736(16)00339-1 [Epub ahead of print]

Transmission cycles

● Non-human and human primates are principal vertebrate hosts

● First isolation (Zika Valley, Uganda, 1947) from a febrile sentinel Rhesus

monkey

● Cercopithecus and Erythrocebus monkeys in Africa (without recognized

disease)

● First reported in humans in 1952

● Aedes spp mosquito vectors

● African species: Ae. africanus, Ae. luteocephalus, Ae. vittatus, Ae.

furcifertaylori, Ae. hensilli, Ae. aegypti, etc.

● Outside of Africa, the principal vector is Ae. aegypti (urban areas), and Ae.

albopictus is a potential vector

● Also can be spread sexually (male to male and male to female) and

potentially through contaminated blood

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Monath, Heinz. 1996. In: Fields virology, 3rd Ed, Flaviviruses, pp. 1961-1034.

Grard et al. Zika Virus in Gabon (Central Africa) – 2007: A New Threat from Aedes albopictus? PLoS

Negl Trop Dis. 2014.

Wong et al. Aedes (Stegomyia) albopictus (Skuse): a potential vector of Zika virus in Singapore.

PLoS Negl Trop Dis. 2013 Aug;7(8):e2348.

Global distribution of Aedes aegypti and Aedesalbopictus

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A. aegypti A. albopictus

Occurrence

Kraemer et al. 2015 Sci Data 2:150035.

Kraemer et al 2015 Elife 4:e08347.

Theoretical dispersion of Zika virusPotential for rapid spread to the United States

● Global spread model integrates

● Global ecological niche data for Aedes aegypti

and albopictus

● Temperature profiles

● Characteristics of international travelers

● Travelers pathway

● 9.9 million travelers from Brazilian airports

• 65% to Americas

• 27% to Europe

• 5% to Asia

● Potential dispersion

● Argentina, Italy and the USA have more than

60% of their populations residing in areas

conducive to seasonal zika virus transmission

► Vaccines are perhaps the best tool to stop the

spread of Zika virus and prevent disease.

Draft for Review | 20

Bogoch. International spread of Zika virus from Brazil, Lancet 387. 2016

Licensed human flavivirus vaccines

● Yellow Fever 17D (Sanofi Pasteur, others)

● Attenuated strain of yellow fever; in use since 1938

● Nearly 100% effective, provides life-long immunity

● Recommended for children over 9 months of age

● Japanese Encephalitis

● IXIARO (Valneva, others)- inactivated, Vero cell produced JEV (3 dose

series)

● Live attenuated vaccine- produced in Asia (2 dose)

● IMOJEV (Sanofi Pasteur) – ChimeriVax-JE vaccine, single dose, at least 5

years protection

● Tick-borne Encephalitis

● Inactivated vaccines (Baxter, others)- 3 doses

● Dengue

● Dengvaxia (Sanofi Pasteur)- Chimerivax-Dengue (4 serotypes) - 3 doses

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Potential vaccine approaches for Zika virus

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Vaccine Type Maturity of

technology

Dose

requirement

Appropriate for

pregnant women during

Brazil-like outbreak?

Durability of immunity (e.g.

for cyclical epidemiology

scenario)

ZIKV INV (+/- adjuvant) Fully

established

2-3 doses for

primary

immunization

Yes Periodic boosters needed to

maintain immunity

ChimeriVax-ZIKV LAV Fully

established

1 dose

potential

Must be assessed Long term (perhaps lifetime)

RNA immunization Preclinical

POC

Unknown Yes Unknown

Subunit based on truncated

E protein or prM-E VLP

Preclinical

POC

Multiple Yes Short - boosters will be

required

DNA immunization Early Unknown Yes Unknown

Viral vectored Early ? ? ?

Rational attenuation of ZIKV,

incl. using NS mutations

Theoretical ? ? ?

Passively delivered antibody Early Multiple Potentially Short, <3 months

Sanofi Pasteur’s Zika vaccine program

• Live ChimeriVax approach for broad use

• Evaluating other technologies in parallel to

mitigate risks and accelerate response

1

STRATEGY

• Preclinical testing starting 2Q’16

• Actively seeking serological diagnostics and

animal models

STATUS

2

• Important need for international and regional

collaborations and partnerships

• Working with governmental agencies to

identify sources of funding

PARTNERS

3

Independent.co.uk

Yellow Fever 17D live attenuated vaccine

● Considered the strongest immunogen in man

● Single dose provides lifelong immunity

● Lost the ability to be transmitted by mosquitoes

● Safe for infants >9 months of age

● ID90 <1 log

● Targets and stimulates antigen-presenting cells

● Strongly activates innate and adaptive immunity

● Well known immune correlates (neutralizing antibody)

● Rapid onset of immunity

● Protective neutralizing antibody levels in 90% and 99% of vaccinees by day 10

and 30, respectively

Can be used to deliver foreign flavivirus proteins

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Construction of ChimeriVax® vaccines

• Dengue-1 PUO-359 (Thailand 1980, human) • Dengue-2 PUO-218 (Thailand 1980, human) • Dengue-3 PaH881 (Thailand 1988, human)• Dengue-4 1228 (Indonesia 1978, human)• JE SA14-14-2 (Chinese vaccine) IMOJEVTM

• WN01 NY99 (veterinary)• WN02 mutated NY99 (human)

C Nonstructural5’

YF 17D

prM-E 3’

ChimeriVax® vaccines

Dengvaxia®

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Sanofi Pasteur’s dengue vaccine, Dengvaxia®

● The Sanofi Pasteur vaccine is a 4-serotype,

recombinant, live, attenuated vaccine.1,2

● Four genetic constructs with 1 for each serotype.

● Genes encoding prM/E structural proteins from

each dengue serotype combined with genes

encoding capsid (C) and nonstructural (NS)

proteins from YFV 17D vaccine strain.

● Combination into a single vaccine.3

● Freeze-dried.

● Without adjuvant or preservatives.

17D Yellow feverDengue

Chimeric Virus

17D yellow feverDengue

Recombinant virus

1. Guirakhoo, 2001, J Virol.

2. Guirakhoo, 2000, J Virol.

3. Guy, 2011, Vaccine.

*Vaccine referred to in the literature as Chimeric Yellow Fever 17D-Tetravalent Dengue Vaccine (CYD-TDV).

C=capsid; DENV=dengue virus; E=envelope; NS=nonstructural; prM=precursor membrane; YFV 17D=yellow fever vaccine 17D.

TWO PHASE III LARGE-SCALE RANDOMIZED EFFICACY STUDIES AND ONE PHASE IIb EFFICACY STUDY OF THE CANDIDATE DENGUE VACCINE INCLUDED >35,000 PARTICIPANTS IN ENDEMIC COUNTRIES

● 2009: first proof-of-concept phase IIb efficacy study; results published September 2012.1

● 2011: 2 large-scale efficacy studies in Asia-Pacific and Latin America; results published in 2014.2,3

1. Sabchareon, 2012, Lancet.

2. Villar, 2015, N Engl J Med.

3. Capeding, 2014, Lancet.

4. Hadinegoro, 2015, N Engl J Med.

Phase III Efficacy Latin America (CYD15)2

• Countries: Colombia, Mexico, Honduras,

Puerto Rico, Brazil

• Age group: 9–16 years

• Sample size: 20,869

• Long-term follow-up: 5 years postdose 3

Phase III Efficacy Asia-Pacific (CYD14)3

• Countries: Thailand, Indonesia, Malaysia,

Vietnam, Philippines

• Age group: 2–14 years

• Sample size: 10,275

• Long-term follow-up: 5 years postdose 3

Phase IIb Efficacy Asia-Pacific (CYD23/57*)

Proof-of-concept study1,4

• Country: Thailand

• Age group: 4–11 years

• Sample size: 4002

• Long-term follow-up: 5 years postdose 3

*CYD57 is the long-term follow–up of CYD23.

Reduction in

symptomatic dengue

65.6%(95% CI: 60.7–69.9)

Reduction in

hospitalized dengue

80.8%(95% CI: 70.1–87.7)

CONSISTENT EFFICACY PROFILE IN SUBJECTS 9–16 YEARS OF AGE DURING THE EFFICACY PHASE

1.Hadinegoro, 2015, N Engl J Med.

Key Efficacy Results25-month active phase* Pooled efficacy analyses‡1

*Data come from the 2 pivotal, phase III, large-scale efficacy trials CYD14 and CYD15, which were designed to fully assess efficacy; postdose 1; 1Full Analysis Set for Efficacy

(FASE): all subjects who received at least one injection. †dengue hemorrhagic fever, World Health Organization 1997 criteria. CI=confidence interval; DENV=dengue virus.

Reduction in

severe dengue †

93.2% (95% CI: 77.3–98.0)

SP

GLB

.DE

NG

.15.0

5.0

092

For each serotype:

DENV-1: 58.4% (95% CI: 47.7–66.9)

DENV-2: 47.1% (95% CI: 31.3–59.2)

DENV-3: 73.6% (95% CI: 64.4–80.4)

DENV-4: 83.2% (95% CI: 76.2–88.2)

By dengue serostatus:

Seropositive: 81.9% (95% CI: 67.2–90.0)

Seronegative: 52.5% (95% CI: 5.9–76.1)

Lessons learned from Dengue are pertinent for

Zika vaccine considerations

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•Need for Clinical studies to “down-select” candidates No animal models for disease

•Clinical studies performed in multiple endemic settings

• Importance of collaborations and partnerships in endemic areas

Dynamic dengue epidemiology

•Vaccine immunogenicity, safety and efficacy tested in a background of seropositive and seronegative subjects to YF, JEV and DEN

Pre-existing immunity to flaviviruses

•Large scale field efficacy trials with lab-confirmed clinical endpoints in 10 endemic countries

No known immune correlate of protection

•Large scale safety trials with long-term follow-up

•Driver for tetravalent formulation

Theoretical risk of immunopotentation

•Early need to invest in manufacturing infrastructure to have capacity ready for endemic regions immediately after licensureManufacturing capabilities

Challenge for Dengue vaccine Lessons Learned for Dengue

Multiple challenges need to be addressed to advance Zika vaccine candidates

Examples include:

● No qualified disease animal models

● No identified correlate of protective immunity

● Unknown incidence rates & risk factors for clinical complications

● Unknown spectrum of clinical complications

● If GBS association is confirmed, what is the basis of the pathology ?

● Unfolding epidemiology

● Clinical trial requirements for accelerated registration

● Need to prevent viremia ?

● Disease endpoints in efficacy trials ?

● The complexities of symptomatic and asymptomatic infection

● The development of diagnostic assays that avoid cross-reactivity with other

flaviviruses

● Need to prospectively assess Zika/Dengue interactions

Timelines for vaccine development

● First vaccines will likely enter the clinic in late 2016

● Inactivated Zika vaccines as well as DNA/RNA vaccines will be available

first

● Initial studies will be in small numbers of healthy individuals to establish

safety and immunogenicity

● Likely to require multiple doses and adjuvants for optimal

immunogenicity

● These vaccines will be best suited to emergency response

● Vaccines based upon live-attenuated or recombinant technologies will

come somewhat later

● These may provide longer term immunity

● Clinical studies will be long, costly and difficult to perform

● Too early to predict when a vaccine will be broadly available

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Conclusions

● Zika virus has emerged as a global health threat requiring a coordinated

response

● Vaccines, therapeutics and mosquito control are all important parts of a

holistic approach to fighting the disease

● Multiple vaccine technologies need to be evaluated including inactivated,

sub-unit and live-attenuated platforms

● Important lessons can be learned from the development of Dengvaxia and

other flavivirus vaccines

● Sustained investment and partnerships are critically important

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Thank you!

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