Overview of Zika

Alan D.T. Barrett Department of Pathology

Sealy Center for Vaccine Development

University of Texas Medical Branch

Galveston TX

Disclaimer

• Over 700 papers in pubmed on Zika in the last 12 months.

• Impossible to stay up to date as field is moving so fast.

• Only including material in public domain.

ZIKV: 1947-2006

• Zika virus (ZIKV) causes Zika fever (ZF), an acute febrile illness

characterized by a rash, conjunctival injection, arthralgia, myalgia

and headache.

• The disease appears in all age groups with an incubation period

on the order of 3-14 days and a symptomatic phase lasting about

2-7 days.

• Treatment is largely symptomatic.

• The illness is mild in nature with a very low rate of hospitalization.

• The vast majority of patients make a full recovery and while death

is rarely reported, it has primarily occurred in the

immunocompromised or those with other complicating medical

conditions.

• Only 14 clinical cases in the literature from 1951-2006.

Map showing the known distribution of Zika virus based on serosurveys, virus

detection, and laboratory-diagnosed cases. Blue arrows show recent patterns

of spread deduced from phylogenetic studies

Weaver et al Antiviral Research, Volume 130, 2016, 69–80

ZIKV in the Americas

Current ZIKV activity

ZIKV transmission

• ZIKV is primarily transmitted by Aedes spp. Mosquitoes.

• Infectious ZIKV particles have been demonstrated in urine, saliva,

semen, blood products, and breast milk possible vehicles of

transmission.

• Male-to-female and male-to-male transmission of ZIKV have been

reported, but only from individuals with clinical signs/symptoms of

ZIKV infection.

• Establishing person-to-person transmission is difficult, as contacts

frequently have the same environmental exposures.

• Epidemiologic relevance of person-to-person transmission has yet to

be elucidated.

• Although viremia during infection is short-lived, typically lasting less

than 4 days, it appears that infectious virus may persist in semen and

body organs much longer (perhaps months) making it much more

difficult to prevent human-to-human transmission.

ZIKV serology

Serologic evidence in Africa of infection in forest-dwelling

birds, horses, cattle, ducks, bats, elephants, goats,

hippopotamuses, impala, kongoni, water buffalo, sheep,

wildebeest, rodents, and zebras

Serologic evidence in the Americas of infection in monkeys

and humans. No other species examined?

ZIKV 2007- present (ADEM and GBS)

• Most ZIKV infections are asymptomatic (75-80%).

• ZIKV outbreaks have been associated with increased rates of rare

autoimmune neurologic disorders such as Acute Disseminated

Encephalomyelitis (ADEM) and Guillain-Barre syndrome (GBS).

• Both conditions can result from a number of infectious diseases.

• The incidence is ADEM around one tenth that of GBS and little is known

about its occurrence following ZIKV infection.

• There appears to be about a 10- to 20-fold increase in GBS during ZIKV

outbreaks from the baseline incidence of 1-2 cases per 100,000 people.

• It has been reported that the clinical onset of ZIKV outbreak associated

GBS cases is more rapid than typical, with a median of 6 days from the

onset of signs of ZIKV infection to GBS symptoms.

Congenital Zika Syndrome

• Vertical transmission from mother to the fetus during pregnancy.

• Outcomes: placental insufficiency, fetal growth restriction, oligohydramnios,

ocular disorders, auditory impairments, CNS injury and fetal death.

• Signs of CNS injury associated with ZIKV include congenital microcephaly,

ventricular calcifications, migration defects, simplified gyral patterns,

dysgenesis of the corpus collosum and cerebellar hypoplasia.

• Congenital microcephaly is present at birth and is defined as having a head

circumference at least two standard deviations smaller than the mean for

gestational age, sex and ethnicity.

• The risk of congenital microcephaly is likely to be highest if women are

infected during the first trimester, and is estimated to be 1-15 in 100

pregnant women.

• Studies have demonstrated poor outcomes including microcephaly from

infections occurring in the second and third trimester. It is currently not

known whether intra-uterine transmission can occur in pregnant women

who have asymptomatic ZIKV infections.

Sub-Region Country / territory Suspected Zika Confirmed Zika Deaths among Zika cases

North America Mexico 0 314 0

Subtotal 0 314 0

Central America Belize 0 2 0

Costa Rica 2,090 58 0

El Salvador 11,631 46 0

Guatemala 1,089 1,162 0

Honduras 21,025 44 2

Nicaragua 0 207 0

Panamá 638 274 0

Subtotal 36,473 1,793 2

Latin Caribbean Cuba 0 1 0

Dominican Republic 2,370 73 0

French Guiana 6,700 483 0

Guadeloupe 6,320 379 0

Haiti 1,777 5 0

Martinique 26,650 12 0

Puerto Rico 10,535 1,170 1

Saint Martin 425 109 0

Subtotal 54,777 2,232 1

Andean Bolivia 99 11 0

Colombia 80,953 6,402 0

Ecuador 393 143 0

Peru 0 5 0

Venezuela 31,224 352 0

Subtotal 112,669 6,913 0

South Cone Argentina 1,613 19 0

Brazil 154,270 39,993 3

Paraguay 273 8 0

Subtotal 156,156 40,020 3

Non Latin

Caribbean

Aruba 0 17 0

Barbados 316 7 0

Bonaire 0 3 0

Curacao 0 73 0

Dominica 203 28 0

Grenada 0 1 0

Guyana 0 6 0

Jamaica 646 14 0

Saint Barthelemy 19 7 0

Saint Lucia 0 2 0

Saint Vincent and the Grenadines 0 2 0

Sint Maarten 0 7 0

Suriname 2,503 527 4

Trinidad and Tobago 0 16 0

United States Virgin Islands 228 21 0

Subtotal 3,915 731 4

Total 363,990 52,003 10

Cumulative Zika suspected and confirmed cases reported by countries and territories in the Americas, 2015-2016

Updated as of 2 June 2016

Countries and territories in the Americas with GBS in the context of Zika virus circulation. (WHO, June 2, 2016)

.

Countries and territories in the Americas reporting confirmed and suspected cases of Zika virus disease in pregnant women (WHO, June 2, 2016)

.

Countries and territories in the Americas with reported congenital syndrome associated with Zika virus infection (WHO, June 2, 2016)

.

Number of investigated cases of microcephaly and other congenital malformation of the CNS in Brazil by epidemiological week, EW 3 – EW 21 of 2016

Source: Data published by the Brazil Ministry of Health and reproduced by PAHO/WHO

Asymptomatic

Infections

1,600,000

West Nile Fever

350,000

Central nervous system

13,204

West Nile Clinical Manifestations (1999-2011)

“The Iceberg”

Death

1,352 0.1%

<1%

20%

80%

Asymptomatic

Infections

?????

Zika Fever

?????

Congenital Zika Syndrome

????

Zika Clinical Manifestations (2015-2016)

“The Iceberg”

Death

10 0.0%?

1-15%

20%

80%

Guillain-Barre syndrome 1%?

Viremia (active virus multiplication)

“FACTS”

• Viremia – low and transient in humans and NHPs (104 pfu/ml; 107

genome equivalents/ml)

• Sometimes can detect viremia by RT-PCR but cannot recover

infectious virus from serum. What does this mean?

• Hard to isolate virus from serum

• Sometimes no isolates from outbreak (Yap) or one (French Polynesia)

QUESTIONS:

• Human - mosquito - human transmission cycle? (like dengue)

• Humans are dead-end hosts? (like West Nile)

• Non-mosquito-transmission important?

• What are we missing?

• Will ZIKV be like “dengue” or “West Nile”?

• What do we do if ZIKV activity disappears in Summer 2016?

Complexities of evaluating flavivirus immune responses

• Flavivirus serology is a “minefield”. Hard to

serologically identify an infection as due to a

particular flavivirus unless the individual is

flavivirus-naïve.

• Karl Johnson called flaviviruses the “Hall of

Mirrors”

• Challenging to assess and interpret

immunological data due to cross-reactivity.

Zika cross-reactivity – flavivirus naive

Lanciotti et al., 2008

Lanciotti et al., 2008

Zika cross-reactivity – Zika as a secondary flavivirus infection

Yap Island results and flavivirus vaccines

• The neutralizing antibody titer may be higher against a previous

(primary?) flavivirus infection rather than the most recent

heterologous flavivirus (secondary?) infection.

• Individuals who have been previously vaccinated against yellow

fever, Japanese encephalitis, (candidate) dengue, or tick-borne

encephalitis can have a rapid and high neutralizing antibody titer

following infection by a heterologous flavivirus. Usually, seen with

mosquito-borne flavivirus infections (JE, YF and DEN vaccine).

• Will be complex interpreting immune responses following

immunization with candidate ZIKV vaccines in areas where other

flaviviruses co-circulate.

Candidate ZIKV vaccines

Candidate ZIKV vaccine candidates

• Estimated to be approximately 40 candidates. Not all in public

domain.

• All either in discovery or preclinical.

• “Advanced” candidates in mice and NHPs for immunogenicity.

• Phase I in Fall 2016?

Candidate ZIKV vaccines

Live attenuated (4): Recombinant ChimeriVax™ – Sanofi Pasteur;

Recombinant dengue-2 – Instituto Butantan/US NIH; Recombinant

chimeric 17DD – Bio-Manguinhos/Fiocruz; Recombinant ZIKV

infectious clone – UTMB/Evandro Chagas Institute/Brazil Ministry

of Health.

Formalin inactivated purified whole virus (5): Bharat Biotech;

ChimeriVax – Sanofi Pasteur; Bio-Manguinhos/Fiocruz; NewLink

Genetics; Instituto Butantan/ US NIH

Live vectored (4): Measles virus vector - Themis Bioscience/Institut

Pasteur; Lentivirus-vector – Institut Pasteur; MVA-VLP – GeoVax;

Simian Adenovirus – Jenner Institute

Candidate ZIKV vaccines

DNA/RNA (4): DNA – Bio-Manguinhos/Fiocruz; DNA plasmid

expressing VLP – US CDC; GLS-5700 DNA – Inovio/GeneOne

Life Science; DNA –prM/E – US NIH

Subunit E protein (5): Recombinant N-terminal 80% E plus Alhydrogel

or proprietary adjuvant from collaborator – Hawaii Biotech;

Synthetic Replikins peptides – Replikins; Recombinant E protein –

Protein Sciences/Sinergium Biotech/Mundo Sano; Recombinant

flagellin/E protein – VaxInnate; E protein/nanoparticles –Novavax

VLP-based (3): VLP - Bharat Biotech; VLP – Bio-

Manguinhos/Fiocruz; VLP - PaxVax

Animal models

Mice deficient in either interferon αβ (A129) and αβγ (AG129) receptors

• ZIKV not lethal (only viremia) in immunocompetent mice.

• Number of mouse strain/virus strain combinations where

ZIKV strains cause 100% lethal infections.

• A129 mouse model: MP1751 (Uganda, mosquito, 1962;

East African lineage), 106 pfu, intraperitoneal route in 6

week old mice.

• AG129 mouse model: 259249 (Panama, 2015; Asian

Lineage), 104 pfu, intraperitoneal route in 12 week old

mice.

Mouse pregnancy models

• Miner et al. Zika Virus Infection during Pregnancy in Mice

Causes Placental Damage and Fetal Demise. Cell

• Cugola et al. The Brazilian Zika virus strain causes birth

defects in experimental models. Nature.

• Li et al. Zika virus disrupts neural progenitor development and

leads to microcephaly in mice. Cell Stem Cell.

Models use immunocompetent mice.

SJL looks best mouse model?

ZIKV in NHPs

• Indian-origin rhesus macaques infected with French Polynesia Asian

lineage Zika virus were rechallenged with 104 PFU of the same virus

In all three animals, there is no evidence of plasma viremia at any

timepoint following rechallenge. Similar data with Uganda MR766

strain. (Wisconsin National Primate Research Center)

Human Challenge Model

• Interest in developing human challenge models as has

been done for dengue.

• Alternative/supplement traditional clinical trials.

• Ethical and safety considerations?

Correlates of Protection for flaviviruses How do we define protection?

Neutralizing antibody – surrogate for licensed flavivirus

vaccines

Lack of clinical disease

Reduced/lack of viremia

Memory? Important for live vaccines…. YF 17D

vaccine appears to give life-long protective immunity

after one dose. Inactivated vaccines need boosters

every 3-5 years.

Surrogate of protection for licensed flavivirus vaccines Flavivirus Live, subunit or

inactivated? Serotypes Test Quantity

Japanese encephalitis

Live and inactivated

1 PRNT/neutralization 1 in 10

Yellow fever Live 1 Log neutralization index PRNT/neutralization

0.7+ 1 in 10-50*

???

Tick-borne encephalitis

Inactivated 1? PRNT/neutralization 1 in 10

dengue Live 4 PRNT/neutralization? ?????

Zika ???? 1? PRNT/neutralization? ????

+ The level of antibody considered to be protective was an log10 neutralization index of 0.7

originally based on studies in nonhuman primates

* Seroprotective levels of neutralizing antibodies, measured by PRNT, have not been determined

Passive protection in animal models

Cell Host & Microbe 2016 19, 696-704DOI: (10.1016/j.chom.2016.04.013)

Dai et al. Cell Host & Microbe 2016 19, 696-704

DOI: (10.1016/j.chom.2016.04.013)

Passive protection of mice demonstrated

with mouse Fab

Flavivirus “serotypes”

• Studies to date indicate each mosquito-borne flavivirus species

has multiple genotypes, but only one serotype…. Except maybe

dengue?

• Yellow fever: 7 genotypes; one serotype

• Japanese encephalitis: 5 genotypes; one serotype

• Zika: 3 genotypes?; One serotype?

• Dengue: “Four serologically and genetically related viruses”; 4

serotypes? 4-6 genotypes per “serotype”.

Is ZIKV one serotype?

Amino Acid ML Tree: E Protein

Can we infer from other flaviviruses?

• Yes… but some caveats?

• Monovalent vaccine

• Induction of neutralizing antibodies could be potentially a surrogate

of protection…. But how would we measure a Zika-specific

neutralizing antibody response in vaccinees?

• Passive protection studies has been used in licensure of JE, TBE

and YF vaccines… what about Zika?

How do you do a flavivirus neutralization test? • De ja vue!

• Cell type, virus strain, and assay type will influence results.

• Plaque reduction neutralization test (PRNT)

PRNT50?

PRNT60?

PRNT80?

PRNT90?

• Micro-neut

• Flow cytometry

• A standardized, validated assay will be important to quantitate

neutralizing antibodies

What would be needed to develop a surrogate marker of protection

• Standards: Antibodies and validated assay to

measure marker of protection (NIBSC).

• Standards: qRT-PCR to measure viremia in

validated assay (PEI)

Use Zika vaccine development as the “blueprint” for “Vaccine development pathway” for emerging diseases?