Download New Insights into Zika virus pathogenesis

New Insights into Zika virus pathogenesis
Michael S. Diamond, M.D., Ph.D.
Washington University School of Medicine
Departments of Medicine, Molecular Microbiology, Pathology & Immunology
Center for Human Immunology and Immunotherapy Programs
Wageningen University
November 17, 2016
Talk Outline
Zika Virus
- Epidemiology/Virology/History
- Animal model development/biology
- The male reproductive tract
- Antibody-mediated protection
Acknowledgements: Jonathan Miner, Jen Govero
Estefania Fernandez
Zika virus is a Flavivirus
5’Cap
C
prM
E
NS1
NS3
NS2a
NS2b
NS5
NS4a
NS4b
§ Flaviviridae; Flavivirus, (Hepacivirus)
§ Enveloped virus
§ (+) ssRNA genome (~10.7kb), single ORF
§ Mosquito/tick-transmitted
§ Encephalitis, shock syndrome, liver failure
§ Microcephaly, Guillain-Barre syndrome
Mukhopadhyay etal.,
Science,2003.
West Nile virus (WNV)
Japanese encephalitis virus
Dengue virus
Zika virus
Yellow fever virus
Tick-borne encephalitis virus
Dengue: 390 million infections
Zika-induced microcephaly
>50,000 WNV cases
since 1999
Zika virus relationship to other Flaviviruses
One Serotype
Asian
American
African
Ferguson et al. Science 2016
3-5% difference at amino acid level
Historical Spread of the Zika Virus
Lessler et al. Science 2016
Zika Virus, 2015-2016
Zika virus: historical syndrome
Aedes
aegypti
Disease: fever, rash, muscle ache, headache, conjunctivitis:
Lessler et al. Science 2016
Zika virus: An emerging flavivirus with new syndromes
and unique transmission
• In utero, sexual, and blood transmission in
addition to mosquito transmission
• Guillain-Barré syndrome
• Microcephaly, congenital malformations
• Meningitis/encephalitis
• Ocular damage in newborns
• Viral persistence: semen, urine, saliva, tears
Four hypotheses as to why Zika virus
causes new clinical syndromes
• Zika virus has evolved at the sequence level
New strain
(African versus Asian)
D in pathogenicity in humans
D in mosquito transmission
• Different host populations: unique human genetics
New people
• Pre-existing immunity to a related flavivirus
predisposes to more severe Zika virus infection
New immune
background
Few animal studies of Zika virus before 2016
1947
Rhesus macaques
Zika Forest
T = 40oC
Blood
Sample
DAY 3 of fever
Normal
Normal
Normal
Normal
Normal
Inject blood
Into brain
All mice get sick
at day 10
Dick, 1952 Trans Roy Soc
1976
Virus found in
brain/spinal cord only
ZIKV MR 766
Infected newborn mice with ZIKV
Infected 4 week-old mice with ZIKV
Way 1976 J Gen Virol
Mice died: no description
Creating a mouse model of ZIKV infection
Helen Lazear, Derek Platt, Matt Gorman
Strains: MR 766 (Uganda 1947)
H/PF/2013 (French Polynesia 2013)
Brazil 2015 (Fortaleza)
Senegal 1982
Zika virus can cause lethal disease in adult mice
lacking type I Interferon immunity (but not in WT mice)
Ifnar1-/- and Irf3-/- Irf5-/- Irf7-/- C57BL/6 mice
Strains: MR 766 (Uganda 1947)
H/PF/2013 (French Polynesia 2013)
Brazil 2015 (Fortaleza)
Senegal 1982
Causes paralysis and encephalitis
Lack of antagonism of mouse IFN signaling?
Lazear Cell Host Microbe 2016
Zika virus infects many tissues in IFNAR-/- mice
with highest levels seen in CNS, kidneys, and testes
Day 6 after ZIKV infection
108
ZIKV FFU equivalents per g or ml
ZIKV FFU equivalents per g or ml
Day 2 after ZIKV infection
107
106
105
104
103
102
101
100
10-1
Serum
Liver
Kidney
Spleen
Testes
Wild type
IFNAR-/-
Brain
Spinal
Cord
108
107
106
105
104
103
102
101
100
10-1
Serum
Liver
Kidney
Spleen
Testes
Brain
Spinal
Cord
Wild type
IFNAR-/-
ZIKV (French Polynesia 2013) can infect testes. Associated with sexual transmission
High levels in kidneys – associated with persistent virus in urine
Lazear Cell Host Microbe 2016
Using IFN immunity to create a model of in utero
transmission of Zika virus infection
Pregnant mice
Infect with Zika virus
Different days
E6, E7, E10
IFNARKO
WT
WT
WT
Analyze
newborn or fetal
IFNAR+/- mice
(young WT mice susceptible)
Blocking IFNAR MAb
Readouts: survival, pathology, virology, immune infiltrates, mother/fetus/neonate
Zika virus infection of pregnant dams
results in intrauterine growth retardation
Infection during pregnancy
ZIKV infection leads to placental
Insufficiency and smaller fetuses
Uninfected
ZIKV
Zika virus infection of pregnant dams
results in placental infection
100-1,000-fold more virus in placenta than in maternal serum
Miner Cell 2016
Trophoblasts
Zika virus infects trophoblasts
and endothelial cells on
fetal side of the barrier
Hofbaeur
Placental
macrophages
Fetal endothelial cells
Miner Cell 2016
Quicke CHM 2016
Infection in fetal brain
(targets NPCs)
Increased cell death in brains
of ZIKV-infected fetuses
Infection in skin
Viremia
Infection of placenta
- Trophoblast
- Fetal endothelial cells
- Hofbauer macrophages
Fetal Viremia
Fetal Brain (NPCs)
- Demise
- Microcephaly
- Congenital Malformations
Coyne, Nat Rev Micro, 2016
Using mouse models to study other clinical
manifestations of Zika virus infection
• Long term consequences at sites of persistence (brain, eyes, kidneys, testes)
ZIKV infects cells of the eye
and can accumulate in tears
Miner Cell Reports 2016
ZIKV infects cells of the eye
and cause uveitis
Miner Cell Reports 2016
Consequences of Zika virus infection in the testis
• ZIKV is sexually-transmitted in humans (e.g., male to female and male to male)
• ZIKV RNA has been detected in semen for 139 days in humans
• ZIKV antigen has been detected in human sperm
• ZIKV persists in testes of mice for months: Testis are an immune-privileged site
Mansuy et al
Lancet Inf Dis
2016
Consequences of Zika virus infection in the testis
ZIKV (subQ)
anti-Ifnar1
(one dose)
Day 7
Male
Day 14
Analyses:
Viral yield – testis, epididymis, sperm
Testis size
Histology
Immunohistochemistry
In situ hybridization for viral RNA
Sperm count
Sex hormones
Fertility
Day 21
Day 35
Testis histological architecture
Leydig cells in interstitium
Blood-testis-barrier
Formed by TJ made
by Sertoli cells
Separate sperm from immune system
Progenitor spermatogonia are before
the BTB
Prevents Abs and immune cells from
getting access to germ cells
(prevents autoimmunity against
sperm antigens)
Day 7: Zika virus in the male reproductive tract
Govero et al, Nature,
2016
Day 7: Zika virus in the male reproductive tract
H&E
IHC
Little effect
Little effect
except CD45+
cells in interstitium
Day 7: Zika virus in the male reproductive tract
In situ hybridization for ZIKV RNA
ZIKV RNA present in
a) Spermatogoinia
b) Sertoli cells
Govero et al, 2016
Day 21: Zika virus in the male reproductive tract
Persistent
infection
H&E
Damage to
Seminiferous tubules
Day 21: Zika virus in the male reproductive tract
Destruction of the
Blood-testis-barrier (BTB)
ZIKV RNA
in spermatocytes
Govero et al, Nature,
2016
Zika virus induced damage to the testis is both
viral and adaptive immune mediated
Partial damage
Loss of BTB
Preservation of interstitium
Consequences of Zika virus infection in the testis
Zika virus infection in the testis of mice
results in:
•
•
•
Decreased sex hormone levels
Decreased sperm counts
Decreased rates of fertility
Govero et al, Nature 2016
Consequences of Zika virus infection in the testis
No reversal of pathology at ~day 42
Persistence in male
reproductive tract
Summary
• ZIKV infects spermatogonia (stem cells), spermatocytes, and Sertoli cells
• Other flaviviruses (e.g., Dengue) do not do this
• Infection results in breakdown of the BTB and damage to seminferous tubules
• Immune cells may contribute to disease pathogenesis
• Damage appears irreversible in mice
• Functional consequences: hormones, sperm counts, fertility
Key question: Does this happen during human persistence? At what frequency?
ZIKV interventions
• Vaccines
• Repurposing of FDA drugs
• Antibody therapy
• Mosquito control measures
Estefania Fernandez, Derek Platt, Matt Gorman
Haiyan Zhao (Daved Fremont), Gopal Sapparapu (James Crowe)
Michael Rossmann
Developing protective antibodies
New Human anti-ZIKV mAbs
Zika virus infection
Test binding
to Zika virus
E protein
and virus
Human mAbs
Mouse mAbs
Zika virus structure
E protein
dimer
Sirohi Science 2016
Neutralizing mouse mAbs bind different
regions of E-DIII of Zika virus
Zhao, Cell 2016
ZIKV type-specific mouse mAbs that bind DIII
lateral ridge epitope are protective in adult mice
Zhao, Cell 2016
ZIKV + Hu ZIKV-117
Strongly neutralizing human antibody
ZIKV-117
cross-links the viral E proteins
10 ⍲
Alanine-scanning mapping
Sapparapu, Nature 2016
Binds across E dimer-dimer
Strongly neutralizing
Broadly neutralizing
Strongly neutralizing
human antibody
ZIKV-117 protects
against fetal and
maternal infection
in mice
Sapparapu, Nature in press
Human anti-ZIKV mAbs therapeutics can prevent
placental infection and disease in mice
PBS
CHK-152
ZIKV-117
Human anti-ZIKV mAbs therapeutics can
prevent placental disease in mice
Sapparapu, Nature 2016
Summary of our ZIKV studies
• Development of new mouse models of ZIKV pathogenesis
• Pregnancy and in utero transmission models
• In utero transmission shows trans-placental transmission
• Placental Trophoblasts are targeted by ZIKV
• Virus can spread to fetal brain and cause cell death
• ZIKV persistence in testis is associated with significant damage in mice
• Loss of architecture of seminiferous tubules
• Infection of sperm and spermatocytes
• Decreased sex hormone production and sperm counts
• New panel of anti-ZIKV specific mouse and human mAbs
• Protective in vivo against lethal infection in adults
• Protection against in utero transmission and fetal disease
Acknowledgements
Diamond Lab (Zika)
Helen Lazear (UNC)
Jonathan Miner
Jen Govero
Estefania Fernandez
Derek Platt
Amber Smith
Julie Fox
Matt Gorman
Vanessa Salazar
Fremont Lab
Haiyan Zhao
Chris Nelson
Vanderbilt University
James Crowe Jr
Gopal Sapparapu
Purdue University
Michael Rossmann
Richard Kuhn
Syed Saif
Indira Mysorekar lab (Bin Cao)
Kelle Moley lab (Praba Esakky, Suzanne Scheaffer, Andrea Drury)
Funding: NIH