# Presentation: 179637

## Site Visit 2007

**Laboratory of Hepatitis and Related Emerging Agents (LHREA)**
- Office of Blood Research and Review (OBRR)
- CBER - FDA

## Mission

- Perform research to advance scientific knowledge and support our mission.

## Current Structure of LHREA

**KAPLAN LAB**

**TAYLOR LAB**

**LHREA**

## Site Visit Presentations

- The Hepatitis A Virus Cellular Receptor 1 (HAVCR1): Role in Viral Cell Entry and Immune Responses by Gerardo Kaplan
-  Determinants of HAV Growth and Development of Ebola Virus reagents for Diagnosis and Vaccine Evaluation by Krishnamurthy Konduru
-   Hepatitis B Virus in Blood Safety and Development of Microarray Multiplex Assay by Chu Chieh Hsia
-  *In vitro* and *in vivo* systems for the study of SARS and HCV           by Deborah Taylor
-  Persistence of serum-derived HCV in interferon-deficient cells        by Erica Silberstein

## The Hepatitis A Virus Cellular Receptor 1 

- The Hepatitis A Virus Cellular Receptor 1
- (HAVCR1):
-  Role in Viral Entry and  Immune Responses
- Gerardo Kaplan
- CBER - FDA

**Notes:**
-My lab has worked for some time in understanding the cell entry mechanism of HAV.
It has become apparent in the last few years that HAV may modulate immune responses.
-The molecular link of this odd associations between a liver infection and the modulation of the immune  response  is via HAVCR1, which belongs to a family of receptors expressed in T cells

## Schematic representation of TIM family members

- Mouse

**Notes:**
MOLECULAR EVIDENCE TO THA HYGENE HYPOTHESIS!!!!!!
In this region, they positionally cloned a gene family that encodes T cells immunoglobulin and mucin domain proteins (tim 1 to Tim8). From these genes, they found major polymorphisms in Tim1 an Tim3. These polimorphisms were strongly related to to Th1 –Th2
Tim 1 is transcribed early during primary antigen stimulation, a moment crucial in T cell differentiation
Remarkable amount of sequence polymorphismin the coding region of Tim1 in mice, money and human suggests its relation to asthma
Tim 1 was also cloned as a kidney injure molecule1 (KIM-1) in rat and humans, as an adhesion molecule that was upregulated in kidney injury.
Tim1 is highly polymorphic as well as Tim3. There had not been polymorphisms identified in TIm2 and very low in Tim4Tim 1 is a critical co stimulatory molecule on CD4 T cells that regulates Th1/Th2 cell diferenciation, tolerance and AHR. Mouse Tim2 is upreulated in TH2 cells , may be involved in pheripheral tolerance providing an inhibitory signal to downregulate Th2 responses during autoimmune immflamation.Two different ligands have been siolated for Tim2: semaforin 4A and H ferritin. Mouse Tim3 is expressed in differenciated Th1 cells and mediates allo and auto immune responses . Galectin 9 has been identified as a ligand for Tim2 and their interaction suppresses TH1 responses. Mouse Tim4, that is expressed inAPCs and mature  DCs binds to Tim1 and delivers a positive signal that co stimulate T cell proliferation.  and  has been identified as a al
Both Tim1 and Tim3 are expressed in Th2 upon activation cells and Tim2 is expressed in Th1 cells. Tim4 is expressed in APCs
Tim 1 is expressed after T cell activation on T cells that differentiate to TH2. Tim1 acts as a co stimulator on CD4+ cells enhancing T cell proliferation and TH2 cytokine production
TIM1 in mice: spleen, lung and thymus (poco)
 In humans, Tim1 has been associated with remission of patiens with multiple sclerosis.
Tim 1 is expressed in activated CD4 cells (activated with anti CD3 and anti CD28). Anti tim1 increase proliferation of T cells in vivo and in vitro by sending a positive signal. T cells + OVA + anti Tim1  is higher than without Tim1
Tim1 coestimulation activatesT cells in vitro and in vivo. TIM1 co estimulation is important in T cell activation and leads to the prevention of T cell tolerance  and in the developing of AHR
TH2 polarized + anti TIM1: increment in proliferation.
TH1 Polarized: No increased. 
Inoculation of anti TIM1 in mice tolerized with OVA by various exposures to OVA was inhibited by inoculation of anti Tim1. These mice developed AHR
All this suggests a role of hHAVcr in immune regulation

Sequence identity among the N-terminal Cys-rich region of TIM homologs is about 40%,
while between the mouse and human orthologs is around 60%. There are however marked
differences in the length of the threonine, serine and proline rich mucin domain, with the
number of O-linked glycosylation sites ranging from 43 in Tim-4 to 1 in TIM-3
The TIM gene family is involved in the regulation of immune responses. Tim-1 is
preferentially expressed in TH2 cells and delivers a signal that enhances T-cell activation
and proliferation, increasing airway inflammation and allergy
In contrast, Tim-2 is an inhibitory molecule of TH2 immune responses
Tim-3 is mainly expressed in TH1 cells and provides a negative costimulatory signal that leads to immune tolerance. Polymorphisms in Tim-1 and Tim-3 confer susceptibility to the development of asthma and allergy. Different ligands have been described for the murine members of the TIM family.
Tim-4 expressed in antigen-presenting cells binds to Tim-1 and triggers T cell proliferation,
preventing development of antigen tolerance (Meyers et al., 2005). Similar phenotype was
observed by crosslinking of Tim-1 receptor molecules with the 3B3 mAb, which activated
T cells and prevented the induction of respiratory tolerance (Umetsu et al., 2005). Two
ligands have been shown to bind specifically to Tim-2, Semaphorin 4A and H-ferritin
(Chen et al., 2005; Kumanogoh et al., 2002). There is less information about the functions
of the human TIM receptors. The HAVCR1 gene that codes for TIM-1 was the first
member of the TIM family identified initially in monkeys as the hepatitis A virus (HAV) HAVCR1 is an important asthma determinant gene in humans (McIntire et al., 2003) and its expression is upregulated in acute kidney diseases and renal
Carcinoma.The N-terminal Cys-rich domain is critical for binding of the TIM receptors to their ligands . The mucin domain is
required for binding of Tim-1 to Tim-4 (Meyers et al., 2005) and for the neutralization of
HAV particles by TIM-1 (Silberstein et al., 2003).

By sequencing Human TIM1 from cDNA from lymphocytes, a 6aa insertion was identified (in the centre of the mucin like region). To study the efffect of the insertion with atoied 375 individuals. They found the HAV protects against atopy only in individuals that have the long form of the receptor (63% of caucasian, 65% of african american and 46% in Asian in the analyzed population). Therefore, the protective effect of HAV depends on the allelic form of the receptor. 
 It is suggested tha polymorphisms in TIM1 alter the susceptibility to asthma.

## Cloning of Perro6D cells expressing an HAVCR1 ligand

- Identification of IgA as a natural ligand of HAVCR1

**Notes:**
Perro6D cells were transfected with a human lymph node cDNA library cloned in pEAK8, an episomal shuttle vector containing an EBV origin of replication and puromycin selectable marker, using conditions that allowed transfection of 1-10 plasmids per cell.   Cell transfectants were selected with 2 μg/ml puromycin, and panned to Petri dishes coated with HAVCR1-Ig.    To enrich for cells that bound specifically to HAVCR1 and not to the Fc tail, transfectants were treated with excess PVR-Fc, which contain the same Fc tail than HAVCR1-Ig, and substracted with mAb to human CD32 bound to paramagnetic beads. After this enrichment process, 20 single-cell clones were isolated and further analyzed for the expression of HAVCR1-L (Fig. 1b). To rule out the possibility of a matrix effect, we tested the single-cell clones by panning to coated Petri dishes and by binding of color latex beads coated with soluble receptors.  Both binding assays gave similar results. 
Ten single-cell clones expressed CD32 and bound HAVCR1-Ig and PVR-Fc (Clone H7 is an example of such cell clones), nine clones neither express CD32 nor bound soluble receptors (Clone D1 is an example of such cell clones), and one clone, termed Clone 1, bound to HAVCR1-Ig but not to PVR-Fc and did not express CD32. These data confirmed that clone 1 expressed an HAVCR1-L at the cell surface. 

## IgA enhances neutralization of HAV 

- by soluble forms of HAVCR1

##  Monocytes activated with HAVCR1 induce

- T cell proliferation

**Notes:**
To evaluate the effector function of HAVCR1-Ig-activated monocytes in T cell proliferation, we performed a mixed lymphocyte reaction (MLR).   Monocytes untreated or treated with HAVCR1-Ig or PVR-Fc control were cultured with purified heterologous T cells from normal human blood donors (Fig. 5). T cells cultured with monocytes activated with HAVCR1-Ig Incorporated approximately 16-fold more [3H]thymidine than those cultured with untreated or PVR-Fc-treated monocytes (Fig. 5a).   As expected, T cells incubated without monocytes incorporated minimal levels of radioactivity.  T cells treated with PHA as a positive control incorporated almost twice the amount of [3H]thymidine than T cells cultured with HAVCR1-Ig activated monocytes (data not shown).   T cell proliferation was also visualized by microscopic examination of the MLR cell cultures previous to the addition of [3H]thymidine.  A increased number of T cell clusters of larger  size was observed in wells containing monocytes treated with HAVCR1-Ig (Fig. 5 b) compared to few small clusters in PVR-Fc-treated (Fig. 5c) and untreated monocytes (Fig. 5d).  These data clearly indicated that the monocytes activated with HAVCR1-Ig were capable of inducing T cell proliferation.

## TIM4 and HAVCR1 are Phosphatidylserine Receptors:

- Role of TIM family members in apoptosis

##                      SUMMARY

-  IgA is a ligand of HAVCR1
- The interaction of HAVCR1 with IgA has a synergistic
-   effect that enhances its HAV-receptor function
-  The activation of antigen presenting cells by HAVCR1
-   may modulate TH2 responses involved in the development
-   of allergy
-   HAVCR1 forms homophilic interaction in *trans*
-  HAVCR1 and TIM4 are PS receptors and may regulate
-   immune responses by uptake of apoptotic cells.

##  Development of Ebola virus GP reagents 

- Visiting Associate
- LHREA, DETTD, OBRR, CBER

**Determinants of HAV growth and pathogenesis**

** ****Development of Ebola virus GP reagents **
** ****for diagnosis and vaccine evaluation**

- and

## Slide 13

## Selection of hepatoma cells that support wt HAV growth

## Development of  full-length HAV vectors containing the  blasticidin selectable marker. 

Isolation of Huh7-A-I cells that support the stable growth of wt HAV in cell culture. 

Selection fast-replicating HAV variants to study determinants of HAV growth.

- Isolation of Huh7-A-I cells that support the stable growth of wt HAV in cell culture.
- Selection fast-replicating HAV variants to study determinants of HAV growth.

- HAV Summary

##  Development of Ebola virus GP reagents 

** ****for diagnosis and vaccine evaluation**

-   EBOV cause hemorrhagic fever with morbidity and mortality
-     rates up to 90%.
-   Category A bioterrorism agent.
-   GP plays a major role in virus cell entry and pathogenesis.
-   There is no licensed vaccine or effective treatment.

## Expression of EBOV GP membrane-bound and soluble forms

- Cell-surface ELISA

## Production of retrovirus pseudotypes carrying the EBOV GP.

Purification of membrane-bound and soluble forms of EBOV GP.

Development a Panel of mAbs to study protective epitopes on GP for vaccine evaluation.  

Use mAbs and proteins to develop point-of-care diagnostic kits for EBOV.  

- Purification of membrane-bound and soluble forms of EBOV GP.
- Development a Panel of mAbs to study protective epitopes on GP for vaccine evaluation.
- Use mAbs and proteins to develop point-of-care diagnostic kits for EBOV.

- EBOV Summary

## Hepatitis B Virus in Blood Safety

**Hepatitis B Virus**** ****in Blood Safety**
**               **** **
**                               ****Chu Chieh Hsia, M.D.**
**     **
**                       ****LHREA, DETTD, OBRR, CBER **

## Slide 20

## Slide 21

## Slide 22

## in vitro and in vivo Systems

***in vitro***** and *****in vivo***** Systems**
** **
**for the Study of SARS and HCV**

##              SARS

- Develop animal model that mimics human disease
- Pathogenesis of SARS-CoV  (PRF)

**             ****SARS**

## Inactivation of SARS-CoV by Solvent/Detergent

**Inactivation of SARS-CoV by Solvent/Detergent**

**0**

**1**

**2**

**3**

**4**

**5 min.**

**6 hours**

**24 hours**

**TCID****50 ****(log****10****)**

**Virus in media**

**Virus in media with S/D**

**Virus in serum**

**Virus in serum with S/D**

**S/D as per NYC Blood Center**
- TNBP + Tween80
- 1:10 dilution

## SARS Vaccine and Virus Neutralization

**vaccinated, pre-challenge**

- 0

- 20

- 40

- 60

- 80

- 100

- 120

- 140

- 160

- 180

- 0

- 7

- 14

- 21

- 29

- 35

- 43

- 48

**Day of bleed**

**Neutralization titer**

** ****post-challenge**

- 0

- 1000

- 2000

- 3000

- 4000

- 5000

- 48

- 50

- 55

- 62

- 71

**Day of bleed**

**Neutralization titer**

***vaccine***

***boost***

***challenge***

***challenge***

**Darnell et al., 2007**
***Journal of Infectious Diseases***

**SARS Vaccine and Virus Neutralization**

## Ferrets: Summary

**No evidence of Antibody-Dependent Enhancement of disease**
**No evidence of disease symptoms such as coughing, sneezing**
**Some elevated temperature and ALT**
**Lung lesions similar to human pathology**
**First publication showing lung histopathology after SARS infection**

## Hepatitis C Virus

- Huh 7.5 infected with HCV 2a

- Family: *Flaviviridae*
- Genus: Hepacivirus
- Enveloped
- Single-strand Positive RNA genome of 9.6 kb

**Hepatitis C Virus**

## {

**Cytoplasm**

**Nucleus**

**Mx**
**MHC**
**2’, 5’OAS**
**PKR**

**ISGs**

** ****ISRE**

**IFN********/******

**JAK/Tyk**

**p**

**STAT1**

**STAT2**

**ADAR1**

**p**

**Interferon-induced Antiviral Signaling **

## siRNA Knock-down of ADAR1 Stimulates the Replicon

- 41±12.7

**Relative HCV RNA**

- -

- +

- 0

- 5

- 10

- 40

- -

- +

- ADAR2

- Actin

- ADAR1

- 30

- 20

- 50

**siRNA**

## Persistence of Serum-derived 

**Hepatitis C virus **
**in Interferon-deficient Cells**

**Erica Silberstein, Ph.D.**
**LHREA Site Visit**
**November 8, 2007**

## VA RNAI

**Adenovirus **_**V**_**irus– **_**a**_**ssociated RNA**
**160 nts**
**Late expression,  RNA Pol III**
**VA RNA****I**** and VA RNA****II****, 40:1**
**Nuclear and cytoplasmic**
**Stimulates translation**
**Binds and inhibits PKR and ADAR1**

## Growth of J6/JFH-1 is stimulated by VA RNAI

**Growth of J6/JFH-1 is stimulated by ****VA RNA****I**

**- VA RNA****I**

**+ VA RNA****I**

## Culture supernatants from persistently-infected Vero cells induce CPE in Huh 7.5 cells 

**HCV infected cells**

- Crystal violet staining

**Mock**

## Summary

**Serum-derived wild-type HCV replicates in hepatic and**
**     ****nonhepatic cells if VA RNA****I**** is present**

**HCV genotypes 1a, 1b and 2a replicate in Vero cells**

**HAV replicates in Vero cells with VA RNA****I**

**Vero cells can support growth of a CPE virus**

**VA RNA****I**** can help establish persistent infection of HCV**

## Summary LHREA 2007 Site Visit Presentations 

-  LHREA developed mission-oriented research
-   programs in HAV, HBV, and HCV.
-  Additional research programs in Filovirus and
-   SARS-Co were developed in support of
-   Counterterrorism and Emerging Infectious
-   Diseases initiatives at CBER.
-  Research programs are basic and applied, and
-   support the regulatory mission of LHREA.

## Slide 37