Presentation of Activities of New Visby Hepatitis C Network 2002-2011 Anders Widell,

Presentation of Activities of New Visby Hepatitis C Network

2002-2011

Anders Widell, MD, PhD, Ass Prof, Network Coordinator, Department of Medicine, Lund University, Sweden

Maria Isaguliants,PhD, Ass ProfMicrobiology and Tumor Biology Center, Karolinska Institutet

Year 2002: 2879/2002 (381/M20)

COORDINATOR Malmö University Hospital, Lund University

Swedish Institute for Infectious Disease Control, Karolinska Institutet

Academy of Medical Sciences, Ivanovsky Institute of Virology, Moscow

Institute of Cytology, St Petersburg

Biomedical Research and Study Center, Latvian University, Riga

Narvac R & D Company, Moscow

Swedish participants:Core HCV Network participants active

since 2002/2003 are shadowed in yellow

Participants from Baltic Region

6 teams; 31 researchers & students

http://www.si.se/templates/StartPage____3.aspx

How we started – a talk given by Network Coordinator Prof Anders Widell, Univesity of Lund, Malmö, on the 7th Annual meeting of the Network eight years later

Year 2003: NNNN

COORDINATOR

Malmö University Hospital, Lund

University



Institute of Cytology, St Petersburg

Biomedical Research and Study Center, Latvian University, Riga

Center for Molecular Medicine, Karolinska Institutet

Moscow State University of Medicine and Dentistry, Moscow


Swedish participants



Year 2005: 01789/2005

COORDINATOR


University



University of Tartu, Institute of Microbiology, Tartu, Estonia

Biomedical Research and Study Center, Latvian University, Riga, Latvia


Moscow State University of Medicine and Dentistry, Moscow, Russia




Belarusian State Medical University, Minsk, Belarus


Year 2006: 01551/2006

COORDINATOR


University












Year 2007: 01420/2007

COORDINATOR


University

Swedish Institute for Infectious Disease Control








8 teams; researchers & students 57; travelling 25



Year 2008: 01191/2008

COORDINATOR


University

Swedish Institute for Infectious Disease Control


National Institute of Health, Tallinn, Estonia





7 teams; 46 travelling researchers & students planned

Research Institute for Epidemiology and Microbiology, Minsk, Belarus


Year 2009: 00894/2009

COORDINATOR


University, Malmö

Swedish Institute for Infectious Disease Control, Stockholm

Academy of Medical Sciences, Ivanovsky Institute of Virology, Moscow, Russia

National Institute of Health, Tallinn, Estonia



Participants from Baltic Region (6)

Swedish participants (2)

7 teams; 46 travelling researchers & students planned

Department of Internal Medicine, University of Tartu, Tartu, EstoniaInstitute of Cytology,

St Petersburg, Russia


Year 2010: 00747/2010

COORDINATOR


University, Malmö

Department of Microbiology, Tumor

and Cell Biology, Karolinska Institutet,

Stockholm

Academy of Medical Sciences,

Ivanovsky Institute of Virology,

Moscow, Russia

National Institute of Health,

Tallinn, Estonia

Biomedical Research and Study Center,

Latvian University,

Riga, Latvia

Moscow State University of Medicine and

Dentistry, Moscow, Russia

Participants from Baltic Region (7)

Swedish participants (2)

9 teams; mobility resources for 54 researchers &

students

Department of Internal Medicine,

University of Tartu, Tartu, EstoniaInstitute of Cytology,

St Petersburg, Russia

Center for Communicable Diseases and

AIDS, Vilnius, Lithuania


SUGGESTED INCOMMING PROJECT:

New Visby Network on Hepatitis C 2011-2012 (two years):

Identification, Control and Prevention of Hepatitis C Utilizing Novel Biomedical Technologies

Network Structure Applied for in 2011: SwedenKarolinska Institutet, MTC (Coordinator)Lund Universitet

Estonia Tartu University, TartuNational Institute for Health Development, Tallinn

LatviaBiomedical Research and Study Center, Riga;

LithuaniaCenter for Communicable Diseases and AIDS, Vilnius

Russia Academy of Medical Sciences, Moscow; State University of Dentistry and Medicine, Moscow; St Petersburg University, Biomedical Center, St PetersburgInstitute of Cytology, St Petersburg

New partner: Biomedical Center, St Petersburg University, St Petersburg, Russia

Selected joint publications of the teams2002-2010

Selected joint publications of the teams 2004


Euro Surveill. 2006 Jan 26;11(1):E060126.3.

Tefanova V, Tallo T, Kutsar K, Priimgi L.Urgent action needed to stop spread of hepatitis B and C in Estonian drug users.National Institute for Health Development, Estonia.









Selected posters from research meetings

2002-2010

IMMUNIZATION WITH HEPATITIS C VIRUS CORE GENE RESULTS IN THE LOSS OF T-CELLS

M Isaguliants1, 2, N Petrakova1, Yu Suzdaltzeva1, V Makhonov1, A Krivonos3, B Wahren2, and E Nordenfelt2

1Ivanovsky Institute of Virology, Russia; 2Swedish Institute for Infectious Disease Control, Sweden; 3Narvac R&D Company, Moscow; Russia

EXPERIMENTALcDNA encoding full-length core of HCV 1b isolate 274933RU was cloned under the control of CMVIE-promoter and HPV16 polyA signal. Eukaryotic expression was verified by Western blotting and IF staining (Fig. 1). Groups of 10 to 12 C57BL/6 (H-2b) mice were immunized by intramuscular injections of 50 g of HCV core gene, or empty vector, or water, four times at 2 week intervals. Mice (3 to 5) were sacrificed every 3 rd week, tissue samples and spleens were withdrawn. Splenocytes were isolated by gradient centrifugation. Percent of CD19 +, CD3+, CD4+, and CD8+ splenocytes was evaluated by flow cytometry. Proliferative responses of spenocytes were assayed by [ 3H]-incorporation test using recombinant core and core-derived peptides. Production of IFN-, IL-2 and IL-4 in response to T-cell stimulation was assessed by sandwich ELISA. Mouse tissue samples were fixed in 10% neutral-buffered formalin. Fixed tissue sections embedded in paraffin were cut into 4-6 m sections, mounted on glass slides and stained by the routine hematoxyline and eosin techniqu. Immunization and follow-up experiments were repeated three times.

BACKGROUND: Highly conserved core protein of HCV presents an attractive target for anti-HCV vaccine design. Numerous attempts to induce immunity against HCV core by genetic immunization met serious difficulties in optimizing core-specific T-helper cell and antibody responses (Vidalin et al., 1999). Immunomodulatory properties of HCV core could be blamed, since in mice it was shown to interfere with vaccinia-specific CTL responses (Large et al., 1999). No demonstrable effects of HCV core were, however, observed for adenovirus-specific immune responses (Liu et al., 2002).

RESULTS No pathological changes were found in muscle, skin, spleen, liver, lungs, heart, brain, or lymph nodes of pCMVcore191-recipients. Immune response was induced in the majority (24/32, 75%) of mice. Strong T-helper cell response to HCV core and core-derived peptides (SI 2.5 to 80) was induced after three pCMVcore191 injections (Fig. 2). Immunodominant T-helper epitope mapped to aa 76-90 was recognized by 70% of pCMVcore191-recipients (Fig. 2). HCV-core and core peptide-stimulated splenocytes of immunized mice responded by strong sustained production of IFN-moderate levels of IL-2 and infrequent low-level production of IL4 (Fig. 3). Antibody response against core and/or its linear epitopes was weak (titer ≤100) and infrequent (6/22, 27%).

RESULTS Surprisingly, after one month, CD3-positive-cell population in pCMVcore191-recipients started to decrease. One month after the last gene injection, the decrease amounted to 7-9% of CD3+-cells, more CD4 + than CD8+ (p<0.05; day 80,Fig. 4).

CONCLUSIONS•Transient expression of HCV core in the gene recipients did not exert any histopathological changes, despite multiple effects of HCV core on cell function (Zhu et al., 1998; Ray & Ray, 2001; Soguero et al., 2002) including the oncogenicity (Ray et al., 1996).

•HCV core gene immunization induced potent T-cell, but only weak B-cell reactivity.

•Repetitive injections of HCV core gene induce a significant (p<0.05) decrease in CD3-positive cell population. Depletion might be linked to the increased apoptosis sensitivity of core-expressing T-lymphocytes , while B-lymphocytes were shown to be unaffected (Giannini et al., 2002). T-cell deficit resulting from the immunomodulatory properties of HCV core can explain its specific immunogenic performance.

Fig. 1 Mouse fibroblasts NIH3T3 transiently transformed with pCMVcore191. Field A, nuclei and immunofluoresent staining (rabbit anti-HCV core rabbit polyclonal antibodies and FITC-conjugated anti-rabbit antibodies); field B, immunofluoresent staining; field C, phase contrast.

each time point is screened 10-12 days after plasmid injection

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Fig. 3 Cytokine secretion in proliferating splenocytes of pCMVcore191-immunized mice

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aa 141-160 aa 155-177 ConA

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CD3+, CD4+%, emptyvectorCD3+, CD4+%, HCV coregeneCD3+, CD8+%, emptyvectorCD3+, CD8+%, HCV coregene

Fig. 2 Proliferation of splenocytes of pCMVcore191-immunized mice in response to HCV core and core-derived peptides

Fig. 4 Dynamical changes in % of CD4+ and CD8+ T-cell populations in pCMVcore191- versus empty vector-immunized mice (each time-point is represented by five mice)

Acknowledgements: This work was financed by European Commision grants ERB IC CT98-0313, INTAS 96-1565.

DNA Vaccine Conference, Monaco, 2006

Immune imprints of hepatitis C identified in clinical cases of hepatitis of unknown origin

Elena Tsyganova1,2* ; Olga Znoiko1,2; Natalia Petrakova2; Tatjana

Petrova3,4; Igor Maljkov4; Maxim Vonsky5; Alexander Ivanov6,10; Birke

Bartosch7; Mikhail Mikhailov8, Nikolai Yushchuk1; Anders Widell9; and

Maria Isaguliants2,10 1 Moscow State University of Dentistry and Medicine, Moscow;

2. DI Ivanovsky Institute of Virology, Moscow;

3 Russian State Institute of Immunology, Moscow;

4. DNA Technology R&D Company, Moscow;

5. Institute of Cytology, St Petersburg;

6 WA Engelhardt Institute of Molecular Biology, Moscow;

7 INSERM U758, Lyon, France;

8. Institute of polyomyelitis, Moscow;

9. Malmö University hospital, University of Lund, Malmo, Sweden

10 Swedish Institute for Infectious Disease Control, Stockholm, Sweden

24th IUSTI-Europe Conference on sexually transmitted infections and HIV/AIDS, Milano September 4-6, 2008, p 79-80, P41

The presence of neutralizing activity against genotype-matched HCV pseudoparticles in acute phase sera correlates with resolution of HCV infection

Isaguliants M1,2, Petrakova N2,3, Jansons J3, Vonsky M3,4, Dudina K2,5, Gutman E3, Znoiko O5, Bartosch B6,7,8, Widell A3

1Swedish Institute for Infectious Disease Control, Stockholm; 2Ivanovsky Institute of Virology, Moscow; 3Malmö University hospital, Malmö; 4St Petersburg Institute of Cytology, St Petersburg; 5Moscow State University of Dentistry and Medicine, Moscow; 6INSERM, U871, 7Université Lyon 1, IFR62 Lyon-Est, and 8Hospices Civils de Lyon, Lyon, France.

Serum neutralizing activity can be assessed using pseuo-typed retroviral particles displaying HCV envelope proteins E1 and E2. We investigated at

the time of jaundice and during follow up the neutralizing capacity in sera of patients clearing HCV infection and patients who developed chronic HCV

infection

Patients and controls:One patient infected with HCV 1b (St Petersburg, Russia) and one with HCV 3a (Malmö, Sweden) with high viral load. Their sera was used as a source of HCV RNA to create HCV pseudoparticles (HCVpp´s).Patients (n=19) with acute hepatitis C were followed from onset of symptoms, at month 1, 3, 6, and 12 to either clearance confirmed by repetitive PCR analysis of sera and PBMC (RE; n=7;) or chronicity (CHR; n=12). Of clearing patients, 4 were infected with HCV 1b, 1 with HCV 1a; of chronizing, 6 had HCV 1b, and 4 - HCV 3a. In totally four, HCV could not be typed. These sera and sera of healthy individuals (n=9) were used to inhibit HCVpp infection.

Assays & Methods:HCV RNA was amplified by RT-PCR from sera of chronic patients infected with HCV 1b (Gene Bank FJ411253) and HCV 3a (Malmö, Sweden). Fragments encoding the last 60 aa of core, E1 and E2 were cloned into eukaryotic expression vector phCMV-7a (Bartosch B et al, 2003) in exchange for the respective HCV 1a sequence. HEK293T/17 cells were co-transfected with HCV E1/E2 encoding plasmids, CMV-Gag-Pol murine leukemia virus (MLV) packaging construct encoding MLV gag and pol genes, and the MLV-GFP plasmid expressing GFP. Supernatents containing pseudoparticles were collected 40 h later. Control particles carried feline endogenous virus RD114 glycoproteins (RD114pp).HCVpp based on HCV 1b FJ411253 (HCV1bRU) and on HCV3a (HCVpp3aSE) were purified by ultracentrifugation in sucrose gradient, precipitated with TCA and analysed by Western blotting.HCVpp1bRU, HCVpp3aSE and control RD114pp were used to infect Huh7 cells in the presence/absence of sera of healthy and HCV-infected individuals. Sera were supplemented in dilutions 60, 240 and 960. All assays were run in triplicates. Efficacy of infection was monitored by FACS using GFP as a reporter. Read outs were % inhibition by test sera compared to uninhibited infection, and the inhibition titer.The levels of serum antibodies against E1, E2 of HCV 1b and the consensus sequence of the hypervariable loop of E2 (HVR1) represented by synthetic degenerate peptides (Isaguliants M et al, 2002) were assessed by in-house ELISA.

Results

Conclusions Already at early time points, sera of patients clearing HCV infection contain

the components (suggestively antibodies) that inhibit HCVpp infection of target cells. No protective components are contained in the sera of patients developing chronic hepatitis C. The nature of inhibition, specifically the role of immunoglobulins, low and high density lipoproteins, is currently under study.

HCVpp neutralizationAt early time points sera of patients clearing HCV had a significantly stronger capacity to inhibit HCVpp infection. Already at day 3 to 10 from jaundice, sera of patients clearing HCV inhibited infection with HCVpp at dilution 300±150, and by month 6, at 400±200. Early sera of patients developing chronic hepatitis C did not inhibit HCVpp infection. Inhibition titers of 450±250 were reached with a delay of 3 to 6 month. Statistically, early sera of clearing patients were different from sera of healthy controls, as they have the capacity to inhibit infection, while early sera of chronizing patients behaved as that of the healthy controls (no inhibition over the background levels; Fig. 3AB). Inhibition was severely diminished by the removal of serum IgG (Fig. 4).

Fig. 2

Inhibition of HCVpp infection by intact and treated with protein G column sera (IgG binded) / IgG content

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Sera treated withprotein G column :IgG Content

Generation of HCV pseudoparticles

HCVpps were generated by co-transfection of HEK293 cells with MLV expressing vector mediating formation of retroviral pseudoparticles, and HCV E1/E2 and GFP expression vectors that provided for incorporation of HCV E1 and E2 onto particle surface and GFP into the particles, respectively (Fig 1). The nature of HCVpps was confirmed by PAGE and Western blotting using anti-E2 monoclonal antibody (Fig 2).

Fig. 1

Boxplot by Group

Variable: HCV 1b Inhibition % 1/60(p=0.0000 at 1/60; p=0.0006 at 1/240)

Mean Mean±SE Mean±1.96*SE

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Percent inhibition of infection with HCVpp 1b (Fig. 3a) and HCVpp 3a (Fig. 3b). RecE, sera of patients clearing HCV collected at month 0-1 (n=7); RecL, sera of patients clearing HCV collected >6 month (n=7); ChrE, sera of patients developing chronic hepatitis C collected at month 0-1 (n=12); ChrL, sera of patients developing chronic hepatitis C collected > 6 month (n=12); HCV negative, sera of healthy individuals (n=9); HCV positive, control sera of chronic hepatitis C patients with strong neutralizing capacity (positive control, n=2; 6 independent assays). All inhibition assays were performed in triplicates.

Fig. 4Inhibition of HCVpp infection by sera of chronizing patients (n=12) correlated with the presence of antibodies against N-terminus of hypervariable loop of E2 (HVR1; R=0.446, p<0.05). Inhibition of HCVpp infection by sera of clearing patients did not depend on either the total level of anti-env, or antibodies against N-terminus of HVR1, while anti-HVR1 C-terminus antibodies were not detected.

Acknowledgements: New Visby program of the Swedish Institute, INTAS and Lund University ALF grants are gratefully acknowledged for financial support. Mabtech (Sweden) is acknowledged for the gift of kits for antibody quantification, and Imtek Ltd.(Russia), for the columns for selective removal of IgG and lipoproteins.

Materials and Methods

16th Conference on hepatitis C, Nice, 2009

Photogallery fromannual meetings and individual researcher

exchange

PhD students Andreas Boberg (SMI, Stockholm) and Liza Starodubova (IVM, Moscow) working in Ivanovsky Institute, Moscow, December 2006

Andreas Boberg and Maria Isaguliants (SMI, Stockholm) presentation in Ivanovsky Institute, Moscow, December 2006

Natalia Petrakova (PhD, researcher), Liza Starodubova (PhD student, now Postdoc), Alexey Morozov (student), mouse experiments in Ivanovsky Institute, Moscow 2006-2007

20080204_Helmut Diepolder (Munich University, Germany) speaking on Moscow meeting 2008

20080204_Anders Widell (Lund University, Sweden) speaking on Moscow meeting 2008

20080204 Moscow meeting 2008 at Ivanovsky Institute of Virology

20080204 Matti Sällberg (Karolinska Institutet, Stockholm) speaking on HCV DNA vaccine, Moscow meeting 2008

Prof Kochetkov and Dr Znoyko (Moscow, University of Dentistry and Medicine), discussions on Moscow meeting 2008

Network members on sight-seeing tour in Kremlin,

5th Moscow meeting 2008

Network members on sight-seeing tour in Kremlin,

5th Moscow meeting 2008

Dr Heléne Norder (Swedish Institute for Infectious Disease Control, Stockholm), Moscow meeting 2008

Prof Lars Magnius and Dr Heléne Norder (Swedish Institute for Infectious Disease Control, Stockholm), discussions on Tallinn meeting 2009

Group photo at closing of the 7th Annual Network meeting in Tartu February 20-23, 2010

Group photo at closing of the 7th Annual Network meeting in Tartu February 20-23, 2010

One of the group photos on 8th Network meeting, Vilnius, February 13-16, 2011

On the lecture course ”Hepatitis ABC” held by the Network members at the Lithuanian Academy of Sciences, February 17, 2011

Heading for the meeting on the very cold Vilnius streets (-24 C)

We hold fingers for our Network application being granted.

If the decision is positive, we will meet for our 9th meeting in St Petersburg,

April 2012!

All are very welcome to join, look for meeting announcements on the

Network web page!

Presentation of Activities of New Visby Hepatitis C Network 2002-2011 Anders Widell,

Documents

latvian university

institute of microbiology

molecular medicine

study center

department of medicine

moscowuniversity of

univesity of lund

karolinska institutet