1 Biomedical Research and Study Centre of Latvia, Riga, Latvia2 Infectology Center of Latvia, Riga, Latvia

3 Institute of Virology, Hospital of Essen, University of Diusburg-Essen, Essen, Germany

Fig.4. Electron microscopy of chimeric HBc/HCV HVR1 VLPs: (A) HBc_modif/HVR1 (1a), (B) HBc_modif/HVR1 (1b) (C) HBc_modif/HVR1 (3a) (D) HBc145/HVR (1a) (E) HBc145/HVR (3a) (F) HBc183/HVR1 (1a) (G) HBc183/HVR1 (3a) VLPs (magnification x 200 000)

Presentation of hepatitis C virus hypervariable region 1 from individual patient isolates on HBc virus-like particles

Sudmale G.1, Sominskaya I.1, Petrovskis I.1., Ose V.1., Skrastina D.1, Arsha F.2, Sondore V.2, Walker A.3, Lange M.3, Pumpens P.1

gunita@biomed.lu.lv

The major aim of the project was development of virus-like particles (VLP) displaying hipervariable region 1 (HVR1) individual isolates on hepatitis B core (HBc) carrier and analysis of anti-HVR antibodies neutralizing effect.

BackgroundHepatitis C virus (HCV) remains one of the most important human pathogens, but no HCV vaccine is available, yet. Globally, hepatitis C virus has infected an estimated 170 million people, most of whom are chronically infected and are at risk for developing of chronic liver disease, cirrhosis, and primary hepatocellular carcinoma. HCV variability is one of the reasons that prevent creation of anti-HCV vaccine. Despite high variability, the most promising vaccine candidates are the envelope proteins E1 and E2 especially the first 27 amino acids of E2 are the most variable region of HCV and are referred as hypervariable region 1 (HVRI) (Fig.1.). It contains an in principal antibobody neutralizing and CTL epitopes.

Acknovledgements

I would like to thank my colleaugues: Dr.biol. I.Petrovskis and his group (Dz.Dreilina, J.Zakova, I.Lieknina) for protein purification and analysis.J.Bogans for protein purification.I.Akopjana for competent cells.G.Zarins for imunobloting.Dr.chem. L.Ignatovica for sequencing analysis;

This work was supported by ERAF project 2010/0224/2DP/2.1.1.1.0/10/APIA/VIAA/164, ESF project 2009/0204/1DP/1.1.1.2.0/09/APIA/VIAA/150.

Fig.1. HCV genome organization, expressed proteins and proteolytic cleaving (Lloyd A R, et all).

VLPs are self-assembled from the proteins that make up a virus' outer coat, and are often used as a component of vaccines. Recombinant hepatitis B virus core protein (HBc) is one of the most promising carriers for foreign epitopes because of the HBc high immunogenicity and possibility to make well-organized particles (Fig.2.).

Fig. 2. Model of Hepatitis B core protein viruse-like particle .

Materials and methodsHCV HVR1 and core sequences were amplified from ten chronically infected viral hepatitis C patient blood serum samples (Infectology Center of Latvia). To define the HCV genotype core region was sequenced and determined that HCV in researh group represents genotype 1b and 3a. HCV HVR1 region was cloned into pJET1.2 vector and ten clones from each sample were sequenced. Two different HVR1 isolates 1b and 3a from patients and additional HVR1 1a were selected. Further HVR1 isolates were fused to C terminus of full lenght HBc with modified C terminus, non-modifid C terminus and in MIR region of HBc145. Recombinant plasmids were sequenced and expressed as fusion proteins in BL21 and K802 cell lines (Fig.3.).

Conclusions

Rrecombinant plasmids led to relatively high levels of expression of chimeric proteins in E.coli.

Seven of the nine constructions are able to form “mature” recombinant viruse-like particles.

Results of the current study have demonstrated the principal possibility of using HVR1 for creation of chimeric VLPs on the basis of HBcAg .

HBc/HVR1 VLP are highly immunogenic in mice.

The induced antibodies are able to neutralize also unrelated HCVpp’s

Anti-HVR1 antibodies neutralize specific HCVpp’s in all dilutions.

Anti-HVR1 antibodies have a virus neutralizing effect.

ResultsAll recombinant plasmids led to relatively high levels of expression of chimeric proteins in E.coli, which in case of seven constructions resulted in the formation of complete “mature” VLPs (Fig.4.). Chimeric HBc/HVR1 VLPs were purified by gel filtration.

G47

G48

G50

G44

G51

G52

G53

G54

G55

H77 (1a) HCVpp

0

10

20

30

40

50

60

70

80

90

100

110

1:25 1:50 1:100 1:200

Serum dilution

% N

eu

trali

zati

on

anti-G44

anti-G47

anti-G53

anti-G55

GT 1a HCVpp

0

10

20

30

40

50

60

70

80

90

100

110

1:25 1:50 1:100 1:200

serum dilutions

% N

eu

tral

izat

ion

anti-G44

anti-G47

anti-G53

anti-G55

GT 3a HVCpp

0

10

20

30

40

50

60

70

80

90

100

110

1:25 1:50 1:100 1:200

serum dilutions

% N

eu

trali

zati

on

anti -G44

anti-G47

anti-G53

anti-G55

65203

40794

7950

31675

69088

100

104450

100

22000

2800

55000

32000

63000

22900

0

20000

40000

60000

80000

100000

120000

titre

G44 G47 G48 G50 G52 G53 G55

Anti-HBc and anti-HVR1 antibody titres

anti-HBc

anti-HVR1

Fig.3. HBc/HVR1 chimeric constructs used in the study

D E F

GF

HVR13a

Modified Hbc 183 HVR11a

Hbc 183

HVR1 1b

Modified Hbc 183 HVR13a

HVR11a Hbc 145 Hbc 145

Hbc 145 Hbc 145HVR11b

HVR11b

HVR13a

Hbc 145Hbc 145

HVR11b

HVR13a

HVR11a

Modified Hbc 183

Hbc 183 Hbc 183

Hbc 183

Hbc 183

Hbc 183

Groups of 4-6 Balb/c mice were immunised with 25 g recombinant protein and 125 g adjuvant- alhydrogel and bosted in day 14 and 28 (Fig.5). At day 42 mice were bleeded and tested for specific antibodies. Serial dilutions were made and dilutions yielded tree times the optical density at 495 obtained with intact mice serum were scored positive (Fig.6)

Fig.5. Immunization sheme of experiment

Fig.6. Specific titer of anti-HBc and anti-HVR antibodies after immunization with HBc/HVR1 VLPs.

G44(1a) , G47(1a), G53(1a) and G55(3a) mice sera samples were selected for viruse neutralization test. HCV pseodo-particles (HCVpp) were generated by contrainfection of 293-T cells with equal amonts of expresion plasmids expresing viral gps, E1E2 containing diferent HVR1 sequences and NL4.3.Luc.R -E-. HCVpp’s were incubated with different concentrations of anti-HVR antibodies prior to infection of naive Huh 7.5. – cells. At day 3 post-infection neutralization was analyzed by luciferase activity (Fig.7,8,9,10).

Fig.7. Neutralization of HCVpp bearing unrelated HVR1 variants. Anti-HVR antibody neutralizing effect was tested with four different HCVpp: YK5829 (1b subtype), G31 consensus sequence (Puntoriero et all, 1998), YK5807 (1b subtype), H77 (1a subtype).

0

10

20

30

40

50

60

70

80

90

100

% N

eu

tralizati

on

YK5829pp (1b) G31pp(consensus seq.)

YK5807 pp (1b) H77 pp (1a)

Neutralization of unrelated HCVpp

anti-G44

anti-G47

anti-G53

anti-G55

Fig.8. Neutralization of HCVpp H77 (1a). HCVpp H77 were incubated with different concentration of anti-HVR antibodies: anti-G44(1a), anti-G47(1a), anti-G53(1a), anti-G55(3a) prior to infection of Huh7.5 and after tree days neutralization was analyzed by luciferase activity.

Fig.10. Neutralization of HCVpp GT 3a. HCVpps GT3a bearing specific HVR 3a isolate sequence from patient were incubated with different concentration of anti-HVR antibodies: anti-G44(1a), anti-G47(1a), anti-G53(1a), anti-G55(3a) prior to infection of Huh7.5 and after tree days neutralization was analyzed by luciferase activity.

Fig.9. Neutralization of HCVpp GT 1a. HCVpp GT1a bearing specific HVR 1a sequence were incubated with different concentration of anti-HVR antibodies: anti-G44(1a), anti-G47(1a), anti-G53(1a), anti-G55(3a) prior to infection of Huh7.5 and after tree days neutralization was analyzed by luciferase activity.