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It is estimated around 170 million people worldwide are carriers of hepatitis C virus (HCV).

Chronic HCV infection is the leading cause of liver transplantation in developed countries due to

virus induced cirrhosis and/or hepatocellular carcinoma. Our lab is developing a HCV vaccine

using recombinant glycoproteins gpE1/E2 as antigen. Similar to many successful vaccines in the

market, our vaccine is intended to induce neutralizing antibodies to confer protection. HCV is a

diverse virus, currently divided into 7 major genotypes. For a successful global vaccine, it is

important to generate cross-neutralizing antibodies against all genotypes. Many cross-neutralizing

antibodies have recently been isolated presumably targeting conserved region(s) on gpE1/E2 that

mediate viral entry. Previously, we immunized two goats with genotype 1a (goat g757) or 2a (goat

g773) derived HCV antigen. As expected, sera from both goats were able to neutralize HCV of the

same genotype as the immunizing antigen (genotype specific neutralization): serum from g757

neutralized entry of genotype 1a (H77c strain) HCV, whereas serum from g773 neutralized entry

of genotype 2a (J6 strain) HCV. Surprisingly, serum from g757, immunized with the genotype 1a

antigen, was much more effective than serum from g773 when tested for neutralization activity

against another genotype 2a HCV strain (JFH). This suggests antibodies from the genotype 1a-

immunized g757 are able to recognize genotype 2a glycoproteins of the JFH strain, thus blocking

infection. Identification of these critical regions in gpE1/E2 that confer recognition by g757 serum

would be helpful for future vaccine design to promote development of cross-neutralizing

antibodies. To that end, in this study we generated pseudotyped chimeric HCV particles

expressing chimeric E1JFHE2J6 or E1J6E2JFH to test their sensitivity to neutralization. Serum from

g757 is able to neutralize HCVpp if the coding region of JFH E2 is present. This suggests the

virus determinants of neutralization by the serum from g757 are located within glycoprotein E2.

Future study will focus on identification of specific residue(s) involved in neutralization.

Determine genotype 2a glycoprotein coding region responsible for neutralization sensitivity by

sera from genotype 1a immunized goat and/or genotype 2a immunized goat. The extent of

neutralization of HCVpp pseudotyped with J6 strain E1E2, JFH strain E1E2, chimeria E1J6E2JFH

or chimeriaE1JFHE2J6 are tested.

Characterization of viral determinants in

HCV glycoproteins conferring vaccine

induced neutralization in goats

Jianqi He, Chao Chen, Darren Hockman, John Law and Michael Houghton

Li Ka Shing Institute of Virology, Department of Medical Microbiology and Immunology,

University of Alberta, Edmonton, AB

Figure 1. Flowchart of experiments used in this study

Figure 2. Generation of HCVpp encoding with different glycoprotein gpE1/gpE2. (A) Illustration

of HCVpp production and subsequent transduction. Producer 293T cells are transfected with

CMV-Gag-Pol, pNL4-3.Luc.R.E and various viral full length glycoprotein gpE1/gpE2 encoding

plasmids (see panel B). HCVpp is a retrovirus based pusedoparticle decorated with HCV

gpE1/gpE2 on the surface and encode a luciferase reporter. The envelope HCV glycoprotein

gpE1/gpE2 are responsible for attachment and receptor-mediated endocytosis of HCVpp into

target Huh 7.5 cell. Upon successful entry, HCVpp transfers a plasmid encoding firefly luciferase

reporter. Expression of luciferase can then be be measured by luminescence intensity. (B)

Schematic for various recombinant glycoprotein gpE1/gpE2 used in this study is shown. They

are generated by mixing the glycoproteins coding region between genotype 2a HCV of J6 strain

and JFH strain. Black boxes indicate for signal peptides or transmembrane domain. Additional

phCMV-VSVG plasmid vector is used as positive control. ( A. is traken from :Bartosch B et. al.

Studying HCV Cell Entry with HCV Pseudoparticles (HCVpp). Hepatitis C: Methods and

Protocols. 2(510): 279-293, 2009.)

Figure 3: HCVpp pseudotyped with various HCV glycoproteins were tested for entry into

human hepatoma Huh 7.5 cell (see figure 1b). VSVG pseudoparticles were used as a positive

control and media collected from 293T producer cells without transfecting glycoprotein expressing

plasmid were used as negative control. HCVpp (undiluted or diluted 1 in 10) were transduced by

spinoculation at 500g for 1 hour at 4 oC. Fresh media were replaced 6 hours post-transduction. 48

hours post-transduction, lysates were made using Bright-glow substrate (Promega Inc.) and

luminescence was then measured by plate reader. HCVpp pseudotype with gpE1/gpE2 of J6,

JFH and chimera E1J6E2JFH are able to support entry, whereas chimera E1JFHE2J6 render non-

functional glycoproteins complex. A representative of two independent experiments done in

triplicate were shown. Error represent standard derivation of triplicate.

a

Introduction Testing Entry of Various HCVpp

Figure 4. Serum from genotype 1a antigen immunized goat (G757) or genotype 2a antigen

immunized goat (G737) was tested to neutralize J6 (A), JFH (B) or chimeria E1J6E2JFH (C)

pseudotyped HCVpp. Three fold serial dilution of goat sera were prepared. Then, they were pre-

incubated with HCVpp 1 hour at 37oC prior to addition to Huh7.5 cells. 48 hours post-

transduction, level of HCVpp entry were monitored by luciferase based luminescence as

described. Antibodies to HCV receptor CD81 were used as positive control to show

neutralization. % neutralization was calculated by (RLUPRE–RLUPOST)/RLUPRE*100, where RLU is

the relative light unit measured. The neutralization activity of post-immunization goat sera were

normalized with pre-immunization sera of the same goat, whereas result of anti-CD81 were

normalized with isotype control. Average of three independent experiments done in triplicates

were shown. Serum from goat 773 effectively neutralized J6 HCVpp, but not JFH HCVpp.

Conversely, serum from goat 757 neutralized JFH HCVpp, but not J6. For chimeria E1J6E2JFH

HCVpp, it was neutralized by serum from g757, but not by serum from g773. This suggests to us

that the viral determinants for neutralization were mainly located within E2 of the glycoproteins.

1. JFH, chimera E1J6E2JFH and E1JFHE2J6 pseudotyped HCV pp were generated and tested for

entry (J6 HCVpp were previously made in the lab).

2. All, but chimera E1JFHE2J6, supports HCVpp entry.

3. Chimeric HCVpp E1J6E2JFH was neutralized by g757 post-immune serum which also is

capable to neutralize JFH HCVpp. This suggests viral determinant(s) of neutralization is

concentrated within the E2 of the glycoprotein complex.

4. Specific residue(s) within E2 confer the sensitive to neutralization will be identified

(a) (b)

(A) (B) (C)

Generation of Chimeric HCVpp

Encoding plasmids

Functional Entry Assay

Neutralization

assay

Transfection In 293T cell

HCV Pseudoparticle Harvesting

Acknowledgement

phCMV-VSVG

VSVG CMV Poly(A)

Objective

Methods

HCVpp Neutralization by Serum from Goat 757 or Goat 773

Discussion/ Future Directions

Generation of HCV pseudoparticles (HCVpp) with various HCV

glycoprotein gpE1/gpE2

I here by thanks to all members in Houghton lab to giving me necessary helps and assists. Also, I

would like to give special thanks to Dr.Pukazki, Dr.Shmulevtiz and Tabitha to put their efforts to arrange

this great summer student program.

Lucife

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