Characterize viral determinants in Hepatitis C virus (HCV) glycoprotein E1E2

confer vaccine induced neutralization Jianqi He, John Lok Man Law,Michael Houghton

1 ABSTRACT

HCV have high genetic diversity and are grouped into seven major genotypes (GTs). A successful global prophylactic vaccine need to elicit a broad cross-neutralization against all genotypes. In previous study, presence of cross-neutralizing antibodies were observed in human volunteers who were immunized with genotype 1a derived recombinant gpE1/gpE2 vaccine. However, volunteer’s antisera were effectively neutralizing on genotype 1a, 4a, 5a and 6a as compared with genotype 2a isolate J6. We also tested presence of cross-neutralizing antibodies in goats that were immunized with GT 1a derived glycoprotein gpE1/gpE2 vaccine. Goat immunized with 1a gpE1/gpE2 antigen was incapable to cross neutralize 2a isolate J6 but another 2 a isolate JFH. To characterize the determinants in viral glycoprotein confer this cross neutralization, we generated two chimeric HCV pseudoparticles (HCVpp) using swapped E1 and E2 from both 2a isolate J6 and JFH. We determined entry viability of our HCVpp constructs. Besides, we performed antiCD81 antibody entry blocking assay to reassured HCVpp entry do have correlation with host CD81. Subsequent goat antisera neutralization assay indicate chimeric HCVpp J6e1JFHe2 is neutralized by 1a gpE1/gpE2 antigen immunized goat not 2a gpE1/gpE2 antigen immunized goat. Thus, we conclude that glycoprotein gpE2 is the targets for our vaccine and it confers to cross neutralization on 2a JFH from 1a immunized goat antisera. Overall, this study identified immunogenic residue(s) on glycoprotein E2 of 2a JFH is the cause for cross neutralization from 1a immunized goat and understanding of this residue(s) can help us to develop a prophylactic HCV global vaccine.

2 INTRODUCTION

HCV has an immense impact on global health with estimated 160 million chronic carrier worldwide1.

Acute phase of viral infection is usually asymptomatic, but chronic infection frequently leads to severe

liver damages. These including hepatic steatosis, cirrhosis and fibrosis. Patients who are persistently

infected with HCV also have a high rate of developing hepatocellular carcinoma and many require liver

transplantation. Pegylated interferon with nucleotide analogue Ribavirin is the standard of care.

However, the effectiveness of this therapy is vary among genotypes and patients. Reported side effects

are also a drawback of this treatment. Recently, a viral RNA polymerase inhibitor Sofosbuvir,

manufactured by Gilead, has been approved for HCV therapy. This drug is effective and has increased

the cure rate of infection. However, the cost is egregiously high making it unaffordable for most patients

and health-care system. Therefore, developing a prophylactic HCV vaccine is an urgent need to control

HCV.

HCV vaccine development has been technically challenging because of HCV genome encodes an error

prone RNA dependent RNA polymerase. Without a proof reading RNA polymerase, HCV is genetically

diversified and is divided into 7 major genotypes and within which subdivided into multiple subtypes.

The variation in nucleotide level between each genotype is around 30%, and 20~25% between each

subtype2. A global vaccine is necessarily to protect vaccinee from all 7 major genotypes of this vast

diverse virus. Previous phase I clinical trial has conducted to test safety and immunogenicity of a GT 1a

derived recombinant glycoprotein gpE1/gpE2 vaccine3. Volunteer’s post immune sera were collected

and tested against representative viruses from all 7 major genotypes. Figure1 showed that immunized

volunteers antisera exhibit a significant cross neutralization effect against many genotypes. In particular

there was a strong neutralization against recombinant viruses of GT1a, 4a, 5a and 6a. In comparison,

patient’s antisera showed a reduced efficiency to neutralize viruses of GT 2a, 3a, 7a. Hence, it indicates

there are presence of highly conserved neutralizing epitopes render intergenotypic cross neutralization.

A genotype 1a HCVpp immunized chimpanzees experiment led by Dr. Meunier et al exhibit a similar

cross neutralization profile as the one in Figure 1. Neutralizing antibody raised from 1a HCVpp infection

could neutralize genotype 4a, 5a, 6a but with limited reactivity again 2a and 3a. They further conclude

that genotype 1, 4, 5 and 6 are serologically more closely related with a more distant serologic

relationship to genotype 2 and 34. Their founding further confirmed cross neutralization antibody can

be raise from genotype 1a glycoprotein gpE1/gpE2 vaccine. Unfortunately, those NtAbs were poorly

neutralizing genotype 2 and 3.

Due to the prevalence of GT 1 and GT 2 in North America, we reproduced previous recombinant

vaccine test in a subset of goat immunization experiment. This time, two goats were immunized with 1a

or 2a derived HCV glycoprotein antigen respectively. Goat antisera were tested for neutralization

activity against GT1a isolate H77c and gt2a isolate J6. It showed a genotype-specific neutralization

(Figure 2A, 2B). Interestingly, when we testing the same 1a goat antisera against another 2a isolate JFH,

a cross neutralization is observed. In another word, 1a derived recombinant gpE1/gpE2 is capable to

cross neutralize 2a isolate JFH (Figure 2C). Relative study shows the HCV envelope glycoprotein gpE1 and

gpE2 are the natural targets of the protective antibody response5. Therefore, our objective this study is

to investigate causes of cross neutralization on 2a JFH from 1a immunized goat antisera. We

hypothesize that there are residue(s) on viral glycoprotein confer the phenomenon of cross neutralizing.

In order to answer our question, we generated two HCV pseudoparticles (HCVpp) using glycoprotein

gpE1 and gpE2 from genotype 2a isolate J6 and JFH respectively (Figure 3). In addition, two chimeric

HCVpp are generated by interchange gpE1/gpE2 from either 2a J6 or 2a JFH. Formation of HCVpp

involves incorporation of the full length hepatitis C virus glycoprotein E1 and E2 onto retroviral core

particles. The exact mechanism for interaction between host receptors and viral heterodimer

glycoprotein gpE1/gpE2 are still unclear, but studies indicate both E1 and E2 are indispensable for viral

entry6, 7. Hence, we performed an entry assay to test the viability of our four HCVpp constructs. In

addition, we determined the amount of assembled HCVpp viral titer based on HIV1 p24 ELISA. Finally,

we used goat antisera which either immunized with GT 1a or GT 2a antigens to test neutralization effect

against our HCVpp of various genotypes. Through exchange of glycoprotein gpE1/gpE2 from

intergenotypic HCV 2a isolate J6/JFH, we reassured glycoprotein E2 is the main immunogenic target for

our neutralizing antibodies. Our study provides a substantial understanding for design and development

of future prophylactic HCV vaccine.

Figure1: Human antisera cross-neutralizes all 7 major HCV genotypes. Sera of volunteer 1,5 and 7 were

tested for neutralization activity against chimeric 1a(H77C), 1b(J4), 2a(J6), 2b(J8), 3a(S52),4a(ED43), 5a(SA13),

6a(HK6a) and 7a(QC69) cell culture based HCV. Virus neutralization assays were performed using pre- and

post-vaccination sera at a concentration of 1 in 50. Levels of neutralization activity of post-vaccination sera

were normalized with the activity of pre-vaccination sera. Representative of two independent experiments

performed in triplicate are shown. Figure adapted from Law, John Lok Man 2013.

Figure 2. Goat antisera neutralization against cell culture based HCV of GT1a H77, GT 2a JFH and GT 2a J6. 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 of the same immunoglobulin. GT 1a E2

immunized goat (1a goat) and GT 2a J6 E2 immunized goat (2a goat) antisera were tested for neutralization

activity against HCV of GT1a isolate H77c (A) or GT2a isolate J6 (B). Sera from 1a goat were tested for

neutralization activity against HCV of GT 1a H77, GT2a JFH or GT2a J6 (C). Anti-CD81 antibody serves as a

comparable positive control to show infectivity of H77, JFH and J6 can be hampered by blocking CD81 co

receptor12. Figure adapted from unpublished data, Law, John Lok Man, et al. 2013

C

Figure 3. Schematic of HCVpp production and various glycoprotein expressing constructs. HEK-293T cells

were transfected with pNL4-3 Luc R.E vector encoding HIV gag-pol and luciferase reporter gene and HCV

glycoprotein expression constructs. Successfully transfected 293T cells assemble HCVpp intracellular and

being secreted into supernatant. From that, HCVpp is collected from supernatant by filtrating through 0.22µm

Millipore vacuum filter. Uptakes of HCVpp into hepatoma Huh7.5 cells can be monitored by adding promega

firefly luciferase substrate (see materials and methods). Figure adapted from Usman Ashfaq et al 2011 and

Sheena saayman et al 2010

3 MATERIALS AND METHODS

Plasmids. The retroviral package plasmid were based on insertion of firefly luciferase gene in nef

position at HIV-1 proviral clone pNL4-3. Addition of firefly luciferase substrate (Promage).The viral

envelope glycoprotein constructs contain J6e1J6e2, JFHe1JFHe2, J6e1JFHe1 and JFHe1J6e2 were

constructed by standard PCR and infusion cloning.

Cell culture. Constructs transfected 293T human embryo kidney cells were used a producer for HCVpp.

Huh-7.5 is highly permissive for HCV replication and was used for viral transduction assay with or

without presence of neutralization antibodies.Huh7.5 cells were culture in Dulbecco’s modified Eagle’s

medium supplemented with 10% FBS, 0.1mM NEAA and 100 ug each of penicillin and streptomycin.

Production of HCVpp. The pseudoparticles were produced as described (ref 8). P10 dish with pre-coated

poly-L-lysine were seeded with 2X106 HEK 293 T cells one day before transfection. Fresh 293 T DMEM

medium is replace into P10 dish at 1 hour prior to transfection. Mixtures of retro viral packaging plasmid

and intact viral glycoprotein plasmid were diluted in 500µL Opti-MEM for 5 minutes at room

temperature. Additional 50µL of lipofectamine 2000 were mixed with 500µL Opti-MEM at room

temperature. Combined plasmid dilutions and lipofectamine 2000 were incubated for 20 mins at RT and

equally dispersed on P10 dish containing 293T cells. Leave solution in P10 dish at 37°C overnight, then

replaced with 3% FBS medium containing 1%penicillin and streptomycin. Both 48 hours and 72 hours

transfected supernatant were vacuum filtrated though 0.22µm filter and combined together. Final

concentration 4µg/ml of polybrene and 20mM of HEPES were added into filtered HCVpp containing

supernatant. All HCVpp supernatants were aliquoted into 2ml cryogenic tube and stored at -80°C for

future usage.

Entry assay. Susceptible human hepatoma Huh7.5 were seed in 96 wells plate pre-coated with PLL one

day before infection (1x104 cells/ well ). Huh7.5 growth medium were removed by vacuum filtration and

filled with 50µL of HCVpp. Mixture were centrifuge at 1400rpm for 1 hour at 37°C. Fresh Huh7.5 DMEM

media is replaced into each well 6 hours after centrifugation. After 48 hours, infected cells were washed

with phosphate buffer saline (Invitrogen) and mixed with Bright GloTM luciferase substrate (Promega).

Viral infectivity were measured as relative luminescence light unit under the Enspire 2300 multilable

reader (Perkin Elmer).

HIV p24 elisa for viral titer quantification. The amount of HCVpp in filtered supernatant was

determined by using the standard curve obtained from HIV-1 p24 ELISA kit (Abnova). Kit positive control

was diluted 50 times as required by user manual. Microtitration plate contain HCVpp and positive

control were read using absorbance values at 450nm.

Anti CD81 antibodies blocking assay. Anti-CD81 Antibody (BD PharmingenTM purified mouse anti-human

CD81 0.5mg/ml) were serial diluted into 1/10, 1/30, 1/90, 1/270 prior to mixing with HCVpp. Later, 50µL

of HCVpp were mixed with 1µL of each Abs dilution. Therefore, we made additional 1/50 dilutions for

each serial diluted AntiCD81 antibodies (1/500, 1/1500,1/4500,1/13500). Pseudoparticle VSVG control

was further diluted 200 times before mixing with antiCD81 antibody. Mixture were incubated for 1hr at

37°C and then added to Huh7.5 cells seeded in 96 well (104cells/well). Cells were then spinoculated at

37°C, 1400rpm for 1 hour. Fresh huh7.5 media were replaced in at 8 hours after spinoculation. We

measured relative light unit by adding luciferase substrate post 48 hours of infection.

Goat sera neutralization assay Seed poly-L-Lysine coated 96 well plate with 104 huh 7.5 cells/well one

day before neutralization. Both 1a and 2a goat antisera were serial diluted into 1, 1/3, 1/9 and 1/27

prior than mix with HCVpp. AntiCD81 antibody and mouse IgG isotype control were diluted into 1/10,

1/30, 1/90 and 1/270. We further mixed 3.2µL sera with 160µL HCVpp respectively (1 in 50 dilution).

Mixture were incubated for 1 hour at 37°C. We add 50µL NtAbs/HCVpp mixture to each well and

spinoculate at 37°C, 1400rpm for 1hr. We replaced medium with huh7.5media 8 hours after infection.

HCVpp entry were measured by adding firefly luciferase substrate post 48 hours of infection.

4 RESULTS

4.1 CONSTRUCTION OF JFH AND CHIMERIC E1E2 EXPRESSION PLASMIDS In order to determine the virus determinant to confer sensitivity of neutralization, we decided to use

HCVpp as a model system to test effect of goat neutralizing antibody on glycoprotein gpE1/E2. To do so,

we first subcloned the coding sequence of JFH E1E2 into the expression plasmid of virus glycoproteins.

In addition, we have constructed two chimeric E1E2 expression constructs. The first chimeric HCVpp is

J6e1JFHe2, where the E1 is derived from J6 sequence and the E2 is derived from JFH. The second

chimeric HCVpp is JFHe1J6e2, where the E1 is derived from JFH sequence and the E2 is derived from J6.

Plasmid expressing glycoproteins derived from J6 was already available in the lab. To test the viability of

HCVpp constructs for cell entry, an entry assay is performed using susceptible hepatoma Huh7.5 cells as

host cell. We observed HCVpp expressing all four constructs of E1E2 exhibit a positive correlation

between infectivity and dilution factors. Pseudoparticle control expressing vesicular stomatitis virus G

protein have the highest infectivity and its relative light unit is around 100 folds higher than in HCVpp

(Figure 4). Three HCVpp expressing J6, JFH and J6e1JFHe2 have a higher signal to noise ratio while

chimeric HCVpp JFHe1J6e2 do not have significant higher signal than media control. Subsequent viral

titer quantification assay using HIV p24 ELISA reflect all HCVpp have substantial amount of virus

secreted in supernatant. Based on p24 ELISA standard curve, HCVpp J6 has 65pg/ml, HCVpp JFH has

126pg.ml. Both chimeric HCVpp J6e1JFHe2 and JFHe1J6e2 have very close read, they are 59pg/ml and

54pg/ml respectively (Figure 5). Based on these results, HCVpp encompassed E1E2 of J6, JFH and

J6E1JFHE2 support functional entry where we can test their sensitivity of neutralization.

4.2 ENTRY OF HCVPP EXPRESSING E1E2 OF J6, JFH AND J6E1JFHE2 IS CD81-DEPENDENT To examine the effect of anti-CD81 antibodies on cell entry, we tested viral entry blocking assay using

anti CD81 antibodies. Figure 6 showed that increasing concentration of antiCD81 antibodies reduces the

entry of HCVpp of J6, JFH and J6e1JFHe2, whereas pseudoparticle expressing VSVG is not affected by

anti-CD81 antibody. This has confirmed the various version of HCVpp constructed in this study are using

a CD81-dependent entry pathway, similar to wild type HCV.

4.3 E2 IS THE VIRAL DETERMINANT CONFER SENSITIVITY TO GENOTYPE SPECIFIC

NEUTRALIZATION HCVpp neutralization assay is carried out using antisera collected from goat immunized with either GT

1a or 2a derived glycoprotein. In figure 7, the sera of goat immunized with J6 derived antigen is unable

to neutralize the HCV pp of GT 2a isolate JFH but effectively neutralize against HCVpp of gt2a J6. On the

other hand, GT 1a E1E2 immunized goat exhibit can cross neutralization against HCVpp expressing GT 2a

JFH E1E2. When use these antisera to test neutralization on chimeric HCVpp expressing J6e1JFHe2, this

chimeric HCVpp is neutralized by sera of 1a immunized goat post immune sera (Figure 7). This suggests

that E2 sequence confer the sensitivity of neutralization by the sera of immunized goats.

Figure 4. Entry assay for four HCVpp constructs. Pseudotypic HCVpp with various HCV glycoproteins were

tested for entry into human hepatoma Huh 7.5 cell. VSVG pseudoparticles were used as a positive control,

media collected from 293T producer cells without transfecting glycoprotein expressing plasmid were used

as negative control. Three fold serial diluted HCVpp were transduced by spinoculation at 500g for 1 hour at

37 oC. Fresh media were replaced 8 hours post-transduction. 48 hours post-transduction, lysates were

made using Bright-glow substrate (Promega Inc.) and luminescence was then measured by plate reader.

Values were derived from average of one independent experiment done in triplets, each error bar indicates

standard error for triplicate readings.

Figure 5. HIV1 p24 ELISA viral titer quantification assay. The amount of HCVpp in filtered supernatant was

determined by using the standard curve obtained from HIV-1 p24 ELISA kit (Abnova). Kit positive control was diluted

50 times as required by user manual. Microtitration plate contain HCVpp and positive control were read using

absorbance values at 450nm. Values were obtained from one independent experiment done in duplicate. Amount of

HCVpp constructs J6,JFH, J6e1JFHe2 and JFHe1J6e2 pseudoparticle p24 quantification were displayed in table 1 as

shown.

Figure 6. Anti-CD81 Abs blocking assay for HCVpp constructs. HCVpp constructs from J6 ,JFH, chimeric

J6e1JFHe2 and JFHe1J6e2 were pre-incubated with serial diluted mouse anti human CD81 antibody(BD

pharmingen). The antiCD81 antibody were diluted at 1/500, 1/1500, 1/4500 and 1/13500 and mixed with

HCVpp including pseudoparticle VSVG control. Mixtures were later introduced into 96 well plate seeded with

104 susceptible Huh7.5 cells. Level of HCVpp viral entry is measured by Bright GloTM luciferase substrate and

expressed as the relative light unit by plate reader. Values were obtained from average of one independent

experiment done in triplicates, error bar indicates standard error for each triplicate.

Figure 7. Goat serum neutralization assay against chimeric HCVpp J6e1JFHe2. Serum from genotype 1a

derived E1E2 immunized goat (1a goat) or genotype 2a derived E2 immunized goat (2a goat) was tested

to neutralize J6 ,JFH or chimeria E1J6E2JFH 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. Result of anti-CD81 were normalized with isotype control. Average of three independent

experiments done in triplicates were shown. Error bar were standard error from each triplicate.

5 DISCUSSION

In our study, we investigated the viral immunogenic residues on glycoprotein confer vaccine induced

neutralization. Besides, we reassured HCV pseudoparticle system is viable to test cell entry and we again

confirmed viral entry have correlation with CD81 receptor.

To test our hypothesis, we employed in vitro cell culture HCV pseudoparticle model. Numerous

researches have validated that HCVpp system closely mimic the functionality of the wild type HCV virus

in both cell entry and neutralization5,6,7. Pseudotypic HCV particles were generated by assembling full

length, intact gpE1 and gpE2 onto retroviral core protein derived from HIV7. Viral glycoprotein gpE1 and

gpE2 are interact non-covalently and form a heterodimer structure on surface of HIV core particle 9.

Thus, a functional HCVpp is capable to express viral gpE1 and E2 on its retroviral core and is able to

infect susceptible host cells. In our HCV entry assay, we confirmed all 4 HCVpp constructs can entry into

host Huh7.5 cell and their viral infectivity are reflected by relative light unit by adding luciferase

substrate. Pseudoparticle control VSVG in this case have a substantial higher signal to noise ratio. The

phCMV-G envelope-expression vector encodes vesicular stomatitis virus G protein displays very high

titers and has a broad tropism7,8. Therefore, the high signal to noise ratio for pseudotype VSVG is

reasonable. To produce HCV virus pseudotypes, 293 T cells were cotransfected with retroviral package

plasmid bear CMV promoter and intact HCV envelope glycoprotein7. According to Figure 4, HCVpp

constructs have significant relative light unit except HCVpp expressing chimeric JFHe1J6e2. We

postulated two reasons for low signal noise ratio of our chimera JFHe1J6e2. One is this chimera cannot

support particle assembly in 293T cells. Another possibility is that this chimera JFHe1J6e2 is defective to

mediate entry into target cell. The HCVpp retroviral core is composed of P24 protein in packaging

plasmid pNL4-3 Luc R.E. In order to quantify amount of HCVpp, we performed a HIV1 p24 ELISA. Based

on HIV p24 ELISA results in figure 5, all HCVpp have a substantial amount of p24 been detected.

Therefore, it is very likely that all pseudoparticles were assembled successfully and thus secreted into

supernatant. The significant low signal to noise ratio of chimeric HCVpp JFHe1J6e2 is likely due to defect

happens post-assembly. One of most possible reason is JFHe1J6e2 has a non-functional gpE1/gpE2

heterodimer. However, without further experiment, we are unable to conclude that low infectivity of

chimera JFHe1J6e2 is surely due to defect in gpE1/gpE2 heterodimer.

After examination of HCVpp entry viability. An anti-CD81 antibody viral blocking assay is conducted.

CD81 is ubiquitously expressed on cell surface involving cell development and growth. CD81 is also the

first HCV co-receptor to be identified and interaction between gpE2 and CD81 mediates a post-

attachment event in HCV entry10. Therefore, by adding anti CD81 antibodies we can simply block viral

entry into host cells11. According to Figure6, pseudoparticle VSVG viral infectivity is not affected by

antiCD81 antibody. However, HCVpp J6, JFH and J6e1JFHe2 all exhibit an increase in infectivity along

with decreasing in antiCD81 antibody titers. Therefore, HCVpp constructs viral entry surely have

correlation with CD81 receptor. In later experiment, the antiCD81 antibody against HCVpp constructs

are normalized with mouse IgG isotype control and thus serve as control group for subsequent goat

antiserum neutralization assay.

Studies demonstrate the HCV glycoprotein E1 and E2 are the natural targets of neutralizing antibody4.

HCV retroviral pseudotype particles expressing gpE1 and gpE2 can be neutralized with protective

antibody from antisera12, 13. In this case, we used GT1a gpE1/gpE2 antigen immunized goat antisera and

2a gpE1/gpE2 antigen immunized goat antisera to test neutralization effect on HCVpp constructs.

Challenge HCVpp 2a J6 can be neutralized by 2a goat not 1g goat antisera. Interestingly, 1a immunized

goat antisera displays cross neutralization on 2a JFH and chimeric HCVpp J6e1JFHe2. In another word, a

simple swap glycoprotein gpE1/gpE2 between 2a J6 and 2a JFH resulted chimera J6e1JFHe2 have a

similar neutralization profile as 2a JFH. The evident is convincing that residue contributes to

neutralization on 2a JFH from 1a immunized goat is located on glycoprotein E2. Many studies have

indicated the majority of antibodies with a broad neutralizing activity are recognized conformational

epitopes on glycoprotein E2. Besides, there is also compelling evidence of E2 binding to CD81 and E2

segment is highly immunogenic4. Based on what we have found on glycoprotein E2, we further aligned

E2 protein sequence between GT 1a H77, GT 2a isolate JFH and J6 (Figure8). In this case, the

intragenotypic sequence identity between 2a JFH and 2a J6 is 87%. On the other hand, the

intergenotypic protein sequence identity between 2aJFH and 1a H77, as well as between 2a J6 and 1a

H77 are both 67%. In another word, there is a higher matched protein sequence in intragenotype than

Figure 8. Protein sequence alignment for 1a H77, 2a J6 and 2a JFH. Viral glycoprotein E2 nucleotide

sequencing result for 1a H77, 2a J6 and 2a JFH were converted into amino acids. The length and

relative position for each HCV glycoprotein were aligned. Red brackets indicate identity between 1a

H77 and 2a JFH but differ in J6.

in intergenotype. Nevertheless, antisera neutralization of 2a JFH from 1a immunized goat suggests

critical residue confer cross neutralization is hiding within 67% protein sequence identity between 1a

H77 and 2a JFH. According to Figure8, we highlighted amino acids that are similar between H77 and JFH,

but differ in J6. In total, 12 possible amino acids are identified. Perhaps region render cross

neutralization between 1a and 2a JFH is within these 12 amino acids. Nevertheless, it is also possible

that two or more amino acids are responsible to cross neutralizing phenomenon. For future experiment,

a site directed mutagenesis can be conducted to determine the critical residue(s) confer cross

neutralization at an amino acid level. Pinpoint identification of this immunogenic residue can allowing us

to preserve 2a JFH neutralizing epitope while we improve structure of our 1a gpE1/gpE2 recombinant

vaccine. Despite genetic heterogeneity of HCV, evidence of cross neutralizing antibody and conserved

epitopes on glycoprotein provide a promising future of prophylactic HCV vaccine.

Conclusively, these studies illustrate the viability of using HCV pseudoparticle system to test entry and

neutralization by addition of antisera. We again tested that entry into host cell by using HCVpp system

exhibit a CD81 dependent mechanism. Hence, blocking interaction between gpE1/gpE2 heterodimer can

hamper HCVpp entry into host cell. Importantly, we determined immunogenic epitope render cross

neutralizing 2a isolate JFH from 1a immunized goat antisera are located within HCV glycoprotein E2. We

further proposed highly possible amino acids that contributing to cross neutralization. A future

identification of this critical epitope can be helpful towards our prophylactic vaccine design.

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