Induction of neutralizing antibody responses to hepatitis C ... - Nature

Immunology and Cell Biology (2007) 85, 169–173 & 2007 Australasian Society for Immunology Inc. All rights reserved 0818-9641/07 $30.00 www.nature.com/icb

ORIGINAL ARTICLE

Induction of neutralizing antibody responses to hepatitis C virus with synthetic peptide constructs incorporating both antibody and T-helper epitopes Joseph Torresi1, Alex Fischer1, Lara Grollo2, Weiguang Zeng2, Heidi Drummer3 and David C Jackson2 We describe a peptide-based strategy for hepatitis C virus (HCV) vaccine design that exploits synthetic peptides representing antibody epitopes of the hypervariable region 1 (HVR1) of the E2 glycoprotein and also less variable regions immediately downstream of HVR1. These epitopes were linked to a T-helper (Th) epitope (KLIPNASLIENCTKAEL) derived from the Morbillivirus canine distemper virus. Antibody titres induced by the two vaccine candidates Th-A (E2 amino acid 384–414) and Th-B (E2 amino acid 390–414) were significantly higher than those produced against vaccines lacking the Th epitope (Po0.05). Mice inoculated with the vaccine candidates Th-C (E2 amino acids 412–423) and Th-F (E2 amino acids 436–447) emulsified in complete Freund’s adjuvant each elicited antibody titres that were significantly higher than those elicited by Th-E (E2 amino acids 396–407) and Th-D (E2 amino acids 432–443) (Po0.01). Antisera obtained from mice inoculated with the epitope vaccines Th-A, Th-B, Th-D and Th-E bound to E2 expressed at the surface of 293T cells that had been transfected with E1E2. Furthermore, IgG from the sera of mice inoculated with four of the vaccine candidates, Th-A, Th-C, Th-D and Th-E, inhibited the entry of HCV/human immunodeficieny virus pseudoparticles (HCVpps) into Huh-7 cells. These results demonstrate the potential of synthetic peptide-based constructs in the delivery of potential neutralizing epitopes that are present within the viral envelope of HCV. Immunology and Cell Biology (2007) 85, 169–173. doi:10.1038/sj.icb.7100021; published online 23 January 2007 Keywords: HCV vaccine; epitope-based vaccine; synthetic vaccine

Hepatitis C virus (HCV) affects 3% of the world’s population and is estimated to cause 500 000 deaths annually.1 Treatments for chronic hepatitis C infection have varying success rates in eradicating the virus2–4 and for this reason a prophylactic vaccine against HCV is essential. The challenge facing vaccine researchers, however, is in solving the problem of HCV genotype and quasispecies diversity and developing a multifaceted vaccine strategy that will induce broad cross-protective antibody, CD8+ and helper T-cell responses.5–7 Humoral antibody responses to HCV provide protection against infection with HCV, although they have been reported to have a limited role in clearance of the virus.8 Antibodies directed to epitopes present within the hypervariable region 1 (HVR1) of the E2 glycoprotein9–11 and also downstream of HVR111,12 are neutralizing. Farci et al.10 demonstrated that a synthetic peptide representing a truncated HVR1 sequence induced a neutralizing antibody response, which was protective in chimpanzees against challenge with homologous virus. Strong evidence for the role for antibody in preventing hepatitis C virus infection in humans has also been demonstrated by the protective effect obtained by immune globulin derived from anti-

HCV-positive serum.13 The recent demonstration that humanized anti-E2 antibodies also have the potential to neutralize HCV encouraged the idea that passive immunization against HCV is possible.14 Nevertheless, for a prophylactic vaccine to successfully provide broad protection against HCV, it will need to include multiple cross-protective neutralizing epitopes. Only a handful of promising neutralizing epitopes have been reported and some have shown the potential to elicit crossreactive antibodies to viral envelope proteins.12,15–19 Monoclonal antibodies (mAb) directed to epitopes on the E2 glycoprotein are able to block cell entry of human immunodeficieny virus (HIV)/HCV pseudotypic particles (HCVpps) bearing the E2 glycoprotein of HCV H77 genotype 1a11 and to significantly neutralize cell culture-derived HCV.12 In this study, we have tested the functional potential of antibodies elicited by synthetic peptides representing epitopes described by Hsu et al.,11 which occur within and downstream of HVR1 as well as two HVR1 sequences reported by Farci and co-workers.10 In order to enhance immunogenicity, the synthetic peptides were ligated to a T-helper (Th) epitope and the antibodies induced were tested for their

1Department of Medicine, Royal Melbourne Hospital, The University of Melbourne, Parkville, Victoria, Australia; 2Department of Microbiology and Immunology, The University of Melbourne, Parkville, Victoria, Australia and 3The Burnet Institute, Alfred Medical Research and Education Precinct, Prahran, Victoria, Australia Correspondence: Dr DC Jackson, Department of Microbiology and Immunology, The University of Melbourne, Parkville, Victoria 3010, Australia. E-mail: [email protected]; Dr J Torresi, Department of Medicine (RMH/WH), The University of Melbourne, Post Office, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia. E-mail: [email protected] Received 13 July 2006; accepted 17 August 2006; published online 23 January 2007

HCV epitope-based vaccines J Torresi et al 170

ability to recognize E2 expressed on the surface of transfected 293T cells and also for their ability to inhibit HCVpp cell entry.

hierarchy of antibody binding to E2 expressed at the cell surface was A4E4D4B44C¼F.

RESULTS Immunogenicity of epitope-based immunogens Synthetic peptides A (full-length HVR1) and B (HVR1 truncated by six amino acids from the N-terminus) when linked to the Th epitope were strongly immunogenic when compared to peptides lacking Th (Figure 1). A second dose of peptide A or B-based vaccine administered in Freund’s incomplete adjuvant failed to produce any further rise in antibody titres (results not shown). In light of the enhanced antibody production following inoculation with HVR1 epitopes ligated to Th only E2 peptides coupled to the helper epitope were analysed further. Significant antibody titres were obtained with vaccine candidates based on epitopes C, D, E and F, although lower antibody titres were obtained with Th-D and Th-E than with Th-C or Th-F. All of the titres obtained with these epitopebased vaccines, however, were lower than those obtained with Th-A and Th-B (Figure 1). It should be noted, however, that the solubilities of peptides C, D, E and F alone were much lower than the solubilities of peptides A and B, which in all likelihood resulted in poor antigen coating in ELISA. For this reason and also to examine the biological activity of any anti-peptide epitope antibodies that were elicited by the six vaccine candidates, we examined the abilities of the antisera to bind to E2 protein in two different biological systems.

HCVpp cell entry assay The anti-peptide sera were also tested for their ability to inhibit the entry of HCVpp’s, containing the homologous E1E2 sequence of HCV H77c, into Huh-7 cells (Figure 2). Inhibition of HCVpp cell entry was achieved with a serum dilution of 1/100, whereas a dilution of 1/200 failed to neutralize the HCVpp’s. Even though the anti-Th-C antibodies failed to bind to E2 protein expressed at the cell surface, in this assay, these antibodies demonstrated inhibitory activity similar to the antibodies that were elicited by epitopes A and D. Antibodies directed against epitopes B and F were least effective in inhibiting HCVpp entry.

59±0.03 28±1.8

Th-C Th-D

None detected 29±4.3

Th-E Th-F

39±2.7 None detected

H53

82±12

Virogen anti-E2 Naı¨ve serum

55±10 3±2.0

antibody dilution of 1/50 was used for each primary antibody.

-D

Th

-E

Th

-F

Th

Figure 1 Antibody titres obtained by ELISA in mice inoculated with various epitope-based vaccine candidates. Groups of five mice were inoculated subcutaneously in the base of tail with three 20 nmol doses of peptides A, B, Th-A, Th-B, Th-C, Th-D, Th-E and Th-F. All mice were bled and reinoculated on days 28 and 38, followed by a final bleed on day 48. Antibody titres were detected in sera by ELISA using the appropriate peptides, but lacking the Th epitope, as coating antigens. Titres have been normalized relative to the appropriate pre-immune serum and are shown for day 28 only. Immunology and Cell Biology

0 T h -F Pr eim m un e

-C

Th

-E

-B

Th

h

-A

Th

T

B

-D

A

10

h

0.0

20

T

0.5

-C

1.0

30

h

1.5

40

T

2.0

50

-B

2.5

60

h

3.0

70

T

aAn

-A

10

Th-A Th-B

h

3.5

% positive cellsa mean (±s.d.)

Inoculating peptide

T

4.0 Antibody Titre (log )

Table 1 Binding of antibodies induced by epitope-based vaccine candidates to HCV E2 protein expressed at the surface of transfected 293 T cells

% inhibition of HCVpp cell entry

Binding of anti-Th-E2 peptide antibodies to E2 expressed at the cell surface The ability of antibodies directed to the E2 peptides to recognize and bind to homologous E2 protein expressed on the surface of E1E2transfected HEK 293T cells was determined by flow cytometry (Table 1). Two positive control antibodies, MAb and H53, which recognize the properly folded form of E2,20 and a polyclonal antibody directed to E2 protein (Virogen) bound to 82 and 55% of transfected cells, respectively, and the level of binding of non-immune mouse serum was 3%. Antisera from mice inoculated with Th-A, Th-B and Th-E all bound strongly to E2 expressed at the cell surface, but no binding was detected with anti-Th-C or anti-Th-F antiserum. The

DISCUSSION Identifying epitopes on the HCV E2 glycoprotein that can elicit neutralizing antibody responses could advance the development of an effective vaccine against HCV. In this study, we have shown that such epitopes can be delivered in an immunogenic form and that

Figure 2 Inhibition of HCVpp entry into Huh-7 cells by antibodies produced against the various epitope-based vaccine candidates. Anti-peptide antiserum were diluted 1/100 and incubated with HCVpps for 1 h at room temperature and then added to Huh-7 cells. HCVpp entry was assessed by measuring luciferase activity. The results show the mean percentage entry from quadruplicate assays. Pre-immune mouse sera served as a negative control.


they are capable of eliciting biologically active antibodies against homologous HCV E2 sequences. Peptides A and B when inoculated alone induced low-titre antibody responses, indicating that they do contain a helper T-cell epitope. Nevertheless, when these peptides were ligated to a helper T-cell epitope Th derived from a non-HCV protein, both induced a much stronger antibody response. Each of the HCV epitopes when ligated to Th were immunogenic in mice. Although Th-D appeared to produce low antibody titres when analyzed by ELISA, this peptide clearly induced antibodies that were able to bind to E2 expressed at the cell surface and also neutralized HCVpp entry into cells. Relatively low-antibody titres in ELISA were detected in response to inoculation with Th-C, Th-D, Th-E and Th-F. These low antibody titres detected, however, could have been due to the poor solubility of the antibody epitopes, which were used to coat ELISA plates. The ability of antibodies elicited by the HVR1 peptide constructs to bind to E2 expressed at the cell surface provides one means of assessing the ability of antibodies to bind to epitopes within the envelope proteins of HCV.11,21 When ligated to Th the full-length HVR1, peptide A and the truncated HVR1 peptide B, each, induced antibodies that were capable of binding to the cell surface of a high proportion of E1E2-transfected cells. These findings are in contrast to those of Farci et al.10 who showed that only antibodies raised to the truncated HVR1 and not antibodies raised to the full-length HVR1, were able to bind to E2 expressed on the cell surface. We have also demonstrated that antibodies produced against the full-length HVR1 sequence, when coupled to Th, inhibited HCVpp entry far more efficiently than antibodies raised to the truncated HVR1 sequence. Farci et al.10 reported that antibodies to the truncated HVR1 protected chimpanzees from infection with HCV, but these investigators did not test the neutralizing potential of full-length HVR1. Our findings, therefore, suggest that the six amino acids at the N-terminus of HVR1 are important in eliciting neutralizing antibodies. The epitope represented by peptide C is highly conserved between genotypes 1a, 1b, 2a, 3a, 4a, 5a, 6a, 10a and 11a (Table 2). Minor sequence variations exist in the first five amino-acid residues, whereas the last seven amino acids of this sequence are highly conserved. The importance of epitope C as a neutralizing epitope has also recently been highlighted by Tarr et al.12 and our results demonstrate that antibodies raised to Th-C were among the best inhibitors of HCVpp entry. The conservation of this epitope sequence across genotypes, together with the potential neutralizing activity that it possesses, argues that this epitope is a useful sequence to include in a vaccine formulation. It is interesting to note, however, that antibodies directed to Th-C did not bind to cells expressing E1E2. Inability to correlate functional antibody activity (in this study the inhibition of

HCVpp entry into cells) with a more general immunoreactivity of antibody (measured in this case by the lack of antibody binding in ELISA and the inability to bind to E1E2-transfected cells) has also been reported by Steinman et al.22 The sequence of peptide E is poorly conserved (Table 2), but mice inoculated with this peptide did elicit antibodies that bound to E2 expressed at the cell surface and also inhibited HCVpp entry. The epitope represented by peptide D is downstream of HVR1 and overlaps epitope F. Antibodies directed to Th-D bound to E2 expressed at the cell surface and also inhibited HCVpp entry. In contrast, antibodies raised to Th-F did not bind to E2 expressed at the cell surface and failed to inhibit HCVpp entry. Together, these results suggest that the amino-acid sequence between amino acids 423–436 could contain important epitopes which elicit potential neutralizing antibodies. In conclusion, we have demonstrated the neutralizing potential that can be achieved with synthetic vaccines based on epitopes of HCV. The vaccine candidate Th-A was soluble, highly immunogenic and antibodies raised to this peptide are potentially neutralizing. However, because of the variation that occurs in the HVR1, this peptide may only provide protection against homologous or closely related HCV.12 Of particular interest are the epitopes represented by peptides C and D, which are conserved between HCV genotypes. Both peptides elicited antibodies that are potentially neutralizing. The fact that these epitope sequences are conserved between genotypes suggests that antibodies to them may be cross protective making both epitopes promising vaccine candidates. METHODS Chemicals All chemicals were of analytical grade or its equivalent. N,N¢-dimethylformamide, piperidine, trifluoroacetic acid (TFA), O’benzotriazole-N,N,N¢,N¢-tetramethyl-uronium-hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBT) and diisopropylethylamine (DIPEA) were obtained from Auspep Pty. Ltd. (Melbourne, Australia) and Sigma-Aldrich Pty. Ltd. (Castle Hill, Australia). Dichloromethane (DCM) and diethylether were from Merck Pty. Ltd. (Kilsyth, Australia). Phenol and tri-isopropylsilane (TIPS) were from Aldrich (Milwaulke, WI, USA) and trinitrobenzylsulfonic acid (TNBSA) is from Fluka, Switzerland and 1,8-diazabicyclo-[5.4.0]undec-7-ene (DBU) was obtained from Sigma.

Peptide synthesis Peptides representing B-cell epitopes from within E2 (Figure 3, Tables 2 and 3) were synthesized, as previously described.23,24 Briefly, peptides were assembled on the solid phase using Fmoc chemistry either manually or using an automatic peptide synthesizer. Each peptide was assembled in the carboxamide form, purified using reverse-phase high-performance liquid chromatography (HPLC) and characterized using analytical reverse-phase HPLC and mass spectrometry. Six peptide sequences (Table 3) derived from the E2 protein of H77 HCV

Table 2 Sequence comparison of HCV genotype 1a H77 12-mer peptide sequences used in this study with other HCV genotypes HCV genotype

Peptide C (E2 aa 412–423)

Peptide D (E2 aa 432–443)

Peptide E (E2 aa 396–407)

Peptide F (E2 aa 436–447)

1a 1b

QLINTNGSWHLN --V---------

SLNTGWLAGLFY --Q--FI-A---

TAGLVGLLTPGA -S---S-FSA--

GWLAGLFYHHKF -FI-A---A-RF

2a 3a

------------V----------

--H--F--S---I---FI-----

ART-T-MFSL--QAFA--FDI-P

-F--S---T-SF -FI-----Y--F

4a 5a

--S----------S----I----

-----F--S----Q--FI---M-

LKS-TSFFN--P ----AN-FSS-S

-F--S---T--F -FI---M-A---

6a

------------

--Q--F--S---

-S-FAS------

-F--S---T--F

10 11a

H-V--------------------

-----FI---L--Q--FI-S- --

AFTITS-FST--QS--SW-SQ-P

-FI---L-Y--F -FI-S---RN-F

Abbreviation: HCV, hepatitis C virus.

Immunology and Cell Biology

HCV epitope-based vaccines J Torresi et al 172 genotype 1a10–12 were made. All peptides were assembled with an N-terminal serine to allow formation of an aldehyde group to facilitate oxime bond formation.25 The purified peptides were ligated to the Th epitope KLIPNASLIENCTKAEL, derived from the fusion protein of the Morbillivirus canine distemper virus26 using oxime chemistry as previously described.25 In this way, vaccine candidates were constructed by the ligation of the N-terminus of an HCV antibody epitope to the N-terminus of the Th epitope. Fidelity of synthesis of the vaccine candidates was determined by mass spectrometry using an Agilent 1100 Series ion trap mass spectrometer configured in the positive mode using electrospray as the ion source. Experimentally determined masses were comparable to the theoretical masses (Table 3), except for the value obtained for peptide E, which indicated dehydration of asparagine and/or glutamine residues with concomitant loss of a water molecule (mass 18).

Immunization protocol Groups of five female BALB/C mice were inoculated by the subcutaneous route into the base of tail with 20 nmol immunogen emulsified in an equal volume of complete Freunds adjuvant (CFA; Sigma) in phosphate-buffered saline (PBS). Incomplete Freund’s adjuvant (IFA) was used to emulsify subsequent doses of immunogen. All mice were bled and boosted on days 28 and 38 followed by a final bleed on day 48. All procedures carried out on animals were approved by the Animal Ethics Committee, The University of Melbourne.

Enzyme-linked immunoabsorbent assay (ELISA) A previously described,27 ELISA was used to determine the antibody responses to the synthetic peptide using peptides as coating antigens. Titres of serum antibody were expressed as the reciprocal of the highest dilution of serum required to obtain an optical density (OD) of 0.2, which represents five times the OD obtained in the presence of preimmune serum.

Immunofluorescence of E2 expressed at the cell surface Human embryonic kidney (HEK) 293T cells were grown in Dulbecco’s modified Eagle’s medium (DMEM), containing 10% fetal calf serum (FCS;

HCVpp cell entry assay

E2

363

1 HVR1 A B

1 29

C D E

27 27

7

40 49

13

CSL Ltd, Parkville, VIC, Australia) at 371C with 5% CO2. We seeded 3105 293T cells into six-well plates (Nunc, Roskilde, Denmark) and incubated overnight at 371C with 5% CO2 (v/v). Cells were then transfected with 2 mg of either pcDNAHisMaxC or pcDNAHisMaxC E1E2 (containing the homologous E2 sequence of HCV H77c), using Fugene 6 Transfection Reagent (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. Transfections were performed with a DNA:Fugene ratio of 1:3. Briefly, Fugene transfection reagent was diluted in OptiMEM (Gibco, Grand Island, NY, USA) and incubated for 5 min at room temperature. DNA (2 mg) was added to the OptiMEM-Fugene mix and incubated at room temperature for 15 min. The DNA–Fugene complex was added dropwise to the cells, followed by incubation at 371C with 5% CO2. After 48 h, cells were harvested in fluorescence-activated cell sorter (FACS) buffer (1% bovine serum albumin, 0.02% sodium azide in PBS), sedimented by centrifugation at 460 g for 4 min at 41C, followed by washing once with FACS buffer. With the appropriate anti-peptide mouse sera diluted 1/25 to 1/100 in FACS buffer to a total volume of 200 ml, 5105 cells were stained for 30 min at 41C and in the dark. For comparison, cells were also stained with either a 1/100 dilution of Virogen goat anti-E2 polyclonal antibody (Virostat, ME, USA) or a 1/100 dilution of mouse monoclonal antibody H53 (obtained from Dr J Dubuisson, Institut de Biologie and Institut Pasteur de Lille, Lille, France), which recognizes a conformational epitope on E2. Following staining, cells were washed twice with FACS buffer and any binding of antibodies detected with a 1/500 dilution of Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes, Leiden, The Netherlands) or in the case of the goat anti-E2 antibody with a 1/500 dilution of Alexa Fluor 488 chicken anti-goat IgG (Molecular Probes). Cells were stained for 30 min at 41C and in the dark, followed by washing twice with FACS buffer. Cells were then resuspended in 200 ml of Buffer 1. Binding was detected by measuring the number of fluorescent cells using flow cytometric analysis with a FACS Calibur (Becton Dickinson, Bedford, MA, USA). Results were analyzed using the software package FlowJo (Tree Star Inc., Ashland, OR, USA) and mean and standard deviation were determined on samples analysed in triplicate.

60

The assay was carried out according to Drummer et al.21 Briefly, 293T cells seeded at 350 000 cells/well in six-well culture dishes were transfected with 1 mg each of NL4–3.LUC.R-E- and either pE1E2H77c or pCDNA4HisMax. Three days later, the culture supernatant containing pseudotyped HIV-1 particles was collected and filtered (0.45 mm). This was added to Huh-7 cells (30 000/well) in 48-well culture plates together with a 1/100 or 1/200 dilution of peptide antiserum. Following 7 h incubation at 371C, the inoculum was removed and the cells were cultured for a further 3 days. Luciferase activity was measured in a Microlumat LB (Berthold) luminometer using the Promega luciferase reagent system.

24

F

53

64

Statistical analyses

Figure 3 Schematic diagram showing the arrangement of the epitope sequences within E2 protein that were used to assemble vaccine candidates.

Statistical analyses were performed using a non-parametric, one-tailed Mann–Whitney’s test with 95% confidence interval.

Table 3 Theoretical and experimentally determined masses of individual synthetic peptides Peptide mass Epitope sequence

E2 amino acid

Final mass (HCV epitope+Th)

Acronym Theoretical

Determined

Theoretical

Determined

ETHVYGGSAGRTTAGLVGLLTPGAKQN

384–414

A

2592.4

2592.9

4559.2

4559.6

GSAGRTTAGLVGLLTPGAKQN QLINTNGSWHIN

390–414 412–423

B C

1968.1 1395.7

1968.2 1394.8

3934.9 3362.5

3935.2 3361.5

SLNTGWLAGLFY TAGLVGLLTPGA

432–443 396–407

D E

1340.7 1068.6

1339.8 1068.7

3307.9 3053.4

3307.5 3034.8

GWLAGLFYQHKF KLIPNASLIENCTKAEL

436–447

F Th

1465.8 1928.0

1465.0 1928.8

3432.6

3432.8

Abbreviation: HCV, hepatitis C virus.



ACKNOWLEDGEMENTS We thank Dr Jean Dubuisson, Institut de Biologie de Lille & Institut Pasteur de Lille, Lille, France, for the generous provision of some antibody reagents. This work was supported by grants from the National Health & Medical Research Council of Australia and the Australian Center for HIV and Hepatitis Virology.

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