Immunization of chimpanzees with an envelope protein-based vaccine enhances specific humoral and cellular immune responses that delay hepatitis C virus infection

Vaccine 22 (2004) 991–1000

Immunization of chimpanzees with an envelope protein-based vaccine enhances specific humoral and cellular immune responses that delay hepatitis C virus infection Montserrat Puig, Marian E. Major, Kathleen Mihalik, Stephen M. Feinstone∗ Laboratory of Hepatitis Viruses, Division of Viral Products, CBER, FDA, Building 29A, Room 1D02, 8800 Rockville Pike, Bethesda, MD 20892, USA Received 7 May 2003; received in revised form 15 August 2003; accepted 3 September 2003

Abstract Two chimpanzees, one na¨ıve (Ch1601) and one recovered from hepatitis C virus (HCV) acute infection (Ch1587), were vaccinated with recombinant envelope glycoproteins (E1E2) and then challenged with 100 CID50 of HCV. Results of the challenge were compared to infection in a non-vaccinated control animal. Immunization generated high antibody titers to E1E2 including antibody specifically directed to the hypervariable region 1 (HVR1) in addition to strong and specific HVR1 T-cell proliferative responses. Upon challenge with HCV, viremia was delayed 3 weeks in both vaccinated animals compared to the non-immunized (control) animal. Ch1601 HCV RNA titers were maintained below 5×104 copies/ml, and alanine aminotransferase levels were only minimally elevated. An increase in intrahepatic cytokine mRNA levels coincided with a fall in HCV RNA to non-quantifiable levels. Despite this apparent control of virus replication the animal became persistently infected. Ch1587 had a significantly shorter and milder viremia, compared to the re-infection of the non-vaccinated control animal. This data indicates that a strategy inducing a T-cell immune response combined with antibody responses to E1E2 would make a viable candidate for an HCV vaccine. Published by Elsevier Ltd. Keywords: Hepatitis C; Vaccine; Envelope

1. Introduction Hepatitis C virus (HCV) is responsible for both acute and chronic infections of the liver. A minority of individuals successfully resolve acute infection, however, up to 85% of patients develop persistent infection possibly due to the lack of control by the innate or the adaptive immune systems [1–4]. These persistent infections may result in chronic hepatitis, cirrhosis, and hepatocellular cancer [5,6]. HCV vaccine development has been difficult for several reasons. There is no small animal model or in vitro replication system available to produce large quantities of virus, to study viral replication and assay immune responses. Additionally, there is an incomplete understanding of the immunologic mechanisms that control the virus during natural infections. HCV is highly heterogeneous with six genotypes and multiple subtypes and the virus exists in chronically infected individuals as a quasi-species population, with multiple, but related sequences. This quasi-species variability as well as de novo mutations may allow for selection of ∗

Corresponding author. +1-301-8271880; fax: +1-301-4025585. E-mail address: [email protected] (S.M. Feinstone).

0264-410X/$ – see front matter Published by Elsevier Ltd. doi:10.1016/j.vaccine.2003.09.010

variants that can escape the host immune response and lead to or maintain chronic infections [7]. Typically, the envelope glycoproteins (E1E2) would be the primary target for antibody induced virus neutralization and there is evidence that antibodies to E1E2 have a capacity to neutralize HCV in the chimpanzee model of infection [8–11]. However, while antibodies to E1E2 are frequently present in the serum of patients and chimpanzees with chronic HCV infection, they are rarely detected in chimpanzees that resolve the infection [12]. The E2 glycoprotein contains a region at the amino terminus consisting of approximately 27 amino acids termed the hypervariable region 1 (HVR1). There are also data suggesting that antibody to this region can neutralize HCV and that the virus can escape this neutralization by the selection of a virus containing non-neutralizable HVR1 variants [13]. We designed an envelope protein (E1E2)-based vaccine to generate antibodies against viral surface epitopes and potential neutralizing antibodies to the HVR1 sequence. In order to study the effect of this vaccine, two chimpanzees were vaccinated and later challenged with a clonal HCV, which perfectly matched the sequence of the proteins used in the vaccine.

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Previous reports and our own experience have shown that re-infection with the same or other viral strains is possible in individuals as well as chimpanzees [14,15], although these second infections are generally of a shorter duration and are often subclinical [16,17]. This would suggest that a memory immune response is induced during primary infection [16,17]. Cellular immune responses are believed to play an important role in the successful resolution of acute infections. An early, strong and multispecific cytotoxic T lymphocyte (CTL) and CD4+ helper T-cell responses are associated with viral clearance in both humans and chimpanzees [18–21]. Therefore, the study of immunization plus HCV infection of a na¨ıve compared to a recovered chimpanzee would assess the role of memory T-cell responses. We hypothesized that if we could generate an anti-envelope antibody response in addition to the memory responses developed during the primary infection of a recovered chimpanzee, the animal might completely resist HCV re-infection. A non-vaccinated, na¨ıve chimpanzee served as the control for comparison of the virological and clinical responses to the post-vaccine HCV challenge.

2. Materials and methods 2.1. Animals Three na¨ıve chimpanzees (Pan troglodytes) were included in the study (Table 1). The housing, maintenance, and care of the chimpanzees used in the study were in compliance with all relevant guidelines and requirements. All protocols were approved by the CBER/FDA Animal Care and Use Committee. 2.1.1. Ch1605: na¨ıve non-vaccinated control Ch1605 was inoculated with 3.2 50% chimpanzee infectious doses (CID50 ) of the clonal HCV derived from HCV-H (see below). The animal became infected, cleared the virus and 10 months later was re-inoculated with 100 CID50 of the same virus. 2.1.2. Ch1601: na¨ıve animal vaccinated with rE1E2 Ch1601 was challenged 2 weeks after the last vaccine dose with 100 CID50 of the clonal HCV.

Table 1 Animals and inoculations

Ch1605 Ch1601 Ch1587 a See

Status

Vaccinea

HCV challenge (CID50 )

HCV re-challenge (CID50 )

Na¨ıve Na¨ıve Recovered

None DNA/peptide rE1E2 rE1E2

3.2 100 100

100

dosage in Section 2.3.

2.1.3. Ch1587: recovered animal vaccinated with rE1E2 Ch1587 was initially infected with HCV-H plasma containing 64 CID50 [11] and cleared this infection. Two years after recovery, Ch1587 was vaccinated with rE1E2 and re-challenged with 100 CID50 of the clonal HCV. 2.2. E1E2-based vaccine Recombinant E1E2 (aa192–715) was produced using a Sindbis virus expression system as described elsewhere [22] and purified using a GNL Lectin column (Vector Laboratories, Inc., Burlingame, CA) and a His-Bind Ni2+ column (Novagen, Inc., Madison, WI). Specificity and purity (>80%) were determined by Western blotting and silver staining of proteins separated on polyacrylamide gels (data not shown). 2.3. Vaccination of chimpanzees 2.3.1. Ch1601 Ch1601 was vaccinated intra-muscularly (i.m.) with 1 mg of plasmid DNA expressing the HCV envelope proteins under the CMV promoter (three doses, at 0, 15 and 20 weeks). HVR1 peptide complexed with alum (amino acid positions 391–410: SAGRTTAGLVGLLTPGAKQN) was given i.m. at 2 mg per dose, at 5 and 10 weeks. This priming regimen generated weak humoral and cellular immune responses. At 6 weeks after the last DNA immunization, Ch1601 was boosted i.m. with 25 ␮g of E1E2 antigen combined with RIBIs adjuvant (Corixa, Hamilton, MT) at weeks 0, 2, 8, 9, 35 and 39. This long-term immunization regimen was employed to assess the stability of the antibody response and the time post boost that peak titers were obtained. 2.3.2. Ch1587 Ch1587 was immunized three times (weeks 0, 4 and 10) with the E1E2 antigen combined with TiterMax adjuvant (CytRx Corp., Norcross, GA). A different adjuvant was used because Ch1601 developed granulomas at the injection site due to RIBIs adjuvant. E1E2-immunizations consisted of 25 ␮g of protein per dose, administered i.m. 2.4. Challenge inoculum The HCV used for the challenge inoculum was obtained from plasma collected during the early acute phase of infection in a chimpanzee inoculated with RNA transcribed from an HCV1a (HCV-H) infectious clone [23]. The virus in the plasma was quantified by reverse titration in a single chimpanzee [17]. The virus in this inoculum was found to be identical to the original infectious RNA by sequence analysis and no quasi-species variation was detected [22]. The same sequence was used to generate the recombinant proteins for the vaccine.

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2.5. Tissue samples and biochemical tests The chimpanzees were monitored for the development of hepatitis by serum ALT levels and HCV RNA in liver and serum by reverse transcription nested RT-PCR and real time quantitative RT-PCR (Perkin-Elmer Applied Biosystems, Foster City) as described elsewhere [22,24]. HCV RNA titers during the first infection in Ch1587 were monitored by quantitative PCR as described by Yu et al. [11]. 2.6. Antibody assays Antibody responses in the chimpanzees were evaluated by several different assays: a commercial EIA antibody test (ORTHO HCV Version 3.0 Ortho Diagnostics, Raritan, NJ), and in-house enzyme-linked immunosorbent assays (ELISA) [22]. Antibody to HVR1 was assessed using a biotinylated peptide ELISA [22]. E1E2 antigens for anti-E1E2 antibody analysis were purified using a GNL Lectin column (Vector Laboratories, Inc.) from BHK cells infected with a recombinant vaccinia virus expressing E1E2 of HCV. Anti-NS3, NS5A and NS5B antibodies were detected using bacterial-origin, purified proteins (MIKROGEN GmbH, Germany). ELISAs were carried out as previously described [22]. 2.7. Peripheral blood mononuclear cells (PBMC) preparation PBMC were isolated from total blood by standard laboratory procedures, using LymphoprepTM (Nycomed Pharma, Oslo, Norway) density gradients. Cells were washed twice with 1× PBS or, alternatively, once with 1× PBS and twice with RPMI-10 [RPMI 1640 (Biofluids, Rockville, MD) supplemented with 10% heat-inactivated fetal calf serum, 2 mM l-glutamine, 100 U/ml penicillin, 100 ␮g/ml streptomycin, 25 mM HEPES, and 50 ␮M ␤-mercaptoethanol]. Cells were frozen at concentrations of 107 cells/ml in cell culture freezing medium-DMSO (Invitrogen, Carlsbad, CA). 2.8. Proliferation assay T-cell proliferation assays were performed using thawed PBMCs in 96-well plates (105 cells/well) in 200 ␮l of RPMI-5 AB [RPMI 1640 (Biofluids) supplemented with 5% heat-inactivated human AB serum, 2 mM l-glutamine, 100 U/ml penicillin, 100 ␮g/ml streptomycin, 25 mM HEPES, and 50 ␮M ␤-mercaptoethanol]. Culture of Ch1601 PBMCs was carried out in RPMI-10 AB [RPMI 1640 (Biofluids) supplemented with 10% heat-inactivated human AB serum (Bio-Whittaker, Walkersville, MD), 2 mM l-glutamine, 100 U/ml penicillin, 100 ␮g/ml streptomycin]. Cells were stimulated with 1 ␮g/ml protein (bacterial-origin, purified NS3, NS5A, NS5B proteins (MIKROGEN) and

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rE1E2), and 10 ␮g/ml HVR1 peptide, in three replicate cultures per stimulation condition. After 5 days of incubation, 1 ␮Ci of [3 H]thymidine (Amersham Biosciences, Buckinghamshire, England) was added to each well and cells were harvested 18 h later. Stimulation index (SI) was calculated by dividing the stimulated culture counts by the non-stimulated culture counts. 2.9. Cytokine assays Intrahepatic levels of interferon gamma (IFN-␥), tumor necrosis factor alpha (TNF-␣), and interleukin (IL)-10 were analyzed using real time PCR as previously described [17]. Relative mRNA quantification was normalized to an endogenous reference (human glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) and expressed relative to a calibrator (a liver biopsy taken prior to HCV inoculation) as previously described [17]. 3. Results 3.1. Effect of the vaccine on primary HCV infection One na¨ıve chimpanzee (Ch1601) was vaccinated with rE1E2 vaccine and challenged with HCV to assess the effect of primary immunization. The outcome of the infection was compared to the disease profile in two non-vaccinated na¨ıve chimpanzees (Ch1605 and Ch1587) (Fig. 1). Ch1605 was inoculated with 3.2 CID50 of a clonal HCV [17]. HCV RNA was first detected in serum at week 1 post-inoculation (p.i.) (3.2 × 104 RNA copies/ml) (Fig. 1A) and peaked at week 7 p.i. (1.1 × 106 RNA copies/ml). The maximum ALT level was 176 IU/l, 2 weeks after the RNA peak, and coincided with a significant control of the infection as assessed by a decrease of almost 3 log10 in the RNA level in peripheral blood. While viremia was quantifiable to week 16 p.i. by real-time PCR (cut-off of 300 RNA copies/ml), low levels of RNA were detected by the more sensitive nested RT-PCR method (cut-off of 40 RNA copies/ml) for more than 1 year (week 58 p.i.). Ch1587 was first inoculated with HCV 2 years before the initiation of this vaccination study [11]. Results are shown in Fig. 1B, HCV RNA was quantified by nested PCR and expressed in PCR equivalents/ml. Following the inoculation with 64 CID50 of HCV-H, HCV RNA was detected at week 1 p.i. The HCV RNA titer peaked at 5 × 105 genome equivalents/ml on week 11 p.i. Immediately after the HCV RNA peak, the ALT levels reached maximum values (150 IU/l), coinciding with the decrease in the viral load in serum. Low levels of RNA were detected intermittently for a period of 34 weeks. By week 60 HCV RNA was consistently undetectable. Ch1587 remained negative for more than 1 year. Ch1605 and Ch1587 seroconverted (EIA 3.0 positive) at the time of the ALT peak, week 9 (Fig. 1A) and week 17 (Fig. 1B), respectively.

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(A)

(B)

Ch1587

Ch1605 200

7

EIA 3.0 (+)

7

EIA 3.0 (+)

4 3

80

2

5 120 ALT (IU/L)

120

Log10 RNA copies/ml

5

6

160

40

4 3

80

2 40

1

1 0

0 0

2

4

6

8

10 12 14 16 18 20 22 24 58

0

90

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90

Week p.i.

Log10 RNA PCR equivalents/ml

6

160

ALT (IU/L)

200

0

Week p.i.

(C)

Ch1601

200

EIA 3.0 (+)

7 6

ALT (IU/L)

5 120

4

80

3 2

40

Log10 RNA copies/ml

160

1 0

0 0

2

4

6

8 10 12 14 16 18 20 22 24 26 28 30

Week p.i.

Fig. 1. Disease profile of two na¨ıve chimpanzees (A and B) vs. one vaccinated na¨ıve chimpanzee (C) after inoculation with HCV. Ch1605 and Ch1601 were inoculated with clonal HCV. Ch1587 was challenged with HCV-H plasma. RNA titers were measured by real-time RT-PCR in Ch1605 and Ch1601, and by quantitative PCR in Ch1587 [11] (triangles). ALT elevations are represented with white circles. HCV seroconversion was analyzed by EIA 3.0 (gray bars). Inoculation of the animals with HCV was carried out at week 0.

Ch1601 was initially immunized i.m. with a DNA plasmid (1 mg) expressing HCV envelope proteins and by an HVR1 peptide complex, as described in the methods section. Those treatments generated weak humoral and cellular immune responses in the animal. Later the animal was vaccinated with the protein-based vaccine, consisting of six consecutive doses of rE1E2. After challenge with 100 CID50 , HCV RNA was not detectable until 3 weeks p.i with a titer of 5 × 104 RNA copies/ml. This appearance of HCV was preceded by a decline in the anti-E1E2 antibody levels (Figs. 1C and 2A). The serum HCV RNA titer in Ch1601 did not continue to increase rapidly as is normally seen in na¨ıve chimpanzees but remained at ∼104 copies/ml for 11 weeks. RNA levels fell and were not quantifiable by real-time PCR (<300 copies/ml) during the subsequent 2 weeks. However, the control of the infection was temporary and the animal became persistently viremic at week 19 p.i. During the follow-up, ALT levels never exceeded 50 IU/l. The animal seroconverted (EIA 3.0 positive) at week 12 p.i.

3.2. Humoral responses generated by the vaccine and control of the disease We assessed the degree of protection obtained by the induction of high levels of anti-E1E2 and anti-HVR1 antibodies in vaccinated animals (Ch1601 and C1587) versus a non-vaccinated animal (Ch1605). Additionally, we compared the vaccination of the na¨ıve chimpanzee (Ch1601) with the recovered chimpanzee (Ch1587) to study the contribution of memory responses coupled with induced antibody on the protection of the animals to infection. Analysis of the pattern of antibody titer in Ch1601 indicated that it reached a maximum value greater than 1:104 , 2 weeks after the fourth immunization with rE1E2 but rapidly declined, reaching background levels after 22 weeks (data not shown). At this point, Ch1601 was boosted twice more, 6 and 2 weeks prior to challenge with virus. The chimpanzee received 100 CID50 of HCV when the antibody titer was again >1:104 (Fig. 2A). Viremia was controlled until week

M. Puig et al. / Vaccine 22 (2004) 991–1000

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7

VC

6

3 10

2

5

0

2

4

6

7

9

6

8 6

7

3 10

2

5 0

6

8

10 12 14 16 18 20 22 24 58 90

1 0 -22-20-18-16-14-12-10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 18

Week p.i.

10

7

9

6

8 7

5

6

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3

4 3

2

2

1

1 0

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Log10 RNA copies/ml

4

15

4

Ch1605 rechallenge

(D)

5

20

0 2

Weeks p.i.

6

25

1

0

P/N anti-E1E2 (1:10)

VC

2

3

0

Log10 RNA copies/ml

P/N anti-HVR1 (1:640)

V V

3

4

8 10 12 14 16 18 20 22 24 26 28

Ch1587 vaccination & rechallenge 30

4

5

Weeks p.i.

(C)

5

7

1

0

-68 -2

10

2

1

0

Ch1605 primary infection

Log10 RNA copies/ml

4

15

Log10 RNA copies/ml

5

20

P/N anti-HVR1 (1:10)

25 P/N anti-HVR1 (1:640)

(B)

Ch1601 vaccination & infection

995

0

Week p.i.

Fig. 2. HCV anti-HVR1 antibodies and RNA profiles during vaccination and challenge with HCV. (A and B) Na¨ıve chimpanzee (Ch1601, Ch1605 inoculation); (C and D) Recovered chimpanzees (Ch1587, Ch1605 re-challenge). Anti-HVR1 (A, B and C) and anti-E1E2 (D) antibodies were analyzed by in-house ELISA and expressed as P/N ratios (gray bars). P/N is calculated by dividing the OD405 of test sera by that obtained for a pre-vaccine sample from the same chimpanzee. Cut-off value is P/N = 2. Serum dilution tested is indicated in brackets. No antibodies were detected against HVR1 during any of the infections. RNA titers were measured by quantitative PCR (black triangles) and nested PCR (white box is negative, black box is positive). “V” represents vaccination with rE1E2. “C” represents challenge with clonal HCV.

3 p.i., and the animal became infected at the time that the HVR1 antibody titer had dropped to 1:5000. The animal showed a temporary control of the infection at week 17–18 p.i. (less than 300 RNA copies/ml in serum), however, at week 19, the viral titer increased and was persistently maintained (Fig. 2A). We did not observe any boost in envelope antibodies during this period. By comparison, the na¨ıve, non-vaccinated chimpanzee (Ch1605) became viremic at week 1 p.i. and did not developed antibodies against HVR1 at any point during the primary infection (Fig. 2B). Ch1587 recovered from a previous HCV infection 3 years prior to the vaccination experiment. At this point, this chimpanzee had antibody to HCV antigens as measured by the commercial EIA, but did not have detectable antibody to E1E2. Ch1587 was immunized with rE1E2 and seroconverted 1 week after the second dose of protein (Fig. 2C). The antibody titer, >1:104 (P/N = 20 at dilution 1:640),

remained stable for the next 14 weeks and was further boosted (P/N = 24.5 at dilution 1:640) when a third dose of vaccine was administered. The anti-envelope antibodies remained at a high level for the duration of the study. Four weeks after, the third dose of vaccine the animal was challenged with 100 CID50 of HCV. The E1E2 antibody response in this animal again delayed infection until 3 weeks post-challenge when a very brief (2 weeks), low-level viremia was observed (Fig. 2C). RNA was quantifiable by real-time PCR on only one date (week 3 p.i., 5 × 103 RNA copies/ml). The peak viral titer was 2log10 lower than the maximum titer observed during the primary infection of Ch1605 (Fig 1A). The ALT level remained normal (<24 IU/l) following challenge (data not shown). In contrast, the non-vaccinated animal Ch1605, after re-challenge, developed viremia at week 1 p.i. (5.7 × 104 RNA copies/ml), started controlling the infection by week 13 p.i., but

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Challenge Ch1601 vaccinated

(A)

V

Ch1605 control

V V C

25

25

20

20

15

N.A.

V

C

N.A.

15 10

5

5 0 -30

20 18 16 14 12 10 8 6 4 2 0

V

HVR-1 E1E2

10

0

Non-structural epitopes S.I.

V

30

Ch1605 control

Ch1587 vaccinated

(B)

-25

V

-18 -6

V

-2

2

6

10

14

-21 -19 -17 -15

VC

N.A.

-30

-25

-18 -6

-2

2

Week p.i.

6

10

14

20 18 16 14 12 10 8 6 4 2 0

V

V

-21 -19 -17 -15

-5 -3 -1

V

1

3

5

7

9 11 13 15

C

-5 -3 -1

20 18 16 14 12 10 8 6 4 2 0 1

Week p.i.

3

5

7

9 11 13 15

C NS3 NS5A NS5B

-5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Week p.i.

Fig. 3. T-cell specific proliferation in challenged (A) and re-challenged (B) animals, during rE1E2 vaccination and infection with HCV. T-cell specific proliferation to HVR1 peptide and E1E2 protein epitopes (upper graphs) and to non-structural proteins (NS3, NS5A and NS5B) (lower graphs). Data is expressed as a stimulation index (SI). Stimulation Index was calculated by dividing the stimulated culture counts by the non-stimulated culture counts. “V” represents vaccination with rE1E2. “C” represents challenge with clonal HCV.

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Structural epitopes S.I.

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remained HCV positive by nested PCR up to week 20 p.i (Fig. 2D). ALT levels were slightly elevated at week 2 p.i. (70 IU/l) but remained at normal levels during the remainder of the follow-up period (data not shown). 3.3. T-cell specific proliferation during vaccination and HCV infection In addition to the humoral response generated by the vaccine, other components of the immune system could be involved in the initial control and resolution of infection. Therefore we analyzed the efficacy of the vaccine to generate proliferative T-cell responses.

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In Ch1601, weak T-cell responses were detected following DNA priming but increased after immunization with rE1E2 proteins although the levels were not sustained (Fig. 3A weeks −30 to −6). Subsequent E1E2 boosts led to the stimulation of strong T helper responses against the HCV HVR1 peptide (SI = 13 at week −2) (Fig. 3A, upper graph). The HVR1 specific T helper responses were further enhanced following infection (Fig. 3A, upper graph), although this response did not correlate with increased levels of anti-E1E2 antibody (compare Figs. 2A and 3A). After the animal became persistently infected, the E1E2 region of the virus isolated from Ch1601 was sequenced and no nucleotide changes were observed (data not shown),

Fig. 4. Levels of cytokines in the liver of challenged (A and B) and re-challenged (C and D) animals. Intrahepatic IFN-␥, TNF-␣ and IL-10 mRNA levels were analyzed by real-time RT-PCR and represented as relative levels to the pre-vaccination sample. Time is expressed in weeks post-inoculation (p.i.). “V” represents vaccination with rE1E2. “C” represents challenge with clonal HCV.

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suggesting no epitope changes that would account for immune escape. Specific T-cell proliferation against non-structural protein epitopes was not detected in this animal (Fig. 3A, lower graph). Lack of PBMC samples from the Ch1605 primary infection made the analysis of T-cell proliferation in this animal impossible. In Ch1587, increases in the E1E2 specific proliferative T-cell response was observed following the second and third vaccine boosts (Fig. 3B, upper graph), coinciding with the increase in anti-HVR1 antibody titer (Fig. 2C). Those responses were not sustained, although the anti-HVR1 antibody levels remained high in the serum. At the time of challenge (week 0), T-cell proliferation detected in the peripheral blood was at basal levels, and did not increase until immediately after viral clearance (week 7 p.i.) (Fig. 3B, upper graph). Ch1587 showed specific NS3 T-cell proliferation post challenge, similar to the response observed in the control animal (Fig. 3B, lower graphs). NS5A and NS5-B did not significantly stimulate proliferation of T-cells in Ch1587, in contrast to what we observed in Ch1605 (Fig 3B, lower right panel). 3.4. Intrahepatic cytokine levels The immune response in the liver was addressed by studying the pattern of intrahepatic cytokines. IFN-␥, TNF-␣ (Th1-type cytokines) and IL-10 (Th2-type cytokine) were quantified by real-time PCR and results are shown in Fig. 4. Ch1601 intrahepatic Th1-type cytokines were low during the initial 13 weeks post challenge (Fig. 4A). However, IL-10 levels increased 4.8-fold immediately after challenge, coinciding with increases in proliferative T-cell responses to E1E2 (Fig. 3A). All the cytokines analyzed peaked at weeks 13–15 p.i. in this animal correlating with a decrease in virus titer to less than 300 copies/ml during weeks 17–18 p.i. (Fig. 2A). The cytokine levels decreased to baseline shortly after this time point, coinciding with a resurgence of virus and a persistent infection in this animal. In contrast, the control chimpanzee Ch1605 (Fig. 4B) maintained consistently elevated levels of IFN-␥ from week 6 p.i. (3.8-fold increase). At week 10, IFN-␥ levels peaked at a value 12 times higher than baseline, coinciding with the decrease of HCV RNA titer in the peripheral blood (Fig. 2B). TNF-␣ and IL-10 were maintained at baseline levels throughout the infection. Ch1587 (Fig. 4C) cytokine levels did not significantly increase prior to challenge (week 0), except for IFN-␥ at week −19 (4 weeks after the first E1E2 vaccine dose was administered). The maximum levels of IFN-␥ (95-fold increase) and TNF-␣ (3.8-fold increase) were observed at week 4 p.i., at the same time that HCV RNA was detected in the serum. The levels of IL-10 were not modified during the follow-up period (Fig. 4C). Following re-challenge of Ch1605, elevated Th1-type cytokine levels were immediately observed at weeks 2, 4 and 6 p.i. (Fig. 4D), the virus replication was controlled during this period although serum samples were intermittently positive for HCV RNA (Fig. 2D).

4. Discussion Successful prevention of many pathogen infections resides in production of efficient neutralizing antibodies against surface epitopes. The presence of neutralizing antibodies has been demonstrated in the plasma of HCV infected patients [11,13]. However, most chimpanzees that resolve infection do not elicit an antibody response to envelope proteins. Moreover, it has been reported that chimpanzees can be re-infected with the same strain of virus [14,15] although these animals have an increased ability to clear the virus possibly due to memory T-cell responses that become active in the liver immediately following infection [16,17,25]. Therefore, adding an antibody response through vaccination to the immune response mounted during the primary infection might be sufficient to completely protect the animal against infection (sterilizing immunity). In order to assess the levels of protection conferred by anti-E1E2 antibodies to HCV infection, we vaccinated chimpanzee 1601 (na¨ıve) and 1587 (recovered) with rE1E2 protein. In both animals, this vaccine was able to induce high levels of antibody and circulating T helper cells against E1E2 (and specifically against HVR1). The generated immune response delayed the infection for 3 weeks but was insufficient to completely protect the animals. In contrast, in both the primary and re-challenge infections of the control, non-vaccinated animal, Ch1605, viremia was quantifiable within 1 week of infection and no anti-envelope antibodies were detected in serum. In our experience, inoculation of na¨ıve or recovered chimpanzees with 100 CID50 of clonal virus has always resulted in infection within 1 week (M. Major, personal communication). Our data indicate that humoral and cellular responses to HVR1 as well as other E1E2 epitopes played a role in preventing infection during this early time period. It was not possible to compare the T cell response in the na¨ıve, vaccinated chimpanzee 1601 with the responses following primary infection in the na¨ıve, non-vaccinated chimpanzee 1605 because PBMCs were not available from this control animal. However, it has been widely reported that strong, broad and early T cell responses are associated with viral clearance [18–21]. Interestingly, Ch1601 became viremic when the antibody levels dropped to 1:5000, at the same time that the T-cell proliferation level decreased 4-fold. We used a clonal virus with no detectable quasi-species variation, and with the exact amino acid sequence as the immunogens used in the vaccine. Sequence analysis indicated that immune escape was not a factor in this infection. This pattern of infection occurring when anti-envelope antibody levels are low is similar to that seen in previously published vaccine experiments [8,10]. In a study published by Forns et al., DNA immunization alone was used to generate immune responses to HCV resulting in no delay of infection in either of the chimpanzees vaccinated [26]. In the liver, during the initial period of viremia in Ch1601, IL-10 mRNA levels were high, suggesting T-helper

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(Th2-type) activity. This could account for the control of virus replication during the acute phase of infection that maintained titers at below 105 RNA copies/ml. Those HCV RNA levels were lower than what we would expect for naive non-vaccinated animals (like Ch1605) in which RNA titers increase during the first 6–8 weeks of infection, reaching peak titers of at least 106 RNA copies/ml. Th1 and Th2-type cytokine mRNA levels peaked in Ch1601 at weeks 13–15 p.i. This correlated with a significant drop in virus titer, below 300 copies/ml. However, when the cytokine levels decreased, the virus relapsed and established persistent infection. That is in contrast to the cytokine levels in the non-vaccinated control animal (Ch1605), where increased IFN-␥ mRNA levels were maintained from week 6 p.i. and no variation in IL10 mRNA levels were observed. In Ch1587, where infection was delayed and shorter lived compared to the non-vaccinated re-challenge animal, both a high and sustained antibody response to HVR1, was developed during the immunization. Additionally, an E1E2 and HVR1 specific T-helper response was enhanced in the peripheral blood of this animal, correlating with the boost in antibody titers. These data indicate that memory T-cell and B-cell responses to the E1E2 antigens exist in recovered animals despite the absence of detectable antibody to these viral proteins at any point during or following primary infection. Interestingly, proliferative T-cell responses generated by the vaccine in the peripheral blood were not sustained following challenge and were maintained low during the first 5 weeks p.i. Not until after clearance of the virus, was T-cell proliferation specific for E1E2 and NS3 proteins enhanced again. Additionally, we observed an immediate and substantial intrahepatic increase in IFN-␥ mRNA levels during the period of low T-cell proliferation in the blood. These data may reflect a migration of T lymphocytes from the blood to the liver at the time of the infection, maximizing the presence of effector T-cells intrahepatically. A drop in peripheral IFN-␥ producing CD4+ T-cells following re-challenge of a recovered chimpanzee with HCV has been previously reported [27] suggesting a migration of memory T-cells from the peripheral blood in response to virus replication in the liver. Once the infection has been controlled intrahepatically, memory cells may no longer be recruited to the liver but accumulate in the peripheral circulation. It has been shown in HCV that memory T-cells in the liver do not return to the circulation [28] but such pathways have been described for other viral infections [29,30]. The goal of vaccines is to completely prevent infection. For many viral diseases that are prevented by vaccination, low-level viral replication is likely to occur upon exposure. It appears from our studies that an antibody/Th-cell response directed against the surface glycoproteins was sufficient to control virus replication for up to 3 weeks following infection in both the na¨ıve and recovered animals. The progression to chronicity in Ch1601 may have been due to the lack of T-cell responses to other viral antigens that are presumably present in recovered chimpanzees. The addition of an

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envelope antibody response to the T-cell responses already present in Ch1587 reduced the acute infection to a very low level, with an entirely sub-clinical disease compared to that usually observed in re-challenged chimpanzees. Due to the expense and lack of availability of chimpanzees for research, it is often difficult to draw strong conclusions from data involving so few animals. However, even with these limitations, much useful information is obtained from studies with this model. Future studies should explore vaccine strategies designed to induce a broad T-cell immune response combined with strong antibody responses to surface antigens.

Acknowledgements We thank Dr. Barbara Rehermann for helpful discussion and advice. We thank Estella Jones and Ray Olsen for expert care and handling of chimpanzees and samples within the FDA facility. These studies were supported by internal FDA funds, a grant from the National Vaccine Program Office and a grant from the National Cancer Institute CA85883. References [1] Alter MJ. The detection, transmission, and outcome of hepatitis C virus infection. Infect Agents Dis 1993;2(3):155–66 (JID-9209834). [2] Ishii K, Rosa D, Watanabe Y, et al. High titers of antibodies inhibiting the binding of envelope to human cells correlate with natural resolution of chronic hepatitis C. Hepatology 1998;28(4):1117–20 (JID-8302946). [3] Gerlach JT, Diepolder HM, Jung MC, et al. Recurrence of hepatitis C virus after loss of virus-specific CD4(+) T-cell response in acute hepatitis C. Gastroenterology 1999;117(4):933–41 (JID-0374630). [4] Lechner F, Gruener NH, Urbani S, et al. CD8+ T lymphocyte responses are induced during acute hepatitis C virus infection but are not sustained. Eur J Immunol 2000;30(9):2479–87 (JID-1273201). [5] Kiyosawa K, Sodeyama T, Tanaka E, et al. Interrelationship of blood transfusion, non-A, non-B hepatitis and hepatocellular carcinoma: analysis by detection of antibody to hepatitis C virus. Hepatology 1990;12(4 Pt 1):671–5 (JID-8302946). [6] Di Bisceglie AM, Goodman ZD, Ishak KG, Hoofnagle JH, Melpolder JJ, Alter HJ. Long-term clinical and histopathological follow-up of chronic posttransfusion hepatitis. Hepatology 1991;14(6):969–74 (JID-8302946). [7] Farci P, Shimoda A, Coiana A, et al. The outcome of acute hepatitis C predicted by the evolution of the viral quasi-species. Science 2000;288(5464):339–44 (JID-0404511). [8] Choo QL, Kuo G, Ralston R, et al. Vaccination of chimpanzees against infection by the hepatitis C virus. Proc Natl Acad Sci USA 1994;91(4):1294–8 (JID-7505876). [9] Rosa D, Campagnoli S, Moretto C, et al. A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells. Proc Natl Acad Sci USA 1996;93(5):1759–63 (JID-7505876). [10] Esumi M, Rikihisa T, Nishimura S, et al. Experimental vaccine activities of recombinant E1 and E2 glycoproteins and hypervariable region 1 peptides of hepatitis C virus in chimpanzees. Arch Virol 1999;144(5):973–80 (JID-7506870). [11] Margolis HS, Alter MJ, Liang TJ, Dienstag JL, editors. Protective antibodies in immune globulins prepared from anti-hepatitis C virus

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