Civacir^® hepatitis C immune globulin broadly neutralizes infection of hepatitis C virus liver graft escape variants and direct-acting antiviral-resistant viruses

Abstract

Hepatitis C virus (HCV)-induced end-stage liver disease is the major indication for liver transplantation (LT). However, re-infection of the liver graft is still common, especially in patients with detectable viral load at the time of transplantation. Limited data is available with direct-acting antivirals (DAAs) in the transplant setting for prevention of graft infection. The human hepatitis C immune globulin (HCIG) Civacir^® is an investigational drug that is currently developed in an ongoing phase III clinical trial assessing safety and efficacy to prevent the HCV recurrence after liver transplant in the US.¹ Using well characterized patient-derived HCV variants selected during LT we aimed to study the molecular mechanism of action of Civacir^®. Inhibition of HCV infection was studied using infectious HCV models including HCV pseudoparticles (HCVpp) and cell culture-derived HCV viruses (HCVcc) containing patient-derived viral envelope glycoproteins from 22 HCV variants isolated from patients before and after LT.² Additionally, we studied neutralization of different HCV genotypes and of DAA-resistant viruses. Our results indicate that Civacir potently, broadly and dose-dependently neutralizes all tested patient variants in HCVpp and HCVcc assays including variants displaying resistance to host neutralizing antibodies and anti-viral monoclonal antibodies. The half maximal inhibitory concentrations were independent of the phenotype of the viral variant indicating that virus neutralization by Civacir is not affected by viral selection. Furthermore, Civacir is equally active against tested DAA-resistant HCV isolates in cell culture. Conclusion: Collectively, these results demonstrate broad neutralizing activity of Civacir against resistant viruses and support its further clinical development for prevention of liver graft infection. The broad neutralizing activity of Civacir is likely due to synergy between anti-HCV antibodies derived from different patient donors.

Hepatitis C virus (HCV) infects over 130-150 million people worldwide³ with 70-80% of infected individuals developing chronic infection. Chronic hepatitis C-related cirrhosis and hepatocellular carcinoma are major indications for liver transplantation (LT). However, re-infection of the liver graft is common and leads to accelerated recurrence of liver disease; 10-30% of patients develop cirrhosis within 5 years and over 40% of patients do so within 10 years.^{4, 5} Furthermore, loss of liver graft and mortality are higher for patients developing recurrent HCV compared to uninfected patients.⁵ Re-infection of the liver graft by HCV is a unique scenario dictated by dynamic virus-host interactions. Indeed, despite the presence of anti-HCV antibodies, re-infection of the liver graft occurs within a few hours of graft reperfusion. It is characterized by rapid spread within days following engraftment and is highly efficient with viral titers rapidly reaching pre-transplantation levels.^6–9 In a longitudinal study of a cohort of HCV-infected patients undergoing LT, we have previously demonstrated that the virus evolution changes following LT with only a fraction of variants infecting the graft.² Selected variants infecting the liver graft were characterized by enhanced cell entry and resistance to autologous and heterologous serum-derived antibodies as well as broadly cross-neutralizing anti-envelope monoclonal antibodies (mAbs).¹⁰

Strategies aiming at preventing re-infection of the graft have relied on three approaches based on the timing of therapeutic intervention: antiviral therapy before LT to cure HCV infection or to reduce viral load, prevention of graft re-infection during LT (prophylactic treatment) and treatment of established HCV infection post-transplantation (preemptive treatment). The 2015 EASL¹¹ and AASLD¹² guidelines for anti-HCV treatment pre- and post-LT recommends the use of pegylated interferon (PEG-IFN)-freeregimen involving combination of the second wave DAAs namely sofosbuvir (NS5B polymerase inhibitor), simeprevir (NS3 inhibitor), ledipasvir, daclatasvir (NS5A inhibitors) and others, with or without ribavirin. Several criteria including HCV genotype, degree of cirrhosis (Child-Pugh score) and treatment before or after LT have been shown to dictate the choice of drug combination and duration of treatment.

Prophylactic treatment is advantageous as it provides a window of opportunity when the virus can be targeted prior to infecting the liver graft, thus preventing possible tissue damage. DAAs block active viral RNA replication and virus production as they target HCV proteins important for these processes. They do not target viral entry into the hepatocytes of the new graft, which is however, most warranted to prevent reinfection of the transplanted organ. In this respect, hepatitis C antibody enriched immunoglobulins (HCIG) can be effectively used prophylactically as they would neutralize HCV particles and block infection of the new graft. This is best exemplified by hepatitis B immunoglobulins (HBIG) that are very effective in preventing recurrence of hepatitis B virus (HBV) infection during LT and their use increased patient survival rates from ~50% to ~75%.¹³ Likewise, patient-derived immunoglobulins (IG) could also be an effective strategy to prevent re-infection in HCV LT patients. Indeed, in a retrospective study, anti-HCV IG were shown to reduce the frequency of chronic hepatitis C post-transplantation in patients that inadvertently received HCIG along with HBIG.¹⁴

The human hepatitis C immune globulin (HCIG) Civacir is an investigational drug that is currently developed in an ongoing phase III clinical trial assessing safety and efficacy to prevent the recurrence of HCV after liver transplant in the USA.¹⁵ Civacir has not been approved for safety and effectiveness by competent regulatory authorities. It is prepared from pooled plasma of hundreds of donors obtained from chronically HCV-infected patients and purified employing several steps of fractionation, virus elimination and inactivation. The first generation, a 5% HCIG preparation, was unable to prevent hepatitis C-recurrence after LT in a clinical trial owing to presumably high viral load at the time of transplantation.¹⁵ Compared to the 5% HCIG, Civacir is produced with an improved process and formulated as a 10% HCIG in an optimized protein formulation. In the currently ongoing phase III randomized controlled clinical trial with Civacir, patients receive any antiviral treatment prior to LT that reduces HCV RNA levels to below 100 IU/mL at the time of transplantation and were randomized to either control (no antiviral treatment post-LT) or active treatment arms to receive Civacir at 200 mg/kg or 300 mg/kg doses for 10 weeks in the peri- and post-LT period to prevent recurrence post-LT. Preliminary data showed a reinfection rate of 32% for the control versus a 5% reinfection rate for the 300 mg/kg dose arm.¹ Aiming to understand the potential role of Civacir for difficult-to-treat patients we investigated the activity of Civacir against viruses resistant to host neutralizing responses and antiviral therapies.

Materials and Methods

Human subjects

Human material including liver tissue from patients undergoing surgical resection for isolation of primary human hepatocytes (PHH) as well as human serum for isolation and amplification of HCV RNA was obtained with informed consent from all patients. The respective protocols were approved by the Ethics Committee of the University of Strasbourg Hospitals, France (CPP 10-17).

Cells

293T cells and Huh7.5.1 cells have been described.^16–18 PHH were isolated and cultured as described.^{2, 19}

Reagents

Civacir is an investigational drug that is currently developed in an ongoing phase III clinical trial (clinical trial identifier: NCT01804829) assessing safety and efficacy to prevent the recurrence of HCV after liver transplantation in the USA.¹⁵ Civacir has not been approved for safety and effectiveness by competent regulatory authorities. Civacir is prepared from pooled plasma obtained from hundreds of donors chronically infected with HCV, which are negative for human immunodeficiency virus (HIV) and HBV. It is produced by Biotest Pharmaceuticals Corp. as a 10% IG solution. It is virus inactivated and efficiency is subsequently analyzed by high sensitivity RT-PCR methods. The anti-HCV antibody level was quantified by Abbott HCV ELISA.

Viral strains

Isolation and characterization of pre- and post-transplant HCV variants has been described.² HCV pseudoparticles (HCVpp) derived from strains H77 (genotype 1a), HCV-J (genotype 1b), JFH1 (genotype 2a) and genotype 2b have been described.^{10, 20–22} For production of genotype 3 (strain gt3SXB1) and genotype 4 (strain gt4SXB1) HCVpp, E1E2 sequences of viral strains isolated from chronically HCV-infected patients followed at the University Hospital Strasbourg were amplified and inserted into the phCMV IRES vector using EcoRV restriction sites as described for HCV-AD78.¹⁷ For genotypes 5 and 6, E1E2 gene sequences derived from strains UKN5.14.4 (genotype 5; GenBank accession no. AY785283) and UKN6.5.340 (genotype 6; GenBank accession no. AY736194.1)²³ were synthesized (Geneart) and subsequently inserted into the phCMV IRES vector using EcoRV restriction sites.¹⁷

HCVpp production, infection and neutralization

HCVpps were produced as described.^{2, 24} Briefly, HCVpp were generated by transfection of 293T cells using a CMV-Gag-Pol HIV packaging construct, a luciferase reporter plasmid, and the expression plasmids encoding the HCV envelope glycoproteins derived from LT variants or from different HCV genotypes. Two fold serial dilutions of Civacir or control IG – from 1250 to 1.22 μg/mL, were mixed with HCVpp and pre-incubated for 1 h at 37°C prior to incubation with Huh7.5.1 cells. HCV entry was determined by measuring luciferase activity in cells harvested 72 hours after infection as described.^{16, 25}

HCVcc production, infection and neutralization

JFH1-based HCVcc chimeras encoding structural proteins of patient isolate P1VL (genotype 1b) and its mutant P1VL_F447L^{2, 10, 26} or of the Jc1 strain (genotype 2a) and containing drug resistance mutations L36M or R155K or A156S in the JFH1 NS3 protease as well as the chimera J4/JFH1 containing the Y2065H mutation conferring resistance to NS5A inhibitors were produced as described.^{10,27, 28} Infection and neutralization assays were carried out as reported earlier.^{29, 30} Briefly, Civacir was pre-incubated with HCVcc for 1 hour at 37° C before adding the mixture to Huh7.5.1 cells for 4 hours at 37°C. Following inoculation, cell culture medium was added to the cells and infection analyzed 72 hours later by measuring luciferase activity²⁹ in case of the NS3-A156S and NS3-R155K mutants or by determining the tissue culture infectious dose 50% (TCID₅₀)/mL³⁰ in case of NS3-L36M and NS5A-Y2065H mutant viruses.

Data analysis

IC₅₀ values were calculated by fitting a non-linear three parameter least squares curve to HCV inhibition data expressed as percentage HCV infection observed with control IG. The infection values were plotted against log₁₀ concentrations of HCIG. Curve fitting was performed using the GraphPad Prism software.

Results

Civacir broadly neutralizes cell entry of HCVpp containing envelopes with neutralizing escape mutations in cell culture model

To decipher the molecular mechanisms of HCV re-infection of the liver graft, we had previously generated HCVpp derived from variants circulating before and early after LT in a cohort of 6 HCV genotype 1b-infected patients undergoing LT². This collection of HCVpp reflecting the natural quasispecies circulating during LT was used to evaluate the neutralization activity of Civacir, a polyclonal IG preparation derived from patients with chronic HCV infection. A total of 22 patient-derived HCV variants, comprised of 13 variants selected during LT (“selected variants”) and 13 variants isolated before LT that disappeared following LT (“non-selected variants”), were tested using an HCVpp neutralization assay. We chose a range of Civacir concentrations between 1250 and 1.22 μg/mL to enable calculation of IC₅₀ values and compare variants with different infectivities. As shown in Figure 1, all 22 viral variants were potently and dose-dependently neutralized by Civacir. Furthermore, the IC₅₀ values (Table 1) indicate that Civacir inhibits selected variants with similar potency as non-selected variants at concentrations in the low nanomolar range (mean IC₅₀: 24 μg/mL; range: 0.5 – 96.1 μg/mL).

Figure 1. Civacir potently and dose-dependently neutralizes HCVpp derived from selected and non-selected HCV variants isolated from patients before and after liver transplantation.

HCVpp containing E1E2 envelope glycoproteins from selected (blue) and non-selected (black) variants isolated from 6 patients before and after liver transplantation (LT), respectively, were produced and tested for neutralization against serial dilutions of Civacir or control IG preparation (1250 – 1.22 μg/mL). HCVpp were incubated with Civacir or the control at 37°C for 1 hour and used to inoculate Huh7.5.1 cells. The level of HCVpp entry was determined after 72 hours by measuring luciferase activity, which is shown as percentage relative to the control.

Table 1. IC₅₀ values (μg/ml) of Civacir^® against 22 HCVpp expressing E1E2 envelope glycoproteins of viral isolates from different patient-derived escape (blue) and non-escape (black) variants. IC₅₀ were calculated by non-linear three parameter least squares analysis using GraphPad Prism.

Patient no.	Viral variant	IC50 (μg/ml)	Correlation coefficient
Patient 1	P1VA	1.7	0.87
	P1VC	0.5	0.78
	P1VK	6.7	0.91
	P1VL	28.7	0.99
Patient 2	P2VA	22.0	0.98
	P2VD	36.6	0.95
	P2VH	3.3	1.00
	P2VI	85.8	0.88
	P2VJ	34.9	0.94
Patient 3	P3VA	37.9	0.97
	P3VB	96.1	0.92
	P3VC	10.4	0.97
Patient 4	P4VB	53.4	0.94
	P4VC	5.7	0.72
	P4VD	1.6	0.93
	P4VE	5.6	0.84
Patient 5	P5VD	34.8	0.95
	P5VE	8.9	0.99
	P5VF	38.8	0.94
Patient 6	P6VD	2.1	0.92
	P6VG	8.6	0.78
	P6VI	12.4	0.96

To further validate our findings we tested Civacir for neutralization of three patient variants in primary human hepatocytes (PHH). As with Huh7.5.1 cells, we found that Civacir was equally active at neutralizing HCVpp entry into PHH with comparable potency (Fig. 2B and table 1; IC₅₀ 13.6 vs 1.7 μg/mL for non-selected variant P1VA and 8.2 vs 28.7 and 1.8 vs 34.9 μg/mL for selected variants P1VL and P2VJ, respectively).

Figure 2. Civacir neutralizes HCVpp entry of selected and non-selected variants into primary human hepatocytes.

(A) HCVpp derived from representative selected (blue line) and non-selected (black line) variants from LT patients were incubated with different concentrations of Civacir or control IG (1.3 – 833 μg/mL) at 37°C for 1 hour and added to primary human hepatocytes. The level of HCVpp entry was determined after 72 hours by measuring luciferase activity, which is shown as percentage relative to the control. (B) IC₅₀ values of Civacir using HCVpp derived from patient variants P2VJ, P1VL and P1VA. IC₅₀ values (μg/mL) were calculated by fitting a non-linear three parameter least squares model.

We also tested Civacir for neutralization of chimeric JFH1-based HCVcc derived from a representative selected variant (P1VL) and a non-selected variant (P1VA) to confirm the results obtained with HCVpp and to investigate whether the low-density lipoproteins associated with HCV particles (but not with HCVpp) might have an impact on the neutralizing activity of Civacir. Molecular characterization of these variants had previously identified phenylalanine at position 447 as a determinant resulting in enhanced cell entry and escape from host neutralizing antibodies¹⁰. Furthermore, replacing this residue by leucine present in the non-selected variant P1VA not only reduced viral entry but also rendered the virus highly susceptible to autologous and heterologous sera¹⁰. Therefore, we tested Civacir against chimeric HCVcc generated from these two variants (VL and VL with leucine at position 447 that behaves like variant P1VA). Civacir strongly neutralized both viral variants (Fig. 3A), with the selected variant VL requiring higher concentrations to neutralize HCVcc infection (Fig. 3B). Notably, neutralizing HCVcc-P1VL required around 10-fold higher concentration of Civacir as compared to HCVpp-P1VL (IC₅₀: 375 vs 28.7 μg/mL for HCVcc and HCVpp, respectively). Since HCVcc, unlike HCVpp, associate with lipoproteins and thus resemble native HCV particles, the poorer neutralization likely reflects the role of lipoproteins in HCV-resistance to antibody-mediated neutralization.^{31, 32} Indeed, lipoprotein association has been shown to increase HCV infectivity and facilitate evasion from antibody mediated neutralization.^{33, 34}

Figure 3. Civacir robustly neutralizes HCVcc JFH-P1VL and JFH-P1VL_F447L chimera.

(A) JFH1-based HCVcc chimeras encoding structural proteins of patient isolates P1VL (genotype 1b; left) and P1VL_F447L (right) were produced as described.¹⁰ Replacing phenylalanine at position 447 by leucine renders the particlesless infectious and increases their susceptibility to neutralization with autologous and heterologous serum; akin to isolate P1VA.¹⁰ HCVcc derived from a representative selected (P1VL) and a non-selected (P1VL_F447L) variant from a LT patient were incubated with different concentrations of Civacir or control IG (0.8 – 2500 μg/mL) at 37° C for 1 hour and added to Huh7.5.1 cells. The level of HCVcc infection was determined after 72 hours by determining the tissue culture infectious dose 50% (TCID₅₀/mL). (B) IC₅₀ values (μg/mL) of Civacir against HCVcc JFH1-P1VL and JFH1-P1VL_F447L chimeras. The values were calculated by non-linear three parameter least squares analysis.

Collectively, these data indicate that Civacir robustly neutralizes selected LT patient variants that are resistant to autologous and heterologous serum-derived antibodies.

Civacir broadly neutralizes cell entry of HCVpp across all major genotypes in cell culture model

To investigate the impact of genotype variability on the neutralization capability of Civacir, we tested a panel of well-characterized HCVpp containing patient-derived E1E2 glycoproteins from genotypes 1 to 6. ^{23, 35} As shown in Figure 4A and 4B, Civacir potently neutralized genotype 1a, 1b, 2b, 2b, 4 and 6 in a dose-dependent manner and with comparable efficacy in cell culture. Genotype 3a required higher concentration to inhibit 50% HCVpp entry, and was thus less neutralized compared to other genotypes (Fig. 4B). Note that for genotype 5, we were unable to calculate the IC₅₀ value due to the low infectivity of the HCVpp (data not shown). Collectively, these results demonstrate that in cell culture model systems, Civacir is broadly neutralizing and support a potential use for prevention of liver graft re-infection in patients infected with various genotypes.

Figure 4. Civacir exhibits robust and broad neutralizing activity against representative isolates of major HCV genotypes.

HCVpp containing E1E2 envelope glycoproteins of HCV genotypes 1 to 6 were produced and tested for neutralization by incubating with two-fold serially diluted Civacir or control IG (1250 – 1.22 μg/mL) at 37° C for 1 hour and adding to Huh7.5.1 cells. The level of HCVpp entry was determined after 72 hours by measuring luciferase activity, which is shown as percentage relative to the control. (B) IC₅₀ values (μg/mL) of Civacir for different HCV genotype isolates. The low infectivity of the genotype 5 isolate (data not shown) did not allow calculation of the IC₅₀ value.

Civacir neutralizes DAA-resistant viruses in cell culture model

Finally, we tested Civacir for its ability to neutralize DAA-resistant viruses.^36–38 We used JFH1-chimeric HCVcc harboring individual resistant mutations, namely L36M and A156 conferring resistance against protease inhibitors telaprevir, boceprevir, simeprevir and danoprevir among others, R155K conferring resistance against all protease inhibitors studied except grazoprevir and Y2065H conferring resistance to NS5A-targeting drugs. DAA-resistant viruses were neutralized by Civacir (Fig. 5A). The observed difference in IC₅₀ between L36M/Y2065H and R155K/A156S single mutants (Fig. 5B) is likely due to the different methods used to evaluate viral infectivity, i.e. TCID₅₀ assay for viruses that do not encode a reporter gene vs luciferase activity for viruses containing such a reporter gene. Taken together, these data indicate that Civacir is highly active against DAA-resistant viruses in cell culture.

Figure 5. Civacir neutralizes DAA-resistant viruses.

Chimeric HCVcc containing individual DAA resistance mutations R155K, A156S, or L36M in NS3 or Y2065H in NS5A were incubated with different concentrations of Civacir or control IG at 37° C for 1 hour and added to Huh7.5.1 cells. The level of HCVcc infection was determined after 72 hours either by measuring luciferase activity or by determining the tissue culture infectious dose 50% (TCID₅₀/mL). The inhibition by Civacir is shown as percentage relative to the control. The R155K and A156S mutations were evaluated in the context of the Luc-Jc1 chimeric J6/JFH1 (genotype 2a/2a) virus whereas the NS3-L36M and NS5A-Y2065H mutations were evaluated in the J4/JFH1 (genotype 1b/2a) chimeric virus. (B) IC₅₀ values (μg/mL) of Civacir against DAA-resistant viruses.

Discussion

The availability of DAAs with improved efficacy and reduced toxicity would enable HCV cure in the vast majority of treated patients, however, prevention of liver graft re-infection with HCV remains a challenge. The combination of sofosbuvir and ribavirin has been approved to treat HCV infection in patients awaiting LT to prevent hepatitis C recurrence. The effect of sofosbuvir and ribavirin treatment before LT on post-transplant HCV recurrence was evaluated in a recent study³⁹. Of the 46 patients that received liver graft, sofosbuvir-ribavirin pre-treatment led to reduction in HCV-RNA level to less than the lower limit of quantification (LLOQ, 25 IU/ml) in 43 patients. 70% of these 43 patients achieved post-transplant virological response at 12 weeks (pTVR12). Alternative strategies are thus needed to increase the rate of pTVR and to prevent HCV recurrence in difficult to treat patients with co-infection and co-morbidities.

Civacir is an enriched IG preparation containing anti-HCV antibodies derived from several hundred patients infected with different HCV genotypes. It is currently being evaluated in a phase III clinical trial (clinical trial identifier: NCT01804829) as a 10% solution in an improved formulation compared to the 5% HCIG. Infusions of 5% HCIG were generally well tolerated in chronic HCV patients undergoing LT but failed to suppress re-infection of the graft because of high pre-transplant viral load.¹⁵ Given that viral variants infecting the liver graft are characterized by enhanced cell entry and increased resistance to host antibodies,^{7, 10, 40} we evaluated the activity of Civacir to neutralize HCV variants isolated from patients before and after LT. Our results demonstrate that in HCV model systems, Civacir neutralizes selected variants that are resistant to neutralization by autologous and heterologous patient antibodies as well as defined mAbs. While IC₅₀ values based on the HCVpp data did not indicate significant differences between the selected and non-selected variants, HCVcc data showed that a selected variant (P1VL) required higher amounts of Civacir as compared to a non-selected variant. These data indicate that the dose of Civacir may be an important factor for its activity. Indeed, preliminary results of the ongoing clinical trial evaluating Civacir revealed high frequency of hepatitis C recurrence (30%) in the low-dose arm (200 mg/kg), which was similar to the control group with 32%. In contrast, recurrence of infection was observed in only 5% patients in the high-dose group (300 mg/kg).¹ Notably, the Civacir concentrations required for inhibition of HCV infection in cell-based models (IC₅₀: 0.4605 – 96.1 μg/mL) are in the range of concentrations used in clinical trials (200-300 mg/kg). The IC₅₀ value of 96.1 μg/ml would translate into 500 mg of Civacir for a 70 kg individual (blood volume of 5.2 liters at ~75 ml/kg), corresponding to 7.1 mg/kg. For potent anti-HCV activity as well as considering the pharmacokinetics, several times higher concentration than the IC₅₀ values would be needed. This, however, seems plausible with the 300 mg/kg dose given to patients in the high-dose group in the ongoing phase III clinical trial (NCT01804829).

Viral quasispecies have long been recognized as an impeding factor in devising effective preventive strategies against HCV recurrence after LT. Thus, although broadly neutralizing mAbs have been identified and characterized in vitro, they have yielded little success when used in clinical trials to prevent graft re-infection.^41–43 Likely, targeting a single neutralizing epitope using a mAb does not allow to prevent viral escape by the highly variable HCV and targeting of multiple neutralizing epitopes will be required to prevent HCV recurrence after LT. Passive immunization with cross-neutralizing (against HCVpp and HCVcc) polyclonal IgG isolated from a patient exhibited limited effect against heterologous virus challenge in the human liver-chimeric uPA-SCID mouse model.⁴⁴ This is probably due to inadequate plasma levels and diversity of the neutralizing antibodies to counter the highly heterogeneous virus population leading to eventual escape and viral breakthrough. Civacir is distinct in that it is a pool of IG purified from several hundreds of chronically HCV-infected patients resulting in a very high antibody diversity and likely synergy between antibodies targeting different epitopes. The high diversity and enrichment of antibodies most likely explains the broad and potent neutralizing activity of Civacir (Fig. 6) and absent viral escape as compared to the poor efficacy of mAbs.

Figure 6. HCV escape and antibody diversity.

(A) The antibody response is ineffective in controlling the continuously evolving HCV quasispecies leading to persistent chronic infection and re-infection of the liver graft post-transplantation. (B) Civacir, a human hepatitis C antibody-enriched immunoglobulin product (HCIG), is a polyclonal antibody preparation derived from human plasma enriched with anti-HCV antibodies and collected from several hundreds of donors. The resulting high antibody diversity and synergy between anti-HCV antibodies targeting different epitopes is translated into active neutralization of transplant-selected variants resistant to autologous and heterologous antibodies. The high antibody diversity also likely explains the broad neutralizing activity against different HCV genotypes.

The HCV genotype is one of the most important factors governing treatment efficacy.^45,46 An ideal therapeutic to prevent hepatitis C recurrence in liver graft should thus be equally effective against different genotypes. We tested Civacir for its ability to neutralize isolates derived from all major HCV genotypes in cell culture model. Our results indicate that Civacir is broadly neutralizing in a pan-genotypic manner given the similar neutralizing capacity against different genotypes, including the more difficult-to-treat genotypes 1 and 3, although for the latter higher doses of Civacir were required. This result highlights the potential to use Civacir to prevent hepatitis C recurrence in LT patients infected with different genotypes including difficult-to-treat genotype 3.^45,46

We also evaluated the effect of Civacir against DAA-resistant HCVcc containing mutations in the NS3 and NS5A regions. The four mutations that we tested were identified in patients undergoing therapy and thus have a direct clinical impact.^36–38 These mutations were shown to confer resistance against individual DAAs which is reflected by more than 10-fold increase of the IC₅₀ values of corresponding DAA.²⁷ Of note, also these DAA-resistant viruses are susceptible to neutralization with Civacir. Considering that patients undergoing LT are likely to have circulating viruses resistant to protease and/or NS5A inhibitors as a result of previous antiviral therapy with these drugs, our results with cell culture model indicate that Civacir may not be affected by pre-existing DAA-resistant viruses.

The safety profile of HBIG and absent drug-drug interactions between HBIG and immunosuppressive therapy required for LT indicate that Civacir may also be well tolerated by LT patients. Preliminary data on tolerability and safety of the ongoing phase III trial with Civacir support this hypothesis. Civacir may also be useful in difficult-to-treat patients where use of DAAs is restricted. Furthermore, the ability of new DAAs to rapidly lower the viral load to undetectable levels^{47, 48} presents a new opportunity for combination therapy with the aim to clear residual viruses with the pan-genotypic HCIG approach. Furthermore, given the potential of host-targeting entry inhibitors (HTEIs) for prevention of acute HCV infection⁴⁹, their ability to reduce viral infection in an additive/synergistic manner when combined with neutralizing antibodies^40,50, and their potential of preventing viral resistance²⁷, it may be speculated whether Civacir may be combined with HTEIs in future clinical trials to further improve their efficacy.

In summary, we demonstrate broad and potent neutralizing activity of Civacir against selected transplant variants, against different HCV genotypes and against DAA-resistant variants in different cell culture models. These results support the further clinical development of Civacir to prevent liver graft infection in HCV-infected patients.

Abbreviations

HCV

hepatitis C virus

LT

liver transplantation

DAA

direct-acting antiviral

HCIG

hepatitis C antibody enriched immunoglobulins

HCVpp

HCV pseudoparticles

HCVcc

cell culture-derived HCV

PEG-IFN

pegylated interferon

HBIG

Hepatitis B immunoglobulins

IG

immunoglobulins

PHH

primary human hepatocytes

IC₅₀

inhibitory concentration 50%

HIV

human immunodeficiency virus

HBV

hepatitis B virus