Controlled Human Infection Model for Hepatitis C Virus Vaccine Development: Trial Design Considerations

Abstract

The design of a clinical trial for a controlled human infection model (CHIM) to accelerate hepatitis C virus (HCV) vaccine development requires careful consideration. The design of a potential approach to HCV CHIM is outlined, involving initial sentinel cohorts to establish the safety and curability of the viral inoculum followed by larger cohorts to establish the spontaneous clearance rate for each inoculum. The primary endpoint would be HCV clearance by 24 weeks post-inoculation, recognizing that the prevention of chronic infection would be the primary goal of HCV vaccine candidates. Additional considerations are discussed, including the populations to be enrolled, the required monitoring approach, indications for antiviral therapy, and the required sample size for different CHIM approaches. Finally, safety considerations for CHIM participants are discussed.

The design of a controlled human infection model (CHIM) for hepatitis C virus (HCV) vaccine development requires careful consideration. Building on prior outlines of key principles of CHIM [1–3], we discuss key elements of how a CHIM protocol could be developed, including consideration of trial endpoints, the population to be studied, timing of treatment, required monitoring, and key safety considerations. Ultimately, we propose a study design, outlined in Figure 1, and discuss elements that must be addressed further in development of a final study protocol.

Figure 1.

CHIM trial design schematic. A) An initial sentinel cohort would be infected with the viral inoculum isolated from an infected patient who had been cured with the CHIM DAA regimen. Participants would be followed closely to evaluate the reliability of infection, course of hepatitis, and curability with DAAs. Treatment would be started at 12 weeks or earlier due to signs of severe hepatitis or at participant request. A higher inoculum would be used if not all members of the initial sentinel cohort were infected. Frequency and type of monitoring are shown. B) CHIM participants either unvaccinated (control) or vaccinated with a vaccine candidate will be infected with the established inoculum and followed for spontaneous clearance with initiation of treatment at 24 weeks or sooner due to severe hepatitis or participant requestion. Abbreviations: AE, adverse event; ALT, alanine aminotransferase; CHIM, controlled human infection model; DAA, direct-acting antiviral; HCV, hepatitis C virus; PBMC, peripheral blood mononuclear cell; q, every; SVR, sustained virological response; ULN, upper limit of normal; –ve, negative. *Subset—optional.

TRIAL ENDPOINTS

Sentinel Cohorts to Characterize Inoculum

Before considering efficacy endpoints for vaccine candidates, HCV CHIM must first establish that the viral inoculum predictably recapitulates natural HCV infection, is safe, and cured with direct-acting antivirals (DAAs). A well-characterized standardized inoculum (see Liang et al [4]) would first be evaluated in small sentinel cohorts (3–6 persons) to establish model reliability and provide an adaptive platform to alter the dose (or inoculum), similar to a phase 1 study of a novel therapeutic agent. Inoculum size may affect the likelihood of infection but not the severity of hepatitis or probability of spontaneous clearance, and therefore initial doses would be based on chimpanzee studies (∼100 chimpanzee infectious doses) to maximize the likelihood of reliable infection rather than to use the minimal infectious dose. Because the focus would be on infectivity, early viral dynamics, and treatment response, treatment could be initiated early in the sentinel cohorts (eg, 4–12 wk). Sequential enrollment would ensure that safety and cure are confirmed before infecting subsequent individuals. Following these small sentinel cohorts, larger unvaccinated cohorts would then be required to characterize the spontaneous clearance rate for each inoculum, with treatment initiated at 24 weeks, as discussed below, if clearance did not occur.

ENDPOINTS FOR VACCINE STUDIES

The design and endpoints of a CHIM study should closely mirror the expected effect of the vaccine. Ideally, a vaccine would generate sterilizing immunity and prevent infection. Other acceptable endpoints would include the prevention of progression to chronicity and/or attenuation of disease course to reduce serious outcomes. HCV-related clinical sequelae do not occur for years or even decades of chronic infection and thus the prevention of chronicity would provide clinical benefit, whereas it is not feasible to consider attenuated disease course as an outcome. Therefore, the focus for HCV vaccine development is either prevention of infection or prevention of chronicity.

The prevention of infection permits the simplest CHIM design and will certainly be evaluated. HCV RNA would be the endpoint and monitored closely (eg, weekly), and those in whom HCV RNA is detected could be designated as prevention “failures” and treated promptly. However, data from chimpanzee vaccine studies have only documented sterilizing immunity to challenges with virus homologous to the vaccine, but notably, while infection with heterologous virus was never prevented, progression to chronic infection was reduced [1]. Designing studies to assess only sterilizing immunity would therefore risk discarding a vaccine candidate that effectively prevents viral persistence but does not block initial infection.

The prevention of viral persistence is the most practical HCV vaccine goal. Data from natural infection of humans also support this goal. The approximately 25–30% of persons who spontaneously clear initial HCV infection are more likely to clear if infected again and do so more rapidly with lower peak viremia, indicating that persistent infection can be prevented and partial immunity “acquired” [2, 3, 5]. (See Shoukry et al for a detailed discussion of the immunological basis for CHIM design). The objective of preventing persistent infection is also supported by the absence of long-term harm associated with self-limited infection [6]).

Although spontaneous clearance of HCV infection may occur as late as 24 months or rarely beyond, the majority (>90%) who clear infection do so within 6 months of exposure. Therefore, for studies designed to detect vaccine effectiveness for the prevention of HCV persistence, the preferred endpoint is absence of HCV RNA detection 6 months after inoculation, which balances the risk of missing late clearance against pragmatic considerations for trial completion and duration of infection for participants. Ideally, surrogate endpoints that predict persistent infection would allow for earlier treatment initiation [7–9]. However, limited available natural infection data, including those from the recent vaccine trial [10], show variable patterns of HCV RNA over time without clear thresholds or viral kinetic patterns that can reliably predict who will or will not clear infection. It is possible that viral clearance/persistence will be easier to interpret from early infection dynamics in a CHIM since the inoculum is standardized and homogenous and reinfection is not an issue; however, this was not obviously the case with chimpanzee studies using the early H77 inoculum. While worth exploring in initial CHIM cohorts, it is also important to consider that vaccine candidates may affect the relationship between clearance/persistence and early viral dynamics in an unexpected way. Thus, the abundance of data now favors a 6-month HCV RNA endpoint.

NEED FOR A PLACEBO/UNVACCINATED COHORT

To understand the effect of a vaccine on the prevention of persistence, the spontaneous clearance rate for the viral inoculum must be well defined. While it is tempting to vaccinate all CHIM participants and use historical data on spontaneous clearance rates to determine efficacy, this approach is not preferred given the variability in clearance rates and lack of knowledge of clearance rates with the specific CHIM inocula. The rate of spontaneous clearance during natural infection varies from 10% to 50%, most notably influenced by human immunodeficiency virus (HIV) status, IFNL4 (interferon lambda 4) genotype, sex, hepatitis B exposure, and possibly age [11–15]. Clearance may also be influenced by HCV genotype [15]. With similar rates of persistence reported after infection from transfused blood products and needlestick injuries or injection drug use, the inoculation size is unlikely to affect the probability of clearance. Ideally, well-characterized inocula (representing different genotypes) could be used for all vaccine candidates evaluated using the CHIM approach, but this would require a clear expectation of the spontaneous clearance rate for each inoculum to determine the required sample size and to interpret vaccine efficacy. A well-characterized unvaccinated CHIM cohort could be used as the control arm for future vaccine candidate studies, provided proper matching to account for host determinants of clearance. If clearance rates are unaffected by viral genotype, data from unvaccinated CHIM participants infected with different inocula could be combined, even if the immunity produced may be strain/genotype specific.

POPULATION

The CHIM study aims to identify candidate HCV vaccines that are safe and effective in specific populations. Ideally, participants in a CHIM study would fully reflect those who would likely receive the vaccine post-approval, including people who inject drugs (PWID) [16]. In high-income countries, most infections (79%) occur among PWID compared with 38% in low- and middle-income countries [17]. People who inject drugs may have lower vaccine immunogenicity, further justifying the importance of their inclusion in this research [18, 19]. Although surveys of PWID suggest a high level of willingness to participate in HCV vaccine trials [20–22], recent qualitative studies addressing the acceptability of coronavirus disease 2019 (COVID-19) vaccination highlighted mistrust in institutions and fear of experimental vaccines as barriers to vaccination in this population [23, 24]. Thus, the inclusion of PWID and other at-risk populations will require meaningful engagement with affected communities throughout every phase of the research, based on the Denver Principles [25] and Nothing About Us Without Us movement [26, 27], to ensure dissemination of research results within communities and to foster their ongoing engagement in developing, testing, implementing, and benefiting from future vaccines and vaccination programs [28, 29].

Nonetheless, exclusion of reinfection when interpreting a CHIM and minimizing the risk of transmission to non–study participants make it more scientifically sound and ethical to characterize vaccine candidates in persons only exposed to the inoculum, thus excluding those engaging in HCV risk activities. We propose a stepwise approach beginning with the recruitment of healthy volunteers to characterize the inoculum and document infectivity and treatment feasibility. In this initial phase, the study population should consist of equal numbers of healthy adult male and female volunteers, aged 18–40 years, to reflect the epidemiology of HCV acquisition [30], from varied ethnic backgrounds. Additional inclusion and exclusion criteria are listed in Table 1. Factors associated with HCV clearance [31], such as Interferon lambda 4 (IFNL4) genotype and race, will not be prespecified in recruitment targets but documented and used to match future vaccinated participants. Extended enrollment may be required to balance the initial sample as needed.

Table 1.

Proposed Inclusion and Exclusion Criteria

Inclusion Criteria

Exclusion Criteria

1. 18–40 years old 2. Willingness and ability to provide written informed consent

1. Detectable HCV antibody and HCV RNA 2. Hepatitis B surface antigen (BsAg)-positive and/or anti–hepatitis B core antibody (anti-HBc) positive 3. Anti-HIV positive 4. Pregnancy 5. Body mass index >35 kg/m2 [50] 6. Chronic medical condition requiring prescription medication 7. ALT >40 U/L, AST >40 U/L 8. Total bilirubin >21 µmol/L, INR >1.3, albumin <38 g/L 9. eGFR <60 cc/min 10. HbA1c >6.0 or diagnosis of diabetes mellitus 11. Hemoglobin <13 g/mL (males) <12 g/mL (females) 12. TSH <0.4 or TSH >5.0 µU/mL 13. WBC <4.0 × 109/L, absolute neutrophil count <1.5 × 109/L, platelet count <150 × 109/L 14. Liver stiffness measurement >7.0 KPa 15. Underlying chronic liver disease 16. Substance-use disorder that may pose a risk of HCV transmission and/or liver injury (alcohol) 17. Evidence of cirrhosis or portal hypertension on abdominal ultrasound 18. Any medical or psychiatric condition that in the opinion of the investigator might be worsened by acute HCV infection and/or affect the response to antiviral therapy for HCV infection

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; eGFR, estimated glomerular filtration rate; HbA1c, glycated hemoglobin; HCV, hepatitis C virus; HIV, human immunodeficiency virus; INR, International Normalized Ratio; TSH, thyroid-stimulating hormone; WBC, white blood cell count.

Using an adaptive strategy, recruitment of participants will later be extended to include individuals at risk of acquiring HCV, particularly if the CHIM approach is used beyond initial proof-of-concept studies. Exclusion criteria will be refined with communities and based on specific risk factors for onward transmission or loss to follow-up, rather than broader identification with a particular group (eg, PWID): for example, future CHIM could be performed in people with past experience of injection drug use on stable opiate agonist therapy. Post-approval studies in at-risk populations, particularly PWID, will be critical.

MONITORING

HCV RNA would be followed regularly to document infection and the time of clearance, with initiation of antiviral therapy at 24 weeks for those who do not clear. Treatment could be initiated earlier in the small sentinel cohorts that establish the validity of the inocula (Figure 1). Liver enzymes (alanine aminotransferase [ALT], aspartate aminotransferase [AST]), liver function (total and direct bilirubin, albumin, and INR) and clinical manifestations of liver failure (eg, ascites, hepatic encephalopathy) would also be monitored carefully (Table 2). Immunological monitoring, including leukapheresis and the use of fine-needle aspirates of the liver in a subset (described in Shoukry et al [32]), would provide detailed immunological data to assess vaccine responses and inform vaccine design if initial candidates prove ineffective.

Table 2.

Proposed Monitoring Following Infection

Parameter	Frequency
HCV RNA	− Every 2 days until confirmed viremia − Twice weekly × 4 weeks and then weekly until spontaneous clearance or treatment initiation
Laboratory parameters: − Liver enzymes (AST/ALT) − Liver function (bilirubin, INR, albumin) − Creatinine, complete blood count	− Weekly with more frequently testing if required clinically
Immunology: − PBMC collection +/− leukapheresis − Anti-HCV antibodies	− Before and weekly after infection until initiation of antiviral therapy or confirmed spontaneous clearance − Additional PBMCs at 3 and 6 months post-treatment or documented spontaneous clearance

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; HCV, hepatitis C virus; INR, Interntional Normalized Ratio; PBMC, peripheral blood mononuclear cell.

ANTIVIRAL TREATMENT

Since they are safe, tolerable, and highly effective for the treatment of individuals with acute or chronic HCV infection, approved, pan-genotypic DAAs (sofosbuvir/velpatasvir × 12 wk or glecaprevir/pibrentasvir × 8 wk) will be used for all those with persistent infection. The combination of 3 pan-genotypic DAAs, sofosbuvir/velpatasvir/voxilaprevir, provides safe and effective (99% HCV cure in people without cirrhosis) treatment in the unlikely event of post-treatment relapse, including those with resistance-associated substitutions (RAS) [33].

Oral antivirals provide a critical safety net for protecting participants without spontaneous clearance or those with severe hepatitis. Rapid access to HCV antivirals is an essential component of an HCV CHIM and must be provided to all participants requesting treatment and those meeting specific safety or efficacy outcomes. The indications to initiate treatment before 24 weeks (eg, severe hepatitis) must balance the safety of the participants against the knowledge that greater liver enzyme elevation and jaundice are predictors of spontaneous clearance. Proposed thresholds for treatment are provided in Table 3. Final parameters would be determined in discussion with regulators and may adapt over time as confidence is gained in the safety of HCV CHIMs. How early treatment should be counted in the analysis requires careful consideration to balance the risk of bias against the assumption that those requiring/requesting early treatment would not have cleared and thus “penalizing” a vaccine candidate.

Table 3.

Treatment Indications

Cohort	Treatment Indication	Treatment Regimen
Sentinel cohorts	1. Persistent detectable HCV RNA 12 weeks postinfection 2. Severe hepatitis (ALT >20× ULN) and/or bilirubin >2× ULN 3. Participant request	1. GLE/PIB × 8 weeks 2. SOF/VEL × 12 weeks
Vaccinated cohorts	1. Persistent detectable HCV RNA 24 weeks postinfection 2. Severe hepatitis a. With clinical signs and symptoms of liver failure (ascites, encephalopathy, jaundice) and laboratory evidence of liver dysfunction (elevated INR > 1.5)a b. Asymptomatic ALT >20× ULN or bilirubin >5× ULN → repeat testing in 3 days. If improving, continued close observation. If worsening, initiate treatment. 3. Participant request	1. GLE/PIB × 8 weeks 2. SOF/VEL × 12 weeks
Treatment failure	1. Relapse post-treatment	SOF/VEL/VOX or regimen based on viral sequencing and assessment of resistance-associated substitutions

Abbreviations: ALT, alanine aminotransferase; GLE/PIB, glecaprevir/pibrentasvir; HCV, hepatitis C virus; INR, International Normalized Ratio; SOF/VEL, sofosbuvir/velpatasvir; SOF/VEL/VOX, sofosbuvir/velpatasvir/voxilaprevir; ULN, upper limit of normal

Participants with severe hepatitis should be treated with SOF/VEL for 12 weeks. Due to concerns about the metabolism of the HCV NS3/4A protease inhibitor, glecaprevir, in patients with impaired liver function, GLE/PIB is not recommended.

FOLLOW-UP AFTER HEPATITIS C VIRUS RNA CLEARANCE

Trial participants who achieve spontaneous or treatment-xinduced HCV RNA clearance will be monitored for recurrent viremia for at least 12 months after the end of antiviral treatment or the date of documented spontaneous clearance. Participants with detectable HCV RNA will have virus sequenced for resistance testing to guide retreatment. Long-term follow-up, even in the absence of recurrent viremia, will also be required to verify the safety of the CHIM approach, which will require enrollment in a post-CHIM registry.

COULD A HEPATITIS C VIRUS CONTROLLED HUMAN INFECTION MODEL APPROACH BE USED FOR VACCINE APPROVAL?

Most challenge studies for vaccine development are used to document an efficacy signal to justify larger phase 2/3 trials. However, with a relatively low incidence of infection, even in at-risk populations, standard randomized controlled trials (RCTs) similar to the vaccine is prevention trial would take several years to complete and would still only identify a limited number of acute infections. As a result, the precision of any protective effect would be low, restricting success to vaccines with a very large effect size.

Alternatively, the CHIM approach could also be used to document vaccine efficacy for regulatory purposes. Because all those who participate in a CHIM study are exposed to HCV, it is conceivable that more acute infections could occur and be evaluated in a phase 2/3 CHIM study with dozens of volunteers than in a large RCT with hundreds of participants. If CHIM could be used as a pathway to regulatory approval, as was done for the oral cholera vaccine [34], it would be critical to document postapproval safety and efficacy in populations at increased risk of HCV, particularly PWID.

SAMPLE SIZE CONSIDERATIONS

Sample size considerations will differ based on whether CHIM studies of vaccines are preliminary trials seeking an efficacy signal or larger studies to demonstrate sufficient efficacy for regulatory approval. Trials could be randomized controlled studies with a primary endpoint of viral clearance with a standard contemporaneous control group. However, as discussed, it is more likely that early studies will be adaptive, beginning with an initial “unvaccinated/placebo” group that serves as a control for future vaccine candidates of that inoculum.

The key determinants of the sample size will be the expected/desired effect size, the spontaneous clearance rate without vaccination, and heterogeneity (especially unmeasured) in the placebo/vaccine groups. The lower the spontaneous clearance rate, the easier it is to show a benefit of a vaccine. As shown in Figure 2, sample sizes in the range of 17–20 participants per arm for each inoculum would be adequate (80% power) if the spontaneous clearance rate is approximately 30% and the clearance rate with the vaccine is approximately 60%. The sample sizes required increase considerably if the spontaneous clearance rate is very high (eg, ∼50%) or if vaccine clearance rates are lower. Additional challenges include if persons voluntarily withdraw (eg, to take early treatment) or if the groups differ in ways that affect the expected clearance rate.

Figure 2.

Sample size estimation. The required sample size per cohort (y axis) to achieve 80% power is shown for various spontaneous clearance rates ranging from 10% to 60% with different vaccine clearance rates ranging from 30% to 90% (x axis).

Although modeling studies show that even a vaccine with only 30% efficacy would significantly impact progress towards elimination [35], given the enormous costs and logistical challenges of vaccinating at-risk groups and/or the general population, justification for proceeding to clinical use of a vaccine would likely require at least a 50%, and preferably approximately 70% protection, particularly given that this might be lower in at-risk populations such as PWID.

If larger phase 2/3 efficacy studies are performed using the CHIM approach, sample size and the composition of the study population (age, sex, ethnicity, IFNL4 genotype) will be informed by the initial efficacy trials. The efficacy studies would be accompanied by a large safety database and require postapproval follow-up to confirm real-world effectiveness and safety, particularly in at-risk populations (eg, PWID).

SAFETY CONSIDERATIONS

Controlled human infection model studies are unique in that risk to a participant is entirely iatrogenic and therefore procedures should be instituted to mitigate known and possibly unknown risks. Key safety concerns of a CHIM HCV vaccine study include risks related to the viral inoculum and acute and chronic HCV infection.

As discussed by Liang et al [4], the preferred source of the inoculum would be a plasma-derived virus from infected individuals. Despite transfusion medicine screening procedures, there would be a small risk of transmission of other blood-borne pathogens, including bacteria (∼1 in 75 000), other viruses (∼1 in 2 million), prions, and parasites [36]. However, if an acute HCV inoculum could be secured, the very small inoculum size required (likely <10 µL), and application of metagenomic sequencing, would further reduce transmission risks.

Most cases of acute HCV are asymptomatic or mild. However, approximately 15–25% of cases cause symptoms ranging from abdominal symptoms and fatigue to jaundice [8, 37]. Extremely rarely, the initial presentation may be acute liver failure or even fibrosing cholestatic hepatitis (FCH), a severe form of HCV-related liver disease that typically occurs in immunosuppressed patients [38]. Importantly, prompt DAA therapy has been successfully used to treat severe acute HCV and even FCH, without complication [39, 40]. Direct-acting antiviral therapy is well tolerated and associated with minimal risk. As an additional risk-mitigation strategy, the viral inoculum would be sequenced and screened for RAS and would not be used in CHIM participants until the donor was cured with the regimen to be used in the CHIM protocol. In the very unlikely circumstance of viral relapse after therapy, salvage regimens are available, with viral eradication expected in all individuals [41, 42]. To date, in trials of DAAs in acute HCV, all participants who relapsed after initial treatment have been cured with retreatment (see Martinello et al [43]). Similarly, in the setting of HCV transmitted from infected organ donors, despite their immunocompromised state, all recipients have been cured, even in the setting of initial relapse, including cases of FCH [39, 44–46]. Thus, the acute infection would be expected to be mild in most individuals, and reliably cured, but participants would need to be aware of potential risks and closely monitored with a plan to institute therapy with signs of a severe course.

With a short duration of infection, substantial liver injury, such as cirrhosis, or extrahepatic conditions associated with chronic HCV, such as essential mixed cryoglobulinemia, vasculitis, or B-cell disorders, would not be expected to occur [38]. Moreover, long-term follow-up studies of people with spontaneous clearance of acute HCV or cure in the absence of cirrhosis reveal overall mortality comparable to that of the general population after controlling for HCV-acquisition risks. Precise estimates of risks are difficult, but data from single-source outbreaks in young woman reporting no cases of long-term immunologic complications or hepatocellular carcinoma suggest that the risk is extremely low (see Hsu et al [47] for details on risks after transient HCV infection) [6].

Other considerations for CHIM participants include risks of persistence of HCV RNA. Reports of recovery of replication-competent HCV at very low levels after curative therapy for chronic infection that was shown to be infectious in a chimpanzee raise concern for the persistence of infection with a potential risk of complications and/or transmission to others [48, 49]. However, these reports are few in number and only reported after interferon-based therapy. Furthermore, long-term follow-up data after spontaneous clearance and sustained virologic response are very reassuring, with no clear evidence of recurrent viremia even with very potent immunosuppression, suggesting that viral persistence is unlikely to be a significant concern. Transmission has not been reported from someone after cure of HCV; however, at least for now, CHIM participants would not be allowed to be blood donors, in line with current guidance for those with past HCV infection.

In addition to the risk to CHIM participants, onward HCV transmission must be considered. Participants would be educated on household transmission risks and those with known substance use would be excluded. Despite a low risk of sexual transmission, participants would be required to inform intimate partners and agree to use condoms during the period of infection. Partners would be offered HCV testing after CHIM completion. Finally, there may be a risk of stigma associated with anti-HCV positivity.

CONCLUSIONS

Based on careful consideration of the many aspects to a CHIM approach, we have outlined a potential trial design. Small sentinel cohorts would be enrolled to evaluate the infectivity and curability of each inoculum, followed by larger cohorts to define the spontaneous clearance rate in unvaccinated healthy populations. Once established, vaccine candidates could be evaluated with the primary endpoint of clearance of viremia by 24 weeks postinfection and compared with an unvaccinated cohort, with the sample size determined by the spontaneous clearance rate in the unvaccinated group and a minimum 50% vaccine efficacy. Sterilizing immunity would also be assessed but would not be the expected or required benchmark for vaccine efficacy. The initial study population would be young, healthy adult volunteers matched on sex and IFNL4 genotype to the initial unvaccinated cohort, with a commitment to document safety, immunogenicity, and efficacy, in at-risk populations, particularly PWID, after the initial healthy volunteer studies. Participants who do not clear infection by 24 weeks or who show signs of severe hepatitis would be started on DAA therapy for the duration used to treat chronic HCV infection. Participants would be monitored carefully during therapy and followed long-term after the CHIM study to ensure there are no untoward effects of transient HCV infection.

Hepatitis C virus CHIM is likely required for timely development of an HCV vaccine. Many factors must be considered in designing a clinical trial to evaluate vaccine candidates using the CHIM approach, and although not the only approach, the strategy outlined addresses key considerations required to carry out a CHIM study for HCV in a safe and ethical manner.

Notes

Financial support. This work was supported in part by US National Institutes of Health grants R01DA048063 and K24DA034621, and an anonymous donor.

Supplement sponsorship . This article appears as part of the supplement “Controlled Human Infection Model for HCV Vaccine Development,” sponsored by Toronto General Research Institute, United States National Institutes of Health, Johns Hopkins University, the Canadian Institutes of Health Research (CIHR), and the Canadian Network on Hepatitis C (CanHepC). CanHepC is funded by a joint initiative of CIHR (HPC-178912) and the Public Health Agency of Canada.