Progress, evolving therapeutic/diagnostic approaches, and challenges in the management of hepatitis C virus infections

Hepatitis C virus (HCV) infections are emerging as one of the foremost challenges in healthcare owing to its chronicity and the virus’s quasispecies nature. Worldwide, over 170 million people are chronically infected with HCV, with an annual mortality of over 500,000 people across the world. The emerging pathophysiological evidence links HCV infections to a risk of developing liver diseases such as cirrhosis and hepatocellular carcinoma. Despite the great strides that have been made towards understanding the pathophysiology of disease progression, the tailored treatments of HCV infection remain to be established. The present review provides an update of the literature pertaining to evolving therapeutic approaches and prophylactic measures for the effective management of HCV infections. An extensive discussion of established and experimental immune prophylactic measures also sheds light on current developments in the design of vaccination strategies against HCV infection. We have also attempted to address the application of nanotechnology in formulating effective therapeutic interventions against HCV. Pointing out the limitations of the existing diagnostic methods and therapeutic approaches against HCV might inspire the design and development of novel, efficient, reliable, and cost-effective diagnostic technologies as well as novel therapeutic and immune prophylactic interventions for the effective management of HCV.


Introduction
Viral hepatitis can be caused by various viruses, including hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV), and hepatitis G virus (HGV) [1]. Since its first detection in 1989 [2], it has become clear that HCV is a major cause of chronic hepatitis. HCV belongs to the genus Hepacivirus in the family Flaviviridae [3]. More than two decades since its discovery, HCV continues to be a major cause of concern and a huge burden on national public health systems worldwide. A recent report estimates that over 71 million people worldwide are chronically infected with HCV [4], and approximately 170 million people are estimated to be at risk of HCV infection. This virus has the highest prevalence in Africa and Asia, where it is four times more prevalent than HIV, and hence, HCV has the potential to cause the next pandemic, as the mortality has reached over 500,000 per annum [5]. The incubation period of HCV ranges from weeks to several months, with an average of around 7 weeks until symptoms occur [6]. HCV infection is often asymptomatic, making it very difficult to detect in its early stage. This is the main reason why patients do not receive early treatment, and HCV is often referred to as a "silent killer" [7]. This disease can have various outcomes, ranging from mild (minimal inflammation of the liver) to severe, and can lead to scar tissue formation. Chronic infection eventually causes cirrhosis, leading to hepatocellular carcinoma and, ultimately, death [8]. A report from the Centers for Disease Control and Prevention (CDC) states that HCV-related cirrhosis is observed in 5-20% of patients experiencing chronic HCV infections over a prolonged period of 20-30 years. Moreover, advanced cirrhosis is associated with end-stage liver disease, liver failure because of portal hypertension, hepatic encephalopathy, hepatorenal syndrome, and hepatocellular carcinoma [9]. As the most commonly observed type of liver cancer, research data from the last three decades 1 3 suggest that hepatocellular carcinoma occurs in approximately 1-3% of HCV patients [10]. Of note, effective anti-HCV treatments can help to reduce the overall mortality rates in HCV patients with compensated cirrhosis [11].
HCV has a single-stranded positive-sense RNA genome of around 9.5 kb that encodes a single polyprotein precursor of 3011 to 3033 amino acids [12,13]. Upon proteasemediated cleavage, the polyprotein precursor yields two distinct types of proteins that can be categorized as structural (Core, E1, E2, and p7) and non-structural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins [14]. Owing to the importance of these proteins in the pathogenesis of HCV, we have reviewed recent advancements in the development of drugs targeting the structural and non-structural proteins of HCV. The evolving genetic heterogeneity of HCV serotypes is one of the important limiting factors in the effective management of HCV patients, and also in the development of novel pan-genotypic treatment modalities. The high genetic variability exhibited by HCV has resulted in the generation of seven to eight major genotypes. These genotypes appear to be quite diverse at the genetic level. For example, the identified genotypes show 30% difference in their genome sequences. Within these major genotypes, 84-86 subtypes have been identified that exhibit differences ranging from 15 to 25% at the nucleotide level [15,16]. In the mainstream of HCV research, there are still questions waiting to be answered. For instance, it is not clear whether HCV genotypes originated from a single cross-species transmission and further diversified within the human population or whether it originated from separate zoonotic sources in different geographical regions [17].
In this review, we provide an update on advancements in the field of therapeutics and diagnosis of HCV. Previous reviews have mainly described diagnosis, therapy, or management of HCV infections [18][19][20][21] and have focused on individual genotypes [22,23]. In contrast, in the present review, we provide an extensive literature survey on current diagnosis and therapeutic modalities for various genotypes of HCV and their associated shortfalls. Taking into consideration the current state of the art, the current review offers updated information on the development of novel drug strategies and factors limiting the effective management of HCV infections.

Attempts to develop new HCV therapeutic modalities: successes and failures
With the advent of the COVID-19 pandemic and the acknowledgment of the HCV research of Michael Houghton, Harvey Alter, and Charles Rice by the Nobel Committee in 2020, the search for novel and effective antiviral agents has accelerated, and some of these agents might also find use in the management of HCV. In HCV drug development programs, a sizable amount of human resources and capital is being invested in developing antiviral drugs. Although there is still no completely effective antiviral therapy tailored specifically for treatment of HCV infection, dozens of small-molecule drugs for stand-alone use or combination therapy have been approved and recommended by the FDA (Table 1), and others are in different phases of development, including clinical trials. These inhibitors mostly target viral nonstructural proteins such as NS3/4A, NS5A, and NS5B, or the internal ribosome entry site (IRES) [24]. Some of the most recently identified HCV antiviral agents are shown in Figure 1.
Setrobuvir is a potent small-molecule, non-nucleoside inhibitor of the HCV genotype 1 NS5B RNA polymerase. This drug reached clinical trials at Roche Pharmaceuticals [25,26]. However, it was removed from the development pipeline and from phase II clinical trials in 2015 due to adverse effects. Patients receiving setrobuvir monotherapy and those receiving setrobuvir in combination with other direct-acting antivirals developed rashes. Danoprevir is a macrocyclic, peptidomimetic, small-molecule compound that was developed at Roche Pharmaceuticals. This drug competitively inhibits the HCV NS3/4A protease [27][28][29]. After successfully completing phase III clinical trials, this drug was launched in the market in China in August 2018. Mericitabine is an oral, small-molecule nucleoside analogue that is phosphorylated intracellularly, resulting in two distinct active triphosphate forms that target the NS5B RNAdependent RNA polymerase of HCV, affecting the viral replication [29][30][31]. However, this drug was discontinued in phase II clinical trials in 2016 because of an inadequate number of HCV patients participating in the trials [32]. Iridoids (such as the aglycones of shanzhiside methyl ester loganin and verbenalin) and amphipathic DNA polymers are novel antiviral molecules that were designed to inhibit entry of HCV into cells [33]. The drug telaprevir (VX-950), which targets the NS3-4A protease, showed some success in clinical trials [34]. It is being marketed worldwide by Janssen R&D Ireland. Different analogues of ribavirin, namely 4-iodo-1-beta-ribofuranosylpyrazole-3-carboxamide, 4-propynyl-1-beta-ribofuranosylpyrazole-3-carboxamide, and 4-phenylethynyl-1-beta-ribofuranosylpyrazole-3-carboxamide, are now being considered as alternative candidate drugs [35].
BMS-824393 is a small molecule that targets the NS5A protein [36][37][38], and it was discontinued in phase II clinical trials due to severe adverse effects. Zinc meso-porphyrin holds immense potential as a novel drug to treat HCV infection [39]. In the last decade, the NS5B protein has been shown to be a good target for developing candidate drugs against HCV, and various NS5B inhibitors, such as NM-283 [40], IDX-184, and MK-0608 have been described. NM-283,  [45,49] developed by Idenix Pharmaceuticals, was found to be associated with severe gastrointestinal toxicity and hence discontinued from the phase II clinical trials. Despite initially promising results, the development of IDX-184 has been kept on clinical hold since February 2013, and no further update is available for this agent. MK-0608, despite encouraging results in animal model studies, was discontinued in 2011 due to unsuccessful clinical trials. Pfizer's PF-00868554 was discontinued from phase II clinical trials in February 2013 due to adverse effects, including headache, fatigue, insomnia, and nausea. GS-9190 (tegobuvir), produced by Gilead Corporation, is an NS5B protease inhibitor that was discontinued due to various adverse effects in phase II clinical trials, including flu-like syndrome, pyrexia, headache, and myalgia [41]. Several other molecules, such as VCH-916 (Viro Chem Pharma) [42], were discontinued from phase II clinical trials due to gastrointestinal disorders, throat irritation, and nausea. GSK-625433 (GlaxoS-mithKline) [43] reached phase I but was later discontinued (Fig. 2). ABT-333/dasabuvir (developed by Abbott Laboratories) is currently being successfully marketed in Israel, the UK, Switzerland, and the USA, and, after completing phase III trials, was approved for monotherapy as well as combination therapy with ribavirin [44].

Evolving approaches in HCV therapeutics
The scientific community and researchers at various pharmaceutical companies are continuously working on developing novel and effective therapeutic interventions against HCV. In the past five years, novel lead compounds with improved efficacy and safety have been discovered [45], and a series of anti-HCV candidate drugs have been proposed. The following section attempts to summarize these new candidates and their respective drug targets. NS3/4A has been found to be an attractive target for series of therapeutic agents, including danoprevir, voxilaprevir, vedroprevir, MK-8831, faldaprevir, ABT493, BMS-605339, and BMS-890068. NS4B has also proven to be a good drug target, as various drugs, including PTC725, 2-oxadiazoloquinoline derivative, imidazo[2,1-b]thiazole derivative, and piperazinone have been proposed based on modulation of NS4B activity. NS5A also appears to be a promising drug target, and various drugs, including ABT-530, ruzasvir, ravidasvir, GSK2336805, EDP-239, samatasvir, biphenylimidazole analogues, and disulfiram target this protein. NS5B has been described as an effective therapeutic target, and various chemical agents, including beclabuvir, GS-9669, thiophene carboxylate allosteric inhibitor, mericitabine, deleobuvir, DAPN-PD1, TMC647055, filibuvir/VX-222, GS-6620, tegobuvir, JNJ-54257099, 6-aminoquinolone derivative, and pyrazolobenzothiazines have been proposed as inhibitors of NS5B. In addition, the structural proteins E1/E2 and p7 are being explored as therapeutic targets for drugs such as benzimidazole derivative, cynaropicrin/grosheimol, saikosaponin b2, chlorcyclizine derivative, flunarizine, monoclonal antibodies and adamantane/rimantadine. In addition to directacting antivirals, immunostimulators such as alisporivir, bis-amide derivative, NIM258, phenylepyrrolidine derivative, isothiazolo [5,4-b] pyridine, ITX-5061, MA026, and soraphen A are also under consideration for repurposing as anti-HCV agents. These drugs have been reviewed extensively by Li and De Clercq [45]. Table 1 summarizes the therapies that are now commonly offered to patients with HCV infection, and Fig. 3 provides a brief overview of the location of the genes for these drug targets in the HCV genome. Table 2 summarizes the profile of clinical trials of promising anti-HCV candidate drugs that are in the early stages of development and has enormous potential to act as effective antivirals.

Current immunoprophylactic approaches for the management of HCV infections
The growing incidence of HCV infections and related public-health complications prompted the World Health Organization to establish a program to eliminate HCV by 2030. To achieve this goal, there is a need for an effective, safe, and affordable vaccine against HCV.
Since the discovery of HCV more than 30 years ago, there have been significant advances in understanding the molecular aspects of HCV pathophysiology, the molecular immunological mechanisms involved in spontaneous viral clearance, and the rational design of vaccines. There is a need to develop effective prophylactic vaccines against HCV, as current HCV treatment approaches are not effective in preventing recurrent infections and are not affordable in economically challenged parts of the world restricting their clinical use to developed countries [73,74]. In general, antiviral therapies require long-term treatment, are expensive and toxic, and are effective only in about 50% of patients against the majority of the HCV genotypes [75]. There have been a number of reports indicating progress toward the development of therapeutic vaccines for the management of HCV infections. For example, IC41, a synthetic peptide vaccine containing HCV T-cell epitopes has been shown recently to be safe and is currently undergoing a phase I clinical trial. IC41 can induce HCV-specific INF-gamma-secreting CD4 + and CD8 + T cells in healthy individuals. It has been shown to induce HCV-specific Th1/Tc1 responses in a subset of difficultto-treat HCV non-responder patients [76]. Another interesting candidate for a peptide vaccine is the HCV-specific HLA-A2-restricted epitope NS3-1073 [77]. Table 3 summarizes the promising candidate vaccines against HCV that are in clinical trials, and Table 4 lists the important CD8 and CD4 epitopes of HCV recognized by T cells that have been identified in the past five years [78]. The epitopes identified before this period have been reviewed by Yu and Chiang [79]. These epitopes have potential for use in peptide-based subunit vaccine formulations.

Challenges in vaccine design
The major challenge in the development of a vaccine against HCV infection is the unusual genetic diversity of the virus [83][84][85]. There are seven or eight known genotypes and more than 84-86 subtypes, making this virus even more diverse than human immunodeficiency virus [15,16,[85][86][87]. HCV has 30% genetic diversity at the genotype level and 15% at the strain level. The error-prone polymerase of HCV and immune selection generate related but genetically diverse virus variants, even within the same infected individual, thereby posing the problem of resistance to antibody and T-cell-mediated immune responses [88][89][90][91]. The use of live-attenuated whole virus particles is a common strategy for vaccine development. However, in the case of HCV, the feasibility of live vaccines is uncertain, as in vitro-grown strains of HCV have a high rate of adaptive mutation, which eventually leads to the generation of highly efficient replicative strains [92][93][94]. However, for unknown reasons, the infectivity of such strains in primate cell lines and primate animal models is very low [95][96][97]. Adaptive mutations in such highly replicative strains of HCV are linked to the nature of viral genotypes [87,93]. Another setback in the field of HCV vaccine development is the lack of immunocompetent animal models. Although chimpanzees can be used as an alternative natural host for HCV, factors such as low chronicity and a lack of clinical signs of infection, limit their utility as an animal model [98]. Other primates such as rhesus macaques (Macaca mulatta), doguera baboons (Papio anubis), chacma baboons (Papio ursinus), Japanese macaques (Macaca fuscata), green monkeys (Chlorocebus sabaeus), and New World monkeys (Saguinus mystax, Saguinus labiatus, and Saguinus oedipus) develop asymptomatic HCV infections [99][100][101][102]. Although HCV-like viruses have been  1 3 found in rats, allowing them to be used as a small-animal model for HCV-like infection, the genetic diversity of rat hepacivirus limits its usefulness in vaccine testing. Surface protein modification through mutations in various regions of the envelope proteins E1 and E2 is one of the ways in which HCV escapes from adaptive immune responses of the host. Mutations in hypervariable region 1 (HVR1) subvert the neutralizing antibody response by masking conserved epitopes. Furthermore, post-translation processes such as glycosylation have been reported to cause steric interference with the binding of neutralizing antibodies [103]. Various experimental studies of the effects of glycans at different positions have demonstrated that changes in glycosylation can lead to antibody resistance [104,105]. This is supported by studies showing that natural and sitedirected mutations at various glycosylation sites on the E2 protein decrease the sensitivity of cell-culture-grown HCV (HCVcc) and HCV pseudoparticles (HCVpp) to neutralizing antibodies [105].
Some attributes of the host also help HCV to escape from antibody neutralization. High-density lipoproteins in human serum are known to interact with scavenger receptor class B type I (SR-BI). Such interactions increase the rate of virus entry into the cell and reduce the time window for neutralizing antibodies to act [106]. Apolipoprotein E is functionally involved in viral transport and was also demonstrated recently to help mature HCV particles to escape neutralization by masking their epitopes [107]. A recent study on HCV-infected cell cultures showed that E2-protein-coated exosomes were able to make HCV less sensitive to neutralizing antibodies. These findings may find applications in the development of an HCV vaccine [108].

Challenges in diagnostics
Serological methods are commonly used for the diagnosis of most bacterial and viral infections. In the case of HCV, ELISA, recombinant immunoblot assay (RIBA), and rapid assays are the most commonly used serological detection methods. These methods have the disadvantage that they can give false-negative results when testing immunocompromised patients and when used during the window period of HCV infection. False-positive results can also occur due to cross-reactivity in individuals with lupus or rheumatoid arthritis. NAAT (nucleic acid amplification testing) is considered the gold standard for diagnosis of various diseases, especially viral diseases, and it is generally found to be advantageous over traditional serological tests. PCR and transcription-mediated amplification are the two prominent technologies currently employed to detect viral genomes. NAAT has the advantage that it can discriminate between actively infected and recovered patients and thus provides an additional safety layer for preventing transfusion-transmitted   [63,78] infections (TTIs). It also has the important advantage that it allows a diagnosis to be made at an earlier stage of infection. This is important because early treatment of an acute HCV infection improves the likelihood of eliminating the virus and preventing the establishment of a chronic infection. Using NAAT, viral RNA can be detected in an HCVinfected sample within one week of infection [109]. The high sensitivity and specificity of NAAT make it more reliable than serological tests, with fewer false-negative results [110]. In a survey published in 2007, 486,676 seronegative blood samples were screened by NAAT, with detection limits of 1:97,000, 1:490,000, and 1:2800 for samples containing HIV, HCV, and HBV, respectively [111]. Although NAAT is a highly sensitive and specific method for HCV detection, its high cost and requirement for skilled workers and sophisticated instrumentation limits its usage on a mass scale in resource-constrained countries. To make the method more affordable, institutions are using a mini-pool model of NAAT (MP-NAAT) for the detection of virus contamination. Moreover, highly sensitive immunological assays are being developed that can ensure blood safety at an affordable cost [112].

Other possible challenges
Another major challenge in the management of HCV-related disease is the large number of undiagnosed and therefore untreated cases of HCV infection in the population. In the USA, about 1.8 million people are unaware that they are HCV positive [113]. This is partially due to healthcare providers not taking time to ask appropriate questions when examining patients, as recommended in the guidelines of AASLD-IDSA (American Association for the Study of Liver Diseases and the Infectious Diseases Society of America). Furthermore, risk-assessment strategies sometimes fail to identify HCV-infected patients, and some infected patients do not want to disclose their risk factors. Furthermore, the cost of modern HCV therapies such as direct-acting antivirals (DAAs) is a hurdle in HCV disease management. In the USA, the initial price of the drug sofosbuvir is about 1000 USD for a single tablet, and a standard 12-week treatment costs approximately 10,000 USD [114]. At the global level, the cost issue is a major obstacle to the HCV eradication program.

Traditional treatment regimens: past and present
In the 1990s, interferon combined with the nucleotide analogue ribavirin was the only therapy available and was the sole standard care for HCV infections [115]. Subsequently, the introduction of pegylated interferons (alfa-2a and alfa-2b) in combination with ribavirin improved the sustained virological response (SVR) up to 40% to 50% for genotype 1. Similarly, the SVR was observed to improve to 70% to 80% for genotypes 2 and 3, which was 7 to 12% higher than the previously used interferon-ribavirin combination [115,116]. However, the use of pegylated interferon is associated with psychological, autoimmune, and blood-related side effects. Moreover, it fails to cure chronic cirrhosis caused by HCV infections [18]. It has been reported that, in some cases, complications such as ischemia and neuropsychiatric emergencies do not completely resolve even after withdrawal of the interferon medication [117]. Interferon treatment has been found to be associated with the suppression of bone marrow, leading to a decline in major blood cell lines [117]. Moreover, medications used in interferon therapy are supplied in the form of prefilled syringes that need to be injected subcutaneously, which can be intimidating for patients in the initial stages of the treatment, thereby requiring trained professionals to remain accessible during the entire course of treatment. Ribavirin can also cause side effects, such as teratogenesis, hemolysis, flu-like symptoms, and rashes on the skin [116]. For these reasons, patients need to be closely monitored throughout their treatment, and there is a need for very careful counseling regarding the risks associated with their medications. These side effects of pegylated interferon-ribavirin (PIR) therapy make it unsuitable for standard care of HCV infections. In 2011, the FDA approved two direct-acting antiviral DAAs, boceprevir and telaprevir, which were marketed by two different companies. SVR was observed to increase to 70% when PIR therapy was combined with either of these DAAs. However, complex dosages, the need for a selective diet, and adverse effects remain major impediments to the success of this therapeutic regimen. DAAs are grouped into three classes based on their target. These include NS3 protease inhibitors, NS5A inhibitors, and NS5B polymerase inhibitors [118]. The NS3 protease is involved in the cleavage and processing of structural and non-structural proteins of HCV. NS5A is a target for drugs due to its important role in the modulation of the interferon response. NS5B is the viral RNA-dependent RNA polymerase. RNA polymerase inhibitors are further classified as nucleotide analogue inhibitors and non-nucleotide analogue inhibitors boceprevir and telaprevir are first-generation drugs, and grazoprevir and voxilaprevir are second-generation NS3 protease inhibitors. The use of first-generation NS3 protease inhibitors is believed to contribute in the emergence of resistant strains in non-responder patients [119]. During monotherapy with telaprevir, although the HCV RNA level decreases significantly in the early phase of treatment, the development of resistance is observed in a large proportion of cases, which has been associated with four mutations in the NS3 region [120]. Similarly, six mutations have been observed to be associated with resistance to boceprevir treatment [121]. Frequent treatment failures due to the acquisition of resistance mutations due to boceprevir and telaprevir therapy have resulted in a shift to triple therapy primarily consisting of PIR and telaprevir. Despite the good results of triple therapy in clinical trials, 96.17% of patients experienced adverse effects, and 34.98% experienced severe adverse effects, including renal failure, skin-related problems, and anaemia [120]. This failure necessitated the quest for an alternative therapy, and a breakthrough came in 2013 with the approval of an NS3 protease inhibitor, simeprevir [122], and an NS5B inhibitor, sofosbuvir (Sovaldi, Gilead Sciences) [122,123]. The combination of these two drugs, with or without PIR, resulted in an increase in SVR to 90%, with a well-tolerated daily dose [18]. Different combinations of sofosbuvir and simeprevir are associated with various adverse effects, including fatigue, headache, nausea, insomnia, anaemia, and decreased appetite. In addition, treatment regimens including sofosbuvir and simeprevir have been reported to generate drug resistance-associated virus variants [124][125][126].
Further research in the field of HCV drug development has added well-tolerated, orally active DAAs that are effect without interferon treatment, and combinations of these can be used to treat patients with comorbidities and chronic HCV infections with various genotypes [18,127]. Clinical trials have been conducted on four DAA drugs (paritaprevir/ritonavir/ombitasvir plus dasabuvir) in patients who had been treated previously with pegylated interferon and in untreated patients. The resulting SVR scores were in the range of 90.2 to 100% for HCV genotypes 1a and 1b [128]. In a metaanalysis of published data on the four-DAA drug regimen, among the 20 cohorts tested in 12 countries, SVR was 96.8% for genotype 1 and 98.9% for genotype 4. The same study reported a 1.3% virologic relapse rate among 5158 patients included in the analysis [129]. Thus, DAA treatments show promise, with improved SVR, good tolerability, and a very narrow range of drug resistance when prescribed in combination. However, combination therapy is also associated with various mild, moderate, and severe adverse effects. Furthermore, virological response failures due to mutations in the target proteins have been reported [130].

Future perspectives
DAAs and other therapies can be successful in the primary stage of HCV infection, provided that the infection is diagnosed at an early stage, and in this regard, the methods currently available for HCV diagnosis are not up to the mark in terms of early diagnosis. The potential of nanotechnology in disease diagnosis is gaining attention because it is simple, reliable, durable, and inexpensive. Gold nanoparticles (AuNP) are a well-studied nanosystem that has advantages over other types of nanoparticles. AuNP can be electrostatically surface-functionalized with various biomolecules including antibody peptides and nucleic acids [131][132][133].
In addition, AuNPs are highly sensitive to surface plasmon resonance (SPR), showing a prominent color switch from red to blue while transforming from the dispersed state to the aggregate state [134]. Acetylcholinesterase E (AChE) enzyme-conjugated AuNP have been found to be highly efficient for the detection of HCV antibodies. RT-PCR can be used for virus detection, but it requires highly skilled personnel and may not be available in many places. Colorimetric detection of HCV RNA in patient samples without amplification using AuNP has been suggested to be a useful approach. [135,136]. In a recent study, 27-nucleotide-long RNA probes specific for the 5'UTR of HCV RNA were used for the detection of HCV RNA, either assembled on metallic nanoparticles or without nanoparticles. If a sample is positive, the RNA probes hybridize to the viral RNA and aggregate upon addition of unmodified AuNP, turning the solution blue [135]. Other nanosystems have been used to detect HCV RNA. For example, chemically labelled HCV-RNA-specific probes conjugated with iron oxide nanoparticles can detect HCV in samples from patients, without interference from cellular DNA and RNA [137]. Cytotoxicity and adverse effects of interferon and pegylated interferon can be minimized by site-directed delivery. Hyaluronic acid (HA) is one of the abundant extracellular polysaccharides that naturally target the liver by binding to cluster determinant 44 (CD 44/HA receptors) and get internalized into the cell. HA-decorated AuNPs combined with interferon through hydrophobic and electrostatic forces have been reported to be delivered efficiently to the liver [80]. Furthermore, the polyanionic nature of HA prevents the adsorption of serum proteins onto the nanoparticle-bound interferon. Using this delivery system, no side effects of interferon or pegylated interferon were observed [138]. Targeted delivery of ribavirin can be used to alter its distribution pattern, thereby reducing its cytotoxicity to erythrocytes. The use of polymeric nanoparticles consisting of poly-D, L-lactic acid (PLA) and arabinogalactan poly-Llysine (AG-PLL) conjugated with ribavirin has been shown not only to reduce cytotoxicity but also to result in targeted delivery to liver cells [138,139].
Despite the success of DAA therapy, there has been a significant decrease in the popularity of DAAs due to the issues of affordability, resistance, and genotype selectivity. This situation is motivating the development of new therapeutic options. Peptide C5A, derived from the NS5A protein of HCV, has demonstrated potent toxicity to HCV in Huh 7.5 cells [141]. Another peptide, from the N-terminal region of the NS5A protein has amphipathic properties and has been observed to lyse lipid vesicles in a model system for lysis of the virus [141]. Peptides from the NS5A protein are highly toxic for HCV at sub-micromolar concentrations. Furthermore, these peptides were found to be non-toxic to the host in vitro and in vivo at doses 100-fold higher than those required for antiviral activity [142].
Nano-vaccines offer several advantages over traditional vaccines, including rapid delivery and penetration across the membrane of targeted cells. Recombinant core and NS3 fusion proteins conjugated with polylactide-co-glycolide nanoparticles (rC-N/PLGA NPs) have been shown to induce a T-cell-mediated immune response [143]. The shape, size, and polydispersity index of rC-N/PLGA NPs determine their penetration capacity. Triblock copolymers (e.g., poly (D, L-lactic acid)-co-poly(ethylene glycol)-co-poly(D, L-lactic acid [PLA-PEG-PLA] and poly(D, L-lactic-coglycolic acid)-b-poly(ethylene glycol)-b-poly(D, L-lacticco-glycolic acid [PLGA-PEG-PLGA]) have been found to be good carriers of DNA encoding multiple viral epitopes [144]. Furthermore, these copolymers increase both cellmediated and humoral immune responses of such multiepitope gene constructs compared to their naked forms [145]. Controlled release of multiepitope antigen genes from such micellar systems is expected to provide immunological benefits in a single-dose immunization [146].
A strong and long-lasting immune response is one of the ideal characteristics of an efficient vaccine. A natural adjuvant (heat shock protein 27) and cell penetration protein (CPP-Caddy2 and HR9) co-expressed with NS3 protein have been shown to increase the cellular Th1 immune response [147]. This study also showed an elevated cytokine profile of IgG2a, IgG2b, and IFN-γ and increased granzyme B secretion when using this system. A stronger immune response was observed in a mouse model when an NS3encoding genetic vaccine and an NS3 protein vaccine were administered together than when administered separately [148]. A potent immune response had been reported to be induced by double-stranded RNA encoding the NS3 protein.
In mice immunized with recombinant NS3 protein (rNS3) and poly(I:C) emulsified in montanide ISA 720 (M720), the CD8 T cell immune response was found to persist for 7 months [149]. These studies collectively show that such formulations can induce a strong and persistent cellular immune response against HCV.

Conclusion
The overall objective of the current review was to provide a bird-eye view of recent advancements in the field of HCV therapeutics and prophylaxis, giving a summary of existing HCV therapies and their limitations. We initially summarized recent efforts to develop new HCV therapeutic modalities, discussing a few examples of successful drugs and failed attempts. We also attempted to summarize the features of existing therapies and their inherent drawbacks to pinpoint the opportunities and challenges to consider when developing novel treatments. We also summarized the features of drugs that are in the early stages of clinical trials, providing their clinical trial identifiers for effective monitoring of new HCV therapies. We also covered the discussion on emerging immune prophylactic measures, including new vaccine candidates and important biologicals that are in early clinical trials, with special emphasis on newly identified CD4 and CD8 epitopes that can be included in peptidebased subunit vaccines. We also discussed the hurdles that the scientific community is facing in terms of effective disease diagnosis and challenges in existing HCV therapies. Finally, we attempted to provide a nanotech perspective on vaccine design. In summary, although we have tried to paint a positive picture regarding the development of novel HCV therapeutics, recent therapies are still far from perfect, and further improvements are still needed.
Author contributions All three authors wrote the paper, RNG conceived the idea and supervised the progress of the project, and RJM and GHK prepared figures and tables.

Conflict of interest
The authors declare that they have no conflict of interest.