HCV DIAGNOSTICS, DRUGS AND VACCINES

Hepatitis C is a serious liver disease that results from infection with Hepatitis C Virus (HCV). It was identified as a causative agent for non-A and non-B viral hepatitis in late 1970s. HCV affects more than 200 million people worldwide. Patients are generally asymptomatic and get diagnosed late. Besides research, the key for effective control and elimination of the virus is early diagnosis which requires increase in awareness and broad diagnostic screening programme. Due to its initial non-specific flu like symptoms the disease is not clinically recognisable and requires broad healthcare screening programmes. Despite high levels of HCV replication, the HCV-specific immune responses remain undetectable in most infected individuals for several weeks suggesting that the virus escapes from the immune system and impair immune responses. HCV diagnosis plays a key role in determining and deciding the treatment procedure and the duration of treatment. HCV diagnostic test include the serologic and the molecular diagnostic testing (Figure 1). The serologic testing detects human antibodies generated against the HCV infection where as the molecular test detects the viral RNA.

• Serologic Testing

1.      Enzyme Immunoassay - The third generation EIA is designed to detect antibodies that bind the core and the nonstructural regions of HCV.

2.      Point of Care Rapid Immunoassays- The OraQuick HCV Rapid antibody test (OraSure Technologies, Bethlehem, PA) was approved by FDA in 2010 as a point-of-care test. The specimen type includes whole blood, serum or plasma. The nitrocellulose membrane is immobilised with synthetic peptides of core, NS3 and NS4 antigens. A reddish purple line is generated within 30 min in the presence of anti-HCV antibodies in the specimen on addition of colloidal gold labelled protein A. These rapid tests are cheap and fast¹

• Molecular HCV RNA test-

Molecular test detects the HCV RNA and is commonly called as Nucleic Acid Testing (NAT). The HCV RNA is detected 1-2 weeks after initial infection resulting in a positive HCV NAT test. The quantitative HCV NAT test is ultrasensitive and can detect as few as 5copies/mL. The detection and quantitative is important as it helps in -

1.      In diagnosing chronic HCV infection

2.      A guide to treatment decisions

3.      Used in monitoring response to therapy

It is also used to identify the stage of infection, if a NAT result is negative for a serologic positive case then it indicates that the patient has a resolved infection or spontaneous resolution. However, in cases of chronic infection a second NAT test should be performed².

Figure 1- Interpretation of HCV results

Source: Centers for Disease Control and Prevention (CDC). Testing for HCV infection: an update of guidance for clinicians and laboratories. MMWR Morb Mortal Wkly Rep. 2013; 62:362-5.

• HCV Genotyping

HCV is highly genetically diversified virus with seven distinct genotypes and numerous subtypes of the virus. Genotype determination is important as it plays a direct role in determining the treatment and making correct therapeutic decisions³. Methods to determine the genotype include-

1.      The VERSANT HCV genotype assay is a probe based assay also called LiPA( Line Probe Assay), in this the 5'UTR is amplified using biotinylated primers followed by its hybridisation to genotype-specific probes impregnated on a membrane and the result is detected with streptavidin based on colorimetry⁴.This method determines the genotypes but can't distinguish between the subtypes.

2.      Restriction Fragment Length Polymorphism (RFLP) of the highly conserved 5' UTR region. This technique is easy, affordable but has disadvantages like low sensitivity for typing low RNA levels and cannot determine all the genotypes^{5, 6}.

3.      Nucleotide sequencing is the gold standard confirmatory test where nucleotide sequencing of the UTR or E1 gene or NS5B gene is compared with the known genotype databases by computational analysis (BLAST), these results will determine the genotype of the HCV patient. This method is expensive and time-consuming but is necessary for therapeutic decisions⁷

4.      Abbott Real time PCR HCV assay uses the TaqMan technology to determine the genotype of the patients⁸. However this is expensive, require expertise and cannot differentiate the subtypes.

HEPATITIS C VIRUS VACCINE PROGESS

Hepatitis C Virus infection is still a major global burden. Since its discovery in the year 1989, till date there is no licensed vaccine for HCV. Development of vaccine for HCV has been challenging due to high genetic diversity of the virus, restricted humoral and cell mediated responses, limited potential of the adjuvant. Despite this, research on developing a successful vaccine is ongoing. The different regimens for drug discovery include use of direct acting antiviral (DAA), targets inducing T cell response, developing neutralizing antibodies and drugs targeting host proteins. The different strategies employed for vaccine development include the recombinant vaccines, DNA vaccines, Vector based vaccines and synthetic peptide vaccines. A major outbreak in HCV treatment occurred with the discovery of the 2 direct acting antivirals Teleprevir and Boceprevir, these protease inhibitors were FDA approved in 2011 but due to their low genetic barrier and side effects, they are used merely in combinations. The discovery of these paved way for the discovery of several direct acting antiviral. In 2014 several DAA have been discovered and some of which have been FDA approved and are currently used for treatment of HCV.

• Direct Acting Antivirals

The direct acting antivirals came into action when FDA approved the two NS3/4A protease inhibitors named telaprevir and boceprevir for treatment in combination with PEG-INF/RBV. The currently developed DAA are against HCV protease NS3, NS5A and NS5B ending with "-previr", "- avir" and "-buvir" respectively and different combinations have been recommended by EASL⁹ (Listed in Table 1).

NS3 protease

Teleprevir and Boceprevir are the first generation protease inhibitors that are FDA approved.  NS3 protease inhibitors work on the fact that NS3 protease undergoes end product cleavage and the P side product remain attached to the protease active site inhibiting the binding of the other substrates ^{10, 11}. NS3 protease inhibitors have low genetic barrier hence these are used often in combinations. Simeprevir is a second generation protease inhibitor effective against genotype 1 but taken in combinations due to low genetic barrier. The other approaches to inhibit the NS3 protease Include

1.      Inhibiting the binding site for the Zinc atom leading to misfolding of the protein¹²

2.      Blocking the interaction of NS3 protease with its cofactor NS4A¹³ eg. ACH-806 halted development

NS5A protein

A membrane associated phosphoprotein, involved in membrane web formation and replication. The research by Gao and colleagues lead to the discovery of the antiviral compound Daclatasvir¹⁴. This is known to inhibit the membrane web formation and thus inhibiting RNA replication ( C.Berger and R. Bartenschlager, unpublished). Daclatasvir has high antiviral activity against Genotypes 1 to 4. Daclatasvir also has low resistance barrier. Ledipasvir is another NS5A inhibitor effective against genotype 1, 4 and 6. It is available only in combination with sofosbuvir known as HARVONI produced by Gilead Sciences.

NS5B protein

NS5B is an RNA dependent RNA polymerase that carries HCV replication. The GDD motif in the palm subdomain constitutes the active site of the polymerase which along with the Mg²⁺ ions is involved in binding the nucleotide substrates and nucleotide polymerisation.

There are 2 classes of NS5B inhibitors- the Nucleotide Inhibitors (NIs) and the Non nucleotide Inhibitors (NNIs). The NI resembles the natural substrate and gets incorporated in the growing chain leading to termination of elongation. On the other hand the NNI bind to the enzyme as allosteric inhibitors and induce conformational change thus inhibiting the polymerase activity. On uptake they need to be converted to di- and triphosphate. The NI(s) have pangenotypic activity and have high genetic barrier. Sofosbuvir is a NI that resembles 5'- monophosphate, has pangenotypic activity and showed high SVR in patients.

The non nucleotide inhibitors exhibit four allosteric binding sites- two sites located in the thumb domain(thumb 1 and thumb 2) and two reside in the palm domain (palm 1 and palm 2) close to the active site¹⁵. NNI work by inducing a conformational change like the hyperclosed active site or can limit the mobility of the enzyme.

Table 1- Treatment recommendation for patients with HCV

• Prophylactic and Therapeutic Vaccines

Different preclinical studies have been carried out in the development of prophylactic and therapeutic vaccines within the last two decades using different strategies and targeting different regions of the HCV polyprotein. The different vaccine strategies employed for clinical trial include the recombinant proteins, synthetic peptides, DNA vaccines and Vector based vaccines (Listed in table 2) some of which are discussed below.

Recombinant proteins

The genes encoding the viral proteins are cloned into yeast and the recombinant proteins are purified and used in vaccine development. The advantage of this strategy is that it offers a pathogen free candidate and it do not require cultivation of the organism. Using this approach HCV envelop proteins have been immunised in rodents, chimpanzees and healthy human volunteers^{16, 17}. These studies showed high neutralising antibody response. A  phase I clinical trial (NCT00500747)  addressing the safety and efficacy of the vaccine in healthy humans showed that the vaccine induce strong humoral and CD4+ T cell response¹⁸.This study did not complete the phase II due to technical difficulties in protein production.  Many studies were carried out using recombinant proteins for vaccine development but they had withdraws due to technical difficulties, low titres of nAb elicited and HCV evasion mechanisms.

Synthetic Peptides

These are viral peptides coupled with adjutants that can induce strong humoral and cell mediated immune response.  Strong cellular immune response was showed in mice immunised with peptides of the core and nonstructural proteins (NS5A and NS4B) ¹⁹. A comparative analysis between the DNA and peptide approaches showed that immunization with peptides of nonstructural regions showed efficient viral clearance, long lasting immune response and increased antibody in comparison with DNA based immunisation ²⁰.

DNA vaccines

In DNA vaccine, the plasmid encoding the antigenic HCV protein(s) is injected, this results in protein expression in vivo which generate humoral and cell mediated immune response (strong Cytotoxic T lymphocytes response)^{21. 22} .The host cells take up the DNA vaccine, transcribe and translate it to yield proteins. These viral proteins are processed via the endogenous pathway. The productions in inexpensive and they are safe in animals and humans ²³. Using this strategy a therapeutic vaccine ChronVac C encoding HCV NS3/4A has been proposed for chronic HCV cases²⁴   and currently it is in Phase II clinical trial (ClinicalTrials.gov Identifier: NCT01335711).

Virus vector Based Vaccines

The main idea behind the use of virus vector based vaccines is to manipulate the virus to deliver foreign genetic material into the mammalian cells. They can induce T cell immunity and humoral immune response. The advantages of these vector vaccines are that they can accommodate large foreign genes and they remain outside the host cells. The most commonly used vectors are Adenovirus, Modified vaccinia Ankara (MVA), alphavirus or paramyxovirus vectors. Adenovirus expressing HCV nonstructural proteins shoed protective T cell responses in chimpanzees and were immunogenic in healthy volunteers²⁵.

However the disadvantages with adenovirus are the pre-existing immunity that results in its clearance before inducing immune response. This issue was overcome by the use of Modified vaccinia Ankara virus due to its minimal pre-existing immunity ^{26, 27.}

Using MVA, a vaccine called TG4040 was developed. This encodes the HCV NS3, NS4 and NS5B genes and the results in phase I clinical trial on 15 chronically infected HCV patients showed decline in the viral load and significant T cell response ²⁸. A phase II clinical trial is completed but no results have been posted yet (ClinicalTrials.gov Identifier: NCT01055821).         

Different strategies for vaccine are in phase I and Phase II clinical trial and research is still continuing in terms of development of a safe and efficient vaccine for HCV.  Proposals to improve the design, selection of immunogens, safety and administration are in progress.