| Abstract|| |
Background: Hepatitis C virus (HCV) is characterized by a high degree of nucleotide sequence variability between genotypes. This variability extends to functional and immunological determinants. Serological tests using antigenic segments derived from the HCV polyprotein have been used for the diagnosis of HCV infection. However, available diagnostic Kits do not necessarily take type variability into consideration and are not optimized for HCV genotype 4a (HCV4a), the predominant genotype in Egypt. Aim: The aim of this study was to express some HCV4a-derived polypeptides in order to identify those with immunodiagnostic utility. Materials and Methods: Six sequential/overlapping genomic segments encoding 100–266 amino acid peptides from the core (peptide 1), envelope 1 (E1; peptide 2), envelope 2 (E2; peptides 4, 5, and 6), and E1/E2 (peptide 3) regions of the HCV4apolyprotein were selected for in vitro expression as glutathione S-transferase-fusion proteins. The immunoreactivity of the expressed peptides was evaluated against sera from HCV-infected/uninfected individuals using dot blot, western blot, and enzyme-linked immunosorbent assay. Results: The expressed polypeptides were recognized by HCV-infected sera from 20 patients, while showing no immunoreactivity toward uninfected serum. Peptide 1 derived from the core protein was found to be the most immunoreactive. Conclusion: Expressed polypeptides hold good potential for use in the development of improved HCV immunodiagnostics.
Keywords: Diagnostic reagents, hepatitis C virus, immune recognition, polypeptide expression
|How to cite this article:|
Halim AS, Mohamed MR, Hamid FF, Karim AM. Expression and immune recognition of polypeptides derived from Hepatitis C virus structural proteins. Indian J Pathol Microbiol 2019;62:43-8
|How to cite this URL:|
Halim AS, Mohamed MR, Hamid FF, Karim AM. Expression and immune recognition of polypeptides derived from Hepatitis C virus structural proteins. Indian J Pathol Microbiol [serial online] 2019 [cited 2019 Apr 25];62:43-8. Available from: http://www.ijpmonline.org/text.asp?2019/62/1/43/251258
| Introduction|| |
Hepatitis C virus (HCV) represents a major worldwide public health problem. There are about 150 million people chronically infected with HCV, corresponding to 2%–2.5% of the world's population according to the world health organization (WHO). The WHO estimated prevalence of HCV in Egypt, the largest reservoir of HCV in the world, is >10%.
Comparisons of HCV nucleotide sequences derived from individuals from different geographical regions revealed the presence of six major HCV genotypes (labeled 1–6), later on, a seventh genotype was characterized. A recent study confirmed the presence of at least 7 different HCV genotypes and 67 subtypes.
HCV genotype 4 is most prevalent in the Middle East and Egypt where it accounts for >80% of all HCV infections, with the 4a subtype responsible for most infections  HCV genotype 4 is a very heterogeneous genotype showing significant genetic divergence and more subtypes compared with other genotypes.
HCV infection usually appears as an asymptomatic disease and is often diagnosed accidentally and, unfortunately, remains heavily underdiagnosed. It is estimated that only 30%–50% of individuals infected with HCV are aware of their disease and can take advantage of treatment options.
Serologic assays that detect specific antibodies to HCV (anti-HCV antibodies) are being utilized for the diagnosis of HCV infection. In current clinical practice, antibodies against multiple HCV epitopes are detected by commercially available second and third generation enzyme-linked immunosorbent assay (ELISA).
It has been reported that HCV infection and its complications are among the leading public health challenges in Egypt. Improved immunodiagnostics requires a deeper understanding of the immunogenicity of the different segments of the viral polyprotein. As a part of studies to construct an epitope map for HCV4a that may be of value in developing improved diagnostics and vaccines, we envisioned an approach for expressing viral polypeptides covering the whole genome. In this study, we demonstrate the utility of this approach through expressing a number of polypeptides from the core, envelope 1 (E1), envelope 2 (E2), and E1/E2 regions of the polyprotein and testing their recognition by sera from infected persons.
| Materials and Methods|| |
Basic molecular biology techniques were conducted according to previously described standard procedures, unless otherwise specified. Diagnosis of chronic infection with HCV was based on biochemical, serological, and molecular findings. The study protocol was reviewed and approved by the scientific ethical committee of our institution. All procedures followed were in accordance with the standards of the scientific ethical committee of our institution on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. Informed consent was obtained from all patients for being included in the study.
Amplification and cloning of HCV-cDNA segments into a TA vector
DNA segments were amplified from HCV type 4a genome isolate ED43 cloned in pUC119 plasmid kindly provided by Dr. Richard Elliot. Six overlapping or sequential segments within the recombinant pUC119 plasmid were amplified by the polymerase chain reaction (PCR) using primer pairs flanked with unique restriction enzyme cloning sites [Table 1].
Fresh PCR products were cloned into the pCR2.1vector and transferred into competent cells using the TA Cloning Kit and One Shot TOP10 Chemically Competent Escherichia coli cells (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The presence and orientation of the PCR products were analyzed by restriction enzyme (Bam HI, Bgl II, and Sal I; Promega, Madison, WI, USA) digestion of the isolated plasmids.
Subcloning and expression in pGEX
The pGEX-4T-1 expression vector (Pharmacia LKB) was used for subcloning the inserts from the TA vector and their subsequent expression as fusion proteins with Schistosoma japonicum 26-kDa glutathione S-transferase (Sj26 GST). Insert 1 cloned in the TA vector was excised by digestion with Bam HI, whereas inserts 2 through 6 were excised by double digestion with either Bgl II and Sal I (inserts 2 and 3) or Bam HI and Sal I (inserts 4, 5, and 6), respectively. The pGEX-4T-1 vector was digested with either Bam HI or BamHI and Sal I. Both the excised inserts and the linearized pGEX-4T-1 vector were purified on 1% agarose gel (Applied Biosystems/Ambion, Austin, TX, USA) and recovered from the gel using the freeze-squeeze method.
Inserts were subsequently ligated into the corresponding BamHI or Bam HI and Sal I cloning sites of the pGEX-4T-1 expression vector. The ligation reactions were then used to transform E. coli Top10 competent cells. Positive recombinants were identified using cracking gel analyses. To verify insert size, small-scale plasmid DNA preparations and digestion with restriction enzymes were performed for pGEX-4T-1 plasmids harboring inserts 1, 4, 5, and 6. As inserts 2 and 3 were excised from the respective TA vectors using a combination of Bgl II and Sal I restriction enzymes and subsequently cloned in Bam HI and Sal I digested pGEX-4T-1, these inserts could not be excised with the same restriction enzymes. Therefore, the presence and size of inserts 2 and 3 were verified by PCR using the flanking primers shown in [Table 1].
Bacterial colonies harboring the pGEX-4T-1 vector and colonies harboring recombinant pGEX-4T-1 with each of the six inserts were inoculated individually, in LB-ampicillin medium and incubated at 37°C overnight. Expression of Sj26 GST and the fusion proteins was induced by Isopropyl β-D-1-thiogalactopyranoside. The proteins were purified by affinity chromatography where the filtered supernatant was loaded onto a polypropylene column (Poly-Prep Chromatography Columns, Bio-Rad Laboratories, Hercules, CA, USA) packed with glutathione cellulose resin (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) according to the manufacturer's instructions. The protein concentration was determined using the Pierce bicinchoninic acid  Protein Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). The size and integrity of the purified fusion protein were then verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 50 μg of the purified fusion protein.
Analysis of the immunoreactivity of the expressed fusion proteins
The immunoreactivity of the expressed fusion proteins was tested against sera from both HCV-infected and uninfected individuals in Egypt as well as serum from a rabbit immunized with Sj26 GST (anti-GST serum) using Dot and western blot analysis as well as ELISA. An alkaline phosphatase secondary antibody conjugate together with a p-nitrophenyl phosphate substrate (pNPP; Sigma-Aldrich, Saint Louis, Missouri, USA) was used for detection.
For dot blots, 5 μL (25 μg) of each purified fusion protein or purified Sj26 GST fusion partner was pipetted onto 0.45-μm pore nitrocellulose membrane (Bio-Rad Laboratories) and allowed to dry, the dot blot was then probed with serum diluted 1:100. For western blots, proteins separated on SDS-PAGE as described above were transferred onto a nitrocellulose membrane in a Trans-Blot Semi-Dry Electrophoretic Transfer Cell (Bio-Rad Laboratories) and probed with serum diluted1:100.
For ELISA, polystyrene microtiter plate wells were coated with the expressed protein antigens diluted to 5 μg/mL in 0.05 M bicarbonate buffer pH 9.5 (100 μL per well) for 2 h at room temperature and then transferred to 4°C overnight. Next day, the antigen solution was discarded and the plate was washed three times with phosphate-buffered saline (PBS). The empty sites in the wells were then blocked with PBS containing 2% bovine serum albumin (215 μL per well) for 30 min at room temperature and washed three times with PBS-0.05% Tween-20. The primary antibody diluted in PBS-0.05% Tween-20 at 1:10 was added (100 μL per well) and the plate was incubated at 37°C for 2 h. The excess unbound primary antibody was washed four times with PBS-0.05% Tween-20. Secondary antibody conjugate diluted 1:5,000 in PBS-0.05% Tween-20 was added (100 μL per well) and incubated for 1 h at room temperature. The excess secondary antibody was washed 4 times with PBS-0.05% Tween-20 followed by an additional wash with PBS. Substrate (pNPP) was added (100 μL per well) and the plate was incubated in the dark for 15 min before O.D. measurement at 405 nm.
| Results|| |
Expression of fusion peptides
SDS-PAGE was used to determine the size of six HCV polypeptides expressed as fusion proteins with Sj26 GST. the observed molecular weight for fusion proteins 1, 2, 3, and 5 of ~48 kDa, whereas the observed molecular weights for fusion proteins 4 and 6 were ~55 and 33 kDa, respectively. Taking into consideration the size of the fusion partner, those molecular weights reflect the relative polypeptide lengths shown in [Table 1].
Analysis of the immunoreactivity of the expressed peptides
The immunoreactivity of Sj26 GST as well as the six HCV fusion proteins was tested by dot blots using sera from HCV-infected and uninfected individuals. As shown in [Figure 1]a, both Sj26 GST and the six HCV fusion proteins reacted with anti-GST serum. On the other hand, fusion proteins 1-6 but not Sj26 GST reacted with sera from 20 HCV-infected individuals, with fusion protein 1 showing highest reactivity with all sera and fusion protein 6 least reactive with most sera. Among the infected sera, serum 7 was the most reactive with most but not all fusion proteins [Figure 1]b. Neither Sj26 GST nor the six HCV fusion proteins showed immunoreactivity toward normal uninfected serum [Figure 1]c.
|Figure 1: Dot blot of Sj26 GST and Sj26 GST-HCV fusion proteins. B: Blank (no antigen); GST: purified Sj26 GST; Ag1-6: purified Sj26 GST-HCV fusion proteins 1–6. Blots were probed with (a) anti-GST serum, (b) sera from HCV-infected patients 1-20, and (c) uninfected serum|
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Western blots for the reactivity of the six fusion proteins and their Sj26 GST fusion partner with anti-GST serum, HCV-infected serum 7, and normal uninfected serum are shown in [Figure 2]. Both Sj26 GST and the six HCV fusion proteins were recognized by anti-GST [Figure 2]a, whereas infected serum reacted with the six fusion proteins but not with Sj26 GST [Figure 2]b. Of the six fusion proteins probed with serum 7, the strongest signal was observed with fusion protein 1 as was the case in the dot blot.
|Figure 2: Western blot of Sj26 GST and Sj26 GST-HCV fusion proteins. M = molecular weight marker, GST = purified Sj26 GST, Lanes 1-6 = purified Sj26 GST-HCV fusion proteins 1–6. Blots were probed with (a) anti-GST serum and (b) HCV-infected serum from patient 7|
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Enzyme-linked immunosorbent assay
The immunoreactivity of fusion proteins 1, 2, 3, and 4 was quantitatively tested by ELISA against uninfected human serum and HCV-infected patient sera (1-20). The immunoreactivity of the fusion proteins was expressed as optical density (OD) at a wavelength of 405 nm [Figure 3]. The quantitative results support the dot blot findings showing a pronounced OD reading for all four fusion proteins with fusion protein 1 consistently producing highest OD and serum 7 showing best overall reactivity with all fusion proteins.
|Figure 3: ELISA reactivity of Sj26 GST-HCV fusion proteins 1-4. Ag 1-4: fusion proteins 1–4 reacted with sera from HCV infected patients (1–20) and uninfected serum. Wells were coated with antigens diluted to 5 μg/mL in 0.05 M bicarbonate buffer (100 μL per well)|
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To investigate potential additive, synergistic or antagonistic immunoreactivities of the expressed HCV fusion proteins against the various sera, we prepared fusion protein cocktails (a cocktail of fusion proteins 1 + 2 and a cocktail of fusion proteins 1 + 2 + 3 + 4). Comparison of the ELISA results of both cocktails with each of the 20 HCV-infected patient sera [Figure 4] does not show consistent higher recognition for either one. Moreover, [Table 2] shows that the addition of fusion protein 1 to fusion protein 2 slightly diminished immunoreactivity as compared with fusion protein 1 alone. Furthermore, mixing fusion proteins 1, 2, 3, and 4 was not in favor of improved immunoreactivity in comparison to fusion proteins 1 and 2 alone or combined.
|Figure 4: ELISA reactivity of Sj26 GST-HCV fusion protein cocktails. Ag 1 + Ag2: Wells were coated with 100-μL solution of fusion proteins 1 and 2 at a concentration of 5 μg/mL for each protein; Ag1 + Ag2 + Ag3 + Ag4: Wells were coated with 100-μL solution of fusion proteins 1 + 2 + 3 + 4 at a concentration of 5 μg/mL for each protein. Antigens were reacted with sera from HCV-infected patients 1–20 and uninfected serum|
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| Discussion|| |
An understanding of the immunological properties of the expressed hepatitis C genome is important for the development of sensitive reagents for immunodiagnosis, genotyping, and protective vaccination and studies aimed at understanding the variabilities that help the virus evade the host's immune response. The availability of a repertoire of overlapping polypeptides covering the viral genome provides important reagents toward achieving this goal. In this study, we report on the cloning and expression of six sequential or overlapping segments of the HCV4a genome isolate ED43, encoding parts of the structural proteins: core, E1, and E2.
The core is one of the most conserved regions of the HCV genome. The core molecule has a structural function since it interacts with the envelope 1 protein (E1) and thus forms the viral capsid that contains the HCV genome. In addition, the HCV core protein is highly antigenic, induces specific cellular and humoral responses, and most probably plays a key role in the pathogenesis of HCV infection. It is one of the main components of the kits used for detecting HCV infection in clinical settings.
The E1 and E2 envelope proteins harbor 6 and 11 putative N-glycosylation sites, respectively, those provide targets for immune recognition. Moreover, the envelope proteins are the first antigens to be exposed to the host cells, and formation of antibodies to the envelope region of a virus begins as early as 2 h after viral infection. The envelope proteins were found to induce only a humoral immune response in the host with E2 being more immunogenic than E1.
Anti-E2 antibodies were detected in > 90% of HCV RNA-positive individuals using E2 antigen  and the E2 protein was suggested as useful for monitoring HCV infection. Zaaijer et al. demonstrated that HCV antibodies could be detected using E2-derived peptides, and therefore, the E2 protein was recommended as a promising antigen for the detection of HCV antibodies.
In our study, the polypeptides [Table 1] were expressed as 100–200 amino acid segments of the viral polyprotein fused to Sj26 GST, their recognition by the human immune system was studied by dot blots, western blots and ELISA. Dot blots probed by sera from 20 different patients show that all polypeptides are recognized by the sera; however, signal strength varied differentially between the peptides [Figure 1]. Peptide 1 was the best recognized by all sera. Interestingly, it is a 200-amino acid peptide derived from the core region incorporated in many commercial ELISA kits., Reactivity of the remaining polypeptides with the 20 sera was variable, a peptide poorly recognized by one serum may be better recognized by another, and conversely, a peptide well recognized by the second serum may be less well recognized by the first, e.g., compare peptide 2(E1) and peptide 5(E2) recognition by serum 7 and 9.
Regarding the three E2 polypeptides, peptide 4 (266 amino acids) encompasses the sequence of peptide 5 (166 amino acids), which, in turn, encompasses the sequence of peptide 6 (100 amino acids). Signal intensity in the dot blot for the three peptides generally appears to decrease with decreased peptide size for most sera probably reflecting fewer epitopes. Surprisingly, signal intensity for four infected sera (13, 14, 15, and 19) is strongest for peptide 5 followed by peptide 6 (the smallest), while peptide 4, the largest peptide, displayed the weakest signal. Possibly those sera due to viral or host genetic factors do not recognize predominant epitope(s), which involve sequences within the N-terminal amino acids of peptide 4; removal of those amino acids in peptides 5 and 6 may expose other epitope(s), which are masked by those N-terminal amino acids and are better recognized by the 4 variant sera.
Western blots [Figure 2] probed with anti-GST and patient serum confirmed fusion protein size and integrity. In contrast to dot blots and ELISA, which recognize conformational epitopes, western blots due to the use of SDS gels in their preparation react primarily but probably not exclusively with linear epitopes. Despite this difference, peptide 1, the antigen best recognized using dot blots, was also the best recognized antigen in western blots.
A quantitative assessment of the affinity of peptides 1–4 to antibodies in the 20 patients' sera using ELISA [Figure 3] reflected the affinities observed in dot blots; furthermore, combining peptides in ELISA [Figure 4] did not appear to enhance signal output above that observed for the most reactive peptide but produced a signal reflecting the relative antibody binding affinity of the component antigens. This is evident on comparing ODs reflecting the average immunoreactivity of patients 1–20 with the four peptides alone and in combinations in [Table 2].
In conclusion, the development of sensitive immunodiagnostic reagents for HCV using recombinant 100- to 200-amino acid peptides is a feasible approach. For best results, the genome needs to be screened for the best reactive peptides as detected by ELISA assays using infected human sera. Combining a number of high affinity peptides is probably necessary in order to compensate for decreased sensitivity resulting from HCV type or patient variability in antigen–antibody recognition.
Financial support and sponsorship
This research has been funded by a research grant from Ain Shams University to Amr M. Karim.
Conflicts of interest
There are no conflicts of interest.
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Alyaa S. Abdel Halim
Department of Biochemistry, Faculty of Science, Ain Shams University, Abbassia, Cairo - 11566
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]