Farzin Roohvand

Initiation of Hepatitis C Virus Infection Requires the Dynamic Microtubule Network: ROLE OF THE VIRAL NUCLEOCAPSID PROTEIN*

Early events leading to the establishment of hepatitis C virus (HCV) infection are not completely understood. We show that intact and dynamic microtubules play a key role in the initiation of productive HCV infection. Microtubules were required for virus entry into cells, as evidenced using virus pseudotypes presenting HCV envelope proteins on their surface. Studies carried out using the recent infectious HCV model revealed that microtubules also play an essential role in early, postfusion steps of the virus cycle. Moreover, low concentrations of vinblastin and nocodazol, microtubule-affecting drugs, and paclitaxel, which stabilizes microtubules, inhibited infection, suggesting that microtubule dynamic instability and/or treadmilling mechanisms are involved in HCV internalization and early transport. By protein chip and direct core-dependent pull-down assays, followed by mass spectrometry, we identified β- and α-tubulin as cellular partners of the HCV core protein. Surface plasmon resonance analyses confirmed that core directly binds to tubulin with high affinity via amino acids 2-117. The interaction of core with tubulin in vitro promoted its polymerization and enhanced the formation of microtubules. Immune electron microscopy showed that HCV core associates, at least temporarily, with microtubules polymerized in its presence. Studies by confocal microscopy showed a juxtaposition of core with microtubules in HCV-infected cells. In summary, we report that intact and dynamic microtubules are required for virus entry into cells and for early postfusion steps of infection. HCV may exploit a direct interaction of core with tubulin, enhancing microtubule polymerization, to establish efficient infection and promote virus transport and/or assembly in infected cells.

Advances in hepatitis C virus vaccines, part two: advances in hepatitis C virus vaccine formulations and modalities

Introduction: Developing a vaccine against HCV is an important medical and global priority. Unavailability and potential dangers associated with using attenuated HCV viral particles for vaccine preparation have resulted in the use of HCV genes and proteins formulated in novel vaccine modalities. Areas covered: In part one of this review, advances in basic knowledge for HCV vaccine design were provided. Herein, a detailed and correlated patents (searched by Espacenet) and literatures (searched by Pubmed) review on HCV vaccine formulations and modalities is provided, including: subunit, DNA, epitopic-peptide/polytopic, live vector- and whole yeast-based vaccines. Less-touched areas in vaccine studies such as mucosal, plant-based, and chimeric HBV/HCV vaccines are also discussed. Furthermore, results of preclinical/clinical studies on selected HCV vaccines as well as pros and cons of different strategies are reviewed. Finally, potential strategies for creation and/or improvement of HCV vaccine formulations are discussed. Expert opinion: Promising outcomes of a few HCV vaccine modalities in phase I/II clinical trials predict the accessibility of at least partially effective vaccines to inhibit or treat the chronic state of HCV infection (specially in combination with standard antiviral therapy). ChronVac-C (plasmid DNA), TG4040 (MVA-based), and GI-5005 (whole yeast-based) might be the most obvious HCV vaccine candidates to be approved in the near future.

Advances in hepatitis C virus vaccines, Part one: Advances in basic knowledge for hepatitis C virus vaccine design.

Introduction: Around 3% of the world population is infected with HCV, with 3 – 4 million newly infected subjects added to this reservoir every year. At least 10% of these people will develop liver cirrhosis or cancer over time, while no approved vaccine against HCV infection is available to date. Areas covered: This paper includes a detailed and correlated patent (selected by HCAPLUS search database) and literature (searched by PubMed) review on the HCV genome, proteins and key epitopes (including underestimated HCV proteins, alternate reading frame proteins), HCV immunology, immunosuppressive mechanisms and protective correlations of immunity in acute and chronic states of infection (features for prophylactic and therapeutic HCV vaccine design), recent HCV cell culture systems (HCV/JFH1) and animal models. In part two of this review, advances in HCV vaccine formulations and modalities as well as a detailed list of the current trials for HCV vaccine and discussion of the pros and cones of different strategies will be provided. Expert opinion: By using the advanced basic knowledge and tools obtained about HCV vaccinology in recent years and the application of novel formulations and modalities, at least partially effective vaccines will become available in the near future to prevent (or treat) the chronic (if not the acute) state of HCV infection. A few of such vaccines are already in clinical trials.

Fusion of HBsAg and prime/boosting augment Th1 and CTL responses to HCV polytope DNA vaccine.

Correlation of hepatitis C virus (HCV) spontaneous resolution with Th1 and CD8(+)CTL responses during natural infection implies the potentiality of poly-CTL-epitopic HCV vaccines. We recently reported in silico design and construction of DNA vaccines (pcPOL-plasmids) harboring HCV CTL epitopes. Herein, we provide data of mice immunization by pcPOL, (encoding; core(132-142) [C], E2(405-414) [E(4)], E2(614-622) [E(6)] and NS3(1406-1415) [N] CD8(+)CTL epitopes as CE(4)E(6)N polytope) and its HBsAg-fused counterpart (pcHPOL), compared to the adjuvant-formulated (Montanide+CpG) CE(4)E(6)N synthetic-peptide immunization. All vaccinated groups developed different levels of cellular responses, however, only the pcHPOL-immunized mice elicited strong CTLs and IFN-gamma-secreting cells that were further augmented towards a Th1 response and partial tumor protection by DNA-prime/peptide-boosting regimen. Priming with HBsAg alone could not afford its augmenting effect indicating the importance of priming by polytope itself. Hence, fusion of immunocarriers like HBsAg conjoined with DNA-prime/peptide-boost immunization regimen seems a strategy to enhance the epitope-specific immune responses towards poly-CTL-epitopic vaccines.

Polytope DNA Vaccine Development Against Hepatitis C Virus: A Streamlined Approach from In Silico Design to In Vitro and Primary In Vivo Analyses in BALB/c Mice

For chronic viral infections like Hepatitis C, CD8-CTLs have emerged as important protective tools. Hence, isolated dominant epitopes arranged as polytope DNA or peptide vaccines represent a promising approach. However, because of controversial rules governing the polytope construction and epitope processing, proper design and primary analysis of such vaccines are prior to the costly transgenic animal studies. In this study, based on in silico epitope selection, four HLA-A2 (C132, E614 and N1406) and H-2d (E405 and C132) immunodominant CD8-epitopes of HCV were selected. The codon optimized nucleotide sequences of the epitopes were assembled by overlap extension PCR in three different sequential tandems for the best proteasomal cleavage predictions and cloned into pcDNA3.1 vector. In addition, to enhance particulate formation, three other plasmids containing the fusion of polytopes with hepatitis B surface antigen gene (HBsAg) were also constructed. Proper expression of all constructs in transfected Cos-7 cells was verified by RT-PCR, immunofluorescence, Western-blot, ELISA and dot blot techniques. Moreover, particle formation of HBsAg-fused polytopes was manifested by their secretion to the culture media albeit in lesser amounts compared to sole HBsAg protein. Finally, the positive delayed-type hypersensitivity (DTH) response of vaccinated mice indicated the in vivo expression of all constructs and efficient stimulation of immune response, which was stronger for HBsAg fusion constructs. In addition, proper processing of the epitopes was evidenced by the DTH response towards H-2d epitopic peptides. These data provide enough support and merit for the further evaluation of the designed constructs in HLA-A2 transgenic mice.

Design, modeling, expression, and chemoselective PEGylation of a new nanosize cysteine analog of erythropoietin

Background: Recombinant human erythropoietin (rhEPO) is considered to be one of the most pivotal pharmaceutical drugs in the market because of its clinical application in the treatment of anemia-associated disorders worldwide. However, like other therapeutic proteins, it does not have suitable pharmacokinetic properties for it to be administrated at least two to three times per week. Chemoselective cysteine PEGylation, employing molecular dynamics and graphics in in silico studies, can be considered to overcome such a problem. Methods: A special kind of EPO analog was elicited based on a literature review, homology modeling, molecular dynamic simulation, and factors affecting the PEGylation reaction. Then, cDNA of the selected analog was generated by site-directed mutagenesis and subsequently cloned into the expression vector. The construct was transfected to Chinese hamster ovary/dhfr− cells, and highly expressed clones were selected via methotrexate amplification. Ion-immobilized affinity and size exclusion (SE) chromatography techniques were used to purify the expressed analog. Thereafter, chemoselective PEGylation was performed and a nanosize PEGylated EPO was obtained through dialysis. The in vitro biologic assay and in vivo pharmacokinetic parameters were studied. Finally, E31C analog Fourier transform infrared, analytical SE-high-performance liquid chromatography, zeta potential, and size before and after PEGylation were characterized. Results: The findings indicate that a novel nanosize EPO31-PEG has a five-fold longer terminal half-life in rats with similar biologic activity compared with unmodified rhEPO in proliferation cell assay. The results also show that EPO31-PEG size and charge versus unmodified protein was increased in a nanospectrum, and this may be one criterion of EPO biologic potency enhancement. Discussion: This kind of novel engineered nanosize PEGylated EPO has remarkable advantages over rhEPO.

Design, modeling, and expression of erythropoietin cysteine analogs in Pichia pastoris: Improvement of mean residence times and in vivo activities through cysteine-specific PEGylation

In this study, the low-cost production of recombinant human erythropoietin cysteine analogs (Cys-rhEPOs) from Pichia pastoris and the potential to increase their serum residency and in vivo activity through cysteine-specific PEGylation were investigated. Three-dimensional structures of several Cys-rhEPOs were generated using homology modeling, and three stable Cys-rhEPOs were selected on the basis of model stability in molecular dynamics simulation and surface accessibility of the inserted cysteine. cDNAs encoding Cys-rhEPOs were constructed by site-directed mutagenesis and expressed as secreted proteins in flask cultures of P. pastoris. The selection of highly expressing clones and the optimization of certain culture parameters resulted in protein expression levels of 100-170 mg/l. Purified Cys-rhEPOs were cysteine-specifically PEGylated using 20 kDa and 30 kDa mPEG-maleimides (methoxy polyethylene glycol-maleimides). The E89CEPO analog with the highest (96.6%) cysteine accessibility was conjugated to PEG-polymers with the largest yields (about 80%). In comparison with rhEPO, 30 kDa PEG-E89CEPO demonstrated a significant (approximately 30%) increase in the mean residence time. Whereas the in vitro activities of 30 kDa PEG-E89CEPO were comparable to those of rhEPO, the in vivo activity of this conjugate was more prolonged compared to rhEPO (12 days vs. 7 days). Our results demonstrate that the site-specific PEGylation of Pichia-expressed EPO analogs may be considered as a promising approach for generating cost-effective and long-acting erythropoiesis-stimulating agents.

Selection of valid reference genes for expression studies of hepatic cell lines under IFN-α treatment

The proper selection of reference genes to normalize the quantitative real-time PCR (RT-qPCR) results under particular experimental conditions is crucial for validation of the gene quantification data. Herein, using SYBR green RT-qPCR, five reference genes (GAPDH, ACTB, HMBS, HPRT-1 and TBP) were evaluated to determine the most stable reference genes in hepatic cell lines (Huh-7 and HepG(2)) under IFN-α treatment conditions. Analyses by geNorm program ranked GAPDH and HPRT-1 in Huh-7 and that of ACTB and HMBS in HepG(2) cells as the most stable reference genes under IFN-α treatment. While, same reference gene pairs were ranked by NormFinder program in Huh-7 cells, GAPDH was assessed as the most stable gene in HepG(2) group by this program, implying the importance of the employed algorithm in comparative interpretation of the data. Finally, cumulative analyses by one-way ANOVA, geNorm and NormFinder programs indicated that use of two reference genes (HMBS and GAPDH) in Huh-7 and three (HMBS, ACTB and GAPDH) in HepG(2) cells would greatly improve the normalization of the RT-qPCR data under IFN-α. Data presented in this paper will aid the selection of the most stable reference genes in RT-qPCR studies on evaluation of hepatic viral proteins and IFN pathway.

Co-expression of hepatitis C virus polytope–HBsAg and p19-silencing suppressor protein in tobacco leaves

Abstract Context: Plants transformed by virus-based vectors have emerged as promising tools to rapidly express large amounts and inexpensive antigens in transient condition. Objective: We studied the possibility of transient-expression of an HBsAg-fused polytopic construct (HCVpc) [containing H-2d and HLA-A2-restricted CD8+CTL-epitopic peptides of C (Core; aa 132-142), E6 (Envelope2; aa 614-622), N (NS3; aa 1406-1415), and E4 (Envelope2; aa 405-414) in tandem of CE6NE4] in tobacco (Nicotiana tabacum) leaves for the development of a plant-based HCV vaccine. Materials and methods: A codon-optimized gene encoding the Kozak sequence, hexahistidine (6×His)-tag peptide, and HCVpc in tandem was designed, chemically synthesized, fused to HBsAg gene, and inserted into Potato virus X (PVX-GW) vector under the control of duplicated PVX coat protein promoter (CPP). The resulted recombinant plasmids (after confirmation by restriction and sequencing analyses) were transferred into Agrobacterium tumefaciens strain GV3101 and vacuum infiltrated into tobacco leaves. The effect of gene-silencing suppressor, p19 protein from tomato bushy stunt virus, on the expression yield of HCVpc–HBsAg was also evaluated by co-infiltration of a p19 expression vector. Results: Codon-optimized gene increased adaptation index (CAI) value (from 0.61 to 0.92) in tobacco. The expression of the HCVpc–HBsAg was confirmed by western blot and HBsAg-based detection ELISA on total extractable proteins of tobacco leaves. The expression level of the fusion protein was significantly higher in p19 co-agroinfiltrated plants. Discussion and conclusion: The results indicated the possibility of expression of HCVpc–HBsAg constructs with proper protein conformations in tobacco for final application as a plant-derived HCV vaccine.

Whole recombinant Pichia pastoris expressing HPV16 L1 antigen is superior in inducing protection against tumor growth as compared to killed transgenic Leishmania

The development of an efficient vaccine against high-risk HPV types can reduce the incidence rates of cervical cancer by generating anti-tumor protective responses. Traditionally, the majority of prophylactic viral vaccines are composed of live, attenuated or inactivated viruses. Among them, the design of an effective and low-cost vaccine is critical. Inactivated vaccines especially heat-killed yeast cells have emerged as a promising approach for generating antigen-specific immunotherapy. Recent studies have indicated that yeast cell wall components possess adjuvant activities. Moreover, a non-pathogenic protozoan, Leishmania tarentolae (L.tar) has attracted a great attention as a live candidate vaccine. In current study, immunological and protective efficacy of whole recombinant killed Pichia pastoris and Leishmania tarentolae expressing HPV16 L1 capsid protein was evaluated in tumor mice model. We found that Pichia-L1, L.tar-L1 and Gardasil groups increase the IgG2a/IgG1 ratio, indicating a relative preference for the induction of Th1 immune responses. Furthermore, subcutaneous injection of killed Pichia-L1 generated the significant L1-specific IFN-γ immune response as well as the best protective effects in vaccinated mice as compared to killed L.tar-L1, killed Pichia pastoris, killed L.tar and PBS groups. Indeed, whole recombinant Leishmania tarentolae could not protect mice against C3 tumor mice model. These data suggest that Pichia-L1 may be a candidate for the control of HPV infections.