Favipiravir, an antiviral drug, in combination with tamoxifen exerts synergistic effect in tamoxifen-resistant breast cancer cells via hTERT inhibition.

Tamoxifen (TAM) is one of the most successful treatments for breast cancer; however, TAM resistance continues to be a significant barrier. TAM resistance has been reported to be associated with increased expression of human telomerase reverse transcriptase (hTERT). This enzyme shares structural similarity with RNA-dependent RNA polymerase (RdRp) enzyme of RNA viruses, suggesting that RdRp inhibitors may also inhibit hTERT. Favipiravir (FAV) is an antiviral drug that inhibits RdRp of RNA viruses. Thus, we propose that FAV may also elicit an antitumor effect by suppressing hTERT. This study aimed to investigate the effect of FAV and TAM on TAM-resistant breast cancer (TAMR-1). The cell viabilities were determined. The levels of CDK1/ hTERT, in addition to regulators of hTERT-targeted signaling pathways were measured. Apoptosis, migration, and cell cycle distribution were also determined. Our data revealed that the combination of TAM and FAV suppressed cell proliferation synergistically (CI < 1) and resulted in a significant change in cell migration and apoptosis. Indeed, this was associated with reduced levels of hTERT and CDK1 and shift in the cell cycle distribution. Our findings suggest that the TAM/FAV combination exhibits synergistic effects against TAMR-1 human breast cancer cells by targeting hTERT.

Glycylglycine promotes the solubility and antigenic utility of recombinant HCV structural proteins in a point-of-care immunoassay for detection of active viremia.

BACKGROUND: Although E. coli is generally a well-opted platform for the overproduction of recombinant antigens as heterologous proteins, the optimization of expression conditions to maximize the yield of functional proteins remains empirical. Herein, we developed an optimized E. coli (BL21)-based system for the overproduction of soluble immunoreactive HCV core/envelope proteins that were utilized to establish a novel immunoassay for discrimination of active HCV infection. METHODS: The core/E1-E2 genes were amplified and expressed in E. coli BL21 (DE3) in the absence/presence of glycylglycine. The antigenic performance of soluble proteins was assessed against 63 HCV-seronegative (Ab-) sera that included normal and interferent sera (HBV and/or chronic renal failure), and 383 HCV-seropositive (Ab+) samples that included viremic (chronic/relapsers) and recovered patients' sera. The color intensity (OD450) and S/Co values were estimated. RESULTS: The integration of 0.1-0.4M glycylglycine in the growth media significantly enhanced the solubility/yield of recombinant core and envelope proteins by ~ 225 and 242 fold, respectively. This was reflected in their immunoreactivity and antigenic performance in the developed immunoassay, where the soluble core/E1/E2 antigen mixture showed 100% accuracy in identifying HCV viremic sera with a viral RNA load as low as 3800 IU/mL, without cross-reactivity against normal/interferent HCV-Ab-sera. The ideal S/Co threshold predicting active viremia (> 2.75) showed an AUC value of 0.9362 (95% CI: 0.9132 to 0.9593), with 87.64, 91.23% sensitivity and specificity, and 94.14, 82.11% positive and negative predictive values, respectively. The different panels of samples assayed with our EIA showed a good concordance with the viral loads and also significant correlations with the golden standards of HCV diagnosis in viremic patients. The performance of the EIA was not affected by the immunocompromised conditions or HBV co-infection. CONCLUSION: The applicability of the proposed platform would extend beyond the reported approach, where glycylglycine, low inducer concentration and post-induction temperature, combined with the moderately-strong constitutive promoter enables the stable production of soluble/active proteins, even those with reported toxicity. Also, the newly developed immunoassay provides a cost-effective point-of-care diagnostic tool for active HCV viremia that could be useful in resource-limited settings.

Advent in proteins, nucleic acids, and biological cell membranes functionalized nanocarriers to accomplish active or homologous tumor targeting for smart amalgamated chemotherapy/photo-therapy: A review.

Conventional cancer mono-therapeutic approaches including radiotherapy, surgery, and chemotherapy don't always achieve satisfactory outcomes and are frequently associated with significant limitations. Although chemotherapy is a vital intervention, its effectiveness is frequently inadequate and is associated with metastasis, multidrug resistance, off-target effect, and normal cells toxicity. Phototherapies are employed in cancer therapy, encompassing photo-dynamic and photo-thermal therapies which under favorable NIR laser light irradiation initiate the included photosensitizers and photo-thermal agents to generate ROS or thermal heat respectively for cancer cells destruction. Photo-therapy is considered noninvasive, posing no resistance, but it still suffers from several pitfalls like low penetration depth and excessive heat generation affecting neighboring tissues. Improved selectivity and tumor-homing capacity could be attained through surface modulation of nanoparticles with targeting ligands that bind to receptors, which are exclusively overexpressed on cancerous cells. Developing novel modified targeted nanoparticulate platforms integrating different therapeutic modalities like photo-therapy and chemotherapy is a topic of active research. This review aimed to highlight recent advances in proteins, nucleic acids, and biological cell membranes functionalized nanocarriers for smart combinatorial chemotherapy/photo-therapy. Nanocarriers decorated with precise targeting ligands, like aptamers, antibody, and lactoferrin, to achieve active tumor-targeting or camouflaging using various biological cell membrane coating are designed to achieve homologous tumor-targeting.

Harnessing immunoinformatics for developing a multiple-epitope peptide-based vaccination approach against SARS-CoV-2 spike protein.

COVID-19 has spread to over 200 countries with variable severity and mortality rates. Computational analysis is a valuable tool for developing B-cell and T-cell epitope-based vaccines. In this study, by harnessing immunoinformatics tools, we designed a multiple-epitope vaccine to protect against COVID-19. The candidate epitopes were designed from highly conserved regions of the SARS-CoV-2 spike (S) glycoprotein. The consensus amino acids sequence of ten SARS-CoV-2 variants including Gamma, Beta, Epsilon, Delta, Alpha, Kappa, Iota, Lambda, Mu, and Omicron was involved. Applying the multiple sequence alignment plugin and the antigenic prediction tools of Geneious prime 2021, ten predicted variants were identified and consensus S-protein sequences were used to predict the antigenic part. According to ElliPro analysis of S-protein B-cell prediction, we explored 22 continuous linear epitopes with high scores ranging from 0.879 to 0.522. First, we reported five promising epitopes: BE1 1115-1192, BE2 481-563, BE3 287-313, BE4 62-75, and BE5 112-131 with antigenicity scores of 0.879, 0.86, 0.813, 0.779, and 0.765, respectively, while only nine discontinuous epitopes scored between 0.971 and 0.511. Next, we identified 194 Major Histocompatibility Complex (MHC) - I and 156 MHC - II epitopes with antigenic characteristics. These spike-specific peptide-epitopes with characteristically high immunogenic and antigenic scores have the potential as a SARS-CoV-2 multiple-epitope peptide-based vaccination strategy. Nevertheless, further experimental investigations are needed to test for the vaccine efficacy and efficiency.

A multiepitope peptide vaccine against HCV stimulates neutralizing humoral and persistent cellular responses in mice.

BACKGROUND: Although DAAs hold promise to significantly reduce rates of chronic HCV infections, its eradication still requires development of an effective vaccine. Prolonged T cell responses and cross neutralizing antibodies are ideal for vaccination against the infection. We aimed to design and synthesize a 6 multi epitope peptide vaccine candidate and provide evidence for production of extended cellular and neutralizing Abs in mice. METHODS: Six peptides derived from conserved epitopes in E1, E2 (n = 2),NS4B, NS5A and NS5B were designed, synthesized in a multiple antigenic peptide (MAP) form and administered w/o adjuvant to BALB/c mice as HCVp6-MAP at doses ranging from 800 ng to 16 µg. Humoral responses to structural epitopes were assayed by ELISA at different times after injection. ELISpot assay was used to evaluate IFN É£ producing CD4+/ CD8+ T- lymphocytes at extended durations i.e. > 20 weeks. Viral neutralization by mice sera was tested for genotypes 2a (JFH1) and a chimeric 2a/4a virus (ED43/JFH1) in HCVcc culture. RESULTS: HCVp6-MAP confers potent viral neutralization and specific cellular responses at > 1600 ng/ animal for at least 20 weeks. CONCLUSION: We report on a promising anti HCV vaccine for future studies on permissive hosts and in clinical trials.

Role of the cytosolic domain of occludin in trafficking and hepatitis C virus infection.

The role of the tight-junction (TJ) protein occludin (OCLN) in hepatitis C virus (HCV) entry remains elusive. Here, we investigated the OCLN C-terminal cytosolic domain in HCV infection. We expressed a series of C-terminal deletion mutants in Huh-7 cells KO for OCLN and characterized their functionality in HCV infection and trafficking. Deleting the OCLN cytosolic domain led to protein instability and intracellular retention. The first 15 residues (OCLN-C15 mutant) of the cytosolic domain were sufficient for OCLN stability, but led to its accumulation in the trans-Golgi network (TGN) due to a deficient cell surface export after synthesis. In contrast, the OCLN-C18 mutant, containing the first 18 residues of the cytosolic domain, was expressed at the cell surface and could mediate HCV infection. Point mutations in the context of C18 showed that I279 and W281 are crucial residues for cell surface expression of OCLN-C18. However, in the context of full-length OCLN, mutation of these residues only partially affected infection and cell surface localization. Importantly, the characterization of OCLN-C18 in human-polarized hepatocytes revealed a defect in its TJ localization without affecting HCV infection. These data suggest that TJ localization of OCLN is not a prerequisite for HCV infection in polarized hepatocytes.

Functional Study of the C-Terminal Part of the Hepatitis C Virus E1 Ectodomain.

In the hepatitis C virus (HCV) envelope glycoproteins E1 and E2, which form a heterodimer, E2 is the receptor binding protein and the major target of neutralizing antibodies, whereas the function of E1 remains less characterized. To investigate E1 functions, we generated a series of mutants in the conserved residues of the C-terminal region of the E1 ectodomain in the context of an infectious clone. We focused our analyses on two regions of interest. The first region is located in the middle of the E1 glycoprotein (between amino acid [aa] 270 and aa 291), which contains a conserved hydrophobic sequence and was proposed to constitute a putative fusion peptide. The second series of mutants was generated in the region from aa 314 to aa 342 (the aa314-342 region), which has been shown to contain two α helices (α2 and α3) by nuclear magnetic resonance studies. Of the 22 generated mutants, 20 were either attenuated or noninfectious. Several mutations modulated the virus's dependence on claudin-1 and the scavenger receptor BI coreceptors for entry. Most of the mutations in the putative fusion peptide region affected virus assembly. Conversely, mutations in the α-helix aa 315 to 324 (315-324) residues M318, W320, D321, and M322 resulted in a complete loss of infectivity without any impact on E1E2 folding and on viral assembly. Further characterization of the W320A mutant in the HCVpp model indicated that the loss of infectivity was due to a defect in viral entry. Together, these results support a role for E1 in modulating HCV interaction with its coreceptors and in HCV assembly. They also highlight the involvement of α-helix 315-324 in a late step of HCV entry.IMPORTANCE HCV is a major public health problem worldwide. The virion harbors two envelope proteins, E1 and E2, which are involved at different steps of the viral life cycle. Whereas E2 has been extensively characterized, the function of E1 remains poorly defined. We characterized here the function of the putative fusion peptide and the region containing α helices of the E1 ectodomain, which had been previously suggested to be important for virus entry. We could confirm the importance of these regions for the virus infectivity. Interestingly, we found several residues modulating the virus's dependence on several HCV receptors, thus highlighting the role of E1 in the interaction of the virus with cellular receptors. Whereas mutations in the putative fusion peptide affected HCV infectivity and morphogenesis, several mutations in the α2-helix region led to a loss of infectivity with no effect on assembly, indicating a role of this region in virus entry.

Mouse monoclonal antibody towards e1 specific epitope blocks viral entry and intracellular viral replication in vitro.

We characterized viral neutralization by a murine monoclonal antibody (mAb315) developed against conserved E1 specific epitope aa 315-323 at pre- and post-binding steps of infection into Huh7 cells. Detection of native virus in infected Huh7 cells by mAb315 were demonstrated by immunostaining. Inhibitions of viral entry by three different concentrations of mAb315 were measured by intracellular amplification of HCV RNA post infection. HCV RNA positive sera from 24 patients were used to infect Huh7 cell line in absence or presence of mouse monoclonal antibody produced in Balb/c mice or culture supernatant of mouse hybrid cells. Monoclonal Ab mAb315 could detect synthetic peptide p315 adsorbed on peripheral human lymphocytes by flow cytometry and showed high immuno reactivity to E1 viral antigen in infected Huh7 cells by immunostaining. Antibody-mediated neutralization assays demonstrated the ability of mAb315 to block HCV binding/entry to target cells at 0.73 mg/mL ascitic fluid or 250 µg/mL culture supernatant of mouse hybrid cells. Sixteen of 24 infected sera could infect Huh7 cells (67%). Binding/entry of HCV was completely blocked by mAb315 in 11/16 cases (69%). These findings suggest that mAb315 can induce HCV neutralization in vitro, which makes it a candidate for developing HCV therapeutic antibodies.

A Study of CC-Chemokine Receptor 5 (CCR5) Polymorphism on the Outcome of HCV Therapy in Egyptian Patients.

BACKGROUND: Chronic hepatitis C virus (HCV) infection is a globally serious public health issue. OBJECTIVES: In this study, we investigated CC chemokine receptor 5 (CCR5-59029) polymorphism which is considered an important component of the immune system in determining the outcome of HCV infection. Its critical role as a marker in response to interferon therapy of HCV infection is also investigated besides its effect on other clinical patient factors. PATIENTS AND METHODS: This study was conducted on 82 Egyptian patients with chronic Hepatitis C Virus (HCV) infection who received PEG-INF + Ribavirin treatment for 48 weeks. The study was also conducted on 50 healthy controls (with negative results for HCV antibody and RNA PCR). Full history of patients in this study was recorded. Clinical and histological examinations, qualitative HCV nested RT-PCR, quantitative real -time PCR, and genotyping of HCV RNA genome were performed. CCR5-59029 polymorphism with nucleotide substitution from G to A was amplified. The amplicons were digested with restriction endonuclease Bsp 1286I, and produced RFLPs of the CCR5 genotypes were determined. RESULTS: The present study showed a significant association between the functional SNP of CCR5 gene and the viral response to interferon in chronic HCV Egyptian patients. It was shown that the higher fibrosis stages (F2-F4) had significant association with nonresponse to treatment compared to the lower fibrosis stages (F0-F1) (95% confidence: 5.497 - 55.074, P = 0.0001). In addition, worse liver activity grade (A2-A3) had a very highly significant association with non-responder HCV patients compared to those with better liver activity grade (A1) (95% confidence: 2.242 - 20.974, P = 0.0007). Most importantly HCV patients with G allele had a high significant association with nonresponse to treatment, higher fibrosis stages and worse liver activity grades, while the A allele had a high significant association with sustained response, low fibrosis stages and relatively better liver activity grade (95% confidence: 3.347 - 15.036, P = 0.0001). CONCLUSIONS: SNPs within the CCR5 gene should be considered as an important factor used in combination with other host gene SNPs when developing a mathematical model for anticipating response to HCV therapy.

Neutralizing activities of caprine antibodies towards conserved regions of the HCV envelope glycoprotein E2.

Anti HCV vaccine is not currently available and the present antiviral therapies fail to cure approximately half of the treated HCV patients. This study was designed to assess the immunogenic properties of genetically conserved peptides derived from the C-terminal region of HVR-1 and test their neutralizing activities in a step towards developing therapeutic and/or prophylactic immunogens against HCV infection. Antibodies were generated by vaccination of goats with synthetic peptides derived from HCV E2. Viral neutralizing capacity of the generated anti E2 antibodies was tested using in vitro assays. Goats immunized with E2 synthetic peptides termed p412 [a.a 412-419], p430 [a.a 430-447] and p517 [a.a 517-531] generated high titers of antibody responses 2 to 4.5 fold higher than comparable titers of antibodies to the same epitopes in chronic HCV patients. In post infection experiments of native HCV into cultured Huh7.5 cells anti p412 and anti p 517 were proven to be neutralizing to HCV genotype 4a from patients' sera (87.5% and 75% respectively). On the contrary anti p430 exhibited weak viral neutralization capacity on the same samples (31.25%). Furthermore Ab mixes containing anti p430 exhibited reduced viral neutralization properties. From these experiments one could predict that neutralization by Abs towards different E2-epitopes varies considerably and success in the enrichment of neutralization epitope-specific antibodies may be accompanied by favorable results in combating HCV infection. Also, E2 conserved peptides p517 and p412 represent potential components of a candidate peptide vaccine against HCV infection.

Conserved peptides within the E2 region of Hepatitis C virus induce humoral and cellular responses in goats.

The reason(s) why human antibodies raised against hepatitis C virus (HCV) E2 epitopes do not offer protection against multiple viral infections may be related to either genetic variations among viral strains particularly within the hypervariable region-1 (HVR-1), low titers of anti E2 antibodies or interference of non neutralizing antibodies with the function of neutralizing antibodies. This study was designed to assess the immunogenic properties of genetically conserved peptides derived from the C-terminal region of HVR-1 as potential therapeutic and/or prophylactic vaccines against HCV infection. Goats immunized with E2-conserved synthetic peptides termed p36 (a.a 430-446), p37(a.a 517-531) and p38 (a.a 412-419) generated high titers of anti-p36, anti-p37 and anti-P38 antibody responses of which only anti- p37 and anti- p38 were neutralizing to HCV particles in sera from patients infected predominantly with genotype 4a. On the other hand anti-p36 exhibited weak viral neutralization capacity on the same samples. Animals super-immunized with single epitopes generated 2 to 4.5 fold higher titers than similar antibodies produced in chronic HCV patients. Also the studied peptides elicited approximately 3 fold increase in cell proliferation of specific antibody-secreting peripheral blood mononuclear cells (PBMC) from immunized goats. These results indicate that, besides E1 derived peptide p35 (a.a 315-323) described previously by this laboratory, E2 conserved peptides p37 and p38 represent essential components of a candidate peptide vaccine against HCV infection.