Correlates of immune protection against human rotaviruses: natural infection and vaccination.

Species A rotaviruses are the leading viral cause of acute gastroenteritis in children under 5 years of age worldwide. Despite progress in the characterization of the pathogenesis and immunology of rotavirus-induced gastroenteritis, correlates of protection (CoPs) in the course of either natural infection or vaccine-induced immunity are not fully understood. There are numerous factors such as serological responses (IgA and IgG), the presence of maternal antibodies (Abs) in breast milk, changes in the intestinal microbiome, and rotavirus structural and non-structural proteins that contribute to the outcome of the CoP. Indeed, while an intestinal IgA response and its surrogate, the serum IgA level, are suggested as the principal CoPs for oral rotavirus vaccines, the IgG level is more likely to be a CoP for parenteral non-replicating rotavirus vaccines. Integrating clinical and immunological data will be instrumental in improving rotavirus vaccine efficacy, especially in low- and middle-income countries, where vaccine efficacy is significantly lower than in high-income countries. Further knowledge on CoPs against rotavirus disease will be helpful for next-generation vaccine development. Herein, available data and literature on interacting components and proposed CoPs against human rotavirus disease are reviewed, and limitations and gaps in our knowledge in this area are discussed.

Alum and a TLR7 agonist combined with built-in TLR4 and 5 agonists synergistically enhance immune responses against HPV RG1 epitope.

To relieve the limitations of the human papillomavirus (HPV) vaccines based on L1 capsid protein, vaccine formulations based on RG1 epitope of HPV L2 using various built-in adjuvants are under study. Herein, we describe design and construction of a rejoined peptide (RP) harboring HPV16 RG1 epitope fused to TLR4/5 agonists and a tetanus toxoid epitope, which were linked by the (GGGS)3 linker in tandem. In silico analyses indicated the proper physicochemical, immunogenic and safety profile of the RP. Docking analyses on predicted 3D model suggested the effective interaction of TLR4/5 agonists within RP with their corresponding TLRs. Expressing the 1206 bp RP-coding DNA in E. coli produced a 46 kDa protein, and immunization of mice by natively-purified RP in different adjuvant formulations indicated the crucial role of the built-in adjuvants for induction of anti-RG1 responses that could be further enhanced by combination of TLR7 agonist/alum adjuvants. While the TLR4/5 agonists contributed in the elicitation of the Th2-polarized immune responses, combination with TLR7 agonist changed the polarization to the balanced Th1/Th2 immune responses. Indeed, RP + TLR7 agonist/alum adjuvants induced the strongest immune responses that could efficiently neutralize the HPV pseudoviruses, and thus might be a promising formulation for an inexpensive and cross-reactive HPV vaccine.

Characterization and immunogenicity of a novel chimeric hepatitis B core-virus like particles (cVLPs) carrying rotavirus VP8*protein in mice model.

Given the efficacy and safety issues of the WHO for approved/prequalified live attenuated rotavirus (RV) vaccines, studies on alternative non-replicating modals and proper RV antigens are actively undertaken. Herein, we report the novel chimeric hepatitis B core-virus like particles (VLPs) carrying RV VP8*26-231 protein of a P [8] strain (cVLPVP8*), as a parenteral VLP RV vaccine candidate. SDS-PAGE and Western blotting analyses indicated the expected size of the E. coli-derived HBc-VP8* protein that self-assembled to cVLPVP8* particles. Immunization in mice indicated development of higher levels of IgG and IgA as well as higher IgG1/IgG2a ratios by cVLPVP8* vaccination compared to the VP8* alone. Assessment of neutralizing antibodies (nAbs) indicated development of heterotypic nAbs with cross-reactivity to a heterotypic RV strain by cVLPVP8* immunization compared to VP8* alone. The observed anti-VP8* cross-reactivity might indicate the possibility of developing a Pan-genomic RVA vaccine based on the cVLPVP8* formulation that deserves further challenge studies.

Adenovirus vector-based vaccines as forefront approaches in fighting the battle against flaviviruses.

Flaviviruses are arthropod-borne viruses (arboviruses) that have been recently considered among the significant public health problems in defined geographical regions. In this line, there have been vaccines approved for some flaviviruses including dengue virus (DENV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), and tick-borne encephalitis virus (TBEV), although the efficiency of such vaccines thought to be questionable. Surprisingly, there are no effective vaccine for many other hazardous flaviviruses, including West Nile and Zika viruses. Furthermore, in spite of approved vaccines for some flaviviruses, for example DENV, alternative prophylactic vaccines seem to be still needed for the protection of a broader population, and it originates from the unsatisfying safety, and the efficacy of vaccines that have been introduced. Thus, adenovirus vector-based vaccine candidates are suggested to be effective, safe, and reliable. Interestingly, recent widespread use of adenovirus vector-based vaccines for the COVID-19 pandemic have highlighted the importance and feasibility of their widespread application. In this review, the applicability of adenovirus vector-based vaccines, as promising approaches to harness the diseases caused by Flaviviruses, is discussed.

Designing vaccine candidates against dengue virus by in silico studies on structural and nonstructural domains.

One-third of the world's population is at risk of Dengue infection. Envelope domain 3 (EDIII) and nonstructural protein1 (NS1) proteins as the potent antigenicity regions for humoral immunity in addition to the bc loop region as a completely conserved region have been used for designing protective vaccines. We aimed to design vaccine candidates according to the bc loop, EDIII, and NS1 regions of Dengue serotype2 to be used as vaccine candidates for all serotypes of Dengue virus especially serotype 2. Firstly the bc loop region with EDII fragments at both ends as well as EDIII and NS1 regions were used which were linked with the GGGGS linker to the bc loop region. In two other strategies, the bc loop with EDII and NS1 fragments at both ends was used to increase its structural stability. Tertiary structure prediction and validation of vaccine constructs indicated that all vaccine constructs were modeled with high quality and stable structure during molecular dynamics simulation. B cell epitope mapping by Bepipred and ElliPro methods confirmed the existence of high potent epitopes in the bc loop, EDIII, and NS1 regions in both linear and conformational B cell epitopes. Furthermore, molecular docking for the bc loop region demonstrated that all designed vaccines have a higher affinity to interact with 1C19 monoclonal antibody than only the bc loop region or bc loop epitope in the protein EII. Our data of in silico studies indicated that the designed vaccines could effectively induce humoral immunity against four dengue serotypes.

Rotavirus VP6: involvement in immunogenicity, adjuvant activity, and use as a vector for heterologous peptides, drug delivery, and production of nano-biomaterials.

The first-generation, live attenuated rotavirus (RV) vaccines, such as RotaTeq and Rotarix, were successful in reducing the number of RV-induced acute gastroenteritis (AGE) and child deaths globally. However, the low efficacy of these first-generation oral vaccines, coupled with safety concerns, required development of improved RV vaccines. The highly conserved structural protein VP6 is highly immunogenic, and it can generate self-assembled nano-sized structures, including tubes and spheres (virus-like particles; VLPs). Amongst the RV proteins, only VP6 shows these features. Interestingly, VP6-assembled structures, in addition to being highly immunogenic, have several other useful characteristics that could allow them to be used as adjuvants, immunological carriers, and drug-delivery vehicles as well as acting a scaffold for production of valuable nano-biomaterials. This review provides an overview of the self-assembled nano-sized structures of VP6-tubes/VLPs and their various functions.

Co-administration of rotavirus nanospheres VP6 and NSP4 proteins enhanced the anti-NSP4 humoral responses in immunized mice.

Inconveniences associated with the efficacy and safety of the World Health Organization (WHO) approved/prequalified live attenuated rotavirus (RV) vaccines, sounded for finding alternative non-replicating modals and proper RV antigens (Ags). Herein, we report the development of a RV candidate vaccine based on the combination of RV VP6 nanospheres (S) and NSP4112-175 proteins (VP6S + NSP4). Self-assembled VP6S protein was produced in insect cells. Analyses by western blotting and transmission electron microscopy (TEM) indicated expression of VP6 trimer structures with sizes of ≥140 kDa and presence of VP6S. Four group of mice were immunized (2-dose formulation) intra-peritoneally (IP) by either¨VP6S + NSP4¨ or each protein alone (VP6S or NSP4112-175) emulsified in aluminium hydroxide or control. Results indicated that VP6S + NSP4 formulation induced significant anti-VP6 IgG (P < 0.001) and IgA (P < 0.05) as well as anti-NSP4 IgG (P < 0.001) and enhancement of protective immunity. Analyses of anti-VP6S and anti-NSP4 IgG subclass (IgG1 and IgG2a) showed IgG1/IgG2a ≥6 and IgG1/IgG2a ≥3 ratios, respectively indicating Th2 polarization of immune responses. The combination of VP6S + NSP4 proteins emulsified in aluminum hydroxide adjuvant might present a dual universal, efficient and cost-effective candidate vaccine against RV infection.

BacMam virus-based surface display for HCV E2 glycoprotein induces strong cross-neutralizing antibodies and cellular immune responses in vaccinated mice.

BACKGROUND: Despite recent advancements, limitations in the treatment and control of hepatitis C virus (HCV) infection reprioritized the studies for invention of an efficient HCV vaccine to elicit strong neutralizing antibodies (NAbs) and cellular responses. METHODS: Herein, we report molecular construction of a BacMam virus-based surface display for a subtype-1a HCV gpE2 (Bac-CMV-E2-gp64; Bac) that both expressed and displayed gpE2 in mammalian cells and bacouloviral envelope, respectively. RESULTS: Assessments by western blotting, Immunofluorescence and Immunogold-electron microscopy indicated the proper expression and incorporation in insect cell and baculovirus envelope, respectively. Mice immunized in three different prime-boost immunization groups of: Bac/Bac, Bac/Pro (bacoulovirus-derived gpE2) and Bac/DNA (plasmid DNA (pCDNA)-encoding gpE2) developed high levels of IgG and IFN-γ (highest for Bac/Bac group) indicating the induction of both humeral and cellular immune responses. Calculation of the IgG2a/IgG1 and IFN-γ/IL-4 ratios indicated a Th1 polarization of immune responses in the Bac/Bac and Bac/DNA groups but a balanced Th1-Th2 phenotype in the Bac/Pro group. Sera of the mice in the Bac/Bac group provided the highest percentage of cross-NAbs against a subtype-2a HCVcc (JFH1) compared to Bac/Pro and Bac/DNA groups (62% versus 41% and 6%). CONCLUSIONS: Results indicated that BacMam virus-based surface display for gpE2 might act as both subunit and DNA vaccine and offers a promising strategy for development of HCV vaccine for concurrent induction of strong humoral and cellular immune responses.

Taguchi array optimization of the reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for sensitive and rapid detection of dengue virus serotype 2.

OBJECTIVES: Serotype 2 of dengue virus (DENV-2) is the most prevalent cause of dengue fevers. In this study, the C-prM gene was used for specific detection of DENV-2 by RT-LAMP assay. The RT-LAMP assay was optimized using the Taguchi design of experiments. RESULTS: The efficiency of the assay in such optimal conditions resulted in 100% sensitivity, 100% specificity, and 100% overall accuracy for detection of 4 copies/µL of the genome of DENV-2. In addition, the detection of 2 copies/µL of the genome of DENV-2 was feasible, although the sensitivity was 50%. Considering the importance of the specific detection of the dengue virus serotypes, the cost-effective RT-LAMP approach can be used for rapid, specific, and sensitive detection of DENV-2. CONCLUSION: RT-LAMP, as a cost-effective method, was optimized using Taguchi array approach for specific and rapid detection of DENV-2. Such methods can facilitate the diagnosis procedure in remote regions.

Oncolytic herpes simplex virus type-1 expressing IL-12 efficiently replicates and kills human colorectal cancer cells.

An increasing attitude towards oncolytic viruses (OVs) is witnessed following T-VEC's approval. In this study, we aimed to delete ICP47 and insert IL-12 in the ICP34.5 deleted HSV-1 backbone to improve the oncolytic properties and provide an immune-stimulatory effect respectively. The wild-type and recombinant viruses infected both cancerous, SW480 and HCT116, and non-cancerous, HUVEC, cell lines. Green-red Δ47/Δ34.5 was constructed by replacing ICP47 with GFP. Both ICP34.5 copies were replaced by hIL12. Cytotoxicity and growth kinetics of Δ47/Δ34.5/IL12 and Δ47/Δ34.5 were comparable to the wild virus in the cancerous cells. Δ47/Δ34.5/IL12 was able to produce IL12 in the infected cell lines. INF-γ production and PBMC proliferation were observed in the PBMCs treated with the lysate of Δ47/Δ34.5/IL12 infected cells. These results demonstrated that Δ47/Δ34.5/IL12 was competent in taking advantage of the cytotoxic effect of HSV-1 plus immune-stimulatory characteristics of IL-12.

Bi/tri-specific antibodies (HN-Fc-CD16 and HN-Fc-IL-15-CD16) cross-linking natural killer (NK)-CD16 and Newcastle Disease Virus (NDV)-HN, enhanced NK activation for cancer immunotherapy.

Cancer/tumor cells infected with the "avian paramyxovirus Newcastle Disease Virus (TC-NDV)" express the viral hemagglutinin-neuraminidase (HN) on the cell surface that is used as both the danger signal and anchor for bi/tri-specific antibodies (bs/tsAbs).We constructed a bs-Ab (HN-Fc-CD16) that bindsto HN and natural killer (NK)-CD16 receptor (FcgRIII)and a ts-Ab (HN-Fc-IL15-CD16) harbouring NK-activating cytokine "IL-15" within the bs-Ab.In silicoand computational predictions indicated proper exposure of both Abs in bs/tsAbs.Properbinding of thebi/tsAbstoHN on surface of TC-NDVandCD16+-cells was demonstrated by flow cytometry.The bi/tsAbstriggeredspecificcytotoxicity of NK cells againstTC-NDVand elicited substantial IFN-γproduction by activated NK cells(higher for ts-Ab) that sound promising for cancer immunotherapy purposes.

HCV Core/NS3 Protein Immunization with "N-Terminal Heat Shock gp96 Protein (rNT (gp96))" Induced Strong and Sustained Th1-Type Cytokines in Immunized Mice.

Feeble cellular responses induced by T cell-based vaccines are a major challenge for the development of an effective vaccine against Hepatitis C virus (HCV) infection. To address this challenge, the potential of N-terminal fragment of gp96 heat shock protein (rNT (gp96) as an adjuvant was evaluated and compared to that of the CpG (as a recognized Th1-type adjuvant) in the formulation of HCV core/NS3 antigens in three immunization strategies of protein/protein, DNA/DNA, and DNA/protein. Immunized mice were evaluated for elicited immune responses in week 3 (W3) and 11 post-immunizations. Our results demonstrated that the protein (subunit) vaccine formulated with rNT (gp96) in protein/protein strategy (core/NS3 + gp96) was significantly more efficient than CpG oligodeoxynucleotides (CpG ODN) formulation and all other immunization strategies in the induction of Th1-type cytokines. This group of mice (core/NS3 + gp96) also elicited a high level of anti-Core-NS3 total immunoglobulin G (IgG) with dominant IgG2a isotype at W3. Thus, the co-administration of recombinant NT (gp96) protein with rHCV proteins might be a promising approach in the formulation of HCV subunit vaccine candidates for induction of high levels of Th1 cytokines and humoral responses.

Severe acute respiratory syndrome-coronavirus-2 spike (S) protein based vaccine candidates: State of the art and future prospects.

Coronavirus disease 2019 (Covid-19) is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) which is responsible for a global pandemic that started in late 2019 in Wuhan, China. To prevent the worldwide spread of this highly pathogenic virus, development of an effective and safe vaccine is urgently needed. The SARS-CoV-2 and SARS-CoV share a high degree of genetic and pathologic identity and share safety and immune-enhancement concerns regarding vaccine development. Prior animal studies with first generation (whole virus-based) preparations of SARS-CoV vaccines (inactivated and attenuated vaccine modalities) indicated the possibility of increased infectivity or eosinophilic infiltration by immunization. Therefore, development of second and third generation safer vaccines (by using modern vaccine platforms) is actively sought for this viral infection. The spike (S) protein of SARS-CoVs is the main determinant of cell entry and tropism and is responsible for facilitating zoonosis into humans and sustained person-to-person transmission. Furthermore, 'S' protein contains multiple neutralizing epitopes that play an essential role in the induction of neutralizing antibodies (nAbs) and protective immunity. Moreover, T-cell responses against the SARS-CoV-2 'S' protein have also been characterized that correlate to the IgG and IgA antibody titres in Covid-19 patients. Thus, S protein is an obvious candidate antigen for inclusion into vaccine platforms against SARS-CoV-2 viral infection. This manuscript reviews different characteristics of S protein, its potency and 'state of the art' of the vaccine development strategies and platforms using this antigen, for construction of a safe and effective SARS-CoV-2 vaccine.

The Role of 3'UTR of RNA Viruses on mRNA Stability and Translation Enhancement.

The central dogma of molecular biology explains the flow of genetic information from DNA to functional products such as proteins. In most cases, a linear relationship with a high correlation coefficient exists between the concentration of mRNA, the middle man, and the functional product. Untranslated regions (UTRs) of RNA form a considerable base pairing that contributes to the secondary and tertiary structures of mRNA. The interaction between the mRNA secondary structures (cis-elements), RNA-binding proteins (RBP) and miRs (trans-element) are critical determinants of mRNAs' fate and stability. Among different viral families, the positive sense (+) RNA viruses use the simplest possible strategy of replication and expression, as the same molecule functions both as a genome and mRNA. Additionally, nucleotide composition and codon usage of +RNA viruses are the closest to human codon adaptation index (CAI). Since the origin of replication of viral intermediate RNA molecules is at the 3'-end of the genome, the 3'UTR plays a role in viral RNA replication. Moreover, the messenger role of RNA likely places functional demands on the 3'UTR to serve a role typical of cellular mRNA. This article reviews the effect of 3'UTR of RNA viruses with positive sense and genomes on mRNA stability and translation improvement. A range of animal (e.g., Dengue, Sindbis, Corona and Polio) and plant (Barley yellow dwarf, Brome mosaic, Turnip crinkle, Tobacco mosaic, Cowpea mosaic and Alfalfa mosaic) viruses are examined to highlight the role of 3'UTR in viral survival and as a potential target for pharmaceutical applications.

Co-administration of 2'3'-cGAMP STING activator and CpG-C adjuvants with a mutated form of HPV 16 E7 protein leads to tumor growth inhibition in the mouse model.

Persistent infection with high-risk genotypes of human papillomavirus (HPV) is the leading cause of cervical cancer. The HPV oncoprotein E7 is constitutively expressed in cervical cancer and considered as an essential target for tumor-specific immunity. The goal of this study was to develop a candidate therapeutic vaccine based on the mutated E7 protein that had possibly reduced transformation capacity while was able to elicit a robust immune response. Therefore, the mutant type of HPV 16 E7 (E7GRG) protein was recombinantly expressed in E. coli. The protein was then purified and formulated with 2'-3'cGAMP CDN and/or CpG-C ODN adjuvants and subcutaneously injected to female C57BL/6 mice. To evaluate the immunogenic response, lymphocyte proliferation, secretion levels of IFN-γ and IL-4 cytokines, granzyme B level, and total IgG and subclasses of IgG antibody were measured. The anti-tumor activity was evaluated in tumor-harboring C57BL/6 mice. The highest rate of cell proliferation, IFN-γ and granzyme B levels, and amount of IgG antibody were found in mice group that were injected by E7GRG + 2'-3'cGAMP + CpG-C. Therapeutic immunization with E7GRG + 2'-3'cGAMP + CpG-C also significantly suppressed TC-1 tumor growth in mice. In conclusion, the results demonstrated that E7GRG + 2'-3'cGAMP + CpG-C induced strong cell-mediated and humoral immune responses that resulted in inhibition of tumor in mouse model.

Canola oilseed- and Escherichia coli- derived hepatitis C virus (HCV) core proteins adjuvanted with oil bodies, induced robust Th1-oriented immune responses in immunized mice.

Induction of broad Th1 cellular immune responses and cytokines is crucial characteristics for vaccines against intracellular infections such as hepatitis C virus (HCV). Plants (especially oilseed tissues) and plant-immunomodulators (like oil bodies) offer cost-effective and scalable possibilities for the production of immunologically relevant and safe vaccine antigens and adjuvants, respectively. Herein, we provide data of the murine immunization by transgenic canola oilseed-derived HCV core protein (HCVcp) soluble extract (TSE) and Escherichia coli- derived rHCVcp in combination with Canola oil bodies (oil) compared to that of the Freund's (FA) adjuvant. Mice immunized by TSE+ oil developed both strong humeral (IgG) and Th1-biased cellular responses, manifested by high levels of IFN-γ and lower IgG1/IgG2a ratio and IL-4 secretion. Results of the intracellular cytokine staining indicated that TSE+ oil immunization in mice triggered both CD4+ and CD8+ T cells to release IFN-γ, while CD4+ cells were mostly triggered when FA was used. Analyses by qRT-PCR indicated that a combination of rHCVcp/TSE with oil body induced high levels of IL-10 cytokines compared to that of the FA adjuvant. These characteristics are important properties for the design of an HCV vaccine candidate and indicate the potential of Canola-derived antigen and oil bodies in addressing these concerns.

Expression and Purification of a Bispecific Antibody against CD16 and Hemagglutinin Neuraminidase (HN) in E. Coli for Cancer Immunotherapy.

BACKGROUND: : Immunotherapy of cancer by bispecific antibodies (bsAb) is an attractive approach for retargeting immune effector cells including natural killer (NK) cells to the tumor if the proper expression and purification of the bsAb for such applications could be addressed. Herein, we describe E. coli expression of a recombinant bsAb (bsHN-CD16) recognizing NK-CD16 and hemagglutinin neuraminidase (HN) of Newcastle Disease Virus (NDV). This bsAb might be efficient for ex vivo stimulation of NK cells via coupling to HN on the surface of the NDV-infected tumor cells. METHODS: A bsAb-encoding pcDNA3.1 vector (anti-HN scFv-Fc-anti-CD16 scFv) was used as a template, and the scFv segments (after enzymatic digestion and cutting of the Fc part) were rejoined to construct the Fc-deprived bsAb (anti-HN scFv-anti-CD16 scFv; bsHN-CD16). The constructed bsHN-CD16 was inserted into the HindIII and BamHI site of the T7 promoter-based pET28a plasmid. Following restriction analyses and DNA sequencing to confirm the cloning steps, bsHN-CD16 encoding pET28a was transformed into the E. coli (Rosetta DE3 strain), induced for protein expression by IPTG, and the protein was purified under native condition by Ni/NTA column using imidazole. RESULTS: Analyses by SDS-PAGE and Western Blotting using Rabbit anti-human whole IgG-HRP conjugate, confirmed the expression and purification of the bsAb with the expected full size of 55 kDa and yields around 8% of the total protein. CONCLUSION: Results showed efficient production of the bsAb in E. coli for future large-scale purification.

Biomedical applications of yeasts - a patent view, part two: era of humanized yeasts and expanded applications.

INTRODUCTION: Yeast humanization, ranging from a simple point mutation to substitution of yeast gene(s) or even a complete pathway by human counterparts has enormously expanded yeast biomedical applications. AREAS COVERED: General and patent-oriented insights into the application of native and humanized yeasts for production of human glycoproteins (gps) and antibodies (Abs), toxicity/mutagenicity assays, treatments of gastrointestinal (GI) disorders and potential drug delivery as a probiotic (with emphasis on Saccharomyces bulardii) and studies on human diseases/cancers and screening effective drugs. EXPERT OPINION: Humanized yeasts cover the classical advantageous features of a 'microbial eukaryote' together with advanced human cellular processes. These unique characteristics would permit their use in the production of functional and stable therapeutic gps and Abs in lower prices compared to mammalian (CHO) production-based systems. Availability of yeasts humanized for cytochrome P450 s will expand their application in metabolism-related chemical toxicity assays. Engineered S. bulardii for expression of human proteins might expand its application by synergistically combining the probiotic activity with the treatment of metabolic diseases such as phenylketonuria via GI-delivery. Yeast models of human diseases will facilitate rapid functional/phenotypic characterization of the disease-producing mutant genes and screening of the therapeutic compounds using yeast-based high-throughput research techniques (Yeast one/two hybrid systems) and viability assays.

Cross talk between alcohol-induced oxidative stress and HCV replication.

Alcohol consumption exacerbates the pathogenesis of hepatitis C virus (HCV) infection and aggravates disease consequences in alcohol-abusing patients. Although the exact reasons by which alcohol consumption affects several cellular pathways in liver cells are not clear, they might be partially attributed to the ability of alcohol to further suppress the innate immunity, modulation of autophagy and also its relationship with reactive oxygen species (ROS) generation. To evaluate these issues, Huh7 cells harboring HCV replicon and Cytochrome p450 (CYP2E1) plasmid were exposed to ethanol and mRNA expression of Beclin-1, interferon-stimulated gene15 (ISG15) genes and HCV NS5B for two different times were relatively quantitated. ROS was determined by flow cytometry. The results showed that alcohol treatment in a short time caused an increase in HCV NS5B and Beclin-1 mRNA and decreased ISG 15 mRNA. Long-lasting alcohol treatment increased ROS production in Huh-7 cells and HCV replication was reduced. In conclusion, acute alcohol treatment might contribute to increase HCV replication by interference in innate immunity and induction of autophagy. Chronic alcohol treatment caused oxidative stress, which disrupts autophagy and thereby increased the rate of Huh7 cell injury.

Affinity Based Nano-Magnetic Particles for Purification of Recombinant Proteins in Form of Inclusion Body

Background: Protein purification is the most complicated issue in the downstream processes of recombinant protein production; therefore, improved selective purification methods are important. Affinity-based protein purification method using polyhistidine-tag (His-tag) and nickel-nitrilotriacetic acid (Ni-NTA) resins is one of the most common strategies. Magnetic nanoparticles (MNPs) can be used as a beneficial alternative for Ni-NTA resins. However, there is no data on the capability of MNPs for protein purification from inclusion bodies; this issue is studied here. Methods: Recombinant His-tagged proteins of enhanced green fluorescent protein (EGFP)-His and streptokinase (SK)-His were expressed in E. coli BL-21 (DE3) in soluble and inclusion body forms, respectively. MNPs including Fe3O4 magnetic core, SiO2 shell, and Ni2+ on the surface were synthesized by sol-gel and hydrothermal reactions and then characterized by X-ray powder diffraction, vibrating sample magnetometer, and scanning electron microscopy imaging. Both synthesized Fe3O4@NiSiO3 and Fe3O4@NixSiOy MNPs were employed to purify EGEP-His and SK-His under native and denaturing conditions, respectively. The quantity and purity of purified proteins were analyzed by micro-Bradford assay and SDS-PAGE, respectively. Results: Both synthesized MNPs were spherical and well-dispersed with the size ranging from 290 to 415 nm. Synthesized MNPs contained Fe3O4, SiO2 shell, and Ni2+ on their structures with suitable magnetization properties. Using Fe3O4@NiSiO3 and Fe3O4@NixSiOy yielded 192 and 188 µg/mg of SK-His, as compared to 207 and 195 µg/mg of EGFP-His, respectively. Conclusion: MNPs containing magnetic Fe3O4 core, SiO2 shell, and Ni2+on their surface are versatile alternatives for Ni-NTA resins in protein purification for proteins expressed in both soluble and inclusion body forms.