Transduction Of Mammalian Cells Research Articles

A Step-by-Step Guide for the Production of Recombinant Fluorescent TAT-HA-Tagged Proteins and their Transduction into Mammalian Cells.

Investigating the function of target proteins for functional prospection or therapeutic applications typically requires the production and purification of recombinant proteins. The fusion of these proteins with tag peptides and fluorescently derived proteins allows the monitoring of candidate proteins using SDS-PAGE coupled with western blotting and fluorescent microscopy, respectively. However, protein engineering poses a significant challenge for many researchers. In this protocol, we describe step-by-step the engineering of a recombinant protein with various tags: TAT-HA (trans-activator of transduction-hemagglutinin), 6×His and EGFP (enhanced green fluorescent protein) or mCherry. Fusion proteins are produced in E. coli BL21(DE3) cells and purified by immobilized metal affinity chromatography (IMAC) using a Ni-nitrilotriacetic acid (NTA) column. Then, tagged recombinant proteins are introduced into cultured animal cells by using the penetrating peptide TAT-HA. Here, we present a thorough protocol providing a detailed guide encompassing every critical step from plasmid DNA molecular assembly to protein expression and subsequent purification and outlines the conditions necessary for protein transduction technology into animal cells in a comprehensive manner. We believe that this protocol will be a valuable resource for researchers seeking an exhaustive, step-by-step guide for the successful production and purification of recombinant proteins and their entry by transduction within living cells. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: DNA cloning, molecular assembly strategies, and protein production Basic Protocol 2: Protein purification Basic Protocol 3: Protein transduction in mammalian cells.

HSPA8 acts as an amyloidase to suppress necroptosis by inhibiting and reversing functional amyloid formation.

Ultra-stable fibrous structure is a hallmark of amyloids. In contrast to canonical disease-related amyloids, emerging research indicates that a significant number of cellular amyloids, termed 'functional amyloids', contribute to signal transduction as temporal signaling hubs in humans. However, it is unclear how these functional amyloids are effectively disassembled to terminate signal transduction. RHIM motif-containing amyloids, the largest functional amyloid family discovered thus far, play an important role in mediating necroptosis signal transduction in mammalian cells. Here, we identify heat shock protein family A member 8(HSPA8) as a new type of enzyme-which we name as 'amyloidase'-that directly disassembles RHIM-amyloids to inhibit necroptosis signaling in cells and mice. Different from its role in chaperone-mediated autophagy where it selects substrates containing a KFERQ-like motif, HSPA8 specifically recognizes RHIM-containing proteins through a hydrophobic hexapeptide motif N(X1)φ(X3). The SBD domain of HSPA8 interacts with RHIM-containing proteins, preventing proximate RHIM monomers from stacking into functional fibrils; furthermore, with the NBD domain supplying energy via ATP hydrolysis, HSPA8 breaks down pre-formed RHIM-amyloids into non-functional monomers. Notably, HSPA8's amyloidase activity in disassembling functional RHIM-amyloids does not require its co-chaperone system. Using this amyloidase activity, HSPA8 reverses the initiator RHIM-amyloids (formed by RIP1, ZBP1, and TRIF) to prevent necroptosis initiation, and reverses RIP3-amyloid to prevent necroptosis execution, thus eliminating multi-level RHIM-amyloids to effectively prevent spontaneous necroptosis activation. The discovery that HSPA8 acts as an amyloidase dismantling functional amyloids provides a fundamental understanding of the reversibility nature of functional amyloids, a property distinguishing them from disease-related amyloids that are unbreakable in vivo.

Evaluation of Malarial Var2CSA-Displaying Baculovirus Vector in Transduction Efficiency in Human Cancer Cells.

Baculovirus vectors (BVs) are able to use for gene transduction in mammalian cells and are recognized as growing viral vectors for cancer gene therapy applications. The transduction efficiency of BVs varies among cancer cell types. To improve the transduction efficiency of BVs in human cancer cells, BV displaying malarial variant surface antigen 2-chondroitin sulfate A (var2CSA) molecules was developed in this study. Var2CSA plays a critical role in the sequestration of Plasmodium falciparum-infected erythrocytes in the placenta. Moreover, var2CSA binds to cancer cells via placenta-like chondroitin sulfate A (CSA), but not to non-cancer cells. Var2CSA BV showed significantly higher gene transduction than control BV in HepG2 and Huh7 cells, human hepatic cancer cells as well as AsPC-1 cells, human pancreatic cancer cells. The transduction efficiency of var2CSA BV was significantly inhibited by the anti-gp64 antibody, free heparin, and CSA. The results of this study suggest that var2CSA BV would be an improved vector for cancer gene therapies, especially in the treatment of hepatic and pancreatic cancers.

Selective optogenetic control of Gq signaling using human Neuropsin

Gq proteins are universally important for signal transduction in mammalian cells. The underlying kinetics and transformation from extracellular stimuli into intracellular signaling, however could not be investigated in detail so far. Here we present the human Neuropsin (hOPN5) for specific and repetitive manipulation of Gq signaling in vitro and in vivo with high spatio-temporal resolution. Properties and G protein specificity of hOPN5 are characterized by UV light induced IP3 generation, Ca2+ transients and inhibition of GIRK channel activity in HEK cells. In adult hearts from a transgenic animal model, light increases the spontaneous beating rate. In addition, we demonstrate light induced contractions in the small intestine, which are not detectable after pharmacological Gq protein block. All-optical high-throughput screening for TRPC6 inhibitors is more specific and sensitive than conventional pharmacological screening. Thus, we demonstrate specific Gq signaling of hOPN5 and unveil its potential for optogenetic applications.

CD9 and folate receptor overexpression are not sufficient for VSV-G-independent lentiviral transduction.

Extracellular vesicles have become a research focus for their potential as therapeutic vehicles that carry cargo substances. Extracellular vesicles may origin from the endosomal compartment and share several characteristics with the envelope of lentiviruses. A previous study reported that constitutive expression of the tetraspanin CD9, an extracellular vesicle marker, not only increases vesicle secretion from cells, but has also a positive effect on lentiviral transduction efficiency. Moreover, it was shown that expression of CD9 on the viral envelope in absence of viral glycoproteins was sufficient for the transduction of mammalian cells. In this study, we investigate the effect of CD9 and folate receptor alpha, a GPI-anchored protein, on biosynthesis and transduction efficiency of vesicles carrying lentiviral vectors. We demonstrate that neither CD9 nor FRα nor the combination of both were able to mediate a significant transduction of therapeutic vesicles carrying lentiviral RNA. Further studies are required to identify endogenous mammalian proteins that can be used for pseudotyping of viral envelopes to improve viral targeting without inducing immune responses.

Baculovirus Transduction in Mammalian Cells Is Affected by the Production of Type I and III Interferons, Which Is Mediated Mainly by the cGAS-STING Pathway.

The baculovirus Autographa californica multiple nucleopolyhedrovirus is an insect virus with a circular double-stranded DNA genome, which, among other multiple biotechnological applications, is used as an expression vector for gene delivery in mammalian cells. Nevertheless, the nonspecific immune response triggered by viral vectors often suppresses transgene expression. To understand the mechanisms involved in that response, in the present study, we studied the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway by using two approaches: the genetic edition through CRISPR/Cas9 technology of genes encoding STING or cGAS in NIH/3T3 murine fibroblasts and the infection of HEK293 and HEK293 T human epithelial cells, deficient in cGAS and in cGAS and STING expression, respectively. Overall, our results suggest the existence of two different pathways involved in the establishment of the antiviral response, both dependent on STING expression. Particularly, the cGAS-STING pathway resulted in the more relevant production of beta interferon (IFN-β) and IFN-λ1 in response to baculovirus infection. In human epithelial cells, IFN-λ1 production was also induced in a cGAS-independent and DNA-protein kinase (DNA-PK)-dependent manner. Finally, we demonstrated that these cellular responses toward baculovirus infection affect the efficiency of transduction of baculovirus vectors.IMPORTANCE Baculoviruses are nonpathogenic viruses that infect mammals, which, among other applications, are used as vehicles for gene delivery. Here, we demonstrated that the cytosolic DNA sensor cGAS recognizes baculoviral DNA and that the cGAS-STING axis is primarily responsible for the attenuation of transduction in human and mouse cell lines through type I and type III IFNs. Furthermore, we identified DNA-dependent protein kinase (DNA-PK) as a cGAS-independent and alternative DNA cytosolic sensor that contributes less to the antiviral state in baculovirus infection in human epithelial cells than cGAS. Knowledge of the pathways involved in the response of mammalian cells to baculovirus infection will improve the use of this vector as a tool for gene therapy.

Phosphoregulated orthogonal signal transduction in mammalian cells

Orthogonal tools for controlling protein function by post-translational modifications open up new possibilities for protein circuit engineering in synthetic biology. Phosphoregulation is a key mechanism of signal processing in all kingdoms of life, but tools to control the involved processes are very limited. Here, we repurpose components of bacterial two-component systems (TCSs) for chemically induced phosphotransfer in mammalian cells. TCSs are the most abundant multi-component signal-processing units in bacteria, but are not found in the animal kingdom. The presented phosphoregulated orthogonal signal transduction (POST) system uses induced nanobody dimerization to regulate the trans-autophosphorylation activity of engineered histidine kinases. Engineered response regulators use the phosphohistidine residue as a substrate to autophosphorylate an aspartate residue, inducing their own homodimerization. We verify this approach by demonstrating control of gene expression with engineered, dimerization-dependent transcription factors and propose a phosphoregulated relay system of protein dimerization as a basic building block for next-generation protein circuits.

Assessment of in-vitro Antioxidant/Enzymes Inhibitory Potentials of Aframomum melegueta [Roscoe] K. Schum (Grains of Paradise) Leaf, Stem Bark, Seed Bark and Seed Extracts

The purpose of this research work is to evaluates the in-vitro antioxidant and antioxidant enzymes inhibition potentials of Aframomum melegueta [Roscoe] K. Schum (Grains of Paradise) Leaf, Stem Bark, Seed Bark and Seed Extracts. Aframomum melegueta is a spice with a similar composition as Ginger, that belongs to the same Zingiberaceae family, used in Nigeria, West Africa and it is used for the treatment of infectious diseases such as urinary tract infections, cancer and diabetes. Antioxidants neutralize the effect of free radicals through different ways and may prevent the body from various diseases. The plant extract were collected from a rain forest in Akugba Akoko, Ondo state, Nigeria. The plant sample was dried, pulverized, filtered with Whatman No 1 filtered paper and the filtrates was concentrated in-vacuum using vacuum rotary evaporator at 40°C and was later concentrated to dryness in a hot-air oven at 40°C. Assessment of antioxidant potentials were performed using Singleton method Total flavonoids (mg/g), Phenol (mg/g), Ferric reducing (FRAP) potentials (mg/g), Free radical scavenging ability (DPPH) (1, 1- diphenyl-2-picryhydrazyl) potentials (%),2,2’-azino-bis(3-ethylbenthiazoline-6-sulphonic acid), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) scavenging potentials (mMol/g), Fe2+ chelation potentials (%) and OH Radical Scavenging potentials (%) were evaluated. Antioxidant enzymes of plant were assessed using Bentller method, Superoxide Dismutase (SOD) (%), Reduced Glutathione (GSH) (%), Catalase (CAT) and Glutathione Peroxidise (GPX) were evaluated. Seed extract among other extracts of Aframomum melegueta has the highest quantity of flavonoids, Phenol, FRAP, DPPH, ABTS, Fe2+ chelation and OH radical scavenging potential. The leaf extract of A. meleguata has the highest percentage of Superoxide Dismutase (SOD) and Catalase (CAT) while stem bark of Aframomum melegueta has the highest percentage of Reduced Glutathione (GSH) and Glutathione Peroxidise (GPX).it can be deduced from this research work that all parts of the A. melegueta has pharmacological and therapeutic activities judging from literature that A. melegueta can be used to prevent oxidative damage by ROS (Reactive oxygen species) reacting with free radical chelating and catalytic metals which can be used to reduce and prevent various diseases like heart disease, cancer, DNA degeneration, pulmonary and neurological disorder. SOD, GPX, GSH help to control cytokins induced peroxidise level and mediate signal transduction in mammalian cells. It can be deduced that Aframomum melegueta should be the focal point in human diet, natural herbal drug supplement and even in addition to conventional drug to improve the potency and quality of natural drug system. The use of Aframomum melegueta should be encouraged. These results show the potential of Aframomum melegueta as a source of bioactive compounds to be used for pharmaceutical, nutraceutical, and cosmeceutical applications.

Next-generation of targeted AAVP vectors for systemic transgene delivery against cancer

Bacteriophage (phage) have attractive advantages as delivery systems compared with mammalian viruses, but have been considered poor vectors because they lack evolved strategies to confront and overcome mammalian cell barriers to infective agents. We reasoned that improved efficacy of delivery might be achieved through structural modification of the viral capsid to avoid pre- and postinternalization barriers to mammalian cell transduction. We generated multifunctional hybrid adeno-associated virus/phage (AAVP) particles to enable simultaneous display of targeting ligands on the phage's minor pIII proteins and also degradation-resistance motifs on the very numerous pVIII coat proteins. This genetic strategy of directed evolution bestows a next-generation of AAVP particles that feature resistance to fibrinogen adsorption or neutralizing antibodies and ability to escape endolysosomal degradation. This results in superior gene transfer efficacy in vitro and also in preclinical mouse models of rodent and human solid tumors. Thus, the unique functions of our next-generation AAVP particles enable improved targeted gene delivery to tumor cells.

Improving Baculovirus Transduction of Mammalian Cells by Incorporation of Thogotovirus Glycoproteins.

Baculovirus can transduce a wide range of mammalian cells and is considered a promising gene therapy vector. However, the low transduction efficiency of baculovirus into many mammalian cells limits its practical application. Co-expressing heterologous viral glycoproteins (GPs), such as vesicular stomatitis virus G protein (VSV G), with baculovirus native envelope protein GP64 is one of the feasible strategies for improving virus transduction. Tick-borne thogotoviruses infect mammals and their GPs share sequence/structure homology and common evolutionary origins with baculovirus GP64. Herein, we tested whether thogotovirus GPs could facilitate the entry of the prototype baculovirus Autographa californica multiple multiple nucleopolyhedrovirus (AcMNPV) into mammalian cells. The gp genes of two thogotoviruses, Thogoto virus and Dhori virus, were inserted into the AcMNPV genome. Both GPs were properly expressed and incorporated into the envelope of the recombinant AcMNPVs. The transduction rates of recombinant AcMNPVs expressing the two thogotovirus GPs increased for approximately 4-12 fold compared to the wild type AcMNPV in six of the 12 tested mammalian cell lines. It seemed that thogotovirus GPs provide the recombinant AcMNPVs with different cell tropisms and showed better performance in several mammalian cells compared to VSV G incorporated AcMNPV. Further studies showed that the improved transduction was a result of augmented virus-endosome fusion and endosome escaping, rather than increased cell binding or internalization. We found the AcMNPV envelope protein GP64-mediated fusion was enhanced by the thogotovirus GPs at relatively higher pH conditions. Therefore, the thogotovirus GPs represent novel candidates to improve baculovirus-based gene delivery vectors.

Dissecting the Cell Entry Pathway of Baculovirus by Single-Particle Tracking and Quantitative Electron Microscopic Analysis.

The budded virus of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infects insect cells through mainly clathrin-mediated endocytosis. However, the cell entry pathway of AcMNPV remains unclear. In this study, by using population-based analysis of single-virus tracking and electron microscopy, we investigated the internalization, fusion behavior, and endocytic trafficking of AcMNPV. AcMNPV internalization into host insect cells was facilitated by actin polymerization and dynamin. After incorporation into early endosomes, the AcMNPV envelope fused with the membranes of early endosome, allowing for nucleocapsid release into the cytoplasm. Microtubules were implicated in the bidirectional and long-range transport of virus-containing endosomes. In addition, microtubule depolymerization reduced the motility of virus-bearing early endosomes, impairing the progression of infection beyond enlarged early endosomes. These findings demonstrated that AcMNPV internalization was facilitated by actin polymerization in a dynamin-dependent manner, and nucleocapsid release occurred in early endosomes in a microtubule-dependent manner. This study provides mechanistic and kinetic insights into AcMNPV infection and enhance our understanding of the infection pathway of baculoviruses.IMPORTANCE Baculoviruses are used widely as environmentally benign pesticides, protein expression systems, and potential mammalian gene delivery vectors. Despite the significant application value, little is known about the cell entry and endocytic trafficking pathways of baculoviruses. In this study, we demonstrated that the alphabaculovirus AcMNPV exhibited actin- and microtubule-dependent transport for nucleocapsid release predominantly from within early endosomes. In contrast to AcMNPV transduction in mammalian cells, its infection in host insect cells is facilitated by actin polymerization for internalization and microtubules for endocytic trafficking within early endosomes, implying that AcMNPV exhibits cell type specificity in the requirement of the cytoskeleton network. In addition, experimental depolymerization of microtubules impaired the progression of infection beyond enlarged early endosomes. This is the first study that dissects the cell entry pathway of baculoviruses in host cells at the single-particle level, which advances our understanding of the early steps of baculovirus entry.

BacMam System for Rapid Recombinant Protein Expression in Mammalian Cells.

Baculovirus expression vector system (BEVS) is an established technology for recombinant protein expression in insect cells. Further, BEVS-mediated gene transduction of mammalian cells (BacMam) is emerging as a technique for high level recombinant protein expression in mammalian cells. Here, we describe generic method in using BEVS as a BacMam for rapid recombinant protein expression in mammalian cells.

Overcoming Challenges of Hepatitis C Virus Envelope Glycoprotein Production in Mammalian Cells.

Posttranslational modifications (PTMs) are often required for proper folding and physiological function of proteins, including the envelope glycoproteins 1 and 2 (E1 and E2) of hepatitis C virus (HCV). Commonly used expression systems such as bacteria, yeast, and baculovirus produce soluble HCV E1 and E2 at very low yields, as the cellular environment and molecular machinery necessary for PTMs may be suboptimal or missing. Here, we describe an expression system for HCV E2 ectodomain (eE2) with 11 N-linked glycans and eight disulfide bonds, which combines lentivirus transduction of mammalian cells and a continuous growth, adherent cell bioreactor. It is environmentally friendly, as well as cost- and time-efficient compared to other methods of recombinant protein expression in mammalian systems with final yields of eE2 approaching 60mg/L of cell culture supernatant. eE2 produced by this system is amenable to a variety of biophysical studies, including structural determination by X-ray crystallography. Considering the ease of use and flexibility, this method can be applied to express an array of difficult target proteins in a variety of mammalian cell lines.

Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins.

Structural, biochemical and biophysical studies of eukaryotic soluble and membrane proteins require their production in milligram quantities. Although large-scale protein expression strategies based on transient or stable transfection of mammalian cells are well established, they are associated with high consumable costs, limited transfection efficiency or long and tedious selection of clonal cell lines. Lentiviral transduction is an efficient method for the delivery of transgenes to mammalian cells and unifies the ease of use and speed of transient transfection with the robust expression of stable cell lines. In this protocol, we describe the design and step-by-step application of a lentiviral plasmid suite, termed pHR-CMV-TetO2, for the constitutive or inducible large-scale production of soluble and membrane proteins in HEK293 cell lines. Optional features include bicistronic co-expression of fluorescent marker proteins for enrichment of co-transduced cells using cell sorting and of biotin ligase for in vivo biotinylation. We demonstrate the efficacy of the method for a set of soluble proteins and for the G-protein-coupled receptor (GPCR) Smoothened (SMO). We further compare this method with baculovirus transduction of mammalian cells (BacMam), using the type-A γ-aminobutyric acid receptor (GABAAR) β3 homopentamer as a test case. The protocols described here are optimized for simplicity, speed and affordability; lead to a stable polyclonal cell line and milligram-scale amounts of protein in 3-4 weeks; and routinely achieve an approximately three- to tenfold improvement in protein production yield per cell as compared to transient transduction or transfection.

Identification and characterization of a large family of superbinding bacterial SH2 domains

Src homology 2 (SH2) domains play a critical role in signal transduction in mammalian cells by binding to phosphorylated Tyr (pTyr). Apart from a few isolated cases in viruses, no functional SH2 domain has been identified to date in prokaryotes. Here we identify 93 SH2 domains from Legionella that are distinct in sequence and specificity from mammalian SH2 domains. The bacterial SH2 domains are not only capable of binding proteins or peptides in a Tyr phosphorylation-dependent manner, some bind pTyr itself with micromolar affinities, a property not observed for mammalian SH2 domains. The Legionella SH2 domains feature the SH2 fold and a pTyr-binding pocket, but lack a specificity pocket found in a typical mammalian SH2 domain for recognition of sequences flanking the pTyr residue. Our work expands the boundary of phosphotyrosine signalling to prokaryotes, suggesting that some bacterial effector proteins have acquired pTyr-superbinding characteristics to facilitate bacterium-host interactions.

Crude extracts of recombinant baculovirus expressing rabbit hemorrhagic disease virus 2 VLPs from both insect and rabbit cells protect rabbits from rabbit hemorrhagic disease caused by RHDV2.

Vaccines against viral pathogens are often composed of recombinant proteins expressed in different systems. Such proteins expressed by recombinant baculoviruses have been proven to be effective for vaccination. Especially, after codon usage optimization high amounts of recombinant viral proteins can be obtained which can assemble to virus like particles (VLPs) spontaneously. In this study we compared two different codon usages of RHDV2-VP1 to improve the expression of recombinant VP1 of RHDV2 by recombinant baculoviruses after infection of insect SF9 cells or transduction of mammalian RK13 cells in order to gain high protein yields. Also the influence on the auto-assembly of RHDV2-VP1 to VLPs was investigated. Finally, the immunogenic potential of such recombinant vaccines against RHDV2 to induce a protective immune response in rabbits against RHDV2 should be characterized. There was no influence of different codon usages on RHDV2-VP1 gene expression in the respective cell lines detected. However, in insect cell line SF9 higher rates of recombinant VP1 were measured in comparison to the transduction of mammalian cells RK13. Auto-assembly of RHDV2-VP1 to VLPs was observed in both cell systems by electron microscopy. Finally, both RHDV-VP1 VLPs derived from mammalian and insect cells were able to induce a protective humoral immune response in rabbits against RHDV2.

Amyloid Fibrils with Positive Charge Enhance Retroviral Transduction in Mammalian Cells.

Amyloid fibrils are cross-β-sheet-rich protein/peptide fibrils that are typically associated with neurodegenerative diseases such as Parkinson's and Alzheimer's disease. Recently, functional amyloids have been discovered where amyloids are implicated in performing normal physiological functions of the host organism rather than creating diseases. The ability of amyloids to interact with the cell membrane and other small biomolecules exhibits its great potential to be used as a biomaterial for cell adhesion and gene delivery system. Given the established ability of semen-derived amyloids to concentrate HIV in semen and that of charged polymers as an enhancer of retroviral gene transfer, we hypothesized that charged amyloid fibrils can augment virus-mediated delivery system. We show that amyloids of α-synuclein formed in the presence and absence of cationic polymers chitosan and amyloid of poly-l-lysine can interact with lentiviral particles and enhance transduction efficiency in cells. The amyloid nanofibrils increase transduction efficiency up to ∼4 fold similar to widely used cationic polymer Polybrene. This study shows that amyloid nanofibril scaffolds may be used as targeted gene delivery systems.

Surface displaying of swine IgG1 Fc enhances baculovirus-vectored vaccine efficacy by facilitating viral complement escape and mammalian cell transduction

Baculovirus-mediated gene transfer has been developed as a vaccine design strategy against a number of diseases without apparent viral replication. However, it has been hampered by complement-dependent inactivation, thus hindering the in vivo application of baculovirus. A variety of approaches have been exploited to bypass the complement system in the serum. In this study, we constructed and screened a series of baculovirus vectors displaying complement interfering factors, of which a baculovirus vector displaying swine IgG1 Fc (pFc) showed the highest complement antagonism (75.6%). Flow cytometry analysis of transduced cells demonstrated that the baculovirus display of pFc had a significant increase in transduction efficiency and transgene expression of reporter genes. On this basis, a VSV-G-pseudotyped with swine IgG1 Fc surface displayed baculovirus vector was developed to express the classical swine fever virus (CSFV) E2 gene. The translational enhancers Syn21 and P10UTR were incorporated to improve the antigen expression. The E2 gene was efficiently expressed in both insect and mammalian cells. Pigs immunized with this recombinant baculovirus developed high levels of E2-specific antibody, CSFV-specific neutralizing antibody and IFN-γ-secreting cellular immune responses. These results demonstrate that the strategy of surface-displaying swine IgG1 Fc has a great potential to improve the efficiency of baculovirus-vectored vaccine for CSFV and other swine pathogens.

Characterization of retroviral infectivity and superinfection resistance during retrovirus-mediated transduction of mammalian cells

Retroviral vectors including lentiviral vectors are commonly used tools to stably express transgenes or RNA molecules in mammalian cells. Their utilities are roughly divided into two categories, stable overexpression of transgenes and RNA molecules, which requires maximal transduction efficiency, or functional selection with retrovirus (RV)-based libraries, which takes advantage of retroviral superinfection resistance. However, the dynamic features of RV-mediated transduction are not well characterized. Here, we engineered two murine stem cell virus-based retroviral vectors expressing dual fluorescence proteins and antibiotic markers, and analyzed virion production efficiency and virion stability, dynamic infectivity and superinfection resistance in different cell types, and strategies to improve transduction efficiency. We found that the highest virion production occurred between 60 and 72 h after transfection. The stability of the collected virion supernatant decreased by >60% after 3 days in storage. We found that RV infectivity varied drastically in the tested human cancer lines, while low transduction efficiency was partially overcome with increased virus titer, prolonged infection duration and/or repeated infections. Furthermore, we demonstrated that RV receptors PIT1 and PIT2 were lowly expressed in the analyzed cells, and that PIT1 and/or PIT2 overexpression significantly improved transduction efficiency in certain cell lines. Thus, our findings provide resourceful information for the optimal conditions of retroviral-mediated gene delivery.

The localization of a heterologous displayed antigen in the baculovirus-budded virion determines the type and strength of induced adaptive immune response.

In the search of strategies of presentation of heterologous antigens to elicit humoral or cellular immune responses that modulate and properly potentiate each type of response, researchers have been studying baculovirus (BV) as vaccine vectors with promising results. For some years, several research groups explored different antigen presentation approaches using the BV AcNPV by expressing polypeptides on the surface of budded virions or by de novo synthesis of heterologous antigens by transduction of mammalian cells. In the case of expression on the surface of budded virions, for example, researchers have expressed polypeptides in peplomers as GP64 glycoprotein fusions or distributed throughout the entire surface by fusions to portions of the G protein of vesicular stomatitis virus, VSV. Recently, our group developed the strategy of cross-presentation of antigens by fusions of GP64 to the capsid protein VP39 (capsid display) for the generation of cytotoxic responses. While the different strategies showed to be effective in raising immune responses, the individuality of each analysis makes difficult the comparison of the results. Here, by comparing the different strategies, we show that localization of the model antigen ovalbumin (OVA) strongly determined the quality and intensity of the adaptive response to the heterologous antigen. Furthermore, surface display favored humoral responses, whereas capsid display favored cytotoxic responses. Finally, capsid display showed a much more efficient strategy to activate CD8-mediated responses than transduction. The incorporation of adjuvants in baculovirus formulations dramatically diminished the immunostimulatory properties of baculovirus.