Hepatitis B virus HBV infection and its sequelae remain a major public health burden, but both HBV basic research and the development of antiviral therapeutics have been hindered by the lack of an efficient in vitro infection system.
Moreover, the infection level gradually increased with accelerated speed of spinoculation up to 1,g tested.
Improved Coinfection with Amphotropic Pseudotyped Retroviral Vectors
However, the enhancement of HBV infection was not significantly dependent upon the duration of centrifugation. Furthermore, covalently closed circular ccc DNA was detected in infected cells under optimized infection condition by conventional Southern blot, suggesting a successful establishment of HBV infection after spinoculation. Our data suggest that spinoculation could serve as a standard protocol for enhancing the efficiency of HBV infection in vitro.
This is an open access article distributed under the terms of the Creative Commons Attribution Licensewhich permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist. HBV mainly infects hepatocytes and establishes a pool of a nuclear episomal covalently closed circular ccc DNA form of the viral genome, which serves as transcription template for all the viral RNAs, including 3. HBV has infected approximately 2 billion people worldwide, resulting in — million chronic infections; this significant epidemic level of hepatitis B is partly due to the high infectivity of HBV in vivo when the prophylactic vaccination is not in place [ 3 ].
Although primary human hepatocytes PHHs and the HepaRG cell line can be used for certain HBV infection experiments, the PHHs are costly with limited supply, and their genetic background and susceptibility to HBV infection vary from donor to donor [ 5 ]; in these regards, the HepaRG system does have advantages over PHH but time-consuming cell proliferation and differentiation steps are required prior to infection [ 6 ].
Thus, the basic and antiviral research of HBV in the context of a complete viral life cycle have been hampered for a long period of time. Now, the reconstitution of NTCP expression in commonly used hepatocyte-derived cells i. Nevertheless, the reported HBV infectivity of NTCP-expressing cells varies among different laboratories under different infection conditions, but the average percentage of HBcAg or HBsAg positive cells remains low as revealed by immunofluorescence [ 7 — 11 ].
Various mechanisms for this enhancement have been proposed, from ultrastructural changes in the host cell that render it more permissive to viruses [ 14 ], to surprisingly for such low centrifugation speeds increased deposition of virions on the cell surface [ 15 ].
Herein, we established a HepG2-based NTCP-expressing cell line which is permissive to HBV infection, and developed a spinoculation method to enhance HBV infection with the optimized cell culture conditions and viral inoculum size.
In addition, the enhanced HBV infection by spinoculation is in an NTCP-dependent manner, suggesting that spinoculation promotes HBV infection through the authentic viral entry mechanism. Therefore, spinoculation could serve as a routine procedure in the protocol of in vitro HBV infection.
The 1. Louis, MO. Twenty four blasticidin-resistant colonies were picked and expanded into cell lines. HBV particles were collected from the supernatant of HepDE19 cells as described previously with modifications [ 17 ]. Briefly, HepDE19 cells were cultured in tetracycline-free medium to induce HBV replication and virion production, supernatant was collected every other day with culture medium replenishment for 18 days.
HBV particle gel assay was performed as previously described [ 17 ]. The DNA-containing viral particles on the membrane were first denatured in a solution containing 0. The amplification setting was 0.
To perform spinoculation, the HBV inoculated plate was immediately centrifuged at room temperature with specified speed and time. After being washed with PBS, the cells were stained with Alexa Fluor dye-conjugated secondary antibody Life Technologies and the nuclei were counterstained with DAPI for 60 min at room temperature.
In addition, transfection of plasmid pHBV1. The parental HepG2 cells served as negative control. In order to calculate the virion genome equivalent vge of the HBV inoculum more accurately, we developed a method to serve this purpose. By using the above virus titration method, the vge of the HBV inoculum we prepared from HepDE19 cells is approximately 7. A HBV virions and naked capsids in the indicated volume of virus stock were separated by native agarose gel electrophoresis, and viral DNA was detected by hybridization.
DMSO was added to the PMM medium 24 h prior to the infection and remained present in the entire culture period until the cells were harvested.In retroviral vector-mediated gene transfer, transduction efficiency can be hampered by inhibitory molecules derived from the culture fluid of virus producer cell lines.
To remove these inhibitory molecules to enable better gene transduction, we had previously developed a transduction method using a fibronectin fragment-coated vessel i. In the present study, we developed a method that combined RetroNectin-bound virus transduction with low-temperature shaking and applied this method in manufacturing autologous retroviral-engineered T cells for adoptive transfer gene therapy in a large-scale closed system.
After the supernatant was removed, activated T cells were added to the bag. The bag transduction method has the advantage of increasing transduction efficiency, as simply flipping over the bag during gene transduction facilitates more efficient utilization of the retroviral vector adsorbed on the top and bottom surfaces of the bag.
In conclusion, we demonstrated that the large-scale closed transduction system is highly efficient for retroviral vector-based T cell manufacturing for adoptive transfer gene therapy, and this technology is expected to be amenable to automation and improve current clinical gene therapy protocols.
Editor: Christopher B. This is an open-access article distributed under the terms of the Creative Commons Attribution Licensewhich permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
RetroNectin is a commercialized product of Takara Bio Inc. Fibronectin FNone of the major extracellular matrix proteins, is a disulfide-linked dimeric glycoprotein that has several functional domains including cell binding properties  — .
FN is a glycoprotein that binds to membrane-spanning receptor proteins called integrins. In addition to integrins, FN also binds to extracellular matrix components such as collagen, fibrin, and heparan sulfate proteoglycans. Retroviral vectors are currently one of the most widely used systems for gene transduction, both in experimental studies and in clinical trials.
In particular, murine leukemia virus MLV has traditionally been used as the vector of choice for clinical gene therapy protocols, and a variety of packaging systems  and viral production systems  —  using MLV have been developed.
The amphotropic envelope from these packaging lines also contained some materials that inhibit viral infection . To overcome these problems, a human-derived packaging cell line that produces high titer viral supernatant was developed .
Receptor-Independent Infection of Murine Coronavirus: Analysis by Spinoculation
Purification of retroviral vector was also attempted using a low-speed centrifugation procedure to remove undesirable substances in the viral supernatant and concentrate the retrovirus vector .
To increase the chance of contact between the viral vector and target cells, a flow-through transduction method involving the convective flow of retroviral particles through the target cell monolayer was also proposed . Alternatively, we and others have demonstrated that RN is an efficient tool for enhancing gene transfer into hematopoietic stem cells  —  and T lymphocytes  using a retroviral vector system.
RN consists of three functional regions: the cell-binding domain C-domainthe heparin-binding domain H-domainand the CS-1 sequence. Thus, retrovirus-mediated gene transfer is enhanced by co-locating target cells and virions on the RN molecules  ; because RN's H-domain can bind retrovirus, preloading the retroviral supernatant on an RN-coated vessel will allow transferable inhibitors from the producing cell line to be washed out RN-bound virus; RBV transduction method.
In contrast, gene transfer efficiency does not increase under passive and static preloading conditions, even if the amount of vector used exceeds 0. Viral vector particles cannot be adsorbed under passive conditions, even if the substratum is coated with RN, as these particles are located far from the surface of the substratum.
To utilize the retroviral vector efficiently, active adsorption of the vector is required. To achieve this adsorption, preloading of the vector into an RN-coated plate in combination with low-speed centrifugation or spin transduction centrifuge cells and vectors together in RN-coated vessel is sometimes proposed  — .MHVR-independent infection is hypothetically thought to be attributed to a naturally occurring fusion activation of the wt JHMV S protein, which did not occur in the case of srr7.
In order to make virions attach to the cell surface without MHVR, we have used spinoculation, namely, the centrifugation of cells together with inoculated virus at 3, rpm for 2 h. This procedure forces viruses to attach to the cell surface, as revealed by quantitative estimation of attached virions by real-time PCR and also facilitated wt JHMV infection to MHVR-negative cells, but failed to do so for srr7.
Virions of both wt and srr7 attached on MHVR-negative cells by spinoculation were facilitated for infection in the presence of a soluble form of MHVR that induces conformational changes of both wt and srr7. It was further revealed that wt JHMV S1, but not srr7, was released from the cell surface when S protein was expressed on cells.
These observations support the hypothesis that attachment of the virion to MHVR-negative cells is a critical step and that a unique feature of wt JHMV S1 to be released from S2 in a naturally occurring event is involved in an MHVR-independent infection.
The binding of virus to its specific receptor on a susceptible cell surface is an initial event in viral infection. A number of molecules classified into the immunoglobulin Ig superfamily are known to serve as receptors for various viruses.
However, additional alternative molecules are thought to be used as a receptor for HIV and measles virus, since they are known to infect cells lacking their specific receptors, CD4 or SLAM 4 MHV is classified as one of the Coronaviridaeconsisting of an enveloped virus with a single, positive-stranded genomic RNA of about 31 kb The characteristic spikes on the virion surface are composed of spike S protein.
S protein is a class I fusion protein of to kDa in molecular mass 5 It is synthesized as a large protein and cleaved by cellular protease into two subunits, N-terminal S1 and C-terminal S2 The N-terminal region of S1 consisting of amino acids S1N is responsible for receptor binding 2538and S2 is not involved in this activity After receptor binding, S1 is dissociated from the membrane-anchored S2 subunit, which triggers the conformational changes of S2 to acquire fusion activity Likewise, the structural features of MHV S2 subunit are very similar to those of the membrane-anchored subunit of HIV envelope protein gp There are four isoforms, two having four immunoglobulin-like domains and the other two having Ig-like domains, one of which has either a long or a short Cy 3.
The N-terminal N domain is responsible for virus binding 13induction of S protein conformational changes, and activation for fusion 30although the N domain linked with TM and Cy and expressed on the cell surface is not functional, which is presumably due to inaccessibility for MHV to short molecules expressed on the cell surface 13 We have isolated from wild-type wt JHMV with this activity, a mutant virus resistant to neutralization by soluble receptor srr7 that lacks this unique feature of infection 34 The mechanism of this MHVR-independent infection and fusion is still speculative, although some unusual feature of wt JHMV S, but not srr7 S, hints that there is a possible mechanism of this unique mode of infection.
Both of these activities occur naturally, without binding to MHVR. These findings suggest that the spontaneous release of S1 from S2 triggers the conformational changes of the S2 in a similar fashion, since it occurs after binding its receptor. If the hypothesis described above is correct, wt JHMV can infect a cell without a receptor under conditions whereby the virion is forced to attach to the cell surface.This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Amphotropic pseudotyped retroviral vectors have typically been used to infect target cells without prior concentration. Although this can yield high rates of infection, higher rates may be needed where highly efficient coinfection of two or more vectors is needed.
In this investigation we used amphotropic retroviral vectors produced by the Plat-A cell line and studied coinfection rates using green and red fluorescent proteins EGFP and dsRed2. Target cells were primary human fibroblasts PHF and 3T3 cells. Improved coinfection should be valuable in protocols that require high transduction by combinations of two or more retroviral vectors. High rates of infection of target cells with two or more retroviral vectors are often required to achieve a desired biological outcome.
Methods are needed to ensure that high coinfection rates are reliably achieved. When introducing reprogramming genes into human fibroblasts, Yamanaka's group used a complex strategy to increase coinfection to a rate sufficient for reprogramming.
They first introduced the ecotropic retrovirus receptor into the target human cells using a lentiviral vector, and then infected the cells with retroviruses produced in the ecotropic Plat-E packaging cell line [ 2 ]. Plat-E and Plat-A cells are retrovirus packaging cell lines derived from the T cell line [ 3 ]. However, the Plat-A cell line, used for amphotropic pseudotyping, produces a titer of only about one-tenth that of the ecotropic packaging cell line Plat-E [ 4 ].
Titers from other amphotropic retrovirus packaging cell lines derived from T, such as Phoenix-Ampho, are similar [ 5 ]. In experiments involving coinfection to achieve cellular reprogramming unconcentrated retroviruses have been used. Generally, unconcentrated preparations produced by amphotropic packaging cells are sufficient for the introduction of single genes into cells. However, higher levels of vector are needed for efficient coinfection with two or more retroviruses. There has been little work on optimizing conditions for coinfection with amphotropic or ecotropic retroviral vectors.
Although it may be assumed that any improvement in the rate of infection will increase the rate of coinfection, this has rarely been tested [ 6 ]. We hypothesized that the use of optimized protocols could result in much higher coinfection rates by amphotropic retroviral vectors produced by packaging cell lines like Plat-A.
Strategies for increasing the amount of amphotropic or ecotropic vector that can be delivered to target cells include a increasing vector production rates by the packaging cells [ 5 ], b concentrating the vector, and c increasing the rate or level of attachment of vector to the target cells. Amphotropic retroviruses can be concentrated by centrifugation, although some loss of titer may be encountered [ 78 ]. Retroviruses pseudotyped with vesicular stomatis virus G VSV-G protein can readily be concentrated by centrifugation, but preparation of this type of vector typically requires cotransfection of T cells with multiple plasmids [ 9 ].
A convenient and efficient method for concentrating amphotropic retroviral vectors was introduced recently; this comprises flocculation of the vector with high concentrations of polymers such as Polybrene followed by a brief centrifugation [ 1011 ].
This process also separates active viral particles from inactive particles that can compete for receptors on target cells [ 12 ]. Polymers, usually Polybrene, are also used in standard infection protocols to increase attachment of viral vectors to target cells [ 13 ].
This is particularly effective in established cell lines in combination with low-speed centrifugation of the vector onto the surface of the target cells, a process termed spinoculation or spin-infection [ 814 ].Lenti webinar -Why use lenti, biosafety \u0026 detailed protocols
This has not been extensively tested in primary human cell types, however. The two processes should be complementary: separation of the vector from the bulk medium by Polybrene flocculation can be followed by resuspension of the vector in culture medium and optimized infection of the target cells by spinoculation.
In the present experiments we tested whether amphotropic retroviral vectors can be concentrated and efficiently delivered to target cells primary human fibroblasts and 3T3 cells by spinoculation. Coinfection rates were determined by the use of retroviral vectors encoding two different fluorescent proteins, EGFP and dsRed2 [ 15 ].
Amphotropic retroviruses were generated by transient transfection of Plat-A cells. After 16 hours the medium was changed to 3. After 24 hours the vector-containing medium was removed and filtered through a 0. A second 24 hour medium collection was also used for infections. We investigated whether the titers of the green and red vectors used were equivalent when tested on target cells, PHF or 3T3. In each experiment reported here the titers were approximately equal. Viral vector-containing medium 3.Analyzed the data: WW BV.
Wrote the paper: WW BV. The function of dendritic cells DCs in the immune system is based on their ability to sense and present foreign antigens. Powerful tools to research DC function and to apply in cell-based immunotherapy are either silencing or overexpression of genes achieved by lentiviral transduction. The protocol can be used both for ectopic gene expression and knock-down. This protocol for MDDCs transduction is robust and free of the potential risk arising from the use of Vpx which creates a virus infection-prone environment, potentially dangerous in clinical setting.
Dendritic cells DCs are crucial actors in the interplay between pathogens and the immune system, linking innate and adaptive immune responses. DCs capture incoming pathogens and present them to T cells [ 1 ]. Their important role in induction of anti-tumor immunological responses raises hope that use of this potential will lead to efficient cell-based immunotherapy [ 2 ].
Understanding mechanisms that shape DC crosstalk between immunogens and components of the immune system, is a prerequisite for successful clinical implementation of such therapeutic approach, both in oncology and infectious diseases. Research is hampered by the difficulties to manipulate DCs gene expression profile, especially when it comes to reduction of gene expression.
Selective knock-down of gene products by RNA interference is a widely used method in the study of gene function [ 3 ]. In our study we chose lentivirus-mediated shRNA expression as it provides stable knock-down levels, while producing fewer off-target effects than transfection-based siRNA delivery [ 45 ]. For DC therapy, efficient gene transfer to express antigen is highly desired. Transduction efficiency at lower vector MOI could be increased by careful timing, spinoculation and by use of agents such as polybrene.
SAMHD1 combines the ability to deplete the cytoplasmic pool of dNTPs necessary for the reverse transcription of viral genome [ 12 ] together with ribonuclease activity which degrades incoming viral RNA [ 13 ], thereby highly decreasing the chances of successful lentiviral integration. Exposure to Vpx loaded virus-like particles as a method to overcome SAMHD1 restriction might confer the DCs with distinct features which can affect the read-out of the genetic manipulation intended by the transduction.
A critical phenotypic alteration of DCs induced by Vpx-induced SAMHD1 block is their subsequent permissiveness to viral infections—a caveat for clinical applications. This method preserves the immature MDDC phenotype, which makes it an important tool in studies of DC function and differentiation. Donor samples were obtained after informed consent. Monocytes were isolated from buffy coats of healthy donors following Lymphoprep Axis-Shield, Dundee, Scotland gradient centrifugation and positive or negative magnetic antibody separation kit Miltenyi Biotec, Leiden, Netherlands.Quantification of retroviruses in cell culture supernatants and other biological preparations is required in a diverse spectrum of laboratories and applications.
Methods based on antigen detection, such as p24 for HIV, or on genome detection are virus specific and sometimes suffer from a limited dynamic range of detection. We consider this test to be an accurate, fast and relatively cheap method for retroviral quantification that is easily implemented for use in routine and research laboratories. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No other conflicts of interest are to be reported by the authors. Retroviral vectors have become an indispensable tool in any modern molecular biology laboratory. They allow stable expression of a gene of interest in dividing cells, as well as stable gene knock-down by expression of short hairpin RNA shRNA. A subset of vectors, derived from lentiviruses such as human immunodeficiency virus HIV- 1 and 2 and feline immunodeficiency virus FIVcan be used for efficient transduction of non-dividing cells and have therefore received increased attention for both basic research and clinical applications .
Although methods for accurate quantification of retroviral vector titers will be indispensable in a clinical setting, also the basic research environment can benefit from a fast and inexpensive method to evaluate the quality of retroviral vector productions. Furthermore, research laboratories investigating the replication of retroviruses, such as HIV, require routine assays to determine retroviral titers after production and during viral infection.
Multiple methods for retroviral titer quantification are currently available see  for an overview of lentiviral titration methodsbut they often have some inherent drawbacks. Other more rapid methods measure both functional and non-functional viral particles in the supernatant, by quantifying the levels of retroviral Gag protein such as the HIV p24 protein and the levels of viral genomic RNA.
The former is often done by enzyme-linked immunosorbent assay ELISA and consequently has a limited linear range and high cost. Furthermore, both methods are still quite labor-intensive. An alternative retroviral titration method involves quantification of the reverse transcriptase RT activity, which is associated with all retroviral particles. In the first generation RT assays, cDNA production was monitored by measurement of labeled nucleotide incorporation .
Sensitivity was highly increased when a PCR amplification step of the synthesized cDNA was introduced prior to product detection. PERT assays are now routinely used for detection of retroviral contaminants in biological products intended for human use .
However, in basic research environment, the implementation of real-time PCR based PERT assays is still limited, despite their low cost and fast procedure. The assay was adapted for use with different commercial ready-to-use SYBR Green I qPCR reaction mixes, to allow an easy implementation of the assay in any research lab with qPCR experience and to avoid possible compositional variation inherent to in house prepared qPCR mixes.
Sensitivity and specificity of the assay were determined, as well as the variation on repeated RT activity measurement within and between runs. We used the assay to evaluate the informative value of the RT activity for lentiviral titer determination, by comparing it to more commonly used titration methods.
We observed excellent correlation with the p24 antigen concentration in both replication-competent HIV-1 virus supernatant and replication-incompetent HIV-based lentiviral vector preparations, as well as with the levels of transducing units or infectious units. This particular assay outperformed p24 ELISA by its lower inter-run variation, lower cost and higher linear range.
Furthermore, it was far less time-consuming than both p24 ELISA and determination of transducing or infectious units. We therefore believe that this assay forms an attractive alternative to routine retroviral and lentiviral titer determination in routine and research laboratories.
Human lymphoblastoid Jurkat E6. Achacoso and Dr G.
Viral supernatant was harvested 48 hours or 72 hours after transfection and centrifugated at g for 10 min, to clarify the supernatant from remaining cells. During infection, culture medium was refreshed every two or three days.
All replication-incompetent lentiviral vectors used in this study were produced using the pLKO. G plasmids  and virus was harvested two days after transfection. Cell-free viral supernatant was generally used without prior dilution as input for the assay.
After brief centrifugation, the lysates were resuspended and used as input for the assay. Fluorescence acquisition was done at the end of annealing phase in our experiments, but can alternatively be done at the end of elongation phase.
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