Screening cDNA libraries from solid human tumors with sera of autologous patients (SEREX) has proven to be a powerful approach to identifying tumor antigens recognized by the humoral arm of the immune system. In many cases, application of this methodology has led to the discovery of novel tumor antigens as unknown gene products. We tried to improve the potency of the SEREX approach by combining it with phage-display technology. We designed a new lambda vector to express protein fragments as N-terminal fusions to the D capsid protein and generated high-complexity cDNA libraries from human breast carcinoma cell lines and solid tumors. Screening these phage-displayed libraries required limited amounts of sera from patients and efficiently identified several tumor antigens specifically reacting with sera from breast cancer patients. © 2003 Wiley-Liss, Inc. Key words: breast cancer; immune response; phage displayTumor-associated antigens recognized by humoral effectors of the immune system are an attractive target for diagnostic and therapeutic approaches to human cancer. A considerable number of this type of antigen have been identified over recent years through screening expression cDNA libraries from human solid tumors with sera of the autologous patients (SEREX, serologic identification of antigens by recombinant expression cloning). [1][2][3][4] This type of screening of a cDNA expression library by conventional methods requires the preparation of a large number of membrane filters blotted with bacteriophage plaques that are then searched with a specific probe. In the case of the SEREX experiments, the screening is performed using sera from cancer patients, which are usually available in very limited quantity. The second limitation is that such immunoscreening procedure does not allow selection of antigens that are recognized by sera from different patients.We tried to overcome these limitations and to simplify the screening procedure by performing affinity selection of cDNA libraries in very small volumes by combining the SEREX approach with that of phage-display technology. Phage-display technology is based on the insertion of foreign nucleotide sequences into genes encoding for various coat proteins of filamentous phage, resulting in a heterogeneous mixture of phages, each displaying the different peptide sequence encoded by a corresponding insert. A physical link between a displayed fusion protein and DNA encoded for it make this phage target selectable.Phage-display technology was introduced in 1985 by Smith 5 and has been widely used since for generating and screening peptide libraries to identify ligands for various kinds of receptor molecules. 6 -8 However, significantly less striking progress has been reported on the use of filamentous phage as display vector for constructing cDNA libraries. In most cases, foreign peptides are displayed on the filamentous phage capsid as N-terminal fusions to the major (pVIII) or the minor (pIII) coat protein. Large protein domains fused to pVIII disturb (with some rare...
Tamarins (Saguinus species) infected by GB virus B (GBV-B) have recently been proposed as an acceptable surrogate model for hepatitis C virus (HCV) infection.The availability of infectious genomic molecular clones of both viruses will permit chimeric constructs to be tested for viability in animals. Studies in cells with parental and chimeric constructs would also be very useful for both basic research and drug discovery. For this purpose, a convenient host cell type supporting replication of in vitro-transcribed GBV-B RNA should be identified. We constructed a GBV-B subgenomic selectable replicon based on the sequence of a genomic molecular clone proved to sustain infection in tamarins. The corresponding in vitro-transcribed RNA was used to transfect the Huh7 human hepatoma cell line, and intracellular replication of transfected RNA was shown to occur, even though in a small percentage of transfected cells, giving rise to antibiotic-resistant clones. Sequence analysis of GBV-B RNA from some of those clones showed no adaptive mutations with respect to the input sequence, whereas the host cells sustained higher GBV-B RNA replication than the original Huh7 cells. The enhancement of replication depending on host cell was shown to be a feature common to the majority of clones selected. The replication of GBV-B subgenomic RNA was susceptible to inhibition by known inhibitors of HCV to a level similar to that of HCV subgenomic RNA.A problem that remains unsolved in the search for new therapeutic agents against hepatitis C virus (HCV) infection (12,20,25) is the availability of a small-animal model suitable for pharmacological studies, since the known host range of this virus includes only humans and chimpanzees. An interesting murine model based on the repopulation of mouse liver with human HCV hepatocytes has recently been proposed as sustaining HCV infection and yielding significantly high serum titers (17). The current version of this model, however, presents several drawbacks, including the difficulty of identifying an unlimited source of human hepatocytes and the low success rate, due in part to the peculiar genetic background of the mice used. Albeit very promising, this model needs improvement to be turned into a method accessible to most of the laboratories concerned.In the absence of a direct and convenient small-animal model, over the last few years a model for HCV infection alternative to the chimp has been proposed by various research groups. That model is based on the use of a surrogate virushost system, tamarins (Saguinus species) infected by GB virus B (GBV-B). A growing body of data about the enzymatic activity of GBV-B proteins has corroborated the hypothesis that useful information for research on anti-HCV drugs can be derived from experiments with GBV-B in tamarins. The identification of in vivo infectious cDNA has provided an indispensable tool to engineer the virus genome by insertion of HCV regions of interest. The availability of a small nonhuman primate GBV-B host and the perspective of using c...
The strong similarity between GB virus B (GBV-B) and hepatitis C virus (HCV) makes tamarins infected by GBV-B an acceptable surrogate animal model for HCV infection. Even more attractive, for drug discovery purposes, is the idea of constructing chimeric viruses by inserting HCV genes of interest into a GBV-B genome frame. To accomplish this, infectious cDNA clones of both viruses must be available. The characterization of several HCV molecular clones capable of infecting chimpanzees has been published, whereas only one infectious GBV-B clone inducing hepatitis in tamarins has been reported so far. Here we describe the infection of tamarins by intrahepatic injection of RNA transcribed from a genomic GBV-B clone (FL-3) and transmission of the disease from infected to naive tamarins via serum inoculation. The disease resulting from both direct and secondary infection was characterized for viral RNA titre and hepatitis parameters as well as for viral RNA distribution in the hepatic tissue. Host humoral immune response to GBV-B antigens was also monitored. The progression of the disease was compared to that induced by intravenous injection of different amounts of the non-recombinant virus.
Page 2469, right hand column, line 29. The amino acid substitutions referred to were transposed ; the correct version of the text is given below. Low-affinity CD46-binding isolates can be converted to the high-affinity CD46-binding phenotype by a substitution of Tyr instead of Asn at position 481 (Hsu et al., 1998). Likewise, a substitution of Gly instead of Ser at site 546 has been shown to confer both the properties of CD46 binding and haemadsorption to isolates that were previously defective for these traits (Li et al., 1999).
Hepatitis C virus (HCV) and GB virus B (GBV-BThe simian flavivirus GB virus B (GBV-B) is in principle a useful surrogate model for human hepatitis C virus (HCV) (3): the genome organization and enzymatic activities are similar in the two viruses (6,7,16,(19)(20)(21)(22)(23), and GBV-B offers the advantage of infecting primates more suitable for research than the chimpanzee, which is the only host for HCV (3,5). A valuable achievement for drug discovery purposes would be the construction of viable chimeric HCV/GBV-B viruses with the host range of GBV-B. An intermediate step, important to further validate the use of GBV-B as a model for HCV and as a scaffold for chimeric constructs, is the development of comparable cell-based systems for GBV-B and for HCV.We have recently described a subgenomic dicistronic GBV-B replicon system (9, 18) working in Huh7-derived human hepatoma cell lines and similar to that available for HCV (2,4,14), which allows analysis of the replication and the synthesis/processing of the viral nonstructural proteins. So far no successful attempt at identifying cells different from Huh7 cells as the host for HCV or GBV-B has been described (1, 14). Nonetheless, the availability of more than one host cell type would be important to study the molecular features underlying cell permissiveness for these viruses and as an additional tool in the drug discovery process.In this paper we describe the identification of cells permissive to GBV-B replicons derived from the human hepatoma Hep3B cell line and the spontaneous occurrence of an interesting adapted mutant replicon lacking the stem-loop structure at the immediate 5Ј terminus. MATERIALS AND METHODSCell lines and culture conditions. Human hepatoma cell lines HepG2, Hep3B, Huh7 (our laboratory stocks), and derived cell lines (9) were grown in highglucose Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 2 mM L-glutamine, 100 U of penicillin per ml, 100 g of streptomycin per ml, and 10% fetal bovine serum. Cells were subcultivated twice a week with a 1:5 split ratio. Cells transfected with neomycin-resistant constructs were selected and used, whenever appropriate, in the presence of 0.250 mg of G418 per ml. Nonetheless they were tested for resistance up to 1 mg of G418 per ml.Plasmids. The neo-RepB plasmid (9) encodes a subgenomic dicistronic GBV-B replicon bearing the neomycin phosphotransferase (neo) gene, which confers resistance to G418. The 4-29⌬-neo-RepB mutant construct was obtained by replacing the BamHI-AscI fragment of neo-RepB with an equivalent fragment produced by primer-based mutagenesis with a sense primer spanning the deletion of nucleotides 4 to 29 identified in the resident replicon molecules of the cell line B6/Hep3B, one of the Hep3B-derived cell lines identified in this study. The GBV-B wild-type replicon bearing the -lactamase reporter gene was described previously (9, 18), and the corresponding nucleotide-4-to-29 deletion mutant was constructed with a similar strategy. HCV replicon plasmids were kindly pr...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.