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PubMed1995

Role of the pyrimidine-rich tract on the translation of 'chimeric' polio-hepatitis A mRNAs with engineered 5'-terminal untranslated regions.

The 5'-terminal untranslated region (5'-UTR) of picornavirus RNA contains a series of cis-acting elements required for the internal initiation of translation, including a pyrimidine-rich tract (PRT), which in entero- and rhinoviruses is located about 20 nts upstream from a silent AUG triplet in the vicinity of the translation initiation site. In hepatitis A virus (HAV) RNA, the PRT is only 12 nts upstream from the legitimate AUG initiation codon, and a second, longer PRT in a region far removed from the translation initiation site. This 5'-distal PRT includes a 'core' sequence 80% homologous to the PRT of poliovirus RNA. A 'chimeric' polio-hepatitis A mRNA was constructed in which the sequences extending between nucleotides 45 and 156 of HAV RNA replaced the corresponding ones in poliovirus 5'-UTR. The construction was extended with poliovirus sequences up to position 1809. The recombinant mRNA so generated carried two copies of the PRT. In vitro translation in lysates of truncated poliovirus mRNAs generated a single peptide of Mr = 39 kDa, while the chimeric mRNA generated a series of short peptides as a result of fortuitous (or aberrant) initiation events. A more extensive substitution in the chimeric 5'-UTR which removed the 3'-most PRT brought by the poliovirus sequences, restored the translation at the authentic initiation site. Point mutations were engineered in the 5'-most PRT of the chimeras, and bi-cistronic plasmids were constructed in which either the parental poliovirus 5'-UTR or the chimeric ones were introduced in the intergenic region. Upon transfection of COS-1 cells, the chimeric polio-hepatitis A 5'-UTR containing two PRTs did not express the reporter gene. Removal of the 3'-distal PRT or point mutations engineered into the 5'-most PRT partially restored the transient expression of the reporter gene, consistent with the notion that a single (and only a single) functionally active PRT in a proper context is required to secure the internal initiation of translation of bi-cistronic mRNAs in vivo.

Base Sequence↗
PubMed1995

Characterization of a murine-human chimeric antibody with specificity for the pre-S2 surface antigen of hepatitis B virus expressed in baculovirus-infected insect cells.

We have produced and characterized a murine-human chimeric antibody with specificity for the pre-S2 surface antigen of hepatitis B virus (HBV) in baculovirus-infected insect cells. Recombinant baculovirus carrying the cDNA coding for the heavy or light chain of the chimeric antibody was constructed and co-infected into insect cells. The chimeric antibody (BV-S2) expressed in the cells was purified by an affinity chromatography on Protein A-Sepharose 4B column and characterized by N-terminal amino acid sequencing, affinity determination for pre-S2 peptide, endoglycosidase digestion and Clq binding assay, which were then compared with those of the chimeric antibody H69K that has the same amino acid sequence as BV-S2, but produced from transfected murine myeloma cells. The N-linked glycosylation of the BV-S2 antibody was also analyzed by culturing the baculovirus-infected cells in the presence of tunicamycin. The results showed that the BV-S2 was secreted following correct removal of the leader peptides, contained N-linked carbohydrate at the heavy chain, and had the same binding affinity and Clq binding ability as H69K, suggesting that the BV-S2 chimeric antibody is functional and thus may be useful in the prevention of HBV infection.

Animals↗
PubMed1990

Anti-human interleukin 2 receptor monoclonal antibody isotypic switching: chimeric rat-human antibodies.

Univalent and bivalent (2Fab'-Fc) chimeric rat-human antibodies were constructed by chemical coupling (thioether bonds) of Fab' fragments from the 33B31 rat anti-human interleukin 2 receptor (alpha chain) monoclonal antibody to human IgG or Fc fragments. The purity of the chimeric antibodies obtained after purification was assessed by the sodium dodecyl sulfide-polyacrylamide gel electrophoresis method. The affinity of chimeric antibodies for the human interleukin 2 receptor was determined. The affinity of the 2Fab'-Fc was better (Kd = 0.87 nM) than that of the Fab'-IgG (Kd = 2.04 nM) or the Fab'-Fc chimera (Kd = 3.93 nM). Binding studies on a T-cell clone showed that the percentage of positive cells recognized by chimeric antibodies was comparable to that obtained with unmodified 33B3.1 IgG2a or its corresponding Fab' fragment. In addition, the inhibition of interleukin 2-induced cell proliferation and allogeneic proliferative response by chimeric antibodies was of the same magnitude as that obtained with the rat IgG2a anti-interleukin 2 receptor monoclonal antibody and Fab' fragments. This study shows the possibility of changing the isotype of monoclonal antibodies without important modification to their binding activity. These reagents may offer an alternative to unmodified monoclonal antibodies for therapeutic application.

Animals↗
PubMed1996

Chimeric anti-tenascin antibody 81C6: increased tumor localization compared with its murine parent.

When labeled using the Iodogen method, a chimeric antibody composed of the human IgG2 constant region and the variable regions of murine anti-tenascin 81C6 exhibited superior uptake in human glioma xenografts compared with its murine parent. In the current study, three paired-label experiments were performed in athymic mice with subcutaneous D-54 MG human glioma xenografts to evaluate further the properties of radioiodinated chimeric 81C6. These studies demonstrated that (a) the enhanced tumor uptake of chimeric 81C6 is specific; (b) when labeling was performed using N-succinimidyl 3-iodobenzoate, chimeric 81C6 again showed preferential accumulation in tumor compared with murine 81C6; and (c) the tumor uptake advantage observed previously with murine 81C6 for N-succinimidyl 3-iodobenzoate compared with Iodogen labeling did not occur with chimeric 81C6.

Animals↗
PubMed1995

Production of active anti-CD6 mouse/human chimeric antibodies in the milk of transgenic mice.

The expression of chimeric genes in the mammary gland of transgenic farm animals has become an alternative for the large-scale production of recombinant proteins and for the modification of milk composition. In this paper, we show that a mouse/human chimeric antibody against the human CD6 leukocyte antigen can be assembled and correctly folded by the mammary gland, and secreted to milk, where it maintains its specificity. The base sequences encoding for the heavy and light chain variable regions of the anti-CD6 mouse monoclonal antibody IOR-T1 were cloned by the polymerase chain reaction from hybridoma cDNA, coupled to human heavy and light chain constant region genes, and inserted in a vector containing the 5' regulatory region of the rabbit whey acidic protein gene. Transgenic mice were produced by conventional pronuclei microinjection techniques. Integration and transgene copy number were determined by Southern blot. Assembled human immunoglobulin was detected in milk using a sandwich ELISA. Expression levels of chimeric antibodies in milk were determined to be around 400 micrograms/ml by Western blot, using CHO-derived chimeric IOR-T1 antibodies as reference. The chimeric antibodies produced in milk recognized human peripheral blood T lymphocytes by indirect immunofluorescence, with the classical patch-like pattern of IOR-T1.

Animals↗
PubMed2006

Immunogenicity of a chimeric hepatitis A virus (HAV) carrying the HIV gp41 epitope 2F5.

Its stable particle structure combined with its high immunogenicity makes the hepatitis A virus (HAV) a perfect carrier to expose foreign epitopes to the host immune system. In an earlier report [Beneduce, F., Kusov, Y., Klinger, M., Gauss-Müller, V., Morace, G., 2002. Chimeric hepatitis A virus particles presenting a foreign epitope (HIV gp41) at their surface. Antiviral Res. 55, 369-377] chimeric virus-like particles (HAV-gp41) were described that carried at their surface the dominant gp41 epitope 2F5 (2F5e) of the human immunodeficiency virus HIV-1. Extending this work, we now report that chimeric virus HAV-gp41 replicates in HAV-susceptible cells as well as in non-human primates. Infected marmosets developed both an anti-HAV and anti-2F5 epitope immune response. Furthermore, an HIV-neutralizing antibody response was elicited in guinea pigs immunized with HAV-gp41 chimeric particles. The results demonstrate that the replication-competent chimeric HAV-gp41 can serve as either a live or a subunit vaccine for eliciting of antibodies directed against a foreign antigenic epitope.

AIDS Vaccines↗
PubMed2005

Evaluation and automation of hematopoietic chimerism analysis based on real-time quantitative polymerase chain reaction.

Abstract Chimerism analysis is an essential tool in the follow-up of patients after allogeneic stem cell transplantation. High-resolution methods for chimerism analysis based on real-time quantitative polymerase chain reaction (RQ-PCR) with a detection limit of 0.1% marker-specific cells are especially valuable in the detection of patient-derived subpopulations for the monitoring of minimal residual disease. Using artificial chimeric mixtures of genotypically different cells, we optimized and evaluated the intrasample variation, accuracy, and detection limit of chimerism analysis based on RQ-PCR of short insertion and deletion polymorphisms. Furthermore, automated setup by robot was evaluated. The results were accurate, with acceptable intrasample variation at and above 0.1% marker-specific cells. The sensitivity was mainly limited by background values. Chimerism results based on RQ-PCR were similar to results based on PCR of short tandem repeats when samples from recipients of transplants with nonmyeloablative conditioning were analyzed. Furthermore, automated setup was feasible in a time-, labor-, and reagent-conserving manner.

Evaluation Studies as Topic↗
PubMed2006

Apoptotic donor leukocytes limit mixed-chimerism induced by CD40-CD154 blockade in allogeneic bone marrow transplantation.

"Mini" allogeneic bone marrow transplants using non-myeloablative conditioning have reduced early treatment-related mortalities, but graft-versus-host disease (GVHD) and graft rejection remain clinical problems. Our preliminary studies indicated that low-dose busulfan conditioning and costimulatory blockade using anti-CD154 monoclonal antibody (mAb) in combination with a pretransplantation "tolerating" dose of bone marrow (BM) cells were sufficient to establish stable mixed-chimerism without GVHD when transplanting moderate doses of T cell depleted (TCD)-BM from major histocompatibility complex (MHC) fully-mismatched donors (Adams AB, Durham MM, Kean L, et al. J Immunol. 2001;167:1103-1111). In this study, donor splenocytes were administered before transplantation as a tolerating cell infusion with a conditioning regimen consisting of low-dose busulfan and anti-CD154 mAb. We compared the ability of viable and apoptotic donor cells of different ex vivo treatments and purified different donor cell populations (CD3(+), CD3(-), CD11b(+), and CD11b(-) splenocytes) to induce tolerance and enhance donor chimerism in a MHC mismatched model of murine bone marrow transplantation. We found that mixed chimerism without GVHD was enhanced by pretransplantation administration of viable allogeneic splenocytes and diminished in mice with prior exposure to apoptotic/necrotic donor splenocytes. CD11b(+)-enriched splenocytes more potently enhanced donor chimerism compared to unfractionated splenocytes or other splenocyte subsets. Mixed lymphocyte cultures demonstrated that apoptotic stimulators overcame the immune-tolerating activity of anti-CD154 mAb and led to increased interferon gamma and tumor necrosis factor alpha synthesis, increased proliferation of responder T cells, and decreased production of interleukin-10. In conclusion, viable donor splenocytes administered before transplantation in combination with costimulatory blockade induced tolerance and enhanced donor chimerism, whereas pretransplantation administration of apoptotic/necrotic donor cells led to host T cell activation and decreased overall donor engraftment.

Animals↗
PubMed2005

Hemispheric metacontrol and cerebral dominance in healthy individuals investigated by means of chimeric faces.

Cerebral dominance and hemispheric metacontrol were investigated by testing the ability of healthy participants to match chimeric, entire, or half faces presented tachistoscopically. The two hemi-faces compounding chimeric or entire stimuli were presented simultaneously or asynchronously at different exposure times. Participants did not consciously detect chimeric faces for simultaneous presentations lasting up to 40 ms. Interestingly, a 20 ms separation between each half-chimera was sufficient to induce detection of conflicts at a conscious level. Although the presence of chimeric faces was not consciously perceived, performance on chimeric faces was poorer than on entire- and half-faces stimuli, thus indicating an implicit processing of perceptual conflicts. Moreover, the precedence of hemispheric stimulation over-ruled the right hemisphere dominance for face processing, insofar as the hemisphere stimulated last appeared to influence the response. This dynamic reversal of cerebral dominance, however, was not caused by a shift in hemispheric specialization, since the level of performance always reflected the right hemisphere specialization for face recognition. Thus, the dissociation between hemispheric dominance and specialization found in the present study hints at the existence of hemispheric metacontrol in healthy individuals.

Adult↗
PubMed2006

Hematopoietic chimerism induces renal and skin allograft tolerance in DLA-identical dogs.

OBJECTIVE: Hematopoietic chimerism, a state where donor and recipient bone marrow cells coexist, is associated with donor-specific tolerance. Nonmyeloablative bone marrow transplantation (BMT) has been shown to induce stable mixed hematopoietic chimerism in dog leukocyte antigen (DLA)-matched dogs. The potential for inducing renal and skin allograft tolerance with nonmyeloablative BMT was investigated in DLA-identical and DLA-haploidentical dogs in this study. MATERIALS AND METHODS: Renal allografts were performed in 8 DLA-identical and 4 DLA-haploidentical dogs with nonmyeloablative conditioning (200 cGy TBI) and transient immunosuppression with cyclosporine (CSP) and mycophenolate mofetil (MMF) with (n = 8) and without (n = 4) simultaneous BMT. Skin allografts were performed in 2 DLA-identical and 4 DLA-haploidentical dogs after stopping CSP and MMF. Two DLA-identical control dogs received renal allografts without TBI, BMT, or immunosuppression with CSP and MMF. Molecular chimerism was determined with a PCR-based DNA microsatellite assay. Serum creatinine (Cr) concentration, urine specific gravity, and sequential renal biopsies were monitored to assess renal allograft function. RESULTS: Donor-type blood cells were first detected 4 weeks posttransplantation in both the myeloid and lymphoid lineages. Donor chimerism was present for at least 76 weeks in the DLA-identical dogs. Mixed chimerism was not observed in the DLA-haploidentical dogs or DLA-identical dogs that did not undergo BMT. The renal allografts were acutely rejected within 14 days in the 2 DLA-identical control dogs. There was long-term (> 5 yrs) renal allograft survival as evidenced by a normal (< 2.0 mg/dL) serum Cr concentration in both the DLA-identical and DLA-haploidentical dogs that underwent 200 cGy TBI and transient immunosuppression with CSP and MMF either with or without simultaneous BMT. Renal allograft inflammation was severe in the control dogs, mild to moderate in the DLA-haploidentical dogs, and minimal in the DLA-identical dogs. Donor-specific skin grafts were accepted in the DLA-identical dogs but rejected in the DLA-haploidentical dogs. Nonmyeloablative conditioning (200 cGy TBI) and transient immunosuppression with CSP and MMF induce renal and skin allograft tolerance in DLA-identical and permit long-term renal allograft survival in DLA-haploidentical dogs. These findings suggest it may possible to obtain long-term allograft survival in DLA-identical and -haploidentical dogs without the need for chronic immunosuppressive therapy.

Animals↗
PubMed2005

Hemopoietic chimerism following stem cell transplantation.

Hematopoietic chimerism is a measure of the number of donor and recipient cells in the host following stem cell transplantation (SCT). The type of conditioning therapy prior to SCT has a major impact on the chimeric status in the recipient. Different techniques of measurement have varying sensitivities. The use of polymerase chain reaction (PCR) of short tandem repeats (STR) using fluorescent amplification permits quantification using Genescan analysis. When SCT is used for malignant haematological disorders, measurement of chimeric status may indicate early relapse and in aplastic anemia graft rejection. Reduced intensity or T-cell depletion is associated with mixed haemopoietic chimerism. SCT for benign haematological disorders does not require complete donor chimerism for a successful outcome.

Anemia, Aplastic↗
PubMed2005

Allogeneic hematopoietic stem-cell transplantation, mixed chimerism, and tolerance in living related donor renal allograft recipients.

OBJECTIVE: We designed a prospective, randomized, and controlled clinical trial to evaluate the efficacy and safety of achieving a mixed chimerism-associated tolerance protocol for recipients of living related donor (LRD) renal allografts. PATIENTS AND METHODS: Sixty-six consecutive patients were divided into two equal groups of 33 patients with end-stage renal disease. They were enrolled for transplantation after negative lymphocytotoxicity cross-matching (LCM). Both groups (treated [Tn] and control [Cn]) showed similar clinical and laboratory parameters and donor HLA match profiles. The Tn group underwent thymic transplantation of donor renal tissue, two donor-specific transfusions, low-intensity conditioning, and high-dose hematopoietic stem-cell transplantation (HSCT) before renal transplantation. The conditioning regimen included low-dose, target-specific irradiation (to abdominal and inguinal lymph nodes, bone marrow [BM] from thoracolumbar vertebrae and part of the pelvis on alternate days, 100 rad x 4), anti-T-cell antibodies (1.5 mg/kg body weight [BW]), cyclophosphamide (10 mg/kg BW x 2 consecutive days), and cyclosporine (CyA; >3 mg/kg BW/d). Unfractionated HSCT procured from the donor marrow was administered into the BM, portal and peripheral circulations, within 24 hours of achieving CD 4+/CD 8+ T-cell count less than 10% of normal. This infusion was supplemented with a dose of peripherally mobilized stem cells (mean total dose of 20 x 10(8) cells/kg recipient BW) administered peripherally. Renal transplantation was performed after negative LCM. Donor-specific cytotoxic antibodies were eliminated with intravenous immunoglobulins and plasmapheresis before renal transplantation. Mixed chimerism was evaluated before and after transplantation at monthly intervals in patients with donors of opposite gender by the FISH technique. Both groups received CyA and prednisolone for immunosuppression; Cn subjects also received mycophenolate mofetil/azathioprine. Rejection was treated with standard treatment. Immunosuppression was withdrawn 6 months after renal transplantation for patients with consistently positive chimerism. Clinical tolerance was defined as stable allograft function for more than 100 days without immunosuppression and confirmed by allograft biopsy. RESULTS: Over a mean follow-up of 210 days, all Tn patients showed stable allograft function with mean serum creatinines (SCr) of 1.20 mg/dL, no rejection/CMV infections/graft or patient loss. A low-level donor-specific cytotoxic antibody was observed in all Tn patients. The CyA toxicity was noted in 10 (30.3%) patients. Persistent mixed hematopoietic chimerism was seen in all 21 patients irrespective of donor-recipient HLA matching (mean 0.5% before and 1 +/- 0.3% after transplantation). All four patients on drug withdrawal have shown donor-specific tolerance at a mean follow-up of 129.8 days. Other Tn patients are in the process of being weaned off immunosuppression. Mean SCr of controls was 1.45 mg/dL over a mean follow-up of 216 days. Acute rejection was observed in 17 (51.5%) patients; no CMV infection/patient loss was noted and one (3.03%) graft was lost in controls. No patient was lost in controls. No graft-versus-host disease was observed in Tn patients. CONCLUSION: We have achieved mixed hematopoietic chimerism-associated tolerance with high-dose HSCT, intrathymic donor renal tissue transplantation, and minimal conditioning without any adverse effects.

Adolescent↗
PubMed2004

Lympho-myeloid chimerism achieved by spleen graft of green fluorescent protein transgenic rat in a combined pancreas transplantation model (TI03-029).

BACKGROUND: Multilineage chimerism is a promising strategy to induce donor-specific tolerance. Because the beneficial effect of splenic grafting on tolerance induction is well known, we studied long-term hematopoietic chimerism and the fate of donor-derived cells after allogenic pancreas/spleen transplantation. METHODS: Green fluorescent protein (GFP) transgenic (Tg) Wistar rats were donors and combined pancreas/spleen transplantation (PST) or pancreas transplantation (PT) alone was performed on recipient LEW rats. Graft survival was compared between these two groups and the fate of donor-derived GFP(+) cells was analyzed by flow cytometry. In this system, the donor-derived cells were clearly defined as having lymphocytic or granulocytic lineage by cell size. T-cell subsets of GFP(+) and GFP(-) cells in long graft-surviving rats were also characterized. RESULTS: The survival period of the grafted pancreas in PST rats was significantly longer than that of PT rats (P<0.001). Three of seven PST rats survived >250 days. The chimeric level of donor-derived GFP(+) cells in the recipient peripheral blood was markedly higher in PST rats. In rats with long-surviving grafts, overall peripheral blood chimerism was more than 5%, and both lymphocytes and granulocytes generated from the grafted spleen were stable. T-cell subsets in the recipient LEW rats varied according to the type of cells. CD4(+)CD8(+) subsets decreased in the GFP(+) cells and CD4(-)CD8(+) subsets increased in the GFP(-) (LEW) cells. CONCLUSION: We confirmed the combination effect of the grafted spleen on pancreatic graft survival. Donor lymphocytic and granulocytic lineages were generated in the recipients with long-surviving graft. It suggested that multilineage chimerism was often induced by the spleen graft and protected the pancreatic graft against rejection for a long period.

Animals↗
PubMed2005

Low dose busulfan facilitates chimerism and tolerance in a murine model.

T cell depletion, sirolimus and "mega" dose donor specific bone marrow (DSBM) infusion promotes stable multilineage chimerism and indefinite survival of skin allografts in completely mismatched mice. The purpose of this study is to determine whether the addition of low dose busulfan can reduce the amount of DSBM required while preserving efficacy. C57BL/6 recipients of BALB/c skin allografts were treated with alphaCD4 and alphaCD8 monoclonal antibodies, DSBM, sirolimus and various doses of busulfan. The kinetics and phenotype of chimerism and the presence of clonal deletion of alloreactive T-cells were defined using flow cytometry. In vitro reactivity was determined using mixed lymphocyte culture. Second skin grafts confirmed the presence of tolerance. All doses of busulfan resulted in engraftment when combined with this regimen using a reduced dose of donor marrow. The level, kinetics and character of chimerism observed were dose related. Chimerism was associated with indefinite allograft acceptance (>200 days). Tolerance was documented both in vitro/in vivo and was associated with clonal deletion. Addition of a single low dose of busulfan to an established tolerance protocol reduced the required DSBM dose by over 80% while still promoting comparable levels of donor chimerism and donor-specific tolerance.

Animals↗
PubMed2006

Chimeric West Nile/dengue virus vaccine candidate: preclinical evaluation in mice, geese and monkeys for safety and immunogenicity.

A live attenuated virus vaccine is being developed to protect against West Nile virus (WN) disease in humans. Previously, it was found that chimeric West Nile/dengue viruses (WN/DEN4 and WN/DEN4Delta30) bearing the membrane precursor and envelope protein genes of WN on a backbone of dengue type 4 virus (DEN4) with or without a deletion of 30 nucleotides (Delta30) in the 3' noncoding region of the DEN4 part of the chimeric genome were attenuated and efficacious in mice and monkeys against WN challenge. Here, we report the generation of a clinical lot of WN/DEN4Delta30 virus and its further preclinical evaluation for safety and immunogenicity in mice, geese and monkeys. The vaccine candidate had lost neuroinvasiveness in highly sensitive immunodeficient mice inoculated intraperitoneally and had greatly reduced neurovirulence in suckling mice inoculated intracerebrally (IC). Compared to the wild-type WN parent, the chimeric virus was highly restricted in replication in both murine and human neuroblastoma cells as well as in brains of suckling mice. The WN/DEN4Delta30 virus failed to infect geese, indicating that chimerization of WN with DEN4 completely attenuated WN for this avian host. This observation suggests that the WN/DEN4 chimeric viruses would be restricted in their ability to be transmitted from vaccinees to domestic or wild birds. In monkeys, the WN/DEN4Delta30 vaccine candidate was highly immunogenic despite its low level of replication with undetectable viremia. Furthermore, the WN/DEN4Delta30 vaccine virus was safe and readily induced neutralizing antibodies against WN in monkeys immune to each of the four serotypes of dengue virus. These studies confirm the attenuation of WN/DEN4Delta30 for non-human primates, including dengue-immune monkeys, and demonstrate both a highly restricted replication (>10(8)-fold decrease) in the brain of mice inoculated IC and an absence of infectivity for birds, findings that indicate this vaccine should be safe for both the recipient and the environment.

Animals↗
PubMed2004

An avirulent chimeric Pestivirus with altered cell tropism protects pigs against lethal infection with classical swine fever virus.

A chimeric Pestivirus was constructed using an infectious cDNA clone of bovine viral diarrhea virus (BVDV) [J. Virol. 70 (1996) 8606]. After deletion of the envelope protein E2-encoding region, the respective sequence of classical swine fever virus (CSFV) strain Alfort 187 was inserted in-frame resulting in plasmid pA/CP7_E2alf. After transfection of in vitro-transcribed CP7_E2alf RNA, autonomous replication of chimeric RNA in bovine and porcine cell cultures was observed. Efficient growth of chimeric CP7_E2alf virus, however, could only be demonstrated on porcine cells, and in contrast to the parental BVDV strain CP7, CP7_E2alf only inefficiently infected and propagated in bovine cells. The virulence, immunogenicity, and "marker vaccine" properties of the generated chimeric CP7_E2alf virus were determined in an animal experiment using 27 pigs. After intramuscular inoculation of 1 x 10(7) TCID(50), CP7_E2alf proved to be completely avirulent, and neither viremia nor virus transmission to contact animals was observed; however, CSFV-specific neutralizing antibodies were detected from day 11 after inoculation. In addition, sera from all animals reacted positive in an E2-specific CSFV-antibody ELISA, but were negative for CSFV-E(RNS)-specific antibodies as determined with a CSFV marker ELISA. After challenge infection with highly virulent CSFV strain Eystrup, pigs immunized with CP7_E2alf were fully protected against clinical signs of CSFV infection, viremia, and shedding of challenge virus, and almost all animals scored positive in a CSFV marker ELISA. From our results, we conclude that chimeric CP7_E2alf may not only serve as a tool for a better understanding of Pestivirus attachment, entry, and assembly, but also represents an innocuous and efficacious modified live CSFV "marker vaccine".

Animals↗
PubMed2005

Analysis of varicella zoster virus attenuation by evaluation of chimeric parent Oka/vaccine Oka recombinant viruses in skin xenografts in the SCIDhu mouse model.

Varicella-zoster virus (VZV) is the only human herpes virus for which a vaccine has been licensed. A clinical VZV isolate, designated the parent Oka (pOka) strain was passed in human and non-human fibroblasts to produce vaccine Oka (vOka). The pOka and vOka viruses exhibit similar infectivity in cultured cells but healthy susceptible individuals given vaccines derived from vOka rarely develop the cutaneous vesicular lesions characteristic of varicella. Inoculation of skin xenografts in the SCIDhu mouse model of VZV pathogenesis demonstrated that vOka had a reduced capacity to replicate in differentiated human epidermal cells in vivo (Moffat, J.F., Zerboni, L., Kinchington, P.R., Grose, C., Kaneshima, H., Arvin A.M., 1998a. Attenuation of the vaccine Oka strain of varicella-zoster virus and role of glycoprotein C in alphaherpesvirus virulence demonstrated in the SCID-hu mouse. J Virol. 72:965-74). In order to investigate the attenuation of vOka in skin, we made chimeric pOka and vOka recombinant viruses from VZV cosmids. Six chimeric pOka/vOka viruses were generated using cosmid sets that incorporate linear overlapping fragments of VZV DNA from cells infected with pOka or vOka. The cosmid sets consist of pOka and vOka DNA segments that have identical restriction sites. As expected, the growth kinetics and plaque morphologies of the six chimeric pOka/vOka viruses were indistinguishable in vitro. However, the chimeric viruses exhibited varying capacities to replicate when evaluated in skin xenografts in vivo. The presence of ORFs 30-55 from the pOka genome was sufficient to maintain wild-type infectivity in skin. Chimeric viruses containing different vOka components retained the attenuation phenotype, suggesting that vOka attenuation is multi-factorial and can be produced by genes from different regions of the vOka genome.

Animals↗
PubMed2003

Increased chimerism of bronchial and alveolar epithelium in human lung allografts undergoing chronic injury.

Chimerism on the parenchymal level has been shown for several human allografts, including liver, heart, and kidney, with the integrated recipient-derived cells most likely originating from multipotent bone marrow precursors. We investigated whether chimerism also occurs within epithelial structures of the lung. For this purpose archival tissue biopsies from seven explanted human lung allografts were obtained. We performed laser microdissection of the target structures with subsequent short tandem repeat analysis to detect chimerism within the isolated cells. We found integration of recipient-derived cells in the bronchial epithelium, in type II pneumocytes and in seromucous glands lying adjacent to larger bronchi in all lung allografts studied. Quantitative analysis revealed that the epithelial structures displaying signs of chronic injury, such as squamous metaplasia, showed a markedly higher degree of chimerism (24% versus 9.5%). We therefore conclude that in human lungs, epithelial chimerism occurs at least within bronchi, type II pneumocytes, and seromucous peribronchial glands. Although a bone marrow origin of immigrating host-derived stem cells has been suggested by previous studies in rodents, analysis of lung biopsies from bone marrow-transplanted patients (n = 3) could not prove such delineation in this study. The observation of an enhanced integration of recipient cells into chronically damaged epithelial structures suggests that extrapulmonary precursor cells are able to contribute to pulmonary regeneration.

Adolescent↗