Evaluation of Bystander Infection of Oncolytic Virus using a Medium Flow Integrated 3D In Vitro Microphysiological System.

Replicable oncolytic viruses (OVs) induce tumor cell lysis and release viral progeny. The released progeny virions and cell debris can spread within surrounding tumor cells or blood vessels. These released molecules may also induce bystander damage in additional tumor cells through spreading within surrounding tumor cells or blood vessels. However, this effect has not been clearly demonstrated due to the difficulty of direct observation. Here, the bystander infection of OVs by vessel delivery and selective infection in 3D multicellular tumoroids (MCTs) in an in vitro microphysiological system (MPS) with integrated medium flow is demonstrated. This study uses replicable vesicular stomatitis virus (VSV)-green fluorescence protein (GFP) to identify the location of infection in 3D MCTs. Using this MPS, the oncoselective infection by VSV-GFP and the spreading by delivery of OVs through flow via block-to-block linkage of the primary infected MPS with uninfected 3D MCTs in an integrated MPS is observed. This MPS enables real-time monitoring and various analysis for the bystander infection of OVs. It is expected that the 3D in vitro MPS can be suitable to investigate the oncoselective spreading and bystander infection of OVs.

Detection of Lactate Dehydrogenase Elevating Virus in a Mouse Vivarium Using an Exhaust Air Dust Health Monitoring Program.

Lactate dehydrogenase elevating virus (LDV) continues to be one of the most common contaminants of cells and cell byproducts. As such, many institutions require that tumor cell lines, blood products, and products derived or passaged in rodent tissues are free of LDV as well as other pathogens that are on institutional exclusion lists prior to their use in rodents. LDV is difficult to detect by using a live-animal sentinel health monitoring program because the virus does not reliably pass to sentinel animals. After switching to an exhaust air dust health monitoring system, our animal resources center was able to detect a presumably long-standing LDV infection in a mouse colony. This health monitoring system uses IVC rack exhaust air dust collection media in conjunction with PCR analysis. Ultimately, the source of the contamination was identified as multiple LDV-positive patient-derived xenografts and multiple LDV-positive breeding animals. This case study is the first to demonstrate the use of environmental PCR testing as a method for detecting LDV infection in a mouse vivarium.

Kaposi's Sarcoma-Associated Herpesvirus Infection Modulates the Proliferation of Glioma Stem-Like Cells.

Glioblastoma multiforme is the most lethal malignant brain tumor. Despite many intensive studies, the prognosis of glioblastoma multiforme is currently very poor, with a median overall survival duration of 14 months and 2-year survival rates of less than 10%. Although viral infections have been emphasized as potential cofactors, their influences on pathways that support glioblastoma progression are not known. Some previous studies indicated that human Kaposi's sarcoma-associated herpesvirus (KSHV) was detected in healthy brains, and its microRNA was also detected in glioblastoma patients' plasma. However, a direct link between KSHV infection and glioblastoma is currently not known. In this study, we infected glioblastoma cells and glioma stem-like cells (GSCs) with KSHV to establish an in vitro cell model for KSHV-infected glioblastoma cells and glioma stem-like cells in order to identify virologic outcomes that overlap with markers of aggressive disease. Latently KSHV-infected glioblastoma cells and GSCs were successfully established. Additionally, using these cell models, we found that KSHV infection modulates the proliferation of glioma stem-like cells.

Survival of Frog Virus 3 in Freshwater and Sediment from an English Lake.

Ranaviruses can be transmitted by contaminated water and sediment but must retain infectivity for a sufficient period to reach and infect a susceptible host. To determine the risk a virus represents once it enters the environment, its persistence in that environment must be determined. We evaluated the survival of frog virus 3 (FV3) in water and sediment from an English lake at temperatures of 4, 15, 20, and 30 C over time. The virus survived in both water and sediment; however, survival times were significantly lower in sediment. The virus lost infectivity in both matrices with a rise in temperature. In water, time required for a 90% reduction in virus titer decreased from 34 d at 4 C to 5 d at 30 C. In sediment, required time for a 90% reduction decreased from 10 d at 4 C to 1 d at 30 C. These results can be used to estimate the persistence of FV3 in the environment and indicate that the virus could remain infectious in temperate locations for extended periods during winter.

[Establishment and identification of A new HPV positive esophageal cancer cell line].

In order to explore the relationship between human papilloma virus ( HPV) and upper gastrointestinal cancer(esophageal cancer), An esophageal squamous cell carcinoma(ESCC) tissue was obtained from a 76 year old Chinese female patient from Anyang city, a high-incidence area for esophageal cancer, in China. Transplanted tumor was formed through direct SCID mouse tumorigenicity experiment and cultured monolayer cells were obtained after several passages and screenings Immunofluorescence test, cell growth curve, soft agar assay, chromosome analysis and tissues HE staining were also performed to confirm the epithelial cell origin. Cell DNA STR typing results showed that no three alleles was observed,indicating no contamination of human cells. DNA analysis revealed the presence of HPV type 18 DNA in this cell line. DOLINK test found the E6 protein expression of HPV virus. We concluded that the established cell line is a new esophageal squamous cell-origincarcinoma cell line with HPV DNA positive and expression of viral oncoprotein. It provides new cytologic material for performing etiology studies on the occurrence and development of esophageal cancer.

[Oncolytic properties of some orthopoxviruses, adenoviruses and parvoviruses in human glioma cells].

UNLABELLED: Currently one of the most promising approaches in development of cancer virotherapy is based on the ability of oncolytic viruses to selective infection and lysis of tumor cells. AIM: The goal of the study was to identify and evaluate perspective oncolytic viruses capable of selectively destroying human glioma cells. PATIENTS AND METHODS: Original GB2m, GA14m and GB22m glioma cell cultures derived from patients were used for evaluating in vitro oncolytic activity of some typical orthopoxviruses, adenoviruses and parvoviruses. RESULTS: The oncolytic activity in the human glioma cell models was confirmed for LIVP and WR strains of vaccinia virus, Adel2 and Ad2del strains with deletions within E1B/55K gene and derived from human adenoviruses type 2 and 5, respectively. CONCLUSIONS: We consider these oncolytic viruses as promising agents for the treatment of human malignant glioma.

Therapeutic implications of the enhanced short and long-term cytotoxicity of radiation treatment followed by oncolytic parvovirus H-1 infection in high-grade glioma cells.

The prognosis of malignant brain tumors remains extremely bad in spite of moderate improvements of conventional treatments. A promising alternative approach is the use of oncolytic viruses. Strategies to improve viral toxicity include the combination of oncolytic viruses with standard therapies. Parvovirus H-1 (H-1PV) is an oncolytic virus with proven toxicity in glioma cells. Recently it has been demonstrated that the combination of ionizing radiation (IR) with H-1PV showed promising results. Previously irradiated glioma cells remained fully permissive for H-1PV induced cytotoxicity supporting the use of H-1PV for recurrent gliomas, which typically arise from irradiated cell clones. When glioma cells were infected with H-1PV shortly (24 h) after IR, cell killing improved and only the combination of both treatments lead to complete long-term tumor cell killing. The latter finding raises the question whether IR in combination with H-1PV exerts an additional therapeutic effect on highly resistant glioma stem cells. A likely translation into current clinical treatment protocols is to use stereotactic radiation of non-resectable recurrent gliomas followed by intratumoral injection of H-1PV to harvest the synergistic effects of combination treatment.

Epstein-Barr virus infection leads to partial phenotypic reversion of terminally differentiated malignant B cells.

The B cell lymphomas associated with Epstein-Barr virus (EBV) are not limited to any specific stage of B cell differentiation but covers widely different B cell phenotypes. In vitro infection of the virus negative tumors with a recombinant EBV strain has provided important insights into virus-tumor interaction. Here, we investigated the interaction between EBV and terminally differentiated tumor derived B cells, namely multiple myeloma (MM). The in vitro EBV infected MM expressed restricted viral latency. Acquisition of the virus was accompanied by a partial reprogramming to a mature B cell phenotype. Thus, the plasma cell markers syndecan-1 (CD138), Blimp1 and MUM1 were downregulated, while expression of HLADR, CIITA and TCL1, which are normally not expressed in plasmacytoid cells, was upregulated. The silenced transcription factor gene encoding Pax5 and its target BLNK were activated. Significantly, the free lambda light chains secreted in the medium were reduced in EBV infected MM clones. Collectively, these results suggest that the restricted EBV latency can cause at least partial phenotypic reversion of terminally differentiated B tumor cells. We suggest that the restricted EBV latent gene expression may not only be the consequence but the cause of the mature B cell phenotype, actively participating in the virus persistence.

The in vitro and in vivo evaluation of ddC as a topical antiviral for ocular adenovirus infections.

PURPOSE: To evaluate the antiviral activity of 2', 3'-dideoxycytidine (ddC) in vitro against a panel of ocular adenovirus serotypes and in vivo in the ocular Ad5/NZW rabbit replication model. METHODS: In vitro, the 50% inhibitory concentrations (IC(50)) of ddC and cidofovir were determined using standard plaque-reduction assays. In vivo, 40 rabbits were topically inoculated in both eyes with Ad5 after corneal scarification. On day 1, the rabbits were equally divided into four topical treatment groups: 3% ddC; 2% ddC; 0.5% cidofovir; and saline. ddC and saline eyes were treated four times daily for 7 days, and cidofovir-treated eyes were treated twice daily for 7 days. Eyes were cultured for virus a multiple times over 2 weeks. RESULTS: The in vitro IC(50) for ddC ranged from 0.18 to 1.85 microg/mL, whereas those for cidofovir ranged from 0.018 to 5.47 microg/mL. ddC was more potent than cidofovir for seven of nine serotypes. In vivo, 3% ddC, 2% ddC, and 0.5% cidofovir significantly reduced the number of Ad5-positive cultures per total (days 1-14), mean Ad5 ocular titer (days 1-5), and duration of shedding (among other outcome measures) compared with the saline control. The 3% and 2% ddC treatments were significantly more efficacious than the 0.5% cidofovir treatment in the parameters listed above. CONCLUSIONS: ddC demonstrated potent antiadenovirus activity in vitro and in vivo. Systemic safety studies after topical ocular administration are needed to evaluate ddC as a topical antiviral treatment for adenoviral ocular infections in the target population.

Oncolytic herpes simplex virus type-1 therapy in a highly infiltrative animal model of human glioblastoma.

We have examined the spread and antitumor efficacy of an oncolytic herpes simplex virus-1-based vector (G207) in glioblastoma biopsy spheroids in vitro and in vivo after local delivery to corresponding intracranial xenografts. Spheroids from three patients were infected with increasing doses of G207 and transgene expression was quantified. Other infected spheroids were followed for 10 days to assess cytotoxic effects. For the in vivo study, spheroids were grafted intracerebrally into Rowett nude rats. The resulting highly infiltrative xenografts were injected with 3.4 x 10(6) plaque-forming units (penetration study) or 6.8 x 10(6) plaque-forming units (therapeutic study) of G207 using microprocessor-controlled stereotaxic delivery. Vector spread was tracked by histochemical staining. In the therapeutic study, tumor volumes were monitored weekly by magnetic resonance imaging, and survival data were collected. In vitro, lacZ expression was seen at the spheroid surfaces 24 h postinfection, whereas the spheroid cores were transgene positive after 96 h. Cytotoxic susceptibility varied between the patients, showing a 36% to 95% lysis 10 days postinfection. Local delivery of G207 into intracranial xenografts resulted in extensive vector spread throughout the lesions. In the therapeutic study, G207 application reduced tumor volumes compared with controls, but did not significantly improve survival of the animals. Histologic analysis revealed infection of host structures such as the ventricular and choroid plexus ependyma. In conclusion, G207 replicates in patient-derived glioblastoma multiforme xenografts and tumor volumes are reduced after intratumoral delivery; however, the survival data suggest that the therapeutic effect could be improved by repeated vector application or through combination with other treatment modalities.

Herpes oncolytic therapy of salivary gland carcinomas.

Oncolytic herpes simplex viruses (HSV) have demonstrated potent antitumoral effects against a variety of human malignancies in preclinical studies and are in early clinical trials. We explored the activity of an attenuated, replication-competent, oncolytic HSV (NV1023) for the treatment of human salivary gland carcinomas. NV1023 was able to successfully enter into 4 mucoepidermoid carcinoma (H292, H3118, HTB-41, UT-MUC-1) and 2 adenocarcinoma (HSY, HSG) cell lines, as measured by lacZ assays after exposure to 5 viral particles per cell (MOI 5). Viral plaque assays showed variation of viral replication within these cell lines, ranging from a 268-fold increase (H292) to a 3-fold increase (HSG) in viral titer. At MOI 5, all cell lines showed >95% cytotoxicity from NV1023 by Day 7, except for HSY (73%). At MOI 0.1, H3118 and UT-MUC-1 remained highly sensitive to NV1023, both showing >95% cytotoxicity by Day 7. The mucoepidermoid carcinomas were more sensitive to NV1023 at low viral concentrations compared with the adenocarcinomas. Flank tumors of H3118, HTB-41 and HSY in nude mice showed significant tumor volume reductions after a single intratumoral injection of NV1023 (2 x 10(7) plaque-forming units). These data suggest that oncolytic herpes viruses have significant efficacy entering, replicating within, and lysing human salivary gland carcinomas. These promising biologic agents should be further investigated as novel therapy for patients with salivary carcinomas failing conventional treatment.

Viral antigen production in cell cultures on microcarriers Bovine parainfluenza 3 virus and MDBK cells.

Viral antigens can be obtained from infected mammalian cells cultivated on microcarriers. We have worked out parameters for the production of bovine parainfluenza 3 (PI-3) virus by Mandin-Darby Bovine Kidney (MDBK) cells cultivated on Cytodex 1 microcarriers (MCs) in spinners flasks and bioreactor using fetal bovine serum (FBS) supplemented Eagle minimal essential medium (Eagle-MEM). Medium renewal during the cell culture was shown to be crucial for optimal MCs loading (>90% MCs with confluent cell monolayers) and cell growth (2.5 x 10(6)cells/mL and a micro(x) (h(-1)) 0.05). Since cell cultures performed with lower amount of MCs (1g/L), showed good performances in terms of cell loading, we designed batch experiments with a lower concentration of MCs in view of optimizing the cell growth and virus production. Studies of cell growth with lower concentrations of MCs (0.85 g/L) showed that an increase in the initial cell seeding (from 7 to 40 cells/MC) led to a different kinetic of initial cell growth but to comparable final cell concentrations ((8-10)x10(5)cells/mL at 120 h) and cell loading (210-270 cells/MC). Upon infection with PI-3 virus, cultures showed a decrease in cell growth and MC loading directly related to the multiplicity of infection (moi) used for virus infection. Infected cultures showed also a higher consumption of glucose and production of lactate. The PI-3 virus and PI-3 antigen production among the cultures was not significantly different and attained values ranging from, respectively, 7-9 log(10) TCID(50)/mL and 1.5-2.2 OD. The kinetics of PI-3 virus production showed a sharp increase during the first 24h and those of PI-3 antigen increased after 24h. The differential kinetics of PI-3 virus and PI-3 antigen can be explained by the virus sensitivity to temperature. In view of establishing a protocol of virus production and based on the previous experiments, MDBK cell cultures performed under medium perfusion in a bioreactor of 1.2L were infected and the PI-3 virus production in 12L attained 12 log(10) TCID(50). Other than establishing a protocol for PI-3 production in MDBK cell cultures on Cytodex 1, the experiments are proposed as a basis for approaching the development of a virus production protocol in mammalian cells cultivated on microcarriers in bioreactors.

Expression of CCR9 in HTLV-1+ T cells and ATL cells expressing Tax.

Adult T-cell leukemia (ATL) is a highly aggressive mature CD4+ T-cell malignancy that is etiologically associated with human T-lymphotropic virus Type 1 (HTLV-1). ATL is characterized by frequent infiltration of lymph nodes, spleen, liver, skin and gut. Previously, we and others have shown that the majority of ATL cases are strongly positive for CCR4, which may explain the frequent skin invasion of ATL. Here, we examined whether ATL cells express CCR9, which is involved in T-cell homing to the gastrointestinal tract. Human T cell lines carrying HTLV-1 consistently expressed CCR9 together with the HTLV-1-encoded transcriptional activator Tax. Although ATL cells freshly isolated from peripheral blood hardly expressed CCR9, ATL cells cultured for 1 day consistently expressed CCR9 in parallel with the upregulation of Tax. Induction of Tax by Cd2+ in JPX-9, a subline of Jurkat human T cell line carrying Tax under the control of metallothionein promoter, led to upregulation of CCR9. A luciferase reporter gene under the control of the CCR9 promoter was expressed by cotransfection of an expression vector for Tax or in Cd2+-treated JPX-9 cells. Furthermore, immunohistochemical staining demonstrated that ATL cells infiltrating gastrointestinal tract were frequently positive for CCR9. Collectively, CCR9 is inducible in ATL cells expressing Tax and may play a role in the gastrointestinal involvement of ATL.

Redox modulation of the hepatitis C virus replication complex is calcium dependent.

Reactive species and perturbation of the redox balance have been implicated in the pathogenesis of many viral diseases, including hepatitis C. Previously, we made a surprising discovery that concentrations of H(2)O(2) that are nontoxic to host cells disrupted the hepatitis C virus (HCV) replication complex (RC) in Huh7 human hepatoma cells in a manner that suggested signaling. Here, we show that H(2)O(2) and interferon-gamma have comparable effects on the HCV subgenomic and genomic RNA replication in Huh7 cells. H(2)O(2) induced a gradual rise in the intracellular calcium concentration ([Ca(2+)](i)). Both rapid and sustained suppression of HCV RNA replication by H(2)O(2) depended on this calcium elevation. The peroxide-induced [Ca(2+)](i) elevation was independent of extracellular calcium and derived, at least in part, from the endoplasmic reticulum. Likewise, the suppression of the HCV RC by H(2)O(2) was independent of extracellular calcium but required an intracellular calcium source. Other agents that elevated [Ca(2+)](i) could also suppress the HCV RC, suggesting that calcium elevation might be sufficient to suppress HCV RNA replication. In conclusion, oxidants may modulate the HCV RC through calcium. Effects on the infectivity and the morphogenesis of HCV remain to be determined. These findings suggest possible regulatory roles for redox and calcium signaling during viral infections.

Differentially regulated interferon response determines the outcome of Newcastle disease virus infection in normal and tumor cell lines.

Newcastle disease virus (NDV) is a negative-strand RNA virus with oncolytic activity against human tumors. Its effectiveness against tumors and safety in normal tissue have been demonstrated in several clinical studies. Here we show that the spread of NDV infection is drastically different in normal cell lines than in tumor cell lines and that the two cell types respond differently to beta interferon (IFN-beta) treatment. NDV rapidly replicated and killed HT-1080 human fibrosarcoma cells but spread poorly in CCD-1122Sk human skin fibroblast cells. Pretreatment with endogenous or exogenous IFN-beta completely inhibited NDV replication in normal cells but had little or no effect in tumor cells. Thus, the outcome of NDV infection appeared to depend on the response of uninfected cells to IFN-beta. To investigate their differences in IFN responsiveness, we analyzed and compared the expression and activation of components of the IFN signal transduction pathway in these two types of cells. The levels of phosphorylated STAT1 and STAT2 and that of the ISGF3 complex were markedly reduced in IFN-beta-treated tumor cells. Moreover, cDNA microarray analysis revealed significantly fewer IFN-regulated genes in the HT-1080 cells than in the CDD-1122Sk cells. This finding suggests that tumor cells demonstrate a less-than-optimum antiviral response because of a lesion in their IFN signal transduction pathway. The rapid spread of NDV in HT-1080 cells appears to be caused by their deficient expression of anti-NDV proteins upon exposure to IFN-beta.

Curcumin targets Akt cell survival signaling pathway in HTLV-I-infected T-cell lines.

The Akt signaling pathway is important for survival and growth of cancer cells. In the present paper we show that the Akt signaling pathway is constitutively activated in human T-cell leukemia virus type I (HTLV-I)-infected T-cell lines and in primary adult T-cell leukemia (ATL) cells. Curcumin, a natural compound present in turmeric, has been studied vigorously as a potent chemopreventive agent for cancer therapy because of its inhibitory effect on proliferation and induction of apoptosis in several tumor cell lines. We investigated the effect of curcumin on Akt activity in HTLV-I-infected T-cell lines and primary ATL cells. Phosphorylated PDK1 is an activator of Akt by phosphorylating Akt. Curcumin reduced phosphorylation of PDK1 and inhibited constitutive activation of Akt. Curcumin activated glycogen synthase kinase (GSK)-3beta, a downstream target of Akt kinase, by inhibiting phosphorylation of this protein. Curcumin reduced the expression of cell cycle regulators, cyclin D1 and c-Myc proteins, which are both degraded by activated GSK-3beta. Our results suggest that activation of the Akt signaling pathway plays an important role in ATL cell survival, and that curcumin may have anti-ATL properties mediated, at least in part, by inhibiting Akt activity. We propose that Akt-targeting agents could be useful for the treatment of ATL. In this regard, curcumin is a potentially promising compound for the treatment of ATL.

Treatment of cholangiocarcinoma with oncolytic herpes simplex virus combined with external beam radiation therapy.

Replication-competent oncolytic herpes simplex viruses (HSV), modified by deletion of certain viral growth genes, can selectively target malignant cells. The viral growth gene gamma(1)34.5 has significant homology to GADD34 (growth arrest and DNA damage protein 34), which promotes cell cycle arrest and DNA repair in response to stressors such as radiation (XRT). By upregulating GADD34, XRT may result in greater oncolytic activity of HSV strains deficient in the gamma(1)34.5 gene. The human cholangiocarcinoma cell lines KMBC, SK-ChA-1 and YoMi were treated with NV1023, an oncolytic HSV lacking one copy of gamma(1)34.5. Viral proliferation assays were performed at a multiplicity of infection (MOI, number of viral particles per tumor cell) equal to 1, either alone or after XRT at 250 or 500 cGy. Viral replication was assessed by plaque assay. In vitro cytotoxicity assays were performed using virus at MOIs of 0.01 and 0.1, with or without XRT at 250 cGy and cell survival determined with lactate dehydrogenase assay. Established flank tumors in athymic mice were treated with a single intratumoral injection of virus (10(3) or 10(4) plaque forming units), either alone or after a single dose of XRT at 500 cGy, and tumor volumes measured. RT-PCR was used to measure GADD34 mRNA levels in all cell lines after a single dose of XRT at 250 or 500 cGy. NV1023 was tumoricidal in all three cell lines, but sensitivity to the virus varied. XRT enhanced viral replication in vitro in all cell lines. Combination treatment with low-dose XRT and virus was highly tumoricidal, both in vitro and in vivo. The greatest tumor volume reduction with combination therapy was seen with YoMi cells, the only cell line with increased GADD34 expression after XRT and the only cell line in which a synergistic treatment effect was suggested. In KMBC and SK-ChA-1 cells, neither of which showed increased GADD34 expression after XRT, tumor volume reduction was less pronounced and there was no suggestion of a synergistic effect in either case. Oncolytic HSV are effective in treating human cholangiocarcinoma cell lines, although sensitivity to virus varies. XRT-enhanced viral replication occurs through a mechanism that is not necessarily dependent on GADD34 upregulation. However, XRT-induced upregulation of GADD34 further promotes tumoricidal activity in viral strains deficient in the gamma(1)34.5 gene, resulting in treatment synergy; this effect is cell type dependent. Combined XRT and oncolytic viral therapy is a potentially important treatment strategy that may enhance the therapeutic ratios of both individual therapies.

Targeting of autonomous parvoviruses to colon cancer by insertion of Tcf sites in the P4 promoter.

The Wnt signaling pathway is activated by mutations in the adenomatous polyposis coli (APC) or beta-catenin genes in most colon cancers, leading to the transactivation of promoters containing binding sites for the Tcf/LEF family of transcription factors. We have previously shown that it is possible to confer colon cancer specificity on autonomous parvoviruses by inserting Tcf sites into the viral P4 promoter. The mutant Tcf promoters were responsive to activation of the Wnt pathway but the viruses replicated poorly. We show here that reduction of the number of Tcf sites from four to two leads to an increase in the efficiency of replication and toxicity of the viruses in Co115 colon cancer cells, with only a small reduction in selectivity for cells with an active Wnt signaling pathway. Despite this improvement, virus production by most colon cancer cells remained low. Analysis of parental phH1 virus infection of SW480 colon cancer cells showed that the nonstructural and capsid proteins were expressed, but single stranded DNA and progeny virus were not produced. This defect reflects the dependence of autonomous parvoviruses on host functions for many steps in their replication cycle and represents a major limitation to the use of selectively replicating parvoviruses for colon cancer therapy.

Conjugation of lentivirus to paramagnetic particles via nonviral proteins allows efficient concentration and infection of primary acute myeloid leukemia cells.

Nonviral producer cell proteins incorporated into retroviral vector surfaces profoundly influence infectivity and in vivo half-life. We report the purification and concentration of lentiviral vectors using these surface proteins as an efficient gene transduction strategy. Biotinylation of these proteins and streptavidin paramagnetic particle concentration enhances titer 400- to 2,500-fold (to 10(9) CFU/ml for vesicular stomatitis virus G protein and 5 x 10(8) for amphotropic murine leukemia virus envelope). This method also uses newly introduced membrane proteins (B7.1 and DeltaLNGFR) directed to lentiviral surfaces, allowing up to 17,000-fold concentrations. Particle conjugation of lentivirus allows facile manipulation in vitro, resulting in the transduction of 48 to 94% of human acute myeloid leukemia blasts.

Development of cell culture (MDCK) live cold-adapted (CA) attenuated influenza vaccine.

Optimal conditions are determined for growing cold-adapted reassortant strains of a live influenza vaccine in MDCK cell line cultivated in a fermenter with a serum-free medium and microcarriers. The studied MDCK cell line meet all national and WHO requirements for the finite cell lines used for the production of biological preparations. CA reassortant vaccine strains grown in such conditions which fully preserve its mutations and the mutations lead to amino acid substitution in all genome segments of the studied CA reassortants. Under optimal cultivation conditions, the output of a monovalent live CA influenza vaccine in a 10-l fermenter may reach 100,000 doses.