Current therapy for multiple myeloma is complex and prolonged. Antimyeloma drugs are combined in induction, consolidation and/or maintenance protocols to destroy bulky disease, then suppress or eradicate residual disease. Oncolytic viruses have the potential to mediate both tumor debulking and residual disease elimination, but this curative paradigm remains unproven. Here we engineered an oncolytic vesicular stomatitis virus to minimize its neurotoxicity, enhance induction of antimyeloma immunity, and facilitate noninvasive monitoring of its intratumoral spread. Using high resolution imaging, autoradiography and immunohistochemistry, we demonstrate that the intravenously administered virus extravasates from tumor blood vessels in immunocompetent myeloma-bearing mice, nucleating multiple intratumoral infectious centers which expand rapidly and necrose at their centers, ultimately coalescing to cause extensive tumor destruction. This oncolytic tumor debulking phase lasts only for 72 hours after virus administration, and is completed before antiviral antibodies become detectable in the bloodstream. Anti-myeloma T cells, cross-primed as the virus-infected cells provoke an antiviral immune response, then eliminate residual uninfected myeloma cells. The study establishes a curative oncolytic paradigm for multiple myeloma where direct tumor debulking and immune eradication of minimal disease are mediated by a single intravenous dose of a single therapeutic agent. Clinical translation is underway.

Background

Mesenchymal stem cells (MSC) can serve as carriers to deliver oncolytic measles virus (MV) to ovarian tumors. In preparation for a clinical trial to use MSC as MV carriers, we obtained cells from ovarian cancer patients and evaluated feasibility and safety of this approach.

Methods

MSC from adipose tissues of healthy donors (hMSC) and nine ovarian cancer patients (ovMSC) were characterized for susceptibility to virus infection and tumor homing abilities.

Results

Adipose tissue (range 0.16-3.96 grams) from newly diagnosed and recurrent ovarian cancer patients yielded about 7.41×106 cells at passage 1 (range 4–9 days). Phenotype and doubling times of MSC were similar between ovarian patients and healthy controls. The time to harvest of 3.0×108 cells (clinical dose) could be achieved by day 14 (range, 9–17 days). Two of nine samples tested had an abnormal karyotype represented by trisomy 20. Despite receiving up to 1.6×109 MSC/kg, no tumors were seen in SCID beige mice and MSC did not promote the growth of SKOV3 human ovarian cancer cells in mice. The ovMSC migrated towards primary ovarian cancer samples in chemotaxis assays and to ovarian tumors in athymic mice. Using non-invasive SPECT-CT imaging, we saw rapid co-localization, within 5–8 minutes of intraperitoneal administration of MV infected MSC to the ovarian tumors. Importantly, MSC can be pre-infected with MV, stored in liquid nitrogen and thawed on the day of infusion into mice without loss of activity. MV infected MSC, but not virus alone, significantly prolonged the survival of measles immune ovarian cancer bearing animals.

Conclusions

These studies confirmed the feasibility of using patient derived MSC as carriers for oncolytic MV therapy. We propose an approach where MSC from ovarian cancer patients will be expanded, frozen and validated to ensure compliance with the release criteria. On the treatment day, the cells will be thawed, washed, mixed with virus, briefly centrifuged and incubated for 2 hours with virus prior to infusion of the virus/MSC cocktail into patients.

The purpose of our study was to validate the ability of pinhole micro-single-photon emission computed tomography/computed tomography (SPECT/CT) to 1) accurately resolve the intratumoral dispersion pattern and 2) quantify the infection percentage in solid tumors of an oncolytic measles virus encoding the human sodium iodide symporter (MV-NIS). NIS RNA level and dispersion pattern were determined in control and MV-NIS infected BxPC-3 pancreatic tumor cells and mouse xenografts using quantitative, real-time, reverse transcriptase, polymerase chain reaction, autoradiography, and immunohistochemistry (IHC). Mice with BxPC-3 xenografts were imaged with 123I or 99TcO4 micro-SPECT/CT. Tumor dimensions and radionuclide localization were determined with imaging software. Linear regression and correlation analyses were performed to determine the relationship between tumor infection percentage and radionuclide uptake (% injected dose per gram) above background and a highly significant correlation was observed (r2 = 0.947). A detection threshold of 1.5-fold above the control tumor uptake (background) yielded a sensitivity of 2.7% MV-NIS infected tumor cells. We reliably resolved multiple distinct intratumoral zones of infection from noninfected regions. Pinhole micro-SPECT/CT imaging using the NIS reporter demonstrated precise localization and quantitation of oncolytic MV-NIS infection and can replace more time-consuming and expensive analyses (eg, autoradiography and IHC) that require animal sacrifice.

Off target binding or vector sequestration can significantly limit the efficiency of systemic virotherapy. We report here that systemically administered oncolytic measles virus (MV) was rapidly sequestered by the mononuclear phagocytic system (MPS) of the liver and spleen in measles receptor CD46-positive and CD46-negative mice. Since scavenger receptors on Kupffer cells are responsible for the elimination of blood-borne pathogens, we investigated here if MV uptake was mediated by scavenger receptors on Kupffer cells. Pretreatment of cells with poly(I), a scavenger receptor ligand, reduced MV expression by 99% in murine (J774A.1) macrophages and by 50% in human (THP-1) macrophages. Pre-dosing of mice with poly(I) reduced MPS sequestration of MV and increased circulating levels of MV by 4 to 15-folds at 2 minutes post virus administration. Circulating virus was still detectable 30 mins post infusion in mice predosed with poly(I) while no detectable MV was found at 5–10 min post infusion if mice did not receive poly(I). MPS blockade by poly(I) enhanced virus delivery to human ovarian SKOV3ip.1 and myeloma KAS6/1 xenografts in mice. Higher gene expression and improved control of tumor growth was noted early post therapy. Based on these results, incorporation of MPS blockade into MV treatment regimens is warranted.

Gene therapy has held promise to correct various disease processes. Prostate cancer represents the second leading cause of cancer death in American men. A number of clinical trials involving gene therapy for the treatment of prostate cancer have been reported. The ability to efficiently transduce tumors with effective levels of therapeutic genes has been identified as a fundamental barrier to effective cancer gene therapy. The approach utilizing gene therapy in prostate cancer patients at our institution attempts to address this deficiency. The sodium-iodide symporter (NIS) is responsible for the ability of the thyroid gland to transport and concentrate iodide. The characteristics of the NIS gene suggest that it could represent an ideal therapeutic gene for cancer therapy. Published results from Mayo Clinic researchers have indicated several important successes with the use of the NIS gene and prostate gene therapy. Studies have demonstrated that transfer of the human NIS gene into prostate cancer using adenovirus vectors in vitro and in vivo results in efficient uptake of radioactive iodine and significant tumor growth delay with prolongation of survival. Preclinical successes have culminated in the opening of a phase I trial for patients with advanced prostate disease which is currently accruing patients. Further study will reveal the clinical promise of NIS gene therapy in the treatment of prostate as well as other malignancies.

Abstract

Toxicology studies were performed in rats and rhesus macaques to establish a safe starting dose for intratumoral injection of an oncolytic vesicular stomatitis virus expressing human interferon-β (VSV-hIFNβ) in patients with hepatocellular carcinoma (HCC). No adverse events were observed after administration of 7.59 × 109 TCID50 (50% tissue culture infective dose) of VSV-hIFNβ into the left lateral hepatic lobe of Harlan Sprague Dawley rats. Plasma alanine aminotransferase and alkaline phosphatase levels increased and platelet counts decreased in the virus-treated animals on days 1 and 2 but returned to pretreatment levels by day 4. VSV-hIFNβ was also injected into normal livers or an intrahepatic McA-RH7777 HCC xenograft established in Buffalo rats. Buffalo rats were more sensitive to neurotoxic effects of VSV; the no observable adverse event level (NOAEL) of VSV-hIFNβ in Buffalo rats was 107 TCID50. Higher doses were associated with fatal neurotoxicity and infectious virus was recovered from tumor and brain. Compared with VSV-hIFNβ, toxicity of VSV-rIFNβ (recombinant VSV expressing rat IFN-β) was greatly diminished in Buffalo rats (NOAEL, >1010 TCID50). Two groups of two adult male rhesus macaques received 109 or 1010 TCID50 of VSV-hIFNβ injected directly into the left hepatic lobe under computed tomographic guidance. No neurological signs were observed at any time point. No abnormalities (hematology, clinical chemistry, body weights, behavior) were seen and all macaques developed neutralizing anti-VSV antibodies. Plasma interleukin-6, tumor necrosis factor-α, and hIFN-β remained below detection levels by ELISA. On the basis of these studies, we will be proposing a cautious approach to dose escalation in a phase I clinical trial among patients with HCC.

Prostate cancer cells overexpress the measles virus (MV) receptor CD46. Herein, we evaluated the antitumor activity of an oncolytic derivative of the MV Edmonston (MV-Edm) vaccine strain engineered to express the human sodium iodide symporter (NIS; MV-NIS virus). MV-NIS showed significant cytopathic effect (CPE) against prostate cancer cell lines in vitro. Infected cells effectively concentrated radioiodide isotopes as measured in vitro by Iodide-125 (125I) uptake assays. Virus localization and spread in vivo could be effectively followed by imaging of 123I uptake. In vivo administration of MV-NIS either locally or systemically (total dose of 9 × 106 TCID50) resulted in significant tumor regression (P < 0.05) and prolongation of survival (P < 0.01). Administration of 131I further enhanced the antitumor effect of MV-NIS virotherapy (P < 0.05). In conclusion, MV-NIS is an oncolytic vector with significant antitumor activity against prostate cancer, which can be further enhanced by 131I administration. The NIS transgene allows viral localization and monitoring by noninvasive imaging which can facilitate dose optimization in a clinical setting.

Breast cancer is the second leading cause of malignant effusions in cancer patients. Pleural effusion indicates incurable disease with limited palliative treatment options and poor outcome. Here, we demonstrate the therapeutic efficacy of measles virus (MV) vaccine strain derivative against malignant pleural effusion in an MDA-MB-231 xenograft model of advanced breast cancer. Both systemic intravenous (i.v.) and intrapleural (t.t.) administered virus caused massive infection and syncytia formation in the pleural tumor deposits. Intrapleural administration of 1.5 × 106 plaque-forming units (PFU) total dose of MV significantly improved median survival by approximately 80% compared to the control animal group. Furthermore, we tested human dendritic cells as carriers for delivery of oncolytic MV infection to breast cancer pleural metastases. Carrier-delivered MV infection prevented accumulation of the pleural exudate and also significantly improved the survival of the treated mice. This is the first demonstration of the therapeutic potential of oncolytic virotherapy against malignant pleural effusions in a pre-clinical model of advanced breast cancer.

The entry process of the avian sarcoma and leukosis virus (ASLV) family of retroviruses requires first a specific interaction between the viral surface (SU) glycoproteins and a receptor on the cell surface at a neutral pH, triggering conformational changes in the viral SU and transmembrane (TM) glycoproteins, followed by exposure to low pH to complete fusion. The ASLV TM glycoprotein has been proposed to adopt a structure similar to that of the Ebola virus GP2 protein: each contains an internal fusion peptide flanked by cysteine residues predicted to be in a disulfide bond. In a previous study, we concluded that the cysteines flanking the internal fusion peptide in ASLV TM are critical for efficient function of the ASLV viral glycoproteins in mediating entry. In this study, replication-competent ASLV mutant subgroup A [ASLV(A)] variants with these cysteine residues mutated were constructed and genetically selected for improved replication capacity in chicken fibroblasts. Viruses with single cysteine-to-serine mutations reverted to the wild-type sequence. However, viruses with both C9S and C45S (C9,45S) mutations retained both mutations and acquired a second-site mutation that significantly improved the infectivity of the genetically selected virus population. A charged-amino-acid second-site substitution in the TM internal fusion peptide at position 30 is preferred to rescue the C9,45S mutant ASLV(A). ASLV(A) envelope glycoproteins that contain the C9,45S and G30R mutations bind the Tva receptor at wild-type levels and have improved abilities to trigger conformational changes and to form stable TM oligomers compared to those of the C9,45S mutant glycoprotein.

Gliomas have a dismal prognosis, with the median survival of patients with the most common histology, glioblastoma multiforme, being only 12–15 months. Development of novel therapeutic agents is urgently needed. We have previously demonstrated that oncolytic measles virus strains derived from the Edmonston vaccine lineage have significant antitumor activity against gliomas [Phuong, L.K., Allen, C., Peng, K.W., Giannini, C., Greiner, S., Teneyck, C.J., Mishra, P.K., Macura, S.I., Russell, S.J., Galanis, E.C. (2003). Cancer. Res. 63, 2462–2469]. MV-CEA is an Edmonston vaccine lineage measles virus strain engineered to express the marker peptide carcinoembryonic antigen (CEA): CEA levels can serve as a correlate of viral gene expression. In support of a phase I clinical trial of intratumoral and resection cavity administration of MV-CEA to patients with recurrent gliomas, we assessed the neurotoxicity of MV-CEA in adult immune male rhesus macaques (Macaca mulatta). The animals' immune status and administration schedule mimicked the trial population and proposed administration schema. Macaca mulatta represents the prototype animal species for assessment of measles neurotoxicity. The animals were stereotactically administered either vehicle (n = 1) or MV-CEA at 2 × 105 or 2 × 106 TCID50 (each, n = 2) in the right frontal lobe in two injections on days 1 and 5. Macaques were closely monitored clinically for neurotoxicity. Body weight, temperature, complete blood count, CEA, clinical chemistries, coagulation, complement levels, immunoglobulin, measles antibody titers, viremia, and shedding (buccal swabs) were tested at multiple time points. Furthermore, cisterna magna spinal taps were performed on day 9 and 1 year after the first viral dose administration, and samples were analyzed for protein, glucose, cell differential, and presence of MV-CEA. Magnetic resonance imaging (MRI) was performed between 4 and 5 months after article administration to assess for subclinical neurotoxicity. To date, 36+ months from study initiation there has been no clinical or biochemical evidence of toxicity, including lack of neurological symptoms, fever, or other systemic symptoms and lack of immunosuppression. Quantitative RT-PCR analysis of blood, buccal swabs, and cerebrospinal fluid (CSF) was negative for MV-CEA at all time points, with the exception of viral genome deletion in the blood of one asymptomatic animal at the 2 × 106 TCID50 dose level on day 85. Vero cell overlays of CSF cells and supernatant were negative for viral recovery. There was no detection of CEA in serum or CSF at any time point. MRI scans were negative for imaging abnormalities and showed no evidence of encephalitis. Our results support the safety of CNS administration of MV-CEA in glioma patients. A clinical trial of intratumoral and resection cavity administration of MV-CEA in patients with recurrent glioblastoma multiforme is currently ongoing.

Abstract

Gliomas have a dismal prognosis, with the median survival of patients with the most common histology, glioblastoma multiforme, being only 12–15 months. Development of novel therapeutic agents is urgently needed. We have previously demonstrated that oncolytic measles virus strains derived from the Edmonston vaccine lineage have significant antitumor activity against gliomas [Phuong, L.K., Allen, C., Peng, K.W., Giannini, C., Greiner, S., Teneyck, C.J., Mishra, P.K., Macura, S.I., Russell, S.J., Galanis, E.C. (2003). Cancer. Res. 63, 2462–2469]. MV-CEA is an Edmonston vaccine lineage measles virus strain engineered to express the marker peptide carcinoembryonic antigen (CEA): CEA levels can serve as a correlate of viral gene expression. In support of a phase I clinical trial of intratumoral and resection cavity administration of MV-CEA to patients with recurrent gliomas, we assessed the neurotoxicity of MV-CEA in adult immune male rhesus macaques (Macaca mulatta). The animals' immune status and administration schedule mimicked the trial population and proposed administration schema. Macaca mulatta represents the prototype animal species for assessment of measles neurotoxicity. The animals were stereotactically administered either vehicle (n = 1) or MV-CEA at 2 × 105 or 2 × 106 TCID50 (each, n = 2) in the right frontal lobe in two injections on days 1 and 5. Macaques were closely monitored clinically for neurotoxicity. Body weight, temperature, complete blood count, CEA, clinical chemistries, coagulation, complement levels, immunoglobulin, measles antibody titers, viremia, and shedding (buccal swabs) were tested at multiple time points. Furthermore, cisterna magna spinal taps were performed on day 9 and 1 year after the first viral dose administration, and samples were analyzed for protein, glucose, cell differential, and presence of MV-CEA. Magnetic resonance imaging (MRI) was performed between 4 and 5 months after article administration to assess for subclinical neurotoxicity. To date, 36+ months from study initiation there has been no clinical or biochemical evidence of toxicity, including lack of neurological symptoms, fever, or other systemic symptoms and lack of immunosuppression. Quantitative RT-PCR analysis of blood, buccal swabs, and cerebrospinal fluid (CSF) was negative for MV-CEA at all time points, with the exception of viral genome deletion in the blood of one asymptomatic animal at the 2 × 106 TCID50 dose level on day 85. Vero cell overlays of CSF cells and supernatant were negative for viral recovery. There was no detection of CEA in serum or CSF at any time point. MRI scans were negative for imaging abnormalities and showed no evidence of encephalitis. Our results support the safety of CNS administration of MV-CEA in glioma patients. A clinical trial of intratumoral and resection cavity administration of MV-CEA in patients with recurrent glioblastoma multiforme is currently ongoing.

We recently identified and cloned the receptor for subgroup C avian sarcoma and leukosis viruses [ASLV(C)], i.e., Tvc, a protein most closely related to mammalian butyrophilins, which are members of the immunoglobulin protein family. The extracellular domain of Tvc contains two immunoglobulin-like domains, IgV and IgC, which presumably each contain a disulfide bond important for native function of the protein. In this study, we have begun to identify the functional determinants of Tvc responsible for ASLV(C) receptor activity. We found that the IgV domain of the Tvc receptor is responsible for interacting with the glycoprotein of ASLV(C). Additional experiments demonstrated that a domain was necessary as a spacer between the IgV domain and the membrane-spanning domain for efficient Tvc receptor activity, most likely to orient the IgV domain a proper distance from the cell membrane. The effects on ASLV(C) glycoprotein binding and infection efficiency were also studied by site-directed mutagenesis of the cysteine residues of Tvc as well as conserved amino acid residues of the IgV Tvc domain compared to other IgV domains. In this initial analysis of Tvc determinants important for interacting with ASLV(C) glycoproteins, at least two aromatic amino acid residues in the IgV domain of Tvc, Trp-48 and Tyr-105, were identified as critical for efficient ASLV(C) infection. Interestingly, one or more aromatic amino acid residues have been identified as critical determinants in the other ASLV(A-E) receptors for a proper interaction with ASLV glycoproteins. This suggests that the ASLV glycoproteins may share a common mechanism of receptor interaction with an aromatic residue(s) on the receptor critical for triggering conformational changes in SU that initiate the fusion process required for efficient virus infection.

We previously showed that the cysteines flanking the internal fusion peptide of the avian sarcoma/leukosis virus subtype A (ASLV-A) Env (EnvA) are important for infectivity and cell-cell fusion. Here we define the stage of fusion at which the cysteines are required. The flanking cysteines are dispensable for receptor-triggered membrane association but are required for the lipid mixing step of fusion, which, interestingly, displays a high pH onset and a biphasic profile. Second-site mutations that partially restore infection partially restore lipid mixing. These findings indicate that the cysteines flanking the internal fusion peptide of EnvA (and perhaps by analogy Ebola virus glycoprotein) are important for the foldback stage of the conformational changes that lead to membrane merger.

The avian sarcoma and leukosis virus (ASLV) family of retroviruses contains five highly related envelope subgroups (A to E) thought to have evolved from a common viral ancestor in the chicken population. Three genetic loci in chickens determine the susceptibility or resistance of cells to infection by the subgroup A to E ASLVs. Some inbred lines of chickens display phenotypes that are somewhere in between either efficiently susceptible or resistant to infection by specific subgroups of ASLV. The tvb gene encodes the receptor for subgroups B, D, and E ASLVs. The wild-type TvbS1 receptor confers susceptibility to subgroups B, D, and E ASLVs. In this study, the genetic defect that accounts for the altered susceptibility of an inbred chicken line, line M, to infection by ASLV(B), ASLV(D), and ASLV(E) was identified. The tvb gene in line M, tvbr2, encodes a mutant TvbS1 receptor protein with a substitution of a serine for a cysteine at position 125 (C125S). Here, we show that the C125S substitution in TvbS1 significantly reduces the susceptibility of line M cells to infection by ASLV(B) and ASLV(D) and virtually eliminates susceptibility to ASLV(E) infection both in cultured cells and in the incidence and growth of avian sarcoma virus-induced sarcomas in chickens. The C125S substitution significantly reduces the binding affinity of the TvbS1 receptor for the subgroup B, D, and E ASLV envelope glycoproteins. These are the first results that demonstrate a possible role of the cysteine-rich domain 3 in the function of the Tvb receptors.

Antibodies to viral surface glycoproteins play a crucial role in immunity to measles by blocking both virus attachment and subsequent fusion with the host cell membrane. Here, we demonstrate that certain immunoglobulin G (IgG) antibodies can also enhance the entry of measles virus (MV) into monocytes and macrophages. Antibody-dependent enhancement of infectivity was observed in mouse and human macrophages using virions opsonized by a murine monoclonal antibody against the MV hemagglutinin (H) glycoprotein, polyclonal mouse anti-MV IgG, or diluted measles-immune human sera. Neither H-specific Fab fragments nor H-specific IgM could enhance MV entry in monocytes or macrophages, indicating involvement of a Fc γ receptor (FcγR)-mediated mechanism. Preincubation with an anti-fusion protein (anti-F) monoclonal antibody or a fusion-inhibitory peptide blocked infection, indicating that a functional F protein was required for viral internalization. Classical complement pathway activation did not promote infection through complement receptors and inhibited anti-H IgG-mediated enhancement. In vivo, antibody-enhanced infection allowed MV to overcome a highly protective systemic immune response in preimmunized IfnarKo-Ge46 transgenic mice. These data demonstrate a previously unidentified mechanism that may contribute to morbillivirus pathogenesis where H-specific IgG antibodies promote the spread of MV infection among FcγR-expressing host cells. The findings point to a new model for the pathogenesis of atypical MV infection observed after immunization with formalin-inactivated MV vaccine and underscore the importance of the anti-F response after vaccination.

The five highly related envelope subgroups of the avian sarcoma and leukosis viruses (ASLVs), subgroup A [ASLV(A)] to ASLV(E), are thought to have evolved from an ancestral envelope glycoprotein yet utilize different cellular proteins as receptors. Alleles encoding the subgroup A ASLV receptors (Tva), members of the low-density lipoprotein receptor family, and the subgroup B, D, and E ASLV receptors (Tvb), members of the tumor necrosis factor receptor family, have been identified and cloned. However, alleles encoding the subgroup C ASLV receptors (Tvc) have not been cloned. Previously, we established a genetic linkage between tvc and several other nearby genetic markers on chicken chromosome 28, including tva. In this study, we used this information to clone the tvc gene and identify the Tvc receptor. A bacterial artificial chromosome containing a portion of chicken chromosome 28 that conferred susceptibility to ASLV(C) infection was identified. The tvc gene was identified on this genomic DNA fragment and encodes a 488-amino-acid protein most closely related to mammalian butyrophilins, members of the immunoglobulin protein family. We subsequently cloned cDNAs encoding Tvc that confer susceptibility to infection by subgroup C viruses in chicken cells resistant to ASLV(C) infection and in mammalian cells that do not normally express functional ASLV receptors. In addition, normally susceptible chicken DT40 cells were resistant to ASLV(C) infection after both tvc alleles were disrupted by homologous recombination. Tvc binds the ASLV(C) envelope glycoproteins with low-nanomolar affinity, an affinity similar to that of binding of Tva and Tvb with their respective envelope glycoproteins. We have also identified a mutation in the tvc gene in line L15 chickens that explains why this line is resistant to ASLV(C) infection.

The subgroup A to E avian sarcoma and leukosis viruses (ASLVs) are highly related and are thought to have evolved from a common ancestor. These viruses use distinct cell surface proteins as receptors to gain entry into avian cells. Chickens have evolved resistance to infection by the ASLVs. We have identified the mutations responsible for the block to virus entry in chicken lines resistant to infection by subgroup A ASLVs [ASLV(A)]. The tva genetic locus determines the susceptibility of chicken cells to ASLV(A) viruses. In quail, the ASLV(A) susceptibility allele tvas encodes two forms of the Tva receptor; these proteins are translated from alternatively spliced mRNAs. The normal cellular function of the Tva receptor is unknown; however, the extracellular domain contains a 40-amino-acid, cysteine-rich region that is homologous to the ligand binding region of the low-density lipoprotein receptor (LDLR) proteins. The chicken tvas cDNAs had not yet been fully characterized; we cloned the chicken tva cDNAs from two lines of subgroup A-susceptible chickens, line H6 and line 0. Two types of chicken tvas cDNAs were obtained. These cDNAs encode a longer and shorter form of the Tva receptor homologous to the Tva forms in quail. Two different defects were identified in cDNAs cloned from two different ASLV(A)-resistant inbred chickens, line C and line 72. Line C tvar contains a single base pair substitution, resulting in a cysteine-to-tryptophan change in the LDLR-like region of Tva. This mutation drastically reduces the binding affinity of TvaR for the ASLV(A) envelope glycoproteins. Line 72 tvar2 contains a 4-bp insertion in exon 1 that causes a change in the reading frame, which blocks expression of the Tva receptor.

A complex interaction between the retroviral envelope glycoproteins and a specific cell surface protein initiates viral entry into cells. The avian leukosis-sarcoma virus (ALV) group of retroviruses provides a useful experimental system for studying the retroviral entry process and the evolution of receptor usage. In this study, we demonstrate that evolutionary pressure on subgroup A ALV [ALV(A)] entry exerted by the presence of a competitive inhibitor, a soluble form of the ALV(A) Tva receptor linked to a mouse immunoglobulin G tag (quail sTva-mIgG), can select different populations of escape variants. This escape population contained three abundant ALV(A) variant viruses, all with mutations in the surface glycoprotein hypervariable regions: a previously identified variant containing the Y142N mutation in the hr1 region; a new variant with two mutations, W141G in hr1 and K261E in vr3; and another new variant with two mutations, W145R in hr1 and K261E. The W141G K261E and W145R K261E viruses escape primarily by lowering their binding affinities for the quail Tva receptor competitive inhibitor while retaining wild-type levels of binding affinity for the chicken Tva receptor. A secondary phenotype of the new variants was an alteration in receptor interference patterns from that of wild-type ALV(A), indicating that the mutant glycoproteins are possibly interacting with other cellular proteins. One result of these altered interactions was that the variants caused a transient period of cytotoxicity. We could also directly demonstrate that the W141G K261E variant glycoproteins bound significant levels of a soluble form of the TvbS3 ALV receptor in a binding assay. Alterations in the normally extreme specificity of the ALV(A) glycoproteins for Tva may represent an evolutionary first step toward expanding viral receptor usage in response to inefficient viral entry.

To better understand retroviral entry, we have characterized the interactions between subgroup A avian leukosis virus [ALV(A)] envelope glycoproteins and Tva, the receptor for ALV(A), that result in receptor interference. We have recently shown that soluble forms of the chicken and quail Tva receptor (sTva), expressed from genes delivered by retroviral vectors, block ALV(A) infection of cultured chicken cells (∼200-fold antiviral effect) and chickens (>98% of the birds were not infected). We hypothesized that inhibition of viral replication by sTva would select virus variants with mutations in the surface glycoprotein (SU) that altered the binding affinity of the subgroup A SU for the sTva protein and/or altered the normal receptor usage of the virus. Virus propagation in the presence of quail sTva-mIgG, the quail Tva extracellular region fused to the constant region of the mouse immunoglobulin G (IgG) protein, identified viruses with three mutations in the subgroup A hr1 region of SU, E149K, Y142N, and Y142N/E149K. These mutations reduced the binding affinity of the subgroup A envelope glycoproteins for quail sTva-mIgG (32-, 324-, and 4,739-fold, respectively) but did not alter their binding affinity for chicken sTva-mIgG. The ALV(A) mutants efficiently infected cells expressing the chicken Tva receptor but were 2-fold (E149K), 10-fold (Y142N), and 600-fold (Y142N/E149K) less efficient at infecting cells expressing the quail Tva receptor. These mutations identify key determinants of the interaction between the ALV(A) glycoproteins and the Tva receptor. We also conclude from these results that, at least for the wild-type and variant ALV(A)s tested, the receptor binding affinity was directly related to infection efficiency.

We previously reported that mitogenic activation of porcine peripheral blood mononuclear cells resulted in production of porcine endogenous retrovirus(es) (PERV[s]) capable of productively infecting human cells (C. Wilson et al., J. Virol. 72:3082–3087, 1998). We now extend that analysis to show that additional passage of isolated virus, named here PERV-NIH, through a human cell line yielded a viral population with a higher titer of infectious virus on human cells than the initial isolate. We show that in a single additional passage on a human cell line, the increase in infectivity for human cells is accounted for by selection against variants carrying pig-tropic envelope sequences (PERV-C) as well as by enrichment for replication-competent genomes. Sequence analysis of the envelope cDNA present in virions demonstrated that the envelope sequence of PERV-NIH is related to but distinct from previously reported PERV envelopes. The in vitro host range of PERV was studied in human primary cells and cell lines, as well as in cell lines from nonhuman primate and other species. This analysis reveals three patterns of susceptibility to infection among these host cells: (i) cells are resistant to infection in our assay; (ii) cells are infected by virus, as viral RNA is detected in the supernatant by reverse transcription-PCR, but the cells are not permissive to productive replication and spread; and (iii) cells are permissive to low-level productive replication. Certain cell lines were permissive for efficient productive infection and spread. These results may prove useful in designing appropriate animal models to assess the in vivo infectivity properties of PERV.

The interactions between the subgroup A avian leukosis virus [ALV(A)] envelope glycoproteins and soluble forms of the ALV(A) receptor Tva were analyzed both in vitro and in vivo by quantitating the ability of the soluble Tva proteins to inhibit ALV(A) entry into susceptible cells. Two soluble Tva proteins were tested: the 83-amino-acid Tva extracellular region fused to two epitope tags (sTva) or fused to the constant region of the mouse immunoglobulin G heavy chain (sTva-mIgG). Replication-competent ALV-based retroviral vectors with subgroup B or C env were used to deliver and express the two soluble tv-a (stva) genes in avian cells. In vitro, chicken embryo fibroblasts or DF-1 cells expressing sTva or sTva-mIgG proteins were much more resistant to infection by ALV(A) (∼200-fold) than were control cells infected by only the vector. The antiviral effect was specific for ALV(A), which is consistent with a receptor interference mechanism. The antiviral effect of sTva-mIgG was positively correlated with the amount of sTva-mIgG protein. In vivo, the stva genes were delivered and expressed in line 0 chicken embryos by the ALV(B)-based vector RCASBP(B). Viremic chickens expressed relatively high levels of stva and stva-mIgG RNA in a broad range of tissues. High levels of sTva-mIgG protein were detected in the sera of chickens infected with RCASBP(B)stva-mIgG. Viremic chickens infected with RCASBP(B) alone, RCASBP(B)stva, or RCASBP(B)stva-mIgG were challenged separately with ALV(A) and ALV(C). Both sTva and sTva-mIgG significantly inhibited infection by ALV(A) (95 and 100% respectively) but had no measurable effect on ALV(C) infection. The results of this study indicate that a soluble receptor can effectively block infection of at least some retroviruses and demonstrates the utility of the ALV experimental system in characterizing the mechanism(s) of viral entry.

Edmonston vaccine strains of measles virus (MV) have shown significant antitumor activity in preclinical models of ovarian cancer. We engineered MV to express the marker peptide carcinoembryonic antigen (MVCEA virus) to also permit real-time monitoring of viral gene expression in tumors in the clinical setting. Patients with Taxol and platinum-refractory recurrent ovarian cancer and normal CEA levels were eligible for this phase I trial. Twenty-one patients were treated with MV-CEA i.p. every 4 weeks for up to 6 cycles at seven different dose levels (103–109 TCID50). We observed no dose-limiting toxicity, treatment-induced immunosuppression, development of anti-CEA antibodies, increase in anti-MV antibody titers, or virus shedding in urine or saliva. Dose-dependent CEA elevation in peritoneal fluid and serum was observed. Immunohistochemical analysis of patient tumor specimens revealed overexpression of measles receptor CD46 in 13 of 15 patients. Best objective response was dose-dependent stable disease in 14 of 21 patients with a median duration of 92.5 days (range, 54–277 days). Five patients had significant decreases in CA-125 levels. Median survival of patients on study was 12.15 months (DELnths; range, 1.3–38.4 months), comparing favorably to an expected median survival of 6 months (DELnth) in this patient population. Our findings indicate that i.p. administration of MV-CEA is well tolerated and results in dose-dependent biological activity in a cohort of heavily pretreated recurrent ovarian cancer patients.

Abstract

Vesicular stomatitis virus (VSV) has shown promise as an oncolytic agent, although unmodified VSV can be neurotoxic. To avoid toxicity, a vector was created by introducing the interferon-β (IFN-β) gene (VSV.IFN-β). We conducted this study to determine the ability of VSV.IFN-β to lyse human cancer (mesothelioma) cells and to evaluate the potential of this recombinant virus for clinical translation. Four normal human mesothelial and 12 mesothelioma cell lines were tested for their susceptibility to VSV vectors in vitro. VSV.hIFN-β did not cause cytotoxicity in any normal lines. Only 4 of 12 lines were effectively lysed by VSV.hIFN-β. In the eight resistant lines, pretreatment with IFN-β prevented lysis of cells by VSV.GFP, and VSV infection or addition of IFN-β protein resulted in the upregulation of double-stranded RNA-dependent protein kinase (PKR), myxovirus resistance A (MxA), and 2′,5′-oligo-adenylate-synthetase (2′5′-OAS) mRNA. In the susceptible lines, there was no protection by pretreatment with IFN-β protein and no IFN- or VSV-induced changes in PKR, MxA, and 2′5′-OAS mRNA. This complete lack of IFN responsiveness could be explained by marked downregulation of interferon alpha receptors (IFNARs), p48, and PKR in both the mesothelioma cell lines and primary tumor biopsies screened. Presence of p48 in three tumor samples predicted responsiveness to IFN. Our data indicate that many mesothelioma tumors have partially intact IFN pathways that may affect the efficacy of oncolytic virotherapy. However, it may be feasible to prescreen individual susceptibility to VSV.IFN-β by immunostaining for the presence of p48 protein.