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1.  Reprogrammed viruses as cancer therapeutics: targeted, armed and shielded 
Nature reviews. Microbiology  2008;6(7):529-540.
Virotherapy is currently undergoing a renaissance, based on our improved understanding of virus biology and genetics and our better knowledge of many different types of cancer. Viruses can be reprogrammed into oncolytic vectors by combining three types of modification: targeting, arming and shielding. Targeting introduces multiple layers of cancer specificity and improves safety and efficacy; arming occurs through the expression of prodrug convertases and cytokines; and coating with polymers and the sequential usage of different envelopes or capsids provides shielding from the host immune response. Virus-based therapeutics are beginning to find their place in cancer clinical practice, in combination with chemotherapy and radiation.
doi:10.1038/nrmicro1927
PMCID: PMC3947522  PMID: 18552863
2.  Hydrophobic and Charged Residues in the Central Segment of the Measles Virus Hemagglutinin Stalk Mediate Transmission of the Fusion-Triggering Signal 
Journal of Virology  2013;87(18):10401-10404.
The pH-independent measles virus membrane fusion process begins when the attachment protein H binds to a receptor. Knowing that the central segment of the tetrameric H stalk transmits the signal to the fusion protein trimer, we investigated how. We document that exact conservation of most residues in the 92 through 99 segment is essential for function. In addition, hydrophobic and charged residues in the 104 through 125 segment, arranged with helical periodicity, are critical for F protein interactions and signal transmission.
doi:10.1128/JVI.01547-13
PMCID: PMC3753981  PMID: 23864629
3.  Targeted Cell Entry of Lentiviral Vectors 
Retargeting of lentiviral vector entry to cell types of interest is a key factor in improving the safety and efficacy of gene transfer. In this study we show that the retargetable envelope glycoproteins of measles virus (MV), namely, the hemagglutinin (H) responsible for receptor recognition and the fusion protein (F), can pseudotype human immunodeficiency virus 1 (HIV-1) vectors when their cytoplasmic tails are truncated. We then pseudotyped HIV-1 vectors with MV glycoproteins displaying on H either the epidermal growth factor or a single-chain antibody directed against CD20, but without the ability to recognize their native receptors. Gene transfer into cells that expressed the targeted receptor was several orders of magnitude more efficient than into cells that did not. High-target versus nontarget cell discrimination was demonstrated in mixed cell populations, where the targeting vector selectively eliminated CD20-positive cells after suicide gene transfer. Remarkably, primary human CD20-positive B lymphocytes were transduced more efficiently by the CD20-targeted vector than by a vector pseudo-typed with the vesicular stomatitis virus G (VSV-G) protein. In addition, the CD20-targeted vector was able to transduce even unstimulated primary B cells, whereas VSV-G pseudotyped vectors were unable to do so. Because MV enters cells through direct fusion at the cell membrane, this novel targeting system should be widely applicable.
doi:10.1038/mt.2008.128
PMCID: PMC3927321  PMID: 18578012
4.  The Measles Virus Hemagglutinin β-Propeller Head β4-β5 Hydrophobic Groove Governs Functional Interactions with Nectin-4 and CD46 but Not Those with the Signaling Lymphocytic Activation Molecule 
Journal of Virology  2013;87(16):9208-9216.
Wild-type measles virus (MV) strains use the signaling lymphocytic activation molecule (SLAM; CD150) and the adherens junction protein nectin-4 (poliovirus receptor-like 4 [PVRL4]) as receptors. Vaccine MV strains have adapted to use ubiquitous membrane cofactor protein (MCP; CD46) in addition. Recently solved cocrystal structures of the MV attachment protein (hemagglutinin [H]) with each receptor indicate that all three bind close to a hydrophobic groove located between blades 4 and 5 (β4-β5 groove) of the H protein β-propeller head. We used this structural information to focus our analysis of the functional footprints of the three receptors on vaccine MV H. We mutagenized this protein and tested the ability of individual mutants to support cell fusion through each receptor. The results highlighted a strong overlap between the functional footprints of nectin-4 and CD46 but not those of SLAM. A soluble form of nectin-4 abolished vaccine MV entry in nectin-4- and CD46-expressing cells but only reduced entry through SLAM. Analyses of the binding kinetics of an H mutant with the three receptors revealed that a single substitution in the β4-β5 groove drastically reduced nectin-4 and CD46 binding while minimally altering SLAM binding. We also generated recombinant viruses and analyzed their infections in cells expressing individual receptors. Introduction of a single substitution into the hydrophobic pocket affected entry through both nectin-4 and CD46 but not through SLAM. Thus, while nectin-4 and CD46 interact functionally with the H protein β4-β5 hydrophobic groove, SLAM merely covers it. This has implications for vaccine and antiviral strategies.
doi:10.1128/JVI.01210-13
PMCID: PMC3754078  PMID: 23760251
5.  Nectin-4-Dependent Measles Virus Spread to the Cynomolgus Monkey Tracheal Epithelium: Role of Infected Immune Cells Infiltrating the Lamina Propria 
Journal of Virology  2013;87(5):2526-2534.
After the contagion measles virus (MV) crosses the respiratory epithelium within myeloid cells that express the primary receptor signaling lymphocytic activation molecule (SLAM), it replicates briskly in SLAM-expressing cells in lymphatic organs. Later, the infection spreads to epithelia expressing nectin-4, an adherens junction protein expressed preferentially in the trachea, but how it gets there is not understood. To characterize the mechanisms of spread, we infected groups of 5 or 6 cynomolgus monkeys (Macaca fascicularis) with either a wild-type MV or its “N4-blind” derivative, which is unable to enter nectin-4-expressing cells because of the targeted mutation of two hemagglutinin residues. As expected, both viruses caused similar levels of immunosuppression, as monitored by reductions in white blood cell counts and lymphocyte proliferation activity. However, monkeys infected with the N4-blind MV cleared infection more rapidly. Wild-type virus-infected monkeys secreted virus, while marginal virus titers were detected in tracheal lavage fluid cells of N4-blind MV-infected hosts. Analyses of tracheal rings obtained at necropsy (day 12) documented widespread infection of individual cells or small cell clusters in the subepithelial lamina propria of monkeys infected with either virus. However, only wild-type MV spread to the epithelium, forming numerous infectious centers comprised of many contiguous columnar cells. Infected CD11c+ myeloid (macrophage or dendritic) cells were frequently observed in the lamina propria below epithelial infectious centers. Thus, MV may use myeloid cells as vehicles not only immediately after contagion but also to infect epithelia of tissues expressing nectin-4, including the trachea.
doi:10.1128/JVI.03037-12
PMCID: PMC3571369  PMID: 23255790
6.  Development of measles virus-based shielded oncolytic vectors: suitability of other paramyxovirus glycoproteins 
Cancer gene therapy  2013;20(2):109-116.
Antibody-mediated neutralization may interfere with the efficacy of measles virus (MV) oncolysis. To circumvent vector neutralization, we sought to exchange the envelope glycoproteins, hemagglutinin (H) and fusion (F), with those from the non-cross reactive Tupaia paramyxovirus (TPMV). To sustain efficient particle assembly, we generated hybrid glycoproteins with the MV cytoplasmic tails and the TPMV ectodomains. Hybrid F-proteins that partially retained fusion function, and hybrid H-proteins that retained fusion support activity, were generated. However, when used in combination, the hybrid proteins did not support membrane fusion. An alternative strategy was developed based on a hybrid F protein and a truncated H protein that supported cell-cell fusion. A hybrid virus expressing these two proteins was rescued, and was able to spread by cell fusion, however was only capable of producing minimal amounts of particles. Lack of specific interactions between the matrix and the H-protein, in combination with sub-optimal F-protein processing and inefficient glycoprotein transport in the rescue cells, accounted for inefficient particle production. Ultimately, this interferes with applications for oncolytic virotherapy. Alternative strategies for the generation of shielded MV are discussed.
doi:10.1038/cgt.2012.92
PMCID: PMC3573219  PMID: 23306608
Tupaia paramyxovirus; measles virus; glycoprotein modification; vector shielding
7.  Broadly Neutralizing Immune Responses against Hepatitis C Virus Induced by Vectored Measles Viruses and a Recombinant Envelope Protein Booster 
Journal of Virology  2012;86(21):11558-11566.
Hepatitis C virus (HCV) infection remains a serious public health problem worldwide. Treatments are limited, and no preventive vaccine is available. Toward developing an HCV vaccine, we engineered two recombinant measles viruses (MVs) expressing structural proteins from the prototypic HCV subtype 1a strain H77. One virus directs the synthesis of the HCV capsid (C) protein and envelope glycoproteins (E1 and E2), which fold properly and form a heterodimer. The other virus expresses the E1 and E2 glycoproteins separately, with each one fused to the cytoplasmic tail of the MV fusion protein. Although these hybrid glycoproteins were transported to the plasma membrane, they were not incorporated into MV particles. Immunization of MV-susceptible, genetically modified mice with either vector induced neutralizing antibodies to MV and HCV. A boost with soluble E2 protein enhanced titers of neutralizing antibody against the homologous HCV envelope. In animals primed with MV expressing properly folded HCV C-E1-E2, boosting also induced cross-neutralizating antibodies against two heterologous HCV strains. These results show that recombinant MVs retain the ability to induce MV-specific humoral immunity while also eliciting HCV neutralizing antibodies, and that anti-HCV immunity can be boosted with a single dose of purified E2 protein. The use of MV vectors could have advantages for pediatric HCV vaccination.
doi:10.1128/JVI.01776-12
PMCID: PMC3486281  PMID: 22896607
8.  Targeted Entry of Enveloped Viruses: Measles and Herpes Simplex Virus I 
Current Opinion in Virology  2011;2(1):43-49.
We compare the receptor-based mechanisms that a small RNA virus and a larger DNA virus have evolved to drive the fusion of viral and cellular membranes. Both systems rely on tight control over triggering the concerted refolding of a trimeric fusion protein. While measles virus entry depends on a receptor-binding protein and a fusion protein only, the herpes simplex virus is more complex and requires four viral proteins. Nevertheless, in both viruses a receptor-binding protein is required for triggering the membrane fusion process. Moreover, specificity domains can be appended to these receptor-binding proteins to target virus entry to cells expressing a designated receptor. We discuss how principles established with measles and herpes simplex virus can be applied to targeting other enveloped viruses, and alternatively how retargeted envelopes can be fitted on foreign capsids.
doi:10.1016/j.coviro.2011.12.002
PMCID: PMC3311990  PMID: 22440965
9.  Canine Distemper Virus Epithelial Cell Infection Is Required for Clinical Disease but Not for Immunosuppression 
Journal of Virology  2012;86(7):3658-3666.
To characterize the importance of infection of epithelial cells for morbillivirus pathogenesis, we took advantage of the severe disease caused by canine distemper virus (CDV) in ferrets. To obtain a CDV that was unable to enter epithelial cells but retained the ability to enter immune cells, we transferred to its attachment (H) protein two mutations shown to interfere with the interaction of measles virus H with its epithelial receptor, human nectin-4. As expected for an epithelial receptor (EpR)-blind CDV, this virus infected dog and ferret epithelial cells inefficiently and did not cause cell fusion or syncytium formation. On the other hand, the EpR-blind CDV replicated in cells expressing canine signaling lymphocyte activation molecule (SLAM), the morbillivirus immune cell receptor, with similar kinetics to those of wild-type CDV. While ferrets infected with wild-type CDV died within 12 days after infection, after developing severe rash and fever, animals infected with the EpR-blind virus showed no clinical signs of disease. Nevertheless, both viruses spread rapidly and efficiently in immune cells, causing similar levels of leukopenia and inhibition of lymphocyte proliferation activity, two indicators of morbillivirus immunosuppression. Infection was documented for airway epithelia of ferrets infected with wild-type CDV but not for those of animals infected with the EpR-blind virus, and only animals infected with wild-type CDV shed virus. Thus, epithelial cell infection is necessary for clinical disease and efficient virus shedding but not for immunosuppression.
doi:10.1128/JVI.06414-11
PMCID: PMC3302517  PMID: 22278252
10.  Adherens junction protein nectin-4 (PVRL4) is the epithelial receptor for measles virus 
Nature  2011;480(7378):530-533.
Measles (MV) is an aerosol-transmitted virus that affects more than 10 million children each year and accounts for approximately 120,000 deaths1,2. While it was long believed to replicate in the respiratory epithelium before disseminating, it was recently shown to initially infect macrophages and dendritic cells of the airways using the signaling lymphocytic activation molecule (SLAM, CD150) as receptor3-6. These cells then cross the respiratory epithelium and ferry the infection to lymphatic organs where MV replicates vigorously7. How and where the virus crosses back into the airways has remained unknown. Based on functional analyses of surface proteins preferentially expressed on virus-permissive epithelial cell lines, we identified nectin-48 (poliovirus-receptor-like-4) as a candidate host exit receptor. This adherens junction protein of the immunoglobulin superfamily interacts with the viral attachment protein with high affinity through its membrane-distal domain. Nectin-4 sustains MV entry and non-cytopathic lateral spread in well-differentiated primary human airway epithelial sheets infected basolaterally. It is down-regulated in infected epithelial cells, including those of macaque tracheas. While other viruses use receptors to enter hosts or transit through their epithelial barriers, we suggest that MV targets nectin-4 to emerge in the airways. Nectin-4 is a cellular marker of several types of cancer9-11, which has implications for ongoing MV-based clinical trials of oncolysis12.
doi:10.1038/nature10639
PMCID: PMC3245798  PMID: 22048310
11.  Measles Virus Selectively Blind to Signaling Lymphocytic Activation Molecule (SLAM; CD150) Is Attenuated and Induces Strong Adaptive Immune Responses in Rhesus Monkeys▿  
Journal of Virology  2010;84(7):3413-3420.
The signaling lymphocytic activation molecule (SLAM; CD150) is the immune cell receptor for measles virus (MV). To assess the importance of the SLAM-MV interactions for virus spread and pathogenesis, we generated a wild-type IC-B MV selectively unable to recognize human SLAM (SLAM-blind). This virus differs from the fully virulent wild-type IC-B strain by a single arginine-to-alanine substitution at amino acid 533 of the attachment protein hemagglutinin and infects cells through SLAM about 40 times less efficiently than the isogenic wild-type strain. Ex vivo, this virus infects primary lymphocytes at low levels regardless of SLAM expression. When a group of six rhesus monkeys (Macaca mulatta) was inoculated intranasally with the SLAM-blind virus, no clinical symptoms were documented. Only one monkey had low-level viremia early after infection, whereas all the hosts in the control group had high viremia levels. Despite minimal, if any, viremia, all six hosts generated neutralizing antibody titers close to those of the control monkeys while MV-directed cellular immunity reached levels at least as high as in wild-type-infected monkeys. These findings prove formally that efficient SLAM recognition is necessary for MV virulence and pathogenesis. They also suggest that the selectively SLAM-blind wild-type MV can be developed into a vaccine vector.
doi:10.1128/JVI.02304-09
PMCID: PMC2838096  PMID: 20071568
12.  Measles Virus Infection of Alveolar Macrophages and Dendritic Cells Precedes Spread to Lymphatic Organs in Transgenic Mice Expressing Human Signaling Lymphocytic Activation Molecule (SLAM, CD150) ▿  
Journal of Virology  2009;84(6):3033-3042.
Recent studies of primate models suggest that wild-type measles virus (MV) infects immune cells located in the airways before spreading systemically, but the identity of these cells is unknown. To identify cells supporting primary MV infection, we took advantage of mice expressing the MV receptor human signaling lymphocyte activation molecule (SLAM, CD150) with human-like tissue specificity. We infected these mice intranasally (IN) with a wild-type MV expressing green fluorescent protein. One, two, or three days after inoculation, nasal-associated lymphoid tissue (NALT), the lungs, several lymph nodes (LNs), the spleen, and the thymus were collected and analyzed by microscopy and flow cytometry, and virus isolation was attempted. One day after inoculation, MV replication was documented only in the airways, in about 2.5% of alveolar macrophages (AM) and 0.5% of dendritic cells (DC). These cells expressed human SLAM, and it was observed that MV infection temporarily enhanced SLAM expression. Later, MV infected other immune cell types, including B and T lymphocytes. Virus was isolated from lymphatic tissue as early as 2 days post-IN inoculation; the mediastinal lymph node was an early site of replication and supported high levels of infection. Three days after intraperitoneal inoculation, 1 to 8% of the mediastinal LN cells were infected. Thus, MV infection of alveolar macrophages and subepithelial dendritic cells in the airways precedes infection of lymphocytes in lymphatic organs of mice expressing human SLAM with human-like tissue specificity.
doi:10.1128/JVI.01559-09
PMCID: PMC2826031  PMID: 20042501
13.  Mechanisms of Protein Kinase PKR-Mediated Amplification of Beta Interferon Induction by C Protein-Deficient Measles Virus▿  
Journal of Virology  2009;84(1):380-386.
The measles virus P gene products V and C antagonize the host interferon (IFN) response, blocking both IFN signaling and production. Using Moraten vaccine strain-derived measles virus and isogenic mutants deficient for either V or C protein production (Vko and Cko, respectively), we observed that the Cko virus was a potent inducer of IFN-β, while induction by Vko virus was an order of magnitude lower than that by the Cko virus. The parental recombinant Moraten virus did not significantly induce IFN-β. The enhanced IFN-inducing capacity of the Cko virus correlated with an enhanced activation of IFN regulatory factor 3 (IRF-3), NF-κB, and ATF-2 in Cko-infected compared to Vko or parental virus-infected cells. Furthermore, protein kinase PKR and mitochondrial adapter IPS-1 were required for maximal Cko-mediated IFN-β induction, which correlated with the PKR-mediated enhancement of mitogen-activated protein kinase and NF-κB activation. Our results reveal multiple consequences of C protein expression and document an important function for PKR as an enhancer of IFN-β induction during measles virus infection.
doi:10.1128/JVI.02630-08
PMCID: PMC2798421  PMID: 19846517
14.  Paramyxovirus Entry and Targeted Vectors for Cancer Therapy 
PLoS Pathogens  2010;6(6):e1000973.
doi:10.1371/journal.ppat.1000973
PMCID: PMC2891830  PMID: 20585633
15.  Protective Anti-Hepatitis B Virus Responses in Rhesus Monkeys Primed with a Vectored Measles Virus and Boosted with a Single Dose of Hepatitis B Surface Antigen▿  
Journal of Virology  2009;83(17):9013-9017.
The widely used hepatitis B virus (HBV) vaccine is based on three doses of hepatitis B surface antigen (HBsAg) protein. We previously showed that vectored measles viruses (MV) expressing HBsAg retain measles vaccine function in monkeys but do not induce a protective anti-HBs response in all animals. We show here that a single dose of HBsAg protein following a three-dose vaccination regimen with an optimized HBsAg-expressing MV elicits protective anti-HBs responses in all four vaccinated Rhesus monkeys. Vaccination strategies coupling the effective, long-term immunity elicited by the high-coverage MV vaccine to prophylactic HBV immunity are discussed.
doi:10.1128/JVI.00906-09
PMCID: PMC2738202  PMID: 19535451
16.  Engineered Measles Virus as a Novel Oncolytic Therapy Against Prostate Cancer 
The Prostate  2009;69(1):82-91.
BACKGROUND
No curative therapy is currently available for locally advanced or metastatic prostate cancer. Oncolytic viruses represent a novel class of therapeutic agents that demonstrates no cross-resistance with existing approaches and can therefore be combined with conventional treatment modalities. Measles virus strains deriving from the Edmonston (MV-Edm) vaccine strain have shown considerable oncolytic activity against a variety of solid tumers and hematologic malignancies. In this study, we investigated the antitumor potential of recombinant MV-Edm derivatives as novel oncolytic agents against prostate cancer.
METHODS
The susceptibility of prostate cancer cell lines (PC-3, DU-145, and LNCaP) to measles virus infection was demonstrated using an MV-Edm derivative expressing green fluorescent protein (GFP). MV-Edm replication in prostate cancer cell lines was assessed by one step viral growth curves. The oncolytic effect of an MV-Edm strain engineered to express the human carcinoembryonic antigen (CEA) was demonstrated in vitro by MTT assays and in vivo in subcutaneous PC-3 xenografts. CEA levels were quantitated in cell supernatants and mouse serum samples.
RESULTS
Recombinant MV-Edm strains can effectively infect, replicate in and kill prostate cancer cells. Intratumoral administration of MV-CEA at a total dose of 6 ×106 TCID50 resulted in statistically significant tumor growth delay (P = 0.004) and prolongation of survival (P = 0.001) in a subcutaneous PC-3 xenograft model. Viral growth kinetics paralleled CEA production.
CONCLUSIONS
MV-CEA has potent antitumor activity against prostate cancer cell lines and xenografts. Viral gene expression during treatment can be determined by monitoring of CEA levels in the serum; the latter could allow dose optimization and tailoring of individualized treatment protocols.
doi:10.1002/pros.20857
PMCID: PMC2737678  PMID: 18973133
CEA; measles virus; MV-CEA; prostate cancer; virotherapy
17.  Protein Kinase PKR Mediates the Apoptosis Induction and Growth Restriction Phenotypes of C Protein-Deficient Measles Virus▿  
Journal of Virology  2008;83(2):961-968.
The measles virus (MV) accessory proteins V and C play important roles in MV replication and pathogenesis. Infection with recombinant MV lacking either V or C causes more cell death than infection with the parental vaccine-equivalent virus (MVvac), and C-deficient virus grows poorly relative to the parental virus. Here, we show that a major effector of the C phenotype is the RNA-dependent protein kinase PKR. Using human HeLa cells stably deficient in PKR as a result of RNA interference-mediated knockdown (PKRkd cells), we demonstrated that a reduction in PKR partially rescued the growth defect of C knockout (Cko) virus but had no effect on the growth of either wild-type (WT) or V knockout (Vko) virus. Increased growth of the Cko virus in PKRkd cells correlated with increased viral protein expression, while defective growth and decreased protein expression in PKR-sufficient cells correlated with increased phosphorylation of PKR and the α subunit of eukaryotic initiation factor 2. Furthermore, infection with WT, Vko, or especially Cko virus caused significantly less apoptosis in PKRkd cells than in PKR-sufficient cells. Although apoptosis induced by Cko virus infection in PKR-sufficient cells was blocked by a caspase antagonist, the growth of Cko virus was not restored to the WT level by treatment with this pharmacologic inhibitor. Taken together, these results indicate that PKR plays an important antiviral role during MV infection but that the virus growth restriction by PKR is not dependent upon the induction of apoptosis. Furthermore, the results establish that a principal function of the MV C protein is to antagonize the proapoptotic and antiviral activities of PKR.
doi:10.1128/JVI.01669-08
PMCID: PMC2612345  PMID: 19004947
18.  The Measles Virus Fusion Protein Transmembrane Region Modulates Availability of an Active Glycoprotein Complex and Fusion Efficiency▿  
Journal of Virology  2008;82(22):11437-11445.
The glycoprotein complex of paramyxoviruses mediates receptor binding and membrane fusion. In particular, the measles virus (MV) fusion (F) protein executes membrane fusion, after receptor binding by the hemagglutinin (H) protein. Structures and single amino acids influencing fusion function have been identified in the F-protein ectodomain and cytoplasmic tail, but not in its transmembrane (TM) region. Since this region influences function of the envelope proteins of other viruses, we examined its role in the MV F protein. Alanine-scanning mutagenesis revealed that an F protein with a single mutation of a central TM region leucine (L507A) was more fusogenic than the unmodified F protein while retaining similar kinetics of proteolytic processing. In contrast, substitution of residues located near the edges of the lipid bilayer reduced fusion activity. This was true not only when the mutated F proteins were coexpressed with H but also in the context of infections with recombinant viruses. Analysis of the H-F complexes with reduced fusion activities revealed that more precursor (F0) than activated (F1+2) protein coprecipitated with H. In contrast, in complexes with enhanced fusion activity, including H-FL507A, the F0/F1+2 ratio shifted toward F1+2. Thus, fusion activity correlated with an active F-H protein complex, and the MV F protein TM region modulated availability of this complex.
doi:10.1128/JVI.00779-08
PMCID: PMC2573273  PMID: 18786999
19.  Attenuation of V- or C-Defective Measles Viruses: Infection Control by the Inflammatory and Interferon Responses of Rhesus Monkeys▿  
Journal of Virology  2008;82(11):5359-5367.
Patients recruited in virus-based cancer clinical trials and immunocompromised individuals in need of vaccination would profit from viral strains with defined attenuation mechanisms. We generated measles virus (MV) strains defective for the expression of either the V protein, a modulator of the innate immune response, or the C protein, which has multiple functions. The virulence of these strains was compared with that of the parental wild-type MV in a natural host, Macaca mulatta. Skin rash, viremia, and the strength of the innate and adaptive immune responses were characterized in groups of six animals. Replication of V- or C-protein-defective viruses was short-lived and reached lower levels in peripheral blood mononuclear cells and lymphatic organs compared to the wild-type virus; none of the mutants reverted to the wild type. The neutralizing antibody titers and MV-specific T-cell responses were equivalent in monkeys infected with the viral strains tested, documenting strong adaptive immune responses. In contrast, the inflammatory response was better controlled by wild-type MV, as revealed by inhibition of interleukin-6 and tumor necrosis factor alpha transcription. The interferon response was also better controlled by the wild-type virus than by the defective viruses. Since V- and C-defective MVs induce strong adaptive immune responses while spreading less efficiently, they may be developed as vaccines for immunocompromised individuals. Moreover, MV unable to interact with single innate immunity proteins may be developed for preferential replication in tumors with specific contexts of vulnerability.
doi:10.1128/JVI.00169-08
PMCID: PMC2395192  PMID: 18385234
20.  Measles virus blind to its epithelial cell receptor remains virulent in rhesus monkeys but cannot cross the airway epithelium and is not shed 
The Journal of Clinical Investigation  2008;118(7):2448-2458.
The current model of measles virus (MV) pathogenesis implies that apical infection of airway epithelial cells precedes systemic spread. An alternative model suggests that primarily infected lymphatic cells carry MV to the basolateral surface of epithelial cells, supporting MV shedding into the airway lumen and contagion. This model predicts that a mutant MV, unable to enter cells through the unidentified epithelial cell receptor (EpR), would remain virulent but not be shed. To test this model, we identified residues of the MV attachment protein sustaining EpR-mediated cell fusion. These nonpolar or uncharged polar residues defined an area located near the binding site of the signaling lymphocytic activation molecule (SLAM), the receptor for MV on lymphatic cells. We then generated an EpR-blind virus maintaining SLAM-dependent cell entry and inoculated rhesus monkeys intranasally. Hosts infected with the selectively EpR-blind MV developed rash and anorexia while averaging slightly lower viremia than hosts infected with wild-type MV but did not shed virus in the airways. The mechanism restricting shedding was characterized using primary well-differentiated human airway epithelial cells. Wild-type MV infected columnar epithelial cells bearing tight junctions only when applied basolaterally, while the EpR-blind virus did not infect these cells. Thus, EpR is probably a basolateral protein, and infection of the airway epithelium is not essential for systemic spread and virulence of MV.
doi:10.1172/JCI35454
PMCID: PMC2430500  PMID: 18568079
21.  A Vectored Measles Virus Induces Hepatitis B Surface Antigen Antibodies While Protecting Macaques against Measles Virus Challenge▿  
Journal of Virology  2007;81(19):10597-10605.
Hepatitis B virus (HBV) acute and chronic infections remain a major worldwide health problem. Towards developing an anti-HBV vaccine with single-dose scheme potential, we engineered infectious measles virus (MV) genomic cDNAs with a vaccine strain background and expression vector properties. Hepatitis B surface antigen (HBsAg) expression cassettes were inserted into this cDNA and three MVs expressing HBsAg at different levels generated. All vectored MVs, which secrete HBsAg as subviral particles, elicited humoral responses in MV-susceptible genetically modified mice. However, small differences in HBsAg expression elicited vastly different HBsAg antibody levels. The two vectors inducing the highest HBsAg antibody levels were inoculated into rhesus monkeys (Macaca mulatta). After challenge with a pathogenic MV strain (Davis87), control naive monkeys showed a classic measles rash and high viral loads. In contrast, all monkeys immunized with vaccine or a control nonvectored recombinant vaccine or HBsAg-expressing vectored MV remained healthy, with low or undetectable viral loads. After a single vaccine dose, only the vector expressing HBsAg at the highest levels elicited protective levels of HBsAg antibodies in two of four animals. These observations reveal an expression threshold for efficient induction of HBsAg humoral immune responses. This threshold is lower in mice than in macaques. Implications for the development of divalent vaccines based on live attenuated viruses are discussed.
doi:10.1128/JVI.00923-07
PMCID: PMC2045491  PMID: 17634218
23.  Canine Distemper Virus Uses both the Anterograde and the Hematogenous Pathway for Neuroinvasion 
Journal of Virology  2006;80(19):9361-9370.
Canine distemper virus (CDV), a member of the Morbillivirus genus that also includes measles virus, frequently causes neurologic complications, but the routes and timing of CDV invasion of the central nervous system (CNS) are poorly understood. To characterize these events, we cloned and sequenced the genome of a neurovirulent CDV (strain A75/17) and produced an infectious cDNA that expresses the green fluorescent protein. This virus fully retained its virulence in ferrets: the course and signs of disease were equivalent to those of the parental isolate. We observed CNS invasion through two distinct pathways: anterogradely via the olfactory nerve and hematogenously through the choroid plexus and cerebral blood vessels. CNS invasion only occurred after massive infection of the lymphatic system and spread to the epithelial cells throughout the body. While at early time points, mostly immune and endothelial cells were infected, the virus later spread to glial cells and neurons. Together, the results suggest similarities in the timing, target cells, and CNS invasion routes of CDV, members of the Morbillivirus genus, and even other neurovirulent paramyxoviruses like Nipah and mumps viruses.
doi:10.1128/JVI.01034-06
PMCID: PMC1617229  PMID: 16973542
24.  Receptor (SLAM [CD150]) Recognition and the V Protein Sustain Swift Lymphocyte-Based Invasion of Mucosal Tissue and Lymphatic Organs by a Morbillivirus 
Journal of Virology  2006;80(12):6084-6092.
Experimental infections of ferrets with canine distemper virus (CDV) recapitulate many hallmarks of measles: rash, high fever, viremia, depression of delayed-type hypersensitivity responses, lowered leukocyte counts, and reduced lymphocyte proliferation activity. To understand how a morbillivirus invades the host and causes immunosuppression, we generated CDV either unable to recognize one of the receptors or incapable of expressing either one or both of the candidate interferon antagonist proteins V and C. Variants of these viruses expressing green fluorescent protein were also generated. Striking similarities between CDV infection of ferrets and human immunodeficiency virus host invasion were documented: first, massive early replication in the gut-associated lymphatic tissue, including intestinal Peyer's patches, followed by extensive infection of lymphatic organs, including thymus and circulating lymphocytes. Moreover, T cells were selectively depleted. Thus, CDV takes advantage of mucosal surfaces for host invasion and lymphocytes for swift dissemination. A CDV unable to recognize the signaling lymphocytic activation molecule (SLAM [CD150]) that is expressed in lymphocytes and other immune cells did not spread. A V-defective CDV multiplied with reduced efficiency in lymphocytes and did not inhibit the interferon and cytokine responses. Protein C affected the severity of rash and digestive symptoms elicited by V-defective CDV, but it was dispensable for the invasion of the lymphatic organs. These findings prove formally that SLAM recognition is necessary for morbillivirus virulence. They also reveal how two viral proteins affect pathogenesis: V sustains the swift lymphocyte-based invasion of mucosal tissue and lymphatic organs, whereas C sustains subsequent infection phases.
doi:10.1128/JVI.00357-06
PMCID: PMC1472607  PMID: 16731947
25.  Measles Virus V Protein Inhibits p53 Family Member p73 
Journal of Virology  2006;80(11):5644-5650.
Paramyxovirus V proteins function as host interference factors that inactivate antiviral responses, including interferon. Characterization of cellular proteins that copurify with ectopically expressed measles virus V protein has revealed interactions with DNA binding domains of p53 family proteins, p53 and p73. Specific transcriptional assays reveal that expression of measles virus V cDNA inhibits p73, but not p53. Expression of measles virus V cDNA can delay cell death induced by genotoxic stress and also can decrease the abundance of the proapoptotic factor PUMA, a p73 target. Recombinant measles virus with an engineered deficiency in V protein is capable of inducing more severe cytopathic effects than the wild type, implicating measles virus V protein as an inhibitor of cell death. These findings also suggest that p73-PUMA signaling may be a previously unrecognized arm of cellular innate antiviral immunity.
doi:10.1128/JVI.02400-05
PMCID: PMC1472123  PMID: 16699046

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