Michael D. Mühlebach

Adherens junction protein nectin-4 is the epithelial receptor for measles virus

Measles virus is an aerosol-transmitted virus that affects more than 10 million children each year and accounts for approximately 120,000 deaths. Although it was long believed to replicate in the respiratory epithelium before disseminating, it was recently shown to infect initially macrophages and dendritic cells of the airways using signalling lymphocytic activation molecule family member 1 (SLAMF1; also called CD150) as a receptor. These cells then cross the respiratory epithelium and transport the infection to lymphatic organs where measles virus replicates vigorously. How and where the virus crosses back into the airways has remained unknown. On the basis of functional analyses of surface proteins preferentially expressed on virus-permissive human epithelial cell lines, here we identify nectin-4 (ref. 8; also called poliovirus-receptor-like-4 (PVRL4)) 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 measles virus entry and non-cytopathic lateral spread in well-differentiated primary human airway epithelial sheets infected basolaterally. It is downregulated in infected epithelial cells, including those of macaque tracheae. Although other viruses use receptors to enter hosts or transit through their epithelial barriers, we suggest that measles virus targets nectin-4 to emerge in the airways. Nectin-4 is a cellular marker of several types of cancer, which has implications for ongoing measles-virus-based clinical trials of oncolysis.

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 pseudotyped 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.

DARPins: An Efficient Targeting Domain for Lentiviral Vectors

We have recently developed a retargeting system for lentiviral vectors (LVs) that relies on the pseudotyping of LVs with engineered measles virus (MV) glycoproteins (hemagglutinin (H) and fusion protein (F)). Specificity is provided through display of a single-chain antibody (scFv) as targeting domain by fusion to the MV-H protein. As an alternative to scFv, designed ankyrin repeat proteins (DARPins) can be selected to become high-affinity binders to any kind of target molecule. In this study six HER2/neu-specific DARPins exhibiting different affinities and binding to different HER2/neu epitopes were applied as targeting domains. All H-DARPin fusion proteins were efficiently expressed on the cell surface. Upon coexpression with F, syncytia formation was observed in HER2/neu positive cells only and correlated directly with the HER2/neu receptor density. All H-DARPin proteins incorporated into LVs, albeit at different levels. The vectors only transduced HER2/neu-positive cells, while HER2/neu-negative cells remained untransduced. Highest titers were observed with one particular DARPin binding to the membrane distal domain of HER2/neu with medium affinity. When applied in vivo systemically, HER2/neu-targeted LVs showed exclusive gene expression in HER2/neu positive tumor tissue, while vesicular stomatitis virus-glycoprotein (VSV-G) pseudotyped vectors mainly transduced cells in spleen and liver. Thus, DARPins are a promising alternative to scFvs for retargeting of LVs.

Lentiviral vectors with measles virus glycoproteins - dream team for gene transfer?

Lentiviral vectors are potent gene transfer vehicles frequently applied in research and lately also in clinical applications. Recent improvements have come from combining lentiviral vectors with engineered envelope proteins, which now allow targeting of cell entry to any cell population of interest, as well as the transduction of quiescent cells of the haematopoietic system. We propose that measles virus envelope glycoproteins are especially well suited for this purpose because they can mediate pH-independent cell entry at the cell surface membrane and can induce cytoskeleton rearrangements that facilitate the transport of lentiviral core particles to the cell nucleus. Lentiviral vectors pseudotyped with measles virus glycoproteins are expected to improve the safety and efficacy of gene transfer to human cells.

Pseudotyping lentiviral vectors with the wild-type measles virus glycoproteins improves titer and selectivity

We pseudotyped HIV-1 vectors with cytoplasmic tail-truncated envelope glycoproteins of a wild-type (WT) measles virus (MV). The particles entered the lymphatic cells exclusively through the signaling lymphocyte activation molecule (SLAM, CD150), whereas particles pseudotyped with the MV vaccine strain glycoproteins also recognized the ubiquitous membrane cofactor protein (CD46) as receptor and had less specific cell entry. MVWT-HIV vectors reached titers of 108 t.u. ml−1, which were up to 10-fold higher than those of MVVac-HIV vectors, and discriminated between SLAM-positive and SLAM-negative cells, also in mixed cell cultures. As these vectors transduce primary human cells more efficiently than vesicular stomatitis virus-G pseudotyped vectors do, they are promising candidates for gene transfer to human lymphocytes and certain epithelial cells.

Specific Elimination of CD133+ Tumor Cells with Targeted Oncolytic Measles Virus

Tumor-initiating cells (TIC) are critical yet evasive targets for the development of more effective antitumoral strategies. The cell surface marker CD133 is frequently used to identify TICs of various tumor entities, including hepatocellular cancer and glioblastoma. Here, we describe oncolytic measles viruses (MV) retargeted to CD133. The viruses, termed MV-141.7 and MV-AC133, infected and selectively lysed CD133(+) tumor cells. Both viruses exerted strong antitumoral effects on human hepatocellular carcinoma growing subcutaneously or multifocally in the peritoneal cavity of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Notably, the CD133-targeted viruses were more effective in prolonging survival than the parental MV-NSe, which is currently assessed as oncolytic agent in clinical trials. Interestingly, target receptor overexpression or increased spreading kinetics through tumor cells were excluded as being causative for the enhanced oncolytic activity of CD133-targeted viruses. MV-141.7 was also effective in mouse models of orthotopic glioma tumor spheres and primary colon cancer. Our results indicate that CD133-targeted measles viruses selectively eliminate CD133(+) cells from tumor tissue, offering a key tool for research in tumor biology and cancer therapy.

DARPin-targeting of Measles Virus: Unique Bispecificity, Effective Oncolysis, and Enhanced Safety

Oncolytic virotherapy is an emerging treatment modality that uses replication-competent viruses to destroy cancers. Many naturally occurring viruses have a preferential, although nonexclusive, tropism for tumors and tumor cells. In addition, specific targeting of cancer cells can be achieved at the virus entry level. We optimized retargeting of cell entry by elongating the measles virus attachment protein with designed ankyrin repeat proteins (DARPins), while simultaneously ablating entry through the natural receptors. DARPin-targeted viruses were strongly attenuated in off-target tissue, thereby enhancing safety, but completely eliminated tumor xenografts. Taking advantage of the unique properties of DARPins of being fused without generating folding problems, we generated a virus simultaneous targeting two different tumor markers. The bispecific virus retained the original oncolytic efficacy, while providing proof of concept for a strategy to counteract issues of resistance development. Thus, DARPin-targeting opens new prospects for the development of personalized, targeted therapeutics.

A Highly Immunogenic and Protective Middle East Respiratory Syndrome Coronavirus Vaccine Based on a Recombinant Measles Virus Vaccine Platform

ABSTRACT In 2012, the first cases of infection with the Middle East respiratory syndrome coronavirus (MERS-CoV) were identified. Since then, more than 1,000 cases of MERS-CoV infection have been confirmed; infection is typically associated with considerable morbidity and, in approximately 30% of cases, mortality. Currently, there is no protective vaccine available. Replication-competent recombinant measles virus (MV) expressing foreign antigens constitutes a promising tool to induce protective immunity against corresponding pathogens. Therefore, we generated MVs expressing the spike glycoprotein of MERS-CoV in its full-length (MERS-S) or a truncated, soluble variant of MERS-S (MERS-solS). The genes encoding MERS-S and MERS-solS were cloned into the vaccine strain MVvac2 genome, and the respective viruses were rescued (MVvac2-CoV-S and MVvac2-CoV-solS). These recombinant MVs were amplified and characterized at passages 3 and 10. The replication of MVvac2-CoV-S in Vero cells turned out to be comparable to that of the control virus MVvac2-GFP (encoding green fluorescent protein), while titers of MVvac2-CoV-solS were impaired approximately 3-fold. The genomic stability and expression of the inserted antigens were confirmed via sequencing of viral cDNA and immunoblot analysis. In vivo, immunization of type I interferon receptor-deficient (IFNAR−/−)-CD46Ge mice with 2 × 105 50% tissue culture infective doses of MVvac2-CoV-S(H) or MVvac2-CoV-solS(H) in a prime-boost regimen induced robust levels of both MV- and MERS-CoV-neutralizing antibodies. Additionally, induction of specific T cells was demonstrated by T cell proliferation, antigen-specific T cell cytotoxicity, and gamma interferon secretion after stimulation of splenocytes with MERS-CoV-S presented by murine dendritic cells. MERS-CoV challenge experiments indicated the protective capacity of these immune responses in vaccinated mice. IMPORTANCE Although MERS-CoV has not yet acquired extensive distribution, being mainly confined to the Arabic and Korean peninsulas, it could adapt to spread more readily among humans and thereby become pandemic. Therefore, the development of a vaccine is mandatory. The integration of antigen-coding genes into recombinant MV resulting in coexpression of MV and foreign antigens can efficiently be achieved. Thus, in combination with the excellent safety profile of the MV vaccine, recombinant MV seems to constitute an ideal vaccine platform. The present study shows that a recombinant MV expressing MERS-S is genetically stable and induces strong humoral and cellular immunity against MERS-CoV in vaccinated mice. Subsequent challenge experiments indicated protection of vaccinated animals, illustrating the potential of MV as a vaccine platform with the potential to target emerging infections, such as MERS-CoV.

The Measles Virus Fusion Protein Transmembrane Region Modulates Availability of an Active Glycoprotein Complex and Fusion Efficiency

ABSTRACT 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.

Liver Cancer Protease Activity Profiles Support Therapeutic Options with Matrix Metalloproteinase–Activatable Oncolytic Measles Virus

Primary and secondary cancers of the liver are a significant health problem with limited treatment options. We sought here to develop an oncolytic measles virus (MV) preferentially activated in liver tumor tissue, thus reducing infection and destruction of healthy tissue. We documented that in primary tumor tissue, urokinase-type plasminogen activator and especially matrix metalloproteinase-2 (MMP-2) are significantly more active than in adjacent nontumorous tissue. We then generated variants of the MV fusion protein by inserting different MMP substrate motifs at the protease cleavage site and identified the motif PQGLYA as the most efficient cleavage site as determined by syncytia formation on protease-positive tumor cells. The corresponding MMP-activatable oncolytic MV-MMPA1 virus was rescued and shown to be strongly restricted on primary human hepatocytes and healthy human liver tissue, while remaining as effective as the parental MV in the tumor tissue sections. Our findings underline the clinical potency of the MMP activation concept as a strategy to generate safer oncolytic viruses for the treatment of primary and secondary cancers of the liver.