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Cases of Middle East respiratory syndrome coronavirus (MERS-CoV) continue to occur, making it one of the WHO´s targets for accelerated vaccine development. One vaccine candidate is based on live-attenuated measles virus (MV) vaccine encoding the MERS-CoV spike glycoprotein (MERS-S). MVvac2-MERS-S(H) induces robust humoral and cellular immunity against MERS-S mediating protection. Here, the induction and nature of immunity after vaccination with MVvac2-MERS-S(H) or novel MVvac2-MERS-N were further characterized. We focused on the necessity for vector replication and the nature of induced T cells, since functional CD8+ T cells contribute importantly to clearance of MERS-CoV. While no immunity against MERS-CoV or MV was detected in MV-susceptible mice after immunization with UV-inactivated virus, replication-competent MVvac2-MERS-S(H) triggered robust neutralizing antibody titers also in adult mice. Furthermore, a significant fraction of MERS CoV-specific CD8+ T cells and MV-specific CD4+ T cells simultaneously expressing IFN-γ and TNF-α were induced, revealing that MVvac2-MERS-S(H) induces multifunctional cellular immunity., Highlights • Measles vaccine virus encoding MERS-CoV N induce N-specific T ell responses. • Only live recombinant vaccine triggers immunity. • MERS-vaccine candidates work also in adult animals. • Multifunctional antigen-specific T cells targeting MERS-S are induced by recombinant MVvac2-MERS-S(H).

An active landslide, affecting a composite sea cliff section of the island of Rugen, Germany was investigated and monitored using terrestrial laser scanning. Biannual surveys were performed from 2006 to 2011. High resolution scans were used to delineate landslide extensions, monitor the spatial and temporal changes, and evaluate the processes of mass wasting along the cliff section. Between May 2006 and May 2011 the multi-temporal analysis documents a strong landward progression of the landslide that resulted in an increase in the planimetric area by 32%. In the same period, 9365 m 3 of soil volume has been removed primarily due to rainfall triggered surface runoff erosion and the resulting slumping. This volume loss correlates with the preferential erosion of weak and unconsolidated sediments building up the major part of the cliff face. This produced a maximum cliff top retreat of 17.32 m with an average retreat rate of 3.46 m yr − 1 . The quantification of the mass wasting processes and cliff top retreat demonstrates the substantial effect of local cliff lithology on slope stability, and supports a prognosis of the near future development of the landslide. Furthermore, the absence of local wave attack indicators or human activity highlights the major role of subaerial processes in the erosion of the sea cliff. These results can be transferred to cliff locations in the urbanised cliff top area nearby that currently suffer similar cliff erosion processes and episodic events of large cliff top retreats. The study therefore provides reliable data for the local authorities in charge of hazard assessment and planning and implementation of adaptive countermeasures.

Differences in fusion activity between measles virus (MV) attenuated, oncolytic strain MVNSe and pathogenic MVwt323 are reflected in amino acid 94 of the fusion (F) proteins. A valine 94 in FNSe (naturally) or Fwt323 (introduced) correlated with enhanced cell–cell fusion activity during transient glycoprotein expression or recombinant MV infections irrespective of the strains’ targeted receptors, whereas the reverse effect was found for methionine 94. Enhanced fusogenicity was determined by weaker glycoprotein interaction and correlated positively with cytotoxicity in both virus strains. Amino acid 94 of F can be used to tailor fusogenicity and cytotoxicity of recombinant MV, while the cellular receptor triggering fusion is not decisive.

To target oncolytic measles viruses (MV) to tumors, we exploit the binding specificity of designed ankyrin repeat proteins (DARPins). These DARPin-MVs have high tumor selectivity while maintaining excellent oncolytic potency. Stability, small size, and efficacy of DARPins allowed the generation of MVs simultaneously targeted to tumor marker HER2/neu and cancer stem cell (CSC) marker EpCAM. For optimization, the linker connecting both DARPins was varied in flexibility and length. Flexibility had no impact on fusion helper activity whereas length had. MVs with bispecific MV-H are genetically stable and revealed the desired double-target specificity. In vitro, the cytolytic activity of bispecific MVs was superior or comparable to mono-targeted viruses depending on the target cells. In vivo, therapeutic efficacy of the bispecific viruses was validated in an orthotopic ovarian carcinoma model revealing an effective reduction of tumor mass. Finally, the power of bispecific targeting was demonstrated on cocultures of different tumor cells thereby mimicking tumor heterogeneity in vitro, more closely reflecting real tumors. Here, bispecific excelled monospecific viruses in efficacy. DARPin-based targeting domains thus allow the generation of efficacious oncolytic viruses with double specificity, with the potential to handle intratumoral variation of antigen expression and to simultaneously target CSCs and the bulk tumor mass.

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 MV vac2 genome, and the respective viruses were rescued (MV vac2 -CoV-S and MV vac2 -CoV-solS). These recombinant MVs were amplified and characterized at passages 3 and 10. The replication of MV vac2 -CoV-S in Vero cells turned out to be comparable to that of the control virus MV vac2 -GFP (encoding green fluorescent protein), while titers of MV vac2 -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 × 10 5 50% tissue culture infective doses of MV vac2 -CoV-S(H) or MV vac2 -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.

International audience; 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.

Differences in fusion activity between measles virus (MV) attenuated, oncolytic strain MV(NSe) and pathogenic MV(wt323) are reflected in amino acid 94 of the fusion (F) proteins. A valine 94 in F(NSe) (naturally) or F(wt323) (introduced) correlated with enhanced cell-cell fusion activity during transient glycoprotein expression or recombinant MV infections irrespective of the strains' targeted receptors, whereas the reverse effect was found for methionine 94. Enhanced fusogenicity was determined by weaker glycoprotein interaction and correlated positively with cytotoxicity in both virus strains. Amino acid 94 of F can be used to tailor fusogenicity and cytotoxicity of recombinant MV, while the cellular receptor triggering fusion is not decisive.

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.

Background The non-pathogenic course of SIV infection in its natural host is characterized by robust viral replication in the absence of chronic immune activation and T cell proliferation. In contrast, acutely lethal enteropathic SIVsmm strain PBj induces a strong immune activation and causes a severe acute and lethal disease in pig-tailed macaques after cross-species transmission. One important pathogenicity factor of the PBj virus is the PBj-Nef protein, which contains a conserved diacidic motif and, unusually, an immunoreceptor tyrosine-based activation motif (ITAM). Results Mutation of the diacidic motif in the Nef protein of the SIVsmmPBj abolishes the acute phenotype of this virus. In vitro, wild-type and mutant PBj (PBj-Nef202/203GG) viruses replicated to similar levels in macaque PBMCs, but PBj-Nef202/203GG no longer triggers ERK mitogen-activated protein (MAP) kinase pathway including an alteration of a Nef-associated Raf-1/ERK-2 multiprotein signaling complex. Moreover, stimulation of IL-2 and down-modulation of CD4 and CD28 were impaired in the mutant virus. Pig-tailed macaques infected with PBj-Nef202/203GG did not show enteropathic complications and lethality as observed with wild-type PBj virus, despite efficient replication of both viruses in vivo. Furthermore, PBj-Nef202/203GG infected animals revealed reduced T-cell activation in periphery lymphoid organs and no detectable induction of IL-2 and IL-6. Conclusions In sum, we report here that mutation of the diacidic motif in the PBj-Nef protein abolishes disease progression in pig-tailed macaques despite efficient replication. These data suggest that alterations in the ability of a lentivirus to promote T cell activation and proliferation can have a dramatic impact on its pathogenic potential.