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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).

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.

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.

The concept of tumor vaccination is based on the induction of robust immune responses against tumor-associated antigens (TAA) selectively expressed within the tumor. Whereas in the case of foreign antigens an immune response can be induced quite easily, induction of appropriate immunity against self-antigens needs potent antigen presentation to break immune tolerance. Tumor vaccines may be further improved by utilizing the properties of oncolytic viruses (OVs), that selectively replicate within and destroy tumor tissue and may further enhance the vaccination effects by supplying danger signals in situ and stimulating the immune system. Immunogenic OVs additionally delivering a specific TAA by in situ antigen presentation can break immmune tolerance, induce a specifically redirected immune response, and thereby may increase therapeutic efficacy. Attenuated measles virus (MV) derived from a vaccine strain is currently tested as oncolytic virotherapeutic in clinical trials. Recombinant MVs additionally reveal excellent vaccine characteristics per se, inducing potent and long lasting immune responses against endogenous and foreign antigens, if the latter are additionally expressed by the recombinant virus. Therefore, we develop and aim to validate novel prototypic replicating MV for simultaneous oncolysis and in situ tumor vaccination. For that purpose, a well characterized tumor-confined TAA of the claudin family is used as vaccination target antigen (GNTP01). Different antigen formats or epitopes of GNTP01 were cloned into the MV genome and recombinant viruses were generated, which express those epitopes in different amounts as expected due to positional effects of the additional transcription units either behind the P (post P) or the H (post H) gene cassettes of Moraten vaccine strain-derived MVvac2 genome, presented on in situ produced particles or not. Based on these data, selected viruses expressing different antigen formats have been chosen for further characterization. The selected viruses were amplified, and unimpaired growth and genetic stability of the inserted TAA were demonstrated. To test their immunogenic properties, MV-susceptible IFNAR-/--CD46Ge mice have been immunized with the different viruses, and humoral as well as cellular immune responses are currently analyzed. In parallel, an immune competent, syngeneic tumor model has been established by employment of transgenic C57/BL6-derived tumor cell lines expressing the target antigen as well as MV-receptors. For three different transgenic tumor cell lines antigen expression and tumorigenicity in MV-susceptible animals has been confirmed. Such tumor-bearing animals will be used for analysis of induced immune responses, anti-tumoral activity, and therapeutic efficacy after immunization with selected viruses that have been shown to induce significant humoral or cellular immune responses. Once the proof-of-concept has been established this innovative oncolytic virus-based approach holds the potential for a novel therapeutic technology platform that synergistically combines the direct tumor-lytic properties of recombinant MVs with the therapeutic induction of potent immune responses depending on the patient´s TAA expression profile. Citation Format: Stefan Hutzler, Stephanie Erbar, Robert A. Jabulowsky, Tim Beissert, Jan Hanauer, Oezlem Tuereci, Rita Mitnacht-Kraus, Sebastian Kreiter, Mustafa Diken, Cedrik M. Britten, Ugur Sahin, Michael D. Muehlebach. Oncolytic measles virus for tumor vaccination. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B040.