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Patients with cancer frequently receive immune-checkpoint inhibitors (ICIs), which may modulate immune responses to COVID-19 vaccines. Recently, cytokine release syndrome (CRS) was observed in a patient with cancer who received BTN162b2 vaccination under ICI treatment. Here, we analyzed adverse events and serum cytokines in patients with 23 different tumors undergoing (n = 64) or not undergoing (n = 26) COVID-19 vaccination under ICI therapy in a prospectively planned German single-center cohort study (n = 220). We did not observe clinically relevant CRS (≥grade 2) after vaccination (95% CI 0-5.6%; Common Terminology of Adverse Events v.5.0) in this small cohort. Within 4 weeks after vaccination, serious adverse events occurred in eight patients (12.5% 95% CI 5.6-23%): six patients were hospitalized due to events common under cancer therapy including immune related adverse events and two patients died due to conditions present before vaccination. Despite absence of CRS symptoms, a set of pairwise-correlated CRS-associated cytokines, including CXCL8 and interleukin-6 was1.5-fold upregulated in 40% (95% CI 23.9-57.9%) of patients after vaccination. Hence, elevated cytokine levels are common and not sufficient to establish CRS diagnosis.

Oncolytic virotherapy is an emerging treatment option for numerous cancers, with several virus families currently being evaluated in clinical trials. More specifically, vaccine-strain measles virus has arisen as a promising candidate for the treatment of different tumour types in several early clinical trials. Replicating viruses, and especially RNA viruses without proofreading polymerases, can rapidly adapt to varying environments by selecting quasispecies with advantageous genetic mutations. Subsequently, these genetic alterations could potentially weaken the safety profile of virotherapy. In this study, we demonstrate that, following an extended period of virus replication in producer or cancer cell lines, the quasispecies consensus sequence of vaccine strain-derived measles virus accrues a remarkably small number of mutations throughout the nonsegmented negative-stranded RNA genome. Interestingly, we detected a nonrandom distribution of genetic alterations within the genome, with an overall decreasing frequency of mutations from the 3' genome start to its 5' end. Comparing the serially passaged viruses to the parental virus on producer cells, we found that the acquired consensus mutations did not drastically change viral replication kinetics or cytolytic potency. Collectively, our data corroborate the genomic stability and excellent safety profile of oncolytic measles virus, thus supporting its continued development and clinical translation as a promising viro-immunotherapeutic.

Measles viruses derived from the live-attenuated Edmonton-B vaccine lineage are currently investigated as novel anti-cancer therapeutics. In this context, tumor specificity and oncolytic potency are key determinants of the therapeutic index. Here, we describe a systematic and comprehensive analysis of a recently developed post-entry targeting strategy based on the incorporation of microRNA target sites (miRTS) into the measles virus genome. We have established viruses with target sites for different microRNA species in the 3′ untranslated regions of either the N, F, H, or L genes and generated viruses harboring microRNA target sites in multiple genes. We report critical importance of target-site positioning with proximal genomic positions effecting maximum vector control. No relevant additional effect of six versus three miRTS copies for the same microRNA species in terms of regulatory efficiency was observed. Moreover, we demonstrate that, depending on the microRNA species, viral mRNAs containing microRNA target sites are directly cleaved and/or translationally repressed in presence of cognate microRNAs. In conclusion, we report highly efficient control of measles virus replication with various miRTS positions for development of safe and efficient cancer virotherapy and provide insights into the mechanisms underlying microRNA-mediated vector control. Keywords: measles virus, oncolytic viruses, vector engineering, vector safety, microRNAs, microRNA target sites, post-entry targeting

Viruses from the diverse family of Paramyxoviridae include important pathogens and are applied in gene therapy and for cancer treatment. The Tupaia paramyxovirus (TPMV), isolated from the kidney of a tree shrew, does not infect human cells and neutralizing antibodies against other Paramyxoviridae do not cross-react with TPMV. Here, we present a vector system for de novo generation of infectious TPMV that allows for insertion of additional genes as well as targeting using antibody single-chain variable fragments. We show that the recombinant TPMV specifically infect cells expressing the targeted receptor and replicate in human cells. This vector system provides a valuable tool for both basic research and therapeutic applications.

Oncolytic viruses are emerging novel anti-cancer therapeutics currently tested in phase I – III clinical trials. Recent data suggests virotherapy to be well tolerated with adverse events and oncolytic activities being dose-dependent. This data along with improvements in the manufacturing process of oncolytic viruses drives further escalation of administered therapeutic doses. Hence, there is a need for viruses which maintain low toxicity profiles while being administered at much higher doses. Here, we show our optimized modification strategy for oncolytic measles viruses (MV) using microRNA target sites. Furthermore, we will discuss our results on the mechanistic basis of vector control.We have previously shown that the cellular tropism of MV can be modified by insertion of target sequences for microRNAs specifically lost during malignant transformation. Using this approach, we could achieve attenuation of microRNA-controlled MV in cell lines or nontransformed human tissue expressing cognate microRNAs, while full anti-tumor efficacy could be demonstrated in xenograft mouse models. In addition, we have shown the possibility of multi-tissue detargeting by integration of target sites for multiple microRNAs within a single virus.To investigate whether microRNA-mediated vector control is based on translational repression or cleavage of targeted RNAs, we used rapid amplification of cDNA ends (RACE) followed by sequencing. We could show that viral mRNAs containing microRNA target sequences (miRTS) are directly cleaved in presence of cognate microRNAs. Moreover, we describe the genetic engineering and analysis of MV with miRTS in the 3’UTR of the N, F, H or L gene. Insertion of the miRTS box into the N 3’UTR resulted in the tightest regulation, while the F and H position showed an intermediate phenotype and the L position resulted in the lowest level of vector control. To explore additional strategies possibly enhancing microRNA-based vector control, we have analyzed the regulation efficiency after insertion of the same microRNA target site box into the 3’ UTR of either a single gene (NmiRTS) or multiple MV genes (NmiRTS+ FmiRTS, NmiRTS+ LmiRTS). We conclude that a careful miRTS insertion pattern profoundly enhances the efficiency of vector control.This data provides the basis for the rational design of tightly controlled, microRNA-sensitive MV for safe and efficient cancer virotherapy.

Oncolytic viruses directly reduce tumor burden by selective lytic replication in malignant cells, which designates these vectors as a potent platform for targeted cancer therapy. Lately the focus of oncolytic research has shifted towards the immunostimulatory potential of viral infection within the tumor microenvironment. Oncolytic viruses can be considered to act as an autologous tumor vaccine with the capacity to induce a tumor-specific T cell response. We hypothesize that oncolytic measles virus (MV) expressing a tumor-associated antigen (TAA) can specifically support T cell activation and persistence after adoptive transfer of TAA-specific T cells. We modified the Schwarz vaccine-derived measles virus to encode either OVA as a model antigen or one of the endogenous melanoma antigens trp-2 or gp100. TAA insertion did not impede viral replication and oncolysis compared to the parental virus. For in vivo studies, we have generated a new immunocompetent mouse model for MV oncolysis, B16-hCD46. Transduction of hCD46-positive B16 tumor cells with the novel viruses induces a strong virus-mediated expression of the tumor antigens, serving as a specific and lasting stimulus for adoptively transferred T cells. With the increasing availability of tumor sequencing data, this study has direct implications for future clinical trials of personalized immunovirotherapy.

Lentiviral vectors (LVs) are vectors of choice for many gene therapy applications since they mediate long term gene expression and can transduce dividing and non-dividing cells. Recently, efficient targeting of LVs pseudotyped with the measles virus (MV) glycoproteins has been reported. However, MV antibodies in patients might limit the clinical use of these vectors. Thus, aim of this study was the development of targeted LVs pseudotyped with the glycoproteins of Tupaia paramyxovirus (TPMV). Since this animal paramyxovirus does not infect humans, no TPMV antibodies in patients are expected. For efficient incorporation in LVs, the TPMV glycoproteins, the hemagglutinin (H) and fusion (F) protein, were modified by truncation of their cytoplasmic tails. Targeting was achieved by displaying a single-chain antibody against the B cell surface marker CD20 on the H protein. The modified proteins were biochemically characterized and tested for their functionality. Unexpectedly, it was observed that an additional proteolytic cleavage of the F protein occurs during activation, resulting in the fragments F1a, F1b and F2. The newly identified fragment F1a was detected in virions and in supernatant of transfected cells. The F1a/F1b cleavage site was mapped and a cysteine protease was identified as likely activating protease. The data indicate that F protein processing is more complex than expected. After characterization, the modified TPMV glycoproteins were screened in all combinations for their ability to form functional pseudotyped LVs. Most efficient pseudotype formation was achieved with CT truncations of 80 amino acids (aa) for H (HΔ80αCD20) and 32 aa for F (FΔ32) (titers ~ 106 t.u./ml). The resulting vectors selectively transduced CD20-positive cells in a mixed cell population. Furthermore, they mediated efficient gene transfer into activated and quiescent primary human B cells. Neutralization assays showed that TPMV-pseudotyped vectors were not neutralized by human sera containing MV antibodies. In conclusion, it was demonstrated that targeted LVs pseudotyped with TPMV glycoproteins can be generated and escape neutralization by MV antibodies. Remarkably, the vectors are able to efficiently transduce even quiescent B cells. Hence, they might be a valuable vector choice when systemic application of targeted lentiviral vectors in humans is required.

AIM: To explore dendritic cells (DCs) multiple functions in immune modulation. METHODS: We used bone-marrow derived dendritic cells from BALB/c mice pulsed with pseudo particles from the hepatitis C virus to vaccinate naive BALB/c mice. Hepatitis C virus (HCV) pseudo particles consist of the genotype 1b derived envelope proteins E1 and E2, covering a non-HCV core structure. Thus, not a single epitope, but the whole “viral surface” induces immunogenicity. For vaccination, mature and activated DC were injected subcutaneously twice. RESULTS: Humoral and cellular immune responses measured by enzyme-linked immunosorbent assay and interferon-gamma enzyme-linked immunosorbent spot test showed antibody production as well as T-cells directed against HCV. Furthermore, T-cell responses confirmed two highly immunogenic regions in E1 and E2 outside the hypervariable region 1. CONCLUSION: Our results indicate dendritic cells as a promising vaccination model for HCV infection that should be evaluated further.

Eradication of chronic Hepatitis B virus (HBV) infection, marked by HBs seroconversion, is very rarely achieved by treatment with nucleoside and nucleotide analogs. Therapeutic cell based approaches, like interferon therapy, have a higher chance of seroconversion. Dendritic cells (DC) are key players in the cellular immune response and have been shown to play an important role in controlling HBV infection. In this study, the potential of ex vivo activated DC to induce specific immune responses against HBV was examined. DC derived from bone-marrow of BALB/c or C56BL/6 mice were pulsed with HBV subviral particles (HBVsvp), derived from the HepG2.2.15 cell line. HepG2.2.15 produces subviral particles consisting of the HBc and HBs proteins. Thus, the entire "viral surface" is presented to DC to induce an immune reaction. In vitro pulsation with HBVsvp successfully activated bone-marrow derived DC, demonstrated by FACS analysis showing increased MHCII, CD 86 and CCR-7. Immunization of mice, via subcutaneous injection of the activated DC, induced HBV specific immune reactions which were measured by ELISA, ELISPOT and T-cell proliferation analysis. Vaccination with ex vivo activated DC may be a promising tool for therapeutic or prophylactic approaches against the Hepatitis B virus.

Current cancer gene therapies aim at the induction of systemic antitumor immune responses. Tumors may deliver antigens to T-cells, but may lack the costimulatory signals necessary for mounting an effective response. The purpose of this study was to evaluate the efficacy of an adenoviral delivery of the B7-H3 costimulatory molecule in mice to induce antitumor immune responses. Colon cancers were established by orthotopic injection of syngeneic colon cancer cells into the cecum on Balb/c mice. After two weeks, these mice were treated by intratumoral injection of an adenovirus expressing mouse B7-H3 (Ad-B7-H3-GFP) or a control virus (Ad-GFP). Ad-B7-H3-GFP treatment resulted in a reduction of tumor size compared to the controls. In addition, the occurrence of secondary metastasis was significantly reduced in B7-H3 treated mice compared to control animals (lymph node 7/10 vs. 10/10; liver 2/10 vs. 8/10, por=0.05). Ad-B7-H3-GFP treated animals showed significantly higher frequencies of tumor-specific interferon-gamma producing CD8+ T-cells (por=0.05) and higher interleukin-12 levels (por=0.01) than control animals. This study demonstrates that adenoviral B7-H3 transfer is able to induce a specific cellular antitumor immune response leading to primary tumor regression and reduction of secondary metastasis in vivo.

Current cancer gene therapies aim at the induction of systemic antitumor immune responses. Tumors may deliver antigens to T-cells, but may lack the costimulatory signals necessary for mounting an effective response. The purpose of this study was to evaluate the efficacy of an adenoviral delivery of the B7-H3 costimulatory molecule in mice to induce antitumor immune responses. Colon cancers were established by orthotopic injection of syngeneic colon cancer cells into the cecum on Balb/c mice. After two weeks, these mice were treated by intratumoral injection of an adenovirus expressing mouse B7-H3 (Ad-B7-H3-GFP) or a control virus (Ad-GFP). Ad-B7-H3-GFP treatment resulted in a reduction of tumor size compared to the controls. In addition, the occurrence of secondary metastasis was significantly reduced in B7-H3 treated mice compared to control animals (lymph node 7/10 vs. 10/10; liver 2/10 vs. 8/10, p

Surrogate infections with HCV-recombinant vaccinia viruses (HCV-rVV) are a standard method to test the efficacy of hepatitis C virus (HCV) vaccine candidates in the mouse model. We established a panel of 16 HCV-rVV expressing the nonstructural protein 3 (NS3) of HCV genotypes 1a, 1b, 2, 3 and 4. Mice immunized with recombinant NS3 protein derived from HCV genotype 1b were challenged with the rVV. rVV-titers decreased up to 54-fold after subtype 1b challenge and up to 8.5-fold after subtype 1a challenge. No change was detected for genotype 2, 3, or 4. Our model is a convenient and reliable tool to analyze the induction of cross-genotype immunity by experimental vaccination of mice.