Mcl-1 Antisense Therapy Chemosensitizes Human Melanoma in a SCID Mouse Xenotransplantation Model

It is well established that high expression of the antiapoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL can significantly contribute to chemoresistance in a number of human malignancies. Much less is known about the role the more recently described Bcl-2 family member Mcl-1 might play in tumor biology and resistance to chemotherapy. Using an antisense strategy, we here address this issue in melanoma, a paradigm of a treatment-resistant malignancy. After in vitro proof of principle supporting an antisense mechanism of action with specific reduction of Mcl-1 protein as a consequence of nuclear uptake of the Mcl-1 antisense oligonucleotides employed, antisense and universal control oligonucleotides were administered systemically in combination with dacarbazine in a human melanoma SCID mouse xenotransplantation model. Dacarbazine, available now for more than three decades, still remains the most active single agent for treatment of advanced melanoma. Mcl-1 antisense oligonucleotides specifically reduced target protein expression as well as the apoptotic threshold of melanoma xenotransplants. Combined Mcl-1 antisense oligonucleotide plus dacarbazine treatment resulted in enhanced tumor cell apoptosis and led to a significantly reduced mean tumor weight (mean 0.16 g, 95% confidence interval 0.08–0.26) compared to the tumor weight in universal control oligonucleotide plus dacarbazine treated animals (mean 0.35 g, 95% confidence interval 0.2–0.44) or saline plus dacarbazine treated animals (mean 0.39 g, 95% confidence interval 0.25–0.53). We thus show that Mcl-1 is an important factor contributing to the chemoresistance of human melanoma in vivo. Antisense therapy against the Mcl-1 gene product, possibly in combination with antisense strategies targeting other antiapoptotic Bcl-2 family members, appears to be a rational and promising approach to help overcome treatment resistance of malignant melanoma

Single HA2 Mutation Increases the Infectivity and Immunogenicity of a Live Attenuated H5N1 Intranasal Influenza Vaccine Candidate Lacking NS1

Our finding suggests that an efficient intranasal vaccination with a live attenuated H5N1 virus may require a certain level of pH and temperature stability of HA in order to achieve an optimal virus uptake by the nasal epithelial cells and induce a sufficient immune response. The pH of the activation of the H5 HA protein may play a substantial role in the infectivity of HPAIVs for mammals

Preclinical Evaluation of a Replication-Deficient Intranasal ΔNS1 H5N1 Influenza Vaccine

We developed a novel intranasal influenza vaccine approach that is based on the construction of replication-deficient vaccine viruses that lack the entire NS1 gene (ΔNS1 virus). We previously showed that these viruses undergo abortive replication in the respiratory tract of animals. The local release of type I interferons and other cytokines and chemokines in the upper respiratory tract may have a “self-adjuvant effect”, in turn increasing vaccine immunogenicity. As a result, ΔNS1 viruses elicit strong B- and T- cell mediated immune responses.), one dose of vaccine delivered intranasally was sufficient for the induction of antibodies against homologous A/Vietnam/1203/04 and heterologous A/Indonesia/5/05 H5N1 strains.Our findings show that intranasal immunization with the replication deficient H5N1 ΔNS1 vaccine candidate is sufficient to induce a protective immune response against H5N1 viruses. This approach might be attractive as an alternative to conventional influenza vaccines. Clinical evaluation of ΔNS1 pandemic and seasonal influenza vaccine candidates are currently in progress

PLOS One / Attenuated Recombinant Influenza A Virus Expressing HPV16 E6 and E7 as a Novel Therapeutic Vaccine Approach

Persistent infection with high-risk human papillomavirus (HPV) types, most often HPV16 and HPV18, causes all cervical and most anal cancers, and a subset of vulvar, vaginal, penile and oropharyngeal carcinomas. Two prophylactic virus-like particle (VLPs)-based vaccines, are available that protect against vaccine type-associated persistent infection and associated disease, yet have no therapeutic effect on existing lesions or infections. We have generated recombinant live-attenuated influenza A viruses expressing the HPV16 oncogenes E6 and E7 as experimental immunotherapeutic vaccine candidates. The influenza A virus life cycle lacks DNA intermediates as important safety feature. Different serotypes were generated to ensure efficient prime and boost immunizations. The immune response to vaccination in C57BL/6 mice was characterized by peptide ELISA and IFN- ELISpot, demonstrating induction of cell-mediated immunity to HPV16 E6 and E7 oncoproteins. Prophylactic and therapeutic vaccine efficacy was analyzed in the murine HPV16-positive TC-1 tumor challenge model. Subcutaneous (s.c.) prime and boost vaccinations of mice with recombinant influenza A serotypes H1N1 and H3N2, followed by challenge with TC-1 cells resulted in complete protection or significantly reduced tumor growth as compared to control animals. In a therapeutic setting, s.c. vaccination of mice with established TC-1 tumors decelerated tumor growth and significantly prolonged survival. Importantly, intralesional vaccine administration induced complete tumor regression in 25% of animals, and significantly reduced tumor growth in 50% of mice. These results suggest recombinant E6E7 influenza viruses as a promising new approach for the development of a therapeutic vaccine against HPV-induced disease.(VLID)492428

Establishment of a Chimeric, Replication-Deficient Influenza A Virus Vector by Modulation of Splicing Efficiency▿

Segment 8 of the influenza A virus codes for two proteins (NS1 and NS2/NEP) via splicing. Here, we developed a viral vector expressing a cytokine or chemokine instead of the interferon antagonist NS1. To achieve both the desired genetic stability and high transgene expression levels, NS2/NEP mRNA splicing efficacy had to be fine-tuned by modification of splicing elements. Expression levels of secreted foreign proteins could be further enhanced by fusing the N-terminal 13 amino acids of NS1 with an IgK-derived secretion signal peptide. Thus, the first start codon was used for translation initiation of both NS2/NEP and the foreign protein

Recombinant 16E6E7 and 16E6E7m influenza viruses elicit HPV-specific CTL responses in mice.

<p>Four groups of mice (4 per group) were primed with PBS (mock), H1N1 16E6E7 virus, H1N1 16E6E7m virus, parental H1N1 delNS1 virus, or 16L1/L2-E7 VLPs, and boosted 10 days later with PBS, corresponding H3N2 serotypes, or VLPs. Mice were sacrificed <b>A</b> 10 days or <b>B</b> 30 days after boosting, splenocytes were isolated and stimulated in triplicates for 24 h with antigen peptides, SEA or medium alone. For mock and influenza A virus-vaccinated animals, NP_311-325 peptide, for mice immunized with 16L1/L2-E7 VLP, 16L1_165-173 peptide were used as positive controls. Shown are numbers of IFN-γ spots, counted under a light microscope, and plotted as mean ± SD of triplicate wells. One representative experiment of two is shown. Statistically significant differences for 16E6E7 or 16E6E7m compared to mock are indicated as asterisks (*** p<0.001, ** p<0.01, * p<0.05, ns not significant).</p

Infection rate and replication kinetics of influenza A viruses.

<p><b>A</b>) Immune staining of infected cells. Cells were infected at MOI 1, 0.1 or 0.01 respectively. Staining for influenza A virus NP antigen expression was performed 24 hours post infection. <b>B</b>) Cells were infected with IVR-116, delNS1, or delNS1-IL-15 (MOI 0.1). At the given time points, aliquots of the supernatants were taken and the TCID₅₀/mL was determined.</p

Mice s.c. vaccinated with recombinant 16E6E7 or 16E6E7m influenza viruses are partially protected from TC-1 induced tumors.

<p><b>A</b> Schematic illustration of the experimental set up. <b>B</b> Groups (n = 8) of C57BL/6 female mice were vaccinated s.c. with PBS (mock), H1N1 16E6E7, H1N1 16E6E7m, parental virus (delNS1), or HPV16 L1/L2-E7 VLP, boosted 20 days later either with PBS, the corresponding H3N2 influenza serotype, or VLPs and challenged s.c. with 5_x10⁴ TC-1 cells 10 days later. Animals were monitored once per week. Mean tumor volumes ± SEM of individual animals of one representative experiment of two are shown. Statistical significances of differences recorded for immunized groups and mock treated animals, or immunized groups and parental virus treated animals, were calculated by 1-way ANOVA and subsequent Dunnett’s multiple comparison test. Days after TC-1 tumor inoculation are indicated.</p