Outcome of experimental porcine circovirus type 1 infections in mid-gestational porcine foetuses

Background: Porcine circovirus type 1 (PCV1) has been described as a non-cytopathic contaminant of the PK-15 cell line. Several experimental infections with PCV1 failed to reproduce disease in pigs. Therefore, PCV1 is generally accepted as non-pathogenic to pigs. To our knowledge, nothing is known about the outcome of PCV1 infections in porcine foetuses. This was examined in the present study. Results: Nine foetuses from three sows were inoculated at 55 days of gestation: three with 10(4.3) TCID50 of the PCV1 cell culture strain ATCC-CCL33, three with 10(4.3) TCID50 of the PCV1 field strain 3384 and three with cell culture medium (mock-inoculated). At 21 days post-inoculation, all 6 PCV1-inoculated and all 3 mock-inoculated foetuses had a normal external appearance. Microscopic lesions characterized by severe haemorrhages were observed in the lungs of two foetuses inoculated with CCL33. High PCV1 titres (up to 10(4.7) TCID50/g tissue) were found in the lungs of the CCL33-inoculated foetuses. All other organs of the CCL33-inoculated foetuses and all the organs of the 3384-inoculated foetuses were negative (< 10(1.7) TCID50/g tissue) by virus titration. PCV1-positive cells (up to 121 cells/10 mm(2) in CCL33-inoculated foetuses and up to 13 cells/10 mm(2) in 3384-inoculated foetuses) were found in the heart, lungs, spleen, liver, thymus and tonsils. PCR and DNA sequencing of Rep recovered CCL33 or 3384 sequences from CCL33- or 3384-inoculated foetuses, respectively. Conclusions: From this study, it can be concluded that cell culture PCV1 can replicate efficiently and produce pathology in the lungs of porcine foetuses inoculated at 55 days of foetal life

Overcoming tumor resistance by heterologous adeno-poxvirus combination therapy

Successful cancer control relies on overcoming resistance to cell death and on activation of host antitumor immunity. Oncolytic viruses are particularly attractive in this regard, as they lyse infected tumor cells and trigger robust immune responses during the infection. However, repeated injections of the same virus promote antiviral rather than antitumor immunity and tumors may mount innate antiviral defenses to restrict oncolytic virus replication. In this article, we have explored if alternating the therapy virus could circumvent these problems. We demonstrate in two virus-resistant animal models a substantial delay in antiviral immune- and innate cellular response induction by alternating injections of two immunologically distinct oncolytic viruses, adenovirus, and vaccinia virus. Our results are in support of clinical development of heterologous adeno-/vaccinia virus therapy of cancer.Peer reviewe

Antigenic characterisation of porcine circovirus 2 strains and outcome of experimental porcine circovirus 1 and 2 infections in porcine foetuses and in pigs

Porcine circoviruses (PCV1 and PCV2) are ubiquitious around the world. Although the genetic diversity among the PCV2 strains has been extensively studied, the antigenic diversity has been much less explored. PCV1 has been isolated from cases of reproductive failure and congenital tremor in newborn pigs. PCV2 is associated with Postweaning Multisystemic Wasting Syndrome (PMWS) and can cause reproductive failure. Despite the isolation of PCV1 and PCV2 from aborted/stillborn piglets and from cases of congenital tremors in newborn piglets, nothing is known about the outcome of experimental PCV1 infections in porcine foetuses and the outcome of experimental PCV2 (PCV2a or PCV2b) infections in porcine foetuses has only been examined with a restricted number of strains. The pathogenesis of PMWS is not fully understood yet and further research is needed. The absence or presence of low levels of neutralising antibodies in PMWS pigs has been reported and it may be hypothesized that this could be due to a certain immunotolerance upon an intrauterine infection with a less virulent PCV2 strain

Oncolytic Virus Encoding a Master Pro-Inflammatory Cytokine Interleukin 12 in Cancer Immunotherapy

Oncolytic viruses (OVs) are genetically modified or naturally occurring viruses, which preferentially replicate in and kill cancer cells while sparing healthy cells, and induce anti-tumor immunity. OV-induced tumor immunity can be enhanced through viral expression of anti-tumor cytokines such as interleukin 12 (IL-12). IL-12 is a potent anti-cancer agent that promotes T-helper 1 (Th1) differentiation, facilitates T-cell-mediated killing of cancer cells, and inhibits tumor angiogenesis. Despite success in preclinical models, systemic IL-12 therapy is associated with significant toxicity in humans. Therefore, to utilize the therapeutic potential of IL-12 in OV-based cancer therapy, 25 different IL-12 expressing OVs (OV-IL12s) have been genetically engineered for local IL-12 production and tested preclinically in various cancer models. Among OV-IL12s, oncolytic herpes simplex virus encoding IL-12 (OHSV-IL12) is the furthest along in the clinic. IL-12 expression locally in the tumors avoids systemic toxicity while inducing an efficient anti-tumor immunity and synergizes with anti-angiogenic drugs or immunomodulators without compromising safety. Despite the rapidly rising interest, there are no current reviews on OV-IL12s that exploit their potential efficacy and safety to translate into human subjects. In this article, we will discuss safety, tumor-specificity, and anti-tumor immune/anti-angiogenic effects of OHSV-IL12 as mono- and combination-therapies. In addition to OHSV-IL12 viruses, we will also review other IL-12-expressing OVs and their application in cancer therapy

Instability in vitro of a PCV2 infectious clone containing an insertion between ORF1 and ORF2

Porcine circovirus 2 (PCV2) is a small circular single-stranded DNA virus that causes postweaning multisystemic wasting syndrome in pigs. We obtained an infectious clone, derived from PCV2 strain 1147 (size 1,767 bp), with a 60 bp insertion in the cap-rep intergenic region at the 3' end of the rep. In vitro propagation and sequencing of virus showed mixed sequence for the cap-rep intergenic region. Cloning of this region of virus from the passages 2 and 20 showed deletions of various size of the 60 bp insertion. Viruses from the passage 20 showed deletions of the 60 bp insertion and had extra (+1 to +5) or less (-1 or -2) bp in the intergenic region as compared to 1,147. These findings suggest that insertion of a 60-bp DNA sequence at the 3' end of the rep is unstable in vitro. This finding can have implications for the genetic engineering of PCV2

Temozolomide antagonizes oncolytic immunovirotherapy in glioblastoma

Background Temozolomide (TMZ) chemotherapy is a current standard of care for glioblastoma (GBM), however it has only extended overall survival by a few months. Because it also modulates the immune system, both beneficially and negatively, understanding how TMZ interacts with immunotherapeutics is important. Oncolytic herpes simplex virus (oHSV) is a new class of cancer therapeutic with both cytotoxic and immunostimulatory activities. Here, we examine the combination of TMZ and an oHSV encoding murine interleukin 12, G47Δ-mIL12, in a mouse immunocompetent GBM model generated from non-immunogenic 005 GBM stem-like cells (GSCs.Methods We first investigated the cytotoxic effects of TMZ and/or G47Δ-IL12 treatments in vitro, and then the antitumor effects of combination therapy in vivo in orthotopically implanted 005 GSC-derived brain tumors. To improve TMZ sensitivity, O6-methylguanine DNA methyltransferase (MGMT) was inhibited. The effects of TMZ on immune cells were evaluated by flow cytometery and immunohistochemistry.Results The combination of TMZ+G47Δ-IL12 kills 005 GSCs in vitro better than single treatments. However, TMZ does not improve the survival of orthotopic tumor-bearing mice treated with G47Δ-IL12, but rather can abrogate the beneficial effects of G47Δ-IL12 when the two are given concurrently. TMZ negatively affects intratumor T cells and macrophages and splenocytes. Addition of MGMT inhibitor O6-benzylguanine (O6-BG), an inactivating pseudosubstrate of MGMT, to TMZ improved survival, but the combination with G47Δ-IL12 did not overcome the antagonistic effects of TMZ treatment on oHSV therapy.Conclusions These results illustrate that chemotherapy can adversely affect oHSV immunovirotherapy. As TMZ is the standard of care for GBM, the timing of these combined therapies should be taken into consideration when planning oHSV clinical trials with chemotherapy for GBM

3D Printing in Solid Dosage Forms and Organ-on-Chip Applications

3D printing (3DP) can serve not only as an excellent platform for producing solid dosage forms tailored to individualized dosing regimens but can also be used as a tool for creating a suitable 3D model for drug screening, sensing, testing and organ-on-chip applications. Several new technologies have been developed to convert the conventional dosing regimen into personalized medicine for the past decade. With the approval of Spritam, the first pharmaceutical formulation produced by 3DP technology, this technology has caught the attention of pharmaceutical researchers worldwide. Consistent efforts are being made to improvise the process and mitigate other shortcomings such as restricted excipient choice, time constraints, industrial production constraints, and overall cost. The objective of this review is to provide an overview of the 3DP process, its types, types of material used, and the pros and cons of each technique in the application of not only creating solid dosage forms but also producing a 3D model for sensing, testing, and screening of the substances. The application of producing a model for the biosensing and screening of drugs besides the creation of the drug itself, offers a complete loop of application for 3DP in pharmaceutics