An Infrared Dye-Conjugated Virus-like Particle for the Treatment of Primary Uveal Melanoma.

The work outlined herein describes AU-011, a novel recombinant papillomavirus-like particle (VLP) drug conjugate and its initial evaluation as a potential treatment for primary uveal melanoma. The VLP is conjugated with a phthalocyanine photosensitizer, IRDye 700DX, that exerts its cytotoxic effect through photoactivation with a near-infrared laser. We assessed the anticancer properties of AU-011 in vitro utilizing a panel of human cancer cell lines and in vivo using murine subcutaneous and rabbit orthotopic xenograft models of uveal melanoma. The specificity of VLP binding (tumor targeting), mediated through cell surface heparan sulfate proteoglycans (HSPG), was assessed using HSPG-deficient cells and by inclusion of heparin in in vitro studies. Our results provide evidence of potent and selective anticancer activity, both in vitro and in vivo AU-011 activity was blocked by inhibiting its association with HSPG using heparin and using cells lacking surface HSPG, indicating that the tumor tropism of the VLP was not affected by dye conjugation and cell association is critical for AU-011-mediated cytotoxicity. Using the uveal melanoma xenograft models, we observed tumor uptake following intravenous (murine) and intravitreal (rabbit) administration and, after photoactivation, potent dose-dependent tumor responses. Furthermore, in the rabbit orthotopic model, which closely models uveal melanoma as it presents in the clinic, tumor treatment spared the retina and adjacent ocular structures. Our results support further clinical development of this novel therapeutic modality that might transform visual outcomes and provide a targeted therapy for the early-stage treatment of patients with this rare and life-threatening disease. Mol Cancer Ther; 17(2); 565-74. ©2017 AACR.

Oral vaccination with vaccinia virus expressing the tick antigen subolesin inhibits tick feeding and transmission of Borrelia burgdorferi.

Immunization with the Ixodes scapularis protein, subolesin, has previously been shown to protect hosts against tick infestation and to decrease acquisition of Anaplsma marginale and Babesia bigemina. Here we report the efficacy of subolesin, a conserved tick protein that can act as a regulator of gene expression, expressed from vaccinia virus for use as an orally delivered reservoir - targeted vaccine for prevention of tick infestation and acquisition/transmission of Borrelia burgdorferi to its tick and mouse hosts. We cloned subolesin into vaccinia virus and showed that it is expressed from mammalian cells infected with the recombinant virus in vitro. We then vaccinated mice by oral gavage. A single dose of the vaccine was sufficient for mice to generate antibody response to subolesin. Vaccination with the subolesin expressing vaccinia virus inhibited tick infestation by 52% compared to control vaccination with vaccinia virus and reduced uptake of B. burgdorferi among the surviving ticks that fed to repletion by 34%. There was a reduction in transmission of B. burgdorferi to uninfected vaccinated mice of 40% compared to controls. These results suggest that subolesin has potential as a component of a reservoir targeted vaccine to decrease B. burgdorferi, Babesia and Anaplasma species infections in their natural hosts.

Development of a baited oral vaccine for use in reservoir-targeted strategies against Lyme disease.

Lyme disease is a major human health problem which continues to increase in incidence and geographic distribution. As a vector-borne zoonotic disease, Lyme disease may be amenable to reservoir targeted strategies for control. We have previously reported that a vaccinia virus (VV) based vaccine expressing outer surface protein A (OspA) of Borrelia burgdorferi, the causative agent of Lyme disease, protects inbred strains of laboratory mice against infection by feeding ticks and clears the ticks of infection when administered by gavage. Here we extend these studies to develop an effective bait formulation for delivery of the VV based vaccine and test its characteristics under simulated environmental conditions. We show that this vaccine is efficacious in decreasing acquisition of B. burgdorferi by uninfected larval ticks as well as in decreasing transmission from infected ticks to its natural reservoir, Peromyscus leucopus, when fed to mice in oral baits. Using live, in vivo imaging techniques, we describe the distribution of vaccinia virus infection after ingestion of the baited vaccines and establish the use of in vivo imaging technology for optimization of bait delivery. In summary, a VV based OspA vaccine is stable in an oral bait preparation and provides protection against infection for both the natural reservoir and the tick vector of Lyme disease.

Development of a vaccinia virus based reservoir-targeted vaccine against Yersinia pestis.

Yersinia pestis, the causative organism of plague, is a zoonotic organism with a worldwide distribution. Although the last plague epidemic occurred in early 1900s, human cases continue to occur due to contact with infected wild animals. In this study, we have developed a reservoir-targeted vaccine against Y. pestis, to interrupt transmission of disease in wild animals as a potential strategy for decreasing human disease. A vaccinia virus delivery system was used to express the F1 capsular protein and the LcrV type III secretion component of Y. pestis as a fusion protein. Here we show that a single dose of this vaccine administered orally, generates a dose-dependent antibody response in mice. Antibody titers peak by 3 weeks after administration and remain elevated for a minimum of 45 weeks. Vaccination provided up to 100% protection against challenge with Y. pestis administered by intranasal challenge at 10 times the lethal dose with protection lasting a minimum of 45 weeks. An orally available, vaccinia virus expressed vaccine against Y. pestis may be a suitable vaccine for a reservoir targeted strategy for the prevention of enzootic plague.

Bacterial chitinases: properties and potential.

Chitin is among the most abundant biomass present on Earth. Chitinase plays an important role in the decomposition of chitin and potentially in the utilization of chitin as a renewable resource. During the previous decade, chitinases have received increased attention because of their wide range of applications. Chito-oligomers produced by enzymatic hydrolysis of chitin have been of interest in recent years due to their broad applications in medical, agricultural, and industrial applications, including antibacterial, antifungal, hypocholesterolemic, and antihypertensive activity, and as a food quality enhancer. Microorganisms, particularly bacteria, form one of the major sources of chitinase. In this article, we have reviewed some of the chitinases produced by bacterial systems that have gained worldwide research interest for their diverse properties and potential industrial uses.

Biocontrol of wood-rotting fungi with Streptomyces violaceusniger XL-2.

During the previous decade, chitinases have received increased attention because of their wide range of applications. Chito-oligomers produced by enzymatic hydrolysis of chitin have been of interest in recent years because of their broad applications in medical, agricultural, and industrial applications, such as antibacterial, antifungal, hypo cholesterolemic, and antihypertensive activity, and as food quality enhancer. Fungal cell walls being rich in chitin also enable the use of chitinases in biocontrol of fungal pathogens, as bio-fungicides. An actinomycete was isolated from the bark of trees of Dehradun in India and was later identified as Streptomyces violaceusniger. This strain exhibits strong antagonism towards various wood-rotting fungi, such as Phanerochaete chrysosporium, Postia placenta, Coriolus versicolor, and Gloeophyllum trabeum. Further, studies showed an extracellular bioactive compound was responsible for the antagonism. The conditions for the production of this biocontrol agent were optimized, and the effects of various stress factors (like nitrogen-deficient media, carbon-deficient media, etc.) were studied. The presence of chitin in the growth media was found to be an essential factor for the active production of the biocontrol agent. The pH and temperature optima for the biocontrol agent were determined. Purification and characterization of this specific biocontrol agent was performed through anion exchange chromatography using a DEAE-cellulose column, and a single protein band was obtained on a 10% sodium dodecyl sulfate-polyacrylamide gel. The protein was later identified as a 28 kDa endo chitinase by MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) and by a chitobiose activity assay.

Nanotechnology and potential of microorganisms.

There is a growing need to develop clean, nontoxic and environmentally friendly ("green chemistry") procedures for synthesis and assembly of nanoparticles. The use of biological organisms in this area is rapidly gaining importance due to its growing success and ease of formation of nanoparticles. Presently, the potential of bio-organisms ranges from simple prokaryotic bacterial cells to eukaryotic fungus and even live plants. In this article we have reviewed some of these biological systems, which have revolutionized the art of nano-material synthesis.