The Oncolytic Virus VSV-GP Is Effective against Malignant Melanoma.

Previously, we described VSV-GP, a modified version of the vesicular stomatitis virus, as a non-neurotoxic oncolytic virus that is effective for the treatment of malignant glioblastoma and ovarian cancer. Here, we evaluate the therapeutic efficacy of VSV-GP for malignant melanoma. All of the human, mouse, and canine melanoma cell lines that were tested, alongside most primary human melanoma cultures, were infected by VSV-GP and efficiently killed. Additionally, we found that VSV-GP prolonged the survival of mice in both a xenograft and a syngeneic mouse model. However, only a few mice survived with long-term tumor remission. When we analyzed the factors that might limit VSV-GP's efficacy, we found that vector-neutralizing antibodies did not play a role in this context, as even after eight subsequent immunizations and an observation time of 42 weeks, no vector-neutralizing antibodies were induced in VSV-GP immunized mice. In contrast, the type I IFN response might have contributed to the reduced efficacy of the therapy, as both of the cell lines that were used for the mouse models were able to mount a protective IFN response. Nevertheless, early treatment with VSV-GP also reduced the number and size of lung metastases in a syngeneic B16 mouse model. In summary, VSV-GP is a potent candidate for the treatment of malignant melanoma; however, factors limiting the efficacy of the virus need to be further explored.

Re-engineering vesicular stomatitis virus to abrogate neurotoxicity, circumvent humoral immunity, and enhance oncolytic potency.

As cancer treatment tools, oncolytic viruses (OV) have yet to realize what some see as their ultimate clinical potential. In this study, we have engineered a chimeric vesicular stomatitis virus (VSV) that is devoid of its natural neurotoxicity while retaining potent oncolytic activity. The envelope glycoprotein (G) of VSV was replaced with a variant glycoprotein of the lymphocytic choriomeningitis virus (LCMV-GP), creating a replicating therapeutic, rVSV(GP), that is benign in normal brain but can effectively eliminate brain cancer in multiple preclinical tumor models in vivo. Furthermore, it can be safely administered systemically to mice and displays greater potency against a spectrum of human cancer cell lines than current OV candidates. Remarkably, rVSV(GP) escapes humoral immunity, thus, for the first time, allowing repeated systemic OV application without loss of therapeutic efficacy. Taken together, rVSV(GP) offers a considerably improved OV platform that lacks several of the major drawbacks that have limited the clinical potential of this technology to date.

VSV-GP: a potent viral vaccine vector that boosts the immune response upon repeated applications.

UNLABELLED: Antivector immunity limits the response to homologous boosting for viral vector vaccines. Here, we describe a new, potent vaccine vector based on replication-competent vesicular stomatitis virus pseudotyped with the glycoprotein of the lymphocytic choriomeningitis virus (VSV-GP), which we previously showed to be safe in mice. In mice, VSV and VSV-GP encoding ovalbumin (OVA) as a model antigen (VSV-OVA and VSV-GP-OVA) induced equal levels of OVA-specific humoral and cellular immune responses upon a single immunization. However, boosting with the same vector was possible only for VSV-GP-OVA as neutralizing antibodies to VSV limited the immunogenicity of the VSV-OVA boost. OVA-specific cytotoxic T-lymphocyte (CTL) responses induced by VSV-GP-OVA were at least as potent as those induced by an adenoviral state-of-the-art vaccine vector and completely protected mice in a Listeria monocytogenes challenge model. VSV-GP is so far the only replication-competent vaccine vector that does not lose efficacy upon repeated application. IMPORTANCE: Although there has been great progress in treatment and prevention of infectious diseases in the past several years, effective vaccines against some of the most serious infections, e.g., AIDS, malaria, hepatitis C, or tuberculosis, are urgently needed. Here, several approaches based on viral vector vaccines are under development. However, for all viral vaccine vectors currently in clinical testing, repeated application is limited by neutralizing antibodies to the vector itself. Here, we have exploited the potential of vesicular stomatitis virus pseudotyped with the glycoprotein of the lymphocytic choriomeningitis virus (VSV-GP) as a vaccine platform. VSV-GP is the first replication-competent viral vector vaccine that does not induce vector-specific humoral immunity, i.e., neutralizing antibodies, and therefore can boost immune responses against a foreign antigen by repeated applications. The vector allows introduction of various antigens and therefore can serve as a platform technology for the development of novel vaccines against a broad spectrum of diseases.

Inoculation of young horses with bovine papillomavirus type 1 virions leads to early infection of PBMCs prior to pseudo-sarcoid formation.

Bovine papillomavirus types 1 and 2 (BPV-1 and BPV-2) are known to induce common equine skin tumours, termed sarcoids. Recently, it was demonstrated that vaccination with BPV-1 virus-like particles (VLPs) is safe and highly immunogenic in horses. To establish a BPV-1 challenge model for evaluation of the protective potential of BPV-1 VLPs, four foals were injected intradermally with infectious BPV-1 virions and with viral genome-based and control inocula, and monitored daily for tumour development. Blood was taken before inoculation and at weekly intervals. BPV-1-specific serum antibodies were detected by a pseudo-virion neutralization assay. Total nucleic acids extracted from tumours, intact skin and PBMCs were tested for the presence of BPV-1 DNA and mRNA using PCR and RT-PCR, respectively. Intralesional E5 oncoprotein expression was determined by immunofluorescence. Pseudo-sarcoids developed exclusively at sites inoculated with virions. Tumours became palpable 11-32 days after virion challenge, reached a size of ≤20 mm in diameter and then resolved in ≤6 months. No neutralizing anti-BPV-1 serum antibodies were detectable pre- or post-challenge. BPV-1 DNA was present in lesions but not in intact skin. In PBMCs, viral DNA was already detectable before lesions were first palpable, in concentrations correlating directly with tumour growth kinetics. PBMCs from two of two foals also harboured E5 mRNA. Immunofluorescence revealed the presence of the E5 protein in tumour fibroblasts, but not in the apparently normal epidermis overlying the lesions. Together with previous findings obtained in horses and cows, these data suggest that papillomavirus infection may include a viraemic phase.

BPV-1 infection is not confined to the dermis but also involves the epidermis of equine sarcoids.

In equids, bovine papillomaviruses of type 1 (BPV-1) and less frequently type 2 induce common, locally aggressive skin tumours termed sarcoids. Whereas BPV infection in cattle usually involves the epidermis and is productive in this skin layer, infection in equids is currently thought to be abortive, with virus solely residing as multiple episomes in dermal fibroblasts. Based on recent observations that do not agree with this assumption, we hypothesised that BPV also infects equid epidermis and is active in this skin layer. To test this hypothesis, we conducted a proof-of-principle study on eight distinct sarcoids. Presence of viral DNA was addressed by qualitative and quantitative BPV-1 PCR from microdissected sarcoid epidermis, and by subsequent amplicon sequencing. Viral activity was assessed by screening sarcoid epidermis for BPV-1 protein expression using immunohistochemistry (IHC) or immunofluorescence (IF). Virus-free equine skin served as negative control throughout the assays. BPV-1 DNA was demonstrated in all sarcoid epidermis samples, with viral DNA loads ranging between 2 and 195 copies/cell. Identical BPV-1 E5 genes were identified in epidermis and dermis of each of two sarcoids, yet different E5 variants were found in individual lesions. IHC/IF revealed the presence of E5 and E7 protein in sarcoid epidermis, and L1 capsomers in the squamous layer of one lesion. These findings indicate that BPV infection also involves the epidermis, where it may occasionally be productive.

Prevalence of bovine papillomavirus and Treponema DNA in bovine digital dermatitis lesions.

Bovine digital dermatitis (BDD) is a common infectious foot disease whose aetiology is not fully understood. Its origin is thought to be multifactorial, with treponemes being involved. Using PCR-based techniques, BDD samples from 45 affected cows and intact skin from 8 BDD-affected and 33 healthy cows were assessed for the presence of bovine papillomavirus and Treponema DNA. BPV DNA (mainly BPV-1/2) was detected in 22% of lesions and one skin sample from affected animals, and in 15% (BPV-1/-2) and 23% (BPV-3/4/6/9/10) of skin samples from healthy cows. Using quantitative PCR, Treponema DNA was demonstrated in 38/45 BDD lesions, with bacterial DNA loads ranging between 2 × 10(3) and 2.78 × 10(5) copies/40 ng of total DNA. Qualitative PCR confirmed this result and revealed Treponema DNA in 4 additional BDD samples, thus leading to an overall infection rate of 93.3%. Sequence analysis of amplified Treponema DNA revealed T. pedis sp. nov. in 51%, T. medium ssp. bovis in 37.7%, and T. phagedenis ssp. vaccae in 4.4% of lesions. T. brennaborense was not detected in any of the samples. Six BDD samples contained type IV oral Treponema strains, 6 other harboured so far unpublished Treponema sequences. To our knowledge, this is the first report providing information on BPV infection in BDD-affected cattle, and the Treponema DNA load and occurrence of type IV treponemes in BDD samples. Our findings further support an etiologic association of treponemes, particularly T. pedis sp. nov., with BDD disease, yet indicate that BPVs do not directly contribute to BDD development.

Identification of two GH18 chitinase family genes and their use as targets for detection of the crayfish-plague oomycete Aphanomyces astaci.

BACKGROUND: The oomycete Aphanomyces astaci is regarded as the causative agent of crayfish plague and represents an evident hazard for European crayfish species. Native crayfish populations infected with this pathogen suffer up to 100% mortality. The existence of multiple transmission paths necessitates the development of a reliable, robust and efficient test to detect the pathogen. Currently, A. astaci is diagnosed by a PCR-based assay that suffers from cross-reactivity to other species. We developed an alternative closed-tube assay for A. astaci, which achieves robustness through simultaneous amplification of multiple functionally constrained genes. RESULTS: Two novel constitutively expressed members of the glycosyl hydrolase (GH18) gene family of chitinases were isolated from the A. astaci strain Gb04. The primary amino acid sequence of these chitinase genes, termed CHI2 and CHI3, is composed of an N-terminal signal peptide directing the post-translational transport of the protein into the extracellular space, the catalytic GH18 domain, a proline-, serine-, and threonine-rich domain and a C-terminal cysteine-rich putative chitin-binding site. The A. astaci mycelium grown in a pepton-glucose medium showed significant temporal changes in steady-state CHI2 and CHI3 mRNA amounts indicating functional constraint. Their different temporal occurrence with maxima at 48 and 24 hours of incubation for CHI2 and CHI3, respectively, is in accordance with the multifunctionality of GH18 family members. To identify A. astaci-specific primer target sites in these novel genes, we determined the partial sequence homologs in the related oomycetes A. frigidophilus, A. invadans, A. helicoides, A. laevis, A. repetans, Achlya racemosa, Leptolegnia caudata, and Saprolegnia parasitica, as well as in the relevant fungi Fusarium solani and Trichosporon cutaneum. An A. astaci-specific primer pair targeting the novel genes CHI2 and CHI3 as well as CHI1 - a third GH18 family member - was multiplexed with primers targeting the 5.8S rRNA used as an endogenous control. A species was typed unambiguously as A. astaci if two peaks were concomitantly detected by melting curve analysis (MCA). For sensitive detection of the pathogen, but also for quantification of agent levels in susceptible crayfish and carrier crayfish, a TaqMan-probe based real-time PCR (qPCR) assay was developed. It targets the same chitinase genes and allows quantification down to 25 target sequences. CONCLUSION: The simultaneous qualitative detection of multiple sequences by qPCR/MCA represents a promising approach to detect species with elevated levels of genetic variation and/or limited available sequence information. The homogenous closed-tube format, reduced detection time, higher specificity, and the considerably reduced chance of false negative detection achieved by targeting multiple genes (CHI1, CHI2, CHI3, and the endogenous control) at least two of which are subject to high functional constraint, are the major advantages of this multiplex assay compared to other diagnostic methods. Sensitive quantification achieved with TaqMan qPCR facilitates to monitor infection status and pathogen distribution in different tissues and can help prevent disease transmission.