Induction of neutralizing antibodies specific for the envelope proteins of the koala retrovirus by immunization with recombinant proteins or with DNA.

BACKGROUND: The koala retrovirus (KoRV) is the result of a transspecies transmission of a gammaretrovirus with fatal consequences for the new host. Like many retroviruses, KoRV induces lymphoma, leukemia and an immunodeficiency that is associated with opportunistic infections in the virus-infected animals. We recently reported the induction of neutralizing antibodies by immunization with the recombinant ectodomain of the transmembrane envelope protein p15E of KoRV. Since the neutralization titers of the p15E-specific sera were only moderate, we investigated the use of the surface envelope protein gp70 to induce neutralizing antibodies. FINDINGS: We immunized rats and goats with the recombinant gp70 protein of the KoRV, an unglycosylated protein of 52kD (rgp70/p52) or with the corresponding DNA. In parallel we immunized with recombinant rp15E or with a combination of rp15E and rgp70/p52. In all cases binding and neutralizing antibodies were induced. The gp70-specific sera had titers of neutralizing antibodies that were 15-fold higher than the p15E-specific sera. Combining rp15E and rgp70/p52 did not significantly increase neutralizing titers compared to rgp70/p52 alone. High titers of neutralizing antibodies specific for gp70 were also induced by immunization with DNA. Since KoRV and PERV are closely related, we investigated cross-neutralization of the antisera. The antisera against p15E and gp70 of PERV and KoRV inhibited infection by both viruses. CONCLUSION: The envelope proteins of the KoRV may therefore form the basis of an effective preventive vaccine to protect uninfected koalas from infection and possibly an immunotherapeutic treatment for those already infected.

Distribution and expression of porcine endogenous retroviruses in multi-transgenic pigs generated for xenotransplantation.

BACKGROUND: Multi-transgenic pigs produced for use in xenotransplantation have to be screened for the presence and expression of porcine endogenous retroviruses (PERV) to select animals with low PERV load. The production of transgenic pigs may also be associated with the integration of the transgene adjacent to or into the locus of a PERV provirus, potentially leading to an enhanced virus expression. METHODS: Non-transgenic animals, single-transgenic, and multi-transgenic pigs were screened for the presence of PERV-A, -B, and -C and recombinant PERV-A/C using polymerase chain reaction (PCR). PERV expression was determined by real time reverse transcriptase-PCR. An assay based on the activation of PERV in peripheral blood mononuclear cells by mitogens was used to discriminate between low and high PERV producer animals. RESULTS: All animals carried PERV-A and -B. A total of 176 from 181 (97.2%) animals carried PERV-C in the germ line and 18 from 64 animals carried PERV-A/C in the genome of lymphoid cells but not in the germ line. The expression of PERV was very low in all animals and not different between transgenic pigs and non-transgenic animals. PERV expression differed between various pig lines. The highest expression was found in mini-pigs and crossing other pig lines with mini-pigs resulted in increased PERV expression in the progeny. However, expression of viral proteins and particle release were not observed in all transgenic animals. CONCLUSIONS: No evidence for elevated PERV expression in (multi-) transgenic pigs was observed. Differences in PERV expression correlated with the genetic background of the animals, not with the specific transgene. Mini-pigs consistently had the highest level of PERV expression and animals with a mini-pig background had a higher level of expression compared with animals without mini-pig background.

Atu027, a liposomal small interfering RNA formulation targeting protein kinase N3, inhibits cancer progression.

We have previously described a small interfering RNA (siRNA) delivery system (AtuPLEX) for RNA interference (RNAi) in the vasculature of mice. Here we report preclinical data for Atu027, a siRNA-lipoplex directed against protein kinase N3 (PKN3), currently under development for the treatment of advanced solid cancer. In vitro studies revealed that Atu027-mediated inhibition of PKN3 function in primary endothelial cells impaired tube formation on extracellular matrix and cell migration, but is not essential for proliferation. Systemic administration of Atu027 by repeated bolus injections or infusions in mice, rats, and nonhuman primates results in specific, RNAi-mediated silencing of PKN3 expression. We show the efficacy of Atu027 in orthotopic mouse models for prostate and pancreatic cancers with significant inhibition of tumor growth and lymph node metastasis formation. The tumor vasculature of Atu027-treated animals showed a specific reduction in lymph vessel density but no significant changes in microvascular density.

Absence of transmission of potentially xenotic viruses in a prospective pig to primate islet xenotransplantation study.

Shortage of human donor organs for transplantation has prompted usage of animals as an alternative donor source. Pigs are the most acceptable candidate animals but issues of xenozoonoses remain. Despite careful monitoring of designated pathogen free pigs there is still a risk that their tissues may carry infectious agents. Thus xenotransplantation requires extensive pre-clinical study on safety of the graft especially for those viruses that are either potentially oncogenic and/or immunosuppressive, or can establish persistent infection. A prospective pig-to-primate islet xenotransplantation study was performed which includes monitoring for potentially xenotic viruses namely porcine endogenous retrovirus (PERV), porcine cytomegalovirus (PCMV), porcine lymphotropic herpesvirus (PLHV), and porcine circovirus (PCV) using both molecular diagnostic-PCR and RT-PCR and serology methods. There was no evidence of pig virus transmission into primate recipients. This preclinical study underlines the information concerning viral safety of islet cell xenograft in pig-to-primate xenotransplantation.

Knockdown of porcine endogenous retrovirus (PERV) expression by PERV-specific shRNA in transgenic pigs.

BACKGROUND: Xenotransplantation using porcine cells, tissues or organs may be associated with the transmission of porcine endogenous retroviruses (PERVs). More than 50 viral copies have been identified in the pig genome and three different subtypes of PERV were released from pig cells, two of them were able to infect human cells in vitro. RNA interference is a promising option to inhibit PERV transmission. METHODS: We recently selected an efficient si (small interfering) RNA corresponding to a highly conserved region in the PERV DNA, which is able to inhibit expression of all PERV subtypes in PERV-infected human cells as well as in primary pig cells. Pig fibroblasts were transfected using a lentiviral vector expressing a corresponding sh (short hairpin) RNA and transgenic pigs were produced by somatic nuclear transfer cloning. Integration of the vector was proven by PCR, expression of shRNA and PERV was studied by in-solution hybridization analysis and real-time RT PCR, respectively. RESULTS: All seven born piglets had integrated the transgene. Expression of the shRNA was found in all tissues investigated and PERV expression was significantly inhibited when compared with wild-type control animals. CONCLUSION: This strategy may lead to animals compatible with PERV safe xenotransplantation.

Expression of porcine endogenous retroviruses (PERVs) in melanomas of Munich miniature swine (MMS) Troll.

Porcine endogenous retroviruses (PERVs) are integrated in the genome of all pig breeds. Since some of them are able to infect human cells, they might represent a risk for xenotransplantation using pig cells or organs. However, the expression and biological role of PERVs in healthy pigs as well as in porcine tumours is largely unknown. Since we and others have recently shown overexpression of a human endogenous retrovirus, HERV-K, in human melanomas, we studied the expression of PERVs in melanomas of selectively bred Munich miniature swine (MMS) Troll. This breeding herd of MMS Troll is characterised by a high prevalence of melanomas, which histologically resemble various types of cutaneous melanomas in humans. Several genetic factors have been defined when studying inheritance of melanomas and melanocytic nevi in MMS Troll. Here we show that the polytropic PERV-A and PERV-B as well as the ecotropic PERV-C are present in the genome of all melanoma bearing MMS Troll investigated. Most interestingly, in the spleen, but not in other organs, recombinant PERV-A/C proviruses were found. PERV expression was found elevated in melanomas when compared to normal skin and viral proteins were expressed in melanomas and pulmonary metastasis-derived melanoma cell cultures. During passaging of these cells in vitro the expression of PERV mRNA and protein increased and virus particles were released as shown by RT activity in the supernatant and by electron microscopy. Genomic RNA of PERV-A, -B and -C were found in pelleted virus particles. Although PERV expression was elevated in melanomas and pulmonary metastasis-derived cell cultures, the function of the virus in tumour development is still unclear.