Impact of porcine cytomegalovirus on long-term orthotopic cardiac xenotransplant survival.

Xenotransplantation using pig organs has achieved survival times up to 195 days in pig orthotopic heart transplantation into baboons. Here we demonstrate that in addition to an improved immunosuppressive regimen, non-ischaemic preservation with continuous perfusion and control of post-transplantation growth of the transplant, prevention of transmission of the porcine cytomegalovirus (PCMV) plays an important role in achieving long survival times. For the first time we demonstrate that PCMV transmission in orthotopic pig heart xenotransplantation was associated with a reduced survival time of the transplant and increased levels of IL-6 and TNFα were found in the transplanted baboon. Furthermore, high levels of tPA-PAI-1 complexes were found, suggesting a complete loss of the pro-fibrinolytic properties of the endothelial cells. These data show that PCMV has an important impact on transplant survival and call for elimination of PCMV from donor pigs.

Human SAMHD1 restricts the xenotransplantation relevant porcine endogenous retrovirus (PERV) in non-dividing cells.

The release of porcine endogenous retrovirus (PERV) particles from pig cells is a potential risk factor during xenotransplantation by way of productively infecting the human transplant recipient. Potential countermeasures against PERV replication are restriction factors that block retroviral replication. SAMHD1 is a triphosphohydrolase that depletes the cellular pool of dNTPs in non-cycling cells starving retroviral reverse transcription. We investigated the antiviral activity of human SAMHD1 against PERV and found that SAMHD1 potently restricts its reverse transcription in human monocytes, monocyte-derived dendritic cells (MDDC), or macrophages (MDM) and in monocytic THP-1 cells. Degradation of SAMHD1 by SIVmac Vpx or CRISPR/Cas9 knock-out of SAMHD1 allowed for PERV reverse transcription. Addition of deoxynucleosides alleviated the SAMHD1-mediated restriction suggesting that SAMHD1-mediated degradation of dNTPs restricts PERV replication in these human immune cells. In conclusion, our findings highlight SAMHD1 as a potential barrier to PERV transmission from pig transplants to human recipients during xenotransplantation.

Antibody Cross-Reactivity between Porcine Cytomegalovirus (PCMV) and Human Herpesvirus-6 (HHV-6).

Porcine cytomegalovirus (PCMV) infection is widely prevalent among pigs, and PCMV is one of the viruses which may be transmitted during xenotransplantation using pig cells, tissues, or organs. While human cytomegalovirus (HCMV) is a major risk factor for allotransplantation, it is still unclear whether PCMV is able to infect human cells or pose a risk for xenotransplantation. Previously, it was shown that transmission of PCMV after pig kidney to non-human primate transplantations resulted in a significantly reduced survival time of the transplanted organ. To detect PCMV, PCR-based and immunological methods were used. Screening of pigs by Western blot analyses using recombinant viral proteins revealed up to 100% of the tested animals to be infected. When the same method was applied to screen human sera for PCMV-reactive antibodies, positive Western blot results were obtained in butchers and workers in the meat industry as well as in normal blood donors. To exclude an infection of humans with PCMV, the sera were further investigated. PCMV is closely related to human herpesvirus-6 (HHV-6) and human herpesvirus-7 (HHV-7), and a sequence alignment of glycoprotein B suggests that the antibodies may cross-react with identical epitope sequences. HCMV is not related with PCMV, and no correlation between antibody reactivity against PCMV and HCMV was detected. These data indicate that antibodies against PCMV found in humans are cross-reactive antibodies against HHV-6.

Detection of koala retrovirus subgroup B (KoRV-B) in animals housed at European zoos.

Many koalas carry an endogenous retrovirus, KoRV-A, in their genome. Recently, a second retrovirus, KoRV-B, was detected in koalas in Japanese and U.S. zoos. However, this virus is not endogenous, differs in the receptor binding site of the surface envelope protein, and uses a receptor different from that of KoRV-A. We describe here a KoRV-B found in koalas at zoos in Germany and Belgium that differs slightly from that found in the Los Angeles zoo.

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.

Lack of antiviral antibody response in koalas infected with koala retroviruses (KoRV).

Many wild koalas are infected with the koala retrovirus, KoRV, some of which suffer from lymphoma and chlamydial disease. Three subgroups, KoRV-A, KoRV-B and KoRV-J, have so far been described. It is well known that other closely related gammaretroviruses can induce tumours and severe immunodeficiencies in their respective hosts and a possible role for KoRV infection in lymphoma and chlamydial disease in koalas has been suggested. In many wild koalas, KoRV-A has become endogenised, i.e., it is integrated in the germ-line and is passed on with normal cellular genes. In this study, sera from koalas in European zoos and from wild animals in Australia were screened for antibodies against KoRV-A. These naturally infected animals all carry endogenous KoRV-A and two zoo animals are also infected with KoRV-B. The antibody response is generally an important diagnostic tool for detecting retrovirus infections. However, when Western blot analyses were performed using purified virus or recombinant proteins corresponding to KoRV-A, none of the koalas tested positive for specific antibodies, suggesting a state of tolerance. These results have implications for koala vaccination, as they suggest that therapeutic immunisation of animals carrying and expressing endogenous KoRV-A will not be successful. However, it remains unclear whether these animals can be immunised against KoRV-B and immunisation of uninfected koalas could still be worthwhile.

A novel small animal model to study the replication of simian foamy virus in vivo.

Preclinical evaluation in a small animal model would help the development of gene therapies and vaccines based on foamy virus vectors. The establishment of persistent, non-pathogenic infection with the prototype foamy virus in mice and rabbits has been described previously. To extend this spectrum of available animal models, hamsters were inoculated with infectious cell supernatant or bioballistically with a foamy virus plasmid. In addition, a novel foamy virus from a rhesus macaque was isolated and characterised genetically. Hamsters and mice were infected with this new SFVmac isolate to evaluate whether hamsters are also susceptible to infection. Both hamsters and mice developed humoral responses to either virus subtype. Virus integration and replication in different animal tissues were analysed by PCR and co-cultivation. The results strongly indicate establishment of a persistent infection in hamsters. These studies provide a further small animal model for studying FV-based vectors in addition to the established models.

Generation of neutralising antibodies against porcine endogenous retroviruses (PERVs).

Antibodies neutralising porcine endogenous retroviruses (PERVs) were induced in different animal species by immunisation with the transmembrane envelope protein p15E. These antibodies recognised epitopes, designated E1, in the fusion peptide proximal region (FPPR) of p15E, and E2 in the membrane proximal external region (MPER). E2 is localised in a position similar to that of an epitope in the transmembrane envelope protein gp41 of the human immunodeficiency virus-1 (HIV-1), recognised by the monoclonal antibody 4E10 that is broadly neutralising. To detect neutralising antibodies specific for PERV, a novel assay was developed, which is based on quantification of provirus integration by real-time PCR. In addition, for the first time, highly effective neutralising antibodies were obtained by immunisation with the surface envelope protein of PERV. These data indicate that neutralising antibodies can be induced by immunisation with both envelope proteins.

Increased neutralizing antibody response after simultaneous immunization with leucogen and the feline leukemia virus transmembrane protein.

To develop improved vaccination strategies against feline leukemia virus (FeLV), rats were immunized with the transmembrane envelope protein p15E of FeLV alone or in combination with the commercial vaccine Leucogen® comprising the nonglycosylated FeLV surface envelope protein. Binding and neutralizing antibodies were induced in both groups and in the group immunized with Leucogen alone. Higher titers of antibodies neutralizing FeLV were induced by simultaneous immunization with Leucogen and p15E compared to the responses using Leucogen or p15E alone, suggesting that combination vaccines should be used in the future. Epitope mapping of p15E-specific antibodies induced by simultaneous immunization with Leucogen and p15E revealed the same pattern of response as obtained after immunization with p15E alone: one epitope was localized in the membrane-proximal external region (MPER) and the other in the fusion peptide-proximal region, and they are related to the epitopes detected after immunization with p15E of the porcine endogenous retrovirus and the koala retrovirus. The data indicate that these epitopes in the MPER are an effective target for neutralization and that antigens containing them may therefore prove to be a useful component of vaccines against retroviruses, including HIV-1.

Novel functions of prototype foamy virus Gag glycine- arginine-rich boxes in reverse transcription and particle morphogenesis.

Prototype foamy virus (PFV) Gag lacks the characteristic orthoretroviral Cys-His motifs that are essential for various steps of the orthoretroviral replication cycle, such as RNA packaging, reverse transcription, infectivity, integration, and viral assembly. Instead, it contains three glycine-arginine-rich boxes (GR boxes) in its C terminus that putatively represent a functional equivalent. We used a four-plasmid replication-deficient PFV vector system, with uncoupled RNA genome packaging and structural protein translation, to analyze the effects of deletion and various substitution mutations within each GR box on particle release, particle-associated protein composition, RNA packaging, DNA content, infectivity, particle morphology, and intracellular localization. The degree of viral particle release by all mutants was similar to that of the wild type. Only minimal effects on Pol encapsidation, exogenous reverse transcriptase (RT) activity, and genomic viral RNA packaging were observed. In contrast, particle-associated DNA content and infectivity were drastically reduced for all deletion mutants and were undetectable for all alanine substitution mutants. Furthermore, GR box I mutants had significant changes in particle morphology, and GR box II mutants lacked the typical nuclear localization pattern of PFV Gag. Finally, it could be shown that GR boxes I and III, but not GR box II, can functionally complement each other. It therefore appears that, similar to the orthoretroviral Cys-His motifs, the PFV Gag GR boxes are important for RNA encapsidation, genome reverse transcription, and virion infectivity as well as for particle morphogenesis.

Regulation of human endogenous retrovirus-K expression in melanomas by CpG methylation.

The overall prognosis of patients with advanced melanoma is poor due to the lack of effective treatment. A key factor for successful therapy is an early detection of disease. Therefore, reliably detection methods and meaningful tumor markers are required. Expression of the human endogenous retrovirus (HERV)-K(HML-2) was found elevated in melanomas and it was shown that HERV-K supports the in vitro transition of melanoma cells from adherent to a more malignant, nonadherent phenotype. Furthermore, the detection of HERV-K-specific antibodies in melanoma patients was found to correlate with reduced survival. However, the reason for HERV-K expression in melanomas still remains unclear and its use as a tumor marker needs further investigation. Therefore, the tumor-specific transcriptional regulation of HERV-K expression in melanoma was studied in detail. Human melanoma cell lines were investigated for HERV-K expression using real-time PCR. Five cell lines showed very high levels of HERV-K mRNA as a result of increased promoter activity. This promoter activity was directly silenced by DNA methylation in reporter gene experiments. Higher levels of long terminal repeat (LTR) methylation in cells not expressing HERV-K compared with cells expressing HERV-K were found using methylation-sensitive PCR and bisulfite sequencing. Treatment of cell lines with the demethylating agent 5-aza-2'-deoxycytidine resulted in increased levels of HERV-K expression in cells previously not expressing HERV-K and it was shown that this increase is not the result of transcription factor activation. These results demonstrate that increased HERV-K expression in melanomas may be due to increased promoter activity and demethylation of the 5'LTR.

Mode of interaction between the HIV-1-neutralizing monoclonal antibody 2F5 and its epitope.

OBJECTIVE: To determine the mechanism of interaction between the HIV-1 gp41-specific broadly neutralizing monoclonal antibody (mAb) 2F5, its epitope in the membrane proximal external region and a domain located in the fusion peptide proximal region in the N-terminal region of gp41. Knowledge of these interactions would be useful for the design of antigens used to induce 2F5-like antibodies. METHODS: The binding and avidity of the mAb 2F5 were analyzed using enzyme-linked immunosorbent assays, epitope mapping and surface plasmon resonance analysis. Inhibition of virus neutralization by 2F5 was analyzed using peptides corresponding to the gp41 sequence. RESULTS: Using transmembrane envelope proteins of gammaretroviruses, we had previously induced neutralizing antibodies that recognize two epitopes, one located in the N-terminal part of the transmembrane protein (designated E1) and the other in the C-terminal membrane proximal external region (E2). The E2 epitope corresponds to the mAb 2F5/4E10 epitope in the gp41 of HIV and we have now identified a corresponding E1 domain in gp41. Although 2F5 did not bind directly to E1, the presence of E1 peptides increased the binding of 2F5 to peptides carrying its epitope. Neutralization of HIV-1 by 2F5 was inhibited more effectively by both gp41-derived peptides E1 and E2 together than with the peptide E2 alone. CONCLUSION: The interaction between the E1 and E2 domains of gp41 increased the efficacy of mAb 2F5 binding to its E2 epitope. Such an interaction may occur after gp41 folding into a six-helix bundle. Antigens containing both domains might be used to induce broadly neutralizing 2F5-like antibodies.

Transspecies transmission of the endogenous koala retrovirus.

The koala retrovirus (KoRV) is a gammaretrovirus closely related to the gibbon ape leukemia virus and induces leukemias and immune deficiencies associated with opportunistic infections, such as chlamydiosis. Here we characterize a KoRV newly isolated from an animal in a German zoo and show infection of human and rat cell lines in vitro and of rats in vivo, using immunological and PCR methods for virus detection. The KoRV transmembrane envelope protein (p15E) was cloned and expressed, and p15E-specific neutralizing antibodies able to prevent virus infection in vitro were developed. Finally, evidence for immunosuppressive properties of the KoRV was obtained.

Neutralizing antibodies against conserved domains of p15E of porcine endogenous retroviruses: basis for a vaccine for xenotransplantation?

Porcine xenotransplants may offer a potential solution to the problem posed by the limited supply of allotransplants. However, xenotransplantation may be associated with the risk of transmission of microorganisms, in particular of porcine endogenous retroviruses (PERVs) that are an integral part of the porcine genome and able to infect human cells in vitro. Possible strategies to prevent virus transmission include the development of PERV knockout animals or of effective vaccines. When antisera prepared against the main structural proteins of PERV were screened, a goat antiserum against the recombinant ectodomain of the transmembrane envelope protein p15E was found to neutralize PERV infectivity. Epitope mapping using overlapping peptides revealed two epitopes, E1 (GPQQLEK) and E2 (FEGWFN). These sequences are identical for all PERVs and are highly conserved among all gammaretroviruses. Interestingly, antibodies isolated from AIDS patients and specific for sequences of HIV-1 partially homologous with E2 (Mab4E10, LWNWFN) or located in close proximity to E2 (Mab2F5, ELDKWA) are known to neutralize several strains of HIV-1. It is the first report showing epitope mapping of gammaretrovirus-specific neutralizing antibodies and demonstrating similarity to corresponding epitopes in HIV. These domains of the transmembrane proteins of different retroviruses are an effective target for neutralizing antibodies and may be a useful antigen to create an antiretroviral vaccine.