Determination of Sequences Required for Human Endogenous Retrovirus K Transduction and Its Recognition by Foreign Retroviral Virions.

Sequences necessary for transduction of human endogenous retrovirus (HERV)-Kcon, a consensus of the HERV-K(HML-2) family, were analyzed and found to reside in the leader/gag region. They act in an orientation-dependent way and consist of at least two sites working together. Having defined these sequences, we exploited this information to produce a simple system to investigate to what extent virions of HERV-Kcon, murine leukemia virus, and HIV-1 have the ability to transduce each other's genomes, leading to potential contamination of gene therapy vectors.

CNS effects of a CCR5 inhibitor in HIV-infected subjects: a pharmacokinetic and cerebral metabolite study.

BACKGROUND: We conducted a pharmacokinetic and in vivo cerebral (1)H magnetic resonance spectroscopy ((1)H-MRS) study to assess CSF exposure and cerebral metabolite ratios (CMRs) following maraviroc intensification. METHODS: HIV-infected neurologically asymptomatic adults receiving tenofovir, emtricitabine and lopinavir/ritonavir with plasma HIV RNA <50 copies/mL were eligible and received intensified therapy with 150 mg of maraviroc twice daily. (1)H-MRS was performed in several cerebral locations, including the right basal ganglia (RBG), to assess CMRs, including N-acetyl aspartate/creatine (NAA/Cr), at baseline and after 14 days. Subsequently, on day 15, blood samples were obtained to determine plasma concentrations of maraviroc pre-dose (C(trough)) and then paired blood and CSF samples were collected at 4 or 6 h post-dose. Associations between maraviroc exposure, clinical parameters and changes to CMRs were evaluated. TRIAL REGISTRY: ClinicalTrials.gov (http://clinicaltrials.gov/ct2/show/NCT00982878). RESULTS: Twelve subjects (75% male) participated with a mean (SD) CD4+ cell count of 503 (199) cells/µL. Mean (SD) maraviroc plasma concentrations at pre-dose, 4 h post-dose and 6 h post-dose were 337 (74), 842 (174) and 485 (100) ng/mL and CSF concentrations at 4 h post-dose and 6 h post-dose were 7.5 (1.3) and 5.1 (1.2) ng/mL. The mean maraviroc CSF : plasma ratio (range) was 1.01% (0.57%-1.61%). An increase of 14.8% was observed for the RBG NAA/Cr ratio, which was significantly associated with higher maraviroc plasma C(trough) (P = 0.05, r = 0.61), but not CSF concentration (P = 0.16, r = 0.46). CONCLUSIONS: After 14 days of maraviroc intensification, small increases in cerebral metabolite markers of neuronal integrity (NAA/Cr ratios) were observed and are associated with maraviroc plasma C(trough).

Mouse DNA contamination in human tissue tested for XMRV.

BACKGROUND: We used a PCR-based approach to study the prevalence of genetic sequences related to a gammaretrovirus, xenotropic murine leukemia virus-related virus, XMRV, in human prostate cancer. This virus has been identified in the US in prostate cancer patients and in those with chronic fatigue syndrome. However, with the exception of two patients in Germany, XMRV has not been identified in prostate cancer tissue in Europe. Most putative associations of new or old human retroviruses with diseases have turned out to be due to contamination. We have looked for XMRV sequences in DNA extracted from formalin-fixed paraffin- embedded prostate tissues. To control for contamination, PCR assays to detect either mouse mitochondrial DNA (mtDNA) or intracisternal A particle (IAP) long terminal repeat DNA were run on all samples, owing to their very high copy number in mouse cells. RESULTS: In general agreement with the US prevalence, XMRV-like sequences were found in 4.8% of prostate cancers. However, these were also positive, as were 21.5% of XMRV-negative cases, for IAP sequences, and many, but not all were positive for mtDNA sequences. CONCLUSIONS: These results show that contamination with mouse DNA is widespread and detectable by the highly sensitive IAP assay, but not always with less sensitive assays, such as murine mtDNA PCR. This study highlights the ubiquitous presence of mouse DNA in laboratory specimens and offers a means of rigorous validation for future studies of murine retroviruses in human disease.

A one-step SYBR Green I-based product-enhanced reverse transcriptase assay for the quantitation of retroviruses in cell culture supernatants.

PCR-enhanced reverse transcriptase assays (PERT) are sensitive tools for the detection of retroviruses in biological samples. The adaptation of real-time PCR techniques based on fluorescent probes (F-PERT) has added a reliable quantitative capacity to the assay. In the interest of economy and time, the SYBR Green I-based real-time detection system was used to establish a convenient one-step PERT assay (SG-PERT). This assay can be completed in 2h, is linear over six orders of magnitude and can be used to quantify retroviruses belonging to divergent species, such as the human immunodeficiency virus type 1 (HIV-1), murine leukemia virus (MLV) and prototypic foamy virus (PFV).

Modified envelope glycoproteins to retarget retroviral vectors.

A conceptual breakthrough in gene therapy would be gene transfer vector that could be systemically applied, allowing targeted gene transfer into a predetermined cell type. The host range of a retroviral vector is determined by the interaction of the viral envelope glycoprotein (Env) and the retrovirus receptor on the surface of the host cell. In this review, we describe the current efforts to engineer targeted envelope glycoproteins, which can be incorporated into retroviral particles and are capable of delivering genes in a highly specific manner.

Murine leukemia virus (MLV) replication monitored with fluorescent proteins.

BACKGROUND: Cancer gene therapy will benefit from vectors that are able to replicate in tumor tissue and cause a bystander effect. Replication-competent murine leukemia virus (MLV) has been described to have potential as cancer therapeutics, however, MLV infection does not cause a cytopathic effect in the infected cell and viral replication can only be studied by immunostaining or measurement of reverse transcriptase activity. RESULTS: We inserted the coding sequences for green fluorescent protein (GFP) into the proline-rich region (PRR) of the ecotropic envelope protein (Env) and were able to fluorescently label MLV. This allowed us to directly monitor viral replication and attachment to target cells by flow cytometry. We used this method to study viral replication of recombinant MLVs and split viral genomes, which were generated by replacement of the MLV env gene with the red fluorescent protein (RFP) and separately cloning GFP-Env into a retroviral vector. Co-transfection of both plasmids into target cells resulted in the generation of semi-replicative vectors, and the two color labeling allowed to determine the distribution of the individual genomes in the target cells and was indicative for the occurrence of recombination events. CONCLUSIONS: Fluorescently labeled MLVs are excellent tools for the study of factors that influence viral replication and can be used to optimize MLV-based replication-competent viruses or vectors for gene therapy.

Gene delivery the key to gene therapy: the case for foamy viruses.

"There have been rare cases of zoonotic transmission of foamy virus from monkeys to humans, but despite keeping these cases under close scrutiny for years no pathology has ever been detected...".

Xenotropic murine leukaemia virus-related virus (XMRV) does not cause chronic fatigue.

The xenotropic murine leukaemia virus-related virus (XMRV), a gammaretrovirus, was discovered in prostate cancer tumours by Virochip technology in 2006. It was subsequently detected in chronic fatigue patients in 2009. The association between XMRV and chronic fatigue has proved to be controversial. No study has confirmed these findings and many have refuted them. Here, we present the evidence for our contention that XMRV is not a human pathogen.

DNA extraction columns contaminated with murine sequences.

Sequences of the novel gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV) have been described in human prostate cancer tissue, although the amounts of DNA are low. Furthermore, XMRV sequences and polytropic (p) murine leukemia viruses (MLVs) have been reported in patients with chronic fatigue syndrome (CFS). In assessing the prevalence of XMRV in prostate cancer tissue samples we discovered that eluates from naïve DNA purification columns, when subjected to PCR with primers designed to detect genomic mouse DNA contamination, occasionally gave rise to amplification products. Further PCR analysis, using primers to detect XMRV, revealed sequences derived from XMRV and pMLVs from mouse and human DNA and DNA of unspecified origin. Thus, DNA purification columns can present problems when used to detect minute amounts of DNA targets by highly sensitive amplification techniques.

Investigation into the presence of and serological response to XMRV in CFS patients.

The novel human gammaretrovirus xenotropic murine leukemia virus-related virus (XMRV), originally described in prostate cancer, has also been implicated in chronic fatigue syndrome (CFS). When later reports failed to confirm the link to CFS, they were often criticised for not using the conditions described in the original study. Here, we revisit our patient cohort to investigate the XMRV status in those patients by means of the original PCR protocol which linked the virus to CFS. In addition, sera from our CFS patients were assayed for the presence of xenotropic virus envelope protein, as well as a serological response to it. The results further strengthen our contention that there is no evidence for an association of XMRV with CFS, at least in the UK.

No Evidence of XMRV or MuLV Sequences in Prostate Cancer, Diffuse Large B-Cell Lymphoma, or the UK Blood Donor Population.

Xenotropic murine leukaemia virus-related virus (XMRV) is a recently described retrovirus which has been claimed to infect humans and cause associated pathology. Initially identified in the US in patients with prostate cancer and subsequently in patients with chronic fatigue syndrome, doubt now exists that XMRV is a human pathogen. We studied the prevalence of genetic sequences of XMRV and related MuLV sequences in human prostate cancer, from B cell lymphoma patients and from UK blood donors. Nucleic acid was extracted from fresh prostate tissue biopsies, formalin-fixed paraffin-embedded (FFPE) prostate tissue and FFPE B-cell lymphoma. The presence of XMRV-specific LTR or MuLV generic gag-like sequences was investigated by nested PCR. To control for mouse DNA contamination, a PCR that detected intracisternal A-type particle (IAP) sequences was included. In addition, DNA and RNA were extracted from whole blood taken from UK blood donors and screened for XMRV sequences by real-time PCR. XMRV or MuLV-like sequences were not amplified from tissue samples. Occasionally MuLV gag and XMRV-LTR sequences were amplified from Indian prostate cancer samples, but were always detected in conjunction with contaminating murine genomic DNA. We found no evidence of XMRV or MuLV infection in the UK blood donors.

Failure to detect the novel retrovirus XMRV in chronic fatigue syndrome.

BACKGROUND: In October 2009 it was reported that 68 of 101 patients with chronic fatigue syndrome (CFS) in the US were infected with a novel gamma retrovirus, xenotropic murine leukaemia virus-related virus (XMRV), a virus previously linked to prostate cancer. This finding, if confirmed, would have a profound effect on the understanding and treatment of an incapacitating disease affecting millions worldwide. We have investigated CFS sufferers in the UK to determine if they are carriers of XMRV. METHODOLOGY: Patients in our CFS cohort had undergone medical screening to exclude detectable organic illness and met the CDC criteria for CFS. DNA extracted from blood samples of 186 CFS patients were screened for XMRV provirus and for the closely related murine leukaemia virus by nested PCR using specific oligonucleotide primers. To control for the integrity of the DNA, the cellular beta-globin gene was amplified. Negative controls (water) and a positive control (XMRV infectious molecular clone DNA) were included. While the beta-globin gene was amplified in all 186 samples, neither XMRV nor MLV sequences were detected. CONCLUSION: XMRV or MLV sequences were not amplified from DNA originating from CFS patients in the UK. Although we found no evidence that XMRV is associated with CFS in the UK, this may be a result of population differences between North America and Europe regarding the general prevalence of XMRV infection, and might also explain the fact that two US groups found XMRV in prostate cancer tissue, while two European studies did not.

The effects of a nucleoside-sparing antiretroviral regimen on the pharmacokinetics of ritonavir-boosted darunavir in HIV type-1-infected patients.

BACKGROUND: Nucleoside-sparing combination antiretroviral therapy (cART) regimens might be an attractive therapeutic option for HIV type-1 (HIV-1)-infected patients; however, the pharmacokinetic profiles of such regimens are frequently unknown. METHODS: Fourteen HIV-1-infected patients (age 21-55 years, 64% male) on stable cART with plasma HIV RNA <50 copies/ml entered this Phase I pharmacokinetic study. In period 1, patients received tenofovir/emtricitabine/-darunavir/ritonavir (300/200/800/100 mg) all once daily. During period 2, raltegravir 400 mg twice daily was added to the regimen and in period 3 tenofovir/emtricitabine was discontinued. At steady state, intensive pharmacokinetic sampling was undertaken. Differences in the geometric mean ratio (GMR) for pharmacokinetic parameters between periods 2 versus 1 and period 3 versus 1 were assessed for darunavir and ritonavir (period 3 versus 2 for raltegravir). RESULTS: No statistically significant differences in pharmacokinetic parameters were observed between period 2 versus period 1. During period 3, darunavir GMR (95% confidence interval) values for trough and maximum plasma concentration (C(trough) and C(max)), area under the plasma concentration-time curve (AUC) and elimination half-life (t(1/2)) were 0.64 ng/ml (0.44-0.93), 1.05 ng/ml (0.90-1.24), 0.92 ng h/ml (0.78-1.08) and 0.69 h (0.46-1.05), respectively, when compared with period 1. No statistically significant changes were observed in ritonavir or raltegravir pharmacokinetic parameters. Darunavir C(trough)<550 ng/ml (the minimum effective concentration for protease-resistant HIV viral isolates) was observed in four patients during period 3 only. No clinically significant safety concerns were reported. CONCLUSIONS: Darunavir C(trough) is reduced by 36% when administered without tenofovir/emtricitabine in HIV-1-infected patients. This interaction might be of clinical significance in the management of individuals with protease-resistant HIV viral isolates.

An HIV-1 clade C DNA prime, NYVAC boost vaccine regimen induces reliable, polyfunctional, and long-lasting T cell responses.

The EuroVacc 02 phase I trial has evaluated the safety and immunogenicity of a prime-boost regimen comprising recombinant DNA and the poxvirus vector NYVAC, both expressing a common immunogen consisting of Env, Gag, Pol, and Nef polypeptide domain from human immunodeficiency virus (HIV)-1 clade C isolate, CN54. 40 volunteers were randomized to receive DNA C or nothing on day 0 and at week 4, followed by NYVAC C at weeks 20 and 24. The primary immunogenicity endpoints were measured at weeks 26 and 28 by the quantification of T cell responses using the interferon gamma enzyme-linked immunospot assay. Our results indicate that the DNA C plus NYVAC C vaccine regimen was highly immunogenic, as indicated by the detection of T cell responses in 90% of vaccinees and was superior to responses induced by NYVAC C alone (33% of responders). The vaccine-induced T cell responses were (a) vigorous in the case of the env response (mean 480 spot-forming units/10(6) mononuclear cells at weeks 26/28), (b) polyfunctional for both CD4 and CD8 T cell responses, (c) broad (the average number of epitopes was 4.2 per responder), and (d) durable (T cell responses were present in 70% of vaccinees at week 72). The vaccine-induced T cell responses were strongest and most frequently directed against Env (91% of vaccines), but smaller responses against Gag-Pol-Nef were also observed in 48% of vaccinees. These results support the development of the poxvirus platform in the HIV vaccine field and the further clinical development of the DNA C plus NYVAC C vaccine regimen.

Chimeric ecotropic MLV envelope proteins that carry EGF receptor-specific ligands and the Pseudomonas exotoxin A translocation domain to target gene transfer to human cancer cells.

Redirecting retroviral vector transduction simply by insertion of a ligand into the envelope (Env) protein has met with several obstacles. For example, virions targeted to epidermal growth factor receptor (EGFR), after receptor binding, rapidly traffic to the lysosomes, where they are degraded. Exotoxin A of Pseudomonas aeruginosa has the ability to translocate from endosomes to the cytoplasm by means of a translocation domain (TLD). We generated a series of chimeric Env proteins of Moloney murine leukemia virus containing EGFR ligands, where TLD was inserted into different regions. These chimeric proteins were successfully produced, if the translocation domain was not located at the immediate N-terminus of Env. The ability to transduce murine cells via the ecotropic receptor varied but correlated with the amount of Env proteins incorporated into the virions. Chimeric vector particles could bind to EGFR, demonstrating the functional exposure of the peptide ligand. However, transduction of human cells expressing EGFR but not the ecotropic receptor by virions carrying the chimeric protein was not observed.

The proline-rich region of the ecotropic Moloney murine leukaemia virus envelope protein tolerates the insertion of the green fluorescent protein and allows the generation of replication-competent virus.

Sequences encoding the green fluorescent protein (GFP) were inserted into the envelope protein (Env) of ecotropic Moloney murine leukaemia virus, MoMLV. Insertion of these sequences into the proline-rich region (PRR) of Env resulted in a chimeric GFP-Env protein that allowed retrovirus vector transduction of murine cells with titres similar to wild-type Env. However, N-terminal extension with GFP did not result in a functional Env protein. GFP sequences were then inserted into the Env PRR of E-MO virus, a MoMLV that carries epidermal growth factor sequences at the N terminus of its Env protein. The resulting virus, GFP-EMO1, replicates to the same titres as the parental virus. In a chronically infected cell culture, GFP-EMO1 was genetically stable. However, additional insertions of sequences that led to recombination or that may have been incompatible with virus replication were deleted and decreased virus titre. In summary, Env PRR can be used to tag individual virus particles with GFP, which leaves other regions available for modification in studies aimed at altering virus tropism.

Cell-cycle dependence of foamy virus vectors.

Retroviruses differ in the extent to which they are dependent on host-cell proliferation for their replication, an aspect of their replication that impacts on their vector potential. Foamy viruses offer distinct advantages over other retroviruses for development as vectors for gene therapy. A vector derived from the prototypic foamy virus (PFV), formerly known as human foamy virus (HFV), transduced aphidicolin-arrested cells five- to tenfold more efficiently than one derived from murine leukemia virus (MLV), but several-fold less efficiently than a human immunodeficiency virus type 1 (HIV-1) vector. The same relative efficiency was found following transduction of cells that had been arrested by gamma-irradiation or with mitomycin C. Cells that were exposed to vector during aphidicolin arrest and were subsequently allowed to cycle were transduced significantly better by PFV than by MLV. Quiescent human CD34+ progenitor cells were transduced as efficiently by PFV as by HIV vectors (40-50 %) when transduction was assayed after the cells were allowed to cycle.