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 γ 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/106 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.

BackgroundIn 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.MethodologyPatients 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.ConclusionXMRV 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.

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BackgroundWe 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.ResultsIn 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.ConclusionsThese 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.

The human foamy virus (HFV) genome possesses three open reading frames (bel 1, 2, and 3) located between env and the 3' long terminal repeat. By analogy to other human retroviruses this region was selected as the most likely candidate to encode the viral transactivator. Results presented here confirmed this and showed further that a deletion introduced only into the bel1 open reading frume of a plasmid derived from an infectious molecular done of HFV abolished transactivation. In contrast, deletions in bel2 and bel 3 had only minor effects on the ability to transactivate. The role of the bel 1 genomic region as a transactivator was further investigated by eukaryotic expression of a genome fragment of HFV spanning the bel 1 open reading frame. A construct expressing bel 1 under control of a heterologous promoter was found to transactivate the HFV long terminal repeat in a dose-dependent fashion. Furthermore, it is shown that the U3 region of the HFV long terminal repeat is sufficient to respond to the HFV transactivator.

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.

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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 c-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.We demonstrated previously that, like murine leukemia virus (MLV), wild-type foamy viruses do not productively infect G 1 /S-or G 2 -arrested cells and that mitosis is a prerequisite for virus protein expression (Bieniasz et al., 1995). It was demonstrated subsequently, however, that foamy virus-derived vectors transduced stationary-phase cells, albeit with low efficiency . Moreover, nuclear import of the foamy virus pre-integration complex (PIC) can take place in G 1 /S phase-arrested cells, possibly facilitated by the fact that the foamy virus Gag and Pol proteins, including the integrase, contain nuclear localization signals (NLSs) (Schliephake & Rethwilm, 1994;Saïb et al., 1997;Imrich et al., 2000), but the transduction efficiency remains low. After uncoating, the Gag protein, in association with the virus genome, targets the centrosome via the microtubule network and enters the nucleus, but no integration or virus gene expression takes place. Out of step with these studies, Mergia et al. (2001) demonstrated efficient transduction of arrested and stationary-phase cells by simian foamy virus type 1 (SFV-1)-based vectors, which the authors suggested was due to the high titre of vector and the cytomegalovirus (CMV) promoter, which is known to be active in arrested cells. Hirata et al. (1996) showed that haematopoietic progenitor cells (HPCs), pre-stimulated into cycling by cytokines, were transduced similarly by PFV-and MLV-based vectors. However, more recent studies have shown that freshly The HIV vector was made by transient transfection using the following constructs: either pH7G, which encodes eGFP under the control of the CMV promoter (Ikeda et al., 2003), or pH7nZ, which is the same as pH7G, but contains the LacZ transgene in place of eGFP, the HIV Gag-Pol construct, pSYNGP (Kotsopoulou et al., 2000) and the VSV-G envelope construct, pRV67 (Mitrophanous et al., 1999).The pMD11 and pMD9 plasmids encode the PFV vector genome (Heinkelein et al., 2002). The pMD...