Real-time reverse transcriptase PCR for the endogenous koala retrovirus reveals an association between plasma viral load and neoplastic disease in koalas

Koala retrovirus (KoRV) is a newly described endogenous retrovirus and is unusual in that inserts comprise a full-length replication competent genome. As koalas are known to suffer from an extremely high incidence of leukaemia/lymphoma, the association between this retrovirus and disease in koalas was examined. Using quantitative real-time reverse transcriptase PCR it was demonstrated that KoRV RNA levels in plasma are significantly increased in animals suffering from leukaemia or lymphoma when compared with healthy animals. Increased levels of KoRV were also seen for animals with clinical chlamydiosis. A significant positive association between viral RNA levels and age was also demonstrated. Real-time PCR demonstrated as much as 5 log variation in KoRV proviral DNA levels in genomic DNA extracted from whole blood from different animals. Taken together these data indicate that KoRV is an active endogenous retrovirus and suggests that it may be causally linked to neoplastic disease in koalas.

Sequence variation in primer targets affects the accuracy of viral quantitative PCR

This study was designed to assess the effect of sequence mismatches between primers and their targets on viral quantitative PCR. Numerous primers were constructed incorporating various mismatches with a target sequence on the BKV T antigen gene. When using these primers in standard Taqman two-step cycling conditions, as few as two mismatches in a single primer increased cycle threshold values and significantly influenced the calculation of viral load. (C) 2005 Elsevier B.V. All rights reserved.

Phase 1 study of HPV16-specific immunotherapy with E6E7 fusion protein and ISCOMATRIX (TM) adjuvant in women with cervical intraepithelial neoplasia

Purpose: Persistent infection of cervical epithelium with high risk human papillomavirus (HPV) results in cervical intraepithelial neoplasia (CIN) from which squamous cancer of the cervix can arise. A study was undertaken to evaluate the safety and immunogenicity of an HPV 16 immunotherapeutic consisting of a mixture of HPV16 E6E7 fusion protein and ISCOMATRIX(TM) adjuvant (HPV16 Immunotherapeutic) for patients with CIN. Experimental design: Patients with CIN (n = 3 1) were recruited to a randomised blinded placebo controlled dose ranging study of immunotherapy. Results: Immunotherapy was well tolerated. Immunised subjects developed HPV16 E6E7 specific immunity. Antibody, delayed type hypersensitivity, in vitro cytokine release, and CD8 T cell responses to E6 and E7 proteins were each significantly greater in the immunised subjects than in placebo recipients. Loss of HPV16 DNA from the cervix was observed in some vaccine and placebo recipients. Conclusions : The HPV16 Immunotherapeutic comprising HPV16E6E7 fusion protein and ISCOMATRIX(TM) adjuvant is safe and induces vaccine antigen specific cell mediated immunity. (C) 2004 Elsevier Ltd. All rights reserved.

Cytotoxic T-lymphocyte epitope vaccination protects against human metapneumovirus infection and disease in mice

Human metapneumovirus (hMPV) has emerged as an important human respiratory pathogen causing upper and lower respiratory tract infections in young children and older adults. In addition, hMPV infection is associated with asthma exacerbation in young children. Recent epidemiological evidence indicates that hMPV may cocircullate with human respiratory syncytial virus (hRSV) and mediate clinical disease similar to that seen with hRSV. Therefore, a vaccine for hMPV is highly desirable. In the present study, we used predictive bioinformatics, peptide immunization, and functional T-cell assays to define hMPV cytotoxic T-lymphocyte (CTL) epitopes recognized by mouse T cells restricted through several major histocompatibility complex class I alleles, including HILA-A*0201. We demonstrate that peptide immunization with hMPV CTL epitopes reduces viral load and immunopathollogy in the lungs of hMPV-challenged mice and enhances the expression of Th1-type cytokines (gamma interferon and interleukin-12 [IL-12]) in lungs and regional lymph nodes. In addition, we show that levels of Th2-type cytolkines (IL-10 and IL-4) are significantly lower in hMPV CTL epitope-vaccinated mice challenged with hMPV. These results demonstrate for the first time the efficacy of an hMPV CTL epitope vaccine in the control of hMPV infection in a murine model.

Plasma Epstein-Barr virus (EBV) DNA is a biomarker for EBV-positive Hodgkin's lymphoma

Purpose: Latent Epstein-Barr virus (EBV) genomes are found in the malignant cells of approximately one-third of Hodgkin's lymphoma (HL) cases. Detection and quantitation of EBV viral DNA could potentially be used as a biomarker of disease activity. Experimental Design: Initially, EBV-DNA viral load was prospectively monitored from peripheral blood mononuclear cells (PBMC) in patients with HL. Subsequently, we analyzed viral load in plasma from a second cohort of patients. A total of 58 patients with HL (31 newly diagnosed, 6 relapsed, and 21 in long-term remission) were tested. Using real-time PCR, 43 PBMC and 52 plasma samples were analyzed. Results: EBV-DNA was detectable in the plasma of all EBV-positive patients with HL prior to therapy. However, viral DNA was undetectable following therapy in responding patients (P = 0.0156), EBV-positive HL patients in long-term remission (P = 0.0011), and in all patients with EBV-negative HL (P = 0.0238). Conversely, there was no association seen for the EBV-DNA load measured from PBMC in patients with active EBV-positive HL patients as compared with EBV-negative HL, or patients in long-term remission. EBV-DNA load in matched plasma/PBMC samples were not correlated. Conclusions: We show that free plasma EBV-DNA has excellent sensitivity and specificity, and can be used as a noninvasive biomarker for EBV-positive HL and that serial monitoring could predict response to therapy. Additional prospective studies are required to further evaluate the use of free plasma EBV-DNA as a biomarker for monitoring response to treatment in patients with EBV-positive HL.