Rabbits immunised with recombinant BCG expressing the cottontail rabbit papillomavirus (CRPV) L2E7E2 genes induces regression of established papillomas.

We previously demonstrated in a cottontail rabbit papillomavirus (CRPV) challenge model that recombinant Bacille Calmette-Guerin (rBCG) could potentially be used as a prophylactic vaccine vehicle to deliver papillomavirus proteins. In this study we investigated whether regression of CRPV-induced papillomas could be achieved following immunisation of out-bred New Zealand White rabbits with rBCG expressing CRPVL2, CRPVE2, CRPVE7 or CRPVL2E7E2 proteins. Rabbits immunised with rBCG/CRPVL2E7E2 had papillomas that were largely suppressed and were significantly smaller compared to the rBCG negative control group (P

Risk factors for grade 3-4 liver enzyme elevation in HIV and hepatitis C coinfected patients on combination antiretroviral therapy.

Although coinfection with hepatitis C (HCV) is an established risk factor for hepatotoxicity in HIV-positive patients receiving combination antiretroviral therapy (cART), specific variables that may be predictive of severe hepatotoxicity among co-infected patients receiving cART remain poorly defined. A retrospective cohort study of HIV/HCV coinfected adults from two HIV treatment centers covering the period between December 1998 and December 2003 was conducted to address this question. The primary endpoint of the study was the occurrence of grade 3 or 4 elevation of serum alanine aminotransferase (ALT) during follow-up and the primary predictors of interest were specific antiretrovirals. One hundred five coinfected patients receiving cART for a median of 70 months (interquartile range [IQR], 37, 83) were included in the analysis. Twenty-three (22%) patients developed a grade 3 or 4 increase in serum ALT at least once in follow-up. In univariate analysis, current receipt of lopinavir/ritonavir (LPV/r) (odds ratio [OR] 3.09, 95% confidence interval [CI] 1.14-8.34, p = 0.03), baseline ALT (OR 1.01, 95% CI 1.00-1.02, p = 0.004), and current use of boosting ritonavir (OR 2.84, 95% CI 1.16-7.00, p = 0.02) were significantly associated with a grade 3 or 4 increase in serum ALT, although most patients receiving boosting ritonavir were on lopinavir/ritonavir based regimens. Patients receiving LPV/r had been previously exposed to significantly more antiretrovirals (p < 0.0001), protease inhibitors (p < 0.0001), and nucleoside analogues (p = 0.0009) compared to the rest of the cohort. Further research to better clarify risk factors for hepatotoxicity in coinfected patients is warranted given the challenges in treating this population.

Strategies for human papillomavirus therapeutic vaccines and other therapies based on the E6 and E7 oncogenes.

High-risk human papillomaviruses (HR-HPVs) are one of the most devastating oncogenic viruses worldwide and have been causally linked with the development of human cervical cancer. Several prophylactic and therapeutic clinical HPV vaccine trials are in progress. Although prophylactic vaccines are useful in preventing the incidence of cervical cancer, the elimination of existing HPV infections needs to be addressed, because cervical cancer is the leading female cancer in developing countries. Several different and encouraging strategies have been investigated in a preclinical and clinical setting for the treatment and elimination of existing HPV-induced infection. This review summarizes the therapeutic clinical trials and the different preclinical research strategies that are under investigation whereby HR-HPV E6 and E7 oncogenes are delivered in a nucleic acid form, in viral and bacterial vectors, or as peptide- and protein-based vaccines.

No relationship observed between human p53 codon-72 genotype and HPV-associated cervical cancer in a population group with a low arginine-72 allele frequency.

Infection with high-risk human papillomavirus (HR-HPV) is a necessary but not a sufficient event in the development of cervical cancer, as most infections regress without intervention. Thus, genetic host factors and cellular immune responses could be potential modifiers for the risk of developing cervical cancer. In particular, p53 is considered as the most critical tumour suppressor gene and is involved in regulating cell division. The polymorphism on p53, which encodes either a proline or an arginine amino acid residue at codon 72, has been reported as a possible risk factor for cervical disease. This polymorphism has been shown to differentially affect the efficiency of degradation of p53 protein mediated by HR-HPV E6 oncoprotein. Women with histologically proven cancer of the cervix (n = 111) and hospital-based controls (n = 143) were included in this study. The patients and controls were from the Western Cape Province in South Africa. Genotyping of the p53 polymorphism was conducted using polymerase chain reaction and restriction fragment-length polymorphism method. The distributions of the allelic frequencies were stratified in both patients and controls into two South African ethnic population groups. In this study, we observed no association between the distribution of p53 polymorphism and susceptibility to cervical cancer in the Western Cape Province populations (P = 0.466). However, the frequency of the Pro/Pro residue at codon 72 was increased in the South African population when compared to Caucasians, Indians and Portuguese population groups. Notably, as the distribution of the Pro/Pro at codon 72 of p53 gene was significantly different (P < 0.05) between the control groups of South Africa and other population groups. This result suggests that ethnic disparity may influence the levels of p53 produced.

Immunisation with recombinant BCG expressing the cottontail rabbit papillomavirus (CRPV) L1 gene provides protection from CRPV challenge.

Recombinant Bacille Calmette-Guerin (rBCG) could potentially be the vaccine vehicle of choice to deliver foreign antigens from multiple pathogens. In this study we have used the cottontail rabbit papillomavirus (CRPV) rabbit model to provide a "proof of concept" that immunisation with rBCG expressing the CRPV major capsid protein, L1 (rBCG/CRPVL1), will protect outbred New Zealand White rabbits against CRPV challenge. Rabbits immunised with rBCG/CRPVL1 (10(7) cfu/ml) were protected 5 weeks post-CRPV challenge. Rabbits immunised with rBCG/CRPVL1 (10(5) cfu/ml) had papillomas, which were smaller and took longer to appear than the control rabbits. None of the negative control rabbits vaccinated with rBCG expressing an irrelevant gene or PBS were protected from CRPV challenge. Sera from rabbits immunised with rBCG/CRPVL1 (10(7) cfu/ml) were able to neutralise 54.5% of CRPV at serum dilutions of 1:200. These results provide evidence that BCG could potentially be used as a vaccine delivery vehicle for human papillomavirus proteins as a possible prophylactic vaccine.

Plant-produced cottontail rabbit papillomavirus L1 protein protects against tumor challenge: a proof-of-concept study.

The native cottontail rabbit papillomavirus (CRPV) L1 capsid protein gene was expressed transgenically via Agrobacterium tumefaciens transformation and transiently via a tobacco mosaic virus (TMV) vector in Nicotiana spp. L1 protein was detected in concentrated plant extracts at concentrations up to 1.0 mg/kg in transgenic plants and up to 0.4 mg/kg in TMV-infected plants. The protein did not detectably assemble into viruslike particles; however, immunoelectron microscopy showed presumptive pentamer aggregates, and extracted protein reacted with conformation-specific and neutralizing monoclonal antibodies. Rabbits were injected with concentrated protein extract with Freund's incomplete adjuvant. All sera reacted with baculovirus-produced CRPV L1; however, they did not detectably neutralize infectivity in an in vitro assay. Vaccinated rabbits were, however, protected against wart development on subsequent challenge with live virus. This is the first evidence that a plant-derived papillomavirus vaccine is protective in an animal model and is a proof of concept for human papillomavirus vaccines produced in plants.

Incidence of and risk factors for tenofovir-induced nephrotoxicity: a retrospective cohort study.

OBJECTIVES: Despite the recent publication of case reports describing various manifestations of tenofovir-related nephrotoxicity, data regarding the incidence of and risk factors for this adverse effect are currently lacking. METHODS: A retrospective cohort study of patients from four centres in Toronto, Canada, enrolled in the tenofovir expanded access programme with a minimum of 3 months follow up, was carried out. RESULTS: A total of 172 patients receiving tenofovir disoproxil fumarate (TDF) for a median of 16 months (range 3-25 months) were included in the study. Seven (4%) patients developed grade 1 (>44 micromol/L from baseline) increases in serum creatinine (SCr) during follow up; no patient developed grade 2 or higher nephrotoxicity. Fifteen (8.7%) patients had an increase in SCr of greater than 1.5 times baseline values during follow up. Four (2.3%) patients discontinued TDF because of an increase in SCr and/or abnormal urinalysis. Of 62 patients with a urinalysis, grade 1 or higher proteinuria (< 3 g/L) was observed in 27 (43%) patients. Only baseline SCr [odds ratio (OR)=0.51 per 10 micromol/L increase; P=0.0005] and baseline creatinine clearance (1.26 per 10 mL/min increase; P=0.01) were significantly associated with ever having a 1.5-fold increase in serum creatinine. Twenty-eight (16%) and 11 (6%) patients developed grade 1 (serum phosphorus < or = 0.71 mmol/L) and grade 2 (serum phosphorus < or = 0.61 mmol/L) hypophosphataemia during follow-up, respectively. CONCLUSIONS: Although slight increases in SCr did occur after starting TDF, clinically significant nephrotoxicity was rare. The clinical significance of TDF-related hypophosphataemia and proteinuria requires further study.

A PCR detection method for rapid identification of Paenibacillus larvae.

American foulbrood is a disease of larval honeybees (Apis mellifera) caused by the bacterium Paenibacillus larvae. Over the years attempts have been made to develop a selective medium for the detection of P. larvae spores from honey samples. The most successful of these is a semiselective medium containing nalidixic acid and pipermedic acid. Although this medium allows the growth of P. larvae and prevents the growth of most other bacterial species, the false-positive colonies that grow on it prevent the rapid confirmation of the presence of P. larvae. Here we describe a PCR detection method which can be used on the colonies that grow on this semiselective medium and thereby allows the rapid confirmation of the presence of P. larvae. The PCR primers were designed on the basis of the 16S rRNA gene of P. larvae and selectively amplify a 973-bp amplicon. The PCR amplicon was confirmed as originating from P. larvae by sequencing in both directions. Detection was specific for P. larvae, and the primers did not hybridize with DNA from closely related bacterial species.

Analysis of the complete genome sequence of acute bee paralysis virus shows that it belongs to the novel group of insect-infecting RNA viruses.

The complete genome sequence of acute bee paralysis virus (ABPV) was determined. The 9470 nucleotide, polyadenylated RNA genome encoded two open reading frames (ORF1 and ORF2), which were separated by 184 nucleotides. The deduced amino acid sequence of the 5' ORF1 (nucleotides 605 to 6325) showed significant similarity to the RNA-dependent RNA polymerase, helicase, and protease domains of viruses from the picornavirus, comovirus, calicivirus, and sequivirus families, as well as to a novel group of insect-infecting RNA viruses. The 3' ORF2 (nucleotides 6509-9253) was proposed as encoding a capsid polyprotein with three major structural proteins (35, 33, and 24 kDa) and a minor protein (9.4 kDa). This was confirmed by N-terminal sequence analysis of two of these proteins. The overall genome structure of ABPV showed similarities to those of Drosophila C virus, Plautia stali intestine virus, Rhopalosiphum padi virus, and Himetobi P virus, which have been classified into a novel group of picorna-like insect-infecting RNA viruses called cricket paralysis-like viruses. It is suggested that ABPV belongs to the cricket paralysis-like viruses.

A PCR detection method for rapid identification of Melissococcus pluton in honeybee larvae.

Melissococcus pluton is the causative agent of European foulbrood, a disease of honeybee larvae. This bacterium is particularly difficult to isolate because of its stringent growth requirements and competition from other bacteria. PCR was used selectively to amplify specific rRNA gene sequences of M. pluton from pure culture, from crude cell lysates, and directly from infected bee larvae. The PCR primers were designed from M. pluton 16S rRNA sequence data. The PCR products were visualized by agarose gel electrophoresis and confirmed as originating from M. pluton by sequencing in both directions. Detection was highly specific, and the probes did not hybridize with DNA from other bacterial species tested. This method enabled the rapid and specific detection and identification of M. pluton from pure cultures and infected bee larvae.