TA-CIN, a vaccine incorporating a recombinant HPV fusion protein (HPV16 L2E6E7) for the potential treatment of HPV16-associated genital diseases

Commercially available prophylactic HPV vaccines for cervical cancer prevention have limited use in women with previous viral exposure. Therefore, a therapeutic HPV vaccine would benefit patients with HPV-associated genital diseases. Being developed by Cancer Research Technology Ltd, under license from Xenova Group plc, TA-CIN (Tissue Antigen - Cervical Intraepithelial Neoplasia) is a fusion protein vaccine comprising the HPV16 viral proteins L2, E6 and E7 for the treatment of HPV16-associated genital diseases. In mouse models, TA-CIN induced dose-dependent HPV16-specific CD4 and CD8 T-cell responses, which were enhanced when boosted with the vaccinia-based vector vaccine TA-HPV (Therapeutic Antigen - HPV). A phase I clinical trial of TA-CIN in healthy volunteers reported no serious adverse events and HPV16-specific cellular immune responses. Phase II trials in patients with anogenital and vulval intraepithelial neoplasia investigated heterologous prime/boost strategies with TA-CIN/TA-HPV and TA-HPV/TA-CIN, but neither of the regimens offered advantages over single-agent TA-HPV. A recent phase II trial investigating imiquimod/TA-CIN in patients with vulval intraepithelial neoplasia demonstrated significant infiltration of CD4 and CD8 T-cells in lesion responders and complete lesion regression in 63% of patients. More comprehensive case-controlled trials are needed to define responders to immunotherapy with TA-CIN and verify its prophylactic and therapeutic properties

Inverse PCR

Polymerase chain reaction (PCR) is widely accepted as one of the most powerful tools in molecular biology capable of amplifying over 106 identical copies of small (few kb), specific DNA regions. It utilizes the thermostability of the enzyme Taq DNA polymerase I, and allows quick and efficient exponential amplification of a target region between known DNA sequences. This removes the need for time‐consuming cloning, although it does have two key limitations. It is only efficient in amplifying relatively small DNA fragments and secondly, but more importantly, PCR can only be used to amplify regions of known DNA sequence. As such, PCR alone cannot be used to amplify DNA sequences adjacent to an unknown sequence, thus making chromosomal walking unfeasible

Blotting techniques

The capacity to separate nucleic acids and proteins by their molecular weight and identify fractions of interest by the hybridization of specific labeled probes is known as blotting. It was originally developed for the analysis of gel‐fractionated DNA and was termed the Southern blot. Northern blotting was soon to follow and permitted the investigation of RNA templates by using an identical procedure. Since then, Western blotting was designed to analyze proteins and a procedure known as Dot/Slot blotting was developed to detect and quantify any nucleic acid sequence without the necessity of processing [restriction fragment length polymorphism (RFLP) and/or separation by electrophoresis] the nucleic acids

Long distance PCR

The polymerase chain reaction (PCR) is a common tool of molecular biology and is utilized in the field of medical diagnostics to identify genetic disorders and the causative agents of human infections. PCR is also used for typing microorganisms, thus playing an important role in both epidemiology and phylogenetics. The PCR method is a starting point that when used together with cloning is influential in the sequencing of novel genes and genomes

Development of optimal liquid based cytology sample processing methods for HPV testing: Minimising the inadequate test result

Incorporation of HPV testing into cervical screening is anticipated and robust methods for DNA extraction from liquidbasedcytology (LBC) samples are required. This study compared QIAamp extraction with Proteinase K digestion and developed methods to address DNA extraction failure (β-globin PCR negative) from clinical specimens. Proteinase K and QIAamp extraction methods in paired LBC samples were comparable with adequate DNA retrieved from 93.3% of clinical specimens. An HPV prevalence cohort (n = 10,000) found 7% (n = 676) LBC samples tested negative for β-globin, and were classified as inadequate. This ‘failure’ rate is unsuitable for population screening, particularly as the sampling method is intrusive. 379/676 samples were assessed to determine the cause of test failure. Re-testing confirmed adequate DNA in 21.6% of the original extracts; re-extraction from stored material identified 56.2% samples contained adequate material; dilution to overcome sample inhibition (1:10) resolved 51.7% cases in original extracts and 28% in new extracts. A standardised approach to HPV testing with an optimal DNA concentration input rather than standard volume input is recommended. Samples failing initial DNA extraction should be repeat extracted and assessed for sample inhibition to reduce the 7% of HPVtests being reported as inadequate and reduce the need for retesting of those women to <1%

Human papillomavirus negative but dyskaryotic cervical cytology: re-analysis of molecular testing

Background Evaluation of molecular Human Papillomavirus (HPV) testing into UK Cervical Screening Programmes is underway. In South Wales the current HPV prevalence in women attending routine screening is 13.5% with 76.3% HR HPV positive in cases with reported dyskaryotic cervical cytology [Hibbitts S, Jones J, Powell N, Dallimore N, McRea J, Beer H, et al. Human papillomavirus prevalence in women attending routine cervical screening in South Wales, UK: a cross-sectional study. Br J Cancer 2008;99(December (11)):1929–33]. Objectives The aim of this study was to re-analyse the 23.7% cases with reported dyskaryotic cytology that were HR HPV negative (n = 52 out of 219 in a population of 10,000). Study design Three procedures were performed: (i) GP5+/GP6+ PCR-EIA repeat on original DNA extracts; (ii) DNA extraction and GP5+/GP6+ HPV PCR-EIA; (iii) DNA extraction and HPV typing using Greiner Bio-One™ PapilloCheck DNA microarray. Results 51 out of 52 samples were re-analysed. Direct repeat HPV PCR-EIA identified 24% (n = 12/51) of samples positive for HR HPV. Re-extracted DNA and PCR-EIA increased detection to 41.2% (n = 21/51) and PapilloCheck detected 78.4% (n = 40/51). HR HPV detection by PapilloCheck was significantly higher compared with the other methods of re-analysis. Eleven samples were persistently HR HPV negative but 4 tested positive for low risk HPV. Conclusions This study identifies that up to 78% of samples with dyskaryotic cervical cytology that test negative for HPV can be found to be HPV positive on re-analysis. The reliance on a single negative HPV test result could lead to missed HPV related disease in a subset of patients, the number dependant on which HPV test is performed. The clinical significance of a false negative HPV result depends on the screening interval and how HPV testing is incorporated into screening