Persistent infection with human papillomaviruses (HPV) is a prerequisite for the development of cervical cancer. Vaccination with virus-like particles (VLP) has demonstrated efficacy in prophylaxis but lacks therapeutic potential. HPV16 L1E7 chimeric viruslike particles (CVLP) consist of a carboxy-terminally truncated HPV16L1 protein fused to the amino-terminal part of the HPV16 E7 protein and self-assemble by recombinant expression of the fusion protein. The CVLP are able to induce L1-and E7-specific cytotoxic T lymphocytes. We have performed a first clinical trial to gain information about the safety and to generate preliminary data on the therapeutic potential of the CVLP in humans. A randomized, double blind, placebo-controlled clinical trial has been conducted in 39 HPV16 mono-infected high grade cervical intraepithelial neoplasia (CIN) patients (CIN 2/3). Two doses (75 lg or 250 lg) of CVLP were applied. The duration of the study was 24 weeks with 2 optional visits after another 12 and 24 weeks. The vaccine showed a very good safety profile with only minor adverse events attributable to the immunization. Antibodies with high titers against HPV16 L1 and low titers against HPV16 E7 as well as cellular immune responses against both proteins were induced. Responses were equivalent for both vaccine concentrations. A trend for histological improvement to CIN 1 or normal was seen in 39% of the patients receiving the vaccine and only 25% of the placebo recipients. Fifty-six percent of the responders were also HPV16 DNA-negative by the end of the study. Therefore, we demonstrated evidence for safety and a nonsignificant trend for the clinical efficacy of the HPV16 L1E7 CVLP vaccine. ' 2007 Wiley-Liss, Inc.Key words: cervical cancer; clinical trial; immunization; antibody; T cell Genital infection with human papillomavirus (HPV) is one of the most common sexually transmitted diseases. Various molecular and epidemiological studies have documented a correlation between infection with ''high risk'' HPV types and premalignant or malignant tumors of the anogenital tract. 1,2 It is widely acknowledged that a causal relationship exists between persistent HPV infection and development of cervical intraepithelial neoplasia (CIN) and cervical cancer. 3,4 There are over 100 known papillomavirus types that are stratified into low and high risk, based on their association with malignant and invasive lesions. More than 95% of invasive cervical cancers are positive for HPV-DNA, mainly from HPV types 16 (50%) and 18 (20%). Moreover, HPV16 can be detected in 30270% of all HPV-positive high grade CIN patients. 5,6 The prevalence of HPV16 in other intraepithelial neoplasias is even higher, e.g., 70280% in high grade vulvar intraepithelial neoplasia. 7 Whereas for low grade CIN a high spontaneous recovery rate is observed 6,8 high grade CIN regress less often particular at higher age when lesions are more persistent. 9 Because of the potential progression of high grade CIN to invasive cancer, 10 a thorough evaluation consisting of colp...

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a-D-Glucuronidases were purified from the xylanolytic thermophiles CIostridium stercorarium and Thermoanaerobacterium saccharolyticum. This enzyme activity was found to be intracellular in each organism, with T. saccharolyticum producing much greater total activity. The specific activities of the purified enzymes (10 U mg-1 T. saccharolyticum; 1.7 U mg-I C. stercorarium) differed by a factor of approximately 5. For the determination of enzyme activities, 4-Olmethyl-a-D-glucuronosyl-xylotriose was used as a substrate and the glucuronic acid released by a-D-glucuronidase action was quantified by a colorimetric procedure. &OlMethyl-a=D-glucuronosyl-xylotriose was the hydrolysis product that accumulated after exhaustive degradation of &O-methyl-a-D-gIucuronoxylan with xylanases of C. stercorarium. Hydrolysis of side chains in high-molecular-mass glucuronoxylan could not be detected. Neither of the enzymes was able to hydrolyse the chromogenic aryl-substrate p-n i trophenyl-a-D-g I ucuronoside. Both a-D-gl ucuron idases have a dimer ic structure, with monomeric molecular masses of 72 and 76 kDa for C. stercorarium and of 71 kDa for T. saccharolyticum. The pl was estimated to be 4.3 for each enzyme. While both enzymes exhibited a similar pH optimum (pH 556.5) they differed in their thermostabilities. A t 60 "C, half-lives of 14 and 2.5 h, respectively, were determined for the a-D-glucuronidases of C. stercorarium and T. saccharolyticurn. This description of a-D=glucuronidase activity in thermophilic anaerobic bacteria extends our knowledge of these enzymes, previously purified and characterized only in fungi.

A novel enzyme acting on starch and malto-oligosaccharides was identified and characterised. The non-hydrolytic enzyme, designated maltosyltransferase (MTase), of the hyperthermophilic bacterium Thermotoga maritima MSB8 disproportionates malto-oligosaccharides via glycosyl transfer reactions. The enzyme has a unique transfer specificity strictly confined to the transfer of maltosyl units. Incubation of MTase with starch or its constituents, i.e. amylose and amylopectin, led to the formation of a set of multiples of maltose (i.e. maltose, maltotetraose, maltohexaose etc.). Malto-oligosaccharides with a degree of polymerization (DP) X were disproportionated to products with a DP of X Ϯ2n (with Xу 3 and n ϭ 0,1, 2, ...). Maximum activity in a 10-min assay was recorded at pH 6.5 and 85Ϫ90°C. The enzyme displayed extraordinary resistance to thermal inactivation. For example, at 90, 85, and 70°C (pH 6.5, 0.34 mg ml Ϫ1 protein), MTase half-lives of about 2.5 h, 17 h, and 21 days, respectively, were recorded.The gene for MTase, designated mmtA, was isolated from a gene library of T. maritima strain MSB8. Analysis of the MTase primary structure as deduced from the nucleotide sequence of mmtA revealed that the enzyme is not closely related to known protein sequences. However, low-level local similarity between MTase and the A-amylase enzyme family (glycosyl hydrolase family 13) was detected, including conserved acidic residues essential for catalysis. Therefore, MTase should be assigned to this family. Based on detailed sequence analyses and comparison with amylolytic enzymes of known crystal structure we propose that MTase contains a (β/A) 8 -fold as the core supersecondary structure which is typical for the Aamylase family. On the other hand, MTase is unique in that it lacks several residues highly conserved throughout this family. Also, MTase possesses an extraordinarily large domain B (a domain typical for the A-amylase family, inserted between β-strand 3 and A-helix 3 of the (β/A) 8 -barrel fold).Keywords : glycosyl transfer; mmtA gene; nucleotide sequence; hyperthermophilic bacterium; thermostability.Enzymes from organisms inhabiting extremely hot environments represent fascinating objects for basic as well as applied research. On the one hand, the industry is interested in thermostable biocatalysts for biotechnological processes demanding elevated temperatures. On the other hand, considerable efforts have been made over the last years to understand how the proteins of extreme thermophiles maintain their structure and function at high temperatures (for a review, see [1]). We have focused our interests on the enzymes involved in carbohydrate breakdown and utilisation of the hyperthermophilic bacterium, Thermotoga maritima. This organism, which represents one of the deepest branches in the phylogenetic tree of the bacterial domain [2], is characterised by a growth range between 55°C and 90°C with an optimum at 80°C [3], making it one of the most thermophilic bacteria known to date. T. maritima MSB8 (DSM 3109), the typ...

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