No abstract

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...

myr 5 is an unconventional myosin (class IX) from rat that contains a Rho-family GTPase-activating protein (GAP) domain. Herein we addressed the specificity of the myr 5 GAP activity, the molecular mechanism by which GAPs activate GTP hydrolysis, the consequences of myr 5 overexpression in living cells, and its subcellular localization. The myr 5 GAP activity exhibits a high specificity for Rho. To achieve similar rates of GTPase activation for RhoA, Cdc42Hs, and Rac1, a 100-fold or 1000-fold higher concentration of recombinant myr 5 GAP domain was needed for Cdc42Hs or Rac1, respectively, as compared with RhoA. Cell lysates from Sf9 insect cells infected with recombinant baculovirus encoding myr 5 exhibited increased GAP activity for RhoA but not for Cdc42Hs or Rac1. Analysis of Rho-family GAP domain sequences for conserved arginine residues that might contribute to accelerate GTP hydrolysis revealed a single conserved arginine residue. Mutation of the corresponding arginine residue in the myr 5 GAP domain to a methionine (M1695) virtually abolished Rho-GAP activity. Expression of myr 5 in Sf9 insect cells induced the formation of numerous long thin processes containing occasional varicosities. Such morphological changes were dependent on the myr 5 Rho-GAP activity, because they were induced by expressing the myr 5 tail or just the myr 5 Rho-GAP domain but not by expressing the myr 5 myosin domain. Expression of myr 5 in mammalian normal rat kidney (NRK) or HtTA-1 HeLa cells induced a loss of actin stress fibers and focal contacts with concomitant morphological changes and rounding up of the cells. Similar morphological changes were observed in HtTA-1 HeLa cells expressing just the myr 5 Rho-GAP domain but not in cells expressing myr 5 M1695. These morphological changes induced by myr 5 were inhibited by coexpression of RhoV14, which is defective in GTP hydrolysis, but not by RhoI117. myr 5 was localized in dynamic regions of the cell periphery, in the perinuclear region in the Golgi area, along stress fibers, and in the cytosol. These results demonstrate that myr 5 has in vitro and in vivo Rho-GAP activity. No evidence for a Rho effector function of the myr 5 myosin domain was obtained.

cleoside analogues, [8][9][10] the only established treatment option Hepatitis B virus (HBV) replicates via an intermediate to prevent reinfection is the administration of polyclonal hep-RNA step. High frequency of polymerase errors with adatitis B surface antigen antibody (anti-HBs) (hepatitis B imditional selection pressure leads to mutations in the mune globulin [HBIG]). HBIG reduces the rate of reinfection HBV genome. We investigated the number, type, and anfrom about 90% to less than 30% and improves the long-term tigenic effects of mutations in the coding region of the outcome of patients who underwent OLT for HBV-related HBV surface antigen in eight patients who underwent disease. 2-4orthotopic liver transplantation (OLT) for HBV-related A number of explanations for graft infection have been end-stage liver disease and were experiencing infection proposed. First, virions from the explanted liver are circulatof the graft and who received hepatitis B surface antigen ing in the blood during or soon after transplantation. Second, antibody (anti-HBs) prophylaxis (hepatitis B immune virions are replicating at extrahepatic sites. HBIG interferes globulin [HBIG]) after OLT. Controls were chronic HBV with this process and probably prevents virions from entering patients who underwent kidney transplantation and rehepatocytes. 11 The mechanisms that lead to reinfection in ceived the same immunosuppressive regime but no 30% of patients receiving HBIG after OLT are not under-HBIG. The S-gene was amplified from serum before and stood. after transplantation, sequenced, and changes in the ge-HBV employs reverse transcriptase for its replication; this nome were analyzed. In the five patients who experilacks proofreading capability, and thus leads to a higher enced reinfection while receiving anti-HBs, clear mutanumber of mutations in the HBV genome. 12 Some of these tions occurred in the S-gene. In the patient who did not variants may have a replication advantage and become domireceive HBIG and those who experienced reinfection nant. From the pool of variants with similar replication poonly after termination of HBIG, no mutations were tential, some will be positively selected by forces such as found in the S-gene. In the kidney recipients, mutations the humoral or cellular immune response 13; these are termed in the S-gene occurred in only one of eight patients. Beescape mutants. Evolution of viruses under antibody prescause the a determinant contains neutralizing epitopes, sure, either monoclonal or polyclonal, has been well studied this region was chosen for antibody binding to quantify both in vitro and in vivo. The addition of neutralizing antibodantigenic effects of the mutations. The two patients who ies to virus cultures regularly results in isolates that are not selected mutations in the a determinant and became reneutralized by the added antibodies. This is particularly true infected while receiving HBIG had reduced antibody with monoclonal reagents, because the change in viral antibinding after OLT. Our results...