Background:More than 100 different human papillomaviruses (HPVs) can cause proliferative diseases, many of which are malignant, such as cervical cancer. HPV serology is complex because infection and disease lead to distinct type-specific antibody responses. Using bead-based technology, we have developed an assay platform that allows the simultaneous detection of antibodies against up to 100 in situ affinity-purified recombinant HPV proteins. Methods: Twenty-seven HPV proteins were expressed as glutathione S-transferase fusion proteins and affinity-purified in one step by incubation of glutathionedisplaying beads in bacterial lysate. Spectrally distinct bead sets, each carrying one particular antigen, were mixed, incubated with serum, and differentiated in a flow cytometer-like analyzer (xMAP; Luminex Corp). Antibodies bound to the antigens were detected via fluorescent secondary reagents. We studied 756 sera from 2 case-control studies of cervical cancer. Results: Glutathione S-transferase fusion proteins bound with high affinity to glutathione-displaying beads (K d ‫؍‬ 6.9 ؋ 10 ؊9 mol/L). The dynamic range of multiplex serology covered 1.5 orders of magnitude, and antibodies

Papillomavirus-like particles (VLPs) are a promising prophylactic vaccine candidate to prevent human papillomavirus (HPV) infections and associated epithelial neoplasia. However, they are unlikely to have therapeutic effects because the virion capsid proteins are not detected in the proliferating cells of the infected epithelia or in cervical carcinomas. To increase the number of viral antigen targets for cell-mediated immune responses in a VLP-based vaccine, we have generated stable chimeric VLPs consisting of the L1 major capsid protein plus the entire E7 (11 kDa) or E2 (43 kDa) nonstructural papillomavirus protein fused to the L2 minor capsid protein. The chimeric VLPs are indistinguishable from the parental VLPs in their morphology and in their ability to agglutinate erythrocytes and elicit high titers of neutralizing antibodies. Protection from tumor challenge was tested in C57BL͞6 mice by using the tumor cell line TC-1, which expresses HPV16 E7, but not the virion structural proteins. Injection of HPV16 L1͞L2-HPV16 E7 chimeric VLPs, but not HPV16 L1͞L2 VLPs, protected the mice from tumor challenge, even in the absence of adjuvant. The chimeric VLPs also induced protection against tumor challenge in major histocompatibility class IIdeficient mice, but not in ␤ 2 -microglobulin or perforin knockout mice implying that protection was mediated by class I-restricted cytotoxic lymphocytes. These findings raise the possibility that VLPs may generally be efficient vehicles for generating cellmediated immune responses and that, specifically, chimeric VLPs containing papillomavirus nonstructural proteins may increase the therapeutic potential of VLP-based prophylactic vaccines in humans.Human papillomaviruses (HPVs) that infect the genital tract are associated with human anogenital tract cancer, particularly cervical cancer (reviewed in ref. 1). HPVs are thought to be the primary causative agent in Ͼ90% of cervical cancers (2), with HPV16 being the type most frequently found in these tumors. Approximately 500,000 women develop cervical cancer each year, and 200,000 women die from it, making this disease the second-most common cause of cancer deaths in women worldwide (3).Significant advances have been made recently in the development of a candidate prophylactic vaccine against papillomavirus infections (reviewed in ref. 4). Expression of the papillomavirus major capsid protein, L1, in eukaryotic cells leads to self-assembly into virus-like particles (VLPs) that are morphologically indistinguishable from native virions and present the conformational epitopes required for the induction of high titer neutralizing antisera (5). L2, the minor capsid protein, coassembles with L1 at a ratio of Ϸ1 L2 molecule to 30 L1 molecules (6). Although L2 presents some epitopes that induce the production of neutralizing antiserum (7), most neutralizing antibodies induced by L1͞L2 VLPs recognize L1 determinants (8). Several studies have shown that L1 and L1͞L2 VLP-based vaccines protect animals against high dose experimental pap...

Vitiligo is an autoimmune disease characterized by destruction of melanocytes, leaving 0.5% of the population with progressive depigmentation. Current treatments offer limited efficacy. We report that modified inducible heat shock protein 70 (HSP70i) prevents T cell–mediated depigmentation. HSP70i is the molecular link between stress and the resultant immune response. We previously showed that HSP70i induces an inflammatory dendritic cell (DC) phenotype and is necessary for depigmentation in vitiligo mouse models. Here, we observed a similar DC inflammatory phenotype in vitiligo patients. In a mouse model of depigmentation, DNA vaccination with a melanocyte antigen and the carboxyl terminus of HSP70i was sufficient to drive autoimmunity. Mutational analysis of the HSP70i substrate-binding domain established the peptide QPGVLIQVYEG as invaluable for DC activation, and mutant HSP70i could not induce depigmentation. Moreover, mutant HSP70iQ435A bound human DCs and reduced their activation, as well as induced a shift from inflammatory to tolerogenic DCs in mice. HSP70iQ435A-encoding DNA applied months before spontaneous depigmentation prevented vitiligo in mice expressing a transgenic, melanocyte-reactive T cell receptor. Furthermore, use of HSP70iQ435A therapeutically in a different, rapidly depigmenting model after loss of differentiated melanocytes resulted in 76% recovery of pigmentation. Treatment also prevented relevant T cells from populating mouse skin. In addition, ex vivo treatment of human skin averted the disease-related shift from quiescent to effector T cell phenotype. Thus, HSP70iQ435A DNA delivery may offer potent treatment opportunities for vitiligo.

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...