Human papillomaviruses trophic for anogenital epithelia cause benign warts, and certain genotypes are closely associated with cervical neoplasia. By using our modifications of the epithelial raft culture system, we were able to recapitulate and modulate the infectious program of a papillomavirus in vitro for the first time. Small pieces of a condyloma containing human papillomavirus type 11 were explanted onto a dermal equivalent consisting of a collagen matrix with fibroblasts and were cultured at the medium-air interface. The infected stem cells proliferated rapidly across the matrix, stratified, and differentiated, as judged by histology. The results correlated with the state of epithelial differentiation, which, in turn, was dependent on the type of fibroblast in the matrix. Under conditions where the epithelial outgrowth underwent terminal differentiation, the entire productive program took place, leading to virion assembly. In contrast, using an alternative condition where the outgrowth failed to achieve terminal differentiation, only the E-region RNAs from the E1 promoter accumulated to any appreciable extent. The proliferating cell nuclear antigen was induced in the differentiated suprabasal cells in the productive cyst growth, which also exhibited high copy viral DNA and abundant E6--E7 RNAs. Comparable cells in the nonproductive cyst outgrowth were negative for all three. These results suggest that the E6 and E7 proteins may play a role in establishing a cellular environment conducive to vegetative viral replication. The culture conditions described should be useful for genetic analysis of this family of important human pathogens and for testing potential pharmacological agents.

The Li coat protein of human papillomavirus type 11 (HPV-11) was expressed in Sf-9 insect cells with the recombinant baculovirus vector AcllLl. Viruslike particles (VLPs) were identified by electron microscopy in the nucleus and cytoplasm of Sf-9 cells infected with AclILl. The Li protein was purified from AcliLlinfected insect cells. The purified protein spontaneously assembled in vitro into various aggregates, including particles appearing similar to empty virions. Reaction of VLP-containing insect cell extracts with antisera directed against either denatured or nondenatured capsid epitopes in Western blot (immunoblot) and immuno-dot blot assays suggested that conformational epitopes present in native HPV-11 infectious virions were also present on the baculovirus-produced HPV-11 VLPs. Immuno-dot blot assays using human sera obtained from individuals with biopsy-proven condyloma accuminatum correlated closely with results previously obtained in HPV-11 whole virus particle-based enzyme-linked immunosorbent assays. These morphologic and immunologic similarities to native HPV-11 virions suggest that recombinant VLPs produced in the baculovirus system may be useful in seroepidemiology and pathogenesis studies of genital HPV infection and that they may also be potential candidates for vaccine development.

Recombinant human papillomavirus type 11 (HPV-11) virus-like particles (VLPs) were tested for their ability to induce the formation of neutralizing antibodies, and were also tested for serodiagnostic capabilities in an ELISA in comparison with HPV-11 whole virions. VLPs, purified by CsC1 density gradient centrifugation from the cell-free supernatant of AcllLl-infected Sf9 suspension cell cultures, were used to immunize rabbits and anti-VLP antibodies were tested in the athymic mouse model of HPV-11 infection. Pretreatment of infectious HPV-11 virions with the immune serum of VLP-treated animals caused a marked reduction of graft growth (P < 10 -4) and viral gene expression (P < 10-4), similar to the effects obtained using whole virion postimmune serum, and consistent with immune neutralization. To assess the serodiagnostic capabilities of VLPs, a VLP ELISA was developed and used to analyse sera that were tested previously in an HPV-11 whole virion ELISA. Specific antibodies were detected in 49 % of patients' sera (P = 2 x 10-4), and individual VLP seroreactivities correlated with those previously obtained using whole virions as the antigen (r = 0.87; P < 10-6). These results indicate that recombinant VLPs can be used to elicit a neutralizing antibody response, and can substitute faithfully for native virions in the development of HPV-serodiagnostic immunoassays.

SUMMARYThe cellular immune response probably plays a pivotal role in determining the clinical outcome after exposure to Mycobacterium tuberculosis. We used multi-parameter flow-cytometry to evaluate the distribution of T-lymphocyte subsets during infection and disease caused by M. tuberculosis. Samples were obtained from 71 volunteers to identify the T CD4 + and CD8 + lymphocyte numbers, and the activation plus memory/naïve phenotypes, as defined by CD38, HLA-DR, CD45RA and CD27 markers. Subjects were divided into 18 healthy volunteers without detectable reaction to purified protein derivative (PPD -), 18 health care workers with a recent conversion to PPD, 20 patients with active pulmonary tuberculosis (TBC) and 15 patients with treated TBC at 6 months of therapy. By multiple-comparison analyses, the T CD4 + lymphocyte number of the TBC group was lower than the PPD -group (P < 0·05). This difference was apparently lost after treatment. The higher and the lower number of naïve T CD4 + cells was observed in the PPD -and TBC group, respectively. CD8 + T lymphocytes were also statistically different among the four groups (P = 0·0002), lower in the TBC group (P < 0·05). CD8 + T lymphocyte activation was evaluated by the CD38 and HLA-DR surface expression. The percentage distribution of these markers was statistically different between the four groups (P = 0·0055). TBC patients had a higher percentage of CD38 + cells and mean fluorescence index, suggesting an overall increase of cell activation. These results suggest that peripheral T lymphocytes reflect cellular activation during TBC, along with possible redistribution of naïve, memory/effector and late differentiated memory/effector phenotypes in the peripheral blood after infection and disease caused by M. tuberculosis.

Viruslike particles (VLPs) produced from the L1 protein of several papillomaviruses have induced protection from infection after live challenge in animal models. In the present study, the safety and immunogenicity of a human papillomavirus (HPV)--11 L1 VLP candidate vaccine were measured in a phase 1, dose-finding trial in humans. The vaccine was well tolerated and induced high levels of both binding and neutralizing antibodies. Marked increases in lymphoproliferation to HPV--11 L1 antigens were noted after the second vaccination. In addition, lymphoproliferation was induced after vaccination in peripheral blood mononuclear cells (PBMC) stimulated with heterologous L1 VLP antigens of HPV types 6 and 16. Statistically significant increases in HPV antigen--specific interferon--gamma and interleukin-5 production were measured from PBMC culture supernatants. This candidate HPV VLP vaccine induced robust B and T cell responses, and T cell helper epitopes appear to be conserved across HPV types.