Impact of γ-secretase inhibitor on infection by PsV of diverse HPV.

<p>PsVs of each indicated HPV type carrying a luciferase reporter gene were transferred to 293TT cells for 72γ-secretase inhibitor XXI (n = 3). After incubation, luciferase activity was measured and percent inhibition of infectivity compared to control calculated. Red and blue bars represent mucosal and cutaneous HPV types, respectively.</p

Summary of the phylogeny, tropism, charge and in vitro neutralization by L2 11-88x8 antiserum of the 34 HPV genotypes tested.

<p>Phylogeny and tropism were taken from de Villiers et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097232#pone.0097232-deVilliers1" target="_blank">[58]</a>, predicted net charge of L1 at pH7.4 was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097232#pone.0097232-Mistry1" target="_blank">[37]</a>. PsVs of each indicated HPV type carrying a luciferase reporter gene were mixed with titrated rabbit L2 α11-88x8 antiserum for two hours at 37°C, then the mixtures were transferred to 293TT cells and cultured for 72 hours. Cells were then lysed and luciferase activity was measured. Neutralization titer and 95% confidence interval are shown.</p

Inhibition of HPV PsV infection by titrations of heparin and carrageenan.

<p>PsVs of each HPV type carrying a luciferase reporter gene were incubated with 1000 µg/mL, 100 µg/mL, 10 µg/ml or 0 µg/ml heparin (A) or carrageenan (B) at 37°C for 1 hour. The mixtures were transferred to 293TT cells for 72 h. After incubation cells were lysed and luciferase activity was measured, and percent infection compared to control was calculated (n = 3). The percent inhibition of infection by each HPV PsV in the presence of 100 µg/mL of heparin (C) or carrageenan (D) was also plotted separately. Red and blue bars and lines represent mucosal and cutaneous HPV types, respectively.</p

Assessment of infectivity, L2 incorporation, and reporter DNA encapsidation by PsV preparations of 34 HPV types.

<p>(A) 250 million particles (as estimated based on L1 concentration) of HPV PsV were transferred to 293TT cells, incubated at 37°C for 72 hours before cell lysis. Luciferase activity was measured in cell lysate (n = 4). (B) Western blot analysis detecting L2 in 100 ng of each HPV PsV preparation using rabbit antiserum to L2 α11-88x8. (C) Reporter plasmid copy number of each PsV preparations for each HPV type sample was measured by quantitative real time PCR. Black and blue bars represent alpha and beta type, respectively, and mean ± standard error plotted.</p

Immunologic Control of <i>Mus musculus</i> Papillomavirus Type 1

<div><p>Persistent papillomas developed in ~10% of out-bred immune-competent SKH-1 mice following MusPV1 challenge of their tail, and in a similar fraction the papillomas were transient, suggesting potential as a model. However, papillomas only occurred in BALB/c or C57BL/6 mice depleted of T cells with anti-CD3 antibody, and they completely regressed within 8 weeks after depletion was stopped. Neither CD4+ nor CD8+ T cell depletion alone in BALB/c or C57BL/6 mice was sufficient to permit visible papilloma formation. However, low levels of MusPV1 were sporadically detected by either genomic DNA-specific PCR analysis of local skin swabs or in situ hybridization of the challenge site with an E6/E7 probe. After switching to CD3+ T cell depletion, papillomas appeared upon 14/15 of mice that had been CD4+ T cell depleted throughout the challenge phase, 1/15 of CD8+ T cell depleted mice, and none in mice without any prior T cell depletion. Both control animals and those depleted with CD8-specific antibody generated MusPV1 L1 capsid-specific antibodies, but not those depleted with CD4-specific antibody prior to T cell depletion with CD3 antibody. Thus, normal BALB/c or C57BL/6 mice eliminate the challenge dose, whereas infection is suppressed but not completely cleared if their CD4 or CD8 T cells are depleted, and recrudescence of MusPV1 is much greater in the former following treatment with CD3 antibody, possibly reflecting their failure to generate capsid antibody. Systemic vaccination of C57BL/6 mice with DNA vectors expressing MusPV1 E6 or E7 fused to calreticulin elicits potent CD8 T cell responses and these immunodominant CD8 T cell epitopes were mapped. Adoptive transfer of a MusPV1 E6-specific CD8+ T cell line controlled established MusPV1 infection and papilloma in RAG1-knockout mice. These findings suggest the potential of immunotherapy for HPV-related disease and the importance of host immunogenetics in the outcome of infection.</p></div

Impact of furin inhibitor on infection by PsV of diverse HPV.

<p>PsVs of each indicated HPV type carrying a luciferase reporter gene were transferred to 293TT cells for 72 hours in the presence or absence of 20 µM furin inhibitor. After incubation, cells were lysed, luciferase activity was measured and percent inhibition of infectivity compared to control calculated. Red and blue bars represent mucosal and cutaneous HPV types, respectively.</p

Control of established papilloma by adoptive transfer of MusPVE6-specific CD8+ T cell line.

<p>RAG1 knockout mice, n = 4 per group, were challenged with MusPV and papillomas were allowed to grow. After 5 weeks, the papilloma-bearing RAG1 knockout mice received by adoptive transfer either 5x10⁶ CD8+ MusPVE6-specific T cell line or 5x10⁶ CD8+ OT-1 specific T cells. Mice were photographed every week thereafter for 10 weeks until the tails were harvested, sectioned and processed for MusPV1 E6/E7 in situ hybridization by RNAscope and hematoxylin staining. Photographs of one representative mouse from each treatment group are shown over the time 10 weeks post adoptive transfer (A). Analysis of MusPV1 E6/E7 transcription in representative tails harvested from mice that had 10 weeks prior received by adoptive transfer either MusPV E6-specific CD8+ T cells (B) or OT-1 T cells (C). Schematic of MusPV infection of RAG1 knock-out mice and subsequent adoptive transfer of MusPVE6 T-cell line or OT-1 cells as a control (D). Spleens were harvested from mice that had 10 weeks prior received by adoptive transfer either MusPV E6-specific CD8+ T cells or OT-1 T cells. A flow cytometric analysis was performed after intracellular cytokine staining of these splenocytes for interferon-γ and CD8 after stimulation with either mE6 or OVA peptide (E).</p

Adoptive transfer of E6-specific CD8+ cytotoxic T cell line one week after MusPV1 challenge prevents papilloma formation in immunodeficient mice.

<p>Schematic of study in RAG1 knock-out mice that received adoptive transfer of either 5x10⁶ CD8+ MusPVE6-specific T cell line, or 5x10⁶ OT-1 cells one week after MusPV1 challenge (A). Photographs and tail sections stained for MusPV1 <i>E6/E7</i> transcripts of RAG1 knock-out mice 5 weeks post-adoptive transfer with either the MusPV1 E6-specific CD8+ T cell line (B) or OT-1 cells (C). Detection of MusPV1 E6 and OVA peptide specific CD8+ T cells in the spleens of RAG1 knock-out mice 5 weeks post adoptive transfer (D).</p

MusPV1 infection and disease in outbred SKH-1 mice and immunocompromised controls.

<p>(A) Papilloma formation on the tail of a SKH-1 mouse 4 weeks-post infection, and (B) persisting on the tail over 6 months (left panel) and spreading along the tail and occasionally to the muzzle (right panel). These papillomas were also probed for MusPV1 E1^E4 transcripts suggesting active infection (C). However, the papillomas on SKH-1 mice were not as florid as compared to those on nude mice (D). MusPV1 virions were harvested from the papillomas of nude mice and visualized using negative stain transmission electron microscopy (E).</p

Intracellular cytokine staining with flow cytometry analysis reveals immunodominant CD8+ T cell epitope in MusPV1 E6 and its MHC class I restriction.

<p>Schematic of immunization schedule of C57BL/6 mice with DNA vaccines expressing CRT (calrecticulin) fused to different MusPV1 proteins and subsequent harvest of splenocytes (A). Bar graph of flow cytometry data after intracellular cytokine staining of splenocytes for interferon-γ and CD8 after harvest from CRT/mE6-vaccinated mice and stimulation with mE6 peptide library pools (B). Bar graph of flow cytometric analysis of intracellular cytokine staining of splenocytes for interferon-γ and CD8 after harvest from CRT/mE6E7L2-vaccinated mice and stimulation with both mE6 and mE7 peptide library pools (C). Bar graph of flow cytometry data after intracellular cytokine staining of splenocytes for interferon-γ and CD8 after harvest from CRT/mE6 and stimulated with candidate 9mer peptides to map the MHC class I epitopes of MusPV1 E6 (D). Bar graph of flow cytometry analysis showing percentages of interferon-γ expressing mE7-specific CD8+ T cells after co-incubation with 293-K^b or 293-K^d cells that were transfected with either CRT/mE6 or CRT-alone plasmid (E). All data was repeated and representative images provided.</p