Immunologic Control of Mus musculus Papillomavirus Type 1

Abstract

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.

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

(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).

Fig 2. Infiltration of T cells into papilloma site associated with MusPV1 papilloma regression.

Immunohistochemical analysis using CD3-specific antibody of a papilloma on SCID mice that had received BALB/c splenocytes by 5 weeks post adoptive transfer and initiated papilloma regression (A) versus control SCID (BALB/c background) with progressive papilloma (B).

Fig 3. Detection of persistent infection of MusPV1 despite absence of papilloma.

Schedules of T cell depletion using anti-CD4/CD8/CD3 monoclonal antibody and the timing of MusPV1 challenge and virus measurements in BALB/c mice. Challenge of nude mice was used as a positive control (A). MusPV1 E6/E7 transcripts were detected in wart bearing nude mice (B) but not wildtype infected mice (no depletion) (C). Low levels of MusPV1 E6/E7 transcripts were present in CD4+ T cell-depleted mice (D), but not those depleted of CD8+ T cells (E).

Fig 4. Re-activation of MusPV1 infection.

(A) Schema of experiment in BALB/c mice, including schedule of T cell depletion using monoclonal antibodies to CD3, CD4 or CD8 before MusPV1 challenge to the tail. At five weeks post challenge, all mice were switched to treatment with CD3 antibody for ten weeks. Representative gross tail images taken 15 weeks post challenge (left panel) in mice that received no depletion during first 5 weeks (B), or T cell subset depletion using anti-CD4 antibody during first 5 weeks (C), or anti-CD8 antibody during first 5 weeks (D). The right panels of B-D show representative histologic images of tail sections collected after the additional 10 weeks of T cell subset depletion with CD3 antibody for groups B-D and upon in situ hybridization for MusPV1 E6/E7 transcripts and hematoxylin staining.

Fig 5. Intracellular cytokine staining with flow cytometry analysis reveals immunodominant CD8+ T cell epitope in MusPV1 E6 and its MHC class I restriction.

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.

Fig 6. Adoptive transfer of E6-specific CD8+ cytotoxic T cell line one week after MusPV1 challenge prevents papilloma formation in immunodeficient mice.

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 E6/E7 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).

Fig 7. Control of established papilloma by adoptive transfer of MusPVE6-specific CD8+ T cell line.

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).