Capsomer Vaccines Protect Mice from Vaginal Challenge with Human Papillomavirus

Capsomers were produced in bacteria as glutathione-S-transferase (GST) fusion proteins with human papillomavirus type 16 L1 lacking the first nine and final 29 residues (GST-HPV16L1Δ) alone or linked with residues 13–47 of HPV18, HPV31 and HPV45 L2 in tandem (GST-HPV16L1Δ-L2x3). Subcutaneous immunization of mice with GST-HPV16L1Δ or GST-HPV16L1Δ-L2x3 in alum and monophosphoryl lipid A induced similarly high titers of HPV16 neutralizing antibodies. GST-HPV16L1Δ-L2x3 also elicited moderate L2-specific antibody titers. Intravaginal challenge studies showed that immunization of mice with GST-HPV16 L1Δ or GST-HPV16L1Δ-L2x3 capsomers, like Cervarix®, provided complete protection against HPV16. Conversely, vaccination with GST-HPV16 L1Δ capsomers failed to protect against HPV18 challenge, whereas mice immunized with either GST-HPV16L1Δ-L2x3 capsomers or Cervarix® were each completely protected. Thus, while the L2-specific response was moderate, it did not interfere with immunity to L1 in the context of GST-HPV16L1Δ-L2x3 and is sufficient to mediate L2-dependent protection against an experimental vaginal challenge with HPV18

Molecular basis of USP7 inhibition by selective small-molecule inhibitors

Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice

<b>Neutralizing antibody responses of mice vaccinated with capsomers.</b>

<p>Two weeks after the third immunization with GST-HPV16L1Δ, GST-HPV16L1Δ-L2x3 or Cervarix®, two-fold dilutions of serum from naïve or immunized mice were tested for <i>in vitro</i> neutralization activity against pseudovirions derived from HPV16 (A), HPV18 (B), HPV31 (C) or HPV45 (D) starting at a dilution of 1∶100. The reciprocal of the highest serum dilution with ≥50% reduction in reporter gene expression signal was defined as the serum virus neutralizing antibody titer, and <100 was considered not detected (ND) and arbitrarily plotted at 10.</p

Vaginal challenge of mice with HPV PsV after immunization with capsomers.

<p>Mice were subcutaneously immunized with GST-HPV16L1Δ, GST-HPV16L1Δ-L2x3 or Cervarix® three times at two-week intervals. Three and five weeks after the last immunization, the mice were challenged with either HPV16 (A) or HPV18 (B) PsV carrying a luciferase reporter, respectively. Three days after challenge, the mice received an intravaginal instillation of 20 µl of D-luciferin and were imaged with a Xenogen IVIS 100 system (image captured for 10 min post-installation of luciferin). Image analysis in each experiment was based on the average measurement (photons/s/cm²) collected from each selected equally-sized area (A, 1.9 cm²; B, 0.6 cm²) in the vaginal tract.</p

<b>L2-specific antibody titers induced by vaccination of mice with capsomers.</b>

<p>Two weeks after the third immunization with GST-HPV16L1Δ, GST-HPV16L1Δ-L2x3 or Cervarix®, two-fold dilutions of serum from immunized mice were tested by ELISA for L2 specific antibodies. Microtiter plates were coated with either HPV16 L2 amino acids 11–200 (A), or the 17–38 L2 peptide (B), or HPV18 L2 full length protein (C), or HPV31 L2 17–36 peptide (D), or HPV31 full length protein (E) or HPV35 L2 17–36 peptide (F), or HPV45 L2 16–35 peptide (G), or HPV58 L2 16–35 peptide (H), and then incubated with diluted serum samples (1∶50 as the initial dilution; two-fold serial dilutions).</p