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

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

Peptide blockade of 11–88×8 antiserum.

<p>A. Sera of Balb/c mice immunized three times with 11–88×8 or 11–88×8Δ using Alum+MPL as adjuvant were harvested two weeks after the final boost. The antisera diluted 1∶100 were reacted with microtiter plates coated with 1 µg of two HPV16 L2 peptides encompassing residues 43–62 and 48–67. After washing, specific reactivity was measured by ELISA using peroxidase-linked anti-mouse IgG. B. Pooled sera of Balb/c mice immunized three times with 11–88×8 using Alum+MPL as adjuvant were harvested either pre-immunization (Pre-bleed) or two weeks after the final boost (×8 Sera). The antiserum to 11–88×8 was diluted 1∶50 in a total volume of 200 µl and incubated with 7 µg of HPV16 L2 peptide encompassing residues 13–32, 18–37, 23–42, 28–47, 33–52, 38–57, 43–62, 48–67, 53–72, 58–77, 63–82 or 71–90 for an hour prior to mixing with HPV16 pseudovirions carrying a SEAP reporter for a further hour at ambient temperature and subsequent infection of 293TT cells. Infection was measured as optical density, and only HPV16 L2 pepitdes 13–32 and 18–37 substantially blocked neutralization by 1∶3200 dilution of antiserum to 11–88×8. C. Sera of Balb/c mice immunized three times with 11–88×8 using Alum+MPL as adjuvant were harvested two weeks after the final boost and pooled (×8). The 11–88×8 antiserum was mixed with two HPV16 L2 peptides encompassing residues 43–62 and 48–67 (+Peptide) in the same ratios as in B. The 11–88×8 antiserum was administered i.p. either alone or pre-mixed with peptide in volumes of 20 µl, 5 µl or 2 µl to naïve mice (in groups of 5). Separate groups of mice received 200 µg each of antibody affinity purified with protein G columns from the sera of rabbits hyper-immunized with HPV16 L2 peptides 17–36, 47–66 or 373–392. All groups of mice (except the Background group) were subsequently challenged intra-vaginally with HPV16 pseudovirions carrying a luciferase reporter. Infection was assessed by measuring bioluminescence three days later.</p

A comparison of antibody responses of mice vaccinated with DNA expressing L1 or L1+L2 of HPV6, 16, or 18, either singly or together at the same or different sites.

<p>Balb/c mice were vaccinated i.m. with electroporation three times at two week intervals with 20 µg each of DNA expressing L1 or L1+L2 of HPV6, HPV16, and HPV18, either individually, or together at the same site or each at a different site (DS), or s.c. with Gardasil. Serum samples were harvested at two weeks after the third vaccination (A–C) or 3 months after the third vaccination (D–F), and neutralizing antibody titers were measured with HPV6 (A,D), HPV16 (B,E), or HPV18 PsV (C,F). Antibody response to L2 was measured by ELISA against full length HPV16 L2 with serum samples collected two weeks after the third vaccination (G).</p

Comparison of protective antibody responses.

<p>Balb/c mice were vaccinated i.m. with electroporation three times at two week intervals with 20 µg each of DNA expressing L1 or L1+L2 of HPV6, HPV16, and HPV18, either individually, or together at the same site or each at a different site, or s.c. with Gardasil. Serum samples were collected two weeks (A,B) and three months (C) after the last vaccination to test their protective efficacy <i>in vivo</i> and <i>in vitro</i>. Naïve Balb/c mice (5 per group) were passively immunized i.v. with 20 µl of pooled serum. Mice were challenged intra-vaginally with HPV16 (A,C) or HPV6 (B) PsV carrying a luciferase reporter construct. Three days later, luciferin was administered intra vaginally, and bioluminescence imaging was performed.</p

HPV in vitro neutralization titers of sera of mice vaccinated with 13–47×15 and 11–88×8 in different adjuvants.

<p>Balb/c mice were vaccinated three times at 2 week intervals with the indicated 13–47×15 and 11–88×8 proteins formulated in alum, alum+MPL, alum+CpG or GPI-0100. Sera were harvested two weeks later for testing in vitro neutralization titers against HPV16 (A), HPV18 (B), HPV45 (C) and HPV58 (D) pseudovirions, or HPV16 at 3 months after the final vaccination (E).</p

Neutralizing antibody titers induced upon vaccination of mice with a pentavalent HPV L1 DNA vaccine.

<p>Balb/c mice were vaccinated i.m. three times at two week intervals with a mixture of L1 vectors of HPV6, HPV16, HPV18, HPV26, and HPV51 (2 µg each), L1 vector of single HPV type (2 µg or 10 µg) utilizing electroporation. Gardasil vaccination was included as a positive control. Sera from 5 mice were pooled together, and <i>in vitro</i> neutralizing antibody titer (IVNT) was measured with HPV6, HPV16, HPV18, HPV26, and HPV51 PsVs. ND: Not detected at 1:50.</p