Generation of chimeric HPV16 L1/L2-E7 VLP.

<p><b>A</b> Sf-9 insect cells were infected with recombinant HPV16 L1/L2-E7 baculovirus and VLP purified by sucrose cushion and CsCl gradient centrifugation. Purified VLP, infected, and uninfected Sf-9 cell lysates were analyzed by SDS-PAGE and Coomassie staining. <b>B</b> Antigenicity of HPV16 L1/L2-E7 VLP was assessed by Western blot using antibodies to HPV16 L1, HPV16 L2, or HPV16 E7. Purified HPV16 L1 VLP and HPV16 L1/L2 VLP were used as controls. <b>C</b> HPV16 L1/L2-E7 VLP were negatively stained by uranyl acetate and visualized by TEM at 30,000 fold magnification.</p

Recombinant 16E6E7 and 16E6E7m influenza viruses elicit HPV-specific CTL responses in mice.

<p>Four groups of mice (4 per group) were primed with PBS (mock), H1N1 16E6E7 virus, H1N1 16E6E7m virus, parental H1N1 delNS1 virus, or 16L1/L2-E7 VLPs, and boosted 10 days later with PBS, corresponding H3N2 serotypes, or VLPs. Mice were sacrificed <b>A</b> 10 days or <b>B</b> 30 days after boosting, splenocytes were isolated and stimulated in triplicates for 24 h with antigen peptides, SEA or medium alone. For mock and influenza A virus-vaccinated animals, NP_311-325 peptide, for mice immunized with 16L1/L2-E7 VLP, 16L1_165-173 peptide were used as positive controls. Shown are numbers of IFN-γ spots, counted under a light microscope, and plotted as mean ± SD of triplicate wells. One representative experiment of two is shown. Statistically significant differences for 16E6E7 or 16E6E7m compared to mock are indicated as asterisks (*** p<0.001, ** p<0.01, * p<0.05, ns not significant).</p

Mice s.c. vaccinated with recombinant 16E6E7 or 16E6E7m influenza viruses are partially protected from TC-1 induced tumors.

<p><b>A</b> Schematic illustration of the experimental set up. <b>B</b> Groups (n = 8) of C57BL/6 female mice were vaccinated s.c. with PBS (mock), H1N1 16E6E7, H1N1 16E6E7m, parental virus (delNS1), or HPV16 L1/L2-E7 VLP, boosted 20 days later either with PBS, the corresponding H3N2 influenza serotype, or VLPs and challenged s.c. with 5_x10⁴ TC-1 cells 10 days later. Animals were monitored once per week. Mean tumor volumes ± SEM of individual animals of one representative experiment of two are shown. Statistical significances of differences recorded for immunized groups and mock treated animals, or immunized groups and parental virus treated animals, were calculated by 1-way ANOVA and subsequent Dunnett’s multiple comparison test. Days after TC-1 tumor inoculation are indicated.</p

Intratumoral (i.t.) vaccination with recombinant 16E6E7 and 16E6E7m influenza viruses delays growth of, or eradicates established TC-1 tumors.

<p><b>A</b> Schematic illustration of the experimental set up <b>B</b> Five groups of female C57BL/6 mice (8 per group) were inoculated with 5x10⁴ TC-1 cells on day 0 and tumor growth monitored. After palpable tumors had developed, mice were primed i.t. on day 13 and 14 with PBS (mock), H1N1 16E6E7, H1N1 16E6E7m, parental influenza virus (delNS1), or HPV16 L1/L2-E7 VLP, and boosted either with PBS, the corresponding H3N2 influenza serotype, or VLPs 10 days later. Animals were monitored once per week and mean tumor volumes ± SEM of individual animals of one representative experiment of two are shown. Statistical significance of differences recorded for immunized groups and mock treated animals, or immunized groups and parental virus treated animals, were calculated by 1-way ANOVA and subsequent Dunnett’s multiple comparison test. Days after TC-1 tumor inoculation are indicated.</p

Characterization of recombinant 16E6E7 and 16E6E7m influenza viruses for transgene stability, infectivity and fusion gene expression.

<p>Following generation of recombinant H1N1 or H3N2 16E6E7/16E6E7m influenza A viruses and passaging for at least five cycles, <b>A</b> whole viral RNA was isolated or <b>B</b> Vero cells were infected with 10⁵ pfu of recombinant viruses for 12 hours and whole RNA isolated. Viral or cellular RNA was subjected to RT-PCR analysis amplifying either the whole delNS1-E6E7 ORF (<b>A</b>) to analyze stability, or the E6E7 transgene (<b>B</b>) to assess fusion gene expression. <b>C,D</b> Vero cells were infected for 12 hours in the presence (<b>D</b>) or absence (<b>C</b>) of proteasome inhibitor MG-132 with H1N1 or H3N2 recombinant or parental influenza viruses as indicated, and whole cell lysates were separated by 10% SDS-PAGE. Viral NP protein (<b>C</b>), FLAG (upper panel), HPV16 E6 (middle panel) and E7 expression (lower panel) were assessed using specific monoclonal antibodies (<b>D</b>).</p

SL immunization with DelNS1 vaccine activates mucosal and extramucosal CD4+ T cells.

<p>CFSE-labeled CD4⁺ T cells from HA-TCR transgenic mice were adoptively transferred into naïve mice. One day later, recipients were given SL 2×10⁷ pfu of Delta H1N1, 2×10⁵ pfu of mouse-adapted wt live or 40 µg of formalin-inactivated A/PR8. Three days after the immunization, proliferating (CFSE stained) HA-TCR CD4⁺ T cells were detected in cervical lymph nodes (CLN), lungs, mediastinal lymph nodes (MdLN) and spleens by FACS analysis. The data are representative of two experiments showing similar results.</p

Induction of HI Abs upon immunization with Delta H1N1.

<p>On the day before challenge as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039921#pone-0039921-g003" target="_blank">Figure 3</a>, the sera were collected and the titers of HI Abs were determined against A/PR8 virus. The values represent the mean + SEM antibody titers of sera from 5 mice per group.</p

Induction of homotypic and HSI upon immunization with Delta H1N1.

<p>BALB/c mice were immunized IN or SL with different doses of Delta H1N1 (▴H1N1), given once (1x) or twice (2x) at 2-week intervals. Four weeks later, animals were intranasally challenged with 5×LD₅₀ of homologous mouse-adapted A/PR8 (Fig. 3A) or heterosubtypic mouse-adapted A/Philippines (A/P) H3N2 (Fig. 3B) virus. Morbidity and mortality were monitored daily. Data are expressed as mean (body weight or % survival) determined on groups of 5–10 mice.</p

Levels of virus-specific IgG induced in mucosal and systemic compartments upon immunization with DeltaNS1 IAV.

<p>BALB/c mice were immunized with different doses of Delta H1N1 (▴ H1N1) or wt live virus A/PR8/34 (PR8) H1N1 (WT) via the sublingual (SL) or intranasal (IN) route. Four weeks later, titers of H1N1 (A/PR8) virus-specific IgG in sera and secretions were determined by ELISA. The values represent mean + SEM ELISA titers determined on groups of 5 mice.</p

Induction of virus-specific IgG and IgA upon immunization with Delta H5N1.

<p>BALB/c mice were immunized with different doses of Delta H5N1 (▴ H5N1) or wt live virus A/PR8/34 (PR8) H1N1 via the sublingual (SL), nasal (N) or intranasal (IN) route. Four weeks later sera were collected and the levels of H5N1 virus-specific IgG and IgA were determined by Delta H5N1 virus-coated ELISA plates. The values represent the mean + SEM (vertical bars) end point ELISA antibody titers determined on 5 mice per group.</p