Anti-tumor therapeutic efficacy of pCTLA4-E7E6.

<p>(<b>A</b>) Effects on tumor growth by therapeutic vaccination. Tumor-carrier mice were vaccinated with pCTLA4-E7E6, pE7E6, pVAX1 or PBS twice on day 10 and 17 after TC-1 cells injection. Tumor size was recorded twice or thrice a week. (<b>B</b>) Statistic analysis of tumor sizes on day 34 among groups. The graph shows means ± SE. (<b>C</b>) Statistic analysis of tumor development between day 10 and day 38 among groups. (<b>D</b>) Kaplan-Meier survival analysis of mice survival in different groups. (<b>E</b>) Tumor specific CTL responses. Spleen cells were collected from alive mice in different groups on day 38 and used as effector (E) cells. TC-1 cells were used as target (T) cells. The cellular immune response against E7E6 was determined by lactate dehydrogenase (LDH) release assay. Various E/T ratios were tested as indicated. The data were presented as means ± SE. (<b>F</b>) The levels of specific anti-E7E6 serum IgG were determined by ELISA assay on day 24 and 38. The antibody levels of each group were presented as mean OD values ± SE.</p

Schematic diagram of vaccination protocols.

<p>Therapeutic immunization: mice were challenged with 2×10⁵ TC-1 cells on day 0 and vaccinated twice on day 10 and 17. Preventive immunization: mice were vaccinated on day 0 and day 14, and then challenged with 6×10⁴ TC-1 cells on day 28. Tumor size was recorded twice or thrice a week.</p

Fusion of CTLA-4 with HPV16 E7 and E6 Enhanced the Potency of Therapeutic HPV DNA Vaccine

<div><p>Preventive anti-HPV vaccines are effective against HPV infection but not against existing HPV-associated diseases, including cervical cancer and other malignant diseases. Therefore, the development of therapeutic vaccines is urgently needed. To improve anti-tumor effects of therapeutic vaccine, we fused cytotoxic T-lymphocyte antigen 4 (CTLA-4) with HPV16 E7 and E6 as a fusion therapeutic DNA vaccine (pCTLA4-E7E6). pCTLA4-E7E6 induced significantly higher anti-E7E6 specific antibodies and relatively stronger specific CTL responses than the nonfusion DNA vaccine pE7E6 in C57BL/6 mice bearing with TC-1 tumors. pCTLA4-E7E6 showed relatively stronger anti-tumor effects than pE7E6 in therapeutic immunization. These results suggest that fusing CTLA-4 with E7E6 is a useful strategy to develop therapeutic HPV DNA vaccines. In addition, fusing the C-terminal of E7 with the N-terminal of E6 impaired the functions of both E7 and E6.</p></div

Anti-tumor preventive efficacy of pCTLA4-E7E6.

<p>(<b>A</b>) Effects on tumor growth by preventive vaccination. Mice were vaccinated with pCTLA4-E7E6, pE7E6, pVAX1 or PBS twice on day 0 and 14. On day 28 mice were challenged with 6×10⁴ TC-1 tumor cells. Tumor size was recorded twice or thrice a week. (<b>B</b>) Kaplan-Meier survival analysis of mice survival in different groups. (<b>C</b>) Tumor specific CTL responses. Spleen cells were collected from alive mice in different groups on day 80 and used as effector (E) cells. TC-1 cells were used as target (T) cells. The cellular immune response against E7E6 was determined by lactate dehydrogenase (LDH) release assay. Various E/T ratios were tested as indicated. The data were presented as means ± SE. (<b>D</b>) The levels of specific anti-E7E6 serum IgG were determined by ELISA assay on day 14, 28, and 60. The antibody levels of each group were presented as mean OD values ± SE.</p

Western blotting analysis of E7E6, p53 and pRb expression.

<p>The lysates of 293 cells transfected with pCTLA4-E7E6, pE7E6, pwCTLA4-E7E6, pwE7E6, pwE7, pwE6, pVAX1 or phosphate buffered saline (PBS, labeled as None) were probed with mouse anti-E7, goat anti-E6, mouse anti-p53, rabbit anti-pRb, or mouse anti-β-actin antibody. β-actin served as loading control. pCTLA4-E7E6 encodes CTLA-4 and mutant E7E6 fusion protein. pE7E6 encodes mutant E7E6 fusion protein. Accordingly, pwCTLA4-E7E6 and pwE7E6 carry wild type E7E6 fusion gene. pwE7 and pwE6 encode wild type E7 and E6 respectively. Expressed recombinant proteins were labeled by oblique arrows. MW: molecular weight. The lowest panel showed the relative expression levels of p53 and pRb by normalizing the signal intensities to actin.</p

Construction of DNA vaccines pCTLA4-E7E6 and pE7E6.

<p>HPV16 E7 gene was fused with E6 gene. The fusion gene was optimized with human biased codons. (<b>A</b>) Mutation introduced into HPV16 E7 and E6 to disrupt transformation activity are labeled in the schema. ²⁴C, ²⁶E and ⁹¹C of E7 and ⁶³C and ¹⁰⁶C of E6 (arrows) were mutated to glycine. (<b>B</b>) pE7E6 was constructed by cloning the modified E7E6 fusion gene into pVAX1 vector at Kpn I and Not I restriction sites. pCTLA4-E7E6 encodes the signal peptide and extracellular domains of CTLA4, the hinge and Fc region of human IgG1, and the E7E6 fusion gene.</p

Core supra-gingival microbiome in root caries and health.

<p>Red field represents health-associated core species with significantly higher prevalence and relative abundance in Healthy_controls than in Patient_cases; green field represents root caries-associated core species with significantly higher prevalence and relative abundance in Patient_cases than in Healthy_controls; brown field represents core species in both health and root caries with no significantly different prevalence and relative abundance between the two groups. In each field, the prevalences of species were at least 1/2. Inner circles labeled 1 contain the species with high prevalence (prevalence ≥ 2/3) and high abundance (average relative abundance ≥ 2%); circles labeled 2 contain species with high prevalence (prevalence ≥ 2/3) but low abundance (average relative abundance < 2%); circles labeled 3 contain species with moderate prevalence (1/2 ≤ prevalence < 2/3) but high abundance (average relative abundance ≥ 2%); circles labeled 4 contain species with moderate prevalence (1/2 ≤ prevalence < 2/3) and low abundance (average relative abundance < 2%).</p

MTT results of the RAW 264.7 cells viability after treating with indicated concentrations of previously prepared PEI/DNA, CS/DNA and AL/CS/DNA NPs.

<p>Mean ± SD (n = 4).</p

Antibody levels of saliva and serum collected from mice immunized with different formulations and routes.

<p>(A) Specific anti-PAc salivary IgA levels (ng/ml); (B) Specific anti-PAc serum IgG levels (µg/ml). Samples were collected at weeks 2, 4, 6, 8 and 12. Compared with the AL/CS/DNA (i.n.) group, *p<0.05, **p<0.01; Mean ± SD (n = 10).</p

TEER decrease and intracellular uptake on Caco-2 cells after CS/DNA and AL/CS/DNA treatment.

<p>(A) TEER decrease of Caco-2 cell monolayer after 1 h exposure to the above formulations. (B) Percentages of intracellular uptake of test formulations after administration for 2 h. (C) Flow cytometry pictures are representatives of each group. (n = 3) *p<0.05, **p<0.01.</p