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Cervical cancer is the third most common cancer among women worldwide, and nearly half a million cases are diagnosed annually. In developing countries, it is the leading cause of cancer mortality in women (
In the current study, an attenuated
Results of the present study showed that the highly attenuated LM1-2-E7 strain induced E7-specific cell-mediated immunity and exhibited significant prophylactic and therapeutic efficacy against cervical cancer in a murine model. In addition, the antitumor efficacy was associated with intratu-moral CD8+ T cell infiltration and preferential accumulation of LM1-2-E7 within tumor xenografts.
HPV16 E749-57 (RAHYNIVTF) peptide for the H-2Db restricted epitope was synthesized by Beijing Scilight Biotechnology, LLC. (Beijing, China). The plasmid pIMK2-SPhly was generously provided by Professor Chakraborty (Justus Liebig University, Giessen, Germany). Strain yzuLM4 (serotype 1/2a) was isolated and preserved in our laboratory. The LM1-2 strain was
C57BL/6 mice (6–8 weeks old) were purchased from the Comparative Medical Center of Yangzhou University. Animals were housed and used in accordance with the protocols approved by the institutional animal experimental committee. The TC-1 cell line was purchased from Beijing Hualisentai Bio-Scientific, Co., Ltd. (Beijing, China). TC-1 cells are C57BL/6 lung tumor epithelial cells immortalized with HPV16 E6/E7 and transformed with the c-Ha-ras oncogene. Cells were cultured in RPMI-1640, supplemented with 10% fetal calf serum (FCS), 1 mmol/l sodium pyruvate, 100 U/ml penicillin and 100
The E7 fragment was amplified and cloned into vector pIMK2-SPhly using primers E7 forward, (GAC
LM1-2-E7 and LM1-2-control were cultured overnight in brain heart infusion (BHI) broth at 37°C and their supernatants were collected by centrifugation at 8,000 rpm for 10 min. Secreted proteins in the culture supernatants were precipitated with trichloroacetic acid (TCA; Sigma-Aldrich, St. Louis, MO, USA) and resuspended in 1.5 mM Tris-HCl buffer. Then, the precipitates were lysed by ultrasonic waves. The secreted proteins and lysates were separated by 4–12% SDS-PAGE, transferred to a nitrocellulose membrane and detected using an anti-E7 monoclonal antibody (Clone 8C9; Invitrogen Life Technologies, Carlsbad, CA, USA). The signals were measured using ECL detection reagents (Thermo Scientific, Rockford, IL, USA) and exposure to Hyperfilm.
The stability of LM1-2-E7 was determined by serially passaging for 40 times without antibiotics and subsequently analyzing the expression of E7 in the 20th, 30th and 40th passages.
C57BL/6 mice (6–8 weeks old) were intraperitoneally immunized with LM1-2-E7 (0.1 LD50, 5×107 CFU) or LM1-2-control (0.1 LD50, 5×107 CFU) or phosphate-buffered saline (PBS) buffer on days 0 and 7. Seven days after the booster immunization, mice were used for ELISPOT assay, cytotoxicity assay
The ELISPOT assay was performed as previously described (
To determine specific cytotoxicity
The sections of the spleens and livers were fixed in 13% neutral buffered formalin. Paraffin-embedded sections were cut at 5
In a prophylactic experiment, C57BL/6 mice (6–8 weeks old, 8 per group) were intraperitoneally immunized with LM1-2-E7 or LM1-2-control or PBS buffer on days 0 and 7 and subcutaneously challenged on day 11 with 2×105 TC-1 cells. The mice were monitored for tumor formation.
In a therapeutic setting, C57BL/6 mice (8 per group) were subcutaneously injected with 2×105 TC-1 cells on the left flank. When the tumor size reached an average diameter of 5 mm on day 7 after tumor cell inoculation, the mice received LM1-2-E7 or LM1-2-control or PBS buffer intraperitoneally on days 7 and 14. The tumors were monitored every 3 days with calipers and the longest and shortest surface diameters were recorded for each individual tumor. Tumor volume was calculated as the following: length × (width)2/2 (
The tumors in the immunized groups were excised on day 7 after the second immunization, minced using a sterile razor blade and digested with a buffer containing 2 mg/ml collagenase type I and 12 U/ml DNase in PBS buffer. After a 2 h incubation at 37°C with agitation, single cell suspensions were harvested after filtration through a nylon mesh, stained with anti-CD3-FITC (clone145-2C11; BD Pharmingen) and anti-CD8-APC monoclonal antibodies (clone53-6.7; BD Pharmingen) and analyzed by FACS.
For immunohistochemistry analysis, tumor specimens in the immunized groups were fixed in 13% neutral buffered formalin. Paraffin-embedded sections were cut at 5
Tumor-bearing mice (7 days after the tumor cell inoculation) received a single intraperitoneal immunization (0.1 LD50 LM1-2-E7). The spleens, livers and tumors of 3 mice were homogenized on days 1, 2, 3 and 5 post-immunization. The bacterial numbers were determined by plating the cell suspensions on BHI agar. The tumor tissues at 1 day post-immunization were cut into smaller sections, fixed in a solution of 2.5% (w/v) glutaraldehyde in 0.1 M cacodylate buffer, dehydrated and embedded in Epon. Ultrathin sections were cut and stained with uranyl acetate and Reynold’s lead citrate. The sections were examined using a Transmission Electron Microscope (TEM; JEOL Ltd., Tokyo, Japan).
Statistical analyses for
The recombinant attenuated LM1-2-E7 strain, which integrated the encoding gene of the HPV16 E7 into the chromosome of the LM1-2 strain using pIMK2-SPhly, was constructed. Western blotting revealed that the recombinant strain LM1-2-E7 expressed and secreted the HPV16 E7 protein (
The preventive effect of the LM1-2-E7 vaccine was assessed in the TC-1 tumor model. Mice were challenged with TC-1 cells on day 4 after the second immunization and were observed for tumor development. In the LM1-2-E7-vaccinated group, 7 mice were tumor-free until day 50, excluding 1 mouse (87.5%) that indicated tumor growth on day 32 after the tumor cell challenge. By contrast, tumors appeared in all of the mice on day 7 in the PBS group and on day 10 in the LM1-2-control group (
To examine the therapeutic efficacy of the LM1-2-E7 vaccine candidate, C57BL/6 mice were subcutaneously injected TC-1 cells and intraperitoneally immunized with LM1-2-E7, LM1-2-control, or PBS buffer on days 7 and 14 and tumor growth were measured. As shown in
In order to measure the cellular immune responses elicited by LM1-2-E7, the splenocytes were isolated on Day 7 from the second immunization, and the number of IFN-γ/IL-4-secreting cells after stimulation with E749-57 peptide was analyzed using an ELISPOT assay. The mean number of IFN-γ-secreting cells in the LM1-2-E7-immunized group was significantly higher than the number of IL-4-secreting cells (P<0.01) and significantly increased compared to those of the control groups (P<0.01). These results indicate that mice immunized with LM1-2-E7 developed cellular immune responses against the E7 peptide (
Cytotoxic T-lymphocytes (CTLs) are a critical component of the immune response to tumors. Thus, the E7-specific CTL responses
Tumor-infiltrating lymphocytes (TILs) are a part of the tumor surveillance system. The CD8+ T cells were analyzed in the tumors from all the groups. A higher number of tumor-infiltrating CD8+ T cells (22.45±2.66%) was detected in the LM1-2-E7 group compared to the controls (17.45±2.1%, LM1-2-control; 8.55±0.96%, PBS group) (
The spleens from the LM1-2-E7-immunized group and LM1-2-control group did not reveal any significant pathological lesions and the liver sections revealed inflammatory cell infiltration in the hepatic lobules. The results demonstrate that LM1-2-E7 and LM1-2-control induce a mild inflammatory response with no necrosis or structure damage (
Tumor-bearing mice were intraperitoneally immunized with a single dose of LM1-2-E7 and the number of viable bacteria was determined in the spleen, liver and tumor homogenates. As shown in
There are currently prophylactic vaccines against HPV for clinical use, however, the cost of these vaccines is prohibitive in developing countries (
To avoid serious toxicity of
In addition to choosing the attenuated strain as backbone, an integration vector pIMK2-SPhly was used for the construction of recombinant strain. The E7 antigen was stably integrated into the
Antigen-specific T cell responses are most critical for the regression of established tumors and protection against tumor challenge. IFN-γ and IL-4 secreted by Th1 cells and Th2 cells in LM1-2-E7 immunized mice were determined by ELISPOT assay. The increased secretion of the Th1 cytokine IFN-γ indicated that the immune response elicited by LM1-2-E7 was biased toward the Th1 type against the E749-57 peptide. These Th1 T cells have a pivotal role in antitumor immunity and contribute to APC maturation and the release of cytokines during CD8+ T cell proliferation and differentiation (
The generation of CD8+ tumor-infiltrating lymphocytes (TILs) can be used as a surrogate marker for cancer vaccine efficacy and activated CD8+ T cells release cytolytic agents that attack and kill tumor cells (
Our results show that the recombinant
In the present study,
The TC-1 cell line which expresses HPV E6 and E7 oncoproteins was applied to establish an HPV16-positive cancer-associated tumor model. The TC-1 tumor model in C57BL/6 mice is a widely-used model for use in vaccine research against cervical cancer (
In conclusion, our results demonstrate that LM1-2 is a possible vaccine vector, which is capable of delivering TAAs for cancer immunotherapy. LM1-2-E7 exerts a prophylactic effect on tumor growth and leads to the regression of established tumors expressing E7 antigen. The antitumor efficacy was associated with E7-specific CTL response and robust cellular immune responses elicited by recombinant strain. The results may be of importance in further investigations of this vaccine to combat cervical cancer and other HPV-associated cancers.
The authors thank Professor T. Chakraborty for providing plasmid pIMK2-SPhly. This study was supported by grants from the National Basic Research Program of China (2012CB518805), the Science and Technology Support Program of Jiangsu Province (BE2012367), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the National Natural Science Foundation of China (no. 31101841) and the Provincial Government of Jiangsu, China (nos. BK2011446 and 2009KJA230001).
Western blot analysis of LM1-2-E7. (A) Western blot analysis of E7 expression in the LM1-2-E7 strain. Both the secreted proteins and lysates were detected using an anti-E7 monoclonal antibody. Lanes 1–2, the lysates and secretory proteins from LM1-2-E7, respectively; lane 3, HPV16 E7 protein expressed in
Preventive effect of immunization with LM1-2-E7. Three groups of 8 mice were intraperitoneally immunized with LM1-2-E7, LM1-2-control and PBS buffer (twice at 1-week intervals), respectively, and challenged with a subcutaneous injection of 2×105 TC-1 cells on the left flank 4 days following the second vaccination. Mice were observed for tumor development. Seven tumor-free mice were rechallenged with TC-1 cells on Day 50 after the first challenge (arrow) and were observed for tumor formation. The experiment was repeated twice showing similar results.
Therapeutic effect of vaccination with LM1-2-E7. Three groups of 8 mice were subcutaneously injected with 2×105 TC-1 cells on the left flank on day 0. The mice were administered LM1-2-E7 or LM1-2-control or PBS buffer on days 7 and 14. Tumors were monitored twice weekly and tumor size was measured until day 36 when the majority of the mice in the PBS group died. The table shows the number of mice in each group which were either tumor-free (upper panel) or survived (lower panel), compared to the total number of mice/group on day 7 or 36 post-tumor cell inoculation. Data were expressed as the means ± SD values from 3 independent experiments. ***P<0.001 vs. the LM1-2-control group and the PBS group.
Cellular immune responses elicited by LM1-2-E7. (A) Cytokine (IFN-γ/IL-4) secretion after intraperitoneal immunization with LM1-2-E7. On day 7 following the second immunization, the splenocytes of the immunized mice (5 mice/group) were isolated, pulsed with the E7 peptide
Analysis of tumor infiltrating CD8+ T cells. (A) The tumors were excised on day 7 after the second immunization (5 mice/group) and single cell suspensions from the tumors were stained with anti-CD3-FITC and anti-CD8-APC monoclonal antibodies and analyzed using FACS. (B) The number of tumor infiltrating CD8+ T cells in different groups. (C) CD8+ T cells of the tumor samples were analyzed for immunohistochemistry with magnification ×200. Images are representative of 9 mice in the immunized groups and the PBS group. Data were expressed as the means ± SD values from 3 independent experiments. **P<0.01 vs. the PBS group.
Histopathological studies of the organs of the immunized mice. On day 7 after the second immunization, the spleens and livers (5 mice/group) were harvested and stained by H&E. Images are representative of 5 mice in the immunized group and the control groups (original magnification, ×400).
LM1-2-E7 translocation study. C57BL/6 mice were subcutaneously injected with TC-1 cells on day 0 and intraperitoneally immunized with a single dose of 0.1 LD50 LM1-2-E7 on day 7. The spleens, livers and tumors of 3 mice were homogenized on days 1, 2, 3 and 5 post-immunization. The bacterial numbers were determined by plating the cell suspension on BHI agar. (A) The kinetics of LM1-2-E7 CFU in the spleens and livers. The y-axis represents the logarithm of viable bacterial CFU to base 10 in the spleens and livers. (B) The data show the kinetics of CFU in the tumors. (C) A TEM analysis indicates that the LM1-2-E7 infected tumors with high efficacy after a single intraperitoneal immunization. Bar, 500 nm (left) and 200 nm (right). White arrows, LM1-2-E7 bacteria. Data were expressed as the means ± SD values from 2 independent experiments.