Acute graft-versus-host disease (aGVHD) still remains one of the life-threatening complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Immunomodulation of alloreactive donor T cell responses, as well as cytokine secretion is a potential therapeutic approach for the prevention of aGVHD. The synthetic triterpenoid, CDDO (2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid), exhibits potent antitumor activity and has also been shown to mediate anti-inflammatory and immunomodulatory effects. We therefore wanted to assess the effects of CDDO on early lethal aGVHD. In this study, we found that CDDO significantly inhibited in vitro mixed lymphocyte responses and preferentially promoted the apoptosis of proliferating but not resting alloreactive T cells. Using a full major histocompatibility complex (MHC)-disparate murine aGVHD model, we found that the administration of CDDO immediately after transplantation significantly decreased liver pathology as determined by histologic assessment and prolonged survival in mice. Importantly, administration of CDDO did not adversely impair donor myeloid reconstitution as determined by peripheral blood cell count and the extent of donor chimerism. These findings indicate that CDDO has a significant immunomodulatory effects in vitro and on early lethal aGVHD development, particularly affecting the liver, in a murine allo-HSCT model.

Objective This study aims to prepare candidate vaccines for cervical cancer immunotherapy by inserting the fused genes of human papillomavirus (HPV)16/18/58 mE6E7 lacking transforming activity into an adenovirus vector and to verify its efficiency in model mice with tumor expressing the associated HPV genes. Methods The E6/E7 genes of HPV16/18/58 were point-mutated to abolish their transforming activity, and adenovirus (AD)-HPV16/18/58 mE6E7 adenovirus vaccine was constructed. The immune effect of the adenovirus vaccine against HPV16/18/58-type tumors was analyzed by tumor morphology, enzyme linked immunosorbent assay, enzyme-linked immunospot and specific cytotoxic T lymphocyte (CTL) and T lymphocyte subsets. Results The HPV16/18/58 mE6E7 plasmid containing point mutations was verified by quantitative real-time polymerase chain reaction (qRT-PCR), enzyme digestion and electrophoresis, and gene sequencing. qRT-PCR and Western blots verified that AD-HPV16/18/58 mE6E7 could express the HPV16 mE6E7, HPV18 mE6E7 and HPV58 mE6E7 fusion genes and proteins in cells. The results of animal experiments were as follows: In the vaccine group, the tumors formed later, the incubation period was longer, the growth was slower, growth was inhibited, and the survival period was significantly prolonged. The immunological results all showed that the vaccine could induce effective humoral and cellular immunity in mice with three types of tumors, compared with the phosphate buffered saline (PBS) group and the adenovirus-negative control (AD-NC) group, the differences were statistically significant (P < 0.05). Conclusion We successfully constructed the HPV16/18/58 trivalent therapeutic adenovirus vaccine AD-HPV16/18/58 mE6E7. The AD-HPV16/18/58 mE6E7 adenovirus vaccine can protect immunized mice to a certain extent from TC-1, U14/LV-HPV18 E6E7 and U14/LV-HPV58 E6E7 cells, which contain HPV16, 18 and 58 E6 and/or E7 genes, respectively.

Persistent infection with high-risk human papilloma virus (HPV) is the primary cause of cervical intraepithelial neoplasia (CIN) and cervical carcinoma. HPV58 is the third most common HPV genotype in China after HPV16 and HPV18. HPV E6 and E7 are oncoproteins and are constitutively expressed in HPV-associated cancer cells, therefore they are considered to be ideal target antigens for immunotherapy, including HPV therapeutic vaccine. In the present study, human leukocyte antigen (HLA)-A2-restricted cytotoxic T lymphocyte (CTL) epitope peptides were predicted and screened from HPV58 E7 antigen and their immunogenicity was subsequently determined. A total of 6 HLA-A2-binding peptides derived from HPV58 E7 were predicted and selected using 3 different prediction programs. A negative control peptide and PBS were used as two negative controls. Peripheral blood mononuclear cells (PBMCs) with HLA-A2(+) allele were used to detect the specific cellular immune response among the 6 predicted peptides by enzyme-linked immunospot assay (ELISOPT). Following preliminary screening for the predicted peptides, the antigenicity of the peptide HPV58 E7 was further assessed by an immunoassay to a vaccine contained HPV58 E7 antigen. Specific humoral and cellular immunity were detected using the peptide HPV58 E7 as the specific antigen. A total of 6 peptides from HPV58 E7 protein were predicted and subsequently named P1 (E7: TLREYILDL), P2 (E7: DLHPEPTDL), P3 (E7: CINSTTTDV), and P4 (E7: STTTDVRTL), P5 (E7: TLQQLLMGT) and P6 (E7: LLMGTCTIV). In the ELISPOT assay on HLA-A2 (+) human PBMCs, interferon (IFN)-γ-production was evident in the P2 and P4 groups. The average numbers of IFN-γ associated spots in the P2 and P4 groups was 50.61±5.37 spot-forming cells (SFC)/1×10 and 266±34.42 SFC/1×10, respectively. The numbers of spots in the two peptides were significantly increased compared with the other 4 peptides and the control groups (P<0.05). In the further antigenicity verification of P4 (HPV58 E7), the peptide only stimulated the humoral immune response of the AD-HPV16/18/58 mE6E7 vaccine containing HPV58 E7 antigen. Compared with the 2 negative control groups (1:400), the antibody titers of the vaccine group (1:25,600) were significantly increased (P<0.05). In cellular immunoassays the average number of IFN-γ associated spots was 143.3±32.13 SFC/1×10 in the vaccine group, which was significantly enhanced compared with the PBS group (8±5.29 SFC/1×10; P<0.01) and the AD-NC group (28±5.13 SFC/1×10; P<0.01). The peptide HPV58 E7 (STTTDVRTL) displayed sufficient antigenicity to a vaccine contained HPV58 E7 antigen. Therefore, HPV58 E7 peptide may be considered as a candidate epitope peptide for the construction of HPV58 peptide vaccines.

The use of hematopoietic stem cell transplantation (HSCT) in cancer treatment is seriously hampered by the occurrence of graft-versus-host disease (GVHD) and cancer relapse. During acute GVHD, inflammatory cytokines play a pivotal role in the amplification of GVHD. Therefore, assessment of agents with known anti-neoplastic activity that also reduce cytokine production may be useful in both the prevention of GVHD and cancer relapse. The synthetic triterpenoid, CDDO (2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid) is multifunctional molecule which has shown potent anti-cancer activities both in vitro and in vivo through the induction of apoptosis. We first examined the effects of CDDO on both human and murine T cell mitogen responses in vitro. CDDO significantly inhibited mitogen responsiveness of both human and murine T cells in vitro with evidence of cell cycle arrest of the human T cells. We then proceeded to examine the effects of CDDO on acute GVHD induction and progression. In these studies, lethally irradiated C57BL/6 mice received 10 million bone marrow cells (BMC) and 40 million spleen cells from fully MHC-mismatched BALB/c donors. All of the control mice succumbed rapidly due to acute GVHD. In contrast, the mice that received CDDO (120 ug/BID) given from days 0-3 following BMT exhibited significant improvement in survival (P &lt; 0.001). Body weights from the treated mice also were significantly increased compared to untreated controls. We found that the timing of CDDO administration was a critical factor for protection from GVHD as protection only occurred when CDDO was administered early after BMT. Importantly, donor myeloid reconstitution was not adversely affected by CDDO treatment as determined by peripheral blood cell count and donor chimerism assessment on day +14 post-transplant. No adverse toxicities or effects on reconstitution were observed in the mice receiving BMC alone with CDDO being administered continuously. Given the reported direct anti-tumor effects of CDDO, it will be of particular interest in examining the effects of CDDO and allogeneic BMT in tumor-bearing recipients. Our findings suggest that CDDO can enhance the efficacy of allogeneic BMT by decreasing acute GVHD in mice.