Fucoidin enhances dendritic cell-mediated T-cell cytotoxicity against NY-ESO-1 expressing human cancer cells.

Scavenger receptor A (SR-A) plays a crucial role in affecting the dendritic cell-mediated presentation of cancer testis antigens to T cells against human cancer cells. Here we use a dendritic cell-mediated model to verify that a sulphated polysaccharide, fucoidin, can regulate the adverse regulatory function of SR-A, and lead to the up-regulation of the anti-tumor immunological response. SR-A is a receptor of calreticulin (CRT) existing on the surface of dendritic cells (DCs). CRT is a specific receptor for a NY-ESO-1 cancer testis antigen, and CRT itself is responsible for the cross-presentation of NY-ESO-1 to CD8+ cells and the induction of anti-tumor immunity. Flow cytometrical analysis (FACS) showed that fucoidin was able to significantly enhance the binding ratio of NY-ESO-1 to human DCs in a concentration dependent manner, and that the addition of fucoidin promoted the DC maturation upon stimulation of NY-ESO-1. Results from a cytotoxicity assay indicated that fucoidin-treated DCs stimulated the CD8+ T cells more effectively than non-treated DCs via a cross-presentation pathway. Furthermore, it was found that after stimulated by fucoidin-treated DCs, the CD8+ T cells can release more IFN-gamma than non-fucoidin-treated cells as detected by intracellular IFN-gamma staining. We conclude that fucoidin enhances the cross-presentation of NY-ESO-1 to T cells leading to an increase of T-cell cytotoxicity against NY-ESO-1 expressing human cancer cells.

Activation of cytotoxic T lymphocytes against CML28-bearing tumors by dendritic cells transduced with a recombinant adeno-associated virus encoding the CML28 gene.

Induction of anti-tumor immune responses by dendritic cells (DCs) transduced with a recombinant adeno-associated virus type 2 (rAAV2) encoding tumor antigens is considered a promising approach for cancer vaccine development. CML28, a novel antigen with the properties of cancer/ testis (CT) antigens, is an attractive target for antigen-specific immunotherapy. Here we investigated the feasibility of inducing CML28-specific cytotoxic T lymphocyte (CTL) responses using DCs transduced with the rAAV2 vectors containing the CML28 gene (rAAV/CML28). Using an adenovirus-free packaging system, rAAV/CML28 was generated. The transduction efficiency of rAAV/CML28 in DCs increased in a multiplicity of infection (MOI)-dependent manner. The rAAV/CML28 transduction did not impair DC maturation, but even enhanced the CD80 expression. The rAAV/CML28-transduced DCs induced CML28-specific CTLs which exhibited a MHC class I-mediated antigen-specific lytic activity against CML28-bearing tumor cell lines (HepG2 and MCF-7) as well as the primary leukemia blasts. These findings suggest that rAAV/CML28-transduced DCs vaccine may serve as a feasible approach for the treatment of CML28-associated cancers.

Induction of cytotoxic T cell response against HCA661 positive cancer cells through activation with novel HLA-A *0201 restricted epitopes.

HCA661 is a cancer-testis (CT) antigen frequently expressed in human hepatocellular carcinoma (HCC). To search for immunogenic peptides of HCA661, bioinformatics analysis and CD8(+) T cell IFN-gamma ELISPOT assay were employed, and two HLA-A *0201 restricted peptides, H110 and H246, were identified. These two HCA661 peptides are naturally processed in dendritic cells (DCs) and when used for DCs loading, they are sufficient to prime autologous CD8(+) T cells to elicit cytotoxic response against HCA661(+) human cancer cells. The HCA661 peptides, H110 and H246, are hence attractive candidates for human cancer immunotherapy.

Induction of T-cell response by a DNA vaccine encoding a novel HLA-A*0201 severe acute respiratory syndrome coronavirus epitope.

The severe acute respiratory syndrome coronavirus nucleocapsid protein (SARS-CoV N) is one of the major targets for SARS vaccine due to its high potency in triggering immune responses. In this study, we have identified a novel HLA-A*0201 restricted epitope, N220 (LALLLLDRL), of the SARS-CoV N-protein through bioinformatics analysis. The N-protein peptide N220 shows a high binding affinity towards human MHC class I in T2-cells, and is capable of activating cytotoxic T-cells in human peripheral blood mononuclear cells (PBMCs). The application of using the N220 peptide sequence with a single-chain-trimer (SCT) approach to produce a potential DNA vaccine candidate was investigated in HLA-A2.1K(b) transgenic mice. Cytotoxicity assay clearly showed that the T-cells obtained from the vaccinated animals were able to kill the N-protein expressing cells with a cytotoxicity level of 86% in an effector cells/target cells ratio of 81:1 one week after the last vaccination, which is significantly higher than other N-protein peptides previously described. The novel immunogenic N-protein peptide revealed in the present study provides valuable information for therapeutic SARS vaccine design.

Plasmid encoding papillomavirus Type 16 (HPV16) DNA constructed with codon optimization improved the immunogenicity against HPV infection.

Human papillomavirus Type 16 (HPV16) infections can cause neoplasia, which is thought to be closely associated with the development of cervical cancers. In the study, we attempted to construct a DNA plasmid encoding a HPV16 capsid protein (L1) and a HPV16 oncoprotein (E7), which was capable of preventing HPV16 infection and eliminating HPV16-infected cells. A plasmid, L1E7hpSCA1, encoding the L1 and E7 genes with the codon usage optimized for mammalian cell expression, was constructed. Mutations were introduced into the E7 gene sequence for reducing its oncogenicity. C57BL/6 mice were intramuscularly immunized at tibialis anterior (TA) muscles with the newly constructed L1E7hpSCA1 plasmid. The immune responses induced by the L1E7hpSCA1 plasmid (with codon optimization) and a control L1E7pSCA1 plasmid (without codon optimization) were compared. It is shown that the L1E7hpSCA1 was able to induce much stronger immune responses than the L1E7pSCA1. Sera obtained from immunized animals were found to contain anti-HPV16 antibodies as detected by ELISA and hemagglutination inhibition (HAI) assays. Cytotoxicity and interferon-gamma assays showed that spleenocytes from immunized animals were able to recognize and lyze E7 expressing tumor TC-1 cells. Moreover, the growth of E7 expressing tumor mass was inhibited in vaccinated mice. In vivo tumor protection test indicated that tumor formation was prevented in the experimental animals (67%) after vaccination with L1E7hpSCA1, while for the control group injected with L1E7pSCA1 only and the animal group injected with pSCA1 only, tumor formation was observed in all experimental animals. Our results suggest that the L1E7h gene (with codon optimization) is more effective against HPV16 than the L1E7 gene (without codon optimization). The L1E7hpSCA1 plasmid was able to provide protection against E7 expressing tumor, and it might have the potential to be a vaccine candidate for HPV prevention.

A DNA vaccine against foot-and-mouth disease elicits an immune response in swine which is enhanced by co-administration with interleukin-2.

A plasmid DNA vaccine candidate (pCEIS) encoding two foot-and-mouth disease virus (FMDV) VP1 epitopes (amino acid residues 141-160 and 200-213) has been demonstrated to have the ability to elicit both FMDV-specific T cell proliferation and neutralizing antibody against FMD in swine. In this study, the efficiency of the pCEIS DNA vaccine when administrated by intramuscularly injection in swine was confirmed, and the immunogenicity of the pCEIS vaccine candidate was found to be enhanced through co-administration with a newly constructed plasmid (pIL2S) encoding the swine interleukin-2 (IL-2) cDNA. The expression of the pIL2S plasmid was driven by a CMV promotor provided by a pcDNA3.1 vector. Swine IL-2 cDNA was cloned by RT-PCR from swine spleen cells. The pIL2S plasmid was expressed in COS-7 cells after 24 and 96h of transfection in vitro. In an animal trial, results from T cell proliferation assay indicated that the stimulation index (SI) in response to stimulation of FMDV proteins in the swine groups injected with pCEIS plus pIL2S (SI ranging from 9.9 to 15.5) were significantly higher than that with pCEIS alone (SI ranging from 3.3 to 6.6). However, there was no significant difference in FMDV-neutralizing antibody level detected in these two swine groups. Mouse protection tests (MPTs) showed that the blood sera from immunized swine injected with either pCEIS alone or pCEIS plus pIL2S were able to protect suckling mice from FMDV challenge, with protection levels ranging from 10(1) to 10(2) lethal dose 50 (LD(50)) M. In a direct FMDV challenge, all swines immunized with either pCEIS plus pIL2S or with pCEIS alone were challenged with 50LD(50)S (50 x lethal dosage in swine) of FMDV. The animals were fully protected (100%) from the FMD viral challenge. These results suggest that co-administration of the plasmids, pCEIS and pIL2S, enhances of the immunogenicity of the pCEIS DNA vaccine candidate, and both intramuscular injection of pCEIS alone and co-administration of the vaccine candidate with pIL2S can protect the swine from direct FMD challenge.