In order to further the present knowledge of the emerging severe acute respiratory syndrome-associated coronavirus (SARS-CoV), 486 different specimens from 54 patients with a clinical diagnosis of SARS were investigated for the presence of viral RNA, and 314 plasma specimens of 73 patients were examined for IgM and IgG antibodies specific against SARS-CoV using an indirect ELISA. Viral RNA was detectable in 28 of the 54 patients tested. Cumulative data showed that 67 of the 73 SARS patients demonstrated seroconversion by week 5 of illness. In contrast, only 1 of 278 healthy subjects enrolled in the study was found to be positive for the IgG antibody. Coexistence of viral RNA in plasma and specific antibodies was simultaneously observed over three consecutive weeks in two critical cases. In three convalescent patients in particular, cultivable SARS-CoV was detected in stool or urine specimens for longer than 4 weeks (29-36 days). These findings suggest that SARS-CoV may remain viable in the excretions of convalescent patients.

Severe acute respiratory syndrome (SARS) is a highly contagious and life-threatening disease that emerged in China in November 2002. A novel SARS-associated coronavirus was identified as its principal etiologic agent; however, the immunopathogenesis of SARS and the role of special CTLs in virus clearance are still largely uncharacterized. In this study, potential HLA-A*0201-restricted spike (S) and nucleocapsid protein-derived peptides were selected from an online database and screened for potential CTL epitopes by in vitro refolding and T2 cell-stabilization assays. The antigenicity of nine peptides which could refold with HLA-A*0201 molecules was assessed with an IFN-γ ELISPOT assay to determine the capacity to stimulate CTLs from PBMCs of HLA-A2+ SARS-recovered donors. A novel HLA-A*0201-restricted decameric epitope P15 (S411–420, KLPDDFMGCV) derived from the S protein was identified and found to localize within the angiotensin-converting enzyme 2 receptor-binding region of the S1 domain. P15 could significantly enhance the expression of HLA-A*0201 molecules on the T2 cell surface, stimulate IFN-γ-producing CTLs from the PBMCs of former SARS patients, and induce specific CTLs from P15-immunized HLA-A2.1 transgenic mice in vivo. Furthermore, significant P15-specific CTLs were induced from HLA-A2.1-transgenic mice immunized by a DNA vaccine encoding the S protein; suggesting that P15 was a naturally processed epitope. Thus, P15 may be a novel SARS-associated coronavirus-specific CTL epitope and a potential target for characterization of virus control mechanisms and evaluation of candidate SARS vaccines.

ABSTRACT

AIM:To investigate the influence of autologous cytokineinduced killer (CIK) cells on the phenotypes of CIK effector cells, peripheral T lymphocyte subsets and dendritic cell subsets in patients with primary hepatocellular carcinoma (HCC). METHODS: Peripheral blood mononuclear cells (PBMC)were collected by a blood cell separator from 13 patients with HCC, then expanded by priming them with interferongamma (IFN-γ) followed by monoclonal antibody (mAb) against CD3 and interleukin-2 (IL-2) the next day. The phenotypic patterns of CIK cells were characterized by flow cytometry on d 0, 4, 7, 10, 13 and 15 of incubation, respectively. Then, 5 mL of venous blood was obtained from HCC patients before or 8-10 d after CIK cells were transfused into patients to assess the influence of CIK cells on the percentages of effector cells, and proportions of DC1 or DC2 in peripheral blood by flow cytometry. + cells began to decrease on d 7 and 13, respectively. The proportions of type I dendritic cell (DC1) and type II dendritic cell (DC2) subsets increased from 0.59±0.23% and 0.26±0.12% before CIK cell therapy to 0.85±0. 27% and 0.43±0.19% (all P<0.01) after CIK cell transfusion, respectively. The symptoms and characteristics of HCC patients were relieved without major side effects. RESULTS: CONCLUSION:Our results indicated that autologous CIK cells can efficiently improve the immunological status in HCC patients, and may provide a potent approach for HCC patients as the adoptive immunotherapy.

In previous reports, systemic administration of a stimulatory monoclonal antibody directed against the 4-1BB receptor had no effect on survival or tumor burden in mice inoculated with the poorly immunogenic B16-F10 melanoma. We combined IL-12 gene transfer with 4-1BB costimulation to explore a previously noted cooperative anti-tumor effect against this model tumor. We hypothesize that the innate immune response mediated by IL-12-activated natural killer (NK) cells initiates the activation of the immune system, leading to the priming of T cells, whereas 4-1BB costimulation enhances the function of primed tumor-specific T cells. The effect of the combination therapy on the growth of subcutaneous (s.c.) tumors and pulmonary metastasis was examined. The combination therapy significantly retarded the growth of subcutaneously-inoculated tumors, and 50% of tumor-bearing mice survived with complete tumor regression. In contrast, neither IL-12 gene transfer nor anti-4-1BB antibody administration alone was as effective. Enhanced CTL activity against both B16-F10 tumor cells and TRP-2-pulsed EL4 syngeneic tumor cells was observed in tumorbearing animals treated with the combination therapy 2 weeks after treatment and, in long-term survivors from this combination therapy, at >120 days. In a pulmonary metastatic model, only the combination therapy generated significant protection against metastasis. In vivo depletion of NK or CD8؉ but not CD4 ؉ subsets eliminated the protective immunity. Furthermore, NK cell depletion significantly reduced both tumor-specific CTL activity and the number of tumor-specific IFN-␥-producing cells, suggesting that this synergistic effect requires the participation of both NK and CD8 ؉ T cells.