A Phase 2a Randomized, Double-Blind, Dose-Optimizing Study to Evaluate the Immunogenicity and Safety of a Bivalent DNA Vaccine for Hemorrhagic Fever with Renal Syndrome Delivered by Intramuscular Electroporation.

Hantaan virus (HTNV) and Puumala virus (PUUV) are pathogenic hantaviruses found in Asia and Europe, respectively. DNA vaccines targeting the envelope glycoproteins of these viruses have been constructed and found to elicit neutralizing antibodies when delivered to humans by various technologies including intramuscular electroporation. Here, we report findings from a Phase 2a clinical trial of a combined HTNV/PUUV DNA vaccine delivered at varying doses and administration schedules using the Ichor Medical Systems TriGrid intramuscular electroporation delivery technology. The study was designed to characterize the effects of DNA vaccine dose and number of administrations on the frequency and magnitude of immunological response. Subjects (n = 120) were divided into four cohorts. Cohorts 1 and 2 received a dose of 2 mg of DNA (1 mg per plasmid), and cohorts 3 and 4 received a dose of 1 mg of DNA (0.5 mg per plasmid) each vaccination. Each of the four cohorts received a series of four administrations (days 0, 28, 56 and 168). For cohorts 1 and 3, the DNA vaccine candidate was delivered at each of the four administrations. For cohorts 2 and 4, in order to maintain blinding, subjects received the DNA vaccine on days 0, 56 and 168, but on day 28 received only the phosphate buffered saline vehicle rather the DNA vaccine. Sera were collected on days 0, 28, 56, 84, 140, 168, 196, 252 and 365 and evaluated for the presence of neutralizing antibodies by PUUV and HTNV pseudovirion neutralization assays (PsVNAs). Day 84 was also evaluated by a plaque reduction neutralization test (PRNT). Overall the PsVNA50 geometric mean titers (GMTs) and seropositivity rates among cohorts were similar. Cohort 3 exhibited the highest frequency of subjects that became seropositive to both PUUV and HTNV after vaccination, the highest peak GMT against both viruses, and the highest median titers against both viruses.

Characterization of the immunophenotype and the metastatic properties of a murine T-lymphoma cell line. Unexpected expression of cytoplasmatic CD4.

We report the first characterization of a mouse T-lymphoma cell line that surprisingly expresses cytoplasmatic (cy) yCD4. Phenotypically, LBC cells are CD5+, CD8+, CD16+, CD24+, CD25+, CD2-/dim, CD3-/dim, TCRbeta-/dim, TCRgammadelta, CD154 , CD40-, and CD45R. Coexpress cyTCRbeta, cyCD3, cyCD4, and yet lack surface CD4 expression. Transplantation of LBC cells into mice resulted in an aggressive T-lymphoblastic lymphoma that infiltrated lymph nodes, thymus, spleen, liver, ovary, and uterus but not peripheral blood or bone marrow. LBC cells display a modal chromosome number of 39 and a near-diploid karyotype. Based on the characterization data, we demonstrated that the LBC cell line was derived from an early T-cell lymphocyte precursor. We propose that the malignant cell transformation of LBC cells could coincide with the transition stage from late double-negative, DN3 (CD4- CD8 CD44-/low, CD25+) or DN4 (CD4-low, CD8-/low, CD44-, CD25-) to double-positive (DP: CD4+CD8+) stage of T-cell development. LBC cells provide a T-lymphoblastic lymphoma model derived from a malignant early T-lymphocyte that can be potentially useful as a model to study both cellular regulation and differentiation of T-cells. In addition, LBC tumor provides a short latency neoplasm to study cellular regulation and to perform preclinical trials of lymphoma-relatel clisorders.

Expression of CD44 splice variants in spontaneous murine tumors.

CD44 is a widely distributed set of cell surface glycoproteins expressed in several types of cells and tissues, implicated in cell-cell and cell-substrate interactions. This molecule plays a major role in cell differentiation, development and activation and has also been described as a potential marker of malignancy and metastasis. In the present study we investigated by RT-PCR followed by exon specific amplification the expression of CD44 splice variants in four different murine tumors as well as in the invaded organs in order to correlate the expression of CD44 variants with potential tumor invasiveness and their implications for growth. Our data showed deregulation in the expression of CD44 isoforms but no discernible correlation in isoform expression pattern. However, in all tumors studied isoforms presented by the primary tumor were detected in the invaded organs before metastasis could be demonstrated by histopathological analysis.

Splice variant expression of CD44 in patients with breast and ovarian cancer.

CD44 is an adhesion molecule involved in many biological functions and has been described to play a role in tumor progression as well as in promotion of metastasis. It has also been suggested that expression of certain CD44 isoforms could be useful for breast and ovarian cancer screening, detection or staging. However, many other reports document no correlation between CD44 isoform expression and tumor malignancy. In light of such contradictory findings, we evaluated by exon-specific RT-PCR whether the expression of CD44 isoforms in breast and ovarian tumors correlated with any of the diagnostic criteria used to assess these diseases. We found a deregulation in the CD44 expression pattern in malignant tumors of both type of cancer compared with the one in benign tumors or normal tissue. However, we could not find a clear correlation between this deregulation or a given CD44 isoform and any diagnostic criteria evaluated, such as age, clinical data, tumor size, hormone receptor status, histological grade or aggressiveness.

Interleukin 2 exerts autocrine stimulation on murine T-cell leukaemia growth.

As it has been suggested that an autocrine mechanism may control tumour cell growth, in this work cells from a spontaneous murine T lymphocyte leukaemia (LB) expressing the interleukin-2 receptor (IL-2R) (CD25) were evaluated in vitro for IL-2-mediated autocrine growth. Cells grew readily in culture and proliferation was enhanced by the addition of recombinant IL-2 but inhibited by monoclonal antibodies against either IL-2 or IL-2 receptor, in the absence of exogenous IL-2. Cyclosporin A also inhibited LB cell growth. However, when exogenous IL-2 was added together with cyclosporin A, cell proliferation proved similar to controls. Using reverse transcription polymerase chain reaction (PCR), mRNA for IL-2 was found to be present in tumour cells. Our findings support the hypothesis that LB tumour cell proliferation is mediated by an autocrine pathway involving endogenous IL-2 generation, despite the fact that these cells are not dependent on exogenous IL-2 to grow in culture.

Enhancement of anti-tumour immunity in syngeneic mice after MHC class II gene transfection.

The relationship between tumorigenicity and expression of MHC class II molecules in a class II-negative murine leukaemia cell line (LBC) was studied. Analysis of structural DNA sequences encoding MHC class II proteins was performed by Southern blot with DNA isolated from both the original LB tumour and LBC cell line, digested with EcoRI, BamHI and HindIII and hybridised with specific probes for I-A alpha d and I-A beta d chains. Similar patterns were obtained for LB, LBC and normal BALB/c lymphocytes. In vitro treatment with IFN-gamma (20 - 1000 IU ml-1) failed to induce the expression of MHC class II antigens in LBC cell line. LBC cells were tri-transfected by a liposome-mediated protocol with I-A alpha d, I-A beta d genes and pSV2neo. Cells were selected for growth in medium containing Geneticin (G418). Surviving transfectants were cloned and three I-A+ clones were obtained after 20 days (LBCT cells). Syngeneic mice inoculated with 1.0 x 10(3) LBCT (I-A+) cells failed to develop a tumour, whereas the DT50 of mice injected with 1.0 x 10(6) LBCT cells was three times the value for mice injected with LBC cells (I-A-). Furthermore, specific CTL response against tumour cells was significantly enhanced upon priming with irradiated LBC-transfected cells (27 +/- 2%) compared with irradiated LBC cells (15 +/- 1.5%) in a 4 h 51Cr-release assay. It is suggested that neoexpression of MHC class II molecules enhances anti-tumour response by transforming tumour cells into professional antigen-presenting cells (APCs), which may be used to improve tumour-specific immunity in the autologous host.

[Immunobiological characterization of murine LB leukemia and the LBC cell line]. / Caracterización inmunobiológica de la leucemìa murina LB y la línea celular LBC.

LB leukemia is a nonimmunogenic T cell tumor which spontaneously arose in a BALB/c mouse; efforts to induce immunological rejection of the leukemic cells have always failed. The leukemic cells grow rapidly and progressively in the syngeneic host invading spleen, lymph nodes and liver. A cell line (LBC) was developed from the original tumor. Both the original tumor and the cell line have been characterized as expressing the Thy 1+, CD3-, CD25+, MHC class I+, class II-, CD4- (original tumor), CD4+ (cell line), CD8+, gp70-, J11d.2+ phenotypes. Immunization of syngeneic mice with irradiated LBC cells induced cytotoxic T lymphocytes as well as anti-LBC antibodies which reacted with components of 14, 16 and 27 kDa present on LB tumor cells, LBC cell line and normal thymocytes but not on normal lymph node cells. Immunization of syngeneic mice with LBC cells partially protected them against subsequent challenge with the original tumor cells. The effect of sera from tumor-bearing mice and the super-natants from short term cultures were studied on cell proliferation. An inhibitory activity was demonstrated in these fluids, which was abrogated by addition of exogenous IL-2. ELISA showed the presence of soluble IL-2R alpha chain both in the conditioned medium as well as in the serum, which was demonstrated to be responsible for the inhibitory activity. The soluble IL-2R was produced by LB leukemic cells and exerted the inhibitory activity blocking cell proliferation and modulating immune response by binding to free IL-2. Using reverse-transcription PCR, mRNA for IL-2 was found to be present in tumor cells. Our findings indicate that LB cell proliferation is mediated by an autocrine pathway involving endogenous IL-2 generation, despite the fact that these cells are not dependent on exogenous IL-2 to grow in culture. The relationship between tumorigenicity and expression of MHC class II was also investigated. In vitro treatment with IFN-gamma failed to induce the expression of class II antigens in LBC cell line. Therefore these cells were tri-transfected by a liposome-mediated protocol with 1-A alpha d, I-A beta d genes and pSV2neo. Cells were selected to grow in medium containing Genetecin (G418) and surviving transfectants were cloned. Three I-A+ clones were obtained (LBCT) and were used to induce a specific CTL response against tumor cells. Syngeneic mice inoculated with 10(3) LBCT cells failed to develop a tumor while the DT50 of mice injected with 10(6) LBCT cells was three times the value for mice injected with LBC cells (I-A-). It is suggested that neoexpression of MHC class II molecules enhances anti-tumor response by transforming tumor cells into professional antigen-presenting cells, which may be used to improve tumor-specific immunity in the autologous host.

Induction of anti-tumour immunity in syngeneic mice by a leukaemic cell line.

Induction of anti-tumour immunity in syngeneic mice by LBC cell line derived from a non-immunogenic T cell leukaemia was studied. The immunization of BALB/c mice with LBC irradiated cells induced in them anti-tumour spleen cells, cytotoxic T lymphocytes and anti-LBC antibodies. The anti-LBC antibodies reacted with components of 14, 16 and 27 kDa present on LB tumour cells, LBC cell line and normal thymocytes, but not with normal lymph node cells. Furthermore, immunization of the autologous hosts with LBC cells partially protected them against subsequent challenge with the original LB leukaemic cells. These findings demonstrate that culture conditions induced modifications in the antigenic properties of the leukaemic cells, allowing LBC cells to stimulate an immune response directed against components expressed at early stages during T cell maturation. These results also suggest that the immune response is responsible for the prolongation of the survival time of the mice inoculated with the parental leukaemic cells.