Method for augmenting the immunogenicity of an antigen
a technology of immunogenicity and antigen, applied in the field of immunogenicity augmenting an antigen, can solve the problems of low efficacy of dna vaccine in large animals and humans, inability to apply lz-8 in vaccination or cancer therapy, and host may become tolerable, etc., to achieve the effect of augmenting the immunogenicity of an antigen and augmenting the immunogenicity of said protein
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Mice and Cell Cultures
[0047]C57BL / 6, C3H / HeN, and C3H / HeJ (TLR4 mutant) mice were purchased from National Laboratory Animal Center (Taipei, Taiwan) or National Cheng-Kung University (Tainan, Taiwan). OT-I and OT-II TCR transgenic mice were provided by Dr. Clifford Lowell (UCSF, San Francisco, Calif.). All mice were housed in the barrier facility at NHRI (Taiwan) under an Institutional Animal Care and Use Committee-approved protocol. Mouse DCs were generated from bone marrow as previously described. The murine bladder tumor cell line MBT-2 has been described and is known to express high levels of p185neu.
Preparation and Inactivation of LZ-8
[0048]LZ-8 was cloned and expressed in Saccharomyces cerevisiae. Cells expressing LZ-8 (or empty vector as control) were disrupted, centrifuged, and the supernatant was passed through filter and molecular sieves to obtain proteins between 10 kDa and 100 kDa. The filtrate was further purified using FPLC with Superdex 75 columns ...
example 2
The Activation of Mouse DCs by Recombinant LZ-8 was not Due to Microbial Contaminants.
[0055]The stimulatory activity of LZ-8 on human DCs has been identified; however, the application of LZ-8 in vaccination and cancer therapy is not studied. Since the present invention used a mouse model to evaluate the adjuvant effect of LZ-8, the present invention first tested whether LZ-8 could activate mouse DCs. As shown in human DCs previously, LZ-8 promoted cytokine and chemokine production and maturation of bone marrow-derived DCs from C57BL / 6 mice (FIG. 1A). The saturated dose of the LZ-8 for maximal TNF-α production was at 5 μg / mL. No significant cytotoxicity was observed at the highest dose of LZ-8 as measured by propidium iodide staining (data not shown). Furthermore, the LZ-8-induced TNF-α production was dramatically reduced in TLR4 mutant (C3H / HeJ) DCs when compared to WT (C3H / HeN) cells (FIG. 1B), consistent to the report in human DCs. A very important issue is that the present invent...
example 3
LZ-8 Facilitated DC-Induced Ag-Specific T Cell Activation In Vitro and In Vivo
[0057]Induction of antigen-specific T cell activation is the primary function of mature DCs, the present invention next tested whether LZ-8-stimulated DCs are able to activate naïve T cells. OT-I or OT-II T cells were co-cultured with LZ-8-treated, OVAP1- or OVAP2-pulsed DCs, and T cell proliferation and IFN-γ production were determined. LZ-8-activated DCs induced more OVA-specific T cell proliferation and IFN-γ production than control cells in vitro (FIG. 2A). Then, a subunit vaccine model was performed to evaluate the adjuvant effect of LZ-8 on T cell priming in vivo. C57BL / 6 mice were immunized with OVAP2 mixed with IFA alone or IFA plus LZ-8, and draining LN cells were collected after 10 days. The LN T cells isolated from LZ-8-immunized mice showed more proliferation and IFN-γ production than cells from control mice in response to OVAp2 (FIG. 2B). These data reveal that LZ-8-stimulated DCs can induce A...
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