Shao-Yan Si

The antitumor immune responses induced by nanoemulsion-encapsulated MAGE1-HSP70/SEA complex protein vaccine following peroral administration route

Previous studies have shown that there are profuse lymphatic tissues under the intestinal mucous membrane. Moreover, vaccine administered orally can elicit both mucous membrane and system immune response simultaneously, accordingly induce tumor-specific cytotoxic T lymphocyte. As a result, the oral route is constituted the preferred immune route for vaccine delivery theoretically. However, numerous vaccines especially protein/peptide vaccines remain poorly available when administered by this route. Nanoemulsion has been shown as a useful vehicle can be developed to enhance the antitumor immune response against antigens encapsulated in it and it is good for the different administration routes. Of particular interest is whether the protein vaccine following peroral route using nanoemulsion as delivery carrier can induce the same, so much as stronger antitumor immune response to following conventional ways such as subcutaneous (sc.) or not. Hence, in the present study, we encapsulated the MAGE1-HSP70 and SEA complex protein in nanoemulsion as nanovaccine NE (MHS) using magnetic ultrasound method. We then immuned C57BL/6 mice with NE (MHS), MHS alone or NE (-) via po. or sc. route and detected the cellular immunocompetence by using ELISpot assay and LDH release assay. The therapeutic and tumor challenge assay were examined then. The results showed that compared with vaccination with MHS or NE (-), the cellular immune responses against MAGE-1 could be elicited fiercely by vaccination with NE (MHS) nanoemulsion. Furthermore, encapsulating MHS in nanoemulsion could delay tumor growth and defer tumor occurrence of mice challenged with B16-MAGE-1 tumor cells. Especially, the peroral administration of NE (MHS) could induce approximately similar antitumor immune responses to the sc. administration, but the MHS unencapsulated with nanoemulsion via po. could induce significantly weaker antitumor immune responses than that via sc., suggesting nanoemulsion as a promising carrier can exert potent antitumor immunity against antigen encapsulated in it and make the tumor protein vaccine immunizing via po. route feasible and effective. It may have a broad application in tumor protein vaccine.

MAGE-1/Heat shock protein 70/MAGE-3 fusion protein vaccine in nanoemulsion enhances cellular and humoral immune responses to MAGE-1 or MAGE-3 in vivo

Tumor vaccines, which can elicit antigen-specific antitumor immunity and play an important role in prevention and therapy of tumor, are regarded as the most attractive method. Its underlying mechanism is that tumor antigen, especially the tumor specific antigen (TSA), can induce tumor-specific cytotoxic T lymphocyte (CTL) accordingly to damage tumor cells. The melanoma antigen (MAGE) was the first reported example of TSAs. MAGE-1 and MAGE-3 are two important members of MAGE. Whereas the expressions of MAGE family proteins deviate in different tumors, MAGE-1 and MAGE-3 are two most common tumor antigens. They are expressed in most tumors, respectively or jointly but not in normal tissues except the testes and placenta. Moreover, MAGE-1 and MAGE-3 antigens are termed as tumorrejection antigens because tumors expressing these antigens on appropriate human leukocyte antigen (HLA) class I molecules are rejected by host CTLs [16]. Heat shock proteins (HSPs), named as ‘‘molecular chaperone’’, participate in the folding, transporting and assembling of proteins. Besides, HSPs are important in the processing and presenting of antigens, which could conjugate proteins or peptides and improve their antigenicity. Moreover, the HSP receptors expressed on the surface of antigen-presenting cells (APCs) can facilitate APCs uptaking tumor antigens and improve the presentation. Related research had shown that HSP70 could be exploited to enhance the cellular and humoral immune responses against any attached tumor-specific antigens [7]. To find more effective and more comprehensive vaccine, our colleagues have constructed and purified a recombinant MAGE-1/HSP70/MAGE-3 (MHM) fusion protein successfully. In recent years, significant effort has been devoted to develop nanotechnology for drug delivery, since it offers a suitable means of delivering drugs with small molecular weights, as well as macromolecules such as proteins, peptides or genes by either localized or targeted delivery to the tissue of interest [10]. A nanoemulsion is a system of water, oil and amphiphile, which is a single optically isotropic and thermodynamically stable liquid solution. Therefore, a nanoemulsion is capable of encapsulating significant amounts of drugs. When compared to available traditional emulsions, a nanoemulsion is characterized by its smaller size (limited to 1–100 nm), higher efficiency, and elegant compatibility with tissues as well as with proteins. In addition, it can be prepared with oil and surfactant components that are generally regarded as safe, having less side effect, and toxicity. Moreover, W/O (water-in-oil) nanoemulsions can improve absorption of water-soluble drugs or peptides following intraduodenal administration. In this study, we used this novel artificial lipoprotein delivery system – nanoemulsion as delivery vehicle of the MHM fusion protein vaccine and determined its humoral and cellular immune responses and the antitumor effects. The goal was to investigate the potential benefits of using W/O nanoemulsion as an alternative carrier of the vaccine. W. Ge Æ Y.-F. Sui (&) Æ Y.-J. Sun Æ G.-S. Chen Æ Z.-S. Li S.-Y. Si Æ P.-Z. Hu Æ Y. Huang Æ X.-M. Zhang State Key Laboratory of Cancer Biology, Department of Pathology, Xi Jing Hospital, Fourth Military Medical University, Xi’an, 710032 Shaanxi Province, China E-mail: suiyanf@fmmu.edu.cn Tel.: +86-29-83374597 Fax: +86-29-83255697

Immunization with truncated sequence of Telomerase Reverse Transcriptase induces a specific antitumor response in vivo

To select the MHC-I-binding epitope-rich sequence of mice telomerase reverse transcriptase (mTERT) and study the antitumor immune response induced by truncated TERT through mRNA-transfected dendritic cells (DCs) immunization in mice. The MHC-I-binding epitopes of TERT were predicted using bioinformatics software. The selected sequence of TERT (Truncated mTERT, TERTt, mTERT cDNA 1776 bp-2942 bp encoding 584 aa-969 aa) was cloned from B16 mouse melanoma cells and inserted into pBluescriptIIKS(+) plasmid downstream of the T7 promoter. TERTt RNA was prepared through in vitro transcription. Bone marrow-derived DCs were electroporated with TERTt RNA and used to immunize syngeneic naïve mice. The quantity and cytotoxic activity of TERT-specific cytotoxic T lymphocytes (CTLs) in mice spleen were evaluated using IFN-γ enzyme-linked immunospot (ELISPOT) and Lactate dehydrogenase release assay. The immunoprophylactic effects against TERT positive tumor induced by TERTt RNA transfected DC in vivo were evaluated through an immunized-challenged mouse model. TERTt was cloned and in vitro transcribed into TERTt mRNA. As shown in FCM analysis, the efficiency of DC electroporation is 35.1% (29.7–41.2%). After electroporation, a subtle increase of costimulator and MHC-II molecules were expressed on the cell surface. Immunization of TERTt mRNA transfected DCs induced IFN-γ-secreting CTLs which manifested specific cytotoxic activity against TERT-positive target cells. In a cancer mouse model, vaccination of TERTt mRNA-transfected DCs suppressed the growth of TERT positive tumors (p=0.001) and prolong the survival time of tumor-bearing animals (p=0.029). TERTt evokes an antitumor immune response in vivo which is targeted to TERT. TERTt can be used as an antigeneic sequence to produce anti-TERT tumor vaccine.