Eukaryotic Expression Plasmid Research Articles

Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity.

In order to develop a secure and competent technique to express the human immune gene for fighting infections, we cloned and expressed the BD2/3 using VR1020 (a eukaryotic expression plasmid). BD2/3 contains human β-defensin 2 (BD2) and human BD3. To explore safe and effective DNA delivery molecules in vitro and in vivo, the fusion genes of BD2/3 were used as an immune-labelled gene to verify transfection effectivness of modified chitosan (CS). Plasmid of VR1020-BD2/3 was packed with biomaterials: CS, average molecular weight: 25000D; polyethylene glycol-O-chitosan-polyethylenimine (PEG-O-CS-PEI); liposomes (LP); polyamine cationic liposomes (PCL); polyamine cationic liposomes of protamine (PCL-protamine) by ionotropic gelation. We observed that BD2/3 fusion gene showed high bioactivity in vitro and in vivo. The BD2/3 fusion protein inhibited the proliferation of bacteria (S. aureus, S. pneumoniae, P. aeruginosa and E. coli). The Kunming mice were immune to these nanoparticles and we analyzed their delivery efficiency and gene expression effect. BD2/3 results in multiple changes of innate and required immune system of mice. BD2/3 increases expression of IgG, IgG1, IgG2a, IL-2, IL-6, IFN-γ, as well as of lymphocytes and monocytes. Following challenge with virulent E. coli, CD4+ and CD8+ positive T-cell counts were highly elevated in the BD2/3 immunized mice, resulting in higher survival rates of mice. These results indicate that nanoparticles containing modified CS and BD2/3 are potentially safe and effective drugs in vivo to improve the immunity against bacterial infection and enhance innate immunity and adaptive immunity against infectious diseases.

Immunogenicity of a plasmid DNA vaccine encoding G1 epitope of bovine ephemeral fever virus G glycoprotein in mice.

The aim of this study was to investigate the immunogenicity of a plasmid deoxyribonucleic acid (DNA) vaccine encoding the G1 epitope of bovine ephemeral fever virus (BEFV) G glycoprotein in mice. A plasmid DNA carrying the G1 gene was constructed and designated as pcDNA3.1-G1. The expression of the target gene was confirmed in human embryonic kidney 293 (HEK 293) cells transfected with pcDNA3.1-G1 by indirect immunofluorescent staining. Immunisation experiments were intramuscularly carried out by vaccinating 6-week-old female mice in four groups, including the pcDNA3.1-G1 construct, pcDNA3.1 (+) plasmid alone, BEF-inactivated vaccine and phosphate-buffered saline (PBS) (1X) three times with 2-week intervals. Fourteen days after the last immunisation, the animals were bled and the resulting sera were tested for anti-G1-specific antibodies by immunoblotting analysis, indirect enzyme-linked immunosorbent assay (ELISA) and virus neutralisation (VN) test. Serological assays showed that the pcDNA3.1-G1 construct expressing G1 protein was able to elicit specific antibodies against this antigen. Virus neutralisation test showed that pcDNA3.1-G1 could induce anti-BEFV-neutralising antibodies in mice. Our findings indicated that a new dimension can be added to vaccine studies for bovine ephemeral fever (BEF) using eukaryotic expression plasmids encoding the G1 antigen in the future.

Overexpression of Neuregulin-1 (NRG-1) Gene Contributes to Surgical Repair of Brachial Plexus Injury After Contralateral C7 Nerve Root Transfer in Rats.

BackgroundSurgeons usually transfer the contralateral C7 to the median nerve on the injured side via a nerve graft to recover sensation and movement in a paralyzed hand. The purpose of our study was to determine whether NRG-1 affects the recovery of nerve function in brachial plexus injury after contralateral C7 nerve root transfer in a rat model.Material/MethodsAn injury model of left brachial plexus and contralateral C7 nerve root transfer was established. Four weeks after the operation, NRG-1 expression was examined by reverse transcription quantitative polymerase chain reaction and Western blot analysis. The diameter rate differences of the healthy limb and affected limb were estimated. The postoperative mass of the left latissimus dorsi, triceps, extensor carpi radialis brevis, and musculus extensor digitorum were examined. The number of nerve fibers and typical area of the affected side were assessed. Postoperative left motor nerve conduction velocity (MNCV) and motor nerve action potential (MNAP) were tested by use of a biological information recording and collecting system.ResultsEukaryotic expression plasmid of pcDNA4/myc/A-NRG-1 was successfully constructed, and NRG-1 was overexpressed. Compared with the model group, the NRG-1 group had a lower rate of differences of the limbs; higher mass of left latissimus dorsi, triceps, extensor carpi radialis brevis, and musculus extensor digitorum; more nerve fibers and larger typical area in the affected side, left MNCV, and MNAP; and wider CSA of the left triceps.ConclusionsThese results demonstrated that NRG-1 can promote recovery of nerve function in brachial plexus injury after contralateral C7 nerve root transfer in rats.

Influence of human recombinant eukaryotic expression plasmid pIRES-bone morphogenetic proteins 2-vascular endothelial growth factor 165 in vitro transfection on osteogenic differentiation of bone marrow mesenchymal stem cells

Objective To observe the influence of human recombinant eukaryotic expression plasmid pIRES-bone morphogenetic proteins 2 (BMP2)-vascular endothelial growth factor 165 (VEGF165) in vitro transfection on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Methods The isolated rabbit BMSCs were used as the research objects, and the recombinant plasmid pIRES-BMP2-VEGF165 was transfected into cells in the presence of liposomes. Then, G418 was used to screen stable cell lines in vitro and mRNA and protein levels were detected for transfection efficiency. Stable cell lines were then cultured and the cells were divided into pIRES group, pIRES-BMP2-VEGF165 transfection group and osteogenic induction group. Western blotting was used to detect the expression of Collagen Ⅰ (ColⅠ) and osteocalcin (OCN) protein at 7th and 21st day after cultivation, respectively. After 14 days of cultivation, gomori modified calcium and cobalt staining was used to detect alkaline phosphatase activity. After 21 days of cultivation, calcified nodules of cells were detected by alizarin red method calcium staining. Results Compared with the empty plasmid pIRES group, the expression of BMP2 and VEGF165 was detected at the mRNA and protein levels of pIRES-BMP2-VEGF165 plasmid group. Compared with empty plasmid group and osteogenic induction group, the expression levels of ColⅠ and OCN protein in recombinant plasmid group were significantly increased (Col Ⅰ: 0.597±0.040 vs. 0.230±0.020, P=0.000; 0.597±0.040 vs. 0.457±0.035, P=0.005; OCN: 0.433±0.038 vs. 0.083±0.021, P=0.000; 0.433±0.038 vs. 0.350±0.036, P=0.046). Positive relative area (%) of alkaline phosphatase staining increased significantly (69.000±5.568 vs. 18.333±3.056, P=0.000; 69.000±5.568 vs. 43.333±5.508, P=0.002). Positive relative area (%) of calcified nodules increased markedly (37.747±3.763 vs. 1.587±0.972, P=0.000; 37.747±3.763 vs. 19.330±4.033, P=0.008). Conclusion Human recombinant eukaryotic expression plasmid pIRES-BMP2-VEGF165 was successfully transfected into BMSCs. As compared with osteogenic induction group, the recombinant plasmid pIRES-BMP2-VEGF165 was more effective in osteogenic differentiation of the cells. Key words: Bone mesenchymal stem cells; pIRES-bone morphogenetic proteins 2 -vascular endothelial growth factor 165 plasmid; Transfection in vitro; Bone morphogenetic proteins 2; Vascular endothelial growth factor 165

Abstract 733: Modulating T cell immunity in tumors by targeting PD-L1 and neoantigens using a live attenuated oral Salmonella platform

Abstract Significant progresses have been achieved recently in cancer vaccines that aim at engaging or reengaging tumor directed T cells, in particular in the rapidly growing field of personalized immunotherapy. Yet novel immunization solutions to produce effective tumor-associated (neo)antigens T cell responses while simultaneously overcoming the immunosuppressive tumor microenvironment are urgently needed. We are developing a unique and versatile oral T-cell vaccination platform based on the FDA-approved live-attenuated Salmonella Typhi strain Ty21a vaccine Vivotif®, capable of delivering tumor-associated antigens encoded in DNA expression construct to the gut-associated lymphoid tissue, breaking immune tolerance and inducing anti-tumor immunity. This study summarizes the immunogenicity and antileukemia efficacy of VXM10 vaccines based on the live-attenuated Salmonella Typhimurium strain SL7207, transformed with a eukaryotic expression plasmid encoding the murine programmed death-ligand 1 (PD-L1) protein. It also describes for the first time the systemic immunogenicity of Salmonella based polyepitope oral vaccines, supporting the design of Salmonella based neoantigen vaccines. The antileukemia activity of VXM10 was evaluated in the FBL-3 disseminated model of leukemia, in which the tumor cells express high levels of PD-L1. Oral administration of VXM10 produced a strong anti-tumor effect in the FBL-3 leukemia model, with 100% of surviving animals 80 days after leukemia challenge in the highest dose groups. In contrast, administration of the empty vector control did not show any anti-cancer effect. Moreover, 100% of long-term surviving mice resisted re-challenge with FBL-3 cells, demonstrating that vaccination with VXM10 generated a potent memory T cell response against the leukemia. Importantly, full leukemia control was achieved in both prophylactic and therapeutic settings. The anti-tumor effect observed in these experiments was as strong as in previous experiments using a Salmonella Typhimurium transformed with a plasmid encoding Wilm's tumor 1 (WT1). The anti-tumor efficacy was accompanied by an increased systemic antibody response, and the activation of T cells directed against PD-L1 epitopes. Finally, different polyepitope vaccines encoding model epitopes from VEGFR2, Mesothelin, WT1, CEA, and Ovalbumin, induced a significant systemic immunogenicity for up to 6 out of 9 epitopes, 10 days after vaccination of healthy C57BL/6 mice via the oral route, as measured in the spleen by flow cytometry using peptide-MHC class I pentamers. These studies demonstrate that the Salmonella Ty21a platform can be used to generate anti PD-L1 antibody and T cell responses, as well as CD8-positive T cell responses against a majority of MHC class I epitopes of a polyepitope construct. We are now designing a clinical neo-antigen based vaccine that employs the concepts presented. Citation Format: Sébastien Wieckowski, Lilli Podola, Heiko Smetak, Anne-Lucie Nugues, Philippe Slos, Amine Adda Berkane, Ming Wei, Klaus Breiner, Albrecht Meichle, Philipp Beckhove, Marc Mansour, Heinz Lubenau. Modulating T cell immunity in tumors by targeting PD-L1 and neoantigens using a live attenuated oral Salmonella platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 733.

Construction of an eukaryotic expression plasmid for AY358935 gene

To construct an eukaryotic expression plasmid for AY358935 gene and explore its function. cDNA of the AY358935 gene was amplified by reverse transcription-PCR and cloned into pGEM-Teasy. The pGEM-T-AY was validated by sequencing and served as a template for the construction of eukaryotic expression plasmid. The pcDNA3.1-AY recombinant was validated by double enzyme digestion and used for transient transfection of M14 cells. Expression of the AY358935 protein and proliferation of the M14 cells were determined respectively by Western blotting and 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) colorimetry. The amplicons of RT-PCR were confirmed to have similar size with the cDNA fragment of the AY358935 gene as well as cloned region of pcDNA3.1-AY. The cloned region of pGEM-T-AY was sequenced to be identical with cDNA sequence of the AY358935 gene. M14 cells were transfected by the AY358935 gene, pcDNA3.1 and liposomes, respectively. After 48 h, expression of the AY358935 protein in M14 cells transfected with the AY358935 gene was significantly higher than other two groups. They also had a significantly higher absorbance value (A=0.74) than other two groups (A=0.39 and 0.46, respectively; P<0.05). An eukaryotic expression plasmid of the AY358935 gene was successfully constructed. Product of the AY358935 gene may promote the proliferation of M14 cells.

The roles and mechanism of platelets with podoplanin over-expressing human mammary fibroblasts in the in situ growth of breast cancer

Objective To confirm the role of podoplanin (PDPN) over-expressing human mammary fibroblasts (HMF) in tumorigenesis of breast cancer cell line MDA-MB-231, and study the effect and mechanism of reducing the peripheral platelet count in the progress of PDPN over-expressing HMF (HMF-P)-induced tumor growth. Methods We firstly constructed PDPN eukaryotic expression plasmid PDPN-pEGFP-LV5 and established HMF-P by lentivirus transfection. Then we investigated the effect of HMF-P on tumorigenesis in nude mice. We constructed a mouse model of thrombocytopenia by antibodies for mouse platelet depletion polyconal purified R300 and investigated the effect of HMF-P again in these special mice. Lastly, the expression of some tumor growth factors was detected by Western blotting. Results MDA-MB-231 cells commixed with HMF-P generated tumors of greatest volume 3.16 cm3; while the volume of HMF+ MDA-MB-231 and MDA-MB-231 groups was 1.93 cm3 and 0.52 cm3 respectively. The tumors volume in HMF-P + MDA-MB-231 group decreased to 1.03 cm3 in mice with thrombocytopenia, while the change in HMF+ MDA-MB-231 groups was not obvious. Western blotting analysis showed that the expression of platelet-derived growth factor-D (PDGF-D) in HMF-P + MDA-MB-231 group was significantly increased, and that in thrombocytopenia group was significantly decreased. Conclusion HMF-P can promote the tumorigenesis of MDA-MB-231. Its mechanism may be related to the interaction of PDPN with infiltrating platelets to promote its activation and release of PDGF-D. Reducing the peripheral blood platelet count to reduce platelet infiltration can inhibit the effect of HMF-P promoting tumor growth. Key words: Breast cancer; Platelets; Podoplanin; Mammary fibroblasts

Effects of Simultaneous Downregulation of PHD1 and Keap1 on Prevention and Reversal of Liver Fibrosis in Mice

Background and Aim: To investigate whether double-knockdown of PHD1 and Keap1 in mice could enhance the resolution of carbon tetrachloride (CCl4)-induced liver fibrosis.Methods: The liver fibrosis model of mice was established by intraperitoneal injection of 25% CCl4 in olive oil (4 ul/g) twice a week for 8 weeks. PHD1shRNA and Keap1shRNA eukaryotic expression plasmids were simultaneously administered from the beginning of the first to fourth week (preventive group) or from the fifth to eighth week of CCl4 injection (therapeutic group) via hydrodynamic-based tail vein injection. Successful transfection was confirmed with the expression of red fluorescent protein and green fluorescent protein in hepatocytes. Western blot was used for determining the expression of PHD1 and Keap1, HE, Sirius red, and Masson staining for evaluating the histopathological stages of fibrosis. Immunohistochemical techniques were applied to evaluate the expression of a-SMA.Results: The fluorescence of red and green were observed mainly in hepatocytes, and downregulation of PHD1 and Keap1 expression in liver was detected by western blot. Meanwhile, double-knockdown of PHD1 and Keap1 in mice alleviated liver fibrosis, and the effect was further enhanced especially in the preventive group. Immunocytochemical staining showed decreased expression of a-SMA when both PHD1 and Keap1 were knockdown.Conclusion: Downregulation of PHD1 and Keap1 expression in the liver could be achieved via hydrodynamic injection of PHD1shRNA and Keap1shRNA, thereby, preventing liver fibrosis.

Detect the presence of LeIF gene in the Leishmania tropica genome and sequence it

Leishmaniasis remains one of the most neglected diseases globally and affects primarily the poor in developing countries. Due to the absence of safe and effective drug, the prevention of infection of this disease is very important as it requires an effective vaccine is not yet available. Leishmania eukaryotic initiation factor (LeIF) protein acts an inhibitor of the Leishmania major growth in murine macrophages and reduces interleukin-4 in lymph nodes. We aimed to detect its presence in Leishmania tropica genome and clone the encoding gene of LeIF antigen of Leishmania tropica into eukaryotic expression plasmid pCI. We used a Syrian strain; LCEB-Syrian Strain 01 of Leishmania tropica promastigotes. Specific primers were designed to amplify the gene by Polymerase Chain Reaction “PCR”. LeIF gene was amplified from both DNA and cDNA after optimization the conditions and then sequenced. LeIF gene was cloned into pCI plasmid. We transformed E. coli Top 10 bacteria by the recombinant plasmid and the cloning was proved by colony PCR and restriction digestion. As a result, LeIF is a part of both DNA genome and RNA of Leishmania tropica LCEB-Syrian Strain 01, and cloned LeIF is a candidate for future studies as a DNA vaccine against leishmaniasis.

Formulation in DDA-MPLA-TDB Liposome Enhances the Immunogenicity and Protective Efficacy of a DNA Vaccine against Mycobacterium tuberculosis Infection.

Despite the vaccine Mycobacterium bovis Bacillus Calmette–Guérin is used worldwide, tuberculosis (TB) remains the first killer among infectious diseases. An effective vaccine is urgently required. DNA vaccine has shown prophylactic as well as therapeutic effects against TB, while its weak immunogenicity hinders the application. As a strong inducer of Th1-biased immune response, DMT, consisting of dimethyldioctadecylammonium (DDA) and two pattern recognition receptor agonists monophosphoryl lipid A and trehalose 6,6′-dibehenate (TDB), was a newly developed liposomal adjuvant. To elucidate the action mechanism of DMT and improve immunological effects induced by DNA vaccine, a new recombinant eukaryotic expression plasmid pCMFO that secretes the fusion of four multistage antigens (Rv2875, Rv3044, Rv2073c, and Rv0577) of Mycobacterium tuberculosis was constructed. pCMFO/DDA and pCMFO/DMT complexes were then prepared and their physicochemical properties were analyzed. The immunogenicity and protection against M. tuberculosis infection in vaccinated C57BL/6 mice were compared. Formulation of DNA and two agonists into the DDA liposome decreased zeta potential but increased the stability of storage, which resulted in a slower and longer-lasting release of DNA from the DNA–DMT complex than the DNA–DDA liposome. Besides Th1-biased responses, pCMFO/DMT vaccinated mice elicited more significantly CFMO-specific IL2+ TCM cell responses in the spleen and provided an enhanced and persistent protection against M. tuberculosis aerosol infection, compared to pCMFO/DDA and pCMFO groups. Therefore, the adjuvant DMT can release DNA and agonists slowly, which might attribute to the improved protection of DMT adjuvanted vaccines. pCMFO/DMT, a very promising TB vaccine, warrants for further preclinical and clinical trials.

Identification and characterization of a type I interferon induced by cyprinid herpesvirus 2 infection in crucian carp Carassius auratus gibelio

Crucian carp (Carassius auratus gibelio) is a popular food fish in Asia, and cyprinid herpesvirus 2 (CyHV-2) is the only known viral pathogen for crucian carp. Type I interferon genes are induced up on host cell recognition of viral nucleic acids and well recognized for their crucial roles in providing local or systemic protection against the viruses in various organisms. In a transcriptome analysis to uncover differentially expressed genes in crucian carp in response to CyHV-2 challenge, a partial interferon transcript was identified to be significantly up-regulated in the kidney of infected fish, which was named as crucian carp IFNc (ccIFNc). The complete ORF of ccIFNc was further determined by RACE technique, which spanned over 546 bp and encoded a polypeptide containing 182 amino acids. Phylogenetic analysis revealed that ccIFNc clustered with known type I IFN genes from other aquatic organisms. Quantitative RT-PCR analysis demonstrated that ccIFNc was constitutively expressed in all investigated tissues with a comparably higher expression level in spleen, gill, kidney, and muscle. Following challenge with CyHV-2, the transcriptional levels of ccIFNc were dramatically up-regulated in all of the tested tissues, especially in the spleen and gill with increased folds of 436 and 158, respectively. The intramuscular (i.m.) injection of a eukaryotic expression plasmid encoding ccIFNc (pEGFP-cIFNc) resulted in increased ccIFNc expression and reduced the mortality after the CyHV-2 challenge significantly. In summary, our data suggested that the ccIFNc belongs to the type I interferon family with a potential role in countering CyHV-2 infection in crucian carp.

Live attenuated oral Salmonella platform for effective T- and B-cell targeting of PD-L1.

74 Background: Significant progresses have been recently achieved in cancer vaccines, yet novel immunization solutions to deliver efficiently tumor-associated antigens to professional antigen-presenting cells, and to overcome the peripheral tolerance and the immunosuppressive tumor microenvironment that prevent the eradication of cancer in most of patients, are urgently needed. VAXIMM is developing a unique and versatile oral T-cell vaccination platform based on the FDA-approved live-attenuated Salmonella Typhi strain Ty21a vaccine Vivotif, capable of delivering tumor-associated antigens encoded in DNA expression construct to the gut-associated lymphoid tissue, breaking immune tolerance and inducing effective anti-tumor immunity. Methods: This study summarizes the immunogenicity and antileukemia efficacy of VXM10 vaccines based on the live-attenuated Salmonella Typhimurium strain SL7207, transformed with a eukaryotic expression plasmid encoding different variants of the murine PD-L1 protein. Results: The antileukemia activity of VXM10 was evaluated in the FBL-3 disseminated model of leukemia, in which the tumor cells strongly express PD-L1. Multiple oral administrations of VXM10 vaccines produced a strong anti-tumor effect, with 100% of surviving animals 80 days after challenge with FBL3 leukemia in the highest dose groups. Moreover, 100% of long-term surviving mice resisted re-challenge with FBL-3 cells, demonstrating that vaccination with VXM10 generated a potent memory T cell response against the leukemia. Importantly, full leukemia control was achieved in both prophylactic and therapeutic settings. Upon immunization with VXM10 vaccines, T cell response was raised against PD-L1 epitopes after in vitro restimulation of the splenocytes, and anti-PD-L1 antibodies were detected in the serum. The precise mechanism of action of VXM10 vaccines is currently being investigated. Conclusions: This study provides further evidence that VAXIMM’s oral T-cell vaccination platform can be employed to stimulate anti-tumor immunity against antigens of the immune checkpoint regulatory protein PD-L1. These data paved the way for advancing the development of VXM10 into clinical development.

Over-expression of human Notch ligand Delta-like 3 promotes proliferation of human gastric cancer cells in vitro

To construct a eukaryotic expression plasmid carrying human full-length Notch ligand Delta-like 3 (DLL3) gene and study the effect of DLL3 knockdown and overexpression on the proliferation of gastric cancer cells in vitro. Human full-length DLL3 gene was amplified by PCR and cloned into the eukaryotic expression vector pCMV-Tag4. After verification by restriction enzymes and sequencing, the recombinant DLL3/pCMV-Tag4 vector was transiently transfected into HEK293T cells, in which the expressions of human DLL3 mRNA and protein were detected using real-time quantitative PCR and Western blotting, respectively. The expression of DLL3 in normal gastric epithelial cells and gastric cancer cell lines was detected by qRT-PCR and Western blotting. DLL3/pCMV-Tag4 was transfected into 3 gastric cancer cell lines, and their proliferation was assessed with MTT assay. Human gastric cancer cells MGC803 and MKN45 were also transfected with a specific human DLL3-siRNA to assess the effect of DLL3 down-expression on the cell proliferation. The recombinant eukaryotic expression vector DLL3/pCMV-Tag4 was successfully constructed and human full-length DLL3 was expressed in HEK293T cells. MTT assay showed that DLL3 over-expression obviously promoted the proliferation and down-regulation of DLL3 inhibited the proliferation of the gastric cancer cells. DLL3 overexpression can promote the proliferation of gastric cancer cells in vitro, and down-regulation of DLL3 inhibits the proliferation of gastrc cancer cells, which provides a novel strategy for targeted thrapy of gastric cancer.

Peyer's Patches as a Portal for DNA Delivery by Lactococcus lactis in Vivo.

Application of food-grade Lactococcus lactis (L. lactis) as a safe delivery tool for DNA vaccines and therapeutic proteins has been well investigated. Although some studies showed that eukaryotic expression plasmids were transferred from L. lactis to enterocytes, the precise mechanism of the DNA transfer remains unknown. In this study, we generated an invasive L. lactis strain that expresses "murinized" Internalin A, an invasin of intracellular bacteria Listeria monocytogenes with two amino acid alterations for invasion into murine cells, and confirmed that this L. lactis strain delivered DNA in an invasin-dependent manner into a monolayer of epithelial cells polarized to mimic the gastrointestinal tract environment. Although invasive L. lactis inoculated orally can deliver DNA into enterocytes in the gastrointestinal tract of mice, the efficiency of DNA transfer was similar to that of non-invasive L. lactis strain, suggesting that the in vivo DNA transfer from L. lactis occurs invasin-independently. A ligated-intestinal loop assay, a method for a short-term culturing of the whole intestine filled with materials to evaluate the interaction of the materials with intestinal cells, demonstrated that both non-invasive and invasive L. lactis strains were present in the Peyer's patches of the small intestine. On the other hand, few L. lactis was detected in the non-Peyer's patch epithelial region. Thus, our observations lead us to speculate that DNA transfer from L. lactis occurs predominantly in the Peyer's patches in an invasin-independent manner.

Abstract B196: Live attenuated oral Salmonella platform for effective targeting of multiple tumor-associated epitopes and PD-L1

Abstract Significant progress has been achieved recently in cancer vaccines that aim at engaging or reengaging the tumor immunity, in particular in the rapidly growing field of personalized immunotherapy. Yet novel immunization solutions to deliver efficiently tumor-associated (neo)antigens to professional antigen-presenting cells, and to overcome the peripheral tolerance and the immunosuppressive tumor microenvironment that prevent the eradication of cancer in most patients, are urgently needed. VAXIMM is developing a unique and versatile oral T-cell vaccination platform based on the FDA-approved live-attenuated Salmonella Typhi strain Ty21a vaccine Vivotif®, capable of delivering tumor-associated antigens encoded in DNA expression construct to the gut-associated lymphoid tissue, breaking immune tolerance and inducing antitumor immunity. This study summarizes the immunogenicity and antileukemia efficacy of VXM10 vaccines based on the live-attenuated Salmonella Typhimurium strain SL7207, transformed with a eukaryotic expression plasmid encoding the murine programmed death-ligand 1 (PD-L1) protein. It also describes for the first time the systemic immunogenicity of Salmonella Typhimurium-based polyepitope oral vaccines in healthy animals. The antileukemia activity of VXM10 was evaluated in the FBL-3 disseminated model of leukemia, in which the tumor cells express high levels of PD-L1. Multiple oral administrations of VXM10 used at 1010 colony-forming units (CFU) were generally well tolerated, and neither sign of toxicity nor body weight loss were observed throughout the study. Oral administration of VXM10 produced a strong antitumor effect in the FBL-3 leukemia model, with 100% of surviving animals 80 days after leukemia challenge in the highest-dose groups. In contrast, administration of the empty vector control did not show any anticancer effect. Moreover, 100% of long-term surviving mice resisted rechallenge with FBL-3 cells, demonstrating that vaccination with VXM10 generated a potent memory T-cell response against the leukemia. Importantly, full leukemia control was achieved in both prophylactic and therapeutic settings. Immunogenicity and antibody response towards PD-L1 are being evaluated to dissect the exact mechanism of action of these novel vaccines. Finally, different polyepitope vaccines encoding dominant epitopes from VEGFR2, Mesothelin, WT1, CEA, and Ovalbumin induced a significant systemic immunogenicity for up to 6 out of 9 epitopes, 10 days after vaccination of healthy C57BL/6J mice via the oral route, with doses up to 1010 CFU, as measured in the spleen by flow cytometry using peptide-MHC class I pentamers. This study provides further evidence that VAXIMM’s oral T-cell vaccination platform can be employed to stimulate antitumor immunity against antigens of the immune checkpoint regulatory protein PD-L1, and also against T-cell antigens encoded by polyepitope constructs. These data pave the way for advancing the development of VXM10 and neoantigen-based vaccines into clinical development. Citation Format: Sébastien Wieckowski, Lilli Podola, Marco Springer, Iris Kobl, Heiko Smetak, Anne-Lucie Nugues, Philippe Slos, Amine Adda Berkane, Ming Wei, Klaus Breiner, Albrecht Meichle, Beckhove Philipp, Marc Mansour, Matthias Schroff, Heinz Lubenau. Live attenuated oral Salmonella platform for effective targeting of multiple tumor-associated epitopes and PD-L1 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B196.

Inhibitory effects of int-2 gene on the invasion and metastasis of oral cancer cells.

The purpose of this study was to investigate the effects of int-2 transfection on the invasiveness and metastasis of oral cancer BcaCD885 cells, and to determine the relevant mechanisms of action. High-purity int-2 eukaryotic expression plasmids were prepared and transfected using a modified cationic liposome-mediated transfection protocol. Nucleoside diphosphate kinase A (NDPKA) expression before and after transfection was examined, as well as changes in cell invasiveness and metastasis capabilities. Int-2 was confirmed to be stably expressed post-transfection into oral cancer cells. Expression of int-2 in BcaCD885 cells was significantly different before and after transfection. The proportion of invasive cells were 70.3%±8.2% and 46.5%±5.7%, and the proportion of chemotaxis cells were 78.5%±7.9% and 49.6%±7.5%, in the in the control and experimental groups respectively. The adhesion capability of cells in the experimental group was also significantly reduced. Upregulation of int-2 expression can significantly inhibit the invasion and metastasis of BcaCD885 cells.

Fused IgY Fc and Polysaccharide Adjuvant Enhanced the Immune Effect of the Recombinant VP2 and VP5 Subunits-A Prospect for Improvement of Infectious Bursal Disease Virus Subunit Vaccine.

Infectious bursal disease virus (IBDV) is a highly contagious pathogen that causes damage in lymphoid organs and remains a threat to the poultry industry worldwide. Currently, subunit vaccines based on VP2 antigen expressed in prokaryotic systems are widely used in clinical settings. However, the immunogenicity of VP2 vaccines is limited because of their inherent defect that the structure of the antigen expressed in Escherichia coli (E. coli) may be different from its natural conformation. In this study, we fused VP2 and VP5 protective antigen genes and linked the chicken IgY Fc gene onto it. The eukaryotic expression plasmid carrying the fusion gene was transformed into Pichia pastoris (P. pastoris) to express the recombinant VP2–VP5–Fc protein. The recombinant protein was used as immunogen for evaluating immune response, and the recombinant VP2–Fc and VP2 proteins expressed in P. pastoris and the commercial VP2 subunit vaccines were used as controls. Moreover, Taishan Pinus massoniana pollen polysaccharide (TPPPS), an immunomodulator found by our laboratory, was used as adjuvant to investigate its immune modulatory effects on immunogens. Chickens were divided into six groups and inoculated with VP2–VP5–Fc+TPPPS, VP2–VP5–Fc, VP2–Fc, VP2 vaccine, commercial VP2 subunit vaccine, and phosphate buffered saline (PBS). The recombinant VP2 subunit vaccine expressed in P. pastoris exhibited higher immunogenicity than the commercial VP2 subunit vaccine. The VP2–Fc protein showed a better effect than the VP2 protein, and the VP2–VP5–Fc subunit further improved the immune effects. In addition, TPPPS was proved to be a good immunopotentiator for the VP2–VP5–Fc subunit vaccine. Hence, the recombinant VP2–VP5–Fc subunit combined with TPPPS adjuvant exhibits potential as efficient IBDV vaccine to prevent infectious bursal disease.

Immune Effect of Newcastle Disease Virus DNA Vaccine with C3d as a Molecular Adjuvant.

Newcastle disease is a serious infectious disease in the poultry industry. The commercial vaccines can only offer limited protection and some of them are expensive and need adjuvants. At present, DNA vaccines are widely used. However, the immune responses induced by DNA vaccines are too slow and low. Here, we constructed the transfer vectors with a different number of C3d as molecular adjuvants (n = 1, 2, 4, or 6), and the vectors were cloned into the optimal eukaryotic expression plasmid (pVAXI-optiF) that expressed the F gene of Newcastle disease virus (NDV), and named pVAXI-F(o)-C3d1, pVAXI -F(o)-C3d2, pVAXI-F(o)-C3d4, and pVAXI-F(o)-C3d6, respectively. Cell transfection test indicated that pVAXI-F(o)-C3d6 showed the highest expression. In vivo immunization showed that the chickens immunized with pVAXI-F(o)-C3d6 intramuscularly induced better immune responses than the chickens immunized with the other plasmids. The protective efficacy of pVAXI-F(o)-C3d6 was 80% after challenge with the highly virulent NDV strain F48E9. The results in this study showed that C3d6 could be used as a molecular adjuvant to quickly induce an effective immune response to control NDV.

Mapping a Type-specific Epitope by Monoclonal Antibody against VP3 Protein of Duck Hepatitis A Type 1 Virus

Duck hepatitis A subtype 1 virus (DHAV-1) infection causes high mortality in ducklings, resulting in significant losses to duck industries. VP3 is a structural protein of DHAV-1. However, B-cell epitopes on VP3 have not been investigated. To stimulate VP3 antibody response, eukaryotic expression plasmid pCI-neo-VP3 was constructed and used as DNA immunogen to prepare mAbs. Western blot showed that 25.5 kDa VP3 could be detected by mAbs in duck embryo fibroblast (DEF) cells transfected with pCI-neo-VP3. Immunofluorescence assay showed that mAbs could specifically bind to DEF cells infected with DHAV-1. DAPI staining indicated that VP3 localizes to the cytoplasm and nucleus of DHAV-1 infected DEF. With neutralizing mAb 3B7, minimal epitope PSNI was mapped. Sequence alignment indicated that 205PSNI208 is highly conserved among DHAV-1, but different from those of DHAV-2 and DHAV-3. Epitope peptide reacted specifically with DHAV-1-positive duck sera by dot blotting, revealing PSNI is DHAV-1 type-specific epitope and the importance of these amino acids in antibody-epitope binding reactivity. These findings provided useful information for understanding the antigenicity of VP3 and might be valuable in the development of epitope-based vaccine or diagnostic kit for DHAV-1 infection and provide insights for understanding the pathogenesis of DHAV-1.

Characterization of Monoclonal Antibodies against HA Protein of H1N1 Swine Influenza Virus and Protective Efficacy against H1 Viruses in Mice.

H1N1 swine influenza viruses (SIV) are prevalent in pigs globally, and occasionally emerge in humans, which raises concern about their pandemic threats. To stimulate hemagglutination (HA) of A/Swine/Guangdong/LM/2004 (H1N1) (SW/GD/04) antibody response, eukaryotic expression plasmid pCI-neo-HA was constructed and used as an immunogen to prepare monoclonal antibodies (mAbs). Five mAbs (designed 8C4, 8C6, 9D6, 8A4, and 8B1) against HA protein were obtained and characterized. Western blot showed that the 70 kDa HA protein could be detected by all mAbs in MDCK cells infected with SW/GD/04. Three mAbs—8C4, 8C6, and 9D6—have hemagglutination inhibition (HI) and neutralization test (NT) activities, and 8C6 induces the highest HI and NT titers. The protection efficacy of 8C6 was investigated in BALB/c mice challenged with homologous or heterologous strains of the H1 subtype SIV. The results indicate that mAb 8C6 protected the mice from viral infections, especially the homologous strain, which was clearly demonstrated by the body weight changes and reduction of viral load. Thus, our findings document for the first time that mAb 8C6 might be of potential therapeutic value for H1 subtype SIV infection.