Improvement and screening of astaxanthin producing mutants of newly isolated Coelastrum sp. using ethyl methane sulfonate induced mutagenesis technique.

Natural astaxanthin is known to be produced by green microalgae, a potent producer of the most powerful antioxidant. To increase the productivity of astaxanthin in microalgae, random mutagenesis has been extensively used to improve the yield of valuable substances. In the presented work, a newly isolated Coelastrum sp. was randomly mutagenized by exposure to ethyl methane sulfonate and further screened using two approaches; an approach for high growth mutant and an approach for high astaxanthin producing mutant with a high-throughput screening method using glufosinate. Among these, mutant G1-C1 that was selected using glufosinate showed the highest of total carotenoids (45.48±1.5 mg/L) and astaxanthin (28.32±2.5 mg/L) production, which was almost 2-fold higher than that of wild type. This study indicates that random mutagenesis via chemical mutation strategy and screening using glufosinate successfully expedited astaxanthin production in a mutated strain of a Coelastrum sp.

Immunogenic modulation of cholangiocarcinoma cells by chemoimmunotherapy.

BACKGROUND/AIM: Chemoimmunotherapy has been used to treat intrahepatic cholangiocarcinoma (ICC). However, little is known about the phenomena underlying the immunomodulation of ICC cells elicited by chemoimmunotherapy. MATERIALS AND METHODS: Primary ICC cells from a patient with ICC who received gemcitabine followed by 5-fluorouracil (5-FU), both combined with dendritic cells pulsed with Wilms' tumor 1 (WT1) peptides were cultured. ICC cells were treated with gemcitabine, 5-FU or interferon (IFN)-γ in vitro. The phenotype of the ICC cells was examined by flow cytometry and quantitative reverse transcription polymerase chain reaction. RESULTS: Stimulation of the ICC cells with gemcitabine resulted in up-regulation of WT1 mRNA, programmed death receptor ligand-1 (PDL1) and calreticulin. Gemcitabine, 5-FU and IFN-γ induced up-regulation of mucin-1. Moreover, human leukocyte antigen (HLA)-ABC, HLA-DR and PDL1 were extremely up-regulated by IFN-γ. CONCLUSION: Chemoimmunomodulating agents alter the immunogenicity of ICC cells, resulting in complex clinical efficacy results.

Apoptosis of antigen-specific CTLs contributes to low immune response in gut-associated lymphoid tissue post vaccination.

The gut-associated lymphoid tissue (GALT) represents a major reservoir of HIV in infected individuals. Vaccines can induce strong systemic immune responses but these have less impact on CD4 T cells activity and numbers in GALT. In this study, we vaccinated mice with an adenovirus vector that expressed the envelope gene from HIV and observed immune responses in the peripheral blood, spleen, liver, mesenteric lymph nodes, and Peyer's patches. We found that (1) the number of HIV-specific CD8 T cells was dramatically lower in GALT than in other tissues; (2) the programmed cell death protein-1 (PD-1) was expressed at high levels in HIV-specific CD8 T cells including memory T cells in GALT; and (3) high levels of HIV-specific CD8 T cell apoptosis were occurring in GALT. These results suggest that contributing to GALT becoming an HIV reservoir during infection is a combination of exhaustion and/or dysfunction of HIV-specific CTLs at that site. These results emphasize the importance of developing of an effective mucosal vaccine against HIV.

Treatment with chemotherapy and dendritic cells pulsed with multiple Wilms' tumor 1 (WT1)-specific MHC class I/II-restricted epitopes for pancreatic cancer.

PURPOSE: We performed a phase I trial to investigate the safety, clinical responses, and Wilms' tumor 1 (WT1)-specific immune responses following treatment with dendritic cells (DC) pulsed with a mixture of three types of WT1 peptides, including both MHC class I and II-restricted epitopes, in combination with chemotherapy. EXPERIMENTAL DESIGN: Ten stage IV patients with pancreatic ductal adenocarcinoma (PDA) and 1 patient with intrahepatic cholangiocarcinoma (ICC) who were HLA-positive for A*02:01, A*02:06, A*24:02, DRB1*04:05, DRB1*08:03, DRB1*15:01, DRB1*15:02, DPB1*05:01, or DPB1*09:01 were enrolled. The patients received one course of gemcitabine followed by biweekly intradermal vaccinations with mature DCs pulsed with MHC class I (DC/WT1-I; 2 PDA and 1 ICC), II (DC/WT1-II; 1 PDA), or I/II-restricted WT1 peptides (DC/WT1-I/II; 7 PDA), and gemcitabine. RESULTS: The combination therapy was well tolerated. WT1-specific IFNγ-producing CD4(+) T cells were significantly increased following treatment with DC/WT1-I/II. WT1 peptide-specific delayed-type hypersensitivity (DTH) was detected in 4 of the 7 patients with PDA vaccinated with DC/WT1-I/II and in 0 of the 3 patients with PDA vaccinated with DC/WT1-I or DC/WT1-II. The WT1-specific DTH-positive patients showed significantly improved overall survival (OS) and progression-free survival (PFS) compared with the negative control patients. In particular, all 3 patients with PDA with strong DTH reactions had a median OS of 717 days. CONCLUSIONS: The activation of WT1-specific immune responses by DC/WT1-I/II combined with chemotherapy may be associated with disease stability in advanced pancreatic cancer.

Immunogenic comparison of chimeric adenovirus 5/35 vector carrying optimized human immunodeficiency virus clade C genes and various promoters.

Adenovirus vector-based vaccine is a promising approach to protect HIV infection. However, a recent phase IIb clinical trial using the vector did not show its protective efficacy against HIV infection. To improve the vaccine, we explored the transgene protein expression and its immunogenicity using optimized codon usage, promoters and adaptors. We compared protein expression and immunogenicity of adenovirus vector vaccines carrying native or codon usage-optimized HIV-1 clade C gag and env genes expression cassettes driven by different promoters (CMV, CMVi, and CA promoters) and adapters (IRES and F2A). The adenovirus vector vaccine containing optimized gag gene produced higher Gag protein expression and induced higher immune responses than the vector containing native gag gene in mice. Furthermore, CA promoter generated higher transgene expression and elicited higher immune responses than other two popularly used promoters (CMV and CMVi). The second gene expression using F2A adaptor resulted in higher protein expression and immunity than that of using IRES and direct fusion protein. Taken together, the adenovirus vector containing the expression cassette with CA promoter, optimized HIV-1 clade C gene and an F2A adaptor produced the best protein expression and elicited the highest transgene-specific immune responses. This finding would be promising for vaccine design and gene therapy.

Vaccination with Human Induced Pluripotent Stem Cells Creates an Antigen-Specific Immune Response Against HIV-1 gp160.

Induced pluripotent stem cells (iPSCs) are artificially derived from somatic cells that have been transduced with defined reprogramming factors. A previous report has indicated the possibility of using iPSCs as an immune stimulator to generate antigen-specific immunity. In our current study, we have investigated whether human iPSCs (hiPSCs) have the ability to enhance specific immune response against a human immunodeficiency virus type 1 (HIV-1) antigen in a xenogenic mouse model. Our results show that BALB/c mice immunized with hiPSCs transduced with an adenoviral vector encoding HIV-1 gp160 exhibited prominent antigen-specific cellular immune responses. We further found that pre-treatment of hiPSCs with ionizing radiation promotes the secretion of pro-inflammatory cytokines such as interleukin-1 alpha (IL-1α), IL-12, and IL-18. These cytokines might promote the activation of antigen-presenting cells and the effective induction of cellular immunity. Our present findings thus demonstrate that a hiPSCs-based vaccine has the potential to generate cellular immunity against viral antigens such as HIV-1 gp160 in a xenogenic condition.

Stimulation of human butyrophilin 3 molecules results in negative regulation of cellular immunity.

The BTN molecule consists of three subfamilies, BTN1, BTN2. and BTN3, and possesses interesting properties for biological regulation. Although the biological significance of BTN1 and BTN2 has been progressively clarified, the receptor function of BTN3 remains to be elucidated as a result of the absence of appropriate agonists. To clarify the participation of BTN3 in immune regulation, BTN3-specific mAb, referred to as 34-7 and 232-5, were generated from BTN3 gene-immunized mice. The 232-5 mAb, specific to the extracellular domain of the BTN3 molecule, stained almost all populations of human PBMCs, including T, NK, NKT, and B cells. Notably, treatment with the 232-5 mAb resulted in phosphorylation of BTN3A3 molecules, leading to attenuated proliferation and cytokine secretion by CD4+ and CD8+ T cells in a CD4+ CD25+ Treg cell-independent manner, demonstrating the agonistic property of the 232-5 mAb in BTN3-mediated negative signal transduction. The magnitude of the cell surface expression of BTN3 molecules correlated inversely with lymphocyte activity, suggesting that BTN3 molecules contribute to the maintenance of the immune system. Taken together, our findings provide new insights for the role of BTN3 as an inhibitor of excessive cellular immune responses.

DNA vaccine expressing HIV-1 gp120/immunoglobulin fusion protein enhances cellular immunity.

In this study, we explored the possibility of augmenting human immunodeficiency virus (HIV) gp120-specific cell-mediated immune responses in mice by means of a DNA vaccine encoding a mouse Ig Fcgamma2a fragment fused with gp120 (gp120-Ig, Ig-gp120). Western blotting analysis revealed that the HIV gp120 protein expression efficiency was higher in cells transfected with the gp120-Ig-coding plasmid (pGp120Ig) than in those transfected with the gp120 and Ig-gp120 expression plasmids (pGp120 and pIgGp120, respectively). pGp120Ig elicited more HIV-specific CD8 T cells and effector memory CD8 T cells than pGp120 in immunized mice. Furthermore, pGp120Ig significantly reduced the viral load after challenge with an HIV Env gp160-expressing vaccinia virus. These results demonstrate that covalent antigen modification with an Ig sequence can modulate antigen-specific cellular immune responses. The approach may be useful for vaccine development.

Co-administration of viral vector-based vaccines suppresses antigen-specific effector CD8 T cells.

In this study, we explored immune responses after intramuscular co-administration of the HIV-1 gp160 Env gene-expressing adenovirus (Ad) vector and modified vaccinia virus Ankara (MVA) vector in a mouse model. Surprisingly, the simultaneous vaccination of the two vaccines, either as a mixture or separately, suppressed responses, when compared with the administration of each vaccine separately. Ad vaccine or MVA vaccine, co-administered with a mock MVA or mock Ad vector, also resulted in suppressing HIV-specific effector T-cell responses, and a part of antigen-specific memory T-cell responses. In an in vitro experiment, the two vectors infected individual cells and MVA suppressed the transgene expression produced by the adenovirus vector. This viral interference may involve soluble factor(s), secreted by virus-infected cells. Our study may help in designing a vaccination regimen and in investigating viral interference.

Adenovirus type 5 with modified hexons induces robust transgene-specific immune responses in mice with pre-existing immunity against adenovirus type 5.

BACKGROUND: Adenovirus type 5 (Ad5) is widely used as a vehicle for vaccine delivery in the treatment of infectious disease and cancer. However, the efficacy of Ad5 vectors has been limited in humans because exposure to Ad5 infections results in most adults having neutralizing antibodies against Ad5. To overcome this limitation, the hexon epitope present in the fifth hypervariable region of Ad5 was modified. METHODS: To evaluate the ability of Ad5 vectors encoding the HIV env protein to induce Ag-specific immune responses in the face of pre-existing anti-Ad5 immunity, mice were administrated intramuscularly with the Ad-Luc vector, and then vaccinated with parental or hexon-modified Ad5 vectors (Ad-HisHIV, Ad-END/AAAHIV or Ad-HIV) at week 8. HIV-specific cell-mediated immune responses were detected through a combination of tetramer assays and intracellular cytokine staining from weeks 8-23. RESULTS: The hexon-modified Ad vector was able to escape from anti-Ad5 neutralizing antibody, and mice with the modified vector generated significantly lower individual neutralizing antibody than those immunized with the parental vector. Furthermore, mice with pre-existing anti-Ad immunity immunized with the modified vector generated significantly stronger cell-mediated anti-env responses than those immunized with the parental vector. CONCLUSIONS: These data demonstrate that Ad5 vector with hexon modification reduce their sensitivity to pre-existing anti-Ad immunity and improve their clinical utility.

Designed recombinant adenovirus type 5 vector induced envelope-specific CD8(+) cytotoxic T lymphocytes and cross-reactive neutralizing antibodies against human immunodeficiency virus type 1.

BACKGROUND: A monoclonal antibody (mAb) 2F5 binds to the membrane-proximal external region (MPER) of the transmembrane subunit gp41 of human immunodeficiency virus type 1 (HIV-1) is known to broadly neutralize HIV-1 strains. The Adenovirus type 5 vector (Ad5) has been widely applied for HIV-1 vaccine, and hexon hypervariable region 5 (HVR5) is exposed on viral surface and easily target host immune responses against Ad5. METHODS: We constructed a recombinant adenovirus type 5 vector (rAd5) with a 2F5-binding epitope (ELDKWA) of MPER on Ad5-HVR5. In addition, we developed rAd5 encoding the HIV-1(IIIB) envelope (Env) gene for the induction of Env-specific cellular immunity. RESULTS: The virus titers of the constructed rAd5 were similar to that of the parental Ad5 vector. Furthermore, high-dose immunization of rAd5 induced Env-specific CD8(+) cells and high levels of anti-ELDKWA antibodies. Moreover, an in vitro HIV-1 neutralization assay indicated that ELDKWA-specific mAbs derived from rAd5-immunized mice neutralized a wide range of HIV-1 strains. CONCLUSIONS: The present study outlines the development of an Ad5-based HIV-1 vaccine targeting the hypervariable regions of Ad5. The constructed rAd5 induced an HIV-1-specific cellular immune response and neutralizing antibodies against various strains of HIV-1 simultaneously.

Preparation of cyanoacrylate nanoparticles using monosaccharides or disaccharides.

By using biomaterials, including monosaccharides and disaccharides, as polymeric stabilizing agents, the cyanoacrylate nanoparticles was prepared respectively, with particles diameter of approximately 200 nm or 300 nm. The new method was applied to load/encapsulate ampicillin (ABPC) and pDNA into nanoparticles. Loading efficiency of ABPC was increased compared to the existing method in which dextran is used as a stabilizer. The pDNA encapusulation rate was 68.7%, by using glucose.

The role of UvrD in RecET-mediated illegitimate recombination in Escherichia coli.

To study the mechanism of RecET-mediated illegitimate recombination, we examined the formation of lambdabio-transducing phage in Escherichia coli in the presence or absence of UV irradiation. We have previously reported that coexpression of RecE and RecT enhances the frequency of recA-independent illegitimate recombination. RecJOR proteins are required for this RecET-mediated illegitimate recombination, and RecQ suppresses it. Here, we showed that the frequencies of both spontaneous and UV-induced RecET-mediated illegitimate recombination events are reduced by a uvrD mutation. It should be noted that UvrD is required for illegitimate recombination only in the presence, but not in the absence, of RecET. In contrast, frequencies of RecET-mediated illegitimate recombination were not affected by ruvAB, ruvC, recG, and recN mutations. The frequency of spontaneous and UV-induced illegitimate recombination in the uvrD recR double mutant was comparable to that of the uvrD single mutant, suggesting that UvrD works at the same step as RecR in the RecET-mediated recombination pathway. Nucleotide sequence analyses of the recombination junctions showed that RecET-mediated illegitimate recombination detected in UvrD-deficient strain is short-homology-dependent. Based on these and previous results, we propose a model for the role of UvrD on RecET-mediated illegitimate recombination.

Rep helicase suppresses short-homology-dependent illegitimate recombination in Escherichia coli.

To study roles of Rep helicase in short-homology-dependent illegitimate recombination, we examined the effect of a rep mutation on illegitimate recombination and found that the frequency of spontaneous illegitimate recombination is enhanced by the rep mutation. In addition, illegitimate recombination was synergistically enhanced by the rep mutation and UV irradiation, showing that Rep helicase plays a role in suppression of spontaneous as well as UV-induced illegitimate recombination. The defect in RecQ helicase also has a synergistic effect on the increased illegitimate recombination in the rep mutant. It was also found that the illegitimate recombination induced by the rep mutation is independent of the RecA function with or without UV irradiation. Nucleotide sequence analyses of the recombination junctions showed that the illegitimate recombination induced by the rep mutation mostly takes place between short homologous sequences. Based on the fact that the defect of Rep helicase induces replication arrest during replication, resulting in the formation of DNA double-strand breaks, we propose a model for illegitimate recombination, in which double-strand breaks caused by defect of Rep helicase promotes illegitimate recombination via short-homology-dependent-end-joining. In addition, the mechanism of synergistic action between the rep mutation and UV irradiation on illegitimate recombination is discussed.