Development of a selection marker-free rice-based oral cholera toxin B-subunit vaccine and determination of genomic location and structure the transgenes by using a whole genome resequencing approach

新潟大学博士（農学）学位の種類: 博士（農学）. 報告番号: 甲第4050号. 学位記番号: 新大院博（農）甲第147号. 学位授与年月日: 平成27年3月23日新大院博（農）甲第147号thesi

Comparative whole-genome analyses of selection marker–free rice-based cholera toxin B-subunit vaccine lines and wild-type lines

BACKGROUND: We have developed a rice-based oral cholera vaccine named MucoRice-CTB (Cholera Toxin B-subunit) by using an Agrobacterium tumefaciens–mediated co-transformation system. To assess the genome-wide effects of this system on the rice genome, we compared the genomes of three selection marker–free MucoRice-CTB lines with those of two wild-type rice lines (Oryza sativa L. cv. Nipponbare). Mutation profiles of the transgenic and wild-type genomes were examined by next-generation sequencing (NGS). RESULTS: Using paired-end short-read sequencing, a total of more than 300 million reads for each line were obtained and mapped onto the rice reference genome. The number and distribution of variants were similar in all five lines: the numbers of line-specific variants ranged from 524 to 842 and corresponding mutation rates ranged from 1.41 × 10(−6) per site to 2.28 × 10(−6) per site. The frequency of guanine-to-thymine and cytosine-to-adenine transversions was higher in MucoRice-CTB lines than in WT lines. The transition-to-transversion ratio was 1.12 in MucoRice-CTB lines and 1.65 in WT lines. Analysis of variant-sharing profiles showed that the variants common to all five lines were the most abundant, and the numbers of line-specific variant for all lines were similar. The numbers of non-synonymous amino acid substitutions in MucoRice-CTB lines (15 to 21) were slightly higher than those in WT lines (7 or 8), whereas the numbers of frame shifts were similar in all five lines. CONCLUSIONS: We conclude that MucoRice-CTB and WT are almost identical at the genomic level and that genome-wide effects caused by the Agrobacterium-mediated transformation system for marker-free MucoRice-CTB lines were slight. The comparative whole-genome analyses between MucoRice-CTB and WT lines using NGS provides a reliable estimate of genome-wide differences. A similar approach may be applicable to other transgenic rice plants generated by using this Agrobacterium-mediated transformation system. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-1285-y) contains supplementary material, which is available to authorized users

A novel M cell–specific carbohydrate-targeted mucosal vaccine effectively induces antigen-specific immune responses

Mucosally ingested and inhaled antigens are taken up by membranous or microfold cells (M cells) in the follicle-associated epithelium of Peyer's patches or nasopharynx-associated lymphoid tissue. We established a novel M cell–specific monoclonal antibody (mAb NKM 16–2-4) as a carrier for M cell–targeted mucosal vaccine. mAb NKM 16–2-4 also reacted with the recently discovered villous M cells, but not with epithelial cells or goblet cells. Oral administration of tetanus toxoid (TT)– or botulinum toxoid (BT)–conjugated NKM 16–2-4, together with the mucosal adjuvant cholera toxin, induced high-level, antigen-specific serum immunoglobulin (Ig) G and mucosal IgA responses. In addition, an oral vaccine formulation of BT-conjugated NKM 16–2-4 induced protective immunity against lethal challenge with botulinum toxin. An epitope analysis of NKM 16–2-4 revealed specificity to an α(1,2)-fucose–containing carbohydrate moiety, and reactivity was enhanced under sialic acid–lacking conditions. This suggests that NKM 16–2-4 distinguishes α(1,2)-fucosylated M cells from goblet cells containing abundant sialic acids neighboring the α(1,2) fucose moiety and from non-α(1,2)-fucosylated epithelial cells. The use of NKM 16–2-4 to target vaccine antigens to the M cell–specific carbohydrate moiety is a new strategy for developing highly effective mucosal vaccines

Nanogel-Based PspA Intranasal Vaccine Prevents Invasive Disease and Nasal Colonization by Streptococcus pneumoniae

ABSTRACT To establish a safer and more effective vaccine against pneumococcal respiratory infections, current knowledge regarding the antigens common among pneumococcal strains and improvements to the system for delivering these antigens across the mucosal barrier must be integrated. We developed a pneumococcal vaccine that combines the advantages of pneumococcal surface protein A (PspA) with a nontoxic intranasal vaccine delivery system based on a nanometer-sized hydrogel (nanogel) consisting of a cationic cholesteryl group-bearing pullulan (cCHP). The efficacy of the nanogel-based PspA nasal vaccine (cCHP-PspA) was tested in murine pneumococcal airway infection models. Intranasal vaccination with cCHP-PspA provided protective immunity against lethal challenge with Streptococcus pneumoniae Xen10, reduced colonization and invasion by bacteria in the upper and lower respiratory tracts, and induced systemic and nasal mucosal Th17 responses, high levels of PspA-specific serum immunoglobulin G (IgG), and nasal and bronchial IgA antibody responses. Moreover, there was no sign of PspA delivery by nanogel to either the olfactory bulbs or the central nervous system after intranasal administration. These results demonstrate the effectiveness and safety of the nanogel-based PspA nasal vaccine system as a universal mucosal vaccine against pneumococcal respiratory infection

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

A novel nanosize protein-carrier for the development of adjuvant- free nasal vaccine (39.33)

Abstract Most of inactivated or subunit type vaccines are poor immunogens to both systemic and mucosal immune compartments when given mucosally without mucosal adjuvant. We developed an effective intracellular vaccine delivery system with self-assembled nanosize cationic hyrogels (nanogel), which composed of a polysaccharide with ethylendiamine group of cholesteryl group-bearing pullulan. When hold protein antigen in a segregated nanomatrix, the nanogel acts as artificial chaperones, which protect against the aggregation of denature proteins and assist in the refolding of protein after release. In this study, we demonstrated that nasally administered nanogel holding a subunit type of C. botulinum antigen (BoHc-nanogel) was effectively taken up by nasophalynx-associated lymphoid tissue and induced brisk levels of antigen-specific systemic and mucosal immune responses even in the absence of mucosal adjuvant. In contrast, nasal administration with commercialized cationic liposome (Pro-ject) holding the same amount of BoHc without adjuvant induces antigen-specific low systemic and no mucosal antibody immune responses. In addition, mice nasally immunized with BoHc-nanogel protected not only systemically challenged but also nasally challenged botulinum toxin. These data suggest that nanogel-based mucosal vaccination is a novel strategy for the induction of protective immunity without the use of mucosal adjuvant.</jats:p