Takahiro Tougan

Direct Metagenomic Detection of Viral Pathogens in Nasal and Fecal Specimens Using an Unbiased High-Throughput Sequencing Approach

With the severe acute respiratory syndrome epidemic of 2003 and renewed attention on avian influenza viral pandemics, new surveillance systems are needed for the earlier detection of emerging infectious diseases. We applied a “next-generation” parallel sequencing platform for viral detection in nasopharyngeal and fecal samples collected during seasonal influenza virus (Flu) infections and norovirus outbreaks from 2005 to 2007 in Osaka, Japan. Random RT-PCR was performed to amplify RNA extracted from 0.1–0.25 ml of nasopharyngeal aspirates (N = 3) and fecal specimens (N = 5), and more than 10 µg of cDNA was synthesized. Unbiased high-throughput sequencing of these 8 samples yielded 15,298–32,335 (average 24,738) reads in a single 7.5 h run. In nasopharyngeal samples, although whole genome analysis was not available because the majority (>90%) of reads were host genome–derived, 20–460 Flu-reads were detected, which was sufficient for subtype identification. In fecal samples, bacteria and host cells were removed by centrifugation, resulting in gain of 484–15,260 reads of norovirus sequence (78–98% of the whole genome was covered), except for one specimen that was under-detectable by RT-PCR. These results suggest that our unbiased high-throughput sequencing approach is useful for directly detecting pathogenic viruses without advance genetic information. Although its cost and technological availability make it unlikely that this system will very soon be the diagnostic standard worldwide, this system could be useful for the earlier discovery of novel emerging viruses and bioterrorism, which are difficult to detect with conventional procedures.

Plasmodium cynomolgi genome sequences provide insight into Plasmodium vivax and the monkey malaria clade

P. cynomolgi, a malaria-causing parasite of Asian Old World monkeys, is the sister taxon of P. vivax, the most prevalent malaria-causing species in humans outside of Africa. Because P. cynomolgi shares many phenotypic, biological and genetic characteristics with P. vivax, we generated draft genome sequences for three P. cynomolgi strains and performed genomic analysis comparing them with the P. vivax genome, as well as with the genome of a third previously sequenced simian parasite, Plasmodium knowlesi. Here, we show that genomes of the monkey malaria clade can be characterized by copy-number variants (CNVs) in multigene families involved in evasion of the human immune system and invasion of host erythrocytes. We identify genome-wide SNPs, microsatellites and CNVs in the P. cynomolgi genome, providing a map of genetic variation that can be used to map parasite traits and study parasite populations. The sequencing of the P. cynomolgi genome is a critical step in developing a model system for P. vivax research and in counteracting the neglect of P. vivax.

Plasmacytoid Dendritic Cells Delineate Immunogenicity of Influenza Vaccine Subtypes

Rare, circulating dendritic cells differentially shape the immunogenicity mechanisms for protection against H1N1 influenza. Eschewing the Flu “Know thyself” is a maxim said to have been inscribed in the temple of Apollo at Delphi, and is viewed as an embodiment of ancient Greek thought. Though obviously referring to self-knowledge in a philosophical sense, this aphorism applies to our bodies, too, which must distinguish “self” from “nonself” and healthy tissue from a wide range of pathogens. To accomplish this feat, the immune system deploys a complex system of cells, receptors, and signaling molecules. For instance, dendritic cells (DCs) act as sentinels, constantly surveying the body for invaders like viruses and bacteria. These cells detect specific pathogen-associated molecules through a variety of different pattern recognition receptors; once such a molecule binds to an appropriate receptor, anti-pathogen responses—like interferon secretion—are triggered. In addition, mature DCs present pathogen proteins to T and B cells, thereby activating these immune cells. Such responses are also invoked, of course, by vaccination, but exactly which mechanisms are induced by different vaccines isn’t necessarily clear. Now, Ishii and colleagues examine the responses generated by vaccines designed to provide protection against H1N1 influenza A. Flu vaccines come in three general forms. Live attenuated vaccines consist of a weakened form of the virus; the nasal spray FluMist vaccine is an example. Most flu vaccines in use today, however, are inactivated. Whole-virus inactivated vaccines consist of viruses that have been killed with heat or chemical treatment, whereas split-virus forms—the most commonly used type—contain some fraction of the whole virus (for instance, the viral surface protein hemagglutinin that binds the virus to the cell being infected). Do these types of vaccines engage the immune system through different mechanisms? Ishii and co-workers addressed this question by using strains of mice lacking individual immune system components. They found that mice require signaling mediated by one particular pattern recognition receptor—Toll-like receptor 7 (TLR7), which recognizes viral genomic RNA as its ligand—but not others to mount an immune response to an inactivated whole-virus flu vaccine. TLR7 also functions in the response against live virus. However, type I interferon production by a rare class of circulating and tissue-residing DCs (plasmacytoid DCs, which express TLR7) was required for the response to the inactivated whole-virus vaccine, but not live virus—but only during the initial (rather than secondary) vaccination. Furthermore, a split vaccine often used in humans did not provide immunity to mice never before exposed to the virus, perhaps because the vaccine lacked the viral genomic RNA, the TLR7 ligand. Indeed, the addition of a ligand known to activate a different pattern recognition receptor (TLR9) on plasmacytoid DCs improved the split vaccine’s performance. In blood from adult humans (presumably previously exposed to seasonal flu virus), though, the split vaccine caused an immune response, again indicating that the plasmacytoid DC pathway is important for initiating but not boosting the immune response. Together, these studies provide information about basic vaccine biology, as well as possible routes for improving vaccine efficacy. A variety of different vaccine types are available for H1N1 influenza A virus infections; however, their immunological mechanisms of action remain unclear. Here, we show that plasmacytoid dendritic cells (pDCs) and type I interferon (IFN)–mediated signaling delineate the immunogenicity of live attenuated virus, inactivated whole-virus (WV), and split-virus vaccines. Although Toll-like receptor 7 acted as the adjuvant receptor for the immunogenicity of both live virus and WV vaccines, the requirement for type I IFN production by pDCs for the immunogenicity of the vaccines was restricted to WV. A split vaccine commonly used in humans failed to immunize naïve mice, but a pDC-activating adjuvant could restore immunogenicity. In blood from human adults, however, split vaccine alone could recall memory T cell responses, underscoring the importance of this adjuvant pathway for primary, but not secondary, vaccination.

Transcutaneous immunization using a dissolving microneedle array protects against tetanus, diphtheria, malaria, and influenza.

Transcutaneous immunization (TCI) is an attractive alternative vaccination route compared to the commonly used injection systems. We previously developed a dissolving microneedle array for use as a TCI device, and reported that TCI with the dissolving microneedle array induced an immune response against model antigens. In the present study, we investigated the vaccination efficacy against tetanus and diphtheria, malaria, and influenza using this vaccination system. Our TCI system induced substantial increases in toxoid-specific IgG levels and toxin-neutralizing antibody titer and induced the production of anti-SE36 IgG, which could bind to malaria parasite. On influenza HA vaccination, robust antibody production was elicited in mice that provided complete protection against a subsequent influenza virus challenge. These findings demonstrate that TCI using a dissolving microneedle array can elicit large immune responses against infectious diseases. Based on these results, we are now preparing translational research for human clinical trials.

The meiotic recombination checkpoint is regulated by checkpoint rad+ genes in fission yeast

During the course of meiotic prophase, intrinsic double‐strand breaks (DSBs) must be repaired before the cell can engage in meiotic nuclear division. Here we investigate the mechanism that controls the meiotic progression in Schizosaccharomyces pombe that have accumulated excess meiotic DSBs. A meiotic recombination‐defective mutant, meu13Δ, shows a delay in meiotic progression. This delay is dependent on rec12+, namely on DSB formation. Pulsed‐field gel electrophoresis analysis revealed that meiotic DSB repair in meu13Δ was retarded. We also found that the delay in entering nuclear division was dependent on the checkpoint rad+, cds1+ and mek1+ (the meiotic paralog of Cds1/Chk2). This implies that these genes are involved in a checkpoint that provides time to repair DSBs. Consistently, the induction of an excess of extrinsic DSBs by ionizing radiation delayed meiotic progression in a rad17+‐dependent manner. dmc1Δ also shows meiotic delay, however, this delay is independent of rec12+ and checkpoint rad+. We propose that checkpoint monitoring of the status of meiotic DSB repair exists in fission yeast and that defects other than DSB accumulation can cause delays in meiotic progression.

Meu10 is required for spore wall maturation in Schizosaccharomyces pombe

Background: Many genes are meiosis and/or sporulation‐specifically transcribed during this process. Isolation and analysis of these genes might help us to understand how meiosis and sporulation are regulated. For this purpose, we have isolated a large number of cDNA clones from Schizosaccharomyces pombe whose expression is up‐regulated during meiosis.

Phase 1b randomized trial and follow-up study in Uganda of the blood-stage malaria vaccine candidate BK-SE36.

Background Up to now a malaria vaccine remains elusive. The Plasmodium falciparum serine repeat antigen-5 formulated with aluminum hydroxyl gel (BK-SE36) is a blood-stage malaria vaccine candidate that has undergone phase 1a trial in malaria-naive Japanese adults. We have now assessed the safety and immunogenicity of BK-SE36 in a malaria endemic area in Northern Uganda. Methods We performed a two-stage, randomized, single-blinded, placebo-controlled phase 1b trial (Current Controlled trials ISRCTN71619711). A computer-generated sequence randomized healthy subjects for 2 subcutaneous injections at 21-day intervals in Stage1 (21–40 year-olds) to 1-mL BK-SE36 (BKSE1.0) (n = 36) or saline (n = 20) and in Stage2 (6–20 year-olds) to BKSE1.0 (n = 33), 0.5-mL BK-SE36 (BKSE0.5) (n = 33), or saline (n = 18). Subjects and laboratory personnel were blinded. Safety and antibody responses 21-days post-second vaccination (Day42) were assessed. Post-trial, to compare the risk of malaria episodes 130–365 days post-second vaccination, Stage2 subjects were age-matched to 50 control individuals. Results Nearly all subjects who received BK-SE36 had induration (Stage1, n = 33, 92%; Stage2, n = 63, 96%) as a local adverse event. No serious adverse event related to BK-SE36 was reported. Pre-existing anti-SE36 antibody titers negatively correlated with vaccination-induced antibody response. At Day42, change in antibody titers was significant for seronegative adults (1.95-fold higher than baseline [95% CI, 1.56–2.43], p = 0.004) and 6–10 year-olds (5.71-fold [95% CI, 2.38–13.72], p = 0.002) vaccinated with BKSE1.0. Immunogenicity response to BKSE0.5 was low and not significant (1.55-fold [95% CI, 1.24–1.94], p = 0.75). In the ancillary analysis, cumulative incidence of first malaria episodes with ≥5000 parasites/µL was 7 cases/33 subjects in BKSE1.0 and 10 cases/33 subjects in BKSE0.5 vs. 29 cases/66 subjects in the control group. Risk ratio for BKSE1.0 was 0.48 (95% CI, 0.24–0.98; p = 0.04). Conclusion BK-SE36 is safe and immunogenic. The promising potential of BK-SE36, observed in the follow-up study, warrants a double-blind phase 1/2b trial in children under 5 years. Trial Registration Controlled-Trials.com ISRCTN71619711 ISRCTN71619711

TLR9 adjuvants enhance immunogenicity and protective efficacy of the SE36/AHG malaria vaccine in nonhuman primate models

The SE36 antigen, derived from serine repeat antigen 5 (SERA5) of Plasmodium falciparum, is a promising blood stage malaria vaccine candidate. Ongoing clinical trials suggest the efficacy of the SE36 vaccine could be increased by the incorporation of more effective adjuvants into the vaccine formulation. In this study, we assessed the safety, immunogenicity and protective efficacy of SE36/AHG formulated with TLR9 ligand adjuvants K3 CpG oligodeoxyribonucleotides (CpG ODNs) (K3 ODN), D3 ODN or synthetic hemozoin, in two non-human primate models. SE36/AHG with or without each adjuvant was administrated to cynomolgus monkeys. A combination of TLR9 ligand adjuvant with SE36/AHG induced higher humoral and cellular immune response compared with SE36/AHG alone. Administration of a crude extract of P. falciparum parasite resulted in the induction of more SE36-specific IgG antibodies in monkeys vaccinated with a combination of SE36/AHG and adjuvant, as opposed to vaccination with SE36/AHG alone. The most effective TLR9 ligand, K3 ODN, was chosen for further vaccine trials in squirrel monkeys, in combination with SE36/AHG. All monkeys immunized with the combined SE36/AHG and K3 ODN formulation effectively suppressed parasitemia and symptoms of malaria following challenge infections. Furthermore, no serious adverse events were observed. Our results show that the novel vaccine formulation of K3 ODN with SE36/AHG demonstrates safety, potent immunogenicity and efficacy in nonhuman primates, and this vaccine formulation may form the basis of a more effective malaria vaccine.

Plasmodium falciparum serine repeat antigen 5 (SE36) as a malaria vaccine candidate.

A devastating disease spread by mosquitoes with high-efficiency, malaria imposes an enormous burden for which no licensed vaccine currently exists. Although the genome complexity of the parasite has made vaccine development tenuous, an effective malaria vaccine would be a valuable tool for control, elimination and eventual eradication. The Plasmodium serine repeat antigen 5 (SERA5) is an abundant asexual blood stage antigen that does not show any antigenic variation and exhibits limited polymorphism, making it a suitable vaccine candidate. Identified by comparing the IgG status of people in endemic areas with protective immunity and those with malaria symptoms, the vaccine potential of the N-terminal domain of Plasmodium falciparum SERA5 is also strongly supported by experimental data and immune responses both measured in vitro and in animal challenge models. The current understanding of SERA5 will be presented, particularly in relation to its path towards clinical development. The review highlights lessons learned and sorts out issues upon which further research efforts are needed.

Mcp6, a meiosis-specific coiled-coil protein of Schizosaccharomyces pombe, localizes to the spindle pole body and is required for horsetail movement and recombination

We report here that a meiosis-specific gene of Schizosaccharomyces pombe denoted mcp6+ (meiotic coiled-coil protein) encodes a protein that is required for the horsetail movement of chromosomes at meiosis I. The mcp6+ gene is specifically transcribed during the horsetail phase. Green fluorescent protein (GFP)-tagged Mcp6 appears at the start of karyogamy, localizes to the spindle-pole body (SPB) and then disappears before chromosome segregation at meiosis I. In the mcp6Δ strain, the horsetail movement was either hampered (zygotic meiosis) or abolished (azygotic meiosis) and the pairing of homologous chromosomes was impaired. Accordingly, the allelic recombination rates of the mcp6Δ strain were only 10-40% of the wild-type rates. By contrast, the ectopic recombination rate of the mcp6Δ strain was twice the wild-type rate. This is probably caused by abnormal homologous pairing in mcp6Δ cells because of aberrant horsetail movement. Fluorescent microscopy indicates that SPB components such as Sad1, Kms1 and Spo15 localize normally in mcp6Δ cells. Because Taz1 and Swi6 also localized with Sad1 in mcp6Δ cells, Mcp6 is not required for telomere clustering. In a taz1Δ strain, which does not display telomere clustering, and the dhc1-d3 mutant, which lacks horsetail movement, Mcp6 localized with Sad1 normally. However, we observed abnormal astral microtubule organization in mcp6Δ cells. From these results, we conclude that Mcp6 is necessary for neither SPB organization nor telomere clustering, but is required for proper astral microtubule positioning to maintain horsetail movement.