Toshihiro Horii

Toll-like receptor 9 mediates innate immune activation by the malaria pigment hemozoin

Malaria parasites within red blood cells digest host hemoglobin into a hydrophobic heme polymer, known as hemozoin (HZ), which is subsequently released into the blood stream and then captured by and concentrated in the reticulo-endothelial system. Accumulating evidence suggests that HZ is immunologically active, but the molecular mechanism(s) through which HZ modulates the innate immune system has not been elucidated. This work demonstrates that HZ purified from Plasmodium falciparum is a novel non-DNA ligand for Toll-like receptor (TLR)9. HZ activated innate immune responses in vivo and in vitro, resulting in the production of cytokines, chemokines, and up-regulation of costimulatory molecules. Such responses were severely impaired in TLR9−/− and myeloid differentiation factor 88 (MyD88)−/−, but not in TLR2, TLR4, TLR7, or Toll/interleukin 1 receptor domain–containing adaptor-inducing interferon β−/− mice. Synthetic HZ, which is free of the other contaminants, also activated innate immune responses in vivo in a TLR9-dependent manner. Chloroquine (CQ), an antimalarial drug, abrogated HZ-induced cytokine production. These data suggest that TLR9-mediated, MyD88-dependent, and CQ-sensitive innate immune activation by HZ may play an important role in malaria parasite–host interactions.

Protective Role of CD40 in Leishmania major Infection at Two Distinct Phases of Cell-Mediated Immunity

CD40-deficient mice are susceptible to Leishmania major infection while their wild-type littermates can resolve the infection. Upon stimulation with L. major antigens, draining lymph node T cells of the mutant mice and the susceptible mice, BALB/c, secrete comparable amounts of IL-4. The mutant mice produce less IFN gamma than wild-type mice. The expression of IL-12 p40 mRNA was significantly lower in L. major antigen-stimulated cells of mutant mice than those of wild-type or BALB/c mice. In normal mice, engagement of CD40 activates macrophages to a leishmanicidal state in vitro in the presence of IFN gamma. The results suggest that the CD40-CD40 ligand interaction plays an important role in two critical steps of cell-mediated immunity to L. major infection: the generation of a Th1 response and activation of macrophages to a leishmanicidal state.

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.

Phase I/IIa Safety, Immunogenicity, and Efficacy Trial of NYVAC-Pf7, a Pox-Vectored, Multiantigen, Multistage Vaccine Candidate for Plasmodium falciparum Malaria

Candidate malaria vaccines have failed to elicit consistently protective immune responses against challenge with Plasmodium falciparum. NYVAC-Pf7, a highly attenuated vaccinia virus with 7 P. falciparum genes inserted into its genome, was tested in a phase I/IIa safety, immunogenicity, and efficacy vaccine trial in human volunteers. Malaria genes inserted into the NYVAC genome encoded proteins from all stages of the parasites life cycle. Volunteers received three immunizations of two different dosages of NYVAC-Pf7. The vaccine was safe and well tolerated but variably immunogenic. While antibody responses were generally poor, cellular immune responses were detected in >90% of the volunteers. Of the 35 volunteers challenged with the bite of 5 P. falciparum-infected Anopheles mosquitoes, 1 was completely protected, and there was a significant delay in time to parasite patency in the groups of volunteers who received either the low or high dose of vaccine compared with control volunteers.

Regulation of SOS functions: Purification of E. coli LexA protein and determination of its specific site cleaved by the RecA protein

The LexA protein of Escherichia coli was purified to more than 96% purity from cells harboring a recombinant plasmid carrying the lexA gene with the lacZ promoter sequence. The amino acid composition of the LexA protein and its amino-terminal sequence were analyzed. The results are in agreement with the prediction from the nucleotide sequence of the lexA gene. The LexA protein is cleaved into two polypeptides by E. coli RecA protein in the presence of ATP and single-stranded DNA. The site of the specific cleavage was determined by analyzing amino acid sequences of the cleaved products at the amino and carboxyl termini. The cleavage of the LexA protein by the RecA protein was found to occur at a single site between Ala84 and Gly85.

Immunogenicity of whole-parasite vaccines against Plasmodium falciparum involves malarial hemozoin and host TLR9.

Although whole-parasite vaccine strategies for malaria infection have regained attention, their immunological mechanisms of action remain unclear. We find that immunization of mice with a crude blood stage extract of the malaria parasite Plasmodium falciparum elicits parasite antigen-specific immune responses via Toll-like receptor (TLR) 9 and that the malarial heme-detoxification byproduct, hemozoin (HZ), but not malarial DNA, produces a potent adjuvant effect. Malarial and synthetic (s)HZ bound TLR9 directly to induce conformational changes in the receptor. The adjuvant effect of sHZ depended on its method of synthesis and particle size. Although natural HZ acts as a TLR9 ligand, the adjuvant effects of synthetic HZ are independent of TLR9 or the NLRP3-inflammasome but are dependent on MyD88. The adjuvant function of sHZ was further validated in a canine antiallergen vaccine model. Thus, HZ can influence adaptive immune responses to malaria infection and may have therapeutic value in vaccine adjuvant development.

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.

Plasmodium falciparum Accompanied the Human Expansion out of Africa

Plasmodium falciparum is distributed throughout the tropics and is responsible for an estimated 230 million cases of malaria every year, with a further 1.4 billion people at risk of infection. Little is known about the genetic makeup of P. falciparum populations, despite variation in genetic diversity being a key factor in morbidity, mortality, and the success of malaria control initiatives. Here we analyze a worldwide sample of 519 P. falciparum isolates sequenced for two housekeeping genes (63 single nucleotide polymorphisms from around 5000 nucleotides per isolate). We observe a strong negative correlation between within-population genetic diversity and geographic distance from sub-Saharan Africa (R(2) = 0.95) over Africa, Asia, and Oceania. In contrast, regional variation in transmission intensity seems to have had a negligible impact on the distribution of genetic diversity. The striking geographic patterns of isolation by distance observed in P. falciparum mirror the ones previously documented in humans and point to a joint sub-Saharan African origin between the parasite and its host. Age estimates for the expansion of P. falciparum further support that anatomically modern humans were infected prior to their exit out of Africa and carried the parasite along during their colonization of the world.

Metagenomic Diagnosis of Bacterial Infections

To test the ability of high-throughput DNA sequencing to detect bacterial pathogens, we used it on DNA from a patient’s feces during and after diarrheal illness. Sequences showing best matches for Campylobacter jejuni were detected only in the illness sample. Various bacteria may be detectable with this metagenomic approach.