伊従 光洋

Among Plasmodium spp. responsible for human malaria, Plasmodium vivax ranks as the second most prevalent and has the widest geographical range; however, vaccine development has lagged behind that of Plasmodium falciparum, the deadliest Plasmodium species. Recently, we developed a multistage vaccine for P. falciparum based on a heterologous prime-boost immunization regimen utilizing the attenuated vaccinia virus strain LC16m8Δ (m8Δ)-prime and adeno-associated virus type 1 (AAV1)-boost, and demonstrated 100% protection and more than 95% transmission-blocking (TB) activity in the mouse model. In this study, we report the feasibility and versatility of this vaccine platform as a P. vivax multistage vaccine, which can provide 100% sterile protection against sporozoite challenge and >95% TB efficacy in the mouse model. Our vaccine comprises m8Δ and AAV1 viral vectors, both harboring the gene encoding two P. vivax circumsporozoite (PvCSP) protein alleles (VK210; PvCSP-Sal and VK247; -PNG) and P25 (Pvs25) expressed as a Pvs25–PvCSP fusion protein. For protective efficacy, the heterologous m8Δ-prime/AAV1-boost immunization regimen showed 100% (short-term; Day 28) and 60% (long-term; Day 242) protection against PvCSP VK210 transgenic Plasmodium berghei sporozoites. For TB efficacy, mouse sera immunized with the vaccine formulation showed >75% TB activity and >95% transmission reduction activity by a direct membrane feeding assay using P. vivax isolates in blood from an infected patient from the Brazilian Amazon region. These findings provide proof-of-concept that the m8Δ/AAV1 vaccine platform is sufficiently versatile for P. vivax vaccine development. Future studies are needed to evaluate the safety, immunogenicity, vaccine efficacy, and synergistic effects on protection and transmission blockade in a non-human primate model for Phase I trials.

Background: To fight the COVID-19 pandemic, immunity against SARS-CoV-2 should be achieved not only through natural infection but also by vaccination. The effect of COVID-19 vaccination on previously infected persons is debatable. Methods: A prospective cohort was undergone to collect sera from unvaccinated survivors and vaccinated persons—with and without COVID-19 pre-infection. The sera were analyzed for the anti-receptor binding domain (RBD) titers by ELISA and for the capacity to neutralize the pseudovirus of the Wuhan-Hu-1 strain by luciferase assays. Results: Neither the antibody titers nor the neutralization capacity was significantly different between the three groups. However, the correlation between the antibody titers and the percentage of viral neutralization derived from sera of unvaccinated survivors was higher than that from vaccinated persons with pre-infection and vaccinated naïve individuals (Spearman correlation coefficient (r) = −0.8558; 95% CI, −0.9259 to −0.7288), p < 0.0001 vs. −0.7855; 95% CI, −0.8877 to −0.6096, p < 0.0001 and −0.581; 95% CI, −0.7679 to −0.3028, p = 0.0002, respectively), indicating the capacity to neutralize the virus is most superior by infection alone. Conclusions: Vaccines induce anti-RBD titers as high as the natural infection with lower neutralization capacity, and it does not boost immunity in pre-infected persons.

Anopheles stephensi is an Asian and Middle Eastern malaria vector, and it has recently spread to the African continent. It is needed to measure how the malaria parasite infection in A. stephensi is influenced by environmental factors to predict its expansion in a new environment. Effects of temperature and food conditions during larval periods on larval mortality, larval period, female wing size, egg production, egg size, adult longevity, and malaria infection rate were studied using a laboratory strain. Larval survival and female wing size were generally reduced when reared at higher temperatures and with a low food supply during the larval period. Egg production was not significantly affected by temperature during the larval period. Egg size was generally smaller in females reared at higher temperatures during the larval period. The infection rate of mosquitoes that fed on blood from malaria-infected mice was not affected by rearing temperature or food conditions during the larval period. Higher temperatures may reduce infection. A. stephensi; however, larger individuals can still be infective. We suggest that routinely recording the body size of adults in field surveys is effective in finding productive larval breeding sites and in predicting malaria risk.

The Malaria Vaccine Technology Roadmap 2013 (World Health Organization) aims to develop safe and effective vaccines by 2030 that will offer at least 75% protective efficacy against clinical malaria and reduce parasite transmission. Here, we demonstrate a highly effective multistage vaccine against both the pre-erythrocytic and sexual stages of Plasmodium falciparum that protects and reduces transmission in a murine model. The vaccine is based on a viral-vectored vaccine platform, comprising a highly-attenuated vaccinia virus strain, LC16m8Δ (m8Δ), a genetically stable variant of a licensed and highly effective Japanese smallpox vaccine LC16m8, and an adeno-associated virus (AAV), a viral vector for human gene therapy. The genes encoding P. falciparum circumsporozoite protein (PfCSP) and the ookinete protein P25 (Pfs25) are expressed as a Pfs25–PfCSP fusion protein, and the heterologous m8Δ-prime/AAV-boost immunization regimen in mice provided both 100% protection against PfCSP-transgenic P. berghei sporozoites and up to 100% transmission blocking efficacy, as determined by a direct membrane feeding assay using parasites from P. falciparum-positive, naturally-infected donors from endemic settings. Remarkably, the persistence of vaccine-induced immune responses were over 7 months and additionally provided complete protection against repeated parasite challenge in a murine model. We propose that application of the m8Δ/AAV malaria multistage vaccine platform has the potential to contribute to the landmark goals of the malaria vaccine technology roadmap, to achieve life-long sterile protection and high-level transmission blocking efficacy.

Viral vectors are a potent vaccine platform for inducing humoral and T-cell immune responses. Among the various viral vectors, replication-competent ones are less commonly used for coronavirus disease 2019 (COVID-19) vaccine development compared with replication-deficient ones. Here, we show the availability of a smallpox vaccine LC16m8Δ (m8Δ) as a replication-competent viral vector for a COVID-19 vaccine. M8Δ is a genetically stable variant of the licensed and highly effective Japanese smallpox vaccine LC16m8. Here, we generated two m8Δ recombinants: one harbouring a gene cassette encoding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein, named m8Δ-SARS2(P7.5-S)-HA; and one encoding the S protein with a highly polybasic motif at the S1/S2 cleavage site, named m8Δ-SARS2(P7.5-SHN)-HA. M8Δ-SARS2(P7.5-S)-HA induced S-specific antibodies in mice that persisted for at least six weeks after a homologous boost immunization. All eight analysed serum samples displayed neutralizing activity against an S-pseudotyped virus at a level similar to that of serum samples from patients with COVID-19, and more than half (5/8) also had neutralizing activity against the Delta/B.1.617.2 variant of concern. Importantly, most serum samples also neutralized the infectious SARS-CoV-2 Wuhan and Delta/B.1.617.2 strains. In contrast, immunization with m8Δ-SARS2(P7.5-SHN)-HA elicited significantly lower antibody titres, and the induced antibodies had less neutralizing activity. Regarding T-cell immunity, both m8Δ recombinants elicited S-specific multifunctional CD8+ and CD4+ T-cell responses even after just a primary immunization. Thus, m8Δ provides an alternative method for developing a novel COVID-19 vaccine.

Hepatocyte infection by malaria sporozoites is a bottleneck in the life-cycle of Plasmodium spp. including P. falciparum, which causes the most lethal form of malaria. Therefore, developing an effective vaccine capable of inducing the strong humoral and cellular immune responses necessary to block the pre-erythrocytic stage has potential to overcome the spatiotemporal hindrances pertaining to parasite biology and hepatic microanatomy. We recently showed that when combined with a human adenovirus type 5 (AdHu5)-priming vaccine, adeno-associated virus serotype 1 (AAV1) is a potent booster malaria vaccine vector capable of inducing strong and long-lasting protective immune responses in a rodent malaria model. Here, we evaluated the protective efficacy of a hepatotropic virus, adeno-associated virus serotype 8 (AAV8), as a booster vector because it can deliver a transgene potently and rapidly to the liver, the organ malaria sporozoites initially infect and multiply in following sporozoite injection by the bite of an infected mosquito. We first generated an AAV8-vectored vaccine expressing P. falciparum circumsporozoite protein (PfCSP). Intravenous (i.v.) administration of AAV8-PfCSP to mice initially primed with AdHu5-PfCSP resulted in a hepatocyte transduction rate ~2.5 times above that seen with intramuscular (i.m.) administration. This immunization regimen provided a better protection rate (100% sterile protection) than that of the i.m. AdHu5-prime/i.m. AAV8-boost regimen (60%, p &amp;lt; 0.05), i.m. AdHu5-prime/i.v. AAV1-boost (78%), or i.m. AdHu5-prime/i.m. AAV1-boost (80%) against challenge with transgenic PfCSP-expressing P. berghei sporozoites. Compared with the i.m. AdHu5-prime/i.v. AAV1-boost regimen, three other regimens induced higher levels of PfCSP-specific humoral immune responses. Importantly, a single i.v. dose of AAV8-PfCSP recruited CD8⁺ T cells, especially resident memory CD8⁺ T cells, in the liver. These data suggest that boost with i.v. AAV8-PfCSP can improve humoral and cellular immune responses in BALB/c mice. Therefore, this regimen holds great promise as a next-generation platform for the development of an effective malaria vaccine.

Intramuscular administration of wild-type baculovirus is able to both protect against Plasmodium sporozoite challenge and eliminate liver-stage parasites via a Toll-like receptor 9-independent pathway. To investigate its effector mechanism(s), the gene expression profile in the liver of baculovirus-administered mice was characterized by cDNA microarray analysis. The ingenuity pathway analysis gene ontology module revealed that the major gene subsets induced by baculovirus were immune-related signaling, such as interferon signaling. A total of 40 genes commonly upregulated in a Toll-like receptor 9-independent manner were included as possible candidates for parasite elimination. This gene subset consisted of NT5C3, LOC105246895, BTC, APOL9a/b, G3BP3, SLC6A6, USP25, TRIM14, and PSMB8 as the top 10 candidates according to the special unit. These findings provide new insight into effector molecules responsible for liver-stage parasite killing and, possibly, the development of a new baculovirus-mediated prophylactic and therapeutic biopharmaceutical for malaria.

Plasmodium falciparum circumsporozoite protein (PfCSP) is the main target antigen in development of pre-erythrocytic malaria vaccines. To evaluate PfCSP vaccines in animal models, challenge by intravenous sporozoite injection is preferentially used. However, in clinical trials, vaccinated human volunteers are exposed to the bites of malaria-infected mosquitoes. In this study, we down-selected Escherichia coli-produced full-length PfCSP (PfCSP-F) and its three truncated PfCSPs based on their abilities to elicit immune response and protection in mice against two challenge models. We showed that immunization with three doses of PfCSP-F elicited high anti-PfCSP antibody titres and 100% protection against the bites of infected mosquitoes. Meanwhile, three-dose truncated PfCSP induced 60%-70% protection after immunization with each truncated PfCSP. Heterologous prime-boost immunization regimen with adenovirus-PfCSP-F and R32LR greatly induced complete protection against intravenous sporozoite injection. Our results suggest that Abs to both anti-repeat and anti-nonrepeat regions induced by PfCSP-F are required to confer complete protection against challenge by the bites of infected mosquitoes, whereas anti-repeat Abs play an important role in protection against intravenous sporozoite injection. Our findings provide a potential clinical application that PfCSP-F vaccine induces potent Abs capable of neutralizing sporozoites in the dermis inoculated by infected mosquitoes and subsequently sporozoites in the blood circulation.

The saliva of hematophagous arthropods is enriched with a complex mixture of antihemostatic molecules, the biological functions of which are largely unknown. Anopheline antiplatelet protein (AAPP) from malaria vector mosquito exhibits strong antiplatelet activity when bound directly to host collagen by its C-terminus and through its N-terminus with Ca2+-binding activity. To investigate the biological functions of AAPP in blood feeding behavior and malaria transmission, we generated transgenic Anopheles stephensi mosquito lines expressing anti-AAPP antibody single-chain fragment (scFv) in their salivary glands. The AAPP-specific collagen-binding activity was completely abolished by AAPP-scFv complex formation in the saliva. Probing and prediuresis time, feeding success, blood meal size, and fecundity, which are all fitness characteristics, were significantly reduced in the transgenic mosquitoes. However, oocysts number in these mosquitoes were not significantly reduced following blood meal intake from Plasmodium berghei-infected mice. These results show that although AAPP plays an important role in mosquito blood feeding, its neutralizing activity did not affect sporogonic development in our laboratory model, but its high fitness cost would pose a survival risk for parasite-infected mosquitoes in nature.

An ideal malaria vaccine platform should potently induce protective immune responses and block parasite transmission from mosquito to human, and it should maintain these effects for an extended period. Here, we have focused on vaccine development based on adeno-associated virus serotype 1 (AAV1), a viral vector widely studied in the field of clinical gene therapy that is able to induce long-term transgene expression without causing toxicity in vivo. Our results show the potential utility of AAV1 vectors as an extremely potent booster vaccine to induce durable immunity when combined with an adenovirus-priming vaccine in a rodent malaria model. We generated a series of recombinant AAV1s and human adenovirus type 5 (AdHu5) expressing either Plasmodium falciparum circumsporozoite protein (PfCSP) or P25 (Pfs25) protein. Heterologous two-dose immunization with an AdHu5-prime and AAV1-boost (AdHu5-AAV1) elicited robust and durable PfCSP- or Pfs25-specific functional antibodies over 280 days. Regarding protective efficacy, AdHu5-AAV1 PfCSP achieved high sterile protection (up to 80% protection rate) against challenge with transgenic Plasmodium berghei sporozoites expressing PfCSP. When examining transmission-blocking (TB) efficacy, we found that immunization with AdHu5-AAV1 Pfs25 maintained TB activity in vivo against transgenic P. berghei expressing Pfs25 for 287 days (99% reduction in oocyst intensity, 85% reduction in oocyst prevalence). Our data indicate that AAV1-based malaria vaccines can confer potent and durable protection as well as TB efficacy when administered following an AdHu5 priming vaccine, supporting the further evaluation of this regimen in clinical trials as a next-generation malaria vaccine platform.

Malaria parasites undergo several stages in their complex lifecycle. To achieve reductions in both the individual disease burden and malaria transmission within communities, a multi-stage malaria vaccine with high effectiveness and durability is a more efficacious strategy compared with a single-stage vaccine. Here, we generated viral-vectored vaccines based on human adenovirus type 5 (AdHu5) and adeno-associated virus serotype 1 (AAV1) expressing a fusion protein of the pre-erythrocytic stage Plasmodium falciparum circumsporozoite protein (PfCSP) and the transmission-blocking sexual stage P25 protein (Pfs25). A two-dose heterologous AdHu5-prime/AAV1-boost immunization regimen proved to be highly effective for both full protection and transmission-blocking activity against transgenic P. berghei parasites expressing the corresponding P. falciparum antigens in mice. Remarkably, the immunization regimen induced antibody responses to both PfCSP and Pfs25 for over 9 months after the boosting and also maintained high levels of transmission-reducing activity (TRA: >99%) during that period, as evaluated by a direct feeding assay. If similar efficacies on P. falciparum can be shown following vaccination of humans, we propose that this multi-stage malaria vaccine regimen will be a powerful tool for malaria control, providing greater overall protection and cost-effectiveness than single-stage vaccines.

Baculovirus (BV), an enveloped insect virus with a circular dsDNA genome, possesses unique characteristics that induce strong innate immune responses in mammalian cells. In this study, we show that BV administration in BALB/c mice not only provides complete protection against a subsequent Plasmodium berghei sporozoite infection for up to 7 d after the injection but also eliminates existing liver-stage parasites completely. The elimination of sporozoites by BV was superior to that by primaquine, and this effect occurred in a TLR9-independent manner. At 6 h after BV administration, IFN-α and IFN-γ were robustly produced in the serum, and RNA transcripts of IFN-stimulated genes were markedly upregulated in the liver compared with control mice. The in vivo passive transfer of serum after BV administration effectively eliminated liver-stage parasites, and IFN-α neutralization abolished this effect, indicating that the BV liver-stage parasite-killing mechanism is downstream of the type I IFN signaling pathway. These findings provide evidence that BV-induced, fast-acting innate immunity completely kills liver-stage parasites and, thus, may lead to new malaria drug and vaccine strategies.

With the increasing prevalence of artemisinin-resistant malaria parasites, a highly efficacious and durable vaccine for malaria is urgently required. We have developed an experimental virus-vectored vaccine platform based on an envelope-modified baculovirus dual-expression system (emBDES). Here, we show a conceptually new vaccine platform based on an adenovirus-prime/emBDES-boost heterologous immunization regimen expressing the Plasmodium falciparum circumsporozoite protein (PfCSP). A human adenovirus 5-prime/emBDES-boost heterologous immunization regimen consistently achieved higher sterile protection against transgenic P. berghei sporozoites expressing PfCSP after a mosquito-bite challenge than reverse-ordered or homologous immunization. This high protective efficacy was also achieved with a chimpanzee adenovirus 63-prime/emBDES-boost heterologous immunization regimen against an intravenous sporozoite challenge. Thus, we show that the adenovirus-prime/emBDES-boost heterologous immunization regimen confers sterile protection against sporozoite challenge by two individual routes, providing a promising new malaria vaccine platform for future clinical use.

Interleukin-12 (IL-12) plays an important role in antigen-specific adaptive immunity against Plasmodium sporozoites, and this requirement allows for a new approach to developing an effective malaria vaccine. In this study, we examined whether IL-12 could enhance protective efficacy of a baculovirus-based malaria vaccine. For this aim, a baculoviral vector expressing murine IL-12 (mIL-12) under the control of CMV promoter (BES-mIL-12-Spider) and a baculoviral vector expressing Plasmodium falciparum circumsporozoite protein (PfCSP) with post-transcriptional regulatory element of woodchuck hepatitis virus (BDES-sPfCSP2-WPRE-Spider) were generated. BES-mIL-12-Spider produced bioactive IL-12 which activates splenocytes, resulting in induction of IFN-γ. When co-immunized with BES-mIL-12-Spider and BDES-sPfCSP2-WPRE-Spider, the mouse number for high IgG2a/IgG1 ratios and the geometric mean in this group were both increased as compared with those of the other groups, indicating a shift towards a Th1-type response following immunization with BES-mIL-12-Spider. Finally, immunization with BDES-sPfCSP2-WPRE-Spider plus BES-mIL-12-Spider had a higher protective efficacy (73%) than immunization with BDES-sPfCSP2-WPRE-Spider alone (30%) against challenge with transgenic Plasmodium berghei sporozoites expressing PfCSP. These results suggest that co-administration of IL-12 expressing baculoviral vector, instead of IL-12 cDNA, with viral-vectored vaccines provides a new feasible vaccine platform to enhance Th1-type cellular immune responses against Plasmodium parasites.

BACKGROUND: Previous studies have shown that the baculovirus-vectored vaccine based on the "baculovirus dual expression system (BDES)" is an effective vaccine delivery platform for malaria. However, a point of weakness remaining for use of this vaccine platform in vivo concerns viral inactivation by serum complement. In an effort to achieve complement resistance, the gene encoding the human decay-accelerating factor (hDAF) was incorporated into the BDES malaria vaccine expressing the Plasmodium falciparum circumsporozoite protein (PfCSP). RESULTS: The newly-developed BDES vaccine, designated BDES-sPfCSP2-Spider, effectively displayed hDAF and PfCSP on the surface of the viral envelope, resulting in complement resistance both in vitro and in vivo. Importantly, upon intramuscular inoculation into mice, the BDES-sPfCSP2-Spider vaccine had a higher protective efficacy (60%) than that of the control vaccine BDES-sPfCSP2-Spier (30%) against challenge with transgenic Plasmodium berghei sporozoites expressing PfCSP. CONCLUSION: DAF-shielded BDES-vaccines offer great potential for development as a new malaria vaccine platform against the sporozoite challenge.

BACKGROUND: The approach of using transgenic rodent malaria parasites to assess the immune system's response to antigenic targets from a human malaria parasite has been shown to be useful for preclinical evaluation of new vaccine formulations. The transgenic Plasmodium berghei parasite line [PvCSP(VK210)/Pb] generated previously expresses the full-length circumsporozoite protein (CSP) VK210 from Plasmodium vivax. The transgenic parasite expresses one of the two most common alleles of CSP, defined by nine amino acids at the central repeat region of this protein. In the present study, a transgenic P. berghei parasite line [PvCSP(VK247)/Pb] expressing the full-length PvCSP(VK247), which is the alternative common allele, was generated and characterized. METHODS: The P. berghei expressing full-length PvCSP(VK247) was generated and examined its applicability to CSP-based vaccine research by examining its biological characteristics in mosquitoes and mice. RESULTS: Similar to PvCSP(VK210)/Pb, PvCSP(VK247)/Pb developed normally in mosquitoes and produced infectious sporozoites equipped to generate patent infections in mice. Invasion of HepG2 cells by PvCSP(VK247)/Pb sporozoites was inhibited by an anti-PvCSP(VK247) repeat monoclonal antibody (mAb), but not by an anti-PvCSP(VK210) repeat mAb. CONCLUSIONS: These two transgenic parasites thus far can be used to evaluate the potential efficacy of PvCSP-based vaccine candidates encompassing the two major genetic variants in preclinical trials.

Anti-malarial transmission-blocking vaccines (TBVs) aim to inhibit the transmission of Plasmodium from humans to mosquitoes by targeting the sexual/ookinete stages of the parasite. Successful use of such interventions will subsequently result in reduced cases of malarial infection within a human population, leading to local elimination. There are currently only five lead TBV candidates under examination. There is a consequent need to identify novel antigens to allow the formulation of new potent TBVs. Here we describe the design and evaluation of a potential TBV (BDES-PbPSOP12) targeting Plasmodium berghei PSOP12 based on the baculovirus dual expression system (BDES), enabling expression of antigens on the surface of viral particles and within infected mammalian cells. In silico studies have previously suggested that PSOP12 (Putative Secreted Ookinete Protein 12) is expressed within the sexual stages of the parasite (gametocytes, gametes and ookinetes), and is a member of the previously characterized 6-Cys family of plasmodial proteins. We demonstrate that PSOP12 is expressed within the sexual/ookinete forms of the parasite, and that sera obtained from mice immunized with BDES-PbPSOP12 can recognize the surface of the male and female gametes, and the ookinete stages of the parasite. Immunization of mice with BDES-PbPSOP12 confers modest but significant transmission-blocking activity in vivo by active immunization (53.1% reduction in oocyst intensity, 10.9% reduction in oocyst prevalence). Further assessment of transmission-blocking potency ex vivo shows a dose-dependent response, with up to a 76.4% reduction in intensity and a 47.2% reduction in prevalence observed. Our data indicates that PSOP12 in Plasmodium spp. could be a potential new TBV target candidate, and that further experimentation to examine the protein within human malaria parasites would be logical.

A multistage malaria vaccine targeting the pre-erythrocytic and sexual stages of Plasmodium could effectively protect individuals against infection from mosquito bites and provide transmission-blocking (TB) activity against the sexual stages of the parasite, respectively. This strategy could help prevent malaria infections in individuals and, on a larger scale, prevent malaria transmission in communities of endemicity. Here, we describe the development of a multistage Plasmodium vivax vaccine which simultaneously expresses P. vivax circumsporozoite protein (PvCSP) and P25 (Pvs25) protein of this species as a fusion protein, thereby acting as a pre-erythrocytic vaccine and a TB vaccine, respectively. A new-concept vaccine platform based on the baculovirus dual-expression system (BDES) was evaluated. The BDES-Pvs25-PvCSP vaccine displayed correct folding of the Pvs25-PvCSP fusion protein on the viral envelope and was highly expressed upon transduction of mammalian cells in vitro. This vaccine induced high levels of antibodies to Pvs25 and PvCSP and elicited protective (43%) and TB (82%) efficacies against transgenic P. berghei parasites expressing the corresponding P. vivax antigens in mice. Our data indicate that our BDES, which functions as both a subunit and DNA vaccine, can offer a promising multistage vaccine capable of delivering a potent antimalarial pre-erythrocytic and TB response via a single immunization regimen.

Blood clotting is a vitally important process that must be carefully regulated to prevent blood loss on one hand and thrombosis on the other. Severe injury and hemophilia may be treated with pro-coagulants, whereas risk of obstructive clotting or embolism may be reduced with anti-coagulants. Anti-coagulants are an extremely important class of drug, one of the most widely used types of medication, but there remains a pressing need for novel treatments, however, as present drugs such as warfarin have significant drawbacks. Nature provides a number of examples of anti-coagulant proteins produced by blood-sucking animals, which may provide templates for the development of new small molecules with similar physiological effects. We have, therefore, studied an Anopheles anti-platelet protein from a malaria vector mosquito and report its crystal structure in complex with an antibody. Overall the protein is extremely sensitive to proteolysis, but the crystal structure reveals a stable domain built from two helices and a turn, which corresponds to the functional region. The antibody raised against Anopheles anti-platelet protein prevents it from binding collagen. Our work, therefore, opens new avenues to the development of both novel small molecule anti-clotting agents and anti-malarials.

We previously identified an anti-platelet protein, anopheline anti-platelet protein (AAPP), from the salivary gland of female Anopheles stephensi (a mosquito vector of human malaria). AAPP specifically blocks platelet adhesion to collagen by binding directly to collagen and subsequently causing platelet aggregation. The aim of this study was to identify the active region of AAPP responsible for the anti-thrombotic activity because we hypothesized that AAPP could be used as a candidate anti-platelet drug. Various truncated forms of AAPP were produced using an Escherichia coli expression system. Each protein was examined for binding activities to soluble/fibrillar collagen and anti-thrombotic activity using a plate assay and platelet/whole blood aggregation study, respectively. Among the truncated forms examined, only a protein encoded by exon 3-4 (rAAPPex3-4) effectively bound to soluble/fibrillar collagen in a concentration-dependent and saturable manner. The EC50 values of full-length AAPP and rAAPPex3-4 for soluble collagen binding were 35 nM and 36 nM, respectively. In contrast to soluble collagen, there was a difference in binding affinity to fibrillar collagen between full-length AAPP and rAAPPex3-4, with EC50 values of 31 nM and 51 nM, respectively. rAAPPex3-4 also inhibited aggregation of platelets/whole blood, and the IC50 values of full-length AAPP and rAAPPex3-4 for platelet aggregation were 35 nM and 93 nM, respectively. These results indicated that the essential moiety of AAPP for collagen binding and anti-thrombotic activity was in the region encoded by exon 3-4, which is highly conserved among the counterpart regions of other mosquito species.

We have previously developed a new malaria vaccine delivery system based on the baculovirus dual expression system (BDES). In this system, expression of malaria antigens is driven by a dual promoter consisting of the baculovirus-derived polyhedrin and mammal-derived cytomegalovirus promoters. To test this system for its potential as a vaccine against human malaria parasites, we investigated immune responses against the newly developed BDES-based Plasmodium falciparum circumsporozoite protein vaccines (BDES-PfCSP) in mice and Rhesus monkeys. Immunization of mice with BDES-PfCSP induced Th1/Th2-mixed type immune responses with high PfCSP-specific antibody (Ab) titers, and provided significant protection against challenge from the bites of mosquitoes infected with a transgenic P. berghei line expressing PfCSP. Next, we evaluated the immunogenicity of the BDES-PfCSP vaccine in a rhesus monkey model. Immunization of BDES-PfCSP elicited high levels of anti-PfCSP Ab responses in individual monkeys. Moreover, the sera from the immunized monkeys remarkably blocked sporozoite invasion of HepG2 cells. Taken together with two animal models, our results indicate that this novel vaccine platform (BDES) has potential clinical application as a vaccine against malaria.

Dendritic cells recognize pathogens through pattern recognition receptors such as Toll-like receptors and phagocytose and digest them by phagocytic receptors for antigen presentation. This study was designed to clarify the cross-talk between recognition and phagocytosis of microbes in dendritic cells. The murine dendritic cell line XS106 cells were stimulated with the murine C-type lectin SIGNR1 ligand lipoarabinomannan and the Toll-like receptor 2 ligand FSL-1. The co-stimulation significantly suppressed FSL-1-mediated activation of NF-κB as well as production of TNF-α, IL-6 and IL-12p40 in a dose-dependent manner. The suppression was significantly but not completely recovered by knock-down of SIGNR1. SIGNR1 was associated with Toll-like receptor 2 in XS106 cells. The co-stimulation upregulated the expression of suppressor of cytokine signalling-1 in XS106 cells, the knock-down of which almost completely recovered the suppression of the FSL-1-mediated cytokine production by lipoarabinomannan. In addition, it was found that the MyD88-adaptor-like protein in XS106 cells was degraded by co-stimulation with FSL-1 and lipoarabinomannan in the absence, but not the presence, of the proteasome inhibitor MG132 and the degradation was inhibited by knock-down of suppressor of cytokine signalling-1. This study suggests that Toll-like receptor 2-mediated signalling is negatively regulated by SIGNR1-mediated signalling in dendritic cells, possibly through suppressor of cytokine signalling-1-mediated degradation of the MyD88-adaptor-like protein.

Dendritic cells (DCs) are critical in initiating immune responses by cross-priming of tumor Ags to T cells. Previous results showed that NK cells inhibited DC-mediated cross-presentation of tumor Ags both in vivo and in vitro. In this study, enhanced Ag presentation was observed in draining lymph nodes in TRAIL(-/-) and DR5(-/-) mice compared with that of wild-type mice. NK cells inhibit DC cross-priming of tumor Ags in vitro, but not direct presentation of endogenous Ags. NK cells lacking TRAIL, but not perforin, were not able to inhibit DC cross-priming of tumor Ags. DCs that lack expression of TRAIL receptor DR5 were less susceptible to NK cell-mediated inhibition of cross-priming, and cross-linking of DR5 receptor led to reduced generation of MHC class I-Ag peptide complexes, followed by attenuated cross-priming of CD8(+) T cells. In addition, key molecules involved in the TRAIL/DR5 pathway during DC/NK cell interactions were determined. In summary, these data indicate a novel alternative pathway for DC/NK cell interactions in antitumor immunity and may reflect homeostasis of both DCs and NK cells for regulation of CD8(+) T cell function in physiological conditions.

Little is known of how Toll-like receptor (TLR) ligands are processed after recognition by TLRs. This study was therefore designed to investigate how the TLR2 ligand FSL-1 is processed in macrophages after recognition by TLR2. FSL-1 was internalized into the murine macrophage cell line, RAW264.7. Both chlorpromazine and methyl-beta-cyclodextrin, which inhibit clathrin-dependent endocytosis, reduced FSL-1 uptake by RAW264.7 cells in a dose-dependent manner but nystatin, which inhibits caveolae- and lipid raft-dependent endocytosis, did not. FSL-1 was co-localized with clathrin but not with TLR2 in the cytosol of RAW264.7 cells. These results suggest that internalization of FSL-1 is clathrin dependent. In addition, FSL-1 was internalized by peritoneal macrophages from TLR2-deficient mice. FSL-1 was internalized by human embryonic kidney 293 cells transfected with CD14 or CD36 but not by the non-transfected cells. Also, knockdown of CD14 or CD36 in the transfectants reduced FSL-1 uptake. In this study, we suggest that (i) FSL-1 is internalized into macrophages via a clathrin-dependent endocytic pathway, (ii) the FSL-1 uptake by macrophages occurs irrespective of the presence of TLR2, and (iii) CD14 and CD36 are responsible for the internalization of FSL-1.

HEK293 cells stably expressing DC-SIGN (293/DC-SIGN) were examined for phagocytosis of Escherichia coli. 293/DC-SIGN stable transfectants were able to mediate phagocytosis of E. coli. The phagocytosis was inhibited by EDTA or several inhibitors specific for Syk kinase, Raf kinase and the transcription factor NF-kappaB. DC-SIGN consists of characteristic domains and motifs such as CRD, neck, incomplete ITAM, dileucine and tri-acidic cluster. HEK293 cells expressing mutants of DC-SIGN were also examined for the phagocytosis. It was found that Ca(2+) binding sites in the CRD of DC-SIGN were involved in phagocytosis of bacteria as well as multimerization of DC-SIGN, and the neck region played a role in efficiency of binding to microbes as well as multimerization of the protein.

Many studies have shown that the pharmacological effects of resveratrol, a phytoalexin polyphenolic compound, include protective effects against cancer and inflammation as well as enhancement of stress resistance. In this study, we examined whether resveratrol affected the phagocytosis of bacteria by macrophages and the activation of the transcription factor NF-kappaB after stimulation with or without the ligand FSL-1 for Toll-like receptor 2 (TLR2). Phagocytosis of Escherichia coli and of Staphylococcus aureus by THP-1 cells and RAW264.7 cells was inhibited by resveratrol in a dose-dependent manner regardless of stimulation with FSL-1. The NF-kappaB activity in HEK293 cells stably expressing TLR2 was also inhibited by resveratrol after stimulation with FSL-1. Resveratrol also inhibited both the translocation of p65 of NF-kappaB into nuclei in the transfectant and tumor necrosis factor alpha production by THP-1 cells or RAW264.7 cells. It has recently been reported that TLR-mediated signaling pathways lead to the upregulation of mRNAs of phagocytic receptors, including scavenger receptors and C-type lectin receptors. This study also demonstrated that FSL-1 induced the upregulation of mRNAs of phagocytic receptors such as macrophage scavenger receptor-1, CD36, DC-SIGN, and Dectin-1 and that the FSL-1-induced upregulation of their mRNAs was inhibited by resveratrol. In addition, it was found that the expression of DC-SIGN in HEK293 cells stably expressing DC-SIGN was reduced by resveratrol and that the phagocytic activity was significantly inhibited by resveratrol. Thus, this study suggests that resveratrol inhibited bacterial phagocytosis by macrophages by downregulating the expression of phagocytic receptors and NF-kappaB activity.

A significant amount of evidence has been accumulated to show that Toll-like receptors (TLRs) function as sensors for microbial invasion. However, little is known about how signalling triggered by TLRs leads to the phagocytosis of pathogens. This study was designed to determine whether stimulation of TLR2 mainly with the lipopeptide FSL-1 plays a role in the phagocytosis of pathogens by macrophages. FSL-1 enhanced the phagocytosis of Escherichia coli to a markedly greater extent than it did that of Staphylococcus aureus, but did not enhance the phagocytosis of latex beads. FSL-1 stimulation resulted in enhanced phagocytosis of bacteria by macrophages from TLR2(+/+) mice but not by those from TLR2(-/-) mice. Chinese hamster ovary cells stably expressing TLR2 failed to phagocytose these bacteria, but the cells expressing CD14 did. FSL-1 induced upregulation of the expression of phagocytic receptors, including MSR1, CD36, DC-SIGN and Dectin-1 in THP-1 cells. Human embryonic kidney 293 cells transfected with DC-SIGN and MSR1 phagocytosed these bacteria. These results suggest that the FSL-1-induced enhancement of phagocytosis of bacteria by macrophages may be explained partly by the upregulation of scavenger receptors and the C-type lectins through TLR2-mediated signalling pathways, and that TLR2 by itself does not function as a phagocytic receptor.

Details of roles of carbohydrates attached to Toll-like receptors (TLRs) in the recognition of pathogen-associated molecular patterns and in the formation of the functional receptor complex still remain unknown. This study was designed to determine whether the glycans linked at Asn114, Asn199, Asn414 and Asn442 residues of TLR2 ectodomain were involved in the recognition of diacylated lipopeptide and lipoprotein. Single and multiple mutants were transfected into human embryonic kidney (HEK) 293 cells together with a NF-kappaB luciferase reporter plasmid. All of these mutants were expressed on the surface. SDS-PAGE of the transfectants demonstrated that these mutants migrated lower than wild-type TLR2 and their molecular masses decreased as the number of mutated Asn residues increased. TLR2(N114A), TLR2(N199A) and TLR2(N414A) as well as wild-type TLR2 induced NF-kappaB activation when stimulated with these ligands, whereas TLR2(N442A) failed to induce NF-kappaB activation. All of triple and quadruple mutants failed to induce NF-kappaB activation, but were associated with both wild-type TLR2 and TLR6 in the transfectants. TLR2(N114A,N199A), TLR2(N114A,N414A) and, to a lesser extent, TLR2(N114A,N442A), in which two N-linked glycans are speculated to be exposed to the concave surface of TLR2 solenoid, not only induce NF-kappaB activation but also are associated with wild-type TLR2 and TLR6. These results suggest that the glycan at Asn442 and at least two N-linked glycans speculated to be exposed to the concave surface of TLR2 solenoid are involved in the recognition of ligands by TLR2 and/or in formation or maturation of a functional TLR2 receptor complex.