A two-dose viral-vectored Plasmodium vivax multistage vaccine confers durable protection and transmission-blockade in a pre-clinical study.

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.

Comparison between Neutralization Capacity of Antibodies Elicited by COVID-19 Natural Infection and Vaccination in Indonesia: A Prospective Cohort.

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.

Effects of Temperature and Nutrition during the Larval Period on Life History Traits in an Invasive Malaria Vector Anopheles stephensi.

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.

Adeno-associated virus-based malaria booster vaccine following attenuated replication-competent vaccinia virus LC16m8Δ priming.

We previously demonstrated that boosting with adeno-associated virus (AAV) type 1 (AAV1) can induce highly effective and long-lasting protective immune responses against malaria parasites when combined with replication-deficient adenovirus priming in a rodent model. In the present study, we compared the efficacy of two different AAV serotypes, AAV1 and AAV5, as malaria booster vaccines following priming with the attenuated replication-competent vaccinia virus strain LC16m8Δ (m8Δ), which harbors the fusion gene encoding both the pre-erythrocytic stage protein, Plasmodium falciparum circumsporozoite (PfCSP) and the sexual stage protein (Pfs25) in a two-dose heterologous prime-boost immunization regimen. Both regimens, m8Δ/AAV1 and m8Δ/AAV5, induced robust anti-PfCSP and anti-Pfs25 antibodies. To evaluate the protective efficacy, the mice were challenged with sporozoites twice after immunization. At the first sporozoite challenge, m8Δ/AAV5 achieved 100% sterile protection whereas m8Δ/AAV1 achieved 70% protection. However, at the second challenge, 100% of the surviving mice from the first challenge were protected in the m8Δ/AAV1 group whereas only 55.6% of those in the m8Δ/AAV5 group were protected. Regarding the transmission-blocking efficacy, we found that both immunization regimens induced high levels of transmission-reducing activity (>99%) and transmission-blocking activity (>95%). Our data indicate that the AAV5-based multistage malaria vaccine is as effective as the AAV1-based vaccine when administered following an m8Δ-based vaccine. These results suggest that AAV5 could be a viable alternate vaccine vector as a malaria booster vaccine.

Sterile protection and transmission blockade by a multistage anti-malarial vaccine in the pre-clinical study.

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.

A replication-competent smallpox vaccine LC16m8Δ-based COVID-19 vaccine.

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.

Liver-Directed AAV8 Booster Vaccine Expressing Plasmodium falciparum Antigen Following Adenovirus Vaccine Priming Elicits Sterile Protection in a Murine Model.

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 < 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.

Characterization of the Gene Expression Patterns in the Murine Liver Following Intramuscular Administration of Baculovirus.

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.

A Viral-Vectored Multi-Stage Malaria Vaccine Regimen With Protective and Transmission-Blocking Efficacies.

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.

Adeno-Associated Virus as an Effective Malaria Booster Vaccine Following Adenovirus Priming.

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.

Down-selecting circumsporozoite protein-based malaria vaccine: A comparison of malaria sporozoite challenge model.

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.

Anopheline antiplatelet protein from mosquito saliva regulates blood feeding behavior.

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.

Baculovirus-Induced Fast-Acting Innate Immunity Kills Liver-Stage Plasmodium.

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.

Adenovirus-prime and baculovirus-boost heterologous immunization achieves sterile protection against malaria sporozoite challenge in a murine model.

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.

DAF-shielded baculovirus-vectored vaccine enhances protection against malaria sporozoite challenge in mice.

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.

Development of a Plasmodium berghei transgenic parasite expressing the full-length Plasmodium vivax circumsporozoite VK247 protein for testing vaccine efficacy in a murine model.

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.

Baculovirus-vectored multistage Plasmodium vivax vaccine induces both protective and transmission-blocking immunities against transgenic rodent malaria parasites.

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.

The crystal structure of the active domain of Anopheles anti-platelet protein, a powerful anti-coagulant, in complex with an antibody.

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.

Protective efficacy of baculovirus dual expression system vaccine expressing Plasmodium falciparum circumsporozoite protein.

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.

Identification of the active region responsible for the anti-thrombotic activity of anopheline anti-platelet protein from a malaria vector mosquito.

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.