scholarly journals Baculovirus-Vectored Multistage Plasmodium vivax Vaccine Induces Both Protective and Transmission-Blocking Immunities against Transgenic Rodent Malaria Parasites

2014 ◽  
Vol 82 (10) ◽  
pp. 4348-4357 ◽  
Author(s):  
Masanori Mizutani ◽  
Mitsuhiro Iyori ◽  
Andrew M. Blagborough ◽  
Shinya Fukumoto ◽  
Tomohiro Funatsu ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Plasmodium Vivax ◽  
Mammalian Cells ◽  
Expression System ◽  
Viral Envelope ◽  
Vaccine Platform ◽  
Transmission Blocking ◽  
Content Type ◽  
Sexual Stages

ABSTRACTA multistage malaria vaccine targeting the pre-erythrocytic and sexual stages ofPlasmodiumcould 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 multistagePlasmodium vivaxvaccine which simultaneously expressesP. vivaxcircumsporozoite 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 cellsin vitro. This vaccine induced high levels of antibodies to Pvs25 and PvCSP and elicited protective (43%) and TB (82%) efficacies against transgenicP. bergheiparasites expressing the correspondingP. vivaxantigens 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.

scholarly journals Salinomycin and Other Ionophores as a New Class of Antimalarial Drugs with Transmission-Blocking Activity

2015 ◽  
Vol 59 (9) ◽  
pp. 5135-5144 ◽  
Author(s):  
Sarah D'Alessandro ◽  
Yolanda Corbett ◽  
Denise P. Ilboudo ◽  
Paola Misiano ◽  
Nisha Dahiya ◽  
...  
Keyword(s):  
Anticancer Agents ◽  
Drug Target ◽  
Stage Iv ◽  
Stage Ii ◽  
Transmission Blocking ◽  
Content Type ◽  
New Class ◽  
Blocking Activity ◽  
Sexual Stages

ABSTRACTThe drug target profile proposed by the Medicines for Malaria Venture for a malaria elimination/eradication policy focuses on molecules active on both asexual and sexual stages ofPlasmodium, thus with both curative and transmission-blocking activities. The aim of the present work was to investigate whether the class of monovalent ionophores, which includes drugs used in veterinary medicine and that were recently proposed as human anticancer agents, meets these requirements. The activity of salinomycin, monensin, and nigericin onPlasmodium falciparumasexual and sexual erythrocytic stages and on the development of thePlasmodium bergheiandP. falciparummosquito stages is reported here. Gametocytogenesis of theP. falciparumstrain 3D7 was inducedin vitro, and gametocytes at stage II and III or stage IV and V of development were treated for different lengths of time with the ionophores and their viability measured with the parasite lactate dehydrogenase (pLDH) assay. The monovalent ionophores efficiently killed both asexual parasites and gametocytes with a nanomolar 50% inhibitory concentration (IC50). Salinomycin showed a fast speed of kill compared to that of standard drugs, and the potency was higher on stage IV and V than on stage II and III gametocytes. The ionophores inhibited ookinete development and subsequent oocyst formation in the mosquito midgut, confirming their transmission-blocking activity. Potential toxicity due to hemolysis was excluded, since only infected and not normal erythrocytes were damaged by ionophores. Our data strongly support the downstream exploration of monovalent ionophores for repositioning as new antimalarial and transmission-blocking leads.

Evaluation of Two Sexual-stage Antigens as Bivalent Transmission-blocking Vaccines in Rodent Malaria

2021 ◽  
Author(s):  
Fan Yang ◽  
Fei Liu ◽  
Xinxin Yu ◽  
Wenqi Zheng ◽  
Yudi Wu ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Chimeric Protein ◽  
Transmission Blocking ◽  
Native Proteins ◽  
Blocking Activity ◽  
Antibody Titers ◽  
Sexual Stages ◽  
Transmission Blocking Vaccines

Abstract Background: Transmission-blocking vaccine (TBV) is a promising strategy for malaria elimination. It is hypothesized that mixing or fusing two antigens targeting different stages of sexual development may provide higher transmission-blocking activity than these antigens are used individually.Methods: We designed a chimeric protein composed of fragments of Pbg37 and PSOP25 and expressed the recombinant protein in Escherichia coli Rosetta-gami B (DE3). After immunizing mice with mixing or fusing recombinant proteins, the antibody titers of sera were analyzed by ELISA. IFA and Western blot were performed to test the reactivity of the antisera with the native proteins of the parasite. The transmission blocking activity were assessed in vitro and in vivo assay. Results: When Pbg37 and PSOP25 were co-administered in a mixture or as a fusion protein, they elicited similar antibody responses in mice as single antigens without causing immunological interference with each other. Antibodies against the mixed or fused antigens recognized the target proteins in the gametocyte, gamete, zygote and ookinete stages. The two bivalent vaccines (mixed proteins or a fusion protein) produced the superior TBA compared to that of the antibodies against individual antigens.Conclusions: There was no immunological interference between the two antigens of bivalent vaccines. And the bivalent vaccines produced significantly stronger transmission-blocking activities than single antigens. Altogether, these data provide the theoretical basis for the development of combination TBVs targeting different sexual stages.

scholarly journals Small-Molecule Xenomycins Inhibit All Stages of the Plasmodium Life Cycle

2014 ◽  
Vol 59 (3) ◽  
pp. 1427-1434 ◽  
Author(s):  
Jessey Erath ◽  
Julio Gallego-Delgado ◽  
Wenyue Xu ◽  
Grasiella Andriani ◽  
Scott Tanghe ◽  
...  
Keyword(s):  
Life Cycle ◽  
Small Molecule ◽  
Mammalian Cells ◽  
Biological Effects ◽  
Parasite Life Cycle ◽  
Content Type ◽  
Effective Inhibition ◽  
Potential Activity ◽  
Sexual Stages

ABSTRACTWidespread resistance to most antimalaria drugs in use has prompted the search for novel candidate compounds with activity againstPlasmodiumasexual blood stages to be developed for treatment. In addition, the current malaria eradication programs require the development of drugs that are effective against all stages of the parasite life cycle. We have analyzed the antimalarial properties of xenomycins, a novel subclass of small molecule compounds initially isolated for anticancer activity and similarity to quinacrine in biological effects on mammalian cells.In vitrostudies show potent activity of Xenomycins againstPlasmodium falciparum. Oral administration of xenomycins in mouse models result in effective clearance of liver and blood asexual and sexual stages, as well as effective inhibition of transmission to mosquitoes. These characteristics position xenomycins as antimalarial candidates with potential activity in prevention, treatment and elimination of this disease.

scholarly journals VP2 Exchange and NS3/NS3a Deletion in African Horse Sickness Virus (AHSV) in Development of Disabled Infectious Single Animal Vaccine Candidates for AHSV

2015 ◽  
Vol 89 (17) ◽  
pp. 8764-8772 ◽  
Author(s):  
Sandra G. P. van de Water ◽  
René G. P. van Gennip ◽  
Christiaan A. Potgieter ◽  
Isabel M. Wright ◽  
Piet A. van Rijn
Keyword(s):  
Mammalian Cells ◽  
Reverse Genetics ◽  
African Horse Sickness ◽  
African Horse Sickness Virus ◽  
Vaccine Platform ◽  
Biting Midges ◽  
Content Type ◽  
Vp2 Protein ◽  
Single Animal

ABSTRACTAfrican horse sickness virus (AHSV) is a virus species in the genusOrbivirusof the familyReoviridae. There are nine serotypes of AHSV showing different levels of cross neutralization. AHSV is transmitted by species ofCulicoidesbiting midges and causes African horse sickness (AHS) in equids, with a mortality rate of up to 95% in naive horses. AHS has become a serious threat for countries outside Africa, since endemicCulicoidesspecies in moderate climates appear to be competent vectors for the related bluetongue virus (BTV). To control AHS, live-attenuated vaccines (LAVs) are used in Africa. We used reverse genetics to generate “synthetic” reassortants of AHSV for all nine serotypes by exchange of genome segment 2 (Seg-2). This segment encodes VP2, which is the serotype-determining protein and the dominant target for neutralizing antibodies. Single Seg-2 AHSV reassortants showed similar cytopathogenic effects in mammalian cells but displayed different growth kinetics. Reverse genetics for AHSV was also used to study Seg-10 expressing NS3/NS3a proteins. We demonstrated that NS3/NS3a proteins are not essential for AHSV replicationin vitro. NS3/NS3a of AHSV is, however, involved in the cytopathogenic effect in mammalian cells and is very important for virus release from cultured insect cells in particular. Similar to the concept of the bluetongue disabled infectious single animal (BT DISA) vaccine platform, an AHS DISA vaccine platform lacking NS3/NS3a expression was developed. Using exchange of genome segment 2 encoding VP2 protein (Seg-2[VP2]), we will be able to develop AHS DISA vaccine candidates for all current AHSV serotypes.IMPORTANCEAfrican horse sickness virus is transmitted by species ofCulicoidesbiting midges and causes African horse sickness in equids, with a mortality rate of up to 95% in naive horses. African horse sickness has become a serious threat for countries outside Africa, since endemicCulicoidesspecies in moderate climates are supposed to be competent vectors. By using reverse genetics, viruses of all nine serotypes were constructed by the exchange of Seg-2 expressing the serotype-determining VP2 protein. Furthermore, we demonstrated that the nonstructural protein NS3/NS3a is not essential for virus replicationin vitro. However, the potential spread of the virus by biting midges is supposed to be blocked, since thein vitrorelease of the virus was strongly reduced due to this deletion. VP2 exchange and NS3/NS3a deletion in African horse sickness virus were combined in the concept of a disabled infectious single animal vaccine for all nine serotypes.

scholarly journals Neurotrophin Receptor TrkC Is an Entry Receptor for Trypanosoma cruzi in Neural, Glial, and Epithelial Cells

2011 ◽  
Vol 79 (10) ◽  
pp. 4081-4087 ◽  
Author(s):  
Craig Weinkauf ◽  
Ryan Salvador ◽  
Mercio PereiraPerrin
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Neuronal Cell ◽  
Host Cells ◽  
Neurotrophin Receptor ◽  
Content Type

ABSTRACTTrypanosoma cruzi, the agent of Chagas' disease, infects a variety of mammalian cells in a process that includes multiple cycles of intracellular division and differentiation starting with host receptor recognition by a parasite ligand(s). Earlier work in our laboratory showed that the neurotrophin-3 (NT-3) receptor TrkC is activated byT. cruzisurfacetrans-sialidase, also known as parasite-derived neurotrophic factor (PDNF). However, it has remained unclear whether TrkC is used byT. cruzito enter host cells. Here, we show that a neuronal cell line (PC12-NNR5) relatively resistant toT. cruzibecame highly susceptible to infection when overexpressing human TrkC but not human TrkB. Furthermore,trkCtransfection conferred an ∼3.0-fold intracellular growth advantage. Sialylation-deficient Chinese hamster ovarian (CHO) epithelial cell lines Lec1 and Lec2 also became much more permissive toT. cruziafter transfection with thetrkCgene. Additionally, NT-3 specifically blockedT. cruziinfection of the TrkC-NNR5 transfectants and of naturally permissive TrkC-bearing Schwann cells and astrocytes, as did recombinant PDNF. Two specific inhibitors of Trk autophosphorylation (K252a and AG879) and inhibitors of Trk-induced MAPK/Erk (U0126) and Akt kinase (LY294002) signaling, but not an inhibitor of insulin-like growth factor 1 receptor, abrogated TrkC-mediated cell invasion. Antibody to TrkC blockedT. cruziinfection of the TrkC-NNR5 transfectants and of cells that naturally express TrkC. The TrkC antibody also significantly and specifically reduced cutaneous infection in a mouse model of acute Chagas' disease. TrkC is ubiquitously expressed in the peripheral and central nervous systems, and in nonneural cells infected byT. cruzi, including cardiac and gastrointestinal muscle cells. Thus, TrkC is implicated as a functional PDNF receptor in cell entry, independently of sialic acid recognition, mediating broadT. cruziinfection bothin vitroandin vivo.

scholarly journals SpyAD, a Moonlighting Protein of Group A Streptococcus Contributing to Bacterial Division and Host Cell Adhesion

2014 ◽  
Vol 82 (7) ◽  
pp. 2890-2901 ◽  
Author(s):  
Marilena Gallotta ◽  
Giovanni Gancitano ◽  
Giampiero Pietrocola ◽  
Marirosa Mora ◽  
Alfredo Pezzicoli ◽  
...  
Keyword(s):  
Cell Division ◽  
Mammalian Cells ◽  
Group A Streptococcus ◽  
Host Cells ◽  
Knockout Mutant ◽  
Content Type ◽  
Moonlighting Protein ◽  
Group A

ABSTRACTGroup A streptococcus (GAS) is a human pathogen causing a wide repertoire of mild and severe diseases for which no vaccine is yet available. We recently reported the identification of three protein antigens that in combination conferred wide protection against GAS infection in mice. Here we focused our attention on the characterization of one of these three antigens, Spy0269, a highly conserved, surface-exposed, and immunogenic protein of unknown function. Deletion of thespy0269gene in a GAS M1 isolate resulted in very long bacterial chains, which is indicative of an impaired capacity of the knockout mutant to properly divide. Confocal microscopy and immunoprecipitation experiments demonstrated that the protein was mainly localized at the cell septum and could interactin vitrowith the cell division protein FtsZ, leading us to hypothesize that Spy0269 is a member of the GAS divisome machinery. Predicted structural domains and sequence homologies with known streptococcal adhesins suggested that this antigen could also play a role in mediating GAS interaction with host cells. This hypothesis was confirmed by showing that recombinant Spy0269 could bind to mammalian epithelial cellsin vitroand thatLactococcus lactisexpressing Spy0269 on its cell surface could adhere to mammalian cellsin vitroand to mice nasal mucosain vivo. On the basis of these data, we believe that Spy0269 is involved both in bacterial cell division and in adhesion to host cells and we propose to rename this multifunctional moonlighting protein as SpyAD (StreptococcuspyogenesAdhesion andDivision protein).

scholarly journals Improving Baculovirus Infectivity by Efficiently Embedding Enhancing Factors into Occlusion Bodies

2017 ◽  
Vol 83 (14) ◽  
Author(s):  
Shili Yang ◽  
Lijuan Zhao ◽  
Ruipeng Ma ◽  
Wei Fang ◽  
Jia Hu ◽  
...  
Keyword(s):  
Protein Expression ◽  
Fluorescent Protein ◽  
Expression System ◽  
Agrotis Segetum ◽  
Foreign Protein ◽  
Content Type ◽  
Occlusion Bodies ◽  
Foreign Proteins ◽  
Novel Strategy

ABSTRACT The relatively low infectivity of baculoviruses to their host larvae limits their use as insecticidal agents on a larger scale. In the present study, a novel strategy was developed to efficiently embed foreign proteins into Autographa californica multiple nucleopolyhedrovirus (AcMNPV) occlusion bodies (OBs) to achieve stable expression of foreign proteins and to improve viral infectivity. A recombinant AcMNPV bacmid was constructed by expressing the 150-amino-acid (aa) N-terminal segment of polyhedrin under the control of the p10 promoter and the remaining C-terminal 95-aa segment under the control of the polyhedrin promoter. The recombinant virus formed OBs in Spodoptera frugiperda 9 cells, in which the occlusion-derived viruses were embedded in a manner similar to that for wild-type AcMNPV. Next, the 95-aa polyhedrin C terminus was fused to enhanced green fluorescent protein, and the recombinant AcMNPV formed fluorescent green OBs and was stably passaged in vitro and in vivo. The AcMNPV recombinants were further modified by fusing truncated Agrotis segetum granulovirus enhancin or truncated Cydia pomonella granulovirus ORF13 (GP37) to the C-terminal 95 aa of polyhedrin, and both recombinants were able to form normal OBs. Bioactivity assays indicated that the median lethal concentrations of these two AcMNPV recombinants were 3- to 5-fold lower than that of the control virus. These results suggest that embedding enhancing factors in baculovirus OBs by use of this novel technique may promote efficient and stable foreign protein expression and significantly improve baculovirus infectivity. IMPORTANCE Baculoviruses have been used as bioinsecticides for over 40 years, but their relatively low infectivity to their host larvae limits their use on a larger scale. It has been reported that it is possible to improve baculovirus infectivity by packaging enhancing factors within baculovirus occlusion bodies (OBs); however, so far, the packaging efficiency has been low. In this article, we describe a novel strategy for efficiently embedding foreign proteins into AcMNPV OBs by expressing N- and C-terminal (dimidiate) polyhedrin fragments (150 and 95 amino acids, respectively) as fusions to foreign proteins under the control of the p10 and polyhedrin promoters, respectively. When this strategy was used to embed an enhancing factor (enhancin or GP37) into the baculovirus OBs, 3- to 5-fold increases in baculoviral infectivity were observed. This novel strategy has the potential to create an efficient protein expression system and a highly efficient virus-based system for insecticide production in the future.

scholarly journals Blocking Hepatitis C Virus Infection with Recombinant Form of Envelope Protein 2 Ectodomain

2009 ◽  
Vol 83 (21) ◽  
pp. 11078-11089 ◽  
Author(s):  
Jillian Whidby ◽  
Guaniri Mateu ◽  
Hannah Scarborough ◽  
Borries Demeler ◽  
Arash Grakoui ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mammalian Cells ◽  
Expression System ◽  
Developed Countries ◽  
Viral Envelope ◽  
Hcv Infection ◽  
Size Exclusion ◽  
Amino Terminal ◽  
Endosomal Acidification

ABSTRACT More than 120 million people worldwide are chronically infected with hepatitis C virus (HCV), making HCV infection the leading cause of liver transplantation in developed countries. Treatment is limited, and efficacy depends upon the infecting strain and the initial viral load. The HCV envelope glycoproteins (E1 and E2) are involved in receptor binding, virus-cell fusion, and entry into the host cell. HCV infection proceeds by endosomal acidification, suggesting that fusion of the viral envelope with cellular membranes is a pH-triggered event. E2 consists of an amino-terminal ectodomain, an amphipathic helix that forms a stem region, and a carboxy-terminal membrane-associating segment. We have devised a novel expression system for the production of a secreted form of E2 ectodomain (eE2) from mammalian cells and performed a comprehensive biochemical and biophysical characterization. eE2 is properly folded, as determined by binding to human CD81, blocking of infection of cell culture-derived HCV, and recognition by antibodies from patients chronically infected with different genotypes of HCV. The glycosylation pattern, number of disulfide bonds, oligomerization state, and secondary structure of eE2 have been characterized using mass spectrometry, size exclusion chromatography, circular dichroism, and analytical ultracentrifugation. These results advance the understanding of E2 and may assist in the design of an HCV vaccine and entry inhibitor.

scholarly journals Acylhydrazones as Antifungal Agents Targeting the Synthesis of Fungal Sphingolipids

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Cristina Lazzarini ◽  
Krupanandan Haranahalli ◽  
Robert Rieger ◽  
Hari Krishna Ananthula ◽  
Pankaj B. Desai ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Antifungal Agents ◽  
Fungal Infections ◽  
Antifungal Drugs ◽  
Fungal Resistance ◽  
Content Type ◽  
Highly Active ◽  
Pharmacokinetic Properties ◽  
Pass Through

ABSTRACTThe incidence of invasive fungal infections has risen dramatically in recent decades. Current antifungal drugs are either toxic, likely to interact with other drugs, have a narrow spectrum of activity, or induce fungal resistance. Hence, there is a great need for new antifungals, possibly with novel mechanisms of action. Previously our group reported an acylhydrazone called BHBM that targeted the sphingolipid pathway and showed strong antifungal activity against several fungi. In this study, we screened 19 derivatives of BHBM. Three out of 19 derivatives were highly active againstCryptococcus neoformansin vitroand had low toxicity in mammalian cells. In particular, one of them, called D13, had a high selectivity index and showed better activity in an animal model of cryptococcosis, candidiasis, and pulmonary aspergillosis. D13 also displayed suitable pharmacokinetic properties and was able to pass through the blood-brain barrier. These results suggest that acylhydrazones are promising molecules for the research and development of new antifungal agents.

scholarly journals Development and Application of TK6-derived Cells Expressing Human Cytochrome P450s for Genotoxicity Testing

2020 ◽  
Vol 175 (2) ◽  
pp. 251-265 ◽  
Author(s):  
Xilin Li ◽  
Si Chen ◽  
Xiaoqing Guo ◽  
Qiangen Wu ◽  
Ji-Eun Seo ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Expression System ◽  
Cytochrome P450s ◽  
Mass Spectrometry Analysis ◽  
Established Cell Line ◽  
Micronucleus Formation ◽  
Tk6 Cells ◽  
Genotoxicity Testing

Abstract Metabolism plays a key role in chemical genotoxicity; however, most mammalian cells used for in vitro genotoxicity testing lack effective metabolizing enzymes. We recently developed a battery of TK6-derived cell lines that individually overexpress 1 of 8 cytochrome P450s (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, and 3A4) using a lentiviral expression system. The increased expression and metabolic function of each individual CYP in each established cell line were confirmed using real-time PCR, Western blotting, and mass spectrometry analysis; the parental TK6 cells and empty vector (EV) transduced cells had negligible CYP levels. Subsequently, we evaluated these cell lines using 2 prototypical polyaromatic hydrocarbon mutagens, 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (B[a]P), that require metabolic activation to exert their genotoxicity. DMBA-induced cytotoxicity, phosphorylation of histone H2A.X, and micronucleus formation were significantly increased in TK6 cells with CYP1A1, 1B1, 2B6, and 2C19 expression as compared with EV controls. B[a]P significantly increased cytotoxicity, DNA damage, and chromosomal damage in TK6 cells overexpressing CYP1A1 and 1B1 when compared with EV controls. B[a]P also induced micronucleus formation in TK6 cells expressing CYP1A2. These results suggest that our CYP-expressing TK6 cell system can be used to detect the genotoxicity of compounds requiring metabolic transformation.

Sign in / Sign up

Export Citation Format

Share Document