Combining Monophosphoryl Lipid A (MPL), CpG Oligodeoxynucleotide (ODN), and QS-21 Adjuvants Induces Strong and Persistent Functional Antibodies and T Cell Responses against Cell-Traversal Protein for Ookinetes and Sporozoites (CelTOS) of Plasmodium falciparum in BALB/c Mice.

Plasmodium falciparum cell-traversal protein for ookinetes and sporozoites (PfCelTOS) is an advanced vaccine candidate that has a crucial role in the traversal of the malaria parasite in both mosquito and mammalian hosts. As recombinant purified proteins are normally poor immunogens, they require to be admixed with an adjuvant(s); therefore, the objective of the present study was to evaluate the capacity of different vaccine adjuvants, monophosphoryl lipid A (MPL), CpG, and Quillaja saponaria Molina fraction 21 (QS-21), alone or in combination (MCQ [MPL/CpG/QS-21]), to enhance the immunogenicity of Escherichia coli-expressed PfCelTOS in BALB/c mice. This goal was achieved by the assessment of anti-PfCelTOS IgG antibodies (level, titer, IgG isotype profile, avidity, and persistence) and extracellular Th1 cytokines using an enzyme-linked immunosorbent assay (ELISA) on postimmunized BALB/c mouse sera and PfCelTOS-stimulated splenocytes, respectively. Also, an assessment of the transmission-reducing activity (TRA) of anti-PfCelTOS obtained from different vaccine groups was carried out in female Anopheles stephensi mosquitoes by using a standard membrane feeding assay (SMFA). In comparison to PfCelTOS alone, administration of PfCelTOS with three distinct potent Th1 adjuvants in vaccine mouse groups showed enhancement and improvement of PfCelTOS immunogenicity that generated more bias toward a Th1 response with significantly enhanced titers and avidity of the anti-PfCelTOS responses that could impair ookinete development in A. stephensi However, immunization of mice with PfCelTOS with MCQ mixture adjuvants resulted in the highest levels of induction of antibody titers, avidity, and inhibitory antibodies in oocyst development (88%/26.7% reductions in intensity/prevalence) in A. stephensi It could be suggested that adjuvant combinations with different mechanisms stimulate better functional antibody responses than adjuvants individually against challenging diseases such as malaria.

Expression of a New Recombinant Collagenase Protein of Lucilia Sericata in SF9 Insect Cell as a Potential Method for Wound Healing.

BACKGROUND: Today, the use of maggot therapy has become widespread due to the increase in chronic ulcers in the world. The recombinant production of secreted enzymes from these larvae is a novel non-invasive method for the treatment of chronic ulcers. Lucilia Sericata (L. sericata) collagenase (MMP-1) has been expressed in insect cells. Collagenase is an enzyme that is widely used in clinical therapy and industry. It has been indicated that collagenase is expressed and secreted in salivary glands of L. sericata while using for maggot debridement therapy. OBJECTIVES: In the present study we decided to produce the recombinant form of collagenase enzyme in Spodoptera frugiperda (SF9) insect cells using the baculovirus expression system (Bac-to-Bac). MATERIALS AND METHODS: cloned the coding sequences (residues 494-1705) of L. sericata collagenase into the pFastBacHTA as donor plasmid. After transposition in the bacmid of DH10Bac host, the bacmid was transfected into the Sf9 cell line, then the expressed recombinant collagenase (MMP-1) was purified using the Ni-NTA agarose. RESULTS: The recombinant protein was verified by Western blotting. Furthermore, the biological activity of purified protein was measured in the presence of its specific substrate and its inhibitor, which was 67 IU.mL-1 based on our results, it was revealed that the characterized gene in our previous study codes L. sericata collagenesa enzyme. CONCLUSION: Considering to the broad applications of collagenase in medical sciences, for the first time, we cloned the L. sericata collagenase (MMP-1) gene into the insect cell line to establish a method for the expression and purification of L. sericata collagenase (MMP-1). The result help for preparing and designing a safe and versatile recombinant drug in future.

Anti-Plasmodial Assessment of Four Different Iranian Propolis Extracts.

BACKGROUND: Eradication of malaria will depend on discovery of new intervention tools such as anti-malarial drugs. Due to the increasing interest in the application of propolis against significant clinical pathogenic agents, the aim of the present investigation was to evaluate the anti-plasmodial effect of Iranian propolis extracts against chloroquine (CQ)-sensitive Plasmodium falciparum 3D7 and Plasmodium berghei (ANKA strain). METHODS: Crude samples of honeybee (Apis mellifera) propolis were collected from four provinces in northern (Kalaleh, Golestan), northeastern (Chenaran, Razavi Khorasan), central (Taleghan, Alborz) and western (Morad Beyg, Hamedan) areas of Iran with different types of flora. The dried propolis samples were extracted with three different solvents, including ethanol 70% (EtOH), ethyl acetate (EA) and dichloromethane (DCM). RESULTS: All extracts were shown to have in vitro anti-plasmodial activity with IC50 ranging from 16.263 to 80.012 µg/mL using parasite lactate dehydrogenase (pLDH) assay. The DCM extract of Morad Beyg propolis indicated the highest anti-plasmodial activity (IC50: 16.263 ± 2.910 µg/mL; P = 0.027, Kruskal-Wallis H-test). The samples were also evaluated in mice for their in vivo anti-plasmodial effect. The curative effect against established infection (Rane test) showed that both extracts at all doses (50, 100, and 200 mg/kgBW) produced anti-plasmodial activity against the parasite. Furthermore, using gas chromatography-mass spectrometry (GC-MS), the quantity of flavonoids in DCM and EtOH 70% extracts were found to be 7.42% and 3.10%, respectively. CONCLUSION: The potent anti-plasmodial activity of both EtOH 70% and DCM extracts of the propolis of Morad Beyg, Hamedan suggests further analyses of individual components to assess its utilization as anti-malarial drugs.

In vitro antiplasmodial and phytochemical study of five Artemisia species from Iran and in vivo activity of two species.

The extract from Artemisia annua, containing artemisinin, has been proven active against multidrug resistant Plasmodium falciparum in previous studies. The purpose of this paper was to study five Artemisia species from Iran for their in vitro and in vivo antimalarial property and detection of artemisinin in the active species by chromatographic and spectroscopic methods including nuclear magnetic resonance (NMR) spectroscopy. Dried plants were extracted by 80% ethanol, and total extracts were investigated for antiplasmodial property and artemisinin content by TLC, HPLC, and (1)H-NMR techniques. Two plants (A. annua L. and Artemisia absinthium L.) showed good antiplasmodial activity against multidrug resistant and sensitive strain of P. falciparum. A. absinthium and A. annua at concentrations of 200 mg/kg for 4 days reduced parasitemia in BALB/C mice infected with Plasmodium bergei by 94.28% and 83.28%, respectively, but we could not detect artemisinin in all plants studied in this research. The antiplasmodial property of these two herbs is possibly related to essential oils that present in high amounts in their extracts.

In vitro and in vivo anti-malarial activity of Boerhavia elegans and Solanum surattense.

BACKGROUND: There is an urgent need to identify new anti-malarial drug targets for both prophylaxis and chemotherapy, due to the increasing problem of drug resistance to malaria parasites. In the present study, the aim was to discover novel, effective plant-based extracts for the activity against malaria. METHODS: Ten plants found in Iran were selected by ethnobotanical survey of medicinal plants. The crude ethanolic extracts were tested for in vitro anti-plasmodial activity against two strains of Plasmodium falciparum: K1 (chloroquine-resistant strain) and CY27 (chloroquine-sensitive strain), using the parasite lactate dehydrogenase (pLDH) assay. The anti-plasmodial activity of the extracts was also assessed in the 4-day suppressive anti-malarial assay in mice inoculated with Plasmodium berghei (ANKA strain). Crude ethanolic extracts showed good anti-plasmodial activity were further fractionated by partitioning in water and dichloromethane. RESULTS: Of 10 plant species assayed, three species: Boerhavia elegans (Choisy), Solanum surattense (Burm.f.) and Prosopis juliflora (Sw.) showed promising anti-plasmodial activity in vitro (IC50 < or = 50 microg/ml) and in vivo with no toxicity. The dichloromethane fraction of three extracts revealed stronger anti-plasmodial activity than the total extracts. CONCLUSION: Anti-plasmodial activities of extracts of B. elegans and S. surattense are reported for the first time.

Monitoring pyrethroid insecticide resistance in major malaria vector Anopheles culicifacies: comparison of molecular tools and conventional susceptibility test.

BACKGROUND: Anopheles culicifacies is a main malaria vector in southeastern part of Iran, bordring Afghanistan and Pakistan. So far, resistance to DDT, dieldrin, malathion and partial tolerance to pyrethroids has been reported in An. stephensi, but nothing confirmed on resistance status of An. culicifacies in Iran. METHODS: In current study, along with WHO routine susceptibility test with DDT (4%), dieldrin (0.4%), malathion (5%), permethrin (0.25%), lambadacyhalothrin (0.1%), and deltamethrin 0.025, we cloned and sequenced segment VI of domain II (SII6) in voltage-gated sodium channel (vgsc) gene of An. culicifacies specimens collected in Sistan and Baluchistan province (Iran). RESULTS: A 221-bp amplified fragment showed 91% and 93% similarity with exon I and exon II of An. gambiae. The size of intron II in An. culicifacies is 62 bp, while in An. gambiae is 57 bp. The major difference within An. culicifacies specimens and also with An. gambiae is in position 29 of exon I, which led to substitution of Leu to His amino acid. CONCLUSION: This data will act as first report on partial sequence of vgsc gene and its polymorphism in An. culicifacies. A Leu to His amino acid substitution detected upstream the formerly known knockdown resistance (kdr) mutation site could be an indication for other possible mutations related to insecticide resistance. However, the result of WHO susceptibility test carried out in Baluchistan of Iran revealed a level of tolerance to DDT and dieldrin, but almost complete susceptibility to pyrethroids in An. culicifacies. We postulate that the molecular diagnostic tool developed for detection and identification of kdr-related mutations in An. culicifacies, could be useful in monitoring insecticide resistance in Iran and neighbouring countries such as Pakistan and Afghanistan. A phylogenetic tree also constructed based on the sequence of exon I and II, which readily separated An. culicifacies populations from An. stephensi, An. fluviatilis and An. gambiae.