BY PARASITES OR INSECTS

Technical field

Vector borne disease control.

Background art

Hematophagy is the practice by certain animals of feeding on blood. Since blood is a fluid tissue rich in nutritious proteins and lipids that can be obtained without much effort, hematophagy is an ideal form of feeding for many faunal groups. For instance, intestinal nematodes (i.e., Ancylostomatids) feed on blood extracted from the capillaries of the gut, and about 75% of all species of leeches (i.e., Hirudo medicinalis) are hematophagous. In addition, some fish species (i.e., lampreys and candirus), mammals (i.e., vampire bats), and birds (i.e., hood mockingbirds, oxpeckers, tristan thrush, and vampire finches,) also practice hematophagy. Furthermore, Sandfly, blackfly, tsetse fly, bedbug, mosquito, tick, louse, mite, midge, assassin bug, and flea are also few examples for the hematophagous insects. Studies have shown that over 14,000 species and 400 genera of arthropods are hematophagous in nature (Ribeiro, 1995). These hematophagous animals have mouth parts and chemical agents for piercing vascular structures in the skin of hosts such as mammals, birds, reptiles, and fishes. The blood is obtained either by sucking action directly from the veins or capillaries, from a pool of escaped blood or by lapping. These blood feeders have the ability to inhibit natural blood coagulation (hemostasis). Hematophagous animals have evolved to release chemical solutions in their saliva by preventing vasoconstriction, inflammation, and pain sensation in the host. For that, they inject solutions that contain anesthetic and anticlotting agents and chemicals that facilitate capillary dilatation (Tanaka-Azevedo et a!., 2010)

Thrombin is a one of the most important multifunctional serine proteinases that plays a vital role in different organisms including hemostasis, thrombosis, inflammation, and proliferative response (Guillin et al. , 1995). It is the main enzyme of the blood coagulation system that responsible for many important biological functions including the activation of platelets, conversion of fibrinogen to fibrin, and feedback amplification of coagulation. Thrombin also plays a key role in the tracing of inflammatory cells into sites of injury and is chemotactic for a number of different cell types including monocytes, macrophages, and neutrophils (Furie and Furie, 1992; Mann et al., 1999). In nature, blood feeding animals have adapted to a diet of fresh blood and evolve some specific mechanisms to control their host coagulation processes. Concerning this issue, a variety of coagulation inhibitors has been isolated from blood-sucking animals such as bats (Gardell et al., 1991), ticks (Waxman et al., 1990), leeches (Sawyer et al., 1986), hookworms (Cappello et al., 1995) and arthropods including mosquitoes (Jacobs et al. , 1990).

Among these inhibitors, hirudin was the first thrombin inhibitor discovered which was isolated from a leech species. It was first isolated from the salivary glands of the medical leech Hirudo medicinalis (Markwardt, 1970). Hirudin is a polypeptide containing 65 amino acids, which tightly and specifically binds to oc-thrombin. It link with thrombin catalytic site and exosite-1, preventing fibrinogen cleavage and consequently clot formation. The leech Hirudinaria manillensis also produces two thrombin inhibitors as hirullin P6 (Steiner et al., 1992) and hirullin P18 (Steiner et al., 1992). Another tight-binding thrombin inhibitor known as haemadin was isolated from the leech Haemadipsia sylvestris. However, it does not inhibit other proteases and does not reveal any homology to known serine protease inhibitors including hirudin (Strube et al., 1993). Theromin is the most effective thrombin inhibitor, which was isolated from the gut of the leech Theromyson tessulatum (Salzet et al., 2000). It is known as a homodimer of 67 amino-acid residues with 16 Cys residues engaged in eight disulfide bridges.

As a rule, blood-suckers' saliva contains at least one anticlotting, one antiplatelet, and one vasodilatory substance (Ribeiro and Francischetti 2003). In many occasions, more than one molecule exists in each category and in some, a molecule alone is responsible for more than one anti-haemostatic effect. For instance, triabin is a new potent thrombin inhibitor isolated from the saliva of the blood-sucking insect Triatoma pallidipenis (Noeske-Jungblut et al., 1995). It is a 142 amino acid residue protein, which specifically binds to thrombin forming a 1: 1 noncovalent complex. Triabin is a highly potent exosite thrombin inhibitor that inhibits thrombin-induced platelet aggregation and prolongs both thrombin clotting time (TT) and activated partial thromboplastin time (APTT). Rhodniin is also another thrombin inhibitor isolated from the assassin bug Rhodinius prolixus (Fridrich et al., 1993). It binds to thrombin with a unusual interaction, presenting multiple interactions between them, forming a 1: 1 complex. Rhodniin contains 103 amino acids and structurally organized into two Kazal-type domains, linked via an acidic extended peptide fragment. Another inhibitor similar to rhodniin was described as dipetalogastin from the insect Dipetalogaster maximus (Mende et al., 1999). The cDNA of dipetalogastin codes for a huge protein which comprises of six Kazal-type domains. In 2002, another inhibitor similar to rhodniin was described as infestin from the kissing bug Triatoma infestans midgut, one of the imperative Chagas disease vectors (Campos et al., 2002). It is a double Kazal-type domain that strongly inhibits thrombin. Furthermore, a potent and specific inhibitor of the human coagulation thrombin activity was purified from salivary gland extracts of the tsetse fly, Glossina morsitans morsitans, and an important vector of African trypanosomiasis. It is a low molecular weight peptide (MW = 3,530 Da) and a potent inhibitor of thrombin-induced platelet aggregation (Cappello et al., 1996; Cappello et al., 1998).

Ticks have also been identified as important group of vectors of disease-causing agents to humans and livestock (Bior et al., 2002). The coagulation inhibitors and platelet aggregation inhibitors have been reported from the saliva of hard and soft ticks (Sauer et al., 1995; Bowman et al., 1997; Nuttall et al., 2000). Ornithodorin and savignin were similar proteins isolated from Ornithodoros moubata and Ornithodoros savignyi, respectively as thrombin inhibitors (Van De Locht et al., 1996; Joubert et al., 1998; Nienaber et al., 1999). Boophilin is also another interesting thrombin inhibitor isolated from the ixodid tick, Rhipicephalus ( Boophilus ) microplus, which has 12 cysteines distributed in two Kunitz-type domains that interact with thrombin by different manner when compared to that of hirudin or rhodniin (Horn et al., 2000). The salivary gland homogenate of the tick Rhodnius prolixus presents a 19kDa protein described Rhodnius prolixus aggregation inhibitor 1 (RPAI-1) that inhibits collagen-induced platelet aggregation by binding to ADP (Francischetti et al. 2000). The same effect has been observed with a molecule with similar sequence and structure named pallidipin isolated from saliva of Triatoma pallidipennis (Noeske-Jungblut et al. 1994). Mosquito saliva contains various anti-clotting agents for this purpose. Anophelin is a peptide from Anopheles albimanus saliva that behaves as an alpha-thrombin inhibitor and contributes for the anti-clotting phenomena observed in experimental essays (Valenzuela et al. 1999). The deerfly (Chrysops spp.) saliva has the potential to induce platelet aggregation triggered by ADP, thrombin and collagen. Also, it inhibits fibrinogen (Grevelink et al. 1993). Specially, ADP has a crucial function in hemostasis through induction of platelet aggregation and derives from activated platelets and injured cells (Vargaftig et al. 1981). Thus, the most common molecule involved in inhibition of platelet aggregation encountered on the majority of blood feeding arthropods are salivary apyrase enzyme, that hydrolyses ATP and ADP to AMP and orthophosphate. Aedes aegypti (Champagne et al. 1995), Anopheles (Area et al. 1999) and Culex mosquitoes (Nascimento et al. 2000) have apyrases in their saliva belonged to a family called 5'-nucleotidases. A new apyrase enzyme sequence has been found in the salivary glands of the haematophagous bed bug Cimex lectulahus (Valenzuela et al. 1998) and homologous sequences have been reported in the sand flies Lutzomia longipalpis (Charlab et al. 1999) and Phlebotomus papatasi (Valenzuela et al. 2001), indicating that this family of enzymes is widespread among arthropod species. The salivary apyrase from Triatoma infestans also belongs to the 5'-nucleotidase family (Faudry et al. 2004). Platelet function can be annoyed by substances that increase platelet cyclic adenosine monophosphate (cAMP) or cyclic guanosine monophosphate (cGMP). Previous study has shown that prostaglandin E2 (PGE2) and prostacyclin taken from tick's saliva can increase platelet cyclic nucleotides (Higgs et al. 1976).

Salivary anticoagulants of blood-feeding arthropods target specific proteases of the blood-coagulation cascade, blocking or delaying the clot formation process until the blood feeder finishes the meal (Ribeiro 1987). Thus, blood sucking insects have evolved diverse molecules responsible for these actions, which effectiveness also varies by species. Most of these salivary anticoagulant molecules are in different phases of molecular characterization and target components in the final common pathway of the coagulation cascade including factors II (thrombin), V and Xa. For instance, anophelin is a unique peptide isolated from the saliva of Anopheles albimanus that functions as a specific and tight-binding thrombin inhibitor (Noeske-Jungblut et al. 1995, Valenzuela et al. 1999). However, Aedes aegypti saliva contains a 48kDa peptide factor Xa inhibitor that was purified, cloned, expressed and shown to be a member of the serpin family of serine protease inhibitors (Stark and James 1998). The salivary gland extract of Culicoides variipennis contains a factor Xa inhibitor similar to all culicine mosquitoes (Perez de Leon et al. 1997). It is proposed that all anophelines have thrombin directed anticoagulants and culicine mosquitoes have factor Xa directed anticoagulants.

Triatomine bugs also evolved potent anticoagulants such as factors V and VIII inhibitors from Triatoma infestans (Pereira et al. 1996) and triabin, a salivary protein with 142 amino acide resides of Triatoma pallidipennis that selectively interacts with thrombin, exclusively via its fibrinogen recognition exosite (Fuentes-Prior et al. 1997). Prolixin S (nitrophorin 2) that Isolated from salivary gland extracts of Rhodnius prolixus inhibits coagulation factor Vlll-mediator activation of factor X and accounts for all the anti-clotting activity observed in its saliva (Ribeiro et al. 1995). Saliva of the hard tick and Lyme disease vector, Ixodes scapularis, was genetically sequenced and inhibitor was identified as ixolaris, with 140 amino acids. Observations of ixolaris function evidenced the blockage of factor Xa production by endothelial cells expressing tissue factor (Francischetti et al. 2002). Heparin was detected in the salivary gland duct, salivary glands, and midgut of many mosquito species including Aedes togoi (Ha et al., 2014). The mean concentration of heparin is higher in blood-fed female mosquitoes compared to non-blood fed females. Usually, it is secreted to induce the coagulation cascade. Heparin levels are largely increased during the salivation of a blood-feeding mosquito (Ha et al., 2014).

Protamine is well known for its action in inhibiting heparin. Protamines are a group of low molecular weight polypeptides similar to histone proteins in function, which binds to the genetic material of spermatids of many animals and in plants which condenses the genome to a genetically inactive state (Balhorn, 2007). Protamine displays strong alkaline properties that could be ascribed to arginine which accounts to more than 67% of the amino acid composition. This polypeptide is mainly used to reverse the action of heparin anticoagulant, contains 32 amino acids and neutralizes the effects of heparin by electrostatically binding with the anionic heparin and producing a salt precipitate (Rossmann et al., 1982). This has also been explained as a binding of alkaline protamine with acidic heparin to form a neutral precipitate (Nybo & Madsen, 2008). Protamines were first extracted from salmon fish sperm where now it is mainly synthesized through recombinant biotechnology (Boer, 2018).

Protamine has been used for decades as an ingredient for preparation of crystalline insulin, named as Neutral Protamine Hagedorn (NPH) insulin to delay absorption and prolong the action of insulin (Nybo & Madsen, 2008; Yip et al., 2000). The other major use of protamine has been its use as a treatment for heparin overdose. For instance, protamine is used to reverse the action of heparin used to delay coagulation of blood during major cardio vascular surgeries (Borchers, 2015).

Since protamine is known to have caused adverse reactions in patients that undergo cardiovascular surgery with prior administrations of heparin, it is shown that maintaining dosing ratios of heparin and protamine at 1:1 has reduced the adverse drug reactions associated (Boer et al., 2018). However, in an experimental study healthy individual have been administered a protamine sulfate dose of 0.5mg /kg as an intravenous infusion over a period of 10 mins to analyze adverse drug reactions, in which the researcher has observed minimal adverse reactions (Butterworth et al., 2002). Apart from its use as a drug for treatment purposes protamine has been used when producing vaccines such as Japanese encephalitis vaccines to decontaminate the vaccine from other contaminating DNA and proteins (Ding et al., 1998).

The half-life of protamine sulphate has shown to be close to 10 mins in normal individuals without a heparin administration, which is substantially short. (Butterworth et al., 2002). This has shown that the circulating levels of protamine falls below detectable levels after around 20 mins of administration. When administered to cardiovascular surgery patients that have received heparin doses of 250 mg, the half-life was highly variable (1.9-18) with a median of 4.5 mins and the plasma clearance rate was observed to be a median of 1.4 liters/ min (Boer et al., 2018). This short half-life in humans limits its use as a drug which is given as a single dose expecting its effects for extensive periods. It has been observed that the action of protamine against heparin is largely dependent on the size of the heparin molecules where smaller fragments of heparin being more difficult to neutralize than larger molecules (Schroeder et al., 2011).

The dose of protamine used for counteracting the effects of heparin has to match the amount of Heparin in 1:1 ratio to prevent the anticoagulant effects the chemical to come into action (Boer et al. , 2018). This anticoagulant property has been ascribed to its inhibition of conversion of prothrombin to thrombin by the excess protamine sulfate that does not bind with heparin (Tocantins, 1943). Further protamine interferes with hemodynamics in several other methods such as inhibition of coagulating factors, and reduction of clot strength through fibrinolysis and by reducing platelet function (Boer et al., 2018).

Vector-borne diseases are among the most important global public health problems and are associated with significant economic burden in many of the affected countries. These diseases are transmitted by hematophagous arthropods, including mosquitoes, ticks, sand flies, and triatomine bugs. Most vector-borne diseases exist in complex zoonotic cycles involving a variety of birds, rodents, and other vertebrate hosts. The emergence and re-emergence of vector-borne diseases in the past 40 years has been driven by population growth, urbanization, globalization, and lack of public health infrastructure. Vector borne diseases are highly prevalent in tropical and subtropical regions of the world. Among those, mosquitos play a major role in transmitting deadly diseases like dengue hemorrhagic fever, Chickungunya, malaria, yellow fever, Japanese encephalitis, Zika virus and West Nile virus. Preventing or reducing the number of bites caused by mosquitos is considered an effective method of controlling the spread of above diseases. The main means of preventing bites have been based on keeping away mosquitos from host using natural and synthetic repellant products. But most of the methods developed have been proven to be ineffective over time.

A mosquito repellant would only repel but not kill mosquitos and this leads to more bites to those who are not using the repellant (Maia et al., 2013). Therefore, a method that would kill mosquitos after a blood meal is more advantageous since it reduces the mosquito number itself. A study performed in USA revealed a novel method to cause death of mosquitos after consuming a blood meal using RNA interference (RNAi) technology. But the safety of this technique involving gene silencing has to be further evaluated and there is a potential for resistant gene development in mosquitos (“ Blood-Sucking Deadly for Mosquitoes | UANews,” 2011.)

Technical problem

Finding a method to inhibit the action of anticoagulants in mosquito saliva to prevent the normal mechanism of blood feeding by mosquitos.

Technical solution

Administering a chemical/substance (e.g. protamine sulfate) that counter-acts the effects of anticoagulants in mosquito saliva.

Advantageous effects

Provides protection against mosquito bites occurring in any part of the user’s body.

Unlike mosquito repellants that only divert mosquitos to unprotected hosts, this product causes death of mosquitos that will reduce the mosquito population. The effective duration is higher compared to topical applications thus leading to less frequent administration especially if combined with nanotechnology where the circulation times of the chemical/substance that counter-acts the effects of anticoagulants in mosquito saliva can be greatly enhanced.

Mode for invention

Protamine sulfate is a known anti-heparin agent used in current medicine and surgery. It has been used commonly to reverse the effects of heparin after cardiovascular surgery (Boer, Meesters, Veerhoek, & Vonk, 2018). Protamine neutralizes the activity of heparin by binding electrostatically and forming a protamine-heparin salt (Boer et al. , 2018).

Anticoagulants like heparin are found in mosquito saliva and are injected to the bite site together with saliva, before and during sucking to facilitate free flow of blood without clotting (Ha et al., 2014). If the anticoagulant function of compounds in mosquito saliva can be counteracted, the normal blood sucking mechanism can be eliminated. Subsequently, death of the mosquito will occur when blood begins to clot inside the mosquito’s gut. Further, blood serves as a vital source of nutrients for egg production in mosquitos, lack of blood will lead to reduction in mosquito population growth. When administered systemically protamine sulfate will be available throughout the body and will counter act the effects of anticoagulants in mosquito saliva that is being injected at any part of the host’s body.

In a preliminary study to determine the effects of protamine, 2 groups of rats with similar age, sex and body weight (>200 g) were selected. To facilitate feeding by mosquitos a square shaped area with dimensions (~10 cm ² ) was shaved in each rat of both groups. Then rats of the test group were injected with a single dose of 1% protamine sulfate subcutaneous at dose rate of 25 mg/kg. Rats of control group were injected with an equal volume of sterile saline. Five days old 40 individual mosquitoes (20 each from Ar. subalbatus and Ae. albopictus) were selected and kept in fine mesh mosquito rearing cages (50x50x50 cm). Two laboratory rats were inserted to these cages separately after one hour and they were kept about four hours inside these cages from 4.00 p.m. to 8.00 p.m. and their feeding behavior was recorded and observed. A 100% reduction in blood feeding by Ar. subalbatus mosquitos in rats with protamine sulfate injection was observed while 62.5% of the Ar. subalbatus mosquitos fed on rats injected with protamine sulfate died. Ar. subalbatus saliva contains heparin whereas Ae. albopictus does not contain heparin in their saliva and normal blood feeding of Ae. albopictus could be observed.

Industrial applicability

Mass production of a product comprising the active ingredient (e.g. protamine sulfate) can be achieved, since the demand for an effective method for control of vector-borne disease is growing rapidly. Also, by encapsulating the active ingredient in a nanoparticle can extend the circulation time of the active ingredient and increase the safety of administering the active ingredient. Further, if the encapsulated active ingredient in a nanoparticle can be loaded in to red blood cells an extended duration of circulation of the active ingredient in blood up to 3 months can be achieved where single administration can protect the host for 3 months against mosquito bites. References

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