Reducing disease transmission of mediator diseases

Abstract

The present application relates to compounds and pharmaceutically acceptable salts thereof, compositions thereof, and their use in reducing or preventing the propagation of parasitic or vector diseases.

Description

Technical Field

[0001] The present application relates to live bacteria and compounds and pharmaceutically acceptable salts thereof, compositions thereof, and their use in reducing or preventing the transmission of parasitic or vector borne diseases. Background Art

[0002] Infectious diseases are responsible for a wide range of medically and veterinary diseases. Many of these diseases are transmitted by insect vectors. Vector-borne diseases are diseases that are transmitted through the bites of infected arthropod species (e.g., mosquitoes, ticks, triatomine bugs, sand flies, black flies) as well as ectoparasites (e.g., ticks and fleas).

[0003] Mosquitoes are vectors of many infectious diseases. Specifically, three medically relevant mosquito genera that transmit disease are Anopheles, Culex, and Aedes. Culex and Aedes belong to the subfamily Culexinae, while Anopheles belongs to the subfamily Anophelesinae. Anopheles is a vector of malaria and filariasis. Culex is a vector of Japanese encephalitis, other viral diseases, and filariasis. Aedes is a vector of dengue virus, chikungunya, Mayaro virus, Zika virus, yellow fever, Japanese encephalitis, West Nile virus, and filariasis.

[0004] Filariasis is a disease caused by roundworm parasites of the order Filariales and is spread by blood-feeding insects such as black flies and mosquitoes. These are divided into several categories based on where they affect. Lymphatic filariasis is caused by Wuchereria bancrofti, Brugia malayi, and Brugia imperialis, which occupy the lymphatic system and can cause elephantiasis. Subcutaneous filariasis is caused by Loa (eye worm), Chain worm, and Onchocerca volvulus, which occupy only a layer under the skin. Severe cavitary filariasis is caused by the worms Mansonella perstans and Mansonella ozzardi, which occupy the serous cavity of the abdomen. Circulating microfilariae can be ingested during the insect vector's blood meal; in the vector, they develop into infective larvae that can be transmitted to others.

[0005] Zika virus disease is caused by a member of the Flaviviridae family. It is transmitted by diurnal Aedes mosquitoes, such as Aedes aegypti and Aedes albopictus. Symptoms include fever, red eyes, joint pain, headache, and a maculopapular rash. Most cases are asymptomatic, but when symptoms occur, they are usually mild, can resemble dengue fever, and typically last less than seven days. Infection during pregnancy may cause microcephaly and other brain malformations in infants.

[0006] West Nile virus is a single-stranded RNA virus that causes West Nile fever. It is a member of the Flaviviridae family and is primarily transmitted by mosquitoes of the genus Culex. About 80% of infected people experience few or no symptoms. Fever, headache, vomiting, or rash occur in 20% of infected people. Fewer than 1% of people develop encephalitis or meningitis, with associated neck stiffness, confusion, or seizures, and the case fatality rate is about 10%. Recovery may take weeks to months, and patients with neurologic infections have a 10% risk of death.

[0007] Chikungunya virus is a member of the genus Alphavirus and the family Togaviridae. It is an RNA virus with a positive-stranded, single-stranded genome of 11.7 kb. Chikungunya virus causes chikungunya disease, a disease characterized by fever and joint pain that typically develops 2-12 days after exposure. The mortality risk is approximately 1 in 1,000. The disease infects an estimated 3 million people annually. Chikungunya disease is primarily found in developing countries, but epidemics in the Indian Ocean, Pacific Islands, and the Americas continue to alter the disease's distribution.

[0008] Mayaro virus disease is caused by a virus of the family Togaviridae, specifically a member of the genus Alphavirus. Symptoms include fever, headache, myalgia, rash, significant pain in large joints, and association with rheumatic disease. It is known to circulate in South America. MAYV transmission is primarily maintained through a sylvatic cycle involving non-human primates and mosquitoes of the genus Haemoglossus.

[0009] Sand flies are vectors of Leishmania. There are three main forms of leishmaniasis: cutaneous leishmaniasis, mucocutaneous leishmaniasis, and visceral leishmaniasis. Cutaneous leishmaniasis is the most common form of leishmaniasis. Visceral leishmaniasis is the most severe form, in which the parasite migrates to vital organs. Visceral leishmaniasis is caused by the parasite Leishmania donovani and can be fatal if untreated. Leishmaniasis is prevalent in developing countries, with approximately 90% of visceral leishmaniasis cases worldwide occurring in India, Bangladesh, Nepal, Sudan, and Brazil. Leishmaniasis affects 12 million people worldwide, with 1.5 million to 2 million new cases each year. The estimated annual incidence of the visceral form of leishmaniasis is 500,000 new cases and 60,000 deaths. Kabul is estimated to be the world's largest epicenter of cutaneous leishmaniasis, with approximately 67,500 cases as of 2004.

[0010] Kissing bugs, also known as trypanosomes, are members of the subfamily Trypanoideae of the family Triatominae. They are the vectors of Chagas disease (also known as American trypanosomiasis). Chagas disease is a parasite caused by the flagellate protozoan Trypanosoma cruzi. Chagas disease is generally prevalent in the Americas and is endemic in poor rural areas of Mexico, Central America, and South America. An estimated 10-15 million people are infected with Chagas disease each year, and approximately 14,000 die each year. The symptoms of Chagas disease vary over the course of the infection. In the early (acute) stage, symptoms are mild, usually localized swelling at the site of infection. After 4-8 weeks, individuals with active infection enter the chronic stage of Chagas disease, and 60%-80% of chronically infected individuals remain asymptomatic throughout their lifetime. However, the remaining 20% ​​to 40% of infected people will develop debilitating and sometimes life-threatening medical problems during their lifetime. Chagas disease is treated with nifurtimox and benznidazole, both of which cause significant side effects and have negligible benefits in chronic disease.

[0011] The tsetse fly (Glossina tsetse) is the vector of human African trypanosomiasis. Human African trypanosomiasis, also known as African sleeping sickness, is a parasitic disease caused by the protozoan Trypanosoma brucei. There are two forms of the disease, depending on the subspecies of the parasite. Trypanosoma brucei gambiense accounts for 95% of reported cases and occurs in West and Central Africa, causing a chronic infection. Trypanosoma brucei rhodesiense is found in eastern and southern Africa and accounts for approximately 5% of cases.

[0012] These diseases are of great medical importance. Many drugs are available to treat and / or prevent some parasitic or vector-borne diseases. However, not all parasitic or vector-borne diseases can be effectively treated. For example, there are currently no chemotherapy drugs or vaccines available for the dengue virus. Furthermore, with regard to antimalarial drugs, treatment with currently available drugs is becoming less effective due to increasing resistance among some Plasmodium strains. Therefore, there is a need for effective control of parasites and disease vectors to prevent transmission. In this regard, mosquitoes can be targeted by a wide range of insecticides and repellents. Mosquitoes can be targeted with insecticides while they are larval or once they have developed into adults. However, mosquitoes have developed widespread resistance to currently used insecticides.

[0013] One approach to addressing this problem is to develop agents that can reduce or prevent the spread of vector-borne diseases without negatively impacting insect vectors, thereby avoiding the development of drug resistance. In this regard, PCT / EP2020 / 069569 (published as WO 2021 / 009050) discloses bacteria of the genus Delftia and their use in reducing the transmission of mosquito-borne malaria. Furthermore, there is an ongoing need to develop new methods for reducing or preventing the spread of vector-borne diseases. Summary of the Invention

[0014] According to a first aspect of the present invention, there is provided a composition for use in a method of reducing or preventing the transmission of a vector-borne disease, wherein the composition comprises 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and wherein the method comprises the step of contacting at least one vector with the composition.

[0015] According to a second aspect of the present invention, there is provided a composition for use in a method of reducing or preventing the transmission of parasites, wherein the composition comprises 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and wherein the method comprises the step of contacting at least one vector with the composition.

[0016] In a third aspect of the present invention, there is provided a method for reducing or preventing vector-borne diseases or parasitic infections, comprising the step of contacting at least one vector of the vector-borne disease with 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof.

[0017] In a further aspect of the present invention, there is provided use of 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof in reducing or preventing vector-borne diseases or parasitic infections.

[0018] In a further aspect of the present invention, there is provided 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof for use in inhibiting the growth of parasites.

[0019] In a further aspect of the present invention, there is provided a nectar feed comprising 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and one or more sugar sources. Suitably, the nectar feed is used to reduce or prevent vector-borne diseases or parasitic diseases.

[0020] In any of the aspects listed above, the vector-borne or parasitic disease can be selected from human African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi disease, Bourbon virus, Borreliamayonii disease, Chikungunya virus, Chagas disease, Cryptosporidium, Dirofilariasis, Eastern equine encephalitis, Ehrlichia muris-like disease, Ehrlichiosis, Filariasis, Heartland Virus, Japanese encephalitis, Leishmaniasis, Lyme disease, Mayaro virus disease, Pacific Coast tick fever, Rocky Mountain spotted fever, St. Louis encephalitis, Tularemia, Western equine encephalitis, West Nile virus, Yellow fever and Zika virus.

[0021] The present invention may be advantageous in many ways. Specifically, the inventors discovered that the compound 1-methyl-9H-pyrido[3,4-b]indole (also known as harman or 1-methyl-β-carboline) is produced by bacteria of the genus Delftia, which is responsible for preventing the spread of vector-borne diseases and parasites in vectors (e.g., mosquitoes, sand flies, tsetse flies, Triatominae, etc.). In some embodiments, the 1-methyl-9H-pyrido[3,4-b]indole, harman, and 1-methyl-β-carboline are produced by bacteria of the genus Delftia. When introduced into an environment containing a vector, the composition of the present invention prevents parasites from developing in the vector and thus interrupts disease transmission. 1-methyl-9H-pyrido[3,4-b]indole can be used to combat the spread of parasites and vector-borne diseases.

[0022] In a further aspect of the invention, there is provided a composition for use in a method of reducing or preventing the transmission of a vector-borne disease, wherein the composition comprises a bacterium of the genus Delftia, and wherein the method comprises the step of contacting at least one vector with the composition such that the vector orally ingests the composition, wherein the vector-borne disease is selected from the group consisting of human African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi, Bourbon virus, Borrelia merini, chikungunya virus, Chagas disease, dirofilariasis, eastern equine encephalitis, murine ehrlichiosis, ehrlichiosis, filariasis, central plain virus, Japanese encephalitis, leishmaniasis, Lyme disease, Mayaro virus disease, Pacific Coast tick fever, Rocky Mountain spotted fever, St. Louis encephalitis, tularemia, western equine encephalitis, West Nile virus, yellow fever, and Zika virus.

[0023] In a further aspect of the invention, a composition for use in a method of reducing or preventing the transmission of a parasite is provided, wherein the composition comprises a bacterium of the genus Delftia, and wherein the method comprises the step of contacting at least one vector with the composition such that the vector orally ingests the composition, wherein the parasitic disease is selected from the group consisting of human African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi, Borrelia meijinii, Chagas disease, cryptosporidiosis, dirofilariasis, murine ehrlichiosis, ehrlichiosis, filariasis, leishmaniasis, Lyme disease, Pacific Coast tick fever, Rocky Mountain spotted fever, and tularemia. The bacterium comprises the genus Delftia.

[0024] In a further aspect of the invention, a bacterium of the genus Delftia is provided for use in reducing or preventing the transmission of a disease selected from the group consisting of human African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi, Bourbon virus, Borrelia merini, Chikungunya virus, Chagas disease, Cryptosporidium, dirofilariasis, Eastern equine encephalitis, murine ehrlichiosis, ehrlichiosis, filariasis, Central Plains virus, Japanese encephalitis, leishmaniasis, Lyme disease, Mayaro virus disease, Pacific Coast tick fever, Rocky Mountain spotted fever, St. Louis encephalitis, tularemia, Western equine encephalitis, West Nile virus, yellow fever, and Zika virus.

[0025] Herein, the bacterium of the genus Delftia may be appropriately referred to as Delftia tsuruhatensis TC1. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The invention is further described with reference to the accompanying drawings, which are non-limiting.

[0027] Figure 1 Shown is the liquid chromatography-high-resolution accurate mass spectrometry trace of the active components of Delftia tsuruhata.

[0028] Figure 2 Heteronuclear single quantum coherence spectra of the active components of Delftia tsuruhada are shown.

[0029] Figure 3 Shows the active ingredients of Delftia tsuruhae 1 H NMR spectrum.

[0030] Figure 4 shows the effect of Delftia tsuruhada on the establishment of Leishmania parasites in the midgut of sand flies.

[0031] Figure 5 shows the effects of i) feeding and (ii) timing of exposure to D. tsuruhada on the establishment of Leishmania parasites in the sand fly midgut.

[0032] Figure 6 The effects of a second blood meal on Leishmania macrophylla subspecies parasites in sand flies fed Delftia tsuruhada are shown.

[0033] Figure 7 The effects of Delftia tsuruhadensis on sand flies naturally infected by biting mice with infected skin lesions are shown.

[0034] FIG8 shows the effect of exposure to Delftia tsuruhata on mortality in sand flies.

[0035] Figure 9Ashowed the effect of other bacteria such as Escherichia coli or Ornithinibacillus massiliensis on the establishment of Leishmania parasites in the midgut of sand flies.

[0036] Figure 9B The effect of Delftia sutsuruhae on the establishment of Leishmania parasites in the midgut of sand flies is shown.

[0037] FIG10 shows the effect of exposing sand flies to Delftia tsuruhada on the transmission of Leishmania to mice.

[0038] Figure 11 The effect on Zika virus proliferation in mosquitoes that ingested Harman was shown. DETAILED DESCRIPTION

[0039] In one aspect, the present invention provides a composition for use in a method of reducing or preventing the transmission of a vector-borne disease, wherein the composition comprises a bacterium of the genus Delftia and / or 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and wherein the method comprises the step of contacting at least one vector with the composition.

[0040] In some embodiments, the composition comprises a bacterium of the genus Delftia. In some embodiments, the composition comprises 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof.

[0041] As used herein, the term "vector-borne zoonotic diseases" refers to diseases that naturally infect wild animals and are then transmitted to humans by carriers or vectors (e.g., mosquitoes, ticks, tsetse flies, black flies, Triatominae, and sand flies). As used herein, the term "vector-borne diseases" refers to diseases that naturally infect wild animals and are then transmitted to animals by carriers or vectors (e.g., mosquitoes, ticks, tsetse flies, black flies, Triatominae, and sand flies). In some embodiments, the animal is a human. In other embodiments, the animal is selected from a dog, cat, hamster, cow, sheep, goat, pig, rabbit, duck, turkey, horse, chicken, yak, donkey, buffalo, camel, or other domesticated animal.

[0042] Delftia is a genus of Gram-negative Aminobacteria belonging to the class Betaproteobacteria and the family Comamonadaceae.

[0043] The bacterium of the genus Delftia can be any bacterium of the genus Delftia. In one embodiment of the present invention, the bacterium of the genus Delftia is Delftia tsuruhada. Bacterial strain TC1 was deposited on May 21, 2019, under the BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland) with accession number NCIMB 43398. The bacterium was isolated and identified as Delftia tsuruhada by 16S rRNA sequencing. It is a Gram-negative bacterium belonging to the class Betaproteobacteria and the family Comamonadaceae. In one embodiment of the present invention, the bacterium of the genus Delftia is a bacterium deposited on May 21, 2019, under the BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA Scotland) with accession number NCIMB 43398, and the whole genome sequence has been deposited on October 14, 2022 with NCBI (National Center for Biotechnological Information, National Library of Medicine, 8600 Rockville Pike, Bethesda, MD 20894, United States) with accession number PRJNA890603.

[0044] 1-Methyl-9H-pyrido[3,4-b]indole is also known as Harman, and is represented by the following structure:

[0045] .

[0046] This compound was found to be an active compound secreted by bacteria of the genus Delftia and is capable of inhibiting the transmission of parasites in a variety of vectors. Thus, the composition of the present invention can reduce or prevent disease or parasite transmission in mosquitoes.

[0047] It should also be understood that the compounds of the present invention, such as compounds of formula (I), may exist in different tautomeric forms. Tautomers refer to isomeric forms of a compound that are in equilibrium with each other. The concentration of the isomeric forms will depend on the environment in which the compound is located.

[0048] The compound may also be protonated or deprotonated depending on the pH of its surroundings. The compound may also be in the form of a pharmaceutically acceptable salt. Pharmaceutically acceptable salts include, but are not limited to, those described in Berge, J. Pharm. Sci., 1977, 66, 1-19, or those listed in P. H. Stahl and C. G. Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection and Use, Second Edition, John Wiley & Sons, March 2011.

[0049] If the function of the compound allows, suitable pharmaceutically acceptable salts of the compound of formula (I) may be formed, including acid addition salts or base addition salts. Acid addition salts may be formed by reacting with a suitable acid, optionally in a suitable solvent (e.g., an organic solvent), to obtain a salt that can be separated by crystallization and filtration. Base addition salts may be formed by reacting with a suitable base, optionally in a suitable solvent (e.g., an organic solvent), to obtain a salt that can be separated by crystallization and filtration.

[0050] Representative pharmaceutically acceptable acid addition salts include, but are not limited to, 4-acetamidobenzoate, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate (besylate), benzoate, bisulfate, bitartrate, butyrate, calcium edetate, camphorate, camphorsulfonate (dextrorotatory camphorsulfonate), caprate (decanoate), caproate (hexanoate), caprylate (caprylate), and octanoate. octanoate), cinnamate, citrate, cyclamate, digluconate, 2,5-dihydroxybenzoate, disuccinate, lauryl sulfate (propionate lauryl sulfate), edetate (ethylenediaminetetraacetate), propionate lauryl sulfate (lauryl sulfate), ethane-1,2-disulfonate (edisylate), ethanesulfonate (ethanesulfonate), formate, fumarate, galactarate (mucates), gentisate (2,5-dihydroxybenzoate), glucoheptonate (glucoheptonate Gluconate, glucuronate, glutamate, glutarate, glycerophosphorate, glycolate, hexylresorcinate, hippurate, hydrabamine (N,N'-di(dehydroabietyl)-ethylenediamine), hydrobromide, hydrochloride, hydroiodide, hydroxynaphthoate, isobutyrate, lactate, lactobionate, laurate, malate, maleate, malonate, mandelate, methanesulfonate (mesylate), methylsulfate, mucate, naphthalene-1,5-disulfonate (naphthalenedisulfonate), naphthalene- 2-sulfonate (naphthalenesulfonate), nicotinate, nitrate, oleate, palmitate, p-aminobenzenesulfonate, p-aminosalicylate, pamoate (spicate), pantothenate, pectinate, persulfate, phenylacetate, phenethylbarbiturate, phosphate, polygalacturonate, propionate, p-toluenesulfonate (toluenesulfonate), pyroglutamate, pyruvate, salicylate, sebacate, stearate, subacetate, succinate, sulfamate, sulfate, tannate, tartrate, teoclate (8-chlorotheophylline salt), thiocyanate, triethyl iodide, undecanoate, undecenoate, and valerate.

[0051] Representative pharmaceutically acceptable base addition salts include, but are not limited to, aluminum, 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS, tromethamine), arginine, phenethamine (N-benzylphenethylamine), benzathine (N,N'-dibenzylethylenediamine), bis-(2-hydroxyethyl)amine, bismuth, calcium, chloroprocaine, choline, clemizole (1-p-chlorobenzyl-2-pyrrolidin-1'-ylmethylbenzimidazole), cyclohexylamine, dibenzylethylenediamine, diethylamine, diethyltriamine, dimethylamine, dimethylethanolamine, dopamine, ethanolamine, ethylenediamine, L-histidine, iron, isoquinoline, lepidine, lithium, lysine, magnesium, meglumine (N-methylglucamine), piperazine, piperidine, potassium, procaine, quinine, quinoline, sodium, strontium, tert-butylamine, and zinc.

[0052] The compound will be administered in an appropriate "effective amount." This effective amount will depend on a variety of factors including, for example, the size and weight of the subject, the precise condition being treated and its severity, the nature of the formulation, and the route of distribution, and will ultimately be determined by one skilled in the art.

[0053] In some embodiments, the vector-borne disease is selected from the group consisting of African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi, Bourbon virus, Borrelia merini, Chikungunya virus, Chagas disease, dirofilariasis, Eastern equine encephalitis, murine ehrlichiosis, ehrlichiosis, filariasis, Central Plains virus, Japanese encephalitis, leishmaniasis, Lyme disease, Mayaro virus disease, Pacific Coast tick fever, Rocky Mountain spotted fever, St. Louis encephalitis, tularemia, Western equine encephalitis, West Nile virus, yellow fever, and Zika virus.

[0054] In some embodiments, the composition comprises a bacterium of the genus Delftia, and the vector-borne disease is selected from the group consisting of African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi, Bourbon virus, Borrelia merini, Chikungunya virus, Chagas disease, dirofilariasis, Eastern equine encephalitis, murine ehrlichiosis, ehrlichiosis, filariasis, Central Plains virus, Japanese encephalitis, leishmaniasis, Lyme disease, Mayaro virus disease, Pacific Coast tick fever, Rocky Mountain spotted fever, St. Louis encephalitis, tularemia, Western equine encephalitis, West Nile virus, yellow fever, and Zika virus.

[0055] In some embodiments, the composition comprises 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and the vector-borne disease is selected from the group consisting of African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi, Bourbon virus, Borrelia merini, Chikungunya virus, Chagas disease, dirofilariasis, Eastern equine encephalitis, murine ehrlichiosis, ehrlichiosis, filariasis, Central Plains virus, Japanese encephalitis, Lyme disease, Mayaro virus disease, Pacific Coast tick fever, Rocky Mountain spotted fever, St. Louis encephalitis, tularemia, Western equine encephalitis, West Nile virus, yellow fever, and Zika virus.

[0056] In a second aspect, the present invention provides a composition for use in a method of reducing or preventing the transmission of parasites, wherein the composition comprises a bacterium of the genus Delftia and / or 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and wherein the method comprises the step of contacting at least one vector with the composition.

[0057] In some embodiments, the parasitic disease is selected from human African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi, Borrelia merini, Chagas disease, cryptosporidium, dirofilariasis, Ehrlichia muris infection, ehrlichiosis, filariasis, leishmaniasis, Lyme disease, Pacific Coast tick fever, Rocky Mountain spotted fever, and tularemia.

[0058] In some embodiments, the composition for use in a method of reducing or preventing the transmission of a parasite comprises a bacterium of the genus Delftia, and the parasitic disease is selected from human African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi, Borrelia meijinii, Chagas disease, Cryptosporidium, dirofilariasis, Ehrlichia muris infection, Ehrlichiosis, filariasis, leishmaniasis, Lyme disease, Pacific Coast tick fever, Rocky Mountain spotted fever, and tularemia.

[0059] In some embodiments, the composition for use in a method of reducing or preventing parasitic transmission comprises 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and the parasitic disease is selected from human African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi, Borrelia merini, Chagas disease, cryptosporidiosis, dirofilariasis, Ehrlichia muris infection, Ehrlichiosis, filariasis, Lyme disease, Pacific Coast tick fever, Rocky Mountain spotted fever, and tularemia.

[0060] Specifically, it has been demonstrated herein that, when introduced into an environment containing a vector, Harman can prevent the transmission of parasites. In some embodiments, introduction into the vector can occur through contact with sugar baits, nectar baits, blood baits, and / or other feeding baits, whereby the Delftia bacteria and / or 1-methyl-9H-pyrido[3,4-b]indole can be transmitted to or enter the vector via epidermal absorption and / or ingestion. Thus, the compositions of the present invention can reduce or prevent disease transmission and / or parasite transmission in the vector. The vector can be any vector capable of transmitting disease, and in some embodiments, can be a mosquito, such as an Anopheles species. It is contemplated that the compositions and methods of the present invention extend to mosquitoes of any Anopheles species. In one embodiment, the mosquito is Anopheles gambiae, Anopheles stephensi, Anopheles cucullatus, or Anopheles coluzzi. In embodiments of the present invention, the mosquito is Anopheles gambiae or Anopheles stephensi. In embodiments, the mosquito is Anopheles stephensi. In another embodiment, the mosquito is Anopheles gambiae.

[0061] In some embodiments, the composition is used to reduce or prevent: (i) Zika virus, and / or (ii) Zika virus transmission. In some embodiments, the composition is used to reduce or prevent: (i) Zika virus, and / or (ii) Zika virus transmission in mosquitoes. In some embodiments, the composition is used to reduce or prevent Zika virus. In some embodiments, the composition is used to reduce or prevent the transmission of Zika virus in mosquitoes. The mosquito can be any mosquito. In some embodiments, the mosquito is a mosquito of the genus Aedes. In some embodiments, the mosquito is Aedes albopictus or Aedes aegypti.

[0062] In some embodiments, the composition is used to reduce or prevent: (i) chikungunya virus, and / or (ii) chikungunya virus transmission. In some embodiments, the composition is used to reduce or prevent: (i) chikungunya virus, and / or (ii) chikungunya virus transmission in mosquitoes. In some embodiments, the composition is used to reduce or prevent chikungunya virus. In some embodiments, the composition is used to reduce or prevent chikungunya virus transmission in mosquitoes. The mosquito can be any mosquito. In some embodiments, the mosquito is a mosquito of the genus Aedes. In some embodiments, the mosquito is Aedes albopictus or Aedes aegypti.

[0063] In some embodiments, the composition is used to reduce or prevent: (i) filariasis or diofilariasis, and / or (ii) the spread of filariasis or diofilariasis. In some embodiments, the composition is used to reduce or prevent: (i) filariasis or diofilariasis, and / or (ii) the spread of filariasis or diofilariasis in mosquitoes. In some embodiments, the composition is used to reduce or prevent filariasis or diofilariasis. In some embodiments, the composition is used to reduce or prevent the spread of filariasis or diofilariasis in mosquitoes. The mosquito can be any mosquito. In some embodiments, the mosquito is a mosquito of the genus Aedes. In some embodiments, the mosquito is Aedes albopictus or Aedes aegypti.

[0064] In some embodiments, the composition is used to reduce or prevent: (i) Mayaro virus, and / or (ii) Mayaro virus transmission. In some embodiments, the composition is used to reduce or prevent: (i) Mayaro virus, and / or (ii) Mayaro virus transmission in mosquitoes. In some embodiments, the composition is used to reduce or prevent Mayaro virus. In some embodiments, the composition is used to reduce or prevent the transmission of Mayaro virus in mosquitoes. The mosquito can be any mosquito. In some embodiments, the mosquito is a mosquito of the genus Aedes. In some embodiments, the mosquito is Aedes albopictus or Aedes aegypti.

[0065] In some embodiments, the composition is used to reduce or prevent: (i) West Nile virus, and / or (ii) West Nile virus transmission. In some embodiments, the composition is used to reduce or prevent: (i) West Nile virus, and / or (ii) West Nile virus transmission in mosquitoes. In some embodiments, the composition is used to reduce or prevent the transmission of West Nile virus in mosquitoes. The mosquito can be any mosquito. In some embodiments, the mosquito is a mosquito of the genus Aedes. In some embodiments, the mosquito is Aedes albopictus or Aedes aegypti.

[0066] In some embodiments, the composition is used to reduce or prevent: (i) Chagas disease, and / or (ii) Chagas disease transmission. In some embodiments, the composition is used to reduce or prevent the transmission of Chagas disease in the subfamily Triatinae. In some embodiments, the composition is used to reduce or prevent the transmission of Chagas disease parasites in the subfamily Triatinae. In some embodiments, the subfamily Triatoma is Triatoma dimidiate.

[0067] In some embodiments, the composition is used to reduce or prevent: (i) human African trypanosomiasis, and / or (ii) transmission of human African trypanosomiasis. In some embodiments, the composition is used to reduce or prevent: (i) human African trypanosomiasis, and / or (ii) transmission of human African trypanosomiasis in tsetse flies. In some embodiments, the composition is used to reduce or prevent human African trypanosomiasis. In some embodiments, the composition is used to reduce or prevent transmission of human African trypanosomiasis in tsetse flies. In some embodiments, the tsetse fly is a member of the genus Glossina. In some embodiments, the tsetse fly is a palpalis tsetse fly.

[0068] In some embodiments, the composition is used to reduce or prevent: (i) leishmaniasis, and / or (ii) the transmission of leishmaniasis. In some embodiments, the composition is used to reduce or prevent: (i) leishmaniasis, and / or (ii) the transmission of leishmaniasis in sand flies. In some embodiments, the composition is used to reduce or prevent leishmaniasis. In some embodiments, the composition is used to reduce or prevent the transmission of leishmaniasis in sand flies. In some embodiments, the sand flies are of the genus Phlebotomus or Lumbago.

[0069] In some embodiments, the composition comprises a bacterium of the genus Delftia, and the composition is used to reduce or prevent: (i) leishmaniasis, and / or (ii) the transmission of leishmaniasis. In some embodiments, the composition is used to reduce or prevent: (i) leishmaniasis, and / or (ii) the transmission of leishmaniasis in sand flies. In some embodiments, the composition is used to reduce or prevent leishmaniasis. In some embodiments, the composition is used to reduce or prevent the transmission of leishmaniasis in sand flies. In some embodiments, the sand fly is of the genus Phlebotomus or Lumbago.

[0070] In some embodiments, the composition is used to reduce or prevent: (i) Cryptosporidium, and / or (ii) the transmission of Cryptosporidium. In some embodiments, the composition is used to reduce or prevent Cryptosporidium. In some embodiments, the composition is used to reduce or prevent the transmission of Cryptosporidium.

[0071] In another aspect, the present invention provides a method for reducing or preventing the transmission of a vector-borne disease or parasite, the method comprising contacting at least one vector of the vector-borne disease or parasite with a Delftia bacterium and / or 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof. The vector-borne disease or parasite may be as defined above.

[0072] The step of contacting the vector or parasite with the composition can be performed in any suitable manner. For example, a person need not physically contact the vector or parasite with the Delftia bacteria and / or 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof. The composition can be placed where it will come into contact with the vector. The composition can be in a form as described above or below.

[0073] In certain embodiments of the present invention, the contacting can be achieved by treating the area with the composition of the present invention, for example, by using a spray formulation, such as an aerosol or pump spray. In certain embodiments of the present invention, the area can be treated, for example, by aerial delivery, by a truck-mounted device, etc. In some embodiments, the composite material is sprayed, for example, by backpack spraying, aerial spraying, spraying / dusting, etc. The vector or parasite can be any parasitic vector capable of transmitting disease. In some embodiments, the vector is a mosquito, a black fly, a subfamily of Triatominae, a sand fly, or a tsetse fly.

[0074] It is contemplated that the compositions and methods of the present invention extend to any species of mosquito. In some embodiments, the mosquito is of the genus Anopheles. In some embodiments, the mosquito is Anopheles gambiae, Anopheles stephensi, Anopheles cucullatus, or Anopheles coluzzi. In some embodiments, the mosquito is Anopheles stephensi or Anopheles gambiae. In one embodiment, the mosquito is Anopheles stephensi. In another embodiment, the mosquito is Anopheles gambiae.

[0075] For filariasis, the method involves contacting a mosquito or blackfly with methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof. The mosquito can be any mosquito or blackfly capable of transmitting filariasis. It is contemplated that the compositions and methods of the present invention extend to any species of mosquito or blackfly.

[0076] In some embodiments, heartworm disease is caused by Dirofilaria immitis, Dirofilaria filariae, or Dirofilaria immitis. In some embodiments, heartworm disease is caused by Dirofilaria immitis. In some embodiments, heartworm disease is caused by Dirofilaria immitis. In some embodiments, heartworm disease is caused by Dirofilaria immitis.

[0077] In some embodiments, the filariasis is caused by a worm of the order Filariales. In some embodiments, the worm is Wuchereria bancrofti, Brugia malayi, or Brugia volvulus. In some embodiments, the worm is Loa loa, Wireworm, or Onchocerca volvulus. In some embodiments, the worm is Mansonella volvulus or Mansonella ostreatus.

[0078] For Chagas disease, the method involves contacting an infected Triatominae with a composition of the present invention. The Triatominae can be any Triatominae that can transmit Chagas disease, such as Triatominae harassinga, Triatominae bifida, or Triatominae rubripes. In some embodiments, the Triatominae is Triatominae harassinga. In some embodiments, the Triatominae is Triatominae bifida. In some embodiments, the Triatominae is Triatominae rubripes. The parasite can be any parasite that causes Chagas disease. In some embodiments, the parasite is Trypanosoma cruzi.

[0079] For leishmaniasis, the methods involve contacting infected sand flies with a composition of the present invention. In some embodiments, the methods involve contacting infected sand flies with a composition comprising bacteria from the genus Delftia. The sand fly can be any sand fly capable of transmitting Leishmania. It is believed that the compositions and methods of the present invention extend to any species of sand fly. In some embodiments, the sand fly is of the genus Phlebotomus or Lophatherum.

[0080] The parasite can be any leishmaniasis parasite. In some embodiments, the parasite is Leishmania braziliensis, Leishmania donovani, Leishmania infantum, Leishmania chagai, Leishmania panama (Leishmaniapanamensis), Leishmania guyanensis (Leishmania guayanensis), Leishmania amazonensis (Leishmania amazonensis), Leishmania mexicanus, Leishmania tropicalis or Leishmania major subspecies (Leishmania major). In some embodiments, the parasite is Leishmania donovani. In some embodiments, the parasite is Leishmania infantum. In some embodiments, the disease treated is visceral leishmaniasis. In another embodiment, the disease treated is cutaneous leishmaniasis.

[0081] For human African trypanosomiasis, the method involves contacting an infected tsetse fly with a composition of the present invention. The tsetse fly can be any tsetse fly capable of transmitting human African trypanosomiasis. It is believed that the compositions and methods of the present invention extend to any species of tsetse fly. In some embodiments, the tsetse fly is a palpable tsetse fly.

[0082] The parasite can be any parasite that causes African trypanosomiasis in humans. In some embodiments, the parasite is selected from Trypanosoma brucei gambiense (TbG) and Trypanosoma brucei rhodesiense (TbR). In some embodiments, the parasite is selected from Trypanosoma brucei gambiense (TbG). In some embodiments, the parasite is Trypanosoma brucei rhodesiense (TbR).

[0083] For Cryptosporidium, the method involves contacting Cryptosporidium with a composition of the present invention. Cryptosporidium is a parasitic cystic worm of the phylum Cryptosporidium that causes respiratory and gastrointestinal diseases. The parasite can be any parasite that causes Cryptosporidium, and it is believed that the compositions and methods of the present invention extend to any Cryptosporidium parasite. In some embodiments, the parasite is selected from Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium canis, Cryptosporidium felis, Cryptosporidium meleagridid, and Cryptosporidium muris.

[0084] In another aspect, the present invention provides the use of a bacterium of the genus Delftia and / or 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof in reducing or preventing vector-borne diseases or parasitic infections.

[0085] Composition

[0086] The composition of the present invention may be in any suitable form and may include any suitable carrier. The composition may be a feed composition, that is, the composition may be in a form that can be provided to a medium for consumption by oral administration. In one embodiment, the feed composition is a sugar source or nectar feed. In some embodiments, the feed composition is a sugar source. The sugar source may be an attractive sugar bait, or contained within an attractive sugar bait. The attractive sugar bait comprises sugar and a toxic component. It is contemplated that the attractive sugar bait according to the present invention comprises the composition of the present invention but not the toxic component, i.e., comprises sugar and the composition of the present invention. In one embodiment, the composition is in the form of a bait. The bait is designed to attract a medium (e.g., a mosquito) to contact the composition. In some embodiments, upon contact with the medium, the composition is then internalized by the medium or parasite (e.g., a mosquito), for example, by ingestion. Attractants may also be used. The attractant may be a pheromone, such as a male or female pheromone. The attractant is used to lure the medium (e.g., a mosquito) to the bait. The bait may be in any suitable form, such as a solid, paste, pellet, or powder.

[0087] The bait can be provided in a suitable "housing" or "trap". Such housings and traps are commercially available, and existing traps can be adapted to include the composition of the present invention. The housing or trap can, for example, be box-shaped and can be provided in a preformed state, or can, for example, be formed from foldable cardboard. Suitable materials for the housing or trap include plastics and cardboard, particularly corrugated cardboard. The inner surface of the trap can be lined with a sticky substance so that once a vector or parasite (e.g., mosquito) is in the trap, its movement is restricted. The housing or trap can contain a suitable groove in which the bait can be held in place. The trap is distinguished from the housing because mosquitoes cannot easily leave the trap after entering, while the housing acts as a "feeding station", which provides a preferred environment for vectors (e.g., mosquitoes) in which they can eat and feel safe from predators.

[0088] In another embodiment, the present invention provides a mosquito nectar diet comprising a bacterium of the genus Delftia and / or 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and one or more sugar sources.

[0089] In a further aspect of the invention, bacteria of the genus Delftia may be used in disease control strategies based on direct exposure of larval or adult stage vectors to the bacteria. Exposure may be achieved by direct administration of the bacteria.

[0090] Alternatively, bacteria can be deployed directly to colonize local vector populations.

[0091] The bacteria can be delivered by any suitable method and combined with the delivery agent in any suitable manner that allows the composition to be applied to the vehicle.For example, the vehicle can be contacted with the bacteria in pure or substantially pure form, such as a solution containing Delftia.

[0092] In specific embodiments, the bacterium of the genus Delftia is in the composition with the delivery agent.

[0093] In another specific embodiment, the larval forms of vectors (eg, mosquitoes) can simply be "dipped" or "sprayed" with a solution containing the bacteria.

[0094] combination

[0095] It is contemplated that the present invention will be deployed in conjunction with other anti-vector or anti-parasitic eradication measures. For example, compositions, methods, and compounds for the present invention may be used together with known anti-vector or anti-parasitic agents (e.g., antimalarial agents). In some embodiments, compositions or compounds for the present invention may be used in combination with one, two, or three other anti-parasitic agents (e.g., antimalarial agents). Integrated vector management (IVM) suggests making full use of existing tools.

[0096] At least one other antimalarial agent may also be selected from ferrocenechloroquine, KAF156, cipargamin, DSM265, artemisone, artemisinone, artefenomel, MMV048, SJ733, P218, MMV253, PA92, DDD498, AN13762, DSM421, UCT947, ACT 451840, OZ609, OZ277 and SAR97276. In the treatment of Plasmodium falciparum infection, at least one, two or three additional antimalarial agents may be selected from the following list, wherein at least one antimalarial agent is an artemisinin-based agent: artemether and lumefantrine, artesunate and amodiaquine, artesunate and mefloquine, dihydroartemisinin and piperaquine, or artesunate and sulfadoxine-pyrimethamine (SP). The above combination therapy is referred to as artemisinin-based combination therapy (ACT). The choice of ACT is generally based on the results of therapeutic efficacy studies against local strains of Plasmodium falciparum malaria. In the treatment of Plasmodium vivax infection, an ACT as described above may be used. Alternatively, at least one other antimalarial agent may be chloroquine, particularly in areas where there are no chloroquine-resistant Plasmodium vivax. In areas where resistant Plasmodium vivax has been identified, the infection may be treated with an ACT as described above. The combination of therapeutic agents may conveniently be presented for use in the form of a pharmaceutical composition or formulation and may be administered together or separately, and when administered separately, this may occur separately or sequentially in any order (via the same or different routes of administration).

[0097] The compositions or bacteria used in the present invention can be used in combination with insecticide-treated nets (ITNs), including long-lasting insecticidal nets (LLINs), and / or IRS (indoor residual sprays). ATSBs (attractive toxic sugar baits) lure mosquitoes to feed on sugars containing toxic mosquito-killing compounds. For increased efficiency, ATSBs can also include the harman compounds discussed above in place of toxic compounds.

[0098] Example

[0099] The present invention will now be described by the following non-limiting examples. Although specific embodiments of the present invention are described below, it will be understood by those skilled in the art that various changes and modifications may be made. Reference to preparations made in a manner similar to other preparations or by other general methods of preparation may encompass variations in conventional parameters (such as time, temperature, post-processing conditions, and slight variations in reagent dosage, etc.).

[0100] Supernatant preparation

[0101] Delftia tsuruhada TC1 was grown overnight in LB liquid medium (200 rpm, 28 °C). The bacteria were washed and resuspended in M9 medium (10 9 / ml) and incubated (200 rpm, 28 ° C) for 8 h, then centrifuged and the supernatant passed through a 0.22 μm filter to produce Delftia tsuruhada TC1 (D-8h). The Delftia tsuruhada TC1 supernatant was passed through a 3 kDa centrifugal filter (Amicon Ultra-3K, REF: UFC500396) to produce a <3 kDa component.

[0102] Example 1: Bioassay-guided purification of active natural components from the supernatant of Delftia tsuruhata TC1

[0103] The fermentation supernatant (10 L) was loaded onto a C-18 reverse-phase silica gel column (160 x 30 mm; Sepra™ C-18-E (50 µm, 65 Å)) for flash fractionation.

[0104] The material not retained on the column (collected for subsequent activity testing) was eluted isocratically (H2O / CH3CN 95:5) from the column, followed by a gradient from 5% to 100% acetonitrile (CH3CN) in water over 40 minutes, with an isocratic step of 100% acetonitrile at 10 mL / min for 20 minutes. 18 mL fractions were collected. UV detection was used at 210 and 280 nm.

[0105] Separately, 100 mL of unfermented medium was loaded onto a 60 × 15 mm C-18 column and eluted with 100 mL of 100% CH 3 CN, which was dried (12.5 mg) and dissolved in 1 mL of MeOH.

[0106] 500 μL of supernatant, 500 μL aliquots of each fraction, 500 μL of subsequent passthrough obtained when 10 L of supernatant was loaded onto the C-18 column, and 100 μL of blank medium extract were transferred to AB-Gene 0765 800 μL 96-well storage plates and dried in an HT-8 Genevac vacuum centrifuge for shipping and activity assessment.

[0107] LC-HRMS deduplication method

[0108] Active fractions were analyzed using an Agilent 1200 Rapid Resolution HPLC coupled to a Bruker maXis mass spectrometer. The injected sample volume was 2 μL. Separation was performed using a Zorbax SB-C8 column (2.1 × 30 mm, 3.5 μm particle size). Two mobile phases were used: Solvent A: H2O:CH3CN 90:10 and Solvent B: Water:CH3CN 10:90, both containing 13 mM ammonium formate and 0.01% TFA. The gradient composition was:

[0109]

[0110] The mass spectrometer was operated in positive ESI mode. Instrument parameters included a capillary voltage of 4 kV, a drying gas flow rate of 11 L / min at 200°C, and a nebulizer pressure of 2.8 bar. Before sample injection, the instrument was mass calibrated using TFA-Na cluster ions. Before each sample analysis, recalibration was performed by injecting the same TFA-Na calibration solution before the chromatographic front.

[0111] NMR deduplication methods

[0112] For NMR analysis, samples were dissolved in CD3OD. After dissolution, each sample was transferred to a 1.7 mm tube. Acquisition was performed on a Bruker AVANCE III 500 MHz spectrometer equipped with a 1.7 mm TCI micro-cryoprobe (1D 1 H spectra and 2D HSQC spectra). All spectra were recorded at 24°C.

[0113] result

[0114] like Figure 1 As shown in Figure 2, the molecular formula of the active ingredient was determined to be C from LC-HRMS. 12 H 10 N2.

[0115] like Figure 2 and Figure 3 As shown, the HSQC and 1 The H NMR spectrum matched that of Harman.

[0116] Example 2: Effect of Delftia tsuruhata on the establishment of Leishmania parasites in the midgut of sand flies.

[0117] Materials and methods for determining the role of Delftia TC1 in blocking Leishmania infection in sand flies

[0118] Parasite

[0119] A clone of Leishmania major subspecies (WR 2885) was used [Cecílio P, Pires ACAM, Valenzuela JG, et al. Exploring Lutzomyia longipalpis sand fly vector competence for Leishmania major parasites, The Journal of Infectious Diseases. 2020. 222(7):1199-1203. https: / / doi.org / 10.1093 / infdis / jiaa203 .].

[0120] Promastigotes were maintained at 26°C in Schneider insect medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 mg / mL streptomycin (all Thermo Fischer Scientific).

[0121] mice

[0122] Six-week-old female BALB / c mice were obtained from Charles River Laboratories and housed under pathogen-free conditions at the NIAID Winbrook Animal Facility (Rockville, MD) with free access to water and food.

[0123] Sandfly

[0124] Phlebotomus duboscqui sand flies were mass reared in the insect rearing laboratory of the Malaria and Vector Research Laboratory as described by Lawyer P, Killick-Kendrick M, Rowland T, et al. Laboratory colonization and mass rearing of phlebotomine sand flies (Diptera, Psychodidae). Parasite. 2017. 24:42.

[0125] Adult female animals were maintained on a 30% sucrose diet and starved for 12 h before feeding.

[0126] Data representation and statistics

[0127] Results are shown for each individual sand fly / mouse, obtained from at least two independent experiments, and are expressed as group mean ± SD. Statistical analysis was performed using GraphPad Prism software v6.01. Statistical differences were assessed using the nonparametric Mann-Whitney test, with a minimum P ≤ .05.

[0128] Figure 4: Effects of the establishment of Leishmania macroparasites in sand flies fed with Delftia tsuruhada TC1 bacteria

[0129] 5-7 day old P. duboscqi sand flies were fed with a suspension of Delftia tsuruhae TC1 bacteria (1x10 8 CFU / ml in 5% sucrose solution) were applied to cotton rolls (denoted as “bacteria” in the figure) for 24 h; the sand flies in the control group (denoted as “control” in the figure) were given only 5% sucrose.

[0130] After an overnight starvation period, the chickens were fed artificially with a diet containing Leishmania subsp. 6 Sand flies were infected with defibrinated rabbit blood (Spring Valley Laboratories, MD, USA) at 4% leishmania / ml, as described in Cecílio P, Oristian J, Meneses C, et al. (Engineering a vector-based pan-Leishmania vaccine for humans: proof of principle). Scientific Reports. 2020. 10:18653. https: / / doi.org / 10.1038 / s41598-020-75410-0 As stated.

[0131] After infection, females fed with blood were sorted out and maintained on a 30% sucrose diet. Sand flies were collected 7 days (and / or 8 days) and 11 days after infection to assess infection status. Briefly, under a stereomicroscope, the sand fly midgut was dissected in PBS and transferred to a single microtube (Denville Scientific) with 50 μL of formalin solution (0.005% in PBS). The midgut was homogenized and 10 μL was loaded onto a disposable Neubauer chamber (Incyto). Slides were observed at 400× magnification under a phase contrast microscope (Zeiss).

[0132] Determining total parasite counts and metacyclic frequencies [Cecílio P, Pires ACAM, Valenzuela JG, et al. Exploring Lutzomyia longipalpis sand fly vector competence for Leishmania major parasites, The Journal of Infectious Diseases. 2020. 222(7):1199-1203. https: / / doi.org / 10.1093 / infdis / jiaa203 .].

[0133] Data are presented as dot plots, with each symbol corresponding to an individual midgut. Statistical differences were assessed using the nonparametric Mann-Whitney test; values ​​of at least P ≤ .05 indicated statistical significance.

[0134] The data illustrated in Figure 4 (A, B, and C) show that exposure of sand flies to Delftia tsuruhada TC1 affects the establishment of Leishmania parasites in the sand fly midgut. As can be seen, the parasite levels of sand flies exposed to the bacteria were significantly reduced compared to sand flies not treated with Delftia tsuruhada TC1 bacteria. Leishmania-infected sand flies exposed to Delftia tsuruhada TC1 showed a significant reduction in parasite burden (p < 0.0001) (Figure 4C) compared to sand flies treated with Delftia tsuruhada TC1 on days 7 (p < 0.5) (Figure 4A) or 8 (p < 0.01) (Figure 4B) after Leishmania infection.

[0135] The data illustrated in Figure 4 (D, E, F, and G) show that exposure of sand flies to D. tsuruhada TC1 did not affect the percentage of metacyclic parasites, but the total number of parasites in the midgut was significantly reduced in sand flies exposed to the bacteria compared to sand flies not exposed to D. tsuruhada TC1 bacteria.

[0136] Figure 5: Effects of i) feeding and (ii) timing of exposure to Delftia tsuruhada TC1 bacteria on the establishment of Leishmania macrospecies in sand flies

[0137] 5-7 day old P. duboscqi sand flies were fed with a suspension of TC1 bacteria (1x10 8 CFU / ml in 5% sucrose solution) for 24 h; the control group of sand flies (denoted as "control" in the figure) was given only 5% sucrose.

[0138] Sand flies were maintained on a 30% sucrose diet with free access for 6 days and then, after an overnight starvation period (7 days after bacterial feeding), artificially fed with a chicken membrane containing Leishmania large subspecies promastigotes (5 × 10 6 Sand flies were infected with defibrinated rabbit blood (Spring Valley Laboratories, MD, USA) at 4 °C / ml as described previously.

[0139] After infection, blood-fed females were sorted and maintained on a 30% sucrose diet. Sand flies were collected 7 and 11 days after infection to assess infection status as described previously.

[0140] Figure 5AThe data presented show that exposure of sand flies to Delftia tsuruhae TC1 affects the establishment of Leishmania parasites in the sand fly midgut, even when exposure to the bacteria occurs one week prior to infection.

[0141] 5-7 day old P. duboscqi sand flies that had been starved overnight were fed artificially with a diet containing Leishmania large subspecies promastigotes (5x10 6 The infection was carried out with defibrinated rabbit blood (Spring Valley Laboratories, MD, USA) containing 100 μg / ml of blood. After infection, the females that had fed blood were sorted and maintained on a 30% sucrose diet. Five days after infection, when all blood residues had been defecate, the sand flies were fed a TC1 bacterial suspension (1×10 8 Sand flies were then placed on cotton rolls impregnated with 5% sucrose (CFU / ml in a 5% sucrose solution) for 24 hours; a control group of sand flies received only 5% sucrose. Sand flies were then maintained on a 30% sucrose diet with free access until the end of the experiment. Sand flies were collected on days 7 and 11 post-infection to assess infection status as described previously.

[0142] Figure 5B The data presented show that exposure of previously infected sand flies to Delftia tsuruhae TC1 affects the establishment of Leishmania parasites.

[0143] 5-7 day old P. duboscqi sand flies that had been starved overnight were fed artificially with a diet containing Leishmania large subspecies promastigotes (5x10 6 The infection was carried out with defibrinated rabbit blood (Spring Valley Laboratories, MD, USA) containing 100 μg / ml of blood. After infection, the females that had fed blood were sorted and maintained on a 30% sucrose diet. Eight days after infection, when the infection began to mature, the sand flies were fed a suspension of TC1 bacteria (1×10 8 Sand flies were then placed on cotton rolls soaked in a 5% sucrose solution (CFU / ml) for 24 hours; a control group of sand flies received only 5% sucrose. Sand flies were then maintained on a 30% sucrose diet with free access until the end of the experiment. Twelve days after infection, sand flies were harvested and assessed for infection status as described previously.

[0144] Figure 5C The data shown show that exposure of sand flies carrying mature infections to Delftia tsuruhae TC1 affects the establishment of Leishmania parasites.

[0145] Figure 6 Effects of a second blood meal on Leishmania macroparasites in sand flies fed with Delftia tsuruhada TC1 bacteria

[0146] 5-7 day old P. duboscqi sand flies were fed with TC1 bacterial suspension (1x108 CFU / ml in 5% sucrose solution) for 24 h; the control group of sand flies was given only 5% sucrose. After overnight starvation, the sand flies were artificially fed with a diet containing promastigotes of Leishmania subsp. 6 / ml) of defibrinated rabbit blood (Spring Valley Laboratories, MD, USA) and infected with sand flies as described previously.

[0147] After infection, females that had blood fed were sorted out and maintained on a 30% sucrose diet. Twelve days after infection, sand flies were allowed a second blood meal. Briefly, sand flies were allowed to feast on blood for 1 hour on anesthetized, naive mice. Females that had blood fed were sorted out and maintained on a 30% sucrose diet until the end of the experiment. Six days after the second, naive blood meal, sand flies were collected to assess infection status as previously described.

[0148] Figure 6 The data presented show that the infection rate in TC1-colonized sand flies that survived the second blood meal remained significantly lower than that of untreated controls, indicating that the second blood meal did not reverse the infection phenotype.

[0149] Figure 7 Effects of bites on Leishmania macrophylla parasite infection in sand flies fed with Delftia tsuruhada TC1 bacteria

[0150] BALB / c mice were intradermally infected with 1000 Leishmania major subspecies metacyclic parasites in the pinnae and maintained with free access to water and food until typical cutaneous leishmaniasis lesions appeared. Subsequently, sand flies were fed a blood meal at the ear with active lesions using a custom-made fixture using a vial with a mesh surface held in place. The blood-fed sand flies were divided into two groups. One group was then allowed to feed bacteria overnight (1x10 8 11 days after infection, sand flies from both groups were dissected to assess their infection status as described previously.

[0151] Figure 7 The data presented show that TC1-colonized sand flies exhibited reduced midgut parasite loads and a lower prevalence of infection when naturally infected by biting mice infected with skin lesions.

[0152] Figure 8: Mortality of sand flies exposed to Delftia tsuruhada TC1 bacteria

[0153] 5-7 day old P. duboscqi sand flies were fed with TC1 bacterial suspension (1x10 8CFU / ml in 5% sucrose solution) for 24 h; the control group of sand flies was given only 5% sucrose.

[0154] Sand flies fed with bacteria or control sand flies, sand flies not fed with blood, sand flies artificially fed with uninfected blood, or sand flies artificially fed with promastigotes of Leishmania subsp. 6 Sand flies fed 10% sucrose / ml of blood were placed into cardboard pints (100 sand flies per pint) and maintained on a 30% sucrose diet for 11-18 days.

[0155] Sandfly mortality was recorded daily, and dead sandflies were also removed daily. In addition, only infected sandflies were given a second blood meal (as described above) on the 12th day after infection, and mortality was also recorded daily, considering only the blood fed to the sandflies in the subsequent 6 days. Data are presented in the form of Kaplan-Meier curves and as dot plots for each individual time point, with each symbol referring to a group of 100 sandflies (one blood meal) or as part of a whole (second blood meal data). Nonparametric Kruskal-Wallis tests were used with post hoc analysis to assess statistical differences; values ​​of at least P≤.05 were considered to be statistically relevant.

[0156] The data shows Figure 8A Exposure to TC1 bacteria slightly increased sand fly mortality but significantly affected infected sand flies' survival. After the second blood meal, mortality was significantly increased only in TCl-colonized sand flies infected with Leishmania macrophylla parasites.

[0157] The data showed that TC1 bacteria not only reduced the infection burden of sand flies, but also reduced the overall number of infected sand flies.

[0158] Figure 9: Experiment to determine whether the reduction in Leishmania macroparasite load in sand flies is specific to live Delftia TC1 (a Gram-negative bacterium) or applies to all Gram-negative bacteria

[0159] 5-7 day old P. duboscqi sand flies were fed with a suspension of Gram-negative ampicillin-resistant Escherichia coli or a suspension of Ornithinibacillus massiliensis isolated from the microbiota of healthy P. duboscqi sand flies from our colony (1x10 8 CFU / ml, dissolved in 5% sucrose solution) for 24 h; the control group of sand flies was given only 5% sucrose. After an overnight starvation period, the sand flies were artificially fed with a diet containing promastigotes of Leishmania subsp. 6Sand flies were infected with defibrinated rabbit blood (Spring Valley Laboratories, MD, USA) at 4% 4% (v / ml) as described previously. After infection, females that had fed blood were sorted and maintained on a 30% sucrose diet.

[0160] After infection (7 days, if applicable) and 11 days, sand flies were collected to assess infection status as described previously. Figure 9A The data presented show that Escherichia coli and Ornithinibacillus massiliensis do not have the same effect as Delftia tsuruhaeida on parasitic infections in the sand fly midgut. The observed effect of TC1 is not general for Gram-negative bacterial infections but is specific to TC1.

[0161] Live TC1 bacteria were heat-inactivated at 95°C for 10 minutes to produce dead TC1 bacteria. 5-7 day old P. duboscqi sand flies were fed with live or dead TC1 bacterial suspensions (1×10 8 CFU / ml in 5% sucrose solution) for 24 h; the control group of sand flies was given only 5% sucrose. After an overnight starvation period, the sand flies were artificially fed with a diet containing promastigotes of Leishmania subsp. 6 Sand flies were infected with 5% (40%) defibrinated rabbit blood (Spring Valley Laboratories, MD, USA) at 4% (1 / ml) of 4% (2 / 10) spherical blood as described previously. After infection, females that had fed blood were sorted and maintained on a 30% sucrose diet.

[0162] Eleven days after infection, sand fly samples were collected to assess infection status as described previously. Figure 9B The data presented show that dead D. tsuruhae did not have the same effect as live D. tsuruhae on parasite infection in the midgut of sand flies.

[0163] FIG10 illustrates the effect of Delftia tsuruhata on blocking the transmission of Leishmania disease to uninfected mice.

[0164] 5-7 day old P. duboscqi sand flies were fed with TC1 bacterial suspension (1x10 8 CFU / ml in 5% sucrose solution) for 24 h; the control group of sand flies was given only 5% sucrose. After an overnight starvation period, the sand flies were artificially fed with a diet containing promastigotes of Leishmania subsp. 6 Sand flies were infected with defibrinated rabbit blood (Spring Valley Laboratories, MD, USA) at 4 °C / ml as described previously.

[0165] After infection, females that had fed blood were sorted and maintained on a 30% sucrose diet. Eleven days after infection, 20 sand flies were applied to each ear of BALB / c mice using a mesh-surfaced vial secured in place with a custom-made clamp and allowed to feed for at least 30 minutes. The number of sand flies that had fed blood was determined by observing them under a stereomicroscope. Thereafter, mice were monitored weekly to track the development of lesions caused by infection with Leishmania major subspecies.

[0166] Images of individual ears were captured weekly using a smartphone ( Figure 10A Animals were euthanized 4 weeks after infection to determine parasite burden by limiting dilution as described in Dey R, Joshi AB, Oliveira F, et al. Gutmicrobes egested during bites of infected sand flies augment severity of leishmaniasis via inflammasome-derived IL-1β. Cell Host Microbe. 2018. 23:134–43.e6. https: / / doi.org / 10.1016 / j.chom.2017.12.002 The data are reported in Figure 10B Data are presented as dot plots, with each symbol corresponding to an individual mouse ear. Statistical differences were assessed using the nonparametric Mann-Whitney test; values ​​of at least P ≤ 0.05 indicated statistical significance.

[0167] Figure 10A : Mice bitten by sand flies infected with Leishmania previously exposed to D. tsuruda showed no lesions in their ears caused by infection with Leishmania major subspecies compared to mice bitten by sand flies infected with D. tsuruda that were not exposed (images in TEST panel).

[0168] Figure 10B : The parasite burden in the ears of mice bitten by Leishmania-infected sand flies previously exposed to Delftia tsuruhada (bacteria) was significantly reduced compared to mice bitten by infected sand flies that were not exposed to Delftia tsuruhada (control).

[0169] Figure 10C : Sand flies colonized with TC1 bacteria were better able to take a second blood meal because the reduced infection caused by TC1 led to less intestinal blockage.

[0170] The data showed that Delftia TC1 bacteria affected the development of Leishmania macroparasites in P. duboscqi sand flies, as demonstrated by a significant (90% reduction) in the number of total parasites and infective, post-circulating forms in bacteria-fed sand flies relative to control sand flies. This phenotype was consistently observed regardless of the timing of bacterial feeding (one week before infection vs. one day before infection vs. five days after infection) and was even enhanced when sand flies were given a second, uninfected blood meal. Furthermore, TC1 bacteria not only reduced the infection load of sand flies, but also reduced the total number of infected sand flies, leading to increased mortality in sand flies infected with Leishmania, but had no effect on sand flies fed sugar or blood (uninfected blood meal). The data also showed that in the context of a mouse model of cutaneous leishmaniasis, sand flies infected with Leishmania and fed TC1 bacteria were less able to transmit Leishmania macroparasites and cause disease (active lesions were observed in 25% of animals bitten by bacteria-fed sand flies compared to 80% of controls; parasites were detected in 27% of animals bitten by bacteria-fed sand flies compared to 100% of controls).

[0171] Figure 11 : Role of Harman in blocking Zika virus replication in Aedes aegypti mosquitoes

[0172] 3- to 5-day-old Aedes aegypti strain SBE mosquitoes were fed with a membrane feeder containing approximately 10 7 PFU / ml Zika virus and a blood meal (rabbit blood) containing the indicated concentrations of Harman dissolved in dimethyl sulfoxide (DMSO). A control group was fed a blood meal without Harman. Fully fed mosquitoes were selected and maintained on a 10% sucrose diet. A total of 30 mosquitoes were analyzed per treatment.

[0173] After 7 days, mosquito abdomens were dissected and placed in 2 mL tubes containing glass beads and 300 µL of DMEM medium. The samples were homogenized. Serial 1:10 dilutions of this solution were distributed onto 96-well plates containing VERO cells. After 5-6 days, the medium was discarded, and viral plaques were fixed and developed with a stain (1% crystal violet in a 1:1 methanol / acetone solution) for 1 hour at room temperature.

[0174] The plate was rinsed with distilled water and air-dried. Virus plaques were counted and multiplied by the corresponding dilution factor to calculate the number of plaque forming units (PFU). The number of PFU is shown in Figure 11 The horizontal red line is the median. A dose-dependent decrease in PFU / mL was observed in Harman-treated samples compared to the control group. Statistical differences were assessed using the nonparametric Mann-Whitney test. No significant differences were observed in the treated groups compared to the DMSO control.

[0175] Figure 11Data from the study showed that levels of Zika virus were reduced in mosquitoes that ingested Harman.

Claims

1. A composition for use in a method of reducing or preventing the transmission of a vector-borne disease, wherein the composition comprises a bacterium of the genus Delftia and / or 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and wherein the method comprises the step of contacting at least one vector with the composition, wherein the vector-borne disease is selected from the group consisting of human African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi, Bourbon virus, Borrelia merini, Chikungunya virus, Chagas disease, dirofilariasis, Eastern equine encephalitis, murine ehrlichiosis, ehrlichiosis, filariasis, Central Plains virus, Japanese encephalitis, leishmaniasis, Lyme disease, Mayaro virus disease, Pacific Coast tick fever, Rocky Mountain spotted fever, St. Louis encephalitis, tularemia, Western equine encephalitis, West Nile virus, yellow fever, and Zika virus.

2. A composition for use in a method of reducing or preventing the transmission of parasites, wherein the composition comprises a bacterium of the genus Delftia and / or 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, and wherein the method comprises the step of contacting at least one agent with the composition, The parasitic disease is selected from the group consisting of human African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi, Borrelia merini, Chagas disease, cryptosporidium, dirofilariasis, murine ehrlichiosis, ehrlichiosis, filariasis, leishmaniasis, Lyme disease, Pacific Coast tick fever, Rocky Mountain spotted fever, and tularemia.

3. The composition for use according to claim 1 or claim 2, wherein the bacterium is Delftia tsuruhada and / or wherein the composition further comprises at least one carrier, delivery vehicle, adjuvant, solvent, stabilizer or preservative.

4. The composition for use according to any one of claims 1 to 3, wherein the composition comprises an attractant.

5. The composition for use according to claim 4, wherein the attractant is a sugar source or a pheromone.

6. The composition for use according to any one of claims 1 to 5, wherein the composition is used to reduce or prevent: (i) Zika virus, and / or (ii) Zika virus transmission.

7. The composition for use according to claim 6, wherein the composition is used to reduce or prevent: (i) Zika virus in mosquitoes, and / or (ii) Zika virus transmission.

8. The composition for use according to any one of claims 1 or 3 to 5, wherein the composition is used to reduce or prevent: (i) Chikungunya virus, and / or (ii) Chikungunya virus transmission.

9. The composition for use according to claim 8, wherein the composition is used to reduce or prevent: (i) Chikungunya virus in mosquitoes, and / or (ii) Chikungunya virus transmission.

10. The composition for use according to any one of claims 1 to 5, wherein the composition is for reducing or preventing: (i) filariasis or dirofilariasis, and / or (ii) the transmission of filariasis or dirofilariasis.

11. The composition for use according to claim 10, wherein the composition is used to reduce or prevent: (i) filariasis or dirofilariasis, and / or (ii) transmission of filariasis or dirofilariasis in mosquitoes.

12. The composition for use according to any one of claims 1 or 3 to 5, wherein the composition is used to reduce or prevent: (i) Mayaro virus, and / or (ii) Mayaro virus transmission.

13. The composition for use according to claim 12, wherein the composition is used to reduce or prevent: (i) Mayaro virus in mosquitoes, and / or (ii) Mayaro virus transmission.

14. The composition for use according to any one of claims 1 or 3 to 5, wherein the composition is used to reduce or prevent: (i) West Nile virus, and / or (ii) West Nile virus transmission.

15. The composition for use according to claim 14, wherein the composition is used to reduce or prevent: (i) West Nile virus in mosquitoes, and / or (ii) West Nile virus transmission.

16. The composition for use according to any one of claims 6 to 15, wherein the mosquito is a mosquito of the genus Aedes.

17. The composition for use according to claim 16, wherein the mosquito is Aedes albopictus or Aedes aegypti.

18. The composition for use according to any one of claims 1 to 5, wherein the composition is for reducing or preventing: (i) Chagas disease, and / or (ii) the transmission of Chagas disease.

19. The composition for use according to claim 18, wherein the composition is for reducing or preventing: (i) Chagas disease, and / or (ii) the transmission of Chagas disease in the subfamily Conophyceae.

20. The composition for use according to claim 18 or 19, wherein the Triatominae is Triatominae bisecta.

21. The composition for use according to any one of claims 1 to 5, wherein the composition is for reducing or preventing: (i) human African trypanosomiasis, and / or (ii) the transmission of human African trypanosomiasis.

22. The composition for use according to claim 21, wherein the composition is for reducing or preventing: (i) human African trypanosomiasis, and / or (ii) transmission of human African trypanosomiasis in tsetse flies.

23. The composition for use according to claim 22, wherein the tsetse fly is Glossina palpalis.

24. The composition for use according to any one of claims 1 to 5, wherein the composition is for reducing or preventing: (i) leishmaniasis, and / or (ii) the transmission of leishmaniasis.

25. The composition for use according to claim 24, wherein the composition is for reducing or preventing: (i) leishmaniasis in sand flies, and / or (ii) the transmission of leishmaniasis.

26. The composition for use according to claim 25, wherein the sand fly is of the genus Phlebotomus or Loblolly.

27. The composition for use according to any one of claims 2, 4 and 5, wherein the composition is used to reduce or prevent: (i) Cryptosporidium, and / or (ii) Cryptosporidium transmission.

28. A method for reducing or preventing the transmission of a vector-borne disease or parasitic disease comprising the step of contacting at least one vector of said vector-borne disease with a bacterium of the genus Delftia and / or 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof, The vector-borne disease or parasitic disease is selected from the group consisting of human African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi, Bourbon virus, Borrelia merini, Chikungunya virus, Chagas disease, Cryptosporidium, dirofilariasis, Eastern equine encephalitis, murine ehrlichiosis, ehrlichiosis, filariasis, Central Plains virus, Japanese encephalitis, leishmaniasis, Lyme disease, Mayaro virus disease, Pacific Coast tick fever, Rocky Mountain spotted fever, St. Louis encephalitis, tularemia, Western equine encephalitis, West Nile virus, yellow fever and Zika virus.

29. Use of a bacterium of the genus Delftia and / or 1-methyl-9H-pyrido[3,4-b]indole or a pharmaceutically acceptable salt thereof for reducing or preventing vector-borne or parasitic diseases. The vector-borne disease or parasitic disease is selected from the group consisting of human African trypanosomiasis, anaplasmosis, babesiosis, Borrelia miyamotoi, Bourbon virus, Borrelia merini, Chikungunya virus, Chagas disease, Cryptosporidium, dirofilariasis, Eastern equine encephalitis, murine ehrlichiosis, ehrlichiosis, filariasis, Central Plains virus, Japanese encephalitis, leishmaniasis, Lyme disease, Mayaro virus disease, Pacific Coast tick fever, Rocky Mountain spotted fever, St. Louis encephalitis, tularemia, Western equine encephalitis, West Nile virus, yellow fever and Zika virus.

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